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DATE DUE DATE DUE DATE DUE 6/01 c;lClRC/DateDue.p65-p.15 MOLECULAR MECHANISMS REGULATING THE MIXED LINEAGE KINASE MLK3 By Panayiotis Orestes Vacratsis A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry and Molecular Biology 2001 ABSTRACT MECHANISMS REGULATING THE MIXED LINEAGE KINASE-3 By Panayiotis Orestes Vacratsis Mixed lineage kinase 3 (MLK3) is an intracellular serine/threonine kinase with a predicted molecular mass of 93 kDa. MLK3 functions as a mitogen activated protein kinase kinase kinase to activate the c-Jun NHz-terminal kinase pathway (INK). In addition to the kinase domain, the sequence of MLK3 encodes several domains that are predicted to be involved in protein-protein interactions, including a Src-homology 3 (SH3) domain, a leucine zipper domain, a Cdc42/Rae interactive binding (CRIB) motif and a C-terminal region of 220 amino acids that is rich in proline, serine, and threonine residues. MLK3 can associate with an activated form of the GTPase Cdc42 and this association requires a functional CRIB motif. Coexpression of MLK3 and activated Cdc42 in cells increases MLKB's catalytic activity. However, relatively little is known about the regulation of MLK3 activity. The work described in this thesis examines the mechanisms that regulate MLK3 activities Coexpression of activated Cdc42 with MLK3 was found to alter the in vivo phosphorylation pattern of MLK3 suggesting that the mechanism by which Cdc42 increases MLK3’s catalytic activity involves a change in the MLK3 in viva phosphorylation state. Interestingly, the activation of MLK3 by Cdc42 could not be recapitulated in an in vitra system, implying the requirement for an additional component or the cellular environment for MLK3 activation by Cdc42. A single point mutation in the leucine zipper domain abrogates zipper-mediated MLK3 oligomerization. Activated Cdc42 fully activates this monomeric MLK3 mutant in terms of both autophosphorylation and histone phosphorylation activity, and induces the same in viva phosphorylation pattern as wild type MLK3. However, this catalytically active MLK3 zipper mutant is unable to activate JNK. My data show that the monomeric MLK3 mutant fails to phosphorylate one of the two activating phosphorylation sites, Thr’”, of MKK4. These studies suggest that zipper-mediated MLK3 oligomerization is not required for MLK3 activation by Cdc42, but instead is critical for proper interaction and phosphorylation of a downstream target, MKK4. Multiple mass spectrometric techniques coupled with comparative phosphopeptide mapping were used to directly identify twelve in viva MLK3 phosphorylation sites including the Cdc42 inducible phosphorylation sites. Of the phosphorylation sites identified, seven are followed immediately by a Pro residue suggesting that MLK3 may be a target of proline-directed kinases. This is the first report offering sequence information regarding in viva phosphorylation sites of a MLK family member, and provides the necessary first step to understanding how phosphorylation regulates MLK3 function. iv T a my family ACKNOWLEDGEMENTS I am very thankful for the working relationships and friendships with all of the members of the Gallo lab during my graduate training, including Barbara Bock, Mary Chao, Hua Zhang, Ritesh Agrawal, Pierofrancesco Vianello, Yan Du, Karen Schachter, and Dr. Kathleen Gallo. I would like to thank the members of my committee; Drs. Kathleen Gallo, Walter Esselman, Douglas Gage, Robert Hausinger, Lee McIntosh and William Smith, for their valuable discussions and encouragement. I am also very appreciative of Drs. Jack Watson, Doug Gage, and Brett Phinney for their instructive roles in teaching me various aspects of mass spectrometry. I am most grateful for the guidance and efforts of my mentor Dr. Kathleen Gallo. I would like to thank Kathy for being my teacher, and providing an atmosphere that promoted scientific creativity and development. TABLE OF CONTENTS Page List of Figures .................................................................................... ix Key to Abbreviations ........................................................................... xii I. Literature Review .............................................................................. 1 1. Eukaryotic signal transduction overview ............................................. l 1.1 Molecular interactions and participants .......................................... l 2. Protein kinases-Structural features of the catalytic domain ........................ 6 3 Mechanisms regulating protein kinases ............................................. 10 3.1 The binding of activating and inhibiting molecules ......................... 10 3.2 Autoinhibition ..................................................................... 11 3.3 Phosphorylation ................................................................... 12. 3.4 Subcellular localization .......................................................... l3 4. The c-Jun NHz-terminal kinase pathway ........................................... 16 4.1 Mammalian MAPK pathways .................................................. 16 4.2 The JNK pathway ................................................................ 17 5. The Mixed Lineage Kinase family .................................................. 21 5.1 MLK family members ........................................................... 21 5.2 MLK3 .............................................................................. 23 6. Objective of Thesis .................................................................... 28 7 References .............................................................................. 30 II. Cdc42-induced Activation of the Mixed Lineage Kinase MLK3 in Viva ........... 36 1. Abstract ................................................................................. 36 2. Introduction ............................................................................ 38 3. Materials and Methods ............................................................... 42 3.1 Construction of mammalian expression vectors and mutagenesis. . . . . ....42 3.2 Expression and purification of recombinant MLK3 ......................... 43 3.3 Cell lines and transfections ...................................................... 43 3.4 Cell lysis and immunoprecipitations .......................................... 44 3.5 Gel electrophoresis and Western blot analysis ............................... 45 3.6 In vitra kinase assays ............................................................ 46 3.7 In viva phosphopeptide mapping ............................................... 47 vi 4. Results ................................................................................... 49 4.1 MLK3’s CRIB motif is necessary for association with Cdc42 and for Cdc42-induced activation of MLK3 .................................. 49 4.2 Effects of deleting the COOH-terminal portion of MLK3’s zipper domain ..................................................................... 51 4.3 The leucine zipper domain is sufficient for MLK3 oligomerization .................................................................. 52 4.4 Activated Cdc42 fails to activate MLK3 in vitra ............................ 53 4.5 Activated Cdc42 alters the in viva phosphorylation pattern of MLK3 .................................................................. 54 5. Discussion .............................................................................. 66 6. References .............................................................................. 73 III. Zipper-mediated Oligomerization of the MLK3 is Not Required for Its Activation by the Small GTPase Cdc42 but is Necessary for Its Activation of the JNK Pathway ........................................................................ 76 1. Abstract ................................................................................. 76 2. Introduction ............................................................................ 78 3. Materials and Methods ............................................................... 81 3.1 DNA constructs and mutagenesis .............................................. 81 3.2 Cell culture, transfections, and lysis ........................................... 82 3.3 Immunoprecipitations and GST pulldown assays ........................... 82 3.4 Gel electrophoresis and Western blot analysis ............................... 83 3.5 In vitra kinase assays ............................................................ 83 3.6 Expression and purification of MBP fusion proteins ........................ 84 3.7 Size exclusion chromatography ................................................ 85 3.8 Phosphopeptide mapping ........................................................ 85 3.9 Phosphoamino acid analysis .................................................... 86 4. Results ................................................................................... 87 4.1 Point mutation in the zipper domain decreases the in vitra kinase activity of MLK3 ......................................................... 87 4.2 A MLK3 leucine zipper mutant fails to oligomerize ........................ 88 4.3 Activated Cdc42 increases the in vitra catalytic activity of both wild type MLK3 and MLK3 L410P ..................................... 89 4.4 MLK3 oligomerization is necessary for MLK3-induced JNK activation .................................................................... 90 4.5 Activated Cdc42 changes the in viva phosphorylation state of MLK3 L410P ................................................................... 90 4.6 The MLK3 leucine zipper mutant has reduced ability to phosphorylate MKK4 ............................................................ 91 5. Discussion .............................................................................. 105 6. References .............................................................................. 11 1 IV. Identification of in viva phosphorylation sites of MLK3 by vii phosphopeptide mapping and mass spectrometry .................................... 113 1. Abstract ................................................................................. 113 2. Introduction ............................................................................. 114 3. Methods and Materials ................................................................ 118 3.1 DNA constructs and mutagenesis .............................................. 118 3.2 Cell culture, transfections, and lysis ........................................... 118 3.3 32P labeling ........................................................................ 119 3.4 Immunoprecipitation and in gel trypsin digestion ........................... 119 3.5 Reverse-phase HPLC ............................................................ 120 3.6 Porous graphitic carbon chromatography ............................. - ........ 1 20 3.7 Phosphopeptide mapping ........................................................ 121 3.8 Phosphoamino acid analysis .................................................... 121 3.9 Mass spectrometry ............................................................... 121 4. Results ................................................................................... 123 4.1 Recombinant MLK3 displays an incomplete phosphopeptide pattern in vitra ..................................................................... 123 4.2 In gel trypsin digestion of MLK3 immunoprecipitated from 293 cells ..................................................................... 124 4.3 Separation of tryptic peptides by reverse-phase HPLC and phosphopeptide mapping ......................................................... 124 4.4 Identification of phosphorylation sites using MALDI-MS and ESI MS/MS .............................................................................. 125 4.5 Porous graphitic carbon chromatography .................................................. 131 4.6 MALDI-MS Analysis of PGC fractions ....................................... 131 4.7 Comparative two dimensional phosphopeptide mapping of in viva labeled MLK3 variants ........................................................... 132 5. Discussion .............................................................................. 153 6. References .............................................................................. 159 V. Concluding Remarks ..................................................................... 161 viii Figure LIST OF FIGURES Page Chapter 1. Model representing activation of signal transduction molecules by cell surface receptors .......................................................................................... 4 Regulation of small GTPases ............................................................... 5 Ribbon diagram representing the structure of the catalytic domain of protein kinases ....................................................................................... 9 Mammalian MAPK scaffold complexes ................................................ 15 The parallel mammalian MAPK pathways ............................................. 20 The mixed lineage kinase family ......................................................... 27 Chapter II. Schematic of MLK3 ........................................................................ 56 Alignments of CRIB motifs and MLK3 CRIB variants .............................. 57 Effects of mutations in the CRIB motif on association of MLK3 with Cdc42V12 .................................................................... 58 Effects of partial deletion of the zipper/basic stretch an association with Cdc42V12 ............................................................... 59 Effects of mutations in the CRIB motif on MLK3 in vitra kinase activity .................................................................... 60 Effects of partial deletion of the zipper/basic stretch on MLK3 in vitra kinase activity ............................................................ 61 Assay for association of the zipper/basic stretch of MLK3 with MLK3 and MLK3Azip in vitra... .62 In vitra kinase assay using purified Cdc42 ............................................. 63 Phosphopeptide mapping of tryptic peptides derived from in viva phosphorylated MLK3 ............................................................ 64 ix 10. 11. Chapter 111. Effect of a point mutation in the MLK3 zipper domain on catalytic activity ........................................................................ 94 Effects of MLK3 L410P on JNK activity ............................................... 95 Size exclusion chromatography analysis of the MLK3 zipper point mutant ........................................................................ 96 Coimmunoprecipitation analysis of the MLK3 zipper point mutant ................................................................................. 97 In vitra kinase assay of MLK3 and MLK3 L410P coexpressed with Cdc42V12 .............................................................. 98 Effect of MLK3 L410P on Cdc42V12 binding of MLK3 ............................ 99 Effect of MLK3 L410P on JNK activation ............................................ 100 Two-dimensional maps of tryptic phosphopeptides derived from in viva phosphorylated MLK3 or MLK3 L410P ............................... 101 Phosphorylation of MKK4 by MLK3 L410P in vitra ............................... 102 Binding of MKK4 by MLK3 L410P ................................................... 103 Phosphoamino acid analysis of MKK4 phosphorylated by MLK3 variants ............................................................................ 104 Chapter IV. Two-dimensional map of MLK3 following an in vitra kinase assay .............. 134 Reverse phase-HPLC fractionation of MLK3 tryptic peptides ..................... 135 One-dimensional phosphopeptide analysis of radiolabeled HPLC fractions ........................................................................... 136 Analysis of HPLC fraction 45 using MALDI-MS in conjunction with alkaline phosphatase treatment ......................................................... 137 Analysis of HPLC fraction 23 using MALDI-MS in conjunction with alkaline phosphatase treatment ......................................................... 138 10. ll. 12. 13. 14. 15. 16. 17. 18. 19. Phosphoamino acid analysis of peptides from selected HPLC fractions” 139 Identification of phosphorylated Ser‘24 in HPLC fraction 31 by MALDI-MS post source decay ........................................................ 140 Analysis of HPLC fraction 27 using MALDI-MS in conjunction with alkaline phosphatase treatment ......................................................... 141 Analysis of HPLC fraction 27 using MALDI-PSD and ESI-CID .................. 142 770 Identification of Ser as the phosphorylation site in a peptide contained in HPLC fraction 17 ......................................................... 143 793 Identification of Ser as the phosphorylation site in a peptide contained in HPLC fraction 20 ......................................................... 144 Identification of Ser"o as the phosphorylation site in a peptide contained in HPLC fraction 15 .......................................................... 145 Fractionation of C18 flowthrough fractions by RP-HPLC on a porous graphitic carbon column ...................................................... 146 One-dimensional phosphopeptide analysis of radiolabeled PGC fractions .............................................................................. 147 Analysis of PGC fraction 23P using MALDI-MS combined with alkaline phosphatase treatment ................................................... 148 Analysis of PGC fraction 28P using MALDI-MS combined with alkaline phosphatase treatment ................................................... 149 Analysis of PGC fraction 22P using MALDI-MS combined with alkaline phosphatase treatment ................................................... 150 Two-dimensional maps of tryptic phosphopeptides derived from in viva phosphorylated MLK3 variants .......................................... 151 Schematic representation of MLK3 phosphorylation sites............... . . . . . . . . . . . . . . 152 xi ATP CAMP CID CRIB DLK ESI-MS ERK GAP GDP GEF GST GTP HA HPK HPLC IKK IL JIP JN K LZK MALDI-MS MAPK MBP MEK MLK PAA PAGE PAK PBS PCR PGC PKA PSD RP PKC SPRK TLC TLE TNF WASP WT Key to Abbreviations Adenosine 5’-triphosphate Adenosine 3’-5’ cyclic monophosphate Collision-induced dissociation Cdc42/Rae interactive binding Dual leucine zipper-bearing kinase Electrospray ionization-mass spectrometry Extracellular signal-regulating kinase GTPase activating protein Guanosine 5’-diphosphate Guanosine nucleotide exchange factor Glutathione S-transferase Guanosine 5’-tn'phosphate Hemagglutinin Human hematopoietic progenitor kinase High pressure liquid chromatography Inhibitor of NF kappaB-kinase Interleukin JNK interacting protein c-Jun NH; terminal kinase Leucine zipper-bearing kinase Matrix-assisted laser desorption/ionization-mass spectrometry Mitogen-activated protein kinase Maltose binding protein MAPK/ERK kinase Mixed lineage kinase Phosphoamino acid analysis Polyacrylamide gel electrophoresis p21-activating kinase Phosphate-buffered saline Polymerase chain reaction Porous graphitic carbon Protein kinase A Post source decay Reverse-phase Protein kinase C Src-homology 3 domain-containing proline-rich kinase Thin layer chromatography Thin layer electrophoresis Tumor necrosis factor Wiskott-Aldrich syndrome protein Wild type xii I. Literature Review 1. Eukaryotic Signal Transduction Overview 1.1 Molecular Interactions and Participants Signal transduction is a term used to describe the cascade of events that culminates in a cellular response to an extracellular signal or environmental stimuli. From the stimulation of cell surface receptors to the activation of protein kinase cascades, this process involves a variety of molecular participants and regulates numerous cellular processes including gene expression, cell survival, cytoskeletal integrity, cell differentiation, and cell proliferation. The binding of a soluble ligand (e. g., growth factor or non-steroidal peptide hormone) to a cell surface receptor induces oligomerization, phosphorylation and activation of the receptor (1-3) (Fig. 1). Receptor phosphorylation is achieved either by receptor autophosphorylation as in the case of receptor tyrosine kinases (4-6) or by receptor-associated kinases (7-9), creating receptor homo-oligomers or hetero-oligomers. Receptor phosphorylation serves to create binding sites on the receptor molecule for downstream signaling components that recognize the phosphorylated tyrosine region of the activated receptor. The best characterized phospho-tyrosine binding module is the Src homology 2 (SH2) domain (IO-12). SH2 domains directly recognize tyrosine phosphorylation sites, and are thereby recruited to activated, phosphorylated receptors. The SH2 domain is a protein module of approximately 100 amino acids and interacts with tyrosine phosphorylated sites through a network of hydrogen bonds and electrostatic interactions (13,14). These interactions, in turn, recruit additional proteins to the activated receptor. Many SH2 domain-containing proteins serve as adaptor molecules. Adaptor proteins are composed of recognition domains and do not posses enzymatic activity (15). Adaptor proteins will often contain an SH2 domain and another type of recognition domain called the SH3 domain. The SH3 domain consists of 55-70 amino acids and is found in many intracellular signaling proteins (16,17). The SH3 domain recognizes proline-containing sequences on its binding target. Adaptor proteins that contain SH2/SH3 domains are crucial in regulating receptor tyrosine kinase signaling pathways. The major function of these adaptors; such as Grb2, ch, and Crk (18); is to recruit proline-rich effector molecules to the plasma membrane so that they are the vicinity of their substrates or effectors (19). In recent years a variety of proteins capable of associating with the SH2 and the SH3 domains of adaptors have been described, including those of the Ras family. The Ras superfamily of GTPases, which include Ras as well as the Rho/Rae subfamily members Rae and Cdc42 act as molecular switches in the control of a variety of cellular processes (20) (Fig. 2). Small GTPases cycle between a GDP bound inactive state (promoted by GTPase activating proteins (GAPS) (21,22)) and a GTP bound active state (promoted by guanine nucleotide exchange factors (GEFs) (23)). Small GTPases can also be modified by the addition of lipid groups. For example, Cdc42 becomes geranylgeranylated at its COOH-tenninus within a so-called CAAX motif (24). This posttranslational lipid modification allows the GTPase to associate with cellular membranes, including the plasma membrane. Many GEFs are recruited to the activated phosphorylated receptor at the plasma membrane either directly through SH2 domains of their own (25) or indirectly through interactions with adaptor proteins such as Grb2 (26). This relocalization to the plasma membrane can stimulate activation of the membrane bound GTPase by converting it to its GTP bound form (27). Activated GTPases regulate signal transduction pathways by binding to and activating protein kinases. For example, activated Ras binds to and activates the cytoplasmic serine/threonine protein kinase Raf-l (28,29). Activated Cdc42 and Rac bind to and activate serine/threonine kinases such as the p21-activated kinases (PAKs) (30) and the MLKs 2 and 3 (31-33) through a small binding region termed the CRIB motif. Protein kinase activation induces the sequential phosphorylation and activation of other protein kinases, which lead to the phosphorylation and modulation of a multitude of cellular proteins. The above description is a simplified version of eukaryotic signal transduction systems. Other important factors not discussed include spatial and temporal regulation of the pathway components, converging and diverging of pathways, and mechanisms that regulate signaling pathways (both positively and negatively). Some of these issues will be introduced in the sections below. In summary, all together this overall sophisticated design transmits diverse signals to complex cellular machineries, allowing the cell and the organism to respond appropriately to its surroundings. Growth factor :" *4 p P21 ras 1133 p p I TRANSCRIPTION Nucleus M... Fig. 1 Model representing activation of signal transduction molecules by cell surface receptors. Ligand binding induces receptor dimerization, which leads to phosphorylation of the receptor. Adaptor proteins bind to phosphorylated receptor sites and recruit additional signaling molecules to the plasma membrane. inacflve TP _P. G GAPS GDP GEFs Fig. 2 Regulation of small GTPases. Small GTPases cycle between a GDP-bound inactive state, and a GTP-bound active state. Guanine nucleotide exchange factors (GEF s) mediate the exchange of GDP to GTP, while the GTPase-activating proteins (GAPS) mediate the hydrolysis of GTP to GDP. 2. Protein Kinases: Structure of the catalytic domain Protein kinases are enzymes that catalyze the transfer of the y-phosphate of ATP to a protein substrate. This post-translational modification is an important mechanism for covalently and reversibly regulating signal transduction pathways. Protein phosphorylation is a reversible mechanism due to the action of protein phosphatases and it is estimated that one in four intracellular proteins undergo phosphorylation. The majority of protein kinases phosphorylate hydroxyl amino acids and consequently fall into one of two categories, those that target serine and threonine residues and those that target tyrosine residues. A few protein kinases, capable of phosphorylating both groups, are named dual specificity kinases (34,35). The catalytic domain is highly conserved amongst serine/threonine protein kinases and tyrosine protein kinases in eukaryotic organisms. Crystallographic and NMR studies have determined the structure of many protein kinases (36,37) and, along with standard biochemical methods, have led to general concepts characterizing the protein kinase domain (Fig. 3). The kinase domain consists of approximately 250 amino acids and is a two lobed structure, separated by a short cleft where ATP binds and catalysis occurs (38). The smaller amino terminal lobe consists mainly of beta strands with one conserved helix called the C-helix and contains the residues necessary for critical interactions with ATP. The larger COOH-terminal lobe consists mainly of helices and predominantly functions in substrate binding and recognition. In addition to the shared structural similarity, invariable residues are present throughout the kinase domain. The majority of these conserved sequence motifs are associated with MgATP binding and active site stability, while sequences that are involved in substrate binding differ between protein kinases. The amino terminal lobe contains three well-conserved sequence motifs that make critical contacts with MgATP. A so-called glycine loop, Gly-Xxx-Gly-Xxx-Xxx-Gly, is located between the first and second beta strands (39). The backbone amides of this region have been shown to interact with the B-phosphate group of ATP (40-42), although mutagenesis studies demonstrate that this region is not always critical for catalysis (43). A conserved lysine residue in the amino terminal lobe is located in the middle of beta-strand 3 in close proximity to the alpha and beta phosphates of MgATP. Unlike the glycine loop, the conserved lysine is essential for catalytic competence. Substitution of this lysine residue renders protein kinases virtually catalytically inactive. A conserved glutamic acid residue located in the C-helix of the amino terminal lobe forms an ion pair with the conserved lysine and is therefore also important for phosphotransfer activity. These two residues apparently are not critical for ATP binding but rather for stabilizing the proper orientation of the phosphate groups of ATP in the active site (44). Conserved amino acids are also present in the COOH-terminal lobe. Most of the conserved residues are charged amino acids that participate in salt bridges and hydrogen bonds to stabilize the active site. A specific example is the aspartic acid residue located in a loop between beta strands 6 and 7. This residue has been shown to act as a catalytic base by forming a hydrogen bond with the target substrate’s hydroxyl group facilitating the nucleophilic displacement of the y-phosphate of ATP by the substrate (44). A highly conserved sequence motif, Asp-Phe-Gly, located in a loop between beta strands 8 and 9 is critical for catalysis (45). The Asp residue makes contacts with the critical Lys residue in the binary complex and chelates the Mg ion in the ternary complex. This motif is also the beginning of the conserved activation segment. The distal portion of the activation segment contains the conserved sequence motif, Ala-Pro-Glu. The loop between these two sequence motifs often contain phosphorylation sites that have been shown to induce significant conformational changes of the activation segment, allowing access of substrate targets to the active site and facilitating phosphotransfer activity. Specific examples include the activating Thr-X-Tyr phosphorylation sites of MAPK family members (46) (Fig. 3) and the stable phosphorylation site Thr 197 of cAMP dependent protein kinase (cAPK) (47). Inactive ERK Active ERK T133 9 0400ng p118 P a \ activation \\ segment Canagarajah, B. J. et al, 1997 Fig. 3 Ribbon diagram representing the structure of the catalytic domain of protein kinases. The structures of unphosphorylated inactive ERK and phosphorylated active ERK are shown as a general overview of the catalytic domain fold. Arrows indicate the invariable sequences. The phosphorylation of the activation segment is shown, demonstrating the conformational changes that occur upon activation segment phosphorylation. 3. Mechanisms Regulating Protein Kinases Protein phosphorylation catalyzed by protein kinases is an essential mechanism regulating signal transduction pathways. Therefore, the activities of protein kinases are believed to be tightly regulated. Within physiological systems protein kinases are generally maintained in an inactive state and, in response to a physiological signal, become activated. The regulation of a protein kinase’s active state is achieved by a variety of mechanisms including allosteric binding of molecules, posttranslational modifications such as phosphorylation, and indirectly by changes in subcellular localization. Also, since the structure of the catalytic core is relatively conserved, often it is the noncatalytic regions that serve to regulate a specific protein kinase. 3.1 The Binding of Activating and Inhibiting Molecules The allosteric binding of activating and inhibiting molecules is a common mechanism regulating the activity of protein kinases. For example, the binding of growth factors to receptor tyrosine kinases induces a conformational change that promotes receptor dimerization, trans-autophosphorylation, and the recruitment of signaling molecules (48-50). Also the catalytic activities of cyclin dependent protein kinases (CDK) are positively regulated by the binding of cyclins while the binding of CDK inhibitors (CDKI) inhibits CDK activity (51,52). The binding of second messengers such as CAMP, cGMP, and Ca2+ also regulate certain protein kinases. A well-characterized example is the activation of cAPK by CAMP (53). The cAPK molecule is kept in an inactive state by the binding of a dimeric 10 regulatory subunit. Binding of CAMP to the regulatory subunit promotes dissociation of the catalytic subunit from the regulatory subunit. The catalytic subunit is then free to phosphorylate target proteins (54,55). 3.2 Autoinhibition Many protein kinases utilize autoinhibition as a negative-regulatory mechanism. The regulatory subunit of cAPK mentioned above contains a short pseudosubstrate region that interacts with the substrate binding site and functions as a competitive inhibitor (56). Furthermore, regulatory regions located on the same polypeptide as the catalytic domain can also utilize this mechanism. The PKC superfamily contains a pseudosubstrate region that maintains the kinase in a catalytically inactive conformation (57,58). Phosphorylation and protease-mediated cleavage removes the pseudosubstrate region, relieves the autoinhibition and, along with the binding of diacylglyercol, promotes a catalytically active conformation. Another interesting example of autoinhibition has been demonstrated for the Src family of non-receptor tyrosine kinase. Phosphorylation of Tyr 527 in the COOH- terminal domain of Src leads to an interaction between the SH2 domain of Src and phosphorylated Tyr 527 (59). This association induces an intramolecular interaction between the SH3 domain of Src and a proline-containing sequence, further stabilizing the low activity conformation. Dephosphorylation of Tyr 527 relieves the autoinhibition and promotes the autophosphorylation of Tyr 416 located in the activation segment of the kinase domain that leads to kinase activation. 11 3.3 Phosphorylation A posttranslational modification is defined as any covalent modification of protein structure after protein synthesis. Probably the most frequent posttranslational modification involved in regulating protein kinases is phosphorylation. Phosphorylation is a widespread mechanism that rapidly and reversibly alters the activities of kinases. Phosphorylation of hydroxyl side chains can have a number of consequences. One possibility is that phosphorylation of amino acids itself may create a binding interface for recognition of a target protein (60). This phenomenon has been well established for receptor tyrosine kinases, wherein specific phosphotyrosines interact electrostatically with the protein-protein interaction domains of target signaling proteins (61). A second possibility is that introducing a phosphate group changes the conformation of the kinase. Phosphorylation induces a rearrangement of hydrogen bonds on the target, and the phosphate moiety is capable of forming two ion pairs with the side chains of basic amino acid residues (46). The resulting structural changes can have positive or negative effects on the function of the kinase (62). Moreover, multisite phosphorylation could potentially give rise to a variety of protein forms, which may have differential functions and/or specificities. Removal of phosphate groups from protein kinases catalyzed by protein phosphatases is also an important mechanism regulating protein kinases (63). Dephosphorylation of a particular site can be either a positive or negative event (64). The homeostatic balance between phosphorylation and dephosphorylation of protein kinases is critical in determining the cellular response to extracellular signals. For example, one 12 of the earliest events following T-cell receptor activation is the tyrosine phosphorylation of a multitude of intracellular signaling proteins that are required for downstream signaling events (65). Members of the Src family of non-receptor tyrosine kinases mediate much of this tyrosine phosphorylation (66). As mentioned above, Src is kept in an inactive conformation under resting conditions through an interaction involving phosphotyrosine 527 and its own SH2 domain. Under stimulating conditions this autoinhibition is relieved. Activation of Src requires first the dephosphorylation of tyrosine 527 by tyrosine phosphatases such as CD45. This dephosphorylation in turn, facilitates the activating autophosphorylation of tyrosine 416 within the kinase domain of Src. 3.4 Subcellular localization Many protein kinases have multiple in viva substrates. Therefore, in addition to protein kinase activation, the specificity of a ligand-mediated response is dependent on the accessibility of the kinases to their physiological targets. This accessibility can be achieved by regulating the subcellular localization of protein kinases (67). A few protein modules have been demonstrated to coordinate protein kinase localization. Cascades of protein kinases can be organized by scaffold proteins in which each participant associates with a specific region on the scaffold to create a signaling complex. For example, the MEK-Partner 1 (MP1) scaffold protein interacts and coordinates ERK and its activator MEK] in a variety of cell types (68,69), while the scaffold protein JIP (INK interactive protein) interacts with specific members of the JNK signaling pathway (70-72). 13 In contrast to organizing a signaling complex, anchoring proteins such as the A- kinase-anchoring proteins (AKAPs) direct the subcellular localization of target proteins through association with subcellular organelles, membranes, or proteins. AKAPS vary in their size and affinity for various subcellular components (73). Each AKAP contains an anchoring motif that interacts with the regulatory subunit of cAPK, and a targeting domain that associates with the subcellular component (74). This mechanism allows a single kinase such as cAPK to mediate a variety of cellular processes in a specific, coordinated manner. 14 MP1 scaffold complex Davis, R.J., 2000 Fig. 4 Mammalian MAPK scaffold complexes. The scaffold protein 11? coordinates specific components of the JNK signaling cascade. The MP1 scaffold protein coordinates members of the ERK pathway. 4. The c-Jun NHz-terminal kinase pathway 4.1 Mammalian MAPK pathways In the mid 1980s Sturgill and Ray, while studying insulin stimulated phosphorylation of intracellular proteins, discovered and purified a 40 kDa protein they called Microtubular Associated Protein-2 (MAP-2) kinase (75). They further demonstrated that MAP-2 kinase activation by insulin involved threonine'and tyrosine phosphorylation of MAP-2 kinase and demonstrated that both phosphorylation events were required for catalytic activity (76). Meanwhile, various groups were characterizing a 40-45 kDa protein that was tyrosine phosphorylated in response to various mitogens (77). After recognizing that this protein was apparently identical to the MAP-2 kinase the molecule was renamed Mitogen Activated Protein Kinase (MAPK). Boulton and colleagues later cloned the gene encoding MAPK, which they called Extracellular signal- Regulated Kinase (ERK) (78). Currently, three parallel MAPK pathways have been well characterized in mammalian cells including the ERK, the reactivating kinase (p3 8/RK), and the INK pathways. The cellular function of the different MAPK pathways is dependent on the cell type and specific stimuli. The ERK pathway seems to be primarily involved in mitogenic responses and cell survival (79), while the JNK and p38 pathways are involved in stress response and apoptosis (80). MAPK pathways are composed of at least three protein kinases that regulate many cellular processes including proliferation, differentiation, and gene expression. Generally, extracellular signals lead to the phosphorylation and activation of a MAPK kinase kinase (MAPKKK). An activated MAPKKK can productively bind, phosphorylate, and, hence, activate a dual specific MAPK kinase (MAPKK), which in 16 turn activates a MAPK by phosphorylating a threonine residue and a tyrosine residue in the Thr-X—Tyr activation motif within the catalytic domain. A second common characteristic of the MAPKs is their proline-directed substrate specificity. Once activated, MAPKS phosphorylate their substrates at sites that contain a serine or threonine residue followed by a proline residue. Full specificity is ensured through a docking site on the substrate that is recognized by the catalytic domain of the appropriate MAPK (81). This sequential kinase cascade module is utilized to respond to a diverse array of extracellular signals and environmental stimuli and mediate the regulation of a variety of cellular targets in the cytosol and the nucleus including various Ser/Thr kinases, cytoskeletal proteins, and transcription factors. 4.2 The JNK Pathway INK, also known as Stress Activated Protein Kinase (SAPK), was discovered in 1990 as the predominant microtubular associated protein kinase that was activated in the liver by the treatment with the protein synthesis inhibitor cyclobeximide (82). INK was initially characterized as being activated by cellular stresses such as heat shock or UV irradiation, agents that cause DNA damage and lead to formation of reactive oxygen species (83). In addition to being activated by cellular stress, INK is now known to be activated in response to receptor activation, such as the activation of inflammatory cytokine receptors, G protein coupled receptors, and the T-cell receptor (84). Three genes encoding INK family members have been cloned. INKl and INK2 are 17 ubiquitously expressed (85), while INK3 is predominately expressed in the brain (86). Furthermore, each gene is alternatively spliced yielding a total of 12 isoforms (87). INK is phosphorylated and activated by two MKKs; MKK4 (88) and MKK7 (89). However, many MAPKKKs have been shown to phosphorylate MKK4/7 and activate INK, including mixed lineage kinase family members (90), apoptosis signal-regulating kinase (91), transforming grth factor activating kinase (91), Tpl/Cot kinase (92), and a mitogen-activated protein/ERK kinase kinase family member (93), demonstrating the diversity and complexity of INK signaling. Although not well understood, it is presumed that specific MKKKS are activated by distinct extracellular stimuli leading to INK activation. The substrates for INK are predominantly transcription factors. The most notable INK substrate is c-Iun. The gene encoding c-Iun is an immediate early gene whose product belongs to the AP-l transcription factor family (94). Activation of INK leads to nuclear translocation, and phosphorylation of the NHz-terminal region of c-Iun. Phosphorylation of Ser 63 and Ser 73 of c-Iun promotes homodimerization and heterodimerization, stabilizing the protein and thus increasing its transactivation activity (95). INK has several additional known substrates such as the transcription factors Elk-1 and ATP-2 (83) and other cellular proteins such as p53 and bcl—2 (96). The INK pathway participates in the regulation of a variety of cellular processes, such as cell survival (apoptosis), cell differentiation, and the inflammatory response (84). Since diverse upstream molecules and events can activate INK, an unresolved issue has been how signaling specificity is attained. At least part of the answer has come with the discovery by Davis et al in 1998 of a INK pathway scaffolding protein termed IIP (70). 18 IIPS can bind to three components of the pathway including MLK, MKK7, and INK. F urtherrnore, other components of the INK pathway such as the MAPKK, MKK4, and other MAPKKKs do not interact with the IIP scaffolding proteins. Currently, three IIP isozymes have been isolated. The IIP] mRNA is ubiquitously expressed in human tissues, while I 1P2 is predominately expressed in the brain. IIP3 is expressed at high levels in the brain, heart and lung, and at lower levels in other tissues. Furthermore, IIPl and 2 are structurally related, while IIP3 is structurally divergent (71 ,97,98). Initial studies have suggested that IIPS potentiate the activation of INK by MLKs and MKK7. In addition to segregating and assembling pathway components, it is possible that binding to the scaffold may modulate the kinase activity and/or alter the subcellular location of the signaling complex participants, although this has not been demonstrated. Also, the finding that the INK scaffold protein interacts with certain INK activators and not others suggests that multiple mechanisms exist to induce INK activation. 19 Stimulus Biological Response MAPK signaling cascades Growth factors Mitogens l is... Reasons!) 1 I‘ new: H :9 l t: MAPK/ERK :9 l Growth Diflerentlation Development Stress lnflammato Cytokines Growth actors I \ new i: 1.5163,.) 7‘“ k. as!“ a (3 annals 3 (I- MKK4/7 I) l l I: pan mm 9 C“""“"" :9 \ / Inflammation Apoptosis Growth Differentiation Cell Signaling, 2000 Fig. 5 The parallel mammalian MAPK pathways. Three well-defined MAPK pathways identified in mammalian cells include the ERK, INK, and p38 MAPK pathways. MAPK pathways are characterized by a three kinase module, which are sequentially activated by phosphorylation. 20 5. Mixed Lineage Kinase Family 5.1 MLK Family Members The search for novel protein kinases involved in signal transduction pathways has resulted in the recent discovery of a group of related eukaryotic intracellular protein kinases named the mixed lineage kinase (MLK) family. The MLK family of kinases all possess leucine zipper oligomerization domains and are characterized by the sequence of their kinase domains having features of both serine/threonine kinases and tyrosine kinases. However, only serine/threonine kinase activity has been demonstrated for this family. The MLK family can be divided into two subfamilies and consists of six members. Dual leucine zipper-bearing kinase (DLK) (99), leucine zipper kinase (LZK) (100), and leucine zipper and sterile-alpha motif kinase (ZAK) (101) form one MLK subgroup, while MLKl (102), MLK2 (103), and MLK3 (104,105) form a second distinct subgroup. The MLK family members have been identified as upstream activators of the INK pathway (90,101,106,107). MLKs function as MAPKKKS and are capable of phosphorylating and activating the INK activators MKK4 and MKK7 (84). In addition, the INK scaffold protein IIP has been shown to associate with the MLK family members, DLK, MLK2, and MLK3 (71). Association with I IP has been implicated in facilitating MLK mediated INK activation. The kinase and zipper domains of MLK1 , MLK2, and MLK3 share approximately 70% sequence identity. The kinase and zipper domains of DLK and LZK, share approximately 90% sequence identity. However, between the two subgroups these domains have only 35% identity. Furthermore, unlike DLK, LZK, and ZAK, the MLK l- 21 3 subgroup have additional protein-protein interaction domains, including an NH; terminal SH3 domain, a Cdc42/Rae interactive binding (CRIB) motif, and a COOH- terminus that is highly rich in serine, threonine, and proline residues. Considering these structural differences, it is likely that these two MLK subgroups will be activated by different upstream molecules and may exhibit altered biological behaviour. Relatively little is known about the biological properties of MLK1, since the full- length clone of MLK1 was just reported in Genebank last year (McNee and Guesdon). Until then only a partial clone, which encoded the first half of the protein, was available to study (103). MLKl mRNA has been detected in epithelial cell lines of breast, colonic and esophageal origin (103) and shows differential mRN A expression in pancreatic B-cell lines at different stages of maturation and embryonic pancreas development. MLK2 mRN A has been shown to be predominately expressed in brain and skeletal muscle, with a lower level of expression in the pancreas. It has been demonstrated that MLK2 can associate with the normal huntingtin protein in rat hippoeampal neuronal (HN 33) cells. This association inhibits MLK2-induced INK activation and apoptosis in HN33 cells. Furthermore, the defective huntingtin protein found in Huntington’s disease (HD) cannot associate with MLK2 suggesting a possible role for MLK2 in neuronal death in HD. Microinjection and immunostaining studies in NIH 3T3 cells showed a punctuate distribution of MLK2 along microtubules, together with phosphorylated INK (33). Yeast two-hybrid analysis identified KIF3X and KAP3 as potential MLK2 binding partners. KIF3X is a putative member of the kinesin superfamily of motor proteins, which transport cargo vesicles along microtubules (108), whereas KAP3 acts as an adaptor molecule 22 between the vesicle and the cargo molecule. Furthermore, it has been demonstrated using the yeast-two hybrid system and coimmunoprecipitation experiments, that kinesin can associate with the INK scaffolding proteins IIPl, IIP2, and IIP3, along with DLK and and a transmembrane receptor molecule ApoER2 (109,110). These findings suggest that MLK2 and other mixed lineage kinases either may be involved in the vesicular transport processes or that motor proteins may regulate their subcellular distribution. 5.2 Mixed Lineage Kinase 3 I In 1994, the mlk3 gene was cloned by three independent laboratories (104,105,111). MLK3, also known as SPRK (SH3 domain-containing proline-rich kinase) and PTKl (Protein Tyrosine Kinase 1) is a serine/threonine protein kinase with a molecular weight of 95 kDa. Northern blot analysis demonstrated that the MLK3 mRNA is widely expressed in human tissues, with lower expression in the brain and heart (104,105,111). Relatively little is known about the precise physiological function of MLK3 in eukaryotic organisms. MLK3 overexpression has been demonstrated to induce cellular transformation and anchorage-independent growth of NIH 3T3 fibroblasts rendering them competent to grow in soft agar (1 12). A few recent reports suggest a role for MLK3 and other MLK members in mediating INK activation in neuronal apoptosis. Kinase-defective mutants of MLK2/3 inhibit neuronal apoptosis induced by the glutamate receptor (GluR6)/post-synaptic density (PSD) complex. The SH3 domain of PSD-95 was shown to bind to MLK2 and MLK3, and a PSD-95 with a deleted SH3 domain failed to induce INK activation and 23 neuronal apoptosis (113). Another pair of studies has characterized a direct inhibitor of the MLK family, CEP-l347 (114). CEP-1347 competitively inhibits the catalytic activity of MLK3 and blocks MLK-mediated apoptosis induced by NGF deprivation of PC12 neuronal cells (115) Recent work has provided evidence that MLK3 may be involved in T cell receptor signaling. Overexpression of MLK3 in human T lymphoma cells induces TNF- a promoter activity mediated by INK (116). MLK3 was also shown to induce interleukin-2 (IL-2) promoter activity in T cells (117), while a catalytically-inactive MLK3 mutant blocks IL-2 expression induced by the guaninine-nucleotide exchange factor Vav (1 18). In addition to its established role as an upstream activator of the JNK pathway, a recent report has shown that overexpressed MLK3 is capable of activating the NF-KB pathway in T cells. The transcription factor NF-KB is sequestered in the cytosol under unstirnulating conditions through association with the inhibitor of NF-xB (IKE). In response to an activating signal, IxB is phosphorylated by IKB kinase (IKK). Phosphorylation of IKB leads to its proteasome-mediated degradation, thus releasing NF- KB. Hehner et al demonstrated that overexpression of MLK3 increased the transcriptional activity of NF «B. This study also showed that MLK3 can phosphorylate Ich kinase (IKK), and thus may act as an IKK kinase to positively regulate the NF-KB pathway (1 19). MLK3 contains several potential protein-protein interaction domains that may be important for regulation of its activity. MLK3 contains an NHz-terrninal SH3 domain, which may interact with proline-containing sequences on target proteins. A potential 24 target is the hematopoietic progenitor protein kinase-1 (HPKl). Yeast two-hybrid analysis and coimmunoprecipitation experiments showed that a proline rich motif in HPKl associates with the SH3 domain of MLK3 (120). However, the biological significance of this interaction remains unclear since no alteration in MLK3 activity by HPKl has been demonstrated. Since MLK3 contains potential SH3 binding sites, it is possible that MLK3 may be regulated through an SH3 mediated self-association. Work in our lab has demonstrated that the SH3 domain of MLK3 interacts with a proline-containing region in MLK3 between the zipper and the CRIB motif. Mutation of either the SH3 domain or its binding site leads to increased catalytic activity compared to wild type MLK3 suggesting this interaction may mediate MLK3 autoinhibition (121). A region of 63 amino acids following the kinase domain of MLK3 is predicted to be a leucine zipper domain. Leucine zippers mediate protein oligomerization by forming coiled coil structures. These structures are stabilized mainly by leucine residues spaced seven residues apart that interact at the interface of opposing helices (122-124). The leucine zipper domain of MLK3 is necessary and sufficient to mediate homo- oligomerization (125). Based on a deletion study, one report has suggested that zipper- mediated MLK3 oligomerization is required for its activation (126,127). However, the function of zipper-mediated MLK3 oligomerization remains unclear. MLK3 contains a CRIB motif that is located COOH-terminal to the zipper domain. The CRIB motif is a short amino acid sequence that has been identified as a binding motif for the GTPases Cdc42 and Rac (128). F ilter-binding assays and coimmunoprecipitation experiments have demonstrated that Cdc42 can associate with a 25 CRIB-containing fragment of MLK3 (128). Furthermore, coexpression of Cdc42 and MLK3 increases the in vitra autophosphorylation activity of MLK3 (129). However, the mechanism of how Cdc42 activates MLK3 remains uncertain. MLK3 is an upstream serine/threonine kinase that contains a unique arrangement of regulatory domains. Identification of extracellular stimuli leading to MLK3 activation as well as understanding the molecular mechanisms that regulate MLK3 should provide more insight into its specific role in physiological processes. 26 MLK3 Glyfig Kinase Elppe EHj Kinase kippelizy MLK] - MLK2 fl Kinase hippo LZK Kinase Lz DLK Kinase M ZAK Kinase E45»! MLK3 l MLK] L MLK2 LZK DLK . ZAK Fig. 6 The Mixed Lineage Kinase family. The schematic domain arrangement of the various members of the mixed lineage kinase (MLK) family is illustrated. Shown below is the organization of the MLK family members into subgroups based on sequence identity. 27 6. Objectives of this Thesis Protein kinases are involved in a variety of cellular processes ranging from regulating gene expression to modulating the activities of enzymes. Thus, mechanisms that turn kinases on or off are as critical to the cell as the function of the catalytic activity of the kinase itself. Regions outside of the catalytic domain often are involved in controlling protein kinase activity and knowledge of the molecular mechanisms that activate and inactivate a protein kinase provides important insights into their biological role. The serine/threonine kinase MLK3 contains several domains that have the potential to participate in protein-protein interactions that may control its activities, including a CRIB motif and a leucine zipper domain. However, relatively little is known about the functional role these regions play. This thesis is comprised of three research chapters that examine various aspects of molecular mechanisms regulating MLK3. Chapter II investigates the functional requirement of the CRIB motif in terms of interacting with the MLK3 effector molecule Cdc42. This study also examines the effect of Cdc42 coexpression with respect to MLK3 in vitra catalytic activity and changes in the MLK3 in viva phosphorylation pattern. 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Cdc42-Induced Activation of the Mixed-Lineage Kinase MLK3 in viva: Requirement of the Cdc42/Rae Interactive Binding Motif and Changes in Phosphorylation 1. Abstract MLK3 is a serine/threonine kinase that upon overexpression in mammalian cells activates the JNK pathway. The mechanisms by which MLK3 activity is regulated are not well understood. The small Rho-family GTPases, Rae and Cdc42, have been shown to bind and modulate the activities of signaling proteins, including MLK3, which contain CRIB motifs. Coexpression of MLK3 and activated Cdc42 increases MLK3’s activity. MLK3’s CRIB-like motif contains six of the eight consensus residues. Using a site- directed mutagenesis approach, we show that MLK3 contains a functional CRIB motif that is required for MLK3’s association with and activation by Cdc42. However, experiments using a MLK3 variant that lacks the COOH-terminal zipper region/basic stretch suggest that this region may also contribute to Cdc42 binding. Unlike the PAK family of protein kinases, we find that the activation of MLK3 by Cdc42 cannot be recapitulated in an in vitra system using purified, recombinant proteins. Comparative phosphopeptide mapping demonstrates that coexpression of activated Cdc42 with MLK3 alters the in viva phosphorylation pattern of MLK3 suggesting that the mechanism by which Cdc42 increases MLK3’s catalytic activity involves a change in the in viva phosphorylation of MLK3. This is, to the best of our knowledge, the first demonstrated example of a Cdc42-mediated change in the in viva phosphorylation of a protein kinase. 40 These studies suggest the requirement for an additional component or the cellular environment for MLK3 activation by Cdc42. 41 2. Introduction The vast majority of mammalian protein kinases catalyze the transfer of the y- phosphate of ATP to serine, threonine, or tyrosine residues of their target proteins. Phosphorylation is rendered reversible in vivo by the action of protein phosphatases. Since phosphorylation is highly regulated in virtually all physiological processes, it follows that the activities of the protein kinases, themselves, should be highly regulated. Binding of activating or inhibitory molecules, including lipids, cyclic nucleotides, or proteins, can modulate the activity of protein kinases. In addition, posttranslational modifications, such as phosphorylation and proteolysis, can regulate protein kinase activity. These regulatory events may alter the specific activity of a protein kinase or may change its stability. Finally, access to physiological substrates may be limited by restricted subcellular localization or translocation of a protein kinase. Small GTPases regulate certain protein kinases. For instance, by binding and recruiting the serine/threonine kinase Raf to the plasma membrane, GTP-bound, farnesylated Ras contributes to the activation of Raf, and consequently activates the ERK pathway (1). Rho family GTPases, which include Rho, Rae, and Cdc42, play crucial roles in diverse cellular processes (2,3), including cytoskeletal rearrangements (4-6), cell cycle progression (7), cellular transformation (8-14), and nuclear signaling (15-20). They can also function as protein kinase activators. One well-characterized target of Cdc42 and Rac is PAK (21-23). The interaction between Cdc42 and the serine/threonine kinase PAK requires the CRIB motif (26), a 14 to 16 amino acid sequence containing eight consensus amino acids. The structural determinants required for GTPase binding and the mechanism of activation of multiple PAK isoforms have been extensively investigated 42 (21, 24, 25, 32, 33, 34). CRIB-dependent interaction of PAK with GTP-bound Rac/Cde42 induces PAK autophosphorylation and activation both in vitro and in viva. Diverse proteins, including the tyrosine kinase, activated Cdc42HS-associated kinase (ACK) (27), and the non kinase, Wiskott-Aldrich Syndrome protein (WASP) (28, 29), also contain CRIB motifs, suggesting that mechanistically diverse regulatory pathways may share this common structural motif. Likewise, not all protein kinases that interact with Cdc42 and Rac do so through a CRIB motif. F or instance, both the 70 kDa ribosomol S6 kinase (30) and MAPK kinase kinase-1 (MEKK-l) (31), which lack CRIB motifs, have been shown to interact with GTP-bound Cdc42 and Rac. Furthermore, MEKK-4 contains a modified CRIB motif whose deletion only partially diminishes binding to Cdc42 and Rac, indicative of a CRIB-independent GTPase binding determinant (31). Thus the role of CRIB motifs and the mechanisms by which many protein kinases are activated by small GTPases remains largely unexplored. MLK-3 (36); also called SPRK (35) or PTK-l (37), is a member of the so-called "mixed-lineage” kinase family. MLK3 contains a CRIB motif bearing six of the eight consensus amino acids, as well as other domains that may mediate protein-protein interactions including an NHz-terminal SH3 domain, a leucine/isoleucine zipper motif, and a large COOH-terrninal region that is rich in serine, threonine, and proline residues (Fig. 1). Upon overexpression in mammalian cell lines MLK3 activates INK through phosphorylation and activation of the dual specific kinase, MKK4 (38) or MKK7 (39), and binds the INK scaffold proteins, IIP], JIPZ and J 1P3 (39, 43, 44). In some cell types, MLK3 has been reported to activate the MAPK p3 8/Reactivating Kinase via MKK-3/6 43 (40) as well as ERK (41). MLK3 associates with Rae and Cdc42 in filter binding assays (26) and coexpression of MLK3, activated Cdc42, and INK increases MLK3 and INK activity (42). Whether activation by Cdc42 of the distantly related PAK and MLK3 involves mechanistically analogous processes is unknown. MLK3 is functionally homologous to Raf, and thus mechanistic aspects of Cdc42-MLK3 and Ras-Raf activation may be shared. Here we show that MLK3 contains a functional CRIB motif that is absolutely required for Cdc42 binding to and activation of MLK3. The zipper region and adjacent basic sequences of MLK3 may also contribute to Cdc42 binding. Binding of Cdc42 to MLK3 does not require MLK3 catalytic activity. Interestingly, GTP-bound Cdc42 has no effect on the activity of purified, catalytically active MLK3, suggesting that an additional cellular component is required for kinase activation. These studies point to an important distinction between PAK and MLK3 in the mode of GTPase-induced kinase activation. Comparative phosphopeptide mapping revealed that coexpression of activated Cdc42 with MLK3 alters the in vivo phosphorylation sites on MLK3. This change in serine/threonine phosphorylation correlates with increased MLK3 activity. These studies represent the first case where a Cdc42-mediated change in the in viva phosphorylation sites of a protein kinase has been documented and provide evidence for the involvement of in viva phosphorylation of MLK3 for Cdc42-induced activation. Thus, the Cdc42-mediated activation of MLK3 is clearly distinct from the mechanism previously described for Cdc42-induced activation of PAK. 3. Materials and Methods 3.1 Construction of mammalian expression vectors and mutagenesis Construction of the cytomegalovirus-based expression vector carrying the cDNA for wild type MLK3 (pRK5-mlk3), has been described elsewhere (35). The expression plasmid encoding the NHz-terminal Flag epitope-tagged constitutively active variant (pRKS-Nflag.cdc42v'2) of Cdc42 was kindly provided by Avi Ashkenazi (Genentech, Inc., So. San Francisco, CA). Variants of the MLK3 gene containing point mutations in the region encoding the CRIB motif were constructed by a modified recombinant polymerase chain reaction (PCR) method described in detail elsewhere (45). For each mutation two different PCRs were performed, one containing a left mutagenesis primer and a left outside primer, and one containing a right mutagenesis primer and a right outside primer. The left and right outside primers used for all mutations were 5’-GATG-AGTCATCTGAATCCAGG-3’ and 5’-CTGTGGCCTATGGCGTAGCTG-3’, respectively. To obtain the three different CRIB mutants the following left and right mutagenesis primers were used, respectively: (MLK3F498A), 5’-GGTGCTIGGCGT-CGAGTGGCAT-3’ and 5’- ACTCGACGCCAAGCACCGCATC-3’; (MLK3H5OOA) 5’- CTTCAAGGCCCGCATCACCGT-T and 5’-GGTGATGCGGGCCTTGAAG-TCG-3’; (MLK3'492NS493A), 5’-GAAGTCGAGTGGCATGGCGGCACGCT-CG-3’ and 5’- CGAGCGTGCCGCCATGCCACTCGAC-3’. The presence of the desired mutation was confirmed by DNA sequencing using the Sanger method, and the absence of PCR- introduced errors was verified by automated sequencing. 45 Deletion of amino acids 430 through 486 of MLK3 to yield MLK3”.p was accomplished by digestion of the expression vector pRK5-mlk3 with BssHII, followed by ligation with T4 DNA ligase. DNA modifying enzymes were purchased from New England Biolabs or Life Technologies, Inc. 3.2 Expression and purification of recombinant MLK3 An Ncol-HindIII fragment, containing the full length mlk3 cDNA, was subcloned from pRK5-mlk3 into the pFastBac HTb baculovirus expression vector (Life Technologies, Inc.) which encodes a hexahistidine tag. Recombinant histidine-tagged MLK3 was expressed in Sf9 cells and purified by nickel affinity chromatography according to the manufacturer's protocol. Fractions containing histidine-tagged MLK3, as determined by SDS-PAGE followed by Coomassie Blue staining, were pooled and concentrated using a Centriprep concentrator (Amicon). 3.3 Cell lines and transfections Human fetal kidney 293 cells were maintained in Ham’s F12zlow glucose Dulbecco’s modified Eagle’s media (1:1) (Life Technologies, Inc.) supplemented with 8% fetal bovine serum (Life Technologies, Inc.), 2 mM glutamine, and penicillin/streptomycin (Life Technologies, Inc.). Plasmids (5 ug each for 60 mm dishes; 10 ug each for 100 mm dishes) were used to transfect 293 cells using the calcium phosphate technique (Gonnan, 1990). Cell monolayers were incubated with the DNA precipitate for 4 h, then washed once with PBS (phosphate-buffered saline), and cultured in the medium described above. After 18 h cells were harvested. 46 3.4 Cell lysis and immunoprecipitation Cells were washed with ice cold PBS and lysed for 5 min on ice by the addition of 1 m1 of lysis buffer (50 mM HEPES 33H 7.5), 150 mM NaCl, 1.5 mM MgCl,, 2 mM EGTA, 1% Triton X-100, 10% glycerol, 10 mM NaF, 1 mM Na.PP,, 100 11M [3- glycerophosphate, 1 mM Na3V04, 2 mM PMSF, and 0.15 U/ml aprotinin). The lysate was clarified by centrifugation for 20 min at 14,000 rpm in an Eppendorf centrifuge at 4 °C. Rabbit polyclonal antiserum was raised against a peptide corresponding to the COOH-terminal eight amino acids of MLK3 and was purified by Protein A-Sepharose chromatography. Antibodies against the proteins of interest were prebound to protein A- agarose beads [MLK3 antiserum (0.25 ug/ul slurry), M2 monoclonal antibody (Kodak 1B1) directed against the Flag epitope (0.45 ug/ul slurry) and INK C-l7 antibody (Santa Cruz Biotechnology) (50 ng/ul slurry) as previously described (35). Clarified lysate (300 pl) was incubated with 20 ul of antibody-bound Protein A-agarose for 90 min at 4 °C. Irnmunoprecipitates were washed with HNTG buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton-X-IOO, 10% glycerol). Irnmunoprecipitates used for kinase assays were washed three times with HNTG buffer containing 1 M LiCl, three times with HNTG buffer, and twice with kinase assay buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MnClz, 10 mM MgC12, 0.1 mM Na3VO4). 3.5 Gel electrophoresis and Western blot analysis Lysates and immunoprecipitates of MLK3 and Cdc42 were resolved by SDS- polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (47). Proteins 47 were transferred to nitrocellulose and immunoblotted using either MLK3 antiserum (1 pg/ml) or M2 Flag monoclonal antibody (9 pg/ml), followed by the appropriate horseradish peroxidase-conj ugated secondary antibody (Life Technologies, Inc.). Western blots were developed by chemiluminescence. Multiple exposures of the Western blots were developed, and densitometry (NIH Image) of unsaturated films was used to determine relative expression levels. 3.6 In vitro kinase assays Kinase assays were performed in 20 pl of kinase assay buffer containing 50 pM ATP and 5 pCi [y-32P]-ATP (3000 Ci/mmol) (NEN Life Science Products). For the MLK3 kinase assay 10 pg of mixed histones (Boehringer Marmheim) was used as a substrate and the reaction was carried out for 30 min at room temperature. Independent experiments showed that the reaction was linear within this time range. The reactions were terminated by the addition Of an equal volume of 2x SDS sample buffer (100 mM Tris (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromphenol blue, 100 mM DTT, 1% B-mercaptoethanol) containing 50 mM EDTA (pH 8.0). For the kinase assays involving purified kinases, recombinant MLK3 or PAK-2 (1 pg and 2 pg, respectively) was incubated in 50 pl of kinase assay buffer containing 4 pg of GST (glutatlrione S-transferase)-Cdc42 that had been preloaded with GTPyS or GDP and assays were performed as above. GST-Cdc42 (19 pM) was preloaded with GTP'yS or GDP (4 mM) in buffer containing 50mM Tris (pH 7.5), 5 mM EDTA, and 1 mM DTT. The mixture was incubated for 15 min at 30 °C. The nucleotide loading reaction was quenched by the addition of 10 mM MgC12. Purified GST-Cdc42 was obtained from an E. 48 coli overexpression system; and purified PAK-2, obtained using the baculovirus expression system, was kindly provided by Dr. Arie Abo (Onyx Pharmaceuticals) (23). For the INK assays, 8 pg of GST-c-Jun was used as the substrate, and the reaction was carried out for 15 min at room temperature. The pGEX-c-jun (1-115) vector was kindly provided by Dr. Ajay Rana (Massachusetts General Hospital, Harvard Medical School, Boston, MA). GST-c-Jun was expressed in XL-l Blue E. coli and purified by glutathione Sepharose chromatography. Following the kinase assay, proteins were separated by SDS-PAGE. Gels were rinsed in PBS, dried, and incorporation of radioactivity into kinase, or substrates was determined by Phosphorlmaging (Molecular Dynamics). To detect INK expression, proteins were transferred from an SDS polyacrylamide gel to a polyvinylidene difluoride (PVDF) membrane and immunoblotted using the INK C-17 antibody (0.5 pg/ml). 3.7 In vivo phosphopeptide mapping After a 24 h transfection with pRKS-mlk3 in the presence or absence of pRKS- Nflag.cdc42v‘2, 293 cells were washed five times with phosphate-free medium (Dulbecco’s modified Eagle’s medium supplemented with 10% dialyzed fetal bovine serum (Summit Biotechnology», and incubated at 37 °C for 2 h. The cells were then incubated in phosphate-free medium containing 3 mCi/ml [”P] orthophosphate (carrier fi'ee; NEN Life Science Products) for 4 h at 37 °C. Cells were washed five times with ice cold PBS and then lysed in 1 ml of lysis buffer. Lysates were clarified by centrifugation for 15 min at 14,000 rpm in an Eppendorf centrifuge at 4 °C. MLK3 was immunoprecipitated with MLK3 antiserum as described 49 above. Irnmunoprecipitated proteins were resolved by SDS-PAGE and transferred to a PVDF membrane. Radiolabeled bands that co-migrated with MLK3, as judged by Western blotting, were excised from the PVDF membrane. After washing three times with methanol and three times with water, the radioactive piece of membrane was blocked with 1 ml of 0.5% polyvinylpyrrolidine-36O (Sigma) containing 100 mM acetic acid for 30 min at 37 °C, and then washed five times with water. Tryptic digestion was performed with 10 pg of sequencing grade trypsin (Boehringer Mannheim) for 2 h in 200 pl of 50 mM NH4HCO3, pH 8.3, at 37 °C. An additional 10 pg of trypsin was added and the digestion mixture was incubated for an additional 2 h at 37 °C. The membrane was then sonicated for 3 min in 300 pl of water to remove additional tryptic peptides. The solution containing the released tryptic peptides was concentrated in a SpeedVac (Savant Instruments). The peptides were separated on cellulose thin layer chromatography (TLC) plates (Kodak, 20 x 20 cm) by thin layer electrophoresis (TLE) in the first dimension in pH 1.9 buffer (formic acid (88% w/v)/glacia1 acetic acid/water, 25:78:897, v/v/v) at 0 °C and 1000 V for 30 min, and separated in the second dimension by TLC in phosphochromatography buffer (n-butanol/pyridine/glacial acetic acid/water, 15:10:3:12, v/v/v/v). The radiolabeled phosphopeptides were visualized and quantitated using a phosphorimager. 50 4. Results 4.1 MLK3’s CRIB motif is necessary for association with Cdc42 and for Cdc42 induced activation of MLK3 Recently Hall and coworkers have shown that several proteins, including MLK3, associate with GTP-bound Cdc42 and Rac in filter binding assays (26). All of these proteins contain a 14-16 amino acid sequence that includes eight consensus residues, which has been coined the CRIB motif. The CRIB motif of MLK3 contains six of the eight consensus amino acids (Fig. 2). In this study we examined the structural requirements and mechanism of Cdc42 binding and activation of MLK3. MLK3 and Flag epitope-tagged Cdc42 expression vectors were transiently transfected in 293 cells. To mimic the GTP-bound state of Cdc42 we used a constitutively active mutant of the GTPase, i.e. Cdc42 V12. To test whether MLK3’s potential CRIB motif actually functions in the binding of Cdc42 we took a site-directed mutagenesis approach. Three different MLK3 variants were generated by mutating DNA encoding conserved amino acids in the CRIB motif to produce alanine residues: MLK3 F498A, MLK3 H500A, and MLK3 I492A/S493A (Fig. 2). While we cannot absolutely rule out the possibility that introduction of these mutations might alter MLK3’s conformation, the expression levels of the MLK3 CRIB mutants in transient transfections of 293 cells are at least as high as that of wildtype MLK3 (Fig. 3 second panel), suggesting that these variants are stable. The CRIB variants were coexpressed with Cdc42 V12 in 293 cells and the cellular lysates were subjected to co-immunoprecipitation experiments. While wildtype MLK3 associates with Cdc42 V12, none of the MLK3 CRIB mutants detectably associates with the activated GTPase (Fig. 3 upper panel). 51 If Cdc42-induced activation of MLK3 is mediated through its interaction with MLK3’s CRIB motif, one would expect that the MLK3 CRIB mutants should exhibit a defect in Cdc42-induced activation. Accordingly, cells were transiently transfected with cDNAs encoding wildtype MLK3 or MLK3 I492A/S493A, in the presence or absence of Cdc42 V12. The activity of the immunoprecipitated MLK3 or MLK3 CRIB mutant was measured in an in vitro kinase assay. In the absence of Cdc42 V12, both wildtype MLK3 and the MLK3 CRIB variant show similar levels of autophosphorylation and substrate phosphorylation (Fig. 4). In the absence of Cdc42 V12, the differences in the activities of MLK3 and the MLK3 CRIB variants were not statistically significant. This further supports the idea that the mutations in the CRIB motif do not grossly perturb MLK3’s structure or inherent catalytic activity. However, both autophosphorylation and substrate phosphorylation of the CRIB variant is markedly lower (3-fold) than that of wildtype MLK3, when each is coexpressed with the activated GTPase. The small increase in the catalytic activity of the MLK3 CRIB mutant when activated Cdc42 is coexpressed may be. due to Cdc42-activated endogenous MLK3. Alternatively there may be residual binding of the CRIB variant to activated Cdc42 in viva, which we do not detect in our in vitro co- immunoprecipitation assay. Taken together these results demonstrate that MLK3 does contain a functional CRIB motif, and that Cdc42-induced activation of MLK3 is mediated via association with this CRIB motif. 4.2 Effects of deleting the COOH-terminal portion of MLK3’s zipper domain In WASP (51), PAK (32, 52) and ACK (27) the CRIB motif is necessary but not sufficient for GTPase binding. Outside of the CRIB motif MLK3 shares no sequence 52 similarity with the minimal GTPase binding domains that have been defined for these proteins. Instead, MLK3 contains two closely spaced leucine/isoleucine zipper motifs, spanning amino acids 400-462, COOH-terrninal to the CRIB motif (Fig. 1). Considering the close vicinity in linear sequence of the zippers and the CRIB motif we asked whether the zipper motif might contribute to the binding of MLK3 to Cdc42 V12. Accordingly, a variant of MLK3, MLK3Azip, which lacks amino acids 430-486, as shown in Fig. l, was constructed. This deletion removes the second half of the zipper region as well as 22 COOH-terminal amino acids which includes a short basic region, but leaves the entire CRIB motif intact. MLK3Azip is expressed in transiently transfected 293 cells at levels comparable to that of wildtype MLK3 (Fig. 5 second panel). The ability of Cdc42 V12 to associate with MLK3Azip was tested in co-immunoprecipitation experiments with cellular lysates harvested from transiently transfected 293 cells. The deletion of the second zipper/basic stretch greatly diminishes the ability of MLK3 to bind to Cdc42 V12, despite the presence of the complete CRIB motif (Fig. 5 upper panel). Based on these and our previous results, both the CRIB motif and the second half of the zipper region (and a stretch of basic amino acids) may contribute to Cdc42 binding. Alternatively, the COOH-terrninal zipper/basic stretch may not directly interact with Cdc42, but may be required for the proper presentation and binding of the CRIB motif to the GTPase. MLK3Azip has approximately 70% of the basal autophosphorylation activity of wildtype MLK3 (Fig. 6). However, in contrast to wildtype MLK3, there is no Cdc42- induced increase in autophosphorylation of MLK3Azip, consistent with the finding that MLK3Azip binds Cdc42 V12 only very weakly. In an in vitro kinase assay, MLK3Azip in 53 the presence or absence of Cdc42 V12 (Fig. 6) lacks the ability to phosphorylate histones. To address whether the lack of histone phosphorylation might be due to some unique feature of histones we performed the same experiment with myelin basic protein as a substrate and obtained the analogous results (data not shown). These data suggest that the zipper domain/basic stretch may be fundamentally required for substrate phosphorylation. 4.3 The leucine zipper domain is sufficient for MLK3 oligomerization To characterize the oligomerization properties of MLK3Azip, we engineered a cDNA construct encoding amino acids 386-47 7 of MLK3 fused to the coding sequence of the monomeric MBP of E. coli, designated MBP-zips, and tested purified MBP and MBP- zips for their ability to associate with full length MLK3 and MLK3Azip. As shown in Fig. 7A, full length MLK3 binds MBP-zips but not MBP, as judged by immunoblotting. Furthermore, MLK3Azip fails to bind either MBP-zips or MBP. Equal amounts of MBP or MBP-zips protein in the assays were verified by Coomassie Blue staining of a duplicate gel (Fig. 7B). Western blotting of cellular lysates using a MLK3 antibody shown in Fig. 7C, reveals that full length MLK3 and MLKBAzip were expressed at similar levels. These data provide direct evidence that the leucine zipper/basic stretch of MLK3 is capable of protein-protein interactions and is sufficient to mediate MLK3 homo-oligomerization. 4.4 Activated Cdc42 fails to activate MLK3 in vitro The small GTPases Rae and Cdc42 can stimulate the autophosphorylation activity of the CRIB-containing serine/threonine kinase PAK-2 in vitro (23). To determine whether 54 Cdc42-induced activation of MLK3 can be recapitulated in a completely in vitro system, hexahistidine NHz-terminal tagged MLK3 was expressed using the baculovirus system, and purified by metal-chelate chromatography. GST-Cdc42 was expressed in and purified from E. coli. The purified MLK3 is catalytically active as judged by its basal autophosphorylation activity. GTPyS- or GDP-loaded GST-Cdc42 was incubated with purified MLK3 or PAK-2 in an in vitro kinase assay (Fig. 8B). While GTPyS-loaded Cdc42 activates purified PAK-2, it fails to activate purified MLK3. Likewise, MLK3 immunoprecipitated from transfected 293 cells cannot be activated in vitra by the addition of GTPyS-loaded Cdc42 (Fig. 8A). These data support the requirement of a cellular context or coactivator for MLK3 activation by Cdc42. 4.5 Cdc42 alters the in viva phosphorylation pattern of MLK3 As described above, co-immunoprecipitation experiments and in vitro kinase assays show that Cdc42 V12 when coexpressed with MLK3 can associate with MLK3 and modulate its catalytic activity within the cell. However, purified, activated Cdc42 cannot stimulate the autophosphorylation of MLK3 in vitro. In order to determine if the presence of activated Cdc42 alters MLK3 phosphorylation in viva, two-dimensional phosphopeptide analysis of MLK3 labeled in vivo, in the absence and in the presence of Cdc42 V12, was performed. The net incorporation of radiolabel into MLK3 increased approximately three-fold when MLK3 was coexpressed with Cdc42 V12. Two-dimensional TLE/T LC revealed that while the basic pattern of phosphopeptides from the two samples is similar (Fig. 9A), there are notable differences. The major changes are observed in the triangular cluster of 55 phosphopeptides b, c, and d. Phosphopeptide a predominates in both samples. Phosphopeptides b and c are detected in the triangular cluster of the MLK3 map, but are low in abundance relative to peptide a. In the corresponding map of MLK3 that had been expressed in the presence of Cdc42 V12, however, phosphopeptide b is not detected. Instead, phosphopeptide c is the prominent phosphopeptide in the triangular cluster, with 70% of the radioactivity of phosphopeptide a. (Fig. 98). Furthermore, phosphopeptide (I, nearly undetectable in the map of MLK3, appears at high levels in the map of MLK3 expressed with Cdc42 V12. For comparison, the level of another peptide (x) relative to peptide a is essentially constant in both maps. The labeling and mapping from three independent experiments yielded the same results. These data indicate that the presence of activated Cdc42 changes the in vivo phosphorylation state of MLK3, which correlates with an increase in MLK3 catalytic activity. 56 1 43 103 115 384 400 487492 506 632 847 C Gly SH3 Kinase 3:32" 'i’ ProlSerlThr-rich B 400-KREQQGLFDELRAKEKELLSREEELTRAARFQRSOAEQLRRREHLLAQWELEVFERELTLLLQQVDRERPHVRRRRGTFKRSKLRAd-AGT Fig. 1. Schematic of MLK3. The numbers in the above diagram represent amino acid number. The glycine-rich region (amino acids 1-42) is denoted by Gly. The region containing the zipper motif and the polybasic stretch of amino acids includes amino acids 400-486. The amino acid sequence corresponding to this region is shown below with the basic stretch of amino acids printed in bold letters and the non-aromatic hydrophobic residues predicted to occupy the d position in the zipper motifs underlined. The sequence deleted in MLK3Azip (amino acids 430-486) is boxed 57 Consensus ISXPXXXXFXHXXHVG MLK2 m-ISXP. .xxrxrrxxrtvc-487 WASP 233-IGXP. .XXFXHXXHVG-251 ACK 5,, - rsxpxxxxrxnxxnco- 527 MLK3 .92 - ISXPXXXXFXI-IXXTVQ- 505 MLK3l492A-S492A AA l MLKSF‘W‘ A MLK3H5°°A A Fig. 2. Alignment of CRIB motifs and MLK3 variants. The consensus sequence for the CRIB motif as defined by Burbelo et al. is shown aligned with the CRIB motif of MLK2, WASP, ACK, and MLK3. Amino acid numbers are indicated to the left and the right of each sequence. Amino acids occupying the consensus residues are indicated by bold, with those that differ from the consensus italicized. Absent amino acids are indicated by period. The mutations in the engineered CRIB variants of MLK3 are shown with the conserved residue to alanine changes indicated by bold letters. 58 MLK3= - wr F H 1 WT F H I V12, Cdc42 - I - I _ " -" + .+ +. + .. immunoblot Flag IP .- f‘ . - ' MLK3 - r'u‘ v -., .4, ‘1 I .o“ - x.-*a . ~.' .auuyv . .. .u.‘ .,. , ' ' — I . ‘r‘v ‘; ' i > ' , I - r, ‘ fl ' ' I ‘ I, I ’ MLK3 E. .1 ‘ I) . - ,-_ . l ‘ l is. < v. . . ,1 ‘ . . . . ‘1‘ t . , - . ~ . . , “ r . ‘6.“ » --w... J- - a...'..-::-_-..'.r.a..!:..*- ..i‘ Flag-Cdc42 Fig. 3. Effects of mutations in the CRIB motif an association of MLK3 with Cdc42v'z. Co-immunoprecipitation experiments of MLK3 or MLK3 CRIB variants with Cdc42V'2. The presence of bound MLK3 or MLK3 CRIB variant was assessed by immunoblotting with a MLK3 antibody as described previously (upper panel). The middle and lower panels show MLK3 and Cdc42 expression, respectively. WT indicates wild type MLK3, F indicates MLK3F498", H indicates MLK3H500A, and 1 indicates MLK31492A-S493A 59 MLK3: - WT I492A- - WT I492A- S493A S493A Cdc42”: _ _ - + + + autoradlogram 4— MLK3 ‘— histones Immunoblot ’ MLK3 Flag-Cdc42 Fig. 4. Effects of mutations in the CRIB motif on MLK3 in vitro kinase activity. In vitro kinase assay of MLK3 and MLK3“92“S‘93A. The autoradiogram (upper panel) shows MLK3 autophosphorylation and histone phosphorylation indicated by the arrows. The middle and lower panels show MLK3 and Cdc42 expression, respectively. 60 MLK3: . wr Azlp wr Azip V12. C6642 - : .'_ - '1’ ’1": Immunoblot ' -..-cut.,-_-....... .8. . I h Flag IP ‘.. : . r, ,H‘... v-vm—rv— ..- —'r‘ t'rh—hbs— - "a a»: w MLK3 r,’ .‘ . ,.-'“."..." 1' .IJ’J‘JI. .‘R‘Z'VJ u'c‘" a - - ' t q _ . . u _u‘ "_""0 hay/e ‘ 'u . .‘ _ _- . ‘J,",‘.\u In I; I,“ ,1 . (g‘.‘ "t, . is, , 7 ‘: If a L; ‘ MLK3 I. x: .- s's'f. a-lb‘fi "l ‘ Flag-Cdc42 Fig. 5. Effects of partial deletion of the zipper/basic stretch an association with Cdc42v'z. Co-immunoprecipitation experiment of MLK3 and MLK3AZip with Cdc42V12. The presence of bound MLK3 or MLK3”ip was assessed by immunoblotting with a MLK3 antibody as described previously (top panel). The middle and lower panels show MLK3 expression, respectively. 61 MLK3: _ WT Azlp _ WI' Azlp Cdc42“: - - - + + + autoradlogram «MLK3 histones <— lmmunoblot ~ ~ MLK3 : . um Flag-Cdc42 Fig. 6. Effects of partial deletion of the zipper/basic stretch on MLK3 in vitro kinase activity. In vitro kinase assay of MLK3 and MLK3Azip using histones as a substrate. The top panel shows an autoradiogram with bands corresponding to autophosphorylated MLK3 and phosphorylated histones indicated by arrows. The lower and middle panels show MLK3 and Cdc42 expression, respectively. A MBP MBP-alps f D r W O. O- MLK3. 5 a 5 3. m a c m’ ‘ MLK3 .- . , ‘ . . ,9 MBP pulldown MLK3 ' E 2 mm B MBP MBP-zips ~ MLK3 r a) f 2 ’ MLK3: - 5 “g - l; '5 new <— use Coomassie stalnlng F ig. 7. Assay for association of the zipper/basic stretch of MLK3 with MLK3 and MLK3Azip in vitro. Lysates from 293 cells expressing MLK3 or MLK3Azip were incubated with amylase resin to which purified MBP or MBP-zips had been prebound. A, After washing of the resin, the presence of bound MLK3 or MLK3Azip was assessed by immunoblotting with the MLK3 antibody. B, Equal loading of MBP and MBP-Azips on the amylose resin was confirmed by Coomassie staining. C, Expression of MLK3 and MLK3Azip was assessed by immunoblotting of cellular lysates with a MLK3 antibody. 63 GDP GTPyS autoradiogram Cdc42: “ n <— MLK3 Immunoblot 44— MLK3 autoradiogram ‘— MLK3 Fig. 8. In vitro kinase assay using purified Cdc42. A, MLK3 was immunoprecipitated from cellular lysates that had been transfected with cDNA encoding MLK3 and was incubated in an in vitro kinase assay in the presence of 4 pg of GST-Cdc42 preloaded with either GDP or GTPyS. The top panel shows an autoradiogram with bands corresponding to MLK3 autophosphorylation indicated by an arrow. The lower panel shows MLK3 expression. B, Purified MLK3 and PAK2(1 and 2 pg, respectively) were incubated in an in vitro kinase assay in the presence of 4 pg of GST-Cdc42 preloaded with either GDP or GTPyS. Shown is an autoradiogram with bands corresponding to MLK3 or PAK2 autophosphorylation. 64 Fig. 9. Phosphopeptide mapping of tryptic peptides derived from in viva phosphorylated MLK3. A, two-dimensional phosphopeptide mapping of 32P-labeled MLK3 from 293 cells transfected with expression vectors for MLK3 (top) or MLK3 and Cdc42Vlz (bottom). MLK3 was immunopurified from cellular lysates, blotted onto polyvinylidene difluoride membrane, and subjected to partial tryptic digestion. Equal amounts of radioactivity, as determined by Cerenkov counting of the resultant tryptic peptides , were analyzed by TLE in the first dimension and TLC in the second dimension. The direction of electrophoresis and chromatography are indicated by long arrows. Phosphopeptides were visualized by Phosphorlrnaging. The phosphopeptides of interest are alphabetically labeled. Shown is a map representative of three independent experiments. B, the percent radioactivity of the indicated phosphopeptides compared with phosphopeptide a, calculated as: [(volume-background)phosphopeptide]/[(volume- background)phosphopeptide a] x 100, using Image Quant software (Molecular Dynamics). 65 chromatography electrophoresis —' % Radioactivity of Phosphopeptide a * Peptides b c d x MLK3 14 1 3 7 56 3:32;. o 71 50 44 66 5. Discussion Small GTPases of the Ras superfamily have been shown to regulate protein kinases. PAK has emerged as the paradigm CRIB-containing serine/threonine kinase that is activated by GTP-bound Cdc42 and/or Rae. The PAKs play roles in diverse processes, including apoptosis, modulation of actin cytoskeleton, gene. transcription, and cell cycle (5 3). MLK3 is a member of the so-called mixed lineage kinases. Except for the presence of a loosely conserved CRIB motif, MLK3 differs dramatically from the PAKs, both structurally and functionally. While the mammalian PAK-l, -2, and -3 share 95% sequence similarity in their catalytic domains, MLK3’s catalytic domain is just 20% similar to those of the mammalian PAKs. The CRIB motif of the PAKs is found NH;- terrninal to the catalytic domain, whereas MLK3’s CRIB motif is COOH-terminal to the catalytic domain. Flanking MLK3’s catalytic domain is an NHz-terminal SH3 domain and a COOH-terminal leucine zipper motif, both lacking in the PAKs. The only well- established function thus far ascribed to MLK3 is as an MKKK in the activation of the INK pathway. Because the MLKs are so different from the PAKS, it is important to determine whether the structural features of their binding to and the mechanisms of activation by Cdc42 and/or Rac also differ from that of the PAKs. Whereas the three mammalian PAK isoforms contain perfect consensus CRIB motifs, as defined by Burbelo et al. (26), MLK3’s CRIB motif contains only six of the eight consensus residues (Fig. 2). We show that mutations in conserved residues of MLK3’s CRIB motif disrupt the ability of the Cdc42 to bind to and activate MLK3, indicating that MLK3 does indeed contain a functional CRIB motif. WASP and ACK, two other proteins whose CRIB-dependent binding to Cdc42 has been well-established, 67 L _. also contain less than perfect CRIB motifs (Fig. 2), with WASP (28, 29) and ACK (27) containing 7 and 6 of the 8 consensus residues, respectively. It may well be that the CRIB consensus motif is biased towards PAK, due to the large number of PAK isoforrns that have been identified. MLK3 and the closely-related MLK2 lack the second of the two conserved histidine residues of the consensus CRIB motif (Fig. 2). We show here that mutation of the first conserved histidine in MLK3 to an alanine residue (H500A) disrupts binding to Cdc42. The fact that both MLK3 (26) and MLK2 (26, 54) have been demonstrated to bind Cdc42 may indicate that the second of the two conserved histidines in the consensus CRIB motif in other CRIB-containing proteins is not required for binding to Cdc42. Further support for this notion is provided by the finding that the conserved Asp 38 in Cdc42 interacts primarily with the first of the two conserved histidine residues (Hi5520) in ACK (55). In addition, mutation of the first of the two conserved histidine residues in N-WASP to aspartate (H208D) decreases the in vitro binding affinity for Cdc42 and Rac, as well as the activity of N-WASP in vivo and in vitro (56). The regions outside of the CRIB motifs of ACK and WASP exhibit low sequence similarity, and, perhaps not surprisingly, low structural similarity when bound to Cdc42 (55, 57). It is likely that the GTPase binding domain of MLK3, with the exception of the CRIB motif, will differ structurally from those of both WASP and ACK. Because of the proximity of MLK3’s zipper and CRIB motifs in linear sequence, and because sequences flanking the CRIB motif in other proteins contribute to Cdc42/Rae binding, we tested whether deletion of the COOH-terminal half of the zipper motif affects Cdc42 binding. The binding of MLK3Azip (Fig. 1), which contains an intact CRIB motif, 68 to activated Cdc42 is reduced more than 10-fold, suggesting that, in addition to MLK3’s CRIB motif, the zipper domain or the following short basic region of MLK3 may contribute to Cdc42 binding. Interestingly, the basal autophosphorylation activity of MLK3Azip is about 70% that of wildtype MLK3. This may indicate some intramolecular autophosphorylation activity. Alternatively, MLK3Azip may homo-oligomerize and undergo intermolecular autophosphorylation. Leung et a1. (58) recently reported a very large reduction in GST-MLK3 autophosphorylation activity in vitra upon deleting the entire zipper region, but leaving the basic stretch intact. Recent site-directed mutagenesis studies indicate that the serine/threonine kinase PAK contains a basic stretch consisting of three contiguous lysine residues upstream of the CRIB motif whose charge is important for binding to Racl and Rac2, and whose presence is required for efficient PAK-l activation by Racl, RacZ, and Cdc42 (33). MLK3 contains four contiguous arginine residues between the zipper domain and the CRIB motif. These basic amino acids are deleted in the MLK3Azip variant, which exhibits greatly reduced binding to activated Cdc42. Thus, it is plausible that the arginine tract in MLK3 may contribute to Cdc42 binding or activation of MLK3. Currently we are defining a minimal Cdc42-binding domain of MLK3 and are assessing the relative contributions of various amino acids within this domain to Cdc42 binding and Cdc42- induced MLK3 activation. We show that MLK3 and activated Cdc42 can be co-immunoprecipitated from cellular lysates. However, under the conditions of our in vitro kinase assay, which clearly show a Cdc42-induced increase in MLK3 autophosphorylation and histone phosphorylation, Cdc42 is not detected. Possibly once MLK3 is activated by Cdc42, 69 MLK3 has a reduced affinity for the GTPase as has been demonstrated with PAK-2 (21). Furthermore the sites of in vitro autophosphorylation of MLK3 expressed with and without Cdc42, and isolated from mammalian cells, are essentially identical as judged by mapping of tryptic phosphopeptides (data not shown). The mechanism by which the highly conserved PAK family members (PAK-1, -2, and -3) are activated by Rae and Cdc42 has been well'studied. Increased autophosphorylation activity is observed upon incubation of purified activated Cdc42 with purified PAK. In contrast to PAK, purified, catalytically active MLK3 cannot be further activated in vitro by the addition of GTP- bound Cdc42. These data are consistent with a catalytic model in which Cdc42 activates MLK3 in viva, but is not required to maintain MLK3 in its activated state. We therefore decided to assess whether Cdc42 induces differential phosphorylation of MLK3 in viva. Two-dimensional tryptic phosphopeptide mapping studies of in viva labeled MLK3, expressed with or without constitutively active Cdc42, showed similar phosphopeptide maps, with major differences observed in a triangular cluster of phosphopeptides b, c, and d (Fig. 9A). In the map of MLK3 alone, phosphopeptides b and c are present, but at low levels. When activated Cdc42 is coexpressed with MLK3, phosphopeptide b is not detected and phosphopeptides c and d appear at high levels. Since the change in in viva phosphorylation of MLK3 with Cdc42 correlates with increased MLK3 activity, it is likely that phosphopeptides c and d contain activating phosphorylation sites. Phosphopeptides c and d may be distinct phosphopeptides. However, since their chromatographic mobilities are essentially identical, it is also possible that phosphopeptides c and d result from differential trypsin digestion. Phosphopeptides b and c lie on a diagonal which slopes toward the anode, characteristic of peptides that are 70 phosphoisomers. Upon phosphorylation, the negative charge and polar nature of the added phosphate group reduces a peptide's mobility in both the electrophoretic and chromatographic dimensions (5 9). Therefore, phosphopeptide c may differ from phosphopeptide b by the addition of a phosphate group(s). This is consistent with the observation that when activated Cdc42 is coexpressed with MLK3, phosphopeptide c emerges while phosphopeptide b disappears. Cdc42 may induce differential MLK3 autophosphorylation in vivo or, alternatively, another MLK3-activating kinase may be responsible for the in viva change in MLK3 phosphorylation. Because Cdc42 is geranylgeranylated (60) and has been localized to cellular membranes (61, 62, 63) as well as to cytoskeletal elements (64), it is possible that Cdc42 recruits MLK3 to the vicinity of an activating kinase. MLK3 and the serine/threonine kinase Raf both appear to function as MKKKs, activating the JNK and ERK pathways, respectively. The idea that Cdc42 may recruit MLK3 to an activating kinase is reminiscent of the proposed mechanism by which the small GTPase Ras activates Raf. Analogous to our findings with Cdc42 and MLK3, the addition of purified, activated Ras to Raf in vitro is not sufficient for full activation of Raf. However, appending a membrane targeting motif to the COOH-terrninus of Raf causes Raf translocation and activation in the absence of activated Ras (1). It has been recently shown that PAK-3 phosphorylates Raf in vivo and in vitro leading to an increase in Raf activity (65), although it has yet to be determined whether this event requires Raf translocation. In yeast, the PAK homolog STE20 functions as an MKKK in the activation of a yeast MAPK pathway. By analogy, potential MLK3-activating kinases may include PAK-related kinases (53) such as hematopoietic protein kinase-1 (HPK-1). 7l In coexpression studies HPK-1 binds to MLK3's SH3 domain and phosphorylates MLK3, but the effect on MLK3 activity is not reported (66). Interestingly, unlike PAK-1, -2, and -3, HPK-1 lacks a CRIB motif. Our finding that catalytically active MLK3 cannot be further activated in vitro by GTP-bound Cdc42 suggests that the GTPase activates MLK3 differently than the PAKs. Whereas the PAKs can be activated in vitro by interaction with unprenylated GTP-bound Cdc42, the activation of MLK3 by Cdc42 appears to require prenylation of Cdc42, the cellular environment, or an as yet unidentified cellular component. Our data support a model in which the in viva CRIB-dependent interaction of MLK3 and Cdc42 either allows MLK3 to adopt a conformation that leads to autophosphorylation or recruits MLK3 to the vicinity of a serine/threonine kinase that phosphorylates and activates MLK3. Determination of the precise sites of Cdc42- induced phosphorylation of MLK3, coupled with subcellular localization studies, should shed further light on the detailed mechanism of MLK3 activation by Cdc42. The work described in sections 4.1 and 4.2 was done by Barbara Bdck Expression and purification of MLK3 was done by Brian Qamirani. 72 References l. Stokoe, D., Macdonald, S. G., Cadwallader, K., Symons, M., and Hancock, J. F. (1994) Science 264, 1463-1467 2. 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Zipper-Mediated Oligomerization of the Serine/Threonine Kinase MLK3 Is Not Required for Its Activation by the GTPase Cdc42 But Is Necessary for Its Activation of the IN K Pathway (The results described in Chapter II were published in the Journal of Biological Chemistry in 2000 — Vacratsis, P.O., and Gallo, K.A., (2000) J. Biol. Chem. 275, 27893- 27900 78 III. Zipper-Mediated Oligomerization of the Serine/Threonine Kinase MLK3 Is Not Required for Its Activation by the GTPase Cdc42 But Is Necessary for Its Activation of the IN K Pathway 1.Abstract MLK3 is a serine/threonine kinase that has been identified as an upstream activator of the INK pathway. MLK3 is capable of activating MKK4 by phosphorylation of serine and threonine residues, and mutant forms of MKK4 that lack the phosphorylation sites Ser’” and Thr258 block MLK3-induced INK activation. A region of 63 amino acids following the kinase domain of MLK3 is predicted to form a leucine zipper. MLK3’s leucine zipper domain has been shown to be necessary and sufficient for MLK3 oligomerization but its role in regulating activation of MLK3 and downstream signaling remains unclear. In this study, we substituted a proposed stabilizing leucine residue in the zipper domain with a helix-disrupting proline to abrogate zipper-mediated MLK3 oligomerization. We demonstrate that constitutively activated Cdc42 fully activates this monomeric MLK3 mutant in terms of both autophosphorylation and histone phosphorylation activity, and induces the same in viva phosphorylation pattern as wild type MLK3. However, this catalytically active MLK3 zipper mutant is unable to activate IN K. Our data show that the monomeric MLK3 mutant fails to phosphorylate one of the two activating phosphorylation sites, Thrz”, of MKK4. These studies suggest that zipper- mediated MLK3 oligomerization is not required for MLK3 activation by Cdc42, but 79 instead is critical for proper interaction and phosphorylation of a downstream target, MKK4. 80 2. Introduction MAPKs are serine/threonine kinases that are regulated by upstream kinase cascades. The MAPK pathways regulate many cellular processes including proliferation, differentiation, and gene expression. The best-characterized MAPK pathways in mammalian cells are the ERK, p38/RK, and the JNK pathways (1, 2). In MAPK pathways, extracellular signals lead to the phosphorylation and activation of a MAPKKK. An activated MAPKKK can productively bind, phosphorylate, and, hence, activate a dual specific MAPKK, which in turn activates a MAPK by phosphorylating a threonine residue and a tyrosine residue within the conserved activation segment located between subdomains VII and VIII of its catalytic domain. Activating phosphorylation sites for the MAPKK family have also been identified within kinase subdomains VII and VIII (3-5). For instance, the dual specific kinase, MKK4, which phosphorylates and activates INK (6), requires phosphorylation on Ser254 and Thr258 for activation (4, 5). These residues are located within the conserved activation segment of kinase subdomains VII and VIII, and MKK4 mutants lacking these two phosphorylation sites fail to phosphorylate and activate INK (3, 4, 7). MLK3 has been identified as an upstream activator of the JNK pathway (4, 7, 8). MLK3 is capable of activating MKK4 by phosphorylation of serine and threonine residues, and mutant forms of MKK4 that lack the phosphorylation sites, Ser’" and Thr’”, block MLK3-induced INK activation (7, 8). MLK3 is an intracellular serine/threonine kinase with a predicted molecular weight of 93 kDa (9). In addition to the kinase domain, the sequence of MLK3 encodes several domains that are predicted to be involved in protein-protein interactions, 81 including an SH3 domain, a leucine zipper domain, a Cdc42/Rae interactive binding (CRIB) motif and a COOH-terminal 220 amino acid region that is rich in proline, serine, and threonine residues. Recently, it has been demonstrated that MLK3 can associate with an activated form of Cdc42 (10, 11) and that this association requires a functional CRIB motif (12). Coexpression of MLK3 and activated Cdc42 in cells increases MLK3's catalytic activity (11, 12). Interestingly, recombinant catalytically active MLK3 is not activated further by the addition of purified GTP-bound Cdc42, suggesting the requirement of an additional cellular component for MLK3 activation by Cdc42 (12). Furthermore, using in viva labeling experiments and comparative two dimensional phosphopeptide mapping, we have shown that coexpression of MLK3 with activated Cdc42 alters the in viva phosphorylation pattern of MLK3 (12). A region of 63 amino acids following the kinase domain of MLK3 is predicted to be a leucine zipper domain. Leucine zippers mediate protein oligomerization by forming coiled coil structures. These structures are stabilized mainly by leucine residues spaced seven residues apart that interact at the interface of opposing helices (13-16). The heptad repeat is the name given to the notation of labeling the amino acids in the leucine zipper domain, a through g, with leucine residues predominately found at position d (17). In addition, electrostatic interactions in the form of salt bridges may also contribute to coiled coil stability and specificity (18, 19). The leucine zipper domain of MLK3 is necessary and sufficient to mediate homo- oligomerization (12, 20). Based on deletion studies, others have suggested that zipper- mediated MLK3 oligomerization is required for its activation (20). We have examined 82 the role of MLK3’s leucine zipper in more detail and arrive at a different conclusion. Rather than deleting the entire leucine zipper, we substituted a helix-disrupting proline for a leucine residue at one of the proposed d positions in MLK3’S zipper motif. While this point mutant fails to oligomerize, constitutively activated Cdc42 fully activates this monomeric MLK3 mutant in terms of both autophosphorylation and histone phosphorylation activity. Moreover, Cdc42 induces the same in viva phosphorylation pattern of the MLK3 zipper mutant and wild type MLK3. However, this catalytically active MLK3 zipper mutant is unable to activate INK. Our data show that MLK3 oligomerization is necessary to phosphorylate Thr 258, one of the two activating phosphorylation sites of MKK4. These studies suggest that zipper-mediated MLK3 oligomerization is not required for the activation of MLK3 by Cdc42, but is required for proper interaction and phosphorylation of a downstream target, MKK4. 83 3. Materials and Methods 3.1 DNA Constructs and Mutagenesis Construction of the cytomegalovirus-based expression vectors carrying the cDNAs for wild type MLK3 (pRK5-mlk3) has been described elsewhere (9). The expression plasmid encoding NHz-terminal Flag epitope-tagged constitutively active variant (pRK5- Nflag.cdc42V12) of Cdc42 was kindly provided by Avi Ashkenazi (Genentech, Inc.). A variant of MLK3 containing a point mutation in the leucine zipper domain (MLK3 L410P) was constructed using the Quick Change Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA.), with the following oligonucleotides: 5’-CT'I'ITCCTT GGCTCGCGGCTCGTCGAAGAGACC- 3’ and 5’-GGTCTCT‘TCGACGAGCCGCGAGCC AAGGAAAAG-3’. The presence of the desired mutation was confirmed by dideoxy DNA sequencing using Sequenase enzyme (Amersham) and the Sanger method. Construction of the COOH-tenninal triple hemagglutinin (3HA) epitope-tagged variant (pRK5-C3HA.mlk3) was generated by PCR amplification of the 3HA epitope from the CLN2T plasmid (21) using the following oligonucleotides: 5’-CGTGAGGTACCGGAAGCGGGGCCTTACCCATACGATGTTCC-3’ and 5’- CGAGGTCTAGATTAGCACTGAGCAGCGTAATCTGG-3’, followed by subcloning of the amplified fragment into the pRK5-mlk3 vector using Nhe I and Xba I. Dr. Ajay Rana (Massachusetts General Hospital, Boston, MA) kindly provided the pEBG-sek/mkk4 expression plasmid encoding murine MKK4 fused to glutathione S-transferase (GST). 84 3.2 Cell Culture, Transfections, and Lysis Human fetal kidney 293 cells were maintained in Ham’s F12:low glucose Dulbecco’s modified Eagle’s media (1:1) (Gibco BRL) supplemented with 8% fetal bovine serum (Gibco BRL), 2 mM glutamine, and penicillin/streptomycin (Gibco BRL). Plasmids (10 pg each for 100 mm dishes) were used to transfect 293 cells using the calcium phosphate technique. Cell monolayers were incubated with the DNA precipitate for 4 h, then washed once with PBS, and cultured in the medium described above. Cells were harvested, washed with ice cold PBS and lysed as described previously (12). 3.3 Immunoprecipitations and GST Pulldown Assays The following antibodies against the proteins of interest were prebound to protein A-agarose beads: MLK3 antiserum (0.25 pg/pl slurry), M2 monoclonal antibody (Kodak IBI) directed against the Flag epitope (0.45 pg/pl slurry) and INK C-l7 antibody (Santa Cruz) (50 ng/pl slurry). Immunoprecipitation experiments were performed as previously described (9). For the GST-pulldown experiment, 293 cells were transiently cotransfected with pEBG-selr/mkk4 or pEBG, and expression vectors for MLK3 variants. Clarified lysate (300 pl) was incubated with 20 p1 of antibody bound Protein A-agarose or 30 pl of glutathione Sepharose resin for 90 min at 4 °C. Immunoprecipitates and the GST-pulldowns were washed with HNTG buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton-X-IOO, 10% glycerol). Immunoprecipitates used for kinase assays were washed three times with HNTG buffer containing 1 M LiCl, three times with HNTG buffer, and twice with kinase assay buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MnClz, 10 mM MgC12, 0.1 mM N33VO4). 85 3.4 Gel Electrophoresis and Western Blot Analysis Lysates and immunoprecipitates of proteins were resolved by SDS-PAGE according to Laemmli (22). Proteins were transferred to nitrocellulose membranes and immunoblotted using MLK3 antiserum (1 pg/ml), 16B 12 HA monoclonal antibody (BabCo) (5 pg/ml), M2 Flag monoclonal antibody (9 pg/ml), INK 017 antibody (0.5 pg/ml), MKK4 antibody (0.5 pg/ml), or phospho-MKK4 antibody (New England Biolabs) (0.5 pg/ml), followed by the appropriate horseradish peroxidase-conjugated secondary antibody (Gibco BRL). Western blots were developed by chemiluminescence. Multiple exposures of the Western blots were developed, and densitometry (NIH Image) of unsaturated films was used to determine relative expression levels. Statistics were calculated using an unpaired Student’s t-test. A p-value smaller that 0.05 was considered statistically significant. 3.5 In Vitro Kinase Assays Kinase assays were performed in 20 pl of kinase assay buffer containing 50 pM ATP and 5 pCi [y-3ZP]-ATP (3000 Ci/mmol). For the MLK3 kinase assay 10 pg of mixed histones (Boehringer Mannheim) or 10 pg of murine GST-MKK4 was used as the substrate, and the reaction was carried out for 30 min at room temperature. GST-MKK4 was expressed from the pGEX-2T vector in E. coli BL-21 cells and purified by glutathione Sepharose chromatography. The reactions were terminated by the addition of an equal volume of 2x SDS sample buffer (100 mM Tris (pH 6.8), 4% SDS, 20% 86 glycerol, 0.2% bromphenol blue, 100 mM DTT, 1% B-mercaptoethanol) containing 50 mM EDTA (pH 8.0). INK assays were performed as described previously, using GST-c-Jun as the substrate (12). The pGEX-c-jun (1-115) vector was kindly provided by Dr. Ajay Rana (Massachusetts General Hospital, Boston, MA). GST-c-Jun was expressed in E. coli XL- 1 Blue cells and purified by glutathione Sepharose chromatography. Following the kinase assay, proteins were separated by SDS-PAGE. Gels were rinsed in PBS, dried, and incorporation of radioactivity into kinase or substrates was determined by phosphorimaging (Molecular Dynamics). 3.6 Expression and Purification of MBP fusion proteins Maltose binding protein (MBP) fusion protein plasmid construction and protein expression were described previously (12). MBP fusion proteins were purified by amylase affinity chromatography according to the manufacturer’s protocol. Fractions containing the MBP-zips or MBP-zips L410P, as determined by SDS-PAGE followed by Coomassie Blue staining, were pooled and concentrated to about 1 mg/ml using a Centriprep concentrator (Amicon). 3.7 Size exclusion chromatography Gel filtration fast pressure liquid chromatography (FPLC) was used to analyze MBP-zips and MBP-zips L410P. The fusion proteins were applied to a Superose 6 HR 10/30 column (25 m1 column volume) (Amersham-Phannacia). The void volume of the column was determined using Blue Dextran (2000 kDa). The column was calibrated with 87 cytochrome c (12 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), amylase (200 kDa), apoferritin (445 kDa), and thyroglobulin (700 kDa) (Sigma). The fusion proteins were eluted with 250 mM sodium phosphate buffer (pH 7.2) containing 125 mM NaCl at room temperature. The flow rate was 0.5 ml/min, and the effluent was continuously monitored at 280 nm. Approximately 30 fractions of 1.0 ml each were collected and the presence of MBP firsion proteins was assessed by SDS-PAGE followed by Coomassie Blue staining. 3.8 Phosphopeptide mapping After a 24 h transfection with pRK5-mlk3 or pRK5-mlk3 L410P in the presence or absence of pRK5-Nflag.cdc42Vl2, 293 cells were washed five times with phosphate-free medium (Dulbecco’s modified Eagle’s medium supplemented with 10% dialyzed FBS (Summit Biotechnology), and incubated at 37 °C for 2 h. The cells were then incubated in phosphate-free medium containing 3 mCi/ml [32P]orthophosphate (NEN Life Science Products) for 4 h at 37 °C. Lysis and subsequent tryptic phosphopeptide mapping of labeled cells has been described in detail elsewhere (12). 3.9 Phosphoamino Acid Analysis Following an in vitro kinase assay in which 10 pg of GST-MKK4 was used as a substrate, the proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Radiolabeled MKK4 bands were excised from the PVDF membrane. After washing three times with methanol and three times with water, the radioactive piece of membrane was hydrolyzed in 200 pl of 6 N HCl for 1 h at 100 °C. 88 The phosphoamino acids were concentrated in a SpeedVac. Unlabeled phosphoamino acid standards (Sigma) and xylene cyanol FF marker dye (Sigma) were added to each sample. The phosphoamino acids were separated by one dimensional thin layer electrophoresis (TLE) in pH 2.5 buffer (66.7% pH 3.5 buffer (glacial acetic acid/pyridine/water, 50:5:945, v/v/v) and 33.3% pH 1.9 buffer on 20 x 20 cm cellulose thin layer chromatography (TLC) plates at 0 °C and 500 V for 1.5 h. The unlabeled phosphoamino acids were visualized by ninhydrin staining and the 32P-labeled phosphoamino acids were visualized and quantitated by phosphorimaging. 89 4 Results 4.1 Point mutation in the zipper domain decreases the in vitro kinase activity of MLK3 Previous studies have shown that MLK3 with an intact zipper domain can self- associate and that large deletions in the zipper domain compromise MLK3’S autophosphorylation activity and abrogate its ability to activate INK (12, 20). To more carefirlly probe the function of MLK3’s zipper domain, a leucine residue at position 410, which is predicted to reside at a coiled coil interface (23), was substituted with a proline residue using site-directed mutagenesis. The catalytic properties of this zipper mutant, MLK3 L410P, were compared with those of wild type MLK3. Cells transiently expressing MLK3 or MLK3 L410P were lysed, and the MLK3 proteins were immunoprecipitated and used in an in vitro kinase assay with a mixture of histones as a substrate. Data from a representative experiment are shown in Fig. 1. Based on three independent experiments, MLK3 L410P has 35% of the autophosphorylation activity and 30% of the histone phosphorylation activity of wild type MLK3. While expression of wild type MLK3 activates INK in 293 cells, MLK3 L410P fails to induce INK activation (Fig. 2). These data suggest that MLK3’s zipper domain is important for its basal phosphorylation activity and for INK activation. 4.2 A MLK3 leucine zipper mutant fails to oligomerize Leucine zipper domains commonly dirnerize or form higher order oligomers. To test whether MLK3 L410P in the context of its zipper domain can oligomerize, fusion proteins consisting of the monomeric maltose binding protein (MBP) from E. coli and either the leucine zipper domain of wild type MLK3 (MBP-zips) or that containing the 90 proline mutation (MBP-zips L410P) were constructed and analyzed by size exclusion chromatography (Fig. 3). MBP-zips elutes as a high molecular weight complex corresponding to a molecular weight of approximately 300 kDa. However, MBP-zips L410P elutes predominantly as a single peak corresponding to a molecular weight of about 55 kDa, the expected size for the monomeric protein. These data demonstrate that the leucine zipper domain of MLK3 is capable of forming multimers of MBP and that a point mutation of one leucine residue is sufficient to disrupt the oligomerization of MLK3’s leucine zipper domain. To examine whether the zipper point mutation affects association with fiill length MLK3, we examined whether MLK3 L410P or wild type MLK3 could associate with epitope-tagged MLK3. After transient coexpression of MLK3 L410P or wild type MLK3 with HA-tagged MLK3 (3HA-MLK3), cellular lysates were immunoprecipitated with the HA antibody. The presence of associated untagged MLK3 or MLK3 L410P was assessed by Western blotting with a MLK3 antibody (Fig. 4). The triple HA epitope adds twenty- seven amino acids to the COOH-terrninus of MLK3 allowing it to be distinguished fi'om untagged MLK3 by its slower mobility on SDS polyacrylamide gels. While wild type MLK3 associates with 3HA-MLK3, MLK3 L410P cannot form a detectable complex with 3HA-MLK3. These results, coupled with the gel filtration experiments, indicate that MLK3 L410P behaves as a monomer. 91 4.3 Activated Cdc42 increases the in vitra catalytic activity of both wild type MLK3 and MLK3 L410P The GTPase Cdc42 in its active form can associate with MLK3 and increase MLK3’s catalytic activity (11, 12). This increased activity measured in vitra correlates with a change in the in viva phosphorylation state of MLK3 (12). We examined the effect of Cdc42V12, a mutant form of the GTPase that renders Cdc42 constitutively active, on the activity of MLK3 L410P in an immunocomplex kinase assay. MLK3 or MLK3 L410P, expressed in the presence or absence of Cdc42V12, was immunoprecipitated from cellular lysates of transiently transfected 293 cells and subjected to an in vitro kinase assay using a mixture of histones as a substrate. After coexpression with Cdc42V12, the in vitro autophosphorylation and histone phosphorylation activities of MLK3 L410P are the same as those of wild type MLK3 coexpressed with Cdc42V12 (Fig. 5). To test the ability of the leucine zipper mutant to associate with activated Cdc42, MLK3 L410P or wild type MLK3 was coexpressed with F lag-tagged Cdc42V12 in 293 cells. As shown in Fig. 6, MLK3 L410P coimmunoprecipitated with Cdc42V12, although to a lesser extent than did wild type MLK3. These data suggest that MLK3 L410P can bind and be fully activated by Cdc42V12. 4.4 MLK3 oligomerization is necessary for MLK3-induced JNK activation Since coexpression with Cdc42V12 renders MLK3 L410P fully active in in vitro kinase assays (Fig. 5) we tested whether this zipper mutant, when coexpressed with the GTPase, was competent in downstream signaling. Cells expressing MLK3 or MLK3 92 L410P, in the presence or absence of Cdc42V12, were lysed and the activity of endogenous INK was measured in an immune complex in vitro kinase assay using GST- c-Jun as a substrate. Wild type MLK3, upon overexpression, activates INK (Fig. 7 lane 2); Cdc42V12 expressed alone moderately activates INK (lane 4). Coexpression of Cdc42V12 with MLK3 further increases INK activity (lane 5). However, MLK3 L410P coexpressed with activated Cdc42 exhibits no increase in INK activation over that of Cdc42V12 alone (lanes 4, 6). Thus, MLK3 L410P, when coexpressed with Cdc42V12, fails to activate endogenous INK even though under these conditions it exhibits wild type phosphotransfer activity in vitro. These findings suggest a role for leucine zipper- mediated MLK3 oligomerization in downstream signaling events. 4.5 Activated Cdc42 changes the in viva phosphorylation state of MLK3 L410P We recently reported that coexpression with Cdc42V 12 induces a differential phosphorylation pattern of MLK3 in vivo (12). To examine the effect of activated Cdc42 on the phosphorylation state of MLK3 L410P in viva, comparative two-dimensional tryptic phosphopeptide analyses of MLK3 and MLK3 L410P labeled in vivo, in the absence or in the presence of Cdc42V12, were performed. As shown in Fig. 8, in the absence of Cdc42V12, MLK3 and MLK3 L410P have similar phosphopeptide patterns. Cdc42V12 induces a differential in viva phosphorylation state of both MLK3 and MLK3 L410P, and the phosphopeptide patterns are alike. The two new phosphopeptides observed after coexpression of Cdc42V12 with MLK3, and with MLK3 L410P, are indicated by arrows in the lower panels of Fig. 8. These data strongly suggest that both MLK3 and MLK3 L4 1 OP undergo the same changes in phosphorylation upon 93 coexpression with Cdc42V12. Furthermore, these changes in in viva phosphorylation correlate with the increased in vitro catalytic activity of both MLK3 and MLK3 L410P (Fig. 5). 4.6 The MLK3 leucine zipper mutant has reduced ability to phosphorylate MKK4 Coexpression with Cdc42V12 yields a fully active MLK3 L410P as judged by in vitra catalytic activity (Fig. 5), yet MLK3 L410P, even when coexpressed with Cdc42V12, fails to activate INK (Fig. 7). To explore these disparate findings we examined whether MLK3 L410P might be defective in phosphorylating an established physiological substrate, MKK4, which can directly phosphorylate and activate INK. MKK4 was expressed in E. coli as a GST fusion and purified using a glutathione Sepharose column. Primary data from an in vitro kinase assay using MKK4 as the substrate are shown in Fig 9. MKK4 displays low basal autophosphorylation activity (Fig. 9, top panel, lane 1). MLK3 alone phosphorylates MKK4 (lane 2) but MLK3 L410P alone does not (lane 3). Coexpression of Cdc42V12 increases MLK3’s phosphorylation of MKK4 in vitro by ~4.5-fold. However, when coexpressed with Cdc42V12, the ability of MLK3 L410P to phosphorylate MKK4 is less than that of wild type MLK3 (lanes 5, 6). Activation of murine MKK4 requires the phosphorylation of Serz“ and Thr’”. An immunoblot of the same gel (Fig. 9, top panel) was probed with an antibody that recognizes phospho- Thr258 of MKK4 (Fig. 9, second panel). When coexpressed with activated Cdc42, the in vitro phosphorylation of Thr258 of MKK4 by wild type MLK3 is at least 6-fold greater that that of MLK3 L410P (Fig. 9, second panel, lanes 5 and 6). Thus after coexpression with the activated GTPase, the zipper mutant of 94 MLK3, compared with wild type MLK3, has reduced MKK4 phosphorylation activity in an in vitro kinase assay, as judged by net phosphorylation of MKK4 as well as by phosphorylation of Thr”8 of MKK4. The ability of MLK3 L410P to associate with MKK4 after coexpression was assessed in GST pulldown experiments with harvested cellular lysates from transiently transfected 293 cells (Fig. 10). MLK3 L410P associates with MKK4, but to a lesser extent than does wild type MLK3. This may suggest that MLK3 oligomerization contributes to MKK4 binding. MKK4 activation requires phosphorylation of Ser254 and Thr”8 by its upstream activators MEKK and MLK3 (3, 7, 8). MLK3 cannot phosphorylate a variant of MKK4 in which these two activating sites have been replaced by nonphospborylatable residues (7, 8). Therefore, to compare the relative abilities of Cdc42V12-activated MLK3 L410P to phosphorylate Serz" and Thr258 of MKK4, phosphoamino acid analysis was performed. Following an in vitro kinase assay as shown in Fig. 9, the radiolabeled MKK4 was subjected to acid hydrolysis and its phosphoamino acid content was analyzed by TLE (Fig. 11). Wild type MLK3 coexpressed with Cdc42V12 phosphorylates serine and threonine of MKK4 at a one to one ratio. This indicates that Cdc42V12-activated wild type MLK3 phosphorylates serine and threonine of MKK4 to the same extent, as would be predicted for full activation of MKK4. However MLK3 L410P coexpressed with Cdc42V12 phosphorylates serine and threonine of MKK4 at a four to one ratio. These data, together with the results from the phospho-Thr258 blot, indicates that the monomeric MLK3 zipper mutant is defective in phosphorylating Thr258 of MKK4, suggesting that MKK4 would remain inactive and unable to activate INK. 95 MLK3: - WT L410P MLK3 lP I MLK3® ‘— histone® ‘— MLK3 we MLK3IP . ~ Fig. 1 Effect of a point mutation in the MLK3 zipper domain on catalytic activity MLK3 variants were immunoprecipitated from cellular lysates and subjected to an in vitro kinase assay using histones as a substrate. The top panel shows an autoradiogram with bands corresponding to MLK3 autophophorylation and histone phophorylation, indicated by arrows. The lower panel shows the MLK3 expression. 96 MLK3: JNK 1P <— GST-c-Jun@ JNK WB Fig. 2. Effects of MLK3 L410P on JNK activity. Endogenous JNK was immunoprecipitated from cellular lysates that had been transiently transfected with cDNAs encoding the specified MLK3 variants. An in vitro kinase assay for INK was performed using GST-c-jun as a substrate. The top panel shows an autoradiogram with bands corresponding to phosphorylated GST-c-jun indicated by an arrow. The second panel shows a INK immunoblot of the same immunoprecipitated samples from the in vitro kinase assay. 97 Fig. 3. Size exclusion chromatography analysis of the MLK3 zipper point mutant. Size exclusion chromatographic analyses of MBP-zips and MBP-zips L410P. A Superose 6 HR 10/30 column was calibrated with cytochrome c (12 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), amylase (200 kDa), apoferritin (445 kDa), and thyroglobulin (700 kDa). The void volume of the column was determined with Blue Dextran (2000 kDa). Each fusion protein (100 pg) was applied to the column and eluted with 250 mM sodium phosphate buffer (pH 7.2) containing 125 mM NaCl at room temperature. The flow rate was 0.5 ml/min, and the effluent was continuously monitored at 280 nm. 98 + HA-MLK3 r \ MLK3 MLK3 ' L410P <— MLK3 HAIP I. l: p .. «HA-MLK3 MLK3WB 1‘ u “- HA 1P HA WB MLK3|P MLK3 we j; Fig. 4. Coimmunoprecipitation analysis of the MLK3 zipper point mutant. Coimmunoprecipitation of MLK3 and MLK3 L410P with 3HA-MLK3. Cellular lysates expressing the MLK3 variants were immunoprecipitated (IP) with the HA antibody and the presence of associated untagged MLK3 or MLK3 L410P was assessed by immunoblotting with a MLK3 antibody. The MLK3 antibody detects untagged MLK3 as well as 3HA-MLK3, which migrates slower than untagged MLK3 during SDS-PAGE. Irnmunoblots of 3HA-MLK3 and MLK3 from the immunoprecipitated samples are shown in the middle and lower panels respectively. 99 - Cdc42V12 r \ r \ MLK3: ' WT -L410P WT L410P ‘— MLK3 ® ‘— histones ® ‘— MLK3 WB Flag-Cdc42 .. W8 Fig. 5. In vitro kinase assay of MLK3 and MLK3L410P coexpressed with Cdc42V12. MLK3 was immunoprecipitated from cellular lysates and subjected to an in vitro kinase assay using histones as a substrate. The top panel shows an autoradiogram with bands corresponding to MLK3 autophosphorylation and histone phosphorylation, indicated by arrows. The middle and lower panels show MLK3 and Cdc42 expression, respectively. 100 ' Cdc42 v12 / \f \ MD. a. -5%’2-%§8 2:3 :23 Flag-Cdc42 IP MLK3 “’3 MLK3 WB Flag-Cdc42 WB Fig. 6. Effect of L410P substitution on MLK3binding to Cdc42V12. Co- immunoprecipitation experiments of MLK3A and MLK3A L410P with Cdc42V12. Flag epitope-tagged Cdc42V12 was immunoprecipitated from cellular lysates using an antibody directed against the Flag epitope. The presence of associated MLK3A or MLK3A 1.410? was determined by immunoblotting with a MLK3A antibody (upper panel). Immunoblots of MLK3A and Cdc42 from cellular lysates are shown in the middle and lower panels, respectively. 101 _ Cdc42 V1 2 f \ f \ MLK3: ' WT L410P ' WT L410P JNK 'P <-—— GST-c-Jun® JNK 1P JNK WB MLK3 WB Flag-Cdc42 WB Fig. 7. Effect of MLK3 L410P on JNK activation. Endogenous INK was immunoprecipitated from cellular lysates expressing the indicated MLK3 variant and/or Cdc42V12, and subjected to an in vitra kinase assay using GST-c-Iun as a substrate. A, An autoradiogram with bands corresponding to phosphorylated GST-c-Jun indicated by an arrow is shown in the upper panel. An immunoblot for INK from the same immunoprecipitated samples and immunoblots for MLK3 and Cdc42V12 from cellular lysates are shown below the autoradiogram 102 chromatography _____._, , MLK3 *9 ’ MLK3 L410P it e Cdc42V12 “ ' + Cdc42V12 ,.-~ ié— * - ,5 fi . 1.. h: 531-: electrophoreels —’ chromatography. 103 Fig. 8. Two-dimensional maps of tryptic phosphopeptides derived from in viva phosphorylated MLK3 or MLK3 L410P. A, Cells expressing the specified MLK3 variants in the presence and absence of Cdc42V12were incubated with [”P]orthophosphate. MLK3 variants were immunoprecipitated from cellular lysates, blotted onto a PVDF membrane, and subjected to partial trypsin digestion. The resultant tryptic phosphopeptides were analyzed by comparative two-dimensional phosphopeptide analysis. Short arrows indicate two new major tryptic phosphopeptides from MLK3, or MLK3 L410P expressed in the presence of Cdc42V12. Phosphopeptides were detected by phosphorimaging. Long arrows indicate the direction of electrophoresis and - Cdc42 V12 l \ l \ O. O. 3 S 3 . E 3 MLK3: MLK3 IP _‘+ MLK3 @ .+ GST-MKK4@ P-Thr 258 MKK4 we MLK3 IP ' 1. MLK3 we ' ' Flag-Cdc42 we Fig. 9 Phosphorylation of MKK4 by MLK3 L410P in vitra. MLK3 was immunoprecipitated from cellular lysates and subjected to an in vitra kinase assay using GST-MKK4 as a substrate. The top panel shows an autoradiogram with bands corresponding to MLK3 autophosphorylation and GST-MKK4 phosphorylation, indicated by arrows. The second panel from the top shows an immunoblot of the samples from the kinase assay using an antibody that recognizes phosphorylated Thr 258 of murine MKK4. Immunoblots of MLK3 from cellular lysates, of recombinant GST- MKK4, and of Cdc42 from cellular lysates are shown in the third, fourth, and bottom panels, respectively. + GST-MKK4 - { \ f \ n. n a con- ?3 x 2 x3 4:: " -' -'v 5.1 E E 2.: GST Pulldown MLK3 WB MLK3 IP MLK3 WB Fig. 10. Binding of MKK4 by MLK3 L410P. GST pulldown experiments of MLK3 and MLK3 L410P with GST-MKK4. Glutathione Sepharose resin was incubated with cellular lysates containing MLK3 or MLK3 L410P, with or without GST-MKK4. The presence of associated MLK3 or MLK3 L410P was determined by immunoblotting with a MLK3 antibody (upper panel). lrnmunoblots of recombinant GST-MKK4 and MLK3 from cellular lysates are shown in the middle and lower panels, respectively. 105 . l . O P-Ser 1' 1* O P-Thr O P-Tyr e a . i ‘ ' origin a as: 1" 1-> N V q- ‘- -J 4% +> n M n N x a: raw Q3 .1 —| .10 .113 E 5 2+ 20 Fig. 11. Phosphoamino acid analysis of MKK4 phosphorylated by MLK3 variants. Following a MLK3 in vitra kinase assay using GST-MKK4 as a substrate as described in Fig. 8, proteins were resolved by SDS-PAGE and transferred to a PVDF membrane. A, Radiolabeled bands corresponding to GST-MKK4 were excised from the membrane and hydrolyzed in 6 N HCl for 1 h at 100 °C. The phosphoamino acids were analyzed using one-dimensional thin layer electrophoresis on TLC plates and visualized by phosphorimaging. The autoradiogram shown is representative of three independent experiments. The positions of the phosphoamino acid standards, as well as the position of free inorganic phosphate (Pi), are indicated. 106 5. Discussion Reversible protein phosphorylation is important in regulating virtually every physiological process. Thus it follows that the activities of the protein kinases and phosphatases that catalyze these events should also be tightly regulated. In response to a particular cellular signal, a protein kinase first is converted into an active form, and then the activated kinase can proceed to phosphorylate its physiological substrate. While the catalytic domains of protein kinases share sequence and structural homology, considerable diversity exists outside of the kinase domains. These non-catalytic regions often mediate interactions with proteins, lipids, or small molecules, which modify the activity of the protein kinase itself or contribute to the binding and phosphorylation of its physiological substrates. We report herein that activation of MLK3 by Cdc42 is independent of zipper-mediated oligomerization, whereas proper phosphorylation of a downstream substrate by Cdc42-activated MLK3 depends on zipper-mediated oligomerization. Numerous intracellular serine/threonine protein kinases have been identified that contain leucine zipper-like motifs that may serve as oligomerization domains, including type I and II COMP-dependent protein kinases (cGK) (24), protein kinase C-related kinase N (25), Tousled kinase (26), and the NIMA-related kinase-2 (27). The predicted leucine zippers of the mixed-lineage kinases, MLK-l (28), MST/MLK-2 (29, 30) and MLK3, are relatively dissimilar to those of the more distantly related DLK/MUK/ZPK (31-33) and LZK (34). In fact, the zippers of MLK3 and DLK fail to interact (35) suggesting that zipper-mediated heterodimerization in vivo is unlikely. MLK3 can homo- oligomerize through its zipper and large deletions of 32 to 60 amino acids of the zipper 107 domain result in a kinase with reduced autophosphorylation and INK activity (12, 20). Based on deletion studies it has been suggested that oligomerization is required for MLK3 activation (20). In order to more precisely decipher the role of MLK3’s zipper domain while keeping MLK3 intact, we engineered a monomeric form of MLK3 that contains a single point mutation in its leucine zipper domain. Leucine zippers are a-helical coiled coil structures characterized by the presence of leucine or another nonaromatic hydrophobic amino acid at every seventh position. While proline residues are almost always absent from short helices or coiled coils, a single proline can be tolerated in some long alpha helices, albeit with disruption of loCal helical geometry (36-38). We replaced the leucine residue at position 410 with a proline in order to deliberately disrupt (at least the local) a-helical structure of the zipper with the aim of destabilizing MLK3 oligomerization. The MBP fusion system (39) has been successfully used to biophysically characterize multimerization of leucine zipper domains (40, 41). Thus fusion proteins between the wild type MLK3 zipper, or the MLK3 L410P mutant zipper, and the monomeric MBP of E. coli were constructed and their native molecular weights were estimated using size-exclusion chromatography. MLK3’s leucine zipper is capable of forming oligomers of MBP (Fig. 3), whereas appending of the mutant zipper yields a monomeric MBP fusion protein. These data indicate that substitution of a single conserved leucine in the zipper domain with proline adequately disrupts zipper-mediated oligomerization. Furthermore, using the full length kinase in coimmunoprecipitation experiments, MLK3 L410P was unable to form a stable complex with wild type MLK3 (Fig. 4). Taken together, these results argue that MLK3 L410P behaves as a monomeric 108 protein. Thus we have constructed an oligomeric loss-of-function MLK3 point mutant by substituting a helix disrupting proline residue within the leucine zipper domain. In addition to its leucine zipper motif, MLK3 contains a CRIB motif that is required for binding and activation by the small GTPase Cdc42 (12). We asked whether MLK3 oligomerization is required for its activation by Cdc42. Coexpression of the monomeric MLK3 L410P with activated Cdc42 resulted in a fully active MLK3 as judged by in vitro autophosphorylation and histone phosphorylation (Fig. 5). Upon coexpression with Cdc42, wild type MLK3 undergoes additional phosphorylation event(s) in viva that correlate with increased MLK3 activity (12). Comparative phosphopeptide mapping experiments presented here indicate that MLK3 L410P undergoes those same changes in in viva phosphorylation upon coexpression with activated Cdc42 (Fig. 8). Taken together these findings demonstrate that zipper-mediated MLK3 oligomerization is not required for activation by Cdc42. Furthermore, the leucine to proline substitution in MLK3 does not compromise the catalytic integrity of the mutant monomeric MLK3, suggesting that the structure of the catalytic domain of the MLK3 L410P is not perturbed. In accord with the ability of Cdc42V12 to activate MLK3 L410P, MLK3 L410P does indeed coimmunoprecipitate with activated Cdc42, but in lesser amounts than does wild type MLK3 (Fig. 6). This may suggest a decreased affinity of Cdc42V12 for MLK3 L410P. Alternatively, a single Cdc42 molecule may bind an oligomer of wild type MLK3 but only a monomer of MLK3 L410P. Without knowledge of the stoichiometry of MLK3 oligomerization or the stoichiometry of the MLK3-Cdc42V12 interaction, we 109 cannot distinguish between these possibilities. Regardless, it is apparent that the in viva affinity of Cdc42V12 for MLK3 L410P is sufficient for full activation of the kinase. As a MAPKKK, MLK3 activates the JNK pathway through phosphorylation of MKK4 or MKK7. Interestingly, both in the absence or presence of activated Cdc42 (Fig 7), MLK3 L410P is unable to activate endogenous INK in 293 cells. These findings indicate that zipper-mediated MLK3 oligomerization is critical for downstream signaling events that culminate in INK activation. Even though the monomeric MLK3 zipper mutant, when coexpressed with activated Cdc42, has high autophosphorylation and histone phosphorylation activities (Fig. 5), we determined that its in vitro activity towards a physiological substrate MKK4 has been compromised (Fig. 9). Interestingly, it has been reported that a monomeric cGK mutant is capable of autophosphorylation and histone phosphorylation but displays a reduced ability to phosphorylate a physiological substrate vimentin (42). Protein kinases of the MK family are activated by dual phosphorylation of two conserved serine/threonine residues in their activation loops. Activation of murine MKK4 requires phosphorylation of both Serz’4 and Thr’", and mutation of these to nonphospborylatable amino acids blocks MLK3 activation of JNK (3, 7, 8). Determination of the ratio of serine to threonine phosphorylation, in combination with the use of an antibody that recognizes phosphorylated Thr258 of MKK4, leads us to conclude that the Cdc42-activated zipper mutant of MLK3 has some serine phosphorylation activity but is defective in phosphorylating Thr258 of MKK4. These results imply that MLK3 L410P cannot activate INK because the zipper mutant fails to properly phosphorylate and activate its physiological substrate, MKK4. 110 Using a monomeric point mutant of MLK3, we have been able to dissociate two discrete steps in the regulation of MLK3. Our results clearly demonstrate that zipper- mediated MLK3 oligomerization is not required for Cdc42 to induce a catalytically active MLK3. However, zipper-mediated MLK3 oligomerization is critical for phosphorylating downstream signaling targets, and is specifically crucial for full activation of MKK4. The mechanistic rationale of why the zipper mutant has a reasonable ability to phosphorylate Serf” of MKK4 but not Thr258 is not clear from these studies. It is possible that monomeric MLK3 has lower affinity for serine phosphorylated MKK4, or that a MLK3 oligomer containing more than one catalytic domain may be needed to bind and phosphorylate both sites of MKK4. 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E., Jr., and Bhatt, R. R. (1997).]. Biol. Chem. 272, 19008-16 114 IV. Identification of MLK3 In vivo Phosphorylation Sites by Phosphopeptide Mapping and Mass Spectrometry 1. Abstract MLK3 is a serine/threonine protein kinase that functions as an upstream activator of the IN K pathway. Previous work has suggested that MLK3 is a multiphosphorylated protein. In this study, mass spectrometry coupled with comparative phosphopeptide mapping was used to directly characterize the MLK3 in viva phosphorylation sites. Various types of mass spectrometry were used to analyze MLK3 tryptic peptides separated by C18 reverse-phase HPLC, leading to the identification of Ser’“, Serm’, Serm, Ser’”, Serm, and Ser793 and a site found on peptide Ser“-ArgJ7 within a Gly-rich region, as MLK3 phosphorylation sites. Additionally, porous graphitic carbon (PGC) chromatography successfully retained and resolved phosphopeptides that had eluted along with nonvolatile salts and buffers in the flowthrough fractions from the C18 column. Following resolution by PGC chromatography, MALDI-MS in conjunction with alkaline phosphatase treatment identified the phosphorylation sites Ser’”, Ser‘", Serm, and Serm. A proline residue immediately follows seven out of the ten unambiguous phosphorylation sites identified, suggesting that MLK3 may be a target of proline- directed kinases. Finally, two-dimensional phosphopeptide mapping confirmed that phosphorylation of Ser’” and Sermis induced by the small GTPase Cdc42. 115 2. Introduction Protein phosphorylation catalyzed by protein kinases is a common regulatory mechanism involved in transmitting extracellular signals to the nucleus to mediate various cellular events. Phosphorylation can activate or inhibit a target protein either by regulating enzymatic activity or by affecting protein-protein interactions (1). Precise identification of phosphorylation sites is a critical first step toward gaining molecular insight concerning the role of a particular phosphorylation event. MLK3 (2-4) is an intracellular serine/threonine kinase that functions as a MAPKKK to activate the JNK pathway (5). MLK3 has also been demonstrated to associate with the JNK scaffolding proteins IIPl, 2, and 3 (6). A recent report has suggested that MLK3 is capable of phosphorylating IKB kinases to positively regulate the NF-xB pathway in response to T-cell costimulation (7). While studies have provided information of MLK3’s role as an upstream kinase, less is known about how MLK3 itself is activated and regulated. MLK3 contains several protein-protein interactions domains that may be important for regulation of the kinase, including an SH3 domain, a leucine zipper domain, a CRIB motif and a COOH-terminal region of 220 amino acids that is rich in proline, serine, and threonine residues. MLK3 can associate with an activated form of the GTPase Cdc42 (8) and coexpression of MLK3 and activated Cdc42 in cells increases the catalytic activity of MLK3(9,10). Furthermore, coexpression of MLK3 with activated Cdc42 alters the in viva phosphorylation pattern of MLK3 (9). 116 The mechanistic understanding of how phosphorylation regulates MLK3 activities first requires the identification of the sites phosphorylated. Based on site directed mutagenesis studies, it has been claimed that Thr277 and Serz‘“ in the activation loop of MLK3 are major phosphorylation sites that are critical for MLK3 activation (11). However, at present there have been no MLK3 phosphorylation sites directly identified. In this study a combination of different mass spectrometric techniques were employed to identify in viva phosphorylation sites of MLK3. Mass spectrometry has proven to be a valuable analytical tool, due to its high mass accuracy, to study post translational protein modifications including identification of phosphorylation sites on proteins. The following is a brief description of the types of mass spectrometry utilized in this project. Mass spectrometry analyzes ionized molecules in the gas phase. The various mass spectrometry instruments are composed of three general components: an ionization section; a mass analyzer/filter section; and a detection system. The two types of mass spectrometry used in this study were matrix assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS) using an ion-trap mass filter. MALDI-MS uses a UV absorbing matrix and laser pulses to induce the formation of intact molecular ions (12). MALDI is often coupled to TOP mass analyzers, where the mass to charge ratio of a molecule is determined by its flight time to the detector in a fi'ee drift field. MALDI-TOF is a relatively simple and extremely sensitive technique that provides very high mass accuracy (+/- 1 Da). However, conventional MALDI-TOF analysis cannot provide protein/peptide sequence information. An extension of MALDI- 117 TOF that does have the potential of obtaining primary sequence information is post source decay (PSD) analysis. PSD is a process whereby the precursor ion fragments via metastable decomposition in the flight tube (13). The precursor ion and the fragment ions have similar velocities and therefore are not differentiated by the detector in linear mode. MALDI-PSD takes advantage of the differing kinetic energies of the precursor ion and the fragment ions. MALDI-PSD analysis involves deflecting the ions using an electrically charged reflectron mirror at the end of the flight tube at various mirror voltages to discriminate the ions by flight time dispersion. The fragment ions reach the detector at different times based on their mass to charge ratio and a resulting spectrum of the precursor ion with its fiagrnent ions is produced, providing sequencing information. In contrast to the desorption/ionization techniques employed in MALDI-MS, ESI- MS produces ions (often multiply charged ions) directly from volatile liquids at atmospheric pressure in the presence of a strong electrical field (14). This allows the ESI machine to be coupled with liquid chromatography instruments and for the ions to be produced continuously. The ion trap mass analyzer uses an alternating electrical field to stabilize (trap the ions) and destabilize (release the ions to be analyzed by the detector) the incoming ions (15,16). Additionally, trapped ions can be fragmented by collision with a neutral gas (e. g. argon). The product ions are ejected out of the trap sequentially, based on their mass to charge ratios, and a resulting collision-induced dissociation (CID) spectrum is produced. This sequencing method has the advantage over MALDI-PSD of on-line coupling to HPLC analyses, and does not rely on the metastable properties of a given peptide sequence. 118 The mass spectra obtained by MALDl-PSD or ESI/CID use a common. nomenclature to characterize NHz-terminal and COOH-terminal ion types produced by fragmentation of the peptide bond(17). NHz-terminal ions are classified as b type ions, while COOH-terminal ions are classified as y type ions. Additionally, loss of phosphate groups due to fragmentation is accompanied by a diagnostic loss of m/z -98, which is due to the neutral loss of H3PO... When a doubly charged peptide loses a phosphate group in ESI/CID, the neutral loss of m/z -49 H3PO. is often seen as the predominant signal in the spectrum. Recent advances have made mass spectrometry sensitive enough to study modified proteins from in viva sources, although phosphorylation site turnover rates, and low amounts of phosphorylated material can still pose a technical challenge. Using MALDI-MS and ESI-MS coupled with phosphopeptide mapping, twelve MLK3 phosphorylation sites were identified, signifying that phosphorylation may play an important role in regulating the biological function of MLK3. 119 3. Material and Methods 3.1 DNA Constructs and Mutagenesis Construction of the cytomegalovirus-based expression vectors carrying the cDNAs for wild type MLK3 (pRK5-mlk3) has been described elsewhere (2). The expression plasmid encoding NHz-terrninal Flag epitope-tagged constitutively active variant (pRK5-Nflag.cdc42 V" 12) of Cdc42 was kindly provided by Avi Ashkenazi (Genentech). The expression plasmid containing the wildtype MLK3 cDNA was used as a template to create two single mutations (S740A, S740E) and four double mutants (SSSSA/SSS6A, SSSSE/S556E, S724A/S727A, and S724E/S727E) using the Quick Change Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA.). The presence of the proper mutation was confirmed by automated DNA sequencing. 3.2 Cell Culture, Transfections, and Lysis Human fetal kidney 293 cells (6 x 10’) were maintained in Ham’s F12:low glucose Dulbecco’s modified Eagle’s media (1:1) (Gibco BRL) supplemented with 8% fetal bovine serum (Gibco BRL), 2 mM glutamine, and penicillin/streptomycin (Gibco BRL). Plasmids (10 pg each) were used to transfect 293 cells using the calcium phosphate technique. Cell monolayers were incubated with the DNA precipitate for 4 h, then washed once with phosphate-buffered saline (PBS), and cultured in the medium described above. Cells were harvested, washed with ice cold PBS and lysed as described in chapter 11. 120 3.3 32P Labeling Following a 14 h transfection with pRK5-mlk3 and pRK5-Nflag.cdc42val 12, 2 x 106 293 cells were washed five times with phosphate-free medium (Dulbecco’s modified Eagle’s medium supplemented with 10% dialyzed F BS (Summit Biotechnology), and incubated at 37 °C for 2 h. The cells were then incubated in phosphate-free medium containing 3 mCi/ml [32P] carrier free orthophosphate (NEN Life Science Products) for 4 h at 37 °C. 3.41mmunoprecipitation and In Gel Trypsin Digestion MLK3 antiserum (0.25 pg/ pl slurry) was prebound to protein A-agarose beads. Immunoprecipitation experiments were performed essentially as described in chapter 11, except that cell lysates fi'om 6 x 107 cells were immunoprecipitated overnight. Following the MLK3 immunoprecipitation experiments, the non-radiolabeled sample (from 6 x 107 cells) and the radiolabeled sample (from 2 x 10° cells) were combined and the proteins were separated by SDS-PAGE. Gels were rinsed in PBS, dried, and incorporation of radioactivity was detected by phosphorimaging (Molecular Dynamics). Radiolabeled MLK3 bands were excised from the dried SDS-PAGE gel and rehydrated with water (4 pl). The gel pieces were washed twice with 500 pl of 0.1 M ammonium bicarbonate containing 50% acetonitrile at 30°C for 50 min. The gel pieces were then completely dried under a gentle stream of N2. The gel pieces were partially rehydrated with 4 pl of 0.1 M ammonium bicarbonate containing 0.02% Tween-20. Sequencing grade trypsin (2 pg) (Roche) was immediately administered to the gel pieces. Complete hydration was achieved by adding 40 pl of digestion buffer (50 mM ammonium bicarbonate). Trypsin digestion was performed overnight at 30 °C. 121 3.5 Reverse-Phase HPLC Following SDS-PAGE and in gel trypsin digestion, the resulting MLK3 peptides were fractionated by micro-bore reverse-phase HPLC (Michrom) on a Vydac C18 column (5 pm, 300 A, 1.0 x 150 mm). Peptides were eluted with a 0-95% linear gradient of acetonitrile in 0.1% aqueous trifluoroacetic acid (TFA) at a flow rate of 50 pl/min. The eluant was monitored by UV absorbance at 214 nm and fractions were collected at 1 min intervals. A 1 pl aliquot of each HPLC fraction was spotted on a TLC plate followed by detection of ”P radioactivity by phosphorimaging analysis. 3.6 Porous Graphitic Carbon Chromatography A Hypercarb reverse-phase PGC column (5 pm, 250 A, 1 x 150mm) (Keystone Scientific) was used to resolve the poorly retained peptides that eluted in the first 5 min off the C18 column. Conditions for PGC chromatography were identical to those for the C18 separation with the exception that the flow rate was reduced to 20 pl/min. A 1 pl aliquot of each PGC fraction was spotted on a TLC plate followed by detection of ”P radioactivity by phosphorimaging analysis. 3.7 Phosphopeptide Mapping Radiolabeled HPLC fractions (1 pl) were analyzed by one-dimensional TLE and one-dimensional TLC. The mobility of the radiolabeled peptides was used to correlate a HPLC fraction with a spot seen on the two-dimensional map from MLK3 labeled in vivo. The conditions for TLE and TLC have been described in detail in chapter 11. 122 3.8 Phosphoamino Acid Analysis Selected radiolabeled HPLC fractions (5 pl) were hydrolyzed in 100 pl of 6 N HCl for l h at 100 °C. The phosphoamino acids were concentrated in a SpeedVac. The phosphoamino acids were separated by One-dimensional TLE in pH 2.5 buffer [66.7% pH 3.5 buffer (glacial acetic acid/pyridine/water, 50:5:945, v/v/v) and 33.3% pH 1.9 buffer] on 20 x 20 cm cellulose TLC plates at 0 °C and 1000 V for 50 min. Unlabeled phosphoamino acid standards (Sigma) and xylene cyanol FF marker dye (Sigma) were spotted on the TLC plate. The unlabeled phosphoamino acids were visualized by ninhydrin staining and the 32P-labeled phosphoamino acids were visualized and quantitated by phosphorimaging. 3.9 Mass Spectrometry MALDI-TOF mass spectrometry was performed on a Voyager-DE STR time of flight instrument (Applied Biosystems), equipped with a nitrogen laser operating at 337 nm. The HPLC fractions were analyzed in either linear positive mode or reflector positive mode (for PSD analysis) using a-cyano-4-hydroxycinnaminic acid (saturated solution in 50% acetonitrile with 0.1% TF A) as the UV absorbing matrix. Samples were prepared by mixing 1 pl of sample and 1 pl of matrix solution on the MALDI plate and allowing them to air dry. All mass spectra were externally calibrated with bradykinin and insulin. Calf intestinal alkaline phosphatase (2 units) (New England Biolabs) was incubated with 5 pl of HPLC fractions containing phosphopeptides in 50 mM NH. HCO, at 37 °C for 3 hrs. The dephosphorylation reaction was terminated by the addition of 50% acetonitrile and the samples were washed with water and concentrated using a 123 SpeedVac. ESI-CID analyses were performed using an electrospray ionization/ion trap mass spectrometer (Finnigan LCQ/Deca, ThermoQuest). The ESI interface was connected in source to a conventional high performance liquid chromatography apparatus (ThermoQuest). A C18 column (1 x 150mm, 5 pm, lOO-A pore material) was used at a flow rate of 300 nl/rnin. The elution was performed using a linear gradient of acetonitrile in the presence of 0.1% TF A. The ion source voltage was set at 3.5 kV, and argon was used as a nebulizer gas. The ESI—CID analysis was carried out in data-dependent mode, where the largest peak in a single scan was subjected to collision-induced dissociation. 124 4. Results 4.1 Recombinant MLK3 Displays an Incomplete Phosphopeptide Pattern In vitra. To evade the limitations of low quantities of endogenous phosphoproteins in mammalian cells, recombinant MLK3 was utilized to identify in vitro autophosphorylation Sites on MLK3. The hypothesis was to correlate sites identified in vitro with those produced in mammalian cells using comparative phosphopeptide mapping. Baculovirus-expressed His-tagged MLK3 was previously purified by affinity chromatography fiom Sf21 insect cells. An in vitro kinase assay was performed using 7- [”P] ATP; and the autophosphorylated MLK3 sample was separated by SDS/PAGE, transferred to a PVDF membrane and analyzed by autoradiography (Fig 1A). The MLK3 band was excised from the membrane, digested with trypsin, and the recovered peptides were analyzed by two-dimensional phosphopeptide mapping (Fig. 1B). The map generated with MLK3 purified from insect cells was found to lack numerous major phosphopeptides when compared to a map generated using MLK3 immunoprecipitated from 293 cells that was subjected to an in vitra kinase assay (Fig. 1C). This striking difference indicates that recombinant MLK3 is not a suitable candidate for identifying autophosphorylation sites. This result also strengthens the argument that an additional cellular component(s) in mammalian cells may be required to fully activate MLK3. 125 4.2 In Gel Trypsin Digestion of MLK3 Immunoprecipitated from 293 cells Comparative two-dimensional phosphopeptide analysis of in viva phosphorylated MLK3 and in vitro kinase assays suggest that MLK3 is a multi-phosphorylated protein whose activity is increased and phosphorylation pattern altered when coexpressed with activated Cdc42 (Cdc42 V12) (see Chapter 11). However, the sites of MLK3 phosphorylation have not been identified. In an attempt to elucidate the exact positions of in viva phosphorylated MLK3 residues, 1 x 108 293 cells expressing MLK3 in the presence of activated Cdc42 were cultured. MLK3 was isolated from cellular lysates by immunoprecipitation with a MLK3 polyclonal antibody. Also, MLK3 was immunoprecipitated from 4 x 106 293 cells labeled with [HP] orthophosphate to be used as a radioactive tracer. The two MLK3 samples were mixed and further purified by SDS/PAGE. The gel was dried and the MLK3 bands were visualized by phosphorimaging. The MLK3 band was excised from the gel and subjected to in-gel digestion with trypsin (Fig. 2A). The tryptic peptides were extracted from the gel with two acetonitrile washes and yielded a 65-80% recovery based on radioactivity using Cerenkov counting. 4.3 Separation of Tryptic Peptides by Reverse-phase HPLC and Phosphopeptide Mapping A small portion of the recovered peptides was saved in order to be analyzed by two-dimensional phosphopeptide mapping (Fig. 3A). The remainder of the tryptic peptides was fractionated by reverse phase HPLC on a C18 microbore column at a flow rate of 50 pl/min (Fig. ZB). The peptides were eluted with an increasing gradient of 126 acetonitrile and monitored by UV absorbance at 214 nm. Peak fractions collected at approximately one-minute intervals were spotted on TLC plates to screen for radioactive peaks (Fig. 2C). A small portion from the total pool of tryptic peptides was analyzed by two- dimensional phosphopeptide mapping to serve as a reference (Fig. 3A). Peptides were separated by TLE in the first dimension and by TLC in the second dimension. The analysis shows the typical in viva phosphorylation pattern observed for MLK3 coexpressed with activated Cdc42 (spots are labeled a-g) including the Cdc42 inducible sites (spots are labeled x and y). Radioactive HPLC fractions were characterized by one-dimensional TLE and one-dimensional TLC (Fig. 33, C). This analysis allowed for major phosphopeptides labeled on the 2-D map in Fig. 3A to be correlated with and assigned to an HPLC fraction number. Also, the analysis makes certain the radioactive spots seen in Fig. 2C are peptides and not free phosphate or background. 4.4 Identification of Phosphorylation Sites using MALDI-MS and ESI-CID Radioactive HPLC fractions were analyzed by MALDI-MS in linear positive-ion mode using a-cyano-4-hydroxycinnamic acid as the UV absorbing matrix. A computer program, MS-Digest, was used to calculate the monoisotopic masses of all MLK3 tryptic peptides including phosphorylated peptides and partially digested peptides and to compare those masses to the data generated by MALDI-MS. Samples that potentially contained phosphorylated peptides were then incubated with alkaline phosphatase and re- examined by MALDI-MS for the loss of multiples of -80 Da, which corresponds to the 127 loss of phosphate groups. MALDI-MS data that correlated to more than one possible phosphopeptide, or peptides that contained multiple phosphorylatable amino acids were considered ambiguous. MALDI-PSD or ESI-CID-analysis was performed on these ambiguous samples to identify the phosphorylation site. Further characterization of certain radioactive HPLC fractions was obtained by phosphoamino acid analysis (Fig. 6). For this analysis, a 5 pl aliquot was incubated in 6 N HCl for 1 h at 37 °C and the phosphoamino acid content was examined by one dimensional TLE. HPLC Fraction 45 - The MALDI-MS spectrum of HPLC fraction 45 (spot i) contained two major peaks, a peak at m/z 2334.59 and a peak at m/z 2254.73 corresponding to the monophosphorylated and unphosphorylated peptide Thr7”-Arg”3, respectively (Fig. 4). Following alkaline phosphatase treatment of the fraction only the unphosphorylated peptide was identified. This peptide contains two threonine residues and one serine residue. Phosphoamino acid analysis (PAA) of this HPLC fiaction revealed that the 705 peptide contained exclusively phosphoserine (Fig. 6). Therefore, Ser was assigned as the phosphorylation Site in HPLC fraction 45. HPLC Fraction 23 - The MALDI-MS spectrum of HPLC fraction 23 (spot e) revealed Signals at m/z 2395.17 and at 2315.94, corresponding to the monophosphorylated and unphosphorylated peptide Ser' '-Arg37 respectively (Fig. 5). Phosphatase treatment produced a shift of 80 Da to yield only the unphosphorylated peak. This peptide contains five serines and no threonines. Ser“-Arg’7 also resides within a glycine-rich motif, and within this 27 amino acid peptide, thirteen glycine residues are present. 128 HPLC Fraction 31 - MALDI-MS analysis of HPLC fraction 31 (spot h) showed a peak at m/z 1699.21 (Fig. 7A). Following phosphatase treatment the peak shifted by -80 Da to 1619.61 (Fig. 7B). The calculated mass values matched two possible mono phosphorylated MLK3 peptides: Asn’”’-Arg”9 and Val'”-Lys‘“. To distinguish between the two possible peptides, MALDI-PSD analysis was performed on fiaction 31 (Fig. 7C). PSD analysis of fraction 31 revealed a series of COOH-terminal fragment ions (y ions) matching peptide Asn5'5-Arg’”. This peptide has one Ser residue (Ser’z‘) and one Thr residue (Thr’z‘). One of the most abundant peaks is y., - 98 Da, which corresponds to the decomposition of a H3PO. from the precursor ion. The y, — 98 Da peak corresponds to the peptide fragment Ser’"-Arg”9 with the loss of H3P04. Furthermore, the mass detected for the y, ion that contains the Thr residue but not the Ser residue, corresponds to unphosphorylated peptide fragments. PAA analysis of HPLC fraction 31 also confirmed only Ser phosphorylation (Fig. 6). Taken together this data allowed assignment of phosphorylation to Ser’". HPLC Fraction 27 - The MALDI-MS spectrum of fraction 27 (spots f and g) contained four monophosphorylated peptide peaks, which shifted by -80 Da following phosphatase treatment (Fig. 8). Phosphoamino acid analysis of fraction 27 revealed only phosphoserine (Fig. 6, lane 2). Analysis of the calculated mass values matched two possible MLK3 monophosphorylated peptides (Ala‘”-Arg’so and Leu“°-Arg‘”) to the value at m/z 2171.93. ESI-CID was unsuccessful at obtaining sequence information on this peptide. Therefore, MALDI-PSD analysis was performed to discern between the two 129 possible peptides (Fig. 9A). The loss of H3PO. (y20 — 98) was readily observable. However, only a few additional fragment ions were detectable. None of the observed fragments matched the peptide Ser’”-Arg”°. On the other hand, three of the fragment peaks matched the monophosphorylated peptide Leu“°-Arg°”. Since PAA analysis revealed that this HPLC fiaction contained only Ser phosphoamino acids, Ser‘“ is the only possible phosphorylation site on the peptide. Phosphorylation at Ser654 is assigned with caution, since the MALDI-PSD data collected was of poor quality, and an improved fragment series is necessary for full confidence of phosphorylation at this site. The remaining three peaks correspond to differential tryptic digestion products of the monophosphorylated peptide Ser7‘8-Arg7“ (at m/z 1940.73, Ser7‘s-Arg7“; at m/z 1272.55, Ser7‘8-Arg’“; and at m/z 1020.44, Ser7’8-Arg7“). This sequence contains three Ser residues and two Thr residues. To determine the site of phosphorylation within this peptide, ESI—CID on fraction 27 was performed using an ion-trap electrospray instrument. Shown in Fig. 9B is the CID spectrum of the doubly charged parent ion of m/z 1020.32. The most prominent peak is the neutral loss of H3PO. (-49 Da) at m/z 461.2. Analysis of the COOH-terrninal y ion fiagrnentation series rules out phosphorylation of Ser’“, since the mass of the ys-ys ions do not contain the additional mass of a phosphate group. Meanwhile, the mass of the b2 ion indicates the presence of a 757 phosphate group at Ser . HPLC Fraction l7 - Fraction 17 (spot c) exhibited one peak in the MALDI-MS spectrum that was reduced by 80 m/z units following phosphatase treatment, corresponding to a phosphorylated peptide (Fig. 10A, B). The m/z 902.46 peak matched 130 two possible phosphopeptide sequences in MLK3. ESI-CID analysis of the doubly charged parent ion produced a mixture of b and y series fragment ions that identified the phosphopeptide as Prom-Arg773 and allowed assignment of phosphorylation at Ser’”, the only possible phosphorylatable residue in this sequence (Fig 10C). The neutral loss of H3PO. was also detected at m/z 402. HPLC Fraction 20 - The MALDI-MS spectrum of fraction 20 (spot d) following phosphatase treatment identified a singly phosphorylated peptide Pro"““-Arg799 (Fig. 11A, B). This sequence contains two Ser residues and no Thr residues. ESI-CID analysis of the doubly charged parent ion of m/z 1323.16 revealed a predominant y series of fragment ions with a couple of b ions (Fig. 11C). The neutral loss of H3PO. was detected at m/z 613.4. The b. ion that contains Ser789 corresponds to a m/z value expected for the unphosphorylated Ser residue. On the other hand, the m/z value of the y7 ion corresponds to the sequence Ser793-Arg7‘” containing a phosphate group, clearly identifying Ser793 as the phosphorylation site in a peptide contained in fiaction 20. HPLC Fraction 15 - Fraction 15 contains spot a, the most intensely radiolabeled peptide on the two-dimensional phosphopeptide map (Fig. 3A). MALDI-MS analysis of this fraction revealed a peak at m/z 1192.93, which shifted by -80 Da to m/z 1112.25 following phosphatase treatment (Fig 12A, B). This value corresponds to peptide Gly736-Arg747 containing a single phosphate moiety. This peptide contains two Thr residues and two Ser residues. ESI-CID of the doubly charged ion revealed a predominant y series of fragment ions with a few b series ions (Fig. 12C). The neutral 131 loss of H3PO. was detected at m/z 547.9. While the value of y7 that contains Thr’" and Ser746 corresponds to the unphosphorylated peptide fragment, the value of ya that contains Set”o corresponds to the phosphorylated peptide fragment. These results identify Ser’” as the phosphorylation site on a peptide contained in fraction 15. 4.5 Fractionation of Unretained C18 Fractions using Porous Graphitic Carbon Chromatography Fractions 1-7 eluted very early off the C- 18 column in a poorly resolved peak that contained contaminants such as salts and detergents. One-dimensional phosphopeptide mapping analysis showed that the Cdc42 inducible sites labeled x and y on the 2-D map eluted within this fiaction (Fig. 3). Obtaining MALDI data for these I-IPLC fractions was not feasible due to signal repression caused by contaminants. To resolve these peptides from the contaminants, reverse phase-HPLC using a more hydrophobic resin, PGC, was performed on the poorly retained C18 fractions. Fig. 13A displays the chromatogram from the PGC analysis. The PGC column was successful in retaining and resolving multiple peaks that were present in the flowthrough fractions from the C18 column, including several that contained radiolabeled peptides (Fig. 138). One dimensional phosphopeptide analysis demonstrated that the radioactive fractions 22P-28P (where P indicates a fraction from the PGC column) contained the Cdc42 inducible sites (spots x and y) as well as spot b (Fig. 14). 132 4.6 MALDI-MS Analysis of PGC Fractions. PGC reverse-phase chromatography was successful in retaining and resolving the hydrophilic peptides, including a few radiolabeled peptides, which were present in the poorly retained eluant from the C18 column. MALDI-MS analysis of these radiolabeled peptides is shown in Figs. 15-17. The MALDI-MS spectrum of fiaction 23P contained a single peak at m/z 805.39. Dephosphorylation of the fraction followed by MALDI-MS showed a loss of —160 Da, corresponding to a loss of two phosphate groups (Fig 15). Analysis of the calculated values indicated that the only possible tryptic phosphopeptide was Serm-Argm. This sequence contains two Ser residues and no Thr residues, thus Ser724 and Ser727 were assigned as the phosphorylation sites in fiaction 23P. Comparison of the mass spectra of fiaction 28P before (Fig 16A) and after phosphatase treatment (Fig. 163) revealed a peak attributable to a phosphopeptide. Based on the calculated values, Leus’z-Arg‘" is the only possible tryptic phosphopeptide for the signal at m/z 1242.36. This peptide contains two Ser residues and no Thr residues, and is an incompletely digested product. The completely digested tryptic peptide was identified in fraction 22P at m/z 1166.86 (Fig. 17), corresponding to Leu’”- Arg’60 containing two phosphate moieties. Therefore, Ser’” and Ser”6 were identified as phosphorylation sites that correspond to the Cdc42 inducible sites. 133 4.7 Comparative Two-Dimensional Phosphopeptide Mapping of In viva Labeled MLK3 Variants. To better correlate the identified phosphorylation Sites from PGC chromatography with the spots on the two dimensional map, phosphopeptide mapping was performed following in viva labeling of wild type MLK3 or of MLK3 variants with mutations in identified phosphorylation sites, including MLK3 S724A/S727A and MLK3 SSSSE/8556E. MLK3 S740A (fraction 15, and spot a) was also analyzed to determine the effect of substituting the amino acid that corresponds to the prominent radiolabeled spot in the phosphopeptide map with a non-phosphorylatable residue. The MLK3 variants and Cdc42V12 were coexpressed in cells labeled with [”P]orthophosphate. MLK3 was immunoprecipitated using an MLK3 polyclonal antibody, separated by SDS-PAGE, and transferred to a nitrocellulose membrane (Fig. 18A). Interestingly, the autoradiogram revealed that substitution of Ser’"o of MLK3 with alanine induces a prominent mobility shift. The mobility shift is also detected by Western blot analysis. On the other hand, changing Ser7"o to Glu in an attempt to mimic phosphorylation does not induce the mobility shift. The two-dimensional map of MLK3 S74OA lacks spot a as expected. Otherwise, the remainder of the phosphorylation pattern of MLK3 S740A resembles the map of wild type MLK3. Finally, analyses of the two-dimensional map of MLK3 S724A/S727A clearly indicates that the peptides containing phosphoSerm and phosphoSer”7 are represented by spot b on the map. Meanwhile, spots x and y are absent in the map of MLK3 S555E/8556E indicating that spots x and y are the products of differential trypsin digestion, and that Cdc42 induced phosphorylation of MLK3 occurs at Ser’” and Ser’“. 134 III vitro phosphorylated A MLK, he <— MLK3 A Purified 8121 cell- 293 cell-expressed expressed MLK3 MLK3 B C r I O TLC . fl 0 .l e + L - r TLE Fig. 1. Two-dimensional map of MLK3 following an in vitro kinase assay. A, autoradiogram of in vitro kinase assay with recombinant MLK3 purified from $121 insect cells. B, two-dimensional phosphopeptide map of MLK3 from A. C, two-dimensional phosphopeptide map of recombinant MLK3 isolated from 293 cells and phosphorylated in vitro. 135 A 32P- labeling . A—MLKB B __ N CO 01 I N ab. 01 I .3 (D (1" I (0 0'1 r absorbance at 214 nm (mAU) I: at 45- L KL 1 l l l l l l I l j l l l J -U -12 5 81114172023262932353841 retention time (nin) 45 20 ' 23 1-7 15 17 Fig. 2 Reverse phase-HPLC fractionation of MLK3 tryptic peptides. — MLK3 was expressed with activated Cdc42 and isolated by immunoprecipitation. MLK3 was also isolated from a smaller scale identical culture that had been labeled with [32P] orthophosphate. A,The two MLK3 samples were mixed and further resolved by SDS/PAGE. The gel was dried and the MLK3 bands were visualized by phosphorimaging. B, The MLK3 band was excised from the gel and subjected to in-gel digestion with trypsin. The tryptic peptides were fractionated by reverse phase-HPLC on a C18 column. C, A 1 p1 aliquot of each HPLC fraction was spotted on a TLC plate and air dried. Radiolabeled fractions were detected by phosphorimaging. 136 A l h‘ {7g TLC e d C 0‘ I i I y b B + TLE ’ ' C 45 i II- a A 31 h or ‘ ‘1 .' 27 fig' 9 ' 23 e a 20 d . if . 17 c Q 15 a .' . ' i 7f E) M x.y.b w '1 " g E!; .. 4- ‘e ’ _ > 1-7 15 17 20 23 27 31 45 TLE x:y,b a C d 8 fig ’1 1 Fig. 3 One-dimensional phosphopeptide analysis of radiolabeled HPLC fractions A 1 pl aliquot of each radiolabeled HPLC fraction was analayzed by TLE and TLC. A, A reference two-dimensional phosphopeptide map of in viva labeled MLK3. B, One- dimensional TLE. C, One-dimensional TLC. For each radiolabeled HPLC fraction, the elution number and the corresponding spot on the two-dimensional phosphopeptide map are labeled. 137 2254.73 A100 2334.59 (l) I l i ‘ l l 96 internally .i, l _ w l ‘ w . . . 1 ~ oW‘WflMWWM 1300 lphosphatase B 2254.96 (1-80) 100; \‘ l Fig. 4 Analysis of HPLC fraction 45 using MALDI-MS combined with alkaline phosphatase treatment. A, HPLC fraction 45 (spot i) was analyzed by MALDI-MS in linear positive ion mode with a-cyano-4-hydroxycinnamic acid as the UV absorbing matrix. The m/z value corresponding to a phosphorylated peptide is indicated in bold. B, Fraction 45 was subjected to alkaline phosphatase treatment and reanalyzed by MALDI-MS. The dephosphorylated peptide is indicated in bold with the change in m/z of -80 corresponding to the loss of a phosphate moiety. 138 100‘ 1313.50 2395.17(e) B 100: l phosphatase a § 1313.66 ,. l l 2315.45 (9 410) l l . l. l I MAMMMMWWWM’WWMMWl‘tl‘lt'mu‘awmlwmw 01200 3600 mix Fig. 5 Analysis of HPLC fraction 23 using MALDI-MS combined with alkaline phosphatase treatment. A, HPLC fraction 23 (spot e) was analyzed by MALDl-MS in linear positive ion mode with a-cyano-4-hydroxycinnamic acid as the UV absorbing matrix. The m/z value corresponding to a phosphorylated peptide is indicated in bold. B, Fraction 23 was subjected to alkaline phosphatase treatment and reanalyzed by MALDI-MS. The dephosphorylated peptide is indicated in bold with the loss of m/z -80 in parenthese. 139 '- 1 ’ Pi ' ' ' ThrP . I Fig. 6 Phosphoamino acid analysis of selected HPLC fractions. A 5 p1 aliquot of selected HPLC fractions were hydrolyzed in 6N HCl at 100 °C for 1 h. The phosphoamino acids were analyzed using one-dimensional thin layer electrophoresis on TLC plates and visualized by phosphorimaging. The position of phosphoamino acid standards stained with ninhydrin as well as the position of free inorganic phosphate (Pi), are indicated. 140 3228.74 A 100i . 1606.21th 2163.55 Silnteneity B t’11’00’ i ' ..J. ' ' " " '3500 (1:00 sifi-V’F-EtVLG-PLG-DEli-ItFLPi'lisn [ l yii Y2 l l 1 v5 “— V15 l ‘ \s g i i , l I . W5.98\‘ . fl \ ‘ l l ye-ee 1m” y11-98 ii i in D2 ’3 l 9, my ' l I \s i l l f wl‘ l / l w. . WWW in... 11th 0 m 2116.0 Fig. 7 Identification of phosphorylated Ser524 in HPLC fraction 31 by MALDI-MS Post Source Decay. HPLC fraction 31(spot h) was analyzed by MALDI-MS before (A) and after phosphatase treatment (B). The phosphorylated and dephosphorylated peptides are indicated in bold. C, MALDI-PSD analysis of fraction 31. Analysis of the y series of fragment ions reveal the presence of a phosphoryl group at Ser’“. The fragment ions whose m/z value corresponds to y ions with the loss of H3PO4 (-98 Da) are labeled. The observed fiagment ions are indicated on the spectrum and peptide sequence. 141 A 1622.64 100 t9:39.57 l i i .. , 1715.57 l ‘ l i i ‘ * 1020.44m * ‘ E 1192.84 1 l l E x 191013” 2111.93lg) 1 272.55“) o. 360 n B eschew-60) l” “pm 1001 , _ I i ‘ 1102.47(r-60) ‘ 1622.38 e . 5 . l 1715.32 8 l , 1861.44(f-80) l l l . \ 2091.29(g-80) ‘ : , i . 1 'llllllll w A i 0. . , . ‘. . L . A , A 660 m 2500 Fig. 8 Analysis of HPLC fraction 27 using MALDI-MS combined with alkaline phosphatase treatment. A, HPLC fraction 27 (spots f and g) was analyzed by MALDI-MS. The m/z value corresponding to a phosphorylated peptide is indicated in bold. B, Fraction 27 was then subjected to alkaline phosphatase treatment and reanalyzed by MALDI-MS. The dephosphorylated peptides are indicated in bold with the loss of m/z -80 in parenthese. The m/z values 1020.44, 1272.55, and 1940.73 (A) correspond to singly phosphorylated peptides resulting from the incomplete trypsin digestion of peptide Serm-Arg’“. The m/z value 2171.98 corresponded to the two possible MLK3 mono phosphorylated tryptic peptides Alam-Arg”0 or Leu“°-Arg°59. 142 A 100 , 64%le Q-R-A-L-L-R-G-T-A-L fishuensny y2 LGLG i/ o M«W-¢--~MMH- .2- 0 w .b 881883388888 t-1.inla_+.._~L._.‘ .. “m-)L--.-424 .....u..~.. . b2+Pl Relative Abundance b & 00! <— 888 8 y2 15 i d 0'0 200 300 1 .,1‘.l11........ .,..'.......ll 11...,“ mlll‘in . -.- 400 500 600 «1.2 (49) y!» w\ . I J .41 hub...“ m/z -L-A-S-L-G-LLG—R659 fl b2 748§|£|PLLLGLLJ|LSR766 10 fl 1711616114 y? l. l ‘ l I I Ul...1.‘la..uc-JLt‘l-.lL.v-l—lb .0» w.l.-‘.-u.l.l.,Jt¢1J.uL~‘§J.J‘.. ..{K—g...”.l.....,.-~r...-.... ...1... 700 000 900 1000 Fig. 9 Analysis of HPLC fraction 27 using MALDI-PSD and LC/MS/MS. A, MALDI-PSD spectrum of the peptide at m/z 2172.98 in fraction 27 (spot f/g). The yl 9-98 fragment ion corresponds to the loss of H3PO,. Just a couple of additional fragment ions were detected. The fragment ions produced matched only the peptide LeuW-Arg659. B, ESI-CIDof m/z 510.19, which corresponds to the phosphorylated peptide Ser758-Arg766 in its doubly charged ion form. The fragment ions whose m/z value corresponds to y or b ions with the addition of a phosphate group (Pi) are labeled. Analysis of the y and b fragment ion series indicates that Ser757 is phosphorylated. The fragment ions that were observed are indicated on the spectrum and peptide sequence (an asterisk indicates a phosphorylated serine). 143 O RuNMeAUxMWxn ousasasasasasasasasa 1 79%: 1494.48 1 002.40 (c) / fihwuuuy 0 ; . . ' 1700 100 . 793 72 l phosphatase 1 name-so) 1 1 A/ , 1 1 e 1 , i . 1 a! 1 1414.99 1 1 1 11 1 1 1 OW“ I; 2- -,-,-:-444;._t.. 2-1 “1... 1 700 1700 “nlw4fl b3 1 707P- R773 fi> w v3 fl {-yS-OPI )Mom 118 . y: 17 l 1 1 yam) ’1 l 1 1 I WPII b5+Pl kw am am um mm «m «w HmI Ian aw Hm an an flmI an mm Fig. 10 Identification of Ser77° as the phosphorylation site in HPLC fraction 17. HPLC fraction 17 (spot c) was analyzed by MALDI-MS before (A) and after phosphatase treatment (B). The phosphorylated and dephosphorylated peptides are indicated in bold. C, ESI-CID of m/z 451.9, which corresponds to the phosphorylated peptide Prom-Arg773 in its doubly charged ion form. The fragment ions whose m/z value corresponds to y or b ions with the addition of a phosphate group (Pi) are labeled. Analysis of the y and b fragment ion series indicates that Ser77° is phosphorylated. The fragment ions that were observed are indicated on the spectrum and peptide sequence (an asterisk indicates a phosphorylated serine). 144 1001 1228.04 2323.10 1 1243.14 1 u/ 1 1 1 1 .1 ' I I II II P I I II I 1500 ' 1243.421-00) 100 \‘ xmuy 1229.36 1 1 0 - -. .. -,__ 1 1050 m 1500 C1 013.4140) 788P-S1P1} P §-P Q P A-P-R799 1H0 v9 v6 y‘ y w4—-Y‘*P' g 115 1 ”(‘31 1 1 ”(4211 . 17m 1 MOOPI I we 1" y10+Pl 000 900 . . 411.1. “.144--- . . 1000 1100 1200 1300 W omaaaasasasaaaaaaasss ; 1 1 1 . 1 ». 1.42:1.l1J.., “.ka b-Ib L-ILI “IJI440-nt- .I-JI‘ u..‘11,LJl-. 4 (no-4.144 200 300 400 500 600 700 mlz Fig. 11 Identification of Ser793 as the phosphorylation site in HPLC fraction 20. HPLC fraction 20 (spot (I) was analyzed by MALDI-MS before (A) and after phosphatase treatment (B). The phosphorylated and dephosphorylated peptides are indicated in bold. C, ESI-CID of m/z 662.5, which corresponds to the peptide Prom- Arg"9 in its doubly charged ion form. The fragment ions whose m/z value corresponds to y or b ions with the addition of a phosphate group (Pi) are labeled. Analysis of the y and b fragment ion series indicates that Ser793 is phosphorylated. The fragment ions that were observed are indicated on the spectrum and peptide sequence (an asterisk indicates a phosphorylated serine). 145 A 100 £24.32 906.49 1011an 1 1 ‘9‘1-‘9 1102.031.) 1 1 on i phosphatase ' 824-“ 906451 11121510 .00) :I1I'1 1 "1111. 1 1 ONIJWW 1"“ ' WRWIl—Qa‘ .-.42444~ 4 4-44 44444 - 44 44441 800 1500 m 100 1 547.9(49) ,7 95 1 90 g ‘ 1 736G- G T HVLSP P-P G-T-S- R747 35 1 1110 119 so ‘ 75 1 7o 65 1 14° = as 1 g 50 1 ”092) 1 g 45 1 1 WP. 1 WPI 4o 1 . 35 3 30 3 ”(+2) 1 1 25 gnyq42111' I174?! y0wl 2° y7(+2\ 1 15 . 1o 3 1 3 ‘ .. ...MJ 0. -.,'l 31.11.11. 11121111111. IdIJulIfilI ; ..:.,l.. I',‘".,...,..,.....,,.,,. 200 300 400 500 600 11112700 800 900 1000 1100 1200 Fig. 12 Identification of Ser74° as the phosphorylation site in HPLC fraction 15. HPLC fraction 15 (spot a) was analyzed by MALDI-MS before (A) and after phosphatase treatment (B). The phosphorylated and dephosphorylated peptides are indicated in bold. C, ESI-CID analysis of m/z 597.1, which corresponds to the phosphorylated peptide Prom-Arg"7 in its doubly charged ion form. The fragment ions whose m/z value corresponds to y or b ions with the addition of a phosphate group (Pi) are labeled. Analysis of the y and b fragment ion series indicates that Ser74° is phosphorylated. The fragment ions that were observed are indicated on the spectrum and peptide sequence (an asterisk indicates a phosphorylated serine). 146 A 245 - S g 195 » E C 3 145 *- N 1'6 8 95 - C (0 E (0 _5 ‘J 1 1 1 L 1 J 1 1 1 1 1 1 1 1 1 1 1 1 2 5 8 11 1417 20232629 32353841 4447 5053 5659 retention time (min) 28? 9 0 22P 23P 24P 27p 16? Fig. 13 Fractionation of C18 flowthrough fractions by RP-HPLC on a Porous Graphitic Carbon column. A, The C18 flowthrough fractions 1-7 were collected (Fig. 2B), combined, and concentrated using a Speed Vac concentrator. The combined flowthrough sample was loaded onto the PGC column. Peptides were eluted by a linear acetonitrile gradient at a flow rate of 20 14 l/min. Fractions were collected at appoximately 1.5 minute intervals. B, A 1 p 1 aliquot of each PGC fraction was spotted on a TLC plate and air dried. Radiolabeled fractions were detected by phosphorimaging. 147 A l h‘ 47g e TLC d 7- C b + 5 - B 28P y o 11 24P x,b . . . TLC 2» x,b . . . s -.: 1 1 22P x o I 1 } 22P 23P 24? 28P TLE x x,b x,b y Fig. 14 One-dimensional phosphopeptide analysis of radiolabeled PGC fractions A 1 1,11 aliquot of each radiolabeled PGC fraction was analayzed by one- dimensional TLE and one-dimensional TLC. A, A reference two-dimensional phosphopeptide map of in viva labeled MLK3. B, One-dimensional TLE. C, One- dimensional TLC. For each radiolabed HPLC fraction, the elution number and the correlating spot on the two-dimensional phosphopeptide map are labeled. 148 100 569.02 18 lrumlty 805.39 1 1 Iflu 1131;. 4111;511:4100: 11.11. 0, . ' I I ~, " , 490.0 1300.0 100 1 645.71(-160) 1 x 568.53 sum-may . 1.. WW“ WWWWIMIWWWWWWMW 1 2100.0 mlz Fig. 15 Analysis of PGC fraction 23P using MALDI-MS combined with alkaline phosphatase treatment. A, PGC fraction 23P was analyzed by MALDI-MS. The phosphorylated peptide are indicated in bold. B, Fraction 23? was then subjected to alkaline phosphatase treatment and reanalyzed by MALDI-MS. The dephosphorylated peptide is indicated in bold with the loss of m/z —160 (two phosphoryl groups) in parenthesis. 149 10° 816.89 1 1242.36 1 1 . 1‘ . , ‘ WWWWmMW-«WWWM‘WMN‘M’1 0. . , . _ 750 2000 B l phosphatase 100‘ 216.05 P 5 I: 1 102.20(-80) 1 1 11 ' 1 0 AM w'ww‘wl W11 _ WMAAWWAWM'NM02110111111111.0011 ‘MMMMMM1 750 2000 ml: Fig. 16 Analysis of PGC fraction 28P using MALDI-MS combined with alkaline phosphatase treatment. A, PGC fraction 28P was analyzed by MALDI-MS. The phosphorylated peptide are indicated in bold. B, Fraction 28P was then subjected to alkaline phosphatase treatment and reanalyzed by MALDI-MS. The dephosphorylated peptide is indicated in bold with the loss of m/z —80 in parenthesis. 150 100 : 1136.04 '5 1mm l phosphatase 100; moan-100) . / Fig. 17 Analysis of PGC fraction 22P using MALDI-MS combined with alkaline phosphatase treatment. A, PGC fraction 22P was analyzed by MALDI-MS. The phosphorylated peptide are indicated in bold. B, Fraction 22P was then subjected to alkaline phosphatase treatment and reanalyzed by MALDI-MS. The dephosphorylated peptide is indicated in bold with the loss of m/z —160 (two phosphoryl groups) in parentheses. 151 Hid << < Hill << < [:1 as as. e m a: g 3 mm} av. 55. a M as. m a x: - _ 32"” 51 O" - 1- 0.5-s- MLK3“ B WT 35551: SSS6E A .. x y b max _y S724A S740A S727A TLC s1 “CI—-> 9‘4- m T .... ._ W.,. -b + p - Fig. 18. Two-dimensional maps of tryptic phosphopeptides derived from in viva phosphorylated MLK3 variants. Cells expressing the specified MLK3 variants in the presence of Cdc42V12 were incubated with [32P]orthophosphate. MLK3 variants were immunoprecipitated from cellular lysates, blotted onto a nitrocellulose membrane (A) TLE and subjected to trypsin digestion. B, The resultant tryptic phosphopeptides were analyzed by comparative two-dimensional phosphopeptide analysis. Short arrows indicate tryptic phosphopeptides that are absent. Phosphopeptides were detected by phosphorimaging. Long arrows indicate the direction of electrophoresis and chromatography. 152 - MW _MH_111+ W HPLC 2D 2521191; sequence Q; Q1; OQL C§l_c Fract. spot 15 a 736GGTVSPPPGTSR747 1192.93 1192.54 1112.25 1112.52 17 c 767PRPSPLR773 902.46 902.49 822.80 822.49 20 d 73338pr SPQPAPR799 1323.16 1323.66 1243.42 1243.66 23 e 1|SPLGSWNGSGS(G) ,RVEGSPK37 2395.17 2395.08 2315.42 2315.08 27 fig 7488APGTPGTPR SPPLGLISR 766 1940.73 1940.99 1861.44 1860.99 758$PPLGLISRPR 768 1272.55 1272.70 1192.47 1192.47 758$PPLGLISR 766 1020.44 1020.65 939.45 940.65 MOLIQRALLRGTALLA SLGLGR659 2171.93 2172.27 2091.29 2092.27 31 h 515NVFEVGPGD SPTFPR529 1699.21 1698.75 1619.61 1618.75 45 i 702TPDSPPTPAPLLLDLGIPVGQR 7261 2334.59 2334.21 2254.96 2234.21 22? x 552LEDSSNGER56| 1166.64 1166.37 1006.87 1006.37 28P y 552LEDSSNGERR562 1242.36 1242.51 1162.20 1162.51 23? b 724SAKSPR729 805.39 805.30 645.71 645.30 1 413 103 ‘115 3641 487”? 506 6? 847 c Gly 8H3 Kinase 1 w E ProISsdThr-rlch $524 $555 $556 S654 S724 S740 S770 S705 S727 S757 S793 Fig. 19. Schematic representation of MLK3 phosphorylation sites. A,Table summarizing the observed and calculated m/z values for the phosphopeptides identified before and after alkaline phosphatase treatment. B, Block diagram of MLK3 indicating the location of the in viva phosphorylation sites. 153 5. Discussion Protein phosphorylation is an important reversible mechanism regulating many protein kinases. Identification of in viva phosphorylation sites is crucial for understanding the function of a particular phosphorylation event. Previous work has indicated that the mixed lineage kinase MLK3 may be regulated by phosphorylation. However, direct identification of MLK3 phosphorylation sites has not been reported. The purpose of this study was to map the in viva sites of phosphorylation on MLK3 as a critical first step to understanding the regulatory role of this post translational modification on MLK3 function. Using a combination of phosphopeptide mapping and mass spectrometry analysis, twelve in viva phosphorylation sites were identified. Previous work has shown that coexpression of activated Cdc42 (Cdc42V12) increases the in vitra catalytic activity of MLK3 and alters its in viva phosphorylation pattern (9). Therefore, in this study HEK 293 cells coexpressing MLK3 and Cdc42 V12 were used as a model system to identify in viva phosphorylation sites on MLK3, including Cdc42 inducible phosphorylation sites. A ponion of the cells were labeled with [’ZP] orthophosphate for use as a radiolabeled tracer. This approach not only simplified selection of phosphopeptides, but also made it possible to assign phosphorylation sites to radiolabeled spots on the two- dimensional map. Indeed, virtually all of the phosphopeptides observed in the two- dimensional map were identified using mass spectrometry. Thus comparative two- dimensional phosphopeptide mapping should serve as a useful tool to study the sites of MLK3 phosphorylation in other model systems, such as the phosphorylation of 154 endogenous MLK3 in response to different stimuli. A limitation of radiolabeled cells is that the resulting radiolabeled phosphopeptides represent MLK3 phosphorylation events that occur only during the labeling period (the last 4h of a 15 h transfection experiment). Since MLK3 expression is usually detected at around 8 h post-transfection, it is possible that very stable phosphorylation sites may be missed. A positive consequence of using the radiolabeled tracer was the realization that the C18 column flowthrough fractions contained phosphorylated peptides, including those that contain Cdc42-inducible phosphorylation sites. Salts and other contaminants commonly found in the flowthrough fi'action from C18 columns suppress the mass spectrometry signal (18). To overcome this problem, reverse-phase HPLC using a PGC column was selected to desalt the C18 flowthrough fractions. The PGC resin has been shown to be a more hydrophobic stationary phase than the C18 resin (19), and has the unique property of retaining very polar compounds. In addition, PGC chromatography has been used to resolve short and very hydrophilic peptides (20,21). The PGC HPLC successfully retained and resolved several peptides fi'om the C18 flowthrough fractions, including the Cdc42-inducible phosphopeptides. Furthermore, the PGC column was used with the same volatile mobile phase as the C18 column, allowing for concentration of the sample and compatibility with mass spectrometry. Following HPLC fractionation the strategy to deduce phosphorylation sites was to first Confirm which peptides from the radiolabel-containing HPLC fractions were phosphopeptides. MALDI-MS data were collected before and after alkaline phosphatase treatment. The loss of -80 Da after phosphatase treatment is diagnostic for loss of phosphate. Many of the data values correlated to more than one potential phosphopeptide 155 or to phosphopeptides that contained multiple Ser and Thr residues. These situations required MALDI-PSD analysis or LC/MS/MS electrospray mass spectrometry to attain sequence information and to assign a phosphorylation site. A surprising result from the LC/MS/MS analysis was the sequencing of three different phosphopeptides that resulted from trypsin cleaving Arg—Pro peptide bonds. Typically, trypsin is reported to not cleave Arg-Pro or Lys-Pro peptide bonds (22-24), and most of the available computer programs used to generate tryptic peptides masses do not include cleavage at Arg/Lys-Pro bonds. This result may indicate that there is something unique surrounding these particular sequences such as modification of the proline residues involved or isomerization of the peptide bond between Arg-Pro. Alternatively, this uncommon cleavage specificity may instead be due to the increased stability of the modified trypsin used in this study. Regardless, these results demonstrate that caution should be taken when calculating trypsin digestion at Arg/Lys-Pro bonds based on the settings of the currently available computer programs. The schematic location of the MLK3 phosphorylation sites is depicted in Fig. 19. Other than the phosphorylation site found in the glycine rich region (between amino acids 11-37), all of the identified phosphorylation sites cluster in two regions. Seven of the twelve sites are positioned between amino acids 650-800 in the COOH-terminal region, while three phosphorylation sites, including the two Cdc42 inducible sites (Set-‘5’ and Ser’“), are situated between amino acids 520-550 in the basic region immediately following the CRIB domain. Interestingly, no phosphorylation sites were found within the catalytic domain. Many protein kinases are regulated by phosphorylation of residues in a conserved region 156 termed the “activation segment” located within the catalytic domain. The phosphate moiety from the phosphorylated residue in the activation segment can interact with the Arg residue of a conserved Arg-Asp sequence located in the catalytic domain (25). This interaction functions in inducing a conformational change of the catalytic domain that often modulates the function of a protein kinase (26-28). However, phosphorylation of the MLK3 activation segment was not detected, suggesting that MLK3 may not be regulated by activation segment phosphorylation. This idea would conflict with a report by Leung et al that claimed Thr277 and Serml in the activation segment are the major MLK3 phosphorylation sites (1 1). In their study, Thrm and Ser’“ were mutated to Ala residues and the MLK3 variants were found to exhibit a decrease in catalytic activity. Anticipating that these activation segment residues might be phosphorylation sites, I previously made the same amino acid substitutions and observed similar results as Leung et a! (data not shown). It is possible that these phosphorylation sites were missed by the methods used in this study. Another possibility is that the decrease in catalytic activity seen with the activation segment mutants was an artifact of site—directed mutagenesis. Regardless, it is clear that direct identification by mass spectrometry is the more credible method for determining phosphorylation sites. It should be noted that not all protein kinases are regulated by activation segment phosphorylation. Crystallographic studies have shown that protein kinases (e. g. phosphorylase kinase) not regulated by activation segment phosphorylation contain an acidic residue within this region, which interacts with the conserved Arg residue (29) in the Arg-Asp sequence. 'MLK3 does contain an acidic residue within the activation segment (Glu’7‘), leaving open the possibility that MLK3 may not be regulated by activation segment phosphorylation. 157 Knowledge of the sequence surrounding the phosphorylation site often provides valuable information as to the identity of the kinase responsible for the phosphorylation event. The majority of the sites identified in this study contain a Pro residue following the phosphorylation site, suggesting that MLK3 may be a target of proline-directed kinases. Praline-directed kinases, which include MAPKs, cyclin dependent protein kinases (CDKs), and glycogen synthase kinase 3 (GSKB) among others, can phosphorylate Ser/Thr residues that are immediately followed by a Pro residue (30). The peptide containing the Cdc42 inducible sites (Ser’ss, Ser‘“) does not contain a phosphoSer-Pro sequence, suggesting that phosphorylation of these sites are catalyzed by a distinct family of kinases or are the result of MLK3 autophosphorylation. The phosphorylation site Ser 7”, located in the COOH-terminal tail of MLK3, resides in the sequence KSPRR and conforms to the stricter consensus sequence for the CDK family (KS/TPXR)(31). The phosphorylation site Ser’“ exists in the sequence PDSP and the phosphorylation site Ser'”7 exists in the sequence PRSP. These arrangements conform to the stricter consensus sequence for MAPK (including JNK) phosphorylation sites (PXS/TP)(31), leaving open the possibility that JNK might phosphorylate MLK3 in a potential feedback mechanism. Evidence for a feedback mechanism has been demonstrated with the ERK pathway, where the MAPKKK Raf has been reported to be phosphorylated by the MAPK ERK (32,33). Also, an in vitra study has shown that activated recombinant JNK can phosphorylate the COOH-terminal region of mixed lineage kinase MLK2 (34), although no sequence studies were undertaken in this study. 158 Due to the low abundance of phosphorylated material in cells, determining the in viva phosphorylation sites of protein kinases has been a difficult challenge. Recent improvements in the resolution and sensitivity of mass spectrometry instruments have made it more feasible to map protein phosphorylation sites from in viva sources. In this study, a variety of mass spectrometry techniques were used to determine in viva phosphorylation sites of the mixed lineage kinase MLK3. Knowledge of the sequences that are modified provides the necessary first step into understanding how phosphorylation regulates MLK3 fimction. 159 6. References 10. 11. 12. 13. 14. 15. 16. Cohen, P. (2000) Trends Biochem Sci. 25, 596-601. Gallo, K. A., Mark, M. R., Scadden, D. T., Wang, Z., Gu, Q., and Godowski, P. J. (1994) J Biol Chem. 269, 15092-100 Ing, Y. L., Leung, I. W., Heng, H. H., Tsui, L. C., and Lassam, N. J. (1994) Oncogene. 9, 1745-50. Ezoe, K., Lee, S. T., Strunk, K. M., and Spritz, R. A. (1994) Oncogene. 9, 935-8. Tibbles, L. A., Ing, Y. L., Kiefer, F., Chan, J ., Iscove, N., Woodgett, J. R., and Lassam, N. J. (1996) Embo J. 15, 7026-35. Davis, R. J. (2000) Cell. 103, 239-52. Hehner, S. P., Hofinann, T. G., Ushmorov, A., Dienz, 0., Wing-Lan Leung, I., Lassam, N., Scheidereit, C., Droge, W., and Schmitz, M. L. (2000) Mol Cell Biol. 20, 2556-68. Burbelo, P. D., Drechsel, D., and Hall, A. (1995) J Biol Chem. 270, 29071-4 Bock, B. C., Vacratsis, P. 0., Qamirani, E., and Gallo, K. A. (2000) J Biol Chem. 275, 14231-41 Teramoto, H., Coso, O. A., Miyata, H., Igishi, T., Miki, T., and Gutkind, J. S. (1996) J Biol Chem. 271, 27225-8 Leung, I. W., and Lassam, N. (2001) J Biol Chem. 276, 1961-7. Hillenkamp, F., Karas, M., Beavis, R. C., and Chait, B. T. (1991) Anal Chem. 63, 1193A-1203A. Spengler, B., Kirsch, D., Kaufrnann, R., and Jaeger, E. (1992) Rapid Commun Mass Spectrom. 6, 105-8. Fenn, J. B., Mann, M., Meng, C., and Whitehouse, C. M. (1990) Mass Spectram Rev. 9, 37-70 Jonscher, K. R., and Yates, J. R., 3rd. (1997) Anal Biochem. 244, 1-15. Johnson, C. G., Caron, S., and Blough, N. V. (1996) Anal Chem. 68, 867-72. 160 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. Roepstorff, P., and Fohlman, J. (1984) Biomed Mass Spectrom. 11, 601. Roboz, J ., Yu, Q., Meng, A., and van Soest, R. (1994) Rapid Commun Mass Spectrom. 8, 621-6. Ross, P., and Knox, J. H. (1997) Adv Chromatogr. 37, 121-62 Yamaki, S., Isobe, T., Okuyama, T., and Shinoda, T. (1996) J Chromatogr A. 729, 143-53. Chin, B. T., and Papac, D. I. (1999) Anal Biochem. 273, 179-85. Boyle, W. J ., van der Geer, P., and Hunter, T. (1991) Methods Enzymal. 201, 110- 49 Hill, R. L. (1965) Adv Protein Chem. 20, 37-107 Carnegie, P. R. (1969) Nature. 223, 958-9. Johnson, L. N., Noble, M. E., and Owen, D. J. (1996) Cell. 85, 149-58. Canagarajah, B. J ., Khokhlatchev, A., Cobb, M. H., and Goldsmith, E. J. (1997) Cell. 90, 859-69. Zheng, J., Knighton, D. R., ten Eyck, L. F ., Karlsson, R., Xuong, N., Taylor, S. S., and Sowadski, J. M. (1993) Biochemistry. 32, 2154-61. Taylor, S. S., Knighton, D. R., Zheng, J ., Ten Eyck, L. F., and Sowadski, J. M. (1992) Annu Rev Cell Biol. 8, 429-62 Owen, D. J ., Noble, M. E., Garman, E. F., Papageorgiou, A. C., and Johnson, L. N. (1995) Structure. 3, 467-82. Hall, F. L., Braun, R. K., Mihara, K., Fung, Y. K., Bemdt, N., Carbonaro—Hall, D. A., and Vulliet, P. R. (1991) J Biol Chem. 266, 17430-40. Songyang, Z., Lu, K. P., Kwon, Y. T., Tsai, L. H., Filhol, 0., Cochct, C., Brickey, D. A., Soderling, T. R., Bartleson, G, Graves, D. J ., DeMaggio, A. J ., Hoekstra, M. F., Blenis, J ., Hunter, T., and Cantley, L. C. (1996) Mal Cell Biol. 16,6486- 93. Lee, R. M., Cobb, M. H., and Blackshear, P. J. (1992) J Biol Chem. 267, 1088-92. Gardner, A. M., Vaillancourt, R. R., Lange-Carter, C. A., and Johnson, G. L. (1994) M01 Biol Cell. 5, 193-201. 161 34. Phelan, D. R., Price, G., Liu, Y. F ., and Dorow, D. S. (2001) J Biol Chem. 276, 10801-10. 162 V. Concluding Remarks The work described in this thesis examined mechanisms regulating the mixed lineage kinase MLK3. In particular, the regulatory role of leucine zipper-mediated MLK3 oligomerization was investigated and identification of in viva MLK3 phosphorylation sites were the foremost aspects studied. With regard to the leucine zipper domain of MLK3, the original hypothesis was that oligomerization mediated by the zipper domain was necessary for the catalytic activity of MLK3. This rationale was partly influenced by previous work in the field of receptor tyrosine kinases, where it has been well established that growth factor-induced receptor dimerization is required for the transautophosphorylation activity of the receptors of the tyrosine kinase family. However, the study presented in chapter 111 suggests that MLK3 phosphotransfer activity does not require oligomerization. A monomeric zipper point mutant retained full autophosphorylation and histone phosphorylation activity when coexpressed with activated Cdc42. Instead, the data demonstrated that zipper-mediated MLK3 oligomerization is important for downstream signaling. In particular, even when coexpressed with Cdc42V12, the zipper point mutant could only phosphorylate one of the two activating phosphorylation sites on its physiological target MKK4, and therefore failed to activate JNK. These findings suggest a slightly more complex role for zipper-mediated oligomerization of MLK3. Rather than being critical for phosphotransfer activity, the function of the zipper domain may be to properly orient the catalytic domain(s) for phosphorylation of specific downstream targets. 163 The discovery that monomeric MLK3 can exhibit full phosphotransfer catalytic activity when coexpressed with activated Cdc42 leaves open the possibility that monomeric MLK3 may be biologically active and participate in pathways distinct fi'orn that of JNK. Interestingly, preliminary results in our lab have shown that the zipper point mutant induced NF-KB promoter activity to a greater extent than wild type MLK3 (Zhang and Du). In addition, under physiological conditions MLK3 oligomerization may be regulated. Recent work in our lab has revealed an autoinhibitory interaction between MLK3’s NHz-terminal SH3 domain and a proline-containing sequence located between the zipper domain and the CRIB motif of MLK3 (Zhang and Gallo). Given the proximity of the zipper and the SH3 binding region, one could imagine that MLK3 oligomerization is influenced by SH3-mediated intramolecular interactions. It would be interesting to explore the prospect that an SH3-mediated intramolecular interaction could inhibit MLK3 oligomerization, while relief of autoinhibition (possibly by activated Cdc42) could promote zipper-mediated oligomerization. Chapter IV describes an analytical project involving the identification of in viva MLK3 phosphorylation sites using a combination of phosphopeptide mapping and mass spectrometry. Seven of the sites identified contained a proline residue immediately following the phosphorylated serine residue. This new information may indicate that MLK3 is regulated by proline-directed kinases, which include the MAPK family of protein kinases. Furthermore, the locations of the Cdc42-inducible phosphorylation sites were determined by correlating the mass spectrometry results with phosphopeptide spots on the two-dimensional phosphopeptide map. Since these sites of MLK3 164 phosphorylation have been identified, the next step will be to understand their effects on MLK3 activities. Understanding the function of the individual phosphorylation sites as well as the net effect of MLK3 phosphorylation will most likely require utilizing a combination of cellular systems. A transfectable system in which the basal activity of wild type MLK3 is moderately law will be useful to study the effects of mutating codons associated with individual phosphorylation sites to nonphosphorylatable residues that mimics phosphorylation (Ser to Glu) or the opposite (Ser to Ala). Besides the effect on catalytic activity, the phosphorylation site mutants should be analyzed in terms of downstream signaling, localization, protein stability, and association with other molecules. Relatively little experimental data has been collected on the biological properties of endogenous MLK3. Identification of the in viva phosphorylation sites may give rise to useful tools for studying endogenous MLK3 in terms of phosphorylation. Antibodies directed against MLK3 phosphorylation sites could be used to study the phosphorylation of endogenous MLK3 in response to various extracellular stimuli or potential activators. Also, two-dimensional phosphopeptide mapping of endogenous MLK3 from in viva labeled cells could also be used as a readout technique since the identified phosphorylation sites were correlated to spots observed on the phosphopeptide map. In conclusion, MLK3 contains several regions that have the potential to serve as regulatory regions. Accumulating evidence from our lab, and from others, suggest that activation of MLK3 may be a multistep process whose regulation is coordinated through numerous mechanisms. The information presented in this thesis contributes important 165 knowledge concerning molecular mechanisms that appear to regulate MLK3 and should advance the understanding of the role of MLK3 in biological processes. 166 111utilizifllfliigiifllill111