.. .55.... A... w.h»WorM....~. h. a... .71.. WW”... u..- .1... p : I) :x .h Illdl umlwnrwnflmnc (Nil. '3 A... Jill it!!! Illiqlllll:n. . . .. . rand. .5 ........ . .. . a. H :51... 31... . I. u... _ . . a s gawéwmflfl ”5..va gnaw... . -E.§m&. .33.»... v 0!. u I . *E p 0 O 11 This is to certify that the dissertation entitled REGULATION OF MIXED LINEAGE KINASE 3 BY SMALL GTPASE, GUANINE NUCLEOTIDE EXCHANGE FACTOR AND PROTEIN-PROTEIN INTERACTIONS presented by Yan Du has been accepted towards fulfillment of the requirements for the Ph.D. degree in Physiology XW/ 442.42 Major Professor’s Signature é/M/Oé / / Date MSU is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DAJEDUE DATEDUE DAIEDUE 6/07 p:/ClRC/DateDue.indd-p.1 REGULATION OF MIXED LINEAGE KINASE 3 BY SMALL GTPASE, GUANINE NUCLEOTIDE EXCHANGE FACTOR AND PROTEIN-PROTEIN INTERACTIONS By Yan Du A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY Department of Physiology 2006 ABSTRACT REGULATION OF MIXED LINEAGE KINASE 3 BY SMALL GTPASE, GUANINE NUCLEOTIDE EXCHANGE FACTOR AND PROTEIN-PROTEIN INTERACTIONS By Yan Du Mixed-lineage kinase 3 (MLK3) is a widely expressed, mammalian serine/threonine protein kinase that functions as a mitogen-activated protein kinase kinase kinase (MAPKKK) and activates multiple MAPK pathways. MLK3 is not only implicated in JNK-mediated neuronal apoptosis in response to trophic factor withdrawal, but also is critical for epidermal growth factor (EGF) induced B-Raf activation and cell proliferation. MLK3 contains multiple protein interaction domains or motifs that may be involved in inter- or intra-molecular interactions, including an N-terminal Src Homology 3 (SH3) domain, a centrally located zipper region, a Cdc42/Rae interactive binding (CRIB) motif and a Pro/Thr/Ser rich COOH-terminus. The data presented in Chapter 2 demonstrate that Cdc42 is a physiological activator of MLK3. The binding of activated Cdc42 induces the activation loop autophosphorylation of MLK3. Prenylation-competent, activated Cdc42 targets MLK3 to cellular membranes, leading to the full activation of MLK3 and signaling to JN K. These data from our lab and other labs help to establish the current working model for Cdc42-induced MLK3 activation. Binding of the activated form of Cdc42 is proposed to disrupt the autoinhibitory conformation of MLK3, promoting the homo-dimerization of MLK3 which leads to the trans-autophosphorylation of MLK3 on its activation loop. The membrane targeting of MLK3 induced by Cdc42 further promotes its full activation as well as the downstream signaling to JNK. Since Rae/Cdc42 promote MLK3 activation, the GEFs specific for these GTPases may regulate MLK3 as well. Vav, a GEF for Rho-GTPases, is demonstrated to increase MLK3 activity as judged by MLK3 autophosphorylation and substrate phosphorylation. Surprisingly, Vav also interacts with MLK3 through at least two distinct binding sites within MLK3, indicating that it regulates MLK3 activity not only through its GEF activity, but also by forming a Vav/GTPase/MLK3 signaling complex. Furthermore, the MLK inhibitor, CEP-l 1004, dramatically blocks JNK activation induced by ectopically expressed Vav, suggesting that MLK family kinases are‘involved in Vav-induced JNK activation. However, the specific disruption of MLK3 expression by siRNA does not impact JNK activation induced by ectopic expression of Vav in HeLa cells. Finally, the Appendix describes the generation of a cell line to inducibly express MLK3 in the human breast cancer cell line, MCF-7. Flag-MLK3 signaling complexes were isolated from this cell line by affinity purification, and were subjected to liquid chromatography/namelectrospray tandem mass spectrometry (LC/MS/MS) analysis to identify proteins in F lag-MLK3 signaling complexes. iv T o my family ACKNOWLEDGMENTS I would like to express my gratitude to my mentor, Dr. Kathy Gallo, for providing me the opportunity to study at Michigan State University. This research work would not be possible without her guidance, her patience and her encouragement. Profound appreciation goes to all my committee members Dr. Susan Conrad, Dr. Laura McCabe, Dr. Cindy Miranti and Dr. William Spielman for their stimulating discussions, advice and support. I also would like to thank every member of Dr. Gallo’s Lab, including Dr. Hua Zhang, Mary Chao, Karen Schatcher and Stancy Liou, for their valuable discussion, support and friendship. I would like to acknowledge Dr. Shirley Owens of the Center for Advanced Microscopy at Michigan State University for her expert assistance with confocal microscopy. 1 thank members of Michigan State University Proteomics Facility for their collaboration. 1 cannot have today without my family. I am grateful to my parents and brothers in China for their unconditional love and encouragement in all of my lifetime. My deepest gratitude belongs to my beloved wife, Xiaoping Cui, for her love, encouragement and her faith in me. TABLE OF CONTENTS Page List of Figures ............................................................................ viii Key to Abbreviations............................................... ................................. ix Chapter 1. Literature Review ............................................................. 1 l.Protein kinases ...................................................................... l 2. The Mitogen activated protein kinase pathways ....................................... 3 2.1. The ERK pathway ............................................................. 4 2.2. The JNK pathway .............................................................. 5 2.3. The p38 pathway ............................................................. 8 3. Mixed Lineage Kinase family ............................................................ 8 3.1. The Members of Mixed lineage kinase family ................................ 9 3.2. Pathways regulated by MLKs ......................................... 11 3.2.1. The JNK pathway ................................................... 11 3.2.2. The p38 pathway .................................................. 11 3.2.3. The ERK pathway ................................................... 12 3.3. The regulation of MLK5 .................................................. 12 3.3.1. Regulation of MLK3 by extracellular signals ........................ 12 3.3.2. Regulation of MLK5 by phosphorylation ....................... 13 3.3.3. Regulation of MLK8 by oligomerization ........................ 14 3.3.4. Regulation of MLK3 by autoinhibition ....................... 15 3.3.5. Regulation of MLK3 by Rho GTPases ....................... 16 3.3.6. Regulation of MLK3 by scaffold proteins ....................... 17 3.3.7. Regulation of MLK5 by heat shock proteins ....................... 19 3.4. The biological functions of MLK5 in mammalian cells .............. 20 3.4.1. MLKs in neuronal apoptosis ......................................... 20 3.4.2. MLKs in cell proliferation ......................................... 21 3.4.3. MLKs in cell cycle .................................................. 21 3.5. The biological functions of MLK3 in Drosophila development ..22 3.6. MLK3 ...................................................................... 23 4. Rho family small GTPases ............................................................ 25 5. Objective of thesis .................................................................... 28 Chapter 2. Cdc42 induces activation loop phosphorylation and membrane targeting of MLK3 .............................................................................. 34 1. Abstract ............................................................................. 34 2. Introduction .............................................................................. 36 3. Materials and Methods ........................................................... 39 3.1 . Antibodies .................................................................... 39 3.2. siRNA ............................................................................. 39 3.3. Expression vectors and site-directed mutagenesis ........................ 39 3.4. Cell Lines and transfections ..................................................... 40 3.5. Subcellular fractionation .................................................... 41 vi 3.6. Cell lysis and immunoprecipitations ............................................ 41 3.7. SDS-PAGE and Western blot Analysis .................................. 42 3.8. lmmunofluorescence and confocal microscopy ......................... 42 3.9. Immune complex kinase assays ............................................ 43 4. Results ............................................................................... 45 4.1. Cdc42-induced JN K activation requires MLK3 ......................... 45 4.2. Membrane targeting-defective variants of activated Cdc42 are able to interact with MLK3 .............................................................. 45 4.3. Activated Cdc42 targets MLK3 to a plasma membrane-enriched fraction.46 4.4. Activated Cdc42 and MLK3 colocalize at the plasma membrane ..47 4.5. Requirements of MLK3 membrane targeting by activated Cdc42 ...... 48 4.6. Cdc42 induces activation Loop (auto)-phosphorylation of MLK3 .......49 4.7. Cdc42 Induces activation Loop phosphorylation independent of membrane targeting ...................................................................... 50 4.8. Membrane targeting contributes to full activation of MLK3 . . 5.1 4.9. Enhanced MLK3 signaling to JNK requires Cdc42-induced membrane targeting ....................................................................... 52 5. Discussion ............................................................................... 54 Chapter 3. Regulation of Mixed Lineage Kinase 3 by Guanine Nucleotide Exchange Factor, Vav ............................................................................... 69 l.Abstract ............................................................................... 69 2. Introduction ............................................................................... 7O 3. Materials and Methods ............................................................. 75 3.1. Plasmids ...................................................................... 75 3.2. Reagents and Antibodies .................................................... 75 3.3. Cell Culture, Transfections, and Lysis .......................................... 76 3.4. Immunoprecipitations ............................................................. 76 3.5. Gel Electrophoresis and Western Blot Analysis ......................... 76 3.6. In Vitro Kinase Assays ............................................................. 77 3.7. siRNA ............................................................................... 77 4. Results ............................................................................... 79 4.1. Vav increases the catalytic activity of MLK3 .................................. 79 4.2. Vavs associate with MLK3 .................................................... 79 4.3. Two distinct regions of MLK3 mediate binding to Vav ............... 80 4.4. Vav-induced JNK activation is largely blocked by the MLK inhibitor, CEP-11004 ...................................................................... 82 4.5. MLK3 is not required for Vav-induced JNK activation ............82 5. Discussion ............................................................................... 84 Chapter 4. Concluding Remarks ................................................... 96 References .............................................................................. 99 vii List of Figures Figure Page Chapter 1. - 1. The mammalian MAPK pathways .................................................... 29 2. Members of the human Mixed-Lineage Kinases (MLK) family . . . . .. . ...30 3. Model of Autoinhibition of MLK3 ..................................................... 31 4. Scaffold proteins that interact with MLKs ........................................... 32 5. The regulation of GTPase .............................................................. 33 Chapter 2. 1. Cdc42-induced JNK activation requires MLK3 .................................. 59 2. Construction of prenylation-defective variants of Cdc42V12 and the ability of Cdc42Vl2 variants to associate with MLK3 ........................................... 60 3. Effect of Cdc42VIZ variants on the subcellular distribution of MLK3 and MLK3 activity in plasma membrane-enriched fractions .................................. 61 4. Subcellular localization of MLK3 .................................................... 63 5. Requirements of MLK3 membrane targeting by activated Cdc42 and activation loop phosphorylation of MLK3 .................................................... 64 6. Cdc42 induces activation loop phosphorylation of MLK3 independent of membrane targeting ....................................................................... 65 7. MLK3 activity induced by Cdc42v'2 variants .................................. 66 8. Effect of Cdc42Vlz variants on MLK3-induced JNK signaling ................ 68 Chapter 3 1. Domain structure of Vav proteins and AVav ........................................... 87 2. Ectopically expressed Vav isoforms potentiate MLK3 catalytic activity in HEK293 cells ................................................................................ 88 3. Full length Vav isoforms associate with MLK3 in HEK293 cells . . .90 4. Interaction of MLK3 with Vav ..................................................... 91 5. CEP-11004 largely blocks Vav-induced JNK activation ......................... 94 6. Effect of MLK3 gene silencing on Vav-induced JNK activation ................ 95 viii AGC ANT-2 AP50 ASK] BSA CAAX CAMK CDK CK] CMGC CRIB DLK DMEM ERK , G1uR6 GST HA HEK HPKl Hsc70 I-ISP 13 Key to Abbreviations containing Protein Kinase A/G/C families adenine nucleotide translocator-2 clathrin assembly protein complex 2 medium chain apoptosis signal- regulating kinase 1 bovine serum albumin; Cys-aliphatic-aliphatic-any amino acid; calcium/calmodulin dependent protein kinases cyclin dependent protein kinases casein kinase 1 containing CDK, MAPK, GSK3, CLK families Cdc42/Rac interactive binding motif dual leucine zipper protein kinase Dulbecco’s Modified Eagle’s Medium extracellular signal-regulated kinase Kainate receptor glutamate receptor glutathione S-transferase hemagglutinin human embryonic kidney; hematopoietic progenitor protein kinase-l heat shock cognate 71 kD protein heat shock protein islet brain immunoglobulin ix J 1P JNK LC LZK MAPK MAPKKK MEK MEKK MKK MLK MS NGF NIMA PAGE PAK PBD PBS PDGF Pin 1 PKA PKC POSH PSD-95 RGC IN K interacting protein c-jun N-terminal kinase liquid chromatography leucine zipper-bearing kinase mitogen activated protein kinase MAPK kinase kinase mitogen-activated protein/ERK kinase MEK kinase MAPK kinase mixed lineage kinase mass spectrometry nerve growth factor never in mitosis A polyacrylamide gel electrophoresis p21 activated protein kinase p21-binding domain; phosphate-buffered saline; platelet-derived growth factor peptidyl-prolyl isomerase Pinl protein kinase A protein kinase C plenty of 81133 post-synaptic density protein 95 receptor guanylate cyclase RNA interference SAM SAPK SDS-PAGE 8H2 8H3 siRNA SPRK STE Syd TAKl TGF TK TKL ZAK sterile alpha motif stress-activated protein kinases SDS-polyacrylamide gel electrophoresis; Ste-homology 2 src-homology 3 small interfering RNA; SH3 domain containing proline rich protein kinase homologs of yeast Sterile 7, ll, 20 kinases Sunday driver TGFB activated protein kinase 1 transforming growth factor tyrosine kinases tyrosine kinase-like tumor necrosis factor leucine-zipper and sterile-alpha motif kinase xi Chapter 1. Literature review 1. Protein kinases Protein phosphorylation is an essential mechanism of cellular signal transduction and protein function regulation. Protein phosphorylation is catalyzed by protein kinases, a family of evolutionarily conserved enzymes that transfer the y-phosphate of ATP to certain Ser, Thr or Tyr residues of their protein substrates. In physiological settings, protein phosphorylation is reversed through the action of protein phosphatases. Protein kinases make up one of the largest protein superfamilies in eukaryotes. The human kinome consists of 5 1 8 protein kinase genes, which represent about 1.7% of all human genes[l]. Protein kinases control a variety of physiological processes, such as metabolism, cell cycle regulation, proliferation, differentiation, gene transcription, cell morphology and motility, and apoptosis. Based on substrate specificity, mammalian protein kinases are mainly divided into three major groups: the tyrosine kinases, which phosphorylate only Tyr residues within their protein substrates; the serine/threonine protein kinases, which phosphorylate Ser or Thr residues; and the dual specificity kinases, which are capable of phosphorylating Tyr, as well as Ser and Thr, residues [2, 3]. The catalytic domains of eukaryotic protein kinases are highly conserved both in their primary amino acid sequence and in the tertiary structure. They consist of approximately 250-300 amino acids, which fold into a two-lobed structure. The N- terminal lobe is smaller and is formed by several antiparallel beta sheets and one conserved helix. In the catalyzed reaction, the binding of Mg2+ and ATP occurs in the N- terrninal lobe. The C-terminal lobe structure, which is mainly composed of alpha helices, primarily functions in protein substrate recognition and binding and also initiates the phosphate transfer from ATP to the substrate [4, 5]. The sequences involved in active site stability and ATP binding are usually highly conserved among protein kinases, whereas the substrate recognition and binding residues diverge, suggesting that protein kinases share a similar catalytic mechanism while having specific substrate specificity [6]. Within the catalytic domains of each protein kinase resides the so-called activation loop, which is located between two highly conserved sequence motifs Asp- Phe-Gly and Ala-Pro-Glu [6]. The phosphorylation of certain sites within this activation loop ofien alters its conformation rendering the kinase catalytically active [7-10]. The regulatory mechanism by which protein kinases are activated through activation-loop phosphorylation is still not completely understood [10-12]. By comparing the structures of the active state to the inactive state of protein kinases, several models have been proposed including formation of a substrate binding site [12-14], regulation of the phosphoryl transfer step [1 1] and control of the active conformation [10]. Based on sequence homology of the catalytic domains, aided by sequence similarity and domain arrangement of the full proteins, and known biological functions, mammalian protein kinases are classified into nine groups: AGC (containing PKA, PKQ, PKQ families), CAMK (galeium/calmodulin dependent protein kinases), CKl (casein kinase 1), CMGC (containing CDK, MAPK, _(_}SK3, QLK families), STE (homologs of yeast Sfirile 7, 11, 20 kinases), TK (girosine kinases), TKL (tyrosine kinase-like), RGC (receptor guanylate cyclase), and atypical kinases [15, 16]. 2. The mitogen-activated protein kinase pathways The mitogen-activated protein kinase (MAPK) pathways play important roles in signal transduction networks in eukaryotic cells. MAPK pathways are involved in transducing a large variety of external signals into cells leading to corresponding cellular responses, such as gene expression, metabolism, cell division, cell morphology, inflammation, cell proliferation, differentiation, and apoptosis. MAPK substrates include transcription factors, other kinases, enzymes, and cytoskeletal proteins. MAPK pathways are organized in three-kinase modules consisting of a terminal MAP kinase (MAPK), which is activated through the phosphorylation of a specific Thr and a Tyr residue within the activation loop of the kinase by a MAPK activator, the dual specific MAPK kinase (MKK). In a similar fashion, a MKK kinase (MAPKKK) phosphorylates and activates the MK (Fig. 1). These three-tiered cascade modules are conserved from yeast to humans [17-19]. In some cases, a MAPKKK kinase (MKKKK) exists and activates the MAPKKK. Upon extracellular stimulation, the MAPKKKK or MAPKKK can ofien be recruited to the plasma membrane. Frequently, this is accomplished through the association with a membrane anchored small GTPase. MAPKs are activated through the dual phosphorylation of conserved Thr and Tyr residues within the activation loop. Once MAPKs are activated, they can either phosphorylate substrates within the cytosol or translocate into the nucleus to phosphorylate nuclear substrates including transcription factors, ultimately leading to appropriate cellular responses. MAPKs are proline-directed kinases whose consensus sequence for substrate phosphorylation is PXT/SP motif, although the first Pro residue is not strictly required [20]. MKKs (MAPKKs) have very high specificity towards their substrate MAPKs. In contrast, there are many different mammalian MAPKKKs, including Raf isoforms, MEKKl-MEKK4, and mixed lineage kinases (MLKl—MLK4, DLK, LZK and ZAK). The MAPKKKs are usually large proteins with many domains or motifs important for inter- or intra-molecular protein interactions that regulate the activity and signaling of the kinase. In mammals, there are five MAPK pathways: the extracellular signal-regulated kinases, ERKl/2 (also known as MAPKs), the c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK), the p38 kinases (p38a, p383, p387, and p386), ERK3/ERK4, and ERKS [18, 19, 21, 22]. The first three have been well studied. 2.1. The ERK pathway The Raf/MEK/ERK signaling pathway was the first MAP kinase cascade to be characterized. It is activated in response to mitogenic stimuli such as growth factors and cytokines, and is involved in regulation of many fundamental cellular processes, such as proliferation, differentiation and cell cycle progression. Depending on the strength and duration of the signal, activation of the ERKllERKZ can lead to either proliferation or differentiation[23]. Targeted disruption of the ERK] gene results in viable mice with modest defects in T-cell development, indicating ERK] is important for T cell responses [24]. ERK1/2 have a large variety of substrates that include the transcription factors such as Elk and NF -KB, kinases such as Rsk, and other structural or signaling proteins such as cell survival regulator, Bel-2, and the cytoskeletal scaffold, paxillin. The activation of ERK is directly mediated by two MKKs, mitogen-activated protein/ERK kinase 1/2 (MEKl and MEK2). Activated MEKl/2 activates ERKl/2 through the phosphorylation of Thr and Tyr within the activation loop of ERK1/2 [25]. The upstream activation of the ERK pathway at the MAPKKK level and above is much more complicated. For instance, growth factors or cytokines bind to cell surface receptor tyrosine kinases (RTKs), G-protein-coupled receptors or cytokine receptors [26]. This leads to activation of Ras. Activated Ras then recruits Raf isoforms to the plasma membrane and induces their activation [27], followed by phosphorylation and activation of MEKl/MEKZ. MAPKKKs that activate the ERK pathway include Raf-l [28], B-Raf [29] and A-Raf [30]. Additionally, the MAPKKKs, MLK3, MEKKl [31], MEKK2, MEKK3 [32], Tp12 [33] have been reported to activate ERK 1/2. Interestingly, it has been reported recently that gene silencing of mixed-lineage kinase 3 (MLK3) blocks EGF-induced B-Raf activation and cell proliferation[34, 35]. It has been proposed that MLK3 is required for maintenance of a MLK3/B-Raf/Raf-l complex[36]. Ras is mutated to an oncogenic form in about 15% of human cancers and mutations in the B-raf gene have been found in more than 60% of malignant melanomas [37]. In addition, activated MEKl/2 transforms fibroblasts and in xenografi studies, MEK-transformed fibroblasts produce tumors in nude mice [38]. Furthermore, activation of ERK2 is sufficient to transform mammalian cells [39]. All of these indicate the critical relationship between ERK pathway and cancer. 2.2. The JN K pathway The JNK family of MAPKs regulates responses to various stresses, and plays crucial roles in inflammation, proliferation, development, apoptosis and metabolism. JNKs are activated by cytokines and environmental stresses, such as heat shock, ultraviolet (UV), radiation, osmotic stress, as well as by growth factors[40, 41]. IN K isoforms are encoded by three genes (JnkI, Jnk2, and Jnk3). Ten JN K isoforms are derived from alternative splicing of the three genes. JNK] and JNK2 are ubiquitously expressed in many tissues, while JNK3 is specifically expressed in the brain [42]. Mice with targeted disruption of only one of the three JNK genes are viable, suggesting that the different JN K isoforms may have overlapping functions, but they do show defects in apoptosis and immune responses regardless of which is knocked out [21, 41]. Jnkl“ and JnkZ” double knockout mice die in early embryogenesis with an open neural tube, which is associated with decreased apoptosis in the hindbrain, and increased apoptosis in the forebrain [43]. However, JnkI'I- Jnk2+ fibroblasts were isolated from these mice, suggesting that JNK is not required for inherent cell viability. The Jnk1+ JnkZ'l- fibroblasts display defects in AP-l transcriptional activation, proliferation, and fail to undergo apoptosis in response to certain stresses such as UV light [44]. The Jnk3 - /- mice show reduced seizure activity and decreased susceptibility to hippocampal neuron apoptosis in response to excitotoxicity [45]. Nuclear substrates of JNK include c-Jun, JunD, Elk] and ATF 2. Activated JN K phosphorylates c-Jun on two sites (Ser63 and Ser73). JNK also phosphorylates anti- apoptotic members of the Bcl family, including Bel-2, Bel-XL and Bim. Two direct activators of IN K have been identified: MKK4 (also call SEKl ) and MKK7 [46, 47]. There is some evidence that MKK4 and MKK7 act synergistically to phosphorylate and activate the JN Ks. MKK4 preferentially phosphorylates Tyrl85 within the INK] activation loop, whereas MKK7 preferentially phosphorylates Thr] 83 [47]. Because dual phosphorylation of JNK on the Tyr and Thr is required for its full activation, both MKK4 and MKK7 are required to achieve the full activation of JNK. Indeed, single knockout of either Mkk4 or Mkk7 results in partial defects in stress-stimulated JN K activation, whereas disruption of both genes eliminates JNK activation [48]. In addition. it has been suggested that these two kinases mediate JNK activation in response to different stimuli; MKK7 is activated primarily by cytokines (such as tumor necrosis factor, TNF. and interleukin-1, lL-l), whereas MKK4 is primarily activated by environmental stresses [2 l ]. When one of these two kinases is preferentially activated by certain stimuli, the basal activity of the other kinase seems to be required for full activation of IN K. For example, targeted disruption studies demonstrate that MKK7 is essential for TNF-stimulated JNK activation whereas MKK4 knockout only results in a partial decrease in JNK activation[48], indicating that TNF-induced .lN K activation is through MKK7, but the basal activity of MKK4 is required for full activation of JNK in response to TNF. The regulation of the JNK pathway at the MAPKKK level is much more complex due to the existence of at least 13 MAPKKKs, including MEKKl-MEKK4 [49], apoptosis signal- regulating kinase 1 (ASKl) [50], transforming growth factor (TGF) [3 activated protein kinase ] (TAKl) [5]], and the MLKs [52]). 2.3. The p38 pathway The p38 MAPKs are activated by numerous physical and chemical stresses, including the cytokines interleukin-1 and tumor necrosis factor, hormones, UV irradiation, ischemia, osmotic shock and heat shock [53]. The p38 MAPK pathway appears to play an important role in apoptosis, cytokine production, transcriptional regulation, and cytoskeletal reorganization. The availability of specific p38 inhibitor, SB 203580, helps to clarify the role that p38 MAPK plays in these processes. Inhibition of p38 by SB 203580, blocks interleukin-2 and -7 (IL-2 and lL-7) induced T cell proliferation. Four p38 isoforms exist: p38a, p383, p387, and p385. Most studies have been performed on p3 8a. Disruption of the p3 8a gene in mice causes embryonic lethality between embryonic days 1 1.5 and 12.5. Those mice which develop past this stage have normal morphology but are anemic due to failure in erythropoiesis [54, 55]. These findings suggest that different p38 isoforms have nonredundant biological fimctions. There are only two MAPKKs, MKK3 and MKK6, that can activate p38 MAPKs. In contrast, numerous MAPKKKs have been demonstrated to participate in p38 pathway regulation, including TAK] [56], ASK] [50], MEKK 4, MLK2, MLK3, and DLK [52]. 3. The mixed lineage kinase family Mixed lineage kinases (MLKs) are a family of widely expressed, serine/threonine protein kinases that act as MAPKKKs. The name “Mixed Lineage Kinase” derives from the sequence similarity of its catalytic domain to both tyrosine and serine/threonine protein kinases [57]. Experimentally, however, only serine/threonine kinase activity has been demonstrated [58]. MLKs have mainly been implicated in the activation of IN K pathway by phosphorylation and activation of the dual specific kinases MKK4/7 [41, 59- 61], and of the p38 pathway through the phosphorylation and activation of MKK3/6[62, 63]. Interestingly, in addition to its function as a MAPKKK in the activation of IN K and p38 pathways, a recent report suggests that MLK3 is involved in mitogen-stimulated B- Raf activation and cell proliferation in cancer cell lines, by forming and maintaining MLK3/B-Raf/Raf-l complex. In mammalian cells, MLKs have been implicated in regulating many physiological processes, including apoptosis, proliferation and differentiation. Studies using small molecule MLK inhibitors implicated MLKs in neuronal apoptosis. The MLKs are therefore potential therapeutic targets for many neurodegenerative diseases such as Parkinson’s disease [52]. 3.1. The Members of Mixed lineage kinase family. According to their domain organization and sequence similarity within the catalytic domains, MLKs can be clustered into three subfamilies: the MLKs; the dual leucine zipper bearing kinases (DLKs); and zipper sterile-a-motif kinase (ZAK) (Fig.2). The MLK subfamily consists of MLK1 , MLK2, MLK3, and MLK4. In addition to the catalytic domain, MLKs contain several conserved domains that are important for inter- or intra- molecular interactions, including an amino terminal Src homology 3 (SH3) domain, a centrally located leucine zipper region and a Cdc42/Rac-interactive binding (CRIB) motif. MLK1-4 share more than 75% sequence identity within their catalytic domains. However, the COOH-terminal regions of MLK8, which are rich in proline, serine and threonine, are highly divergent, indicating that this region might be involved in regulating isoforrn specific functions of MLK. MLK] mRNA and/or protein expression have been detected in epithelial tumor cell lines of colonic, breast and esophageal origin [57], and in an immature B-cell line (RIN-SAH), but not in the more mature RIN-A12 cells, suggesting that MLK] may be involved in the differentiation of B-cells [64]. MLK2 mRNA is present in brain, skeletal muscle and testis [65, 66], whereas MLK3 transcripts are detected in wide variety of adult and fetal human tissues [58]. The DLK subfamily contains DLK (also called zipper protein kinase (ZPK) [67]) and leucine-zipper kinase (LZK). DLKs lack an SH3 domain and CRIB motif. Follow the catalytic domain of the DLKs are two consecutive leucine-zipper motifs separated by a 31 amino acid spacer. Like MLKl-4, the COOH-terminal regions of DLK and LZK are proline rich, but lack significant sequence similarity. DLK mRNA is present at high levels in the brain of adult mice and also in the epithelial layers of various organ systems, including stomach, small intestine, liver, pancreas, and the seminiferous tubules of mature testes [67, 68]. Based on northern blot analysis, LZK is widely expressed, with the highest expression level in the pancreas [69]. The third subfamily, ZAK, contains a leucine zipper and a sterile-or motif (SAM) [70-72]. There are two alternatively spliced isoforms of ZAK (ZAKa and B) [71, 72]. MLKs are absent from yeast, but MLK homologues are found in Drosophila and C. elegans [52]. The Drosophila MLK Slipper (Slpr) contains a similar domain arrangement as MLKl-4 including a SH3 domain, a kinase domain, a leucine zipper and a CRIB motif, and shows significant sequence similarity. Slipper is required for 10 basket/JNK activation during dorsal closure. C. elegans has DLK and ZAK homologues [52]. 3.2. Pathways regulated by MLKs. 3.2.1. The JN K pathway. When ectopically expressed in mammalian cell lines, MLK2 [6]], MLK3 [59], DLK [73], LZK [69], and ZAK [70, 7]] have all been shown to activate JNK. The data presented in Chapter 2 demonstrates that knockdown of the MLK3 expression by siRNA blocks Cdc42-induced JN K activation, suggesting that Cdc42 regulates endogenous JNK through endogenous MLK3. A number of in vitro experiments have been performed to assess the ability of different MLKs to phosphorylate and activate MKK4/7. MLK2 and MLK3 phosphorylate both MKK4 and MKK7[59, 61]. In contrast, DLK phosphorylates and activates MKK7, but not MKK4[73], suggesting that DLK regulates JNK activation solely through MKK7. Co-expression of dominant-negative MKK7 but not MKK4 dramatically blocks ZAK induced JN K activation, indicating that ZAK activates JN K pathway primarily through MKK7[74]. 3.2.2. The p38 pathway. The importance of MLK5 in the activation of p38 pathway is not defined. Upon overexpression, MLK2, MLK3, DLK and ZAKB have been shown to modestly activate the p38 pathway[59, 72, 75, 76]. Unpublished data from our lab show that ectopically expressed MLK3 dramatically activates both JN K and p38 in human embryonic kidney (HEK) 293 cells, but induces the activation of JNK, but not p38, in HeLa cells. MLK3’s ll role in p38 activation might be cell-type dependent. Indeed, J 1P2 (JNK Interacting Protein 2), which is specifically expressed in brain, has been shown to associate with MLK3, MKK3 and p38 to facilitate MLK-mediated activation of p38 [77, 78]. It should be note that these data also relied on ectopic expression. 3.2.3. The ERK pathway. The function of MLK5 in ERK activation is controversial. In certain cell lines, overexpression of MLK2 and ZAK, but not DLK or MLK3, has been shown to activate ERK [59, 71, 72, 75, 76]. However, MLK3 has also been shown to contribute to ERK pathway activation as well [79]; and recent reports have demonstrated that gene silencing of MLK3 using siRNA blocks B-Raf-mediated ERK activation and proliferation [34, 35]. Interestingly, the activity of MLK3 is not required for B-Raf activation, but rather instead of functioning as a MAPKKK, MLK3 is involved in mitogen stimulated B-Raf activation and cell proliferation, by forming and maintaining a MLK3/B-Raf/Raf-1 complex [36]. 3.3 The regulation of MLKs 3.3.1. Regulation of MLK3 by extracellular signals The extracellular stimuli that activate endogenous MLKs in mammalian cells are not completely known. Tumor necrosis factor a (TNF-a) activates endogenous MLK3 in Jurkat T lymphocytes [80] and in the human breast cancer cell line (MCF-7) [81], as judged by in vitro kinase assays using recombinant GST-MKK4/7 as substrates. Consistent with these finding, MLK3 -/- murine fibroblasts display decreased TNF- 12 induced JNK activation. Cerarnide is also reported to increase endogenous MLK3 activity in vivo and in vitro [80]. Finally, transforming growth factor beta (TGF-l3), as well as camptothecin, has shown to induce the phosphorylation of MLK3 in hepatoma cell lines and HeLa cells, respectively [82, 83]. 3.3.2. Regulation of MLK3 by phosphorylation The majority of protein kinases are regulated by phosphorylation. Within the catalytic domain of protein kinases resides an activation loop, which often contains highly conserved regulatory phosphorylation sites. Phosphorylation within the activation loop alters its conformation rendering the kinase catalytically active [7-10]. Indeed, site- directed mutagenesis studies suggest that phosphorylation of Thr277 and Ser28l within the activation loop of MLK3 is critical for its activity [84]. Because the residues analogous to Thr277 and Ser281 are conserved in the activation loops of all mammalian MLKs, the other MLKs may be regulated by activation loop phosphorylation as well. A phosphospecific antibody directed against the phosphorylated activation loop- phosphorylation sites of MLK3 (pThr277/pSer281) serves as a valuable tool to assess MLK3 activation status. In vivo labeling coupled with mass spectrometry has revealed eleven phosphorylation sites within activated MLK3, most of which are clustered at the COOH- terminus [85]. Seven of these eleven identified sites are serines followed immediately by a proline, and thus correspond to the consensus motif for phosphorylation by proline- directed kinases. Indeed, recent data from our lab has demonstrated that activated JNK phosphorylates MLK3 in a positive feedback-loop to regulate MLK3’s distribution to 13 Triton X-100 soluble fractions of cells [86]. IN K can phosphorylate overexpressed MLK2 at its COOH terminal region in vitro as well [87]. Finally, MLK3 is reported to be negatively regulated by Akt phosphorylation [88]. 3.3.3. Regulation of MLK3 by oligomerization Protein oligomerization is often involved in protein kinase regulation. For instance, receptor tyrosine kinases are activated through growth factor induced dimeriztion and transautophophorylation [89]. All MLKs contain some type of leucine zipper domain, suggesting that leucine zipper-mediated oligomerization might be important in regulating MLKs. No evidence for heterodimerizations of MLK3 currently exists [52]. Indeed, formation of disulfide-linked MLK3 dimers requires its leucine zipper region and deletion of the leucine zipper domain abolishes its ability to autophosphorylate and to activate the IN K pathway [90]. Further data suggests that zipper-mediated homo-oligomerization is required for full activity of MLK3, proper dual phosphorylation of the downstream MK, and subsequent activation of the JNK pathway [91]. The leucine zipper of DLK mediates its homodimerization, which is important for its phosphorylation, activation and stimulation of the JNK pathway [92]. Enforced dimerization of DLK using an engineered dimerization strategy is sufficient to induce DLK autophosphorylation and JN K activation [93]. Consistent with the model that homodimerization of DLK promotes its transphosphorylation and activation, overexpression of the DLK leucine zipper effectively inhibits the dimerization and activation of full-length DLK, presumably by forming inactive leucine zipper-DLK hetero-complexes [93]. LZK has been reported to form dimers as well, and deletion of a zipper from LZK prevents the induction of JNK activity [94], suggesting its leucine- zipper domain might have the same function as that in other MLKs. In summary, the model, in which leucine zipper-mediated dimerization/o]igomierization leads to trans- autophosphorylation and subsequent JN K activation, most likely applies to all MLKs. 3.3.4. Regulation of MLKs by autoinhibition Most protein kinases, including the MLKs, contain multiple regulatory domains and motifs, which not only are critical in interacting with other signaling proteins, but also may be involved in intramolecular interactions to regulate catalytic activity. For example, PKC (protein kinase C) contains a pseudosubstrate autoinhibitory domain, which binds the active site of PKC kinase domain, therefore blocks substrates access [95, 96]. Another well known example of intramolecular autoinhibition is tyrosine kinases of the Src family. The SH2 domain of Src associates with a COOH-terminal phosphorylated Tyr, and the SH3 domain of Src binds to a sequence located between the SH2 and kinase domains. Src is kept in an autoinhibitory inactive state by these two intramolecular interactions, because these interactions restrict the movement of the kinase domain of Src, thus block the binding of ATP and substrates [97, 98]. MLK3 is autoinhibited through an interaction between its SH3 domain and a short proline-containing sequence located between its leucine zipper and the CRIB motif (Fig. 3) [99] . Mutation of either the conserved Tyr52 in the SH3 domain or the single proline residue that involved in SH3 binding disrupts the autoinhibition leads to increased catalytic activity of MLK3. The sole proline residue in the SH3-binding region of MLK3 15 is conserved in MLK] , MLK2, MLK4 and the Drosophila MLK, Slpr, suggesting that SH3-mediated autoinhibition is a common regulatory mechanism among these kinases [52, 99]. 3.3.5. Regulation of MLK3 by Rho GTPases Rho family GTPases regulate MAPK pathways. Like Ras, Rho GTPases act as binary switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) conformation [100, 101]. Only in the active GTP-bound state can these GTPases associate with their downstream effector proteins. The Rho family of small GTPases, which includes Rho and Rac isoforms, Cdc42, and TCIO [102], are involved in regulating many eukaryotic processes, such as proliferation, gene transcription, cellular transport, cell motility and cytoskeletal remodeling [103]. Constitutively active forms of Rae and Cdc42 have been shown to activate the JNK and p38 pathways [104]. _ MLK1-4 contain a so called Cdc42/Rae interactive binding (CRIB) motif. The CRIB motif is composed of 14-16 amino acids, eight of which are consensus residues that mediate the binding of GTP bound-Cdc42/Rac [105]. The CRIB motif of MLK3 contains six of the eight conserved residues, and the activated forms of Cdc42 and Rac have been shown to interact with MLK3 in a CRIB motif-dependent manner to increase MLK3’s autophosphorylation and substrate phosphorylation activity [105-107], and to potentiate MLK3-induced activation of JNK [63, 106, 107]. In addition, it has been suggested that activated Cdc42 promotes dimerization of MLK3 [93, 108]. The detailed mechanism through which MLK3 is activated by GTPases will be addressed in Chapter 2. 3.3.6. Regulation of MLK3 by scaffold proteins By assembling signaling complexes, scaffold proteins often facilitate and provide specificity for the signal transduction of MAPK pathways. Scaffolds may not only target the MAPK modules to different subcellular loci, but increasing evidence supports the idea that scaffolds contribute to kinase activation and/or substrate selection. For example: the scaffold protein KSR (Kinase Suppressor of Ras) links the three core kinases of Raf, MEK, and ERK to Ras, thereby facilitating ERK activation [109]. Other scaffold proteins involved in the activation of the ERK pathway include B-arrestin1/2 and MP-l [110]. JIPs (JNK Interacting Protein) are a group of scaffold proteins that can interact with components of the JNK and/or p38 signaling pathway to activate these pathways. In mammalian cells, four JIP proteins have been identified: JIP] , JIP2, JIP3, and JIP4 (fig. 4). JIP] is ubiquitously expressed, while JIP2 is specifically expressed in brain [78]. JIP3 and the newly identified JIP4 are most closely related based on their primary sequence and are structurally distinct from JIP1/2. JIP3 is selectively expressed at high levels in brain and at low levels in heart and testis [111]. In contrast, JIP4 is widely expressed in many tissues including brain, kidney, liver, heart, testis and other tissues[1 12]. .IIP] has been demonstrated to bind several MLKs, including MLK2, MLK3, DLK and LZK [78, I 13]. MKK7, but not MKK4, has been shown to interact with HP] [113], indicating that JIP] may regulate JNK activation specifically through MKK7. Another report suggests that JIP] regulates DLK activation by preventing its oligomerization, because JIP] bound DLK is monomeric, unphosphorylated and inactive, and the recruitment of JNK to HP] promotes the dimerization, phosphorylation and activation of JIP-associated DLK [93]. Originally JIP2 was reported to bind JNKI but its affinity is relatively weak[78]. Subsequently it was shown that JIP2 interacts with p38 and apparently functions as a scaffold protein in MLK-mediated activation of p38 pathway. JIP2 has been shown to associate with MLK3, MKK3 and p38 [77, 78]. JIP2 has also been shown to associate with the upstream activators of the p38 pathway, including Tiaml and Ras-GRF ] , which are Rae-specific guanine nucleotide exchange factors [77]. Expression of JIP2 potentiates Tiaml- or Ras-GRFI- induced p38 activation [77]. JIP3 and JIP4 are structurally distinct from HP] and JIP2. Still, both of them contain a JNK binding domain [112]. JIP3 interacts with MLK3, MKK7 and multiple JNK isoforms to facilitate INK signaling [1 1 1]. However, even though JIP4 associates with JN K, it does not bind MKK7 or MLK3 and does not facilitate the activation JNK pathway. In contrast, I 1P4 interacts with p38 MAP kinase to potentiate the activation of the p38 pathway [1 12]. Interestingly, none of the MAPKKs that activate JNK or p38 kinase (MKK4/7 and MKK3/6) has been shown to interact with JIP4. However, MKK3/6 are required for the regulation of p38 by JIP4, because JIP4 fails to activate p38 in Mkk3—/— Mkk6—/— compound mutant fibroblasts [1 12]. All JlPs interact with kinesin light chain, a component of the microtubule motor protein kinesin-1[78, 1 12], indicating that JIPs might serve as the loading dock for JNK signaling components moving along microtubules and cargoes for kinesin-mediated 18 transport [1 14]. Indeed, genetic studies showed that the Drosophila homologue of JIP3, named as Sunday driver (Syd), is important for kinesin-dependent axonal transport [115]. POSH (Plenty of SH3s) is another scaffold protein involved in the regulation of the W K pathway (fig. 4). Coimmunoprecipitation and pulldown assays demonstrated that POSH interacts directly with activated Racl, MLK1-3 and DLK, and indirectly with MKK4/7 and JN K, suggesting that POSH may act as a scaffold for Rac-mediated JN K activation. Furthermore, POSH-induced apoptosis in P012 cells can be blocked by dominant negative forms of MLKs, MKK4/7 or c-Jun, and by the MLK inhibitor, CEP- 1347 [116]. Finally, Akt2 has been reported to associate with POSH and negatively regulate assembly of the POSH/MLK/MKK/JNK signaling complex[l 17]. 3.3.7. Regulation of MLKs by heat shock proteins Heat shock proteins (HSPs) are ubiquitously expressed highly conserved molecular chaperones that are involved in many cellular functions such as protein folding, assembly, secretion, trafficking and maintaining protein homeostasis. Many heat shock proteins interact with multiple key components of signal transduction pathways [118]. Hsp90 and its kinase-specific co-chaperone p50°d°37 have been identified as MLK3 associating proteins by affinity purification coupled with mass spectrometry [8] ]. Further experiments demonstrate that endogenous MLK3 complexes with Hsp90 and p50‘d‘37 and the Hsps interact through the catalytic domain of MLK3 independent of MLK3 activity. Treatment of MCF-7 cells with the Hsp90 inhibitor, geldanamycin, l9 dramatically decreases MLK3 protein levels in cell lines, suggesting that MLK3 is stabilized through association with Hsp90. 3.4. The biological functions of MLK3 in mammalian cells. 3.4.1. MLKs in neuronal apoptosis. Nerve growth factor (N GF ) withdrawal-induced apoptosis of superior cervical ganglion (SCG) sympathetic neurons requires both the activity of the small GTPase Cdc42 and the activation of .IN K pathway [119], indicating that CRIB motif containing MLKs might be involved in NGF deprivation-induced apoptosis. Indeed, ectopic expression of MLK family members (MLK1-3 or DLK) induces apoptosis in neuronal- like PC 12 cells and sympathetic neurons. Furthermore, NGF deprivation-induced apoptosis can be blocked by expression of dominant negative forms of MLKs [120, 121] or by the MLK inhibitor, CEP-l347 [116]. Another study demonstrated the post-synaptic density protein (PSD-95) associates with MLK2 and MLK3 in HN33 cells and rat brain preparations. This association, which requires the SH3 domain of PSD-95, recruits MLK2/3 to kainate receptor glutamate receptor 6 (GluR6) complex [122]. Co-expression of catalytically inactive forms of MLK2 or MLK3 significantly blocks JN K activation and neuronal apoptosis mediated by GluR6/PSD-95. Co-expression of a mutant form of PSD-95, in which its SH3 domain has been deleted, also inhibits GluR6-induced JNK activation and neuronal toxicity, suggesting that MLK2/3 are involved in GluR6-mediated JNK activation and neuronal apoptosis. 20 Finally, apoptosis of hippocampal neurons induced by gplZO, the major coat protein of the HIV-1 virus, can be inhibited by CEP-1347 [123] . Overexpression of MLK3 in hippocampal pyramidal neurons enhances gpl2OIIIB-induced neuronal apoptosis, whereas overexpression of kinase dead form of MLK3 blocks gpl2OIIIB- induced toxic effects. These data indicate that MLKs may be involved in gpl2OIIIB- induced neuronal apoptosis and are potential targets for inhibiting AIDS-related dementia [1 23]. 3.4.2. MLKs in cell proliferation MLKs have been largely implicated in JNK-mediated apoptosis of neuronal cells. However, recent reports reveal that MLK3 might also contribute to ERK pathway activation and proliferation [36, 79]. Knockdown of MLK3 using siRNA blocks B-Raf- mediated ERK activation and proliferation [34, 35]. Further experiments demonstrated that catalytic activity of MLK3 is not required for its ability to regulate B-Raf activation. Instead, it is proposed that MLK3 regulates B-Raf and ERK activation is through forming a MLK3/B-Raf/Raf-1 signaling complex. Interestingly MLK3, B-Rafl, and Raf] are all chaperoned by Hsp90/Ccd37, suggesting that these MAPKKKs may form multiprotein signaling complexes and that removal of a component may result in disassembly of the entire complex. These new findings also suggest that there might be some cross talk between ERK and W K pathway to regulate the balance between survival and apoptosis. 3.4.3. MLKs in cell cycle. Based on homology of the noncatalytic region, MLK3 was identified as a NIMA (never in mitosis A) like protein kinase [124]. NIMA is a fungal Ser/Thr kinase that is 21 involved in regulating G2/M transition during cell cycle regulation [124, 125]. During the G2/M phase, MLK3 is localized to the centrosome and has increased phosphorylation and activity. However, JN K activity remains low during the cell cycle, suggesting that MLK3 may function in a JNK-independent manner to regulate cell cycle progression. In addition, when overexpressed, MLK3 localizes to the centrosomal region, and induces profound disruption of cytoplasmic microtubules and a nuclear distortion phenotype [124]. It is also reported that cells treated with the MLK inhibitor CEP-l 1004 are defective in exiting mitosis, due to disruption of mitotic spindle formation[]26]. The overexpression of MLK3 can partially rescue the CEP-11004-induced accumulation of cells in G2/M. Expression of ZAK has been reported to increase the population of cells in GZ/M phase [72, 74], whereas overexpression of catalytically inactive ZAK attenuates y- radiation-induced GZ arrest [72], suggesting that ZAK, like MLK3 is involved in cell cycle regulation. 3.5. The biological functions of MLKs in Drosophila development. The Drosophila MLK, called Slipper (Slpr), is critical for the JNK-dependent process of dorsal closure during fly embryogenesis [127]. Dorsal closure is the process in which the dorsal ectodenn moves from a lateral position to the dorsal midline and ultimately fuses at the dorsal midline to enclose the embryo in a continuous protective epidermis [I28]. Genetic studies of the dorsal closure process in flies led to identification of a complete MAPK pathway composed of the small GTPase dRacI, an MKKKK called Misshapen (Msn), the MKK7 homologue Hemipterous (Hep), the JNK homologue 22 Basket (Bsk) and the transcription factors dim: and dFos [129]. Slpr functions at the downstream of dRacI and Msn, and upstream of hep and bsk in the Drosophila JNK pathway [127]. The slpr mutant embryos fail to maintain the proper cell shape within the dorsal epithelium resulting in a dorsal open cuticle phenotype. These data suggest a role for MLKs in controlling cell morphogenesis and development. 3.6. MLK3 MLK3, formerly called SH3 domain-containing proline-rich protein kinase (SPRK) and protein tyrosine kinase 1 (PTK-l), is a serine/threonine kinase consisting of 847 amino acids, with a predicted molecular weight of 93 kDa [58, 130, 131]. MLK3 mRNA is widely expressed in various tissues [58]. MLK3 contains multiple domains and motifs that may be playing regulatory roles, including a unique NHz-terminal glycine-rich region, followed by an SH3 domain and the catalytic domain, a centrally located leucine zipper followed by a CRIB motif and a stretch of basic amino acids. The COOH-terminal 220 amino acids of MLK3 are rich in proline (24%), serine (12%) and threonine (13%) [58]. In addition to activating JNK, ectopically expressed MLK3 in certain experimental settings has also been shown to activate ERK, p38 and NF-KB pathways [59, 63, 73, 132, 133]. MLKs have been implicated in promoting apoptosis and the MLK inhibitor, CEP-1347, partially rescues cells from neuronal apoptosis induced by nerve growth factor withdrawal [52, 121]. Recent evidence suggests that MLK3 may play critical roles in B-Raf-induced ERK activation and, therefore, cell proliferation, by 23 serving as a scaffold protein to hold together a MLK/B-Raf/Raf-l complex that is required for Raf transphosphorylation and activation [34-36]. Our lab has focused on understanding the mechanisms that regulate MLK3 activity and signaling. Zipper-mediated homo-oligomerization is required for full activation and proper substrate phosphorylation of MLK3 [91, 108]. Work from our lab indicates that MLK3 is autoinhibited through an intramolecular interaction between its SH3 domain and a short proline-containing sequence located between its zipper and CRIB motif (fig. 3) [99]. In accord with this model, mutation of a conserved tyrosine residue within the SH3 domain or mutation of the proline residue involved in the interaction disrupts the autoinhibition and results in increased MLK3 activity. Phosphorylation is also important in the regulation of MLK3. Site-directed mutagenesis indicates that phosphorylation of Thr277 and Ser28l within the activation loop is critical for MLK3 activity [84]. In addition, MLK3 is reported to be negatively regulated by Akt phosphorylation [88]. Finally, MLK3 has a large COOH-terminus that is rich in proline, serine and threonine. Previous data from our lab has revealed multiple phosphorylation sites of MLK3, most of which are clustered at the COOH-terminal region [85]. Recent work from our lab suggests JNK can phosphorylate MLK3 in vitro and in vivo. Inhibition of JNK results in a hypophosphorylated form of MLK3, which is inactive, and redistributes to a Triton X-lOO-insoluble fraction. Recovery of JNK activity leads to phosphorylation of MLK3, which restores its solubility and activity [86]. The upstream activators of MLK3 are less defined. Activated forms of the small GTPases Cdc42 and Rac interact with MLK3 through the CRIB motif, increase 24 autophosphorylation and substrate phosphorylation activity of MLK3 [105-107] and potentiate MLK3-induced activation of JNK [63, 106, 107]. However, the detailed mechanism of how GTPases activate MLK3 is still incomplete. 4. Rho family small GTPases Rho (Ras-homologous) family GTPases are key regulators of numerous cellular functions, including morphogenesis, polarity, motility, vesicular transport, endocytosis, and cell cycle progression [134-138]. They belong to a subfamily of the superfamily of Ras-related small GTPases and are found in all eukaryotic cells. In mammalian cells, 22 Rho GTPases have been identified, including Rho isoforms A, B, and C; Rac isoforms 1, 2, and 3; Cdc42, RhoD, Rnd], Rnd2, RhoE/Rnd3, RhoG, TCIO, and TCL; RhoH/TT F; Chp and Wrch-l; Rif, RhoBTB], and 2; and Miro-1 and 2 [139, 140]. Like other GTPases, Rho GTPases act as binary switches cycling between an inactive GDP-bound state and an active GTP-bound state (Fig. 5) [100, 101, 140]. Only in the GTP-bound active state, do these GTPases adapt a conformation that allows them to bind downstream effectors to transmit signals. To date, over 50 effectors have been identified for Rho, Rac, and Cdc42, including kinases, lipases, oxidases, and scaffold proteins [140]. Structural analysis suggests that the binding of the small GTPase usually causes conformational changes in the effector proteins to release the effector from a usually closed, inactive conformation [14]]. However, it is possible that GTPases might also regulate the subcellular localization of effectors by targeting them to specific locations. The GTPase switch is regulated by three types of proteins: guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPS), and guanine nucleotide 25 dissociation inhibitors (GDIs). GEFs catalyze the exchange of GDP for GTP to generate the activated form of the GTPase. GAPs stimulate the intrinsic GTPase activity of Rho family members to inactivate the switch. Finally, GDIs interact with the prenylated, GDP-bound form of GTPase to block spontaneous activation of the GTPases and to regulate GTPases cycling between membrane and cytosol [142]. Since Ras is the most extensively characterized GTPase, the knowledge from Ras has been applied to study other GTPases. Ras is an oncogene which is mutated in about 30% of human tumors [6]. Oncogenic forms of Ras are created by point mutations that results in amino acid substitutions at position 12, 13 or 61, which render Ras insensitive to GAPS and thus unable to hydrolyze GTP, resulting in a constitutively activated GTPase [143, 144]. Ras G12V is the most common one among these mutations. A so- called “dominant negative” form of Ras, generated by substitution of Ser17 with Asn, is inactive due to its preferential binding of GDP. Based on sequence and structural homology, the analogous amino acid substitutions have been made for many other small GTPases, including Rae and Cdc42, in order to generate constitutively active or inactive forms of GTPases; and these GTPases variants have become indispensable tools in studying the signaling of these GTPases. Like all members of the Ras superfamily of GTPases, Rho family GTPases are able to associate with cellular membranes through the posttranslational prenylation of the Cys residues of their COOH-terminal CAAX (Cys-a1iphatic-aliphatic-any amino acid) motifs [145, 146]. Protein prenylation is a type of lipid modification in which either a 15-carbon famesyl or 20-carbon geranylgeranyl lipid is attached to the Cys of the CAAX via a thioester bond. This process is catalyzed by one of the two prenyltransferases 26 (famesyltransferase or geranylgeranyltransferase). The residue in the X position of the CAAX motif determines whether a famesyl or a geranylgeranyl lipid group will be attached[146]. If the X is a leucine or a methionine residue, a geranylgeranyl group is added. After the prenylation, the AAX residues are cleaved by a specific protease. The prenylcysteine at the new COOH terminus is then recognized by a prenylcysteine carboxyl methyltransferase (chMT) that methylesterifies the a carboxyl group [146]. A second signal for membrane localization, found in the so-called hypervariable region immediately upstream of the CAAX motif, is often present as well. It typically contains either palmitoylation sites [147] or a series of basic residues [148]. For example, in N-Ras or H-Ras, the hypervariable region immediately upstream of the CAAX motif contains two cysteine residues, which can be reversibly modified by palmitic acid via a labile thioester linkage to generate a hydrophobic COOH terminus [145, 147]. In contrast, the hypervariable region in K-Ras does not have the palmitoylation sites; instead it contains a polybasic sequence, serving as the second membrane targeting signal [148]. Similar to K-Ras, Cdc42 contains four basic residues in its COOH-terminal hypervariable region, and Rac has six basic residues. Prenylation within the CAAX motif coupled with the secondary palmitoylation sites or polybasic sequences in the hypervariable region is sufficient for membrane targeting of the GTPase[145]. 27 5. Objective of thesis MLK3 is an activator of multiple MAPK pathways. MLKs have been implicated in INK-mediated apoptosis in some cells including neuronal cells. However, recent evidence suggests MLK3 is also critical for ERK activation and cell proliferation. Indeed, ample levels of MLK3 are present in human cancer cell lines, including breast cancer cell lines. Therefore, understanding how MLK3 and its signaling pathways are regulated may open up new therapeutic avenues for treating not only neurodegenerative diseases, such as Parkinson’s disease (PD), but also cancers. Activated forms of the small GTPases Cdc42 and Rac interact with MLK3 in a CRIB motif-dependent manner to increase MLK3 activity [105-107] and to potentiate MLK3-induced activation of JNK [63, 106, 107]. However, little is known about the detailed mechanism by which small GTPases activate MLK3. In Chapter 2, the molecular mechanisms through which Cdc42 regulates MLK3, is investigated. Upstream stimuli/activator of MLK3 are less defined. Since GEFs are the direct activators of the small GTPases, Cdc42 and Rac, the regulation of MLK3 by one of the GEFs, Vav, is examined in Chapter 3. Finally, proteomic/mass spectrometry methods have been used to identify the components of MLK3 signaling complexes isolated from breast cancer cells. These data are presented in the Appendix. 28 MEKK1-4, TAK1,ASK1, ASKI, TAKl, ' MLKs MLK1-4, DLK ~ MKKK ; Raf, MEKK1-3, Tp12 WK“ MAPK (5116333 «1111ch :1" Cigss :V' Fig. 1. The mammalian MAPK pathways. In mammalian cells, the three well defined MAPK pathways include ERK, JNK and p38 pathways. (Adapted from [52]) See text for details. 29 SH3 Kinase LZ CRIB MLKl MLK2 [: 1" 17 1 4| D :] Gly MLK MLK3 cm MLK4a [_ r; r I - D :1 MLK4fl firm—u...— --‘ In" DLK Em DLK LZKI: F III:- :1 ZAKa L l' “'1 [:1 :1 z [ ZAK]: r:::=n:] Fig. 2. Members of the human Mixed-Lineage Kinases (MLK) family. This diagram demonstrates the relative positions of the Src-homology-3 (SH3) domain(light blue), kinase domain (red), leucine-zipper (LZ) (green) and Cdc42/Rac-interactive binding (CRIB) motif (dark blue), and the sterile-o: motif (SAM) (pink). (Adapted from [52]) 30 NHz ‘ = "rm ‘F‘J >COOH W ; CRIB fl..- 7 , ; Fig. 3. Model of autoinhibition of MLK3. MLK3 is autoinhibited through the association between its SH3 domain and a proline-containing sequence located between the leucine zipper and the CRIB motif [99]. See text for details. 31 Fig. 4. Scaffold proteins that interact with MLKs. JIPl specifically binds several MLKs, MKK7, but not MKK4 and JNK isoforms to facilitate JNK activation. JIP2 associates with MLK3, MKK3, p38 and the upstream activators of p38 pathways, including Tiaml and Ras-GRF 1. JIP2 binds to MLKs, MKK3 and p38. POSH interacts with activated Rae and MLKs directly. JIP4 interacts with p38 MAP kinase and potentiate the activation of p38 pathway. None of the MAP2Ks that activates JNK or p38 kinase (MKK4/7 and MKK3/6) interact with J 1P4. POSH interacts directly with activated Rae] , MLK1-3 and DLK, and indirectly with MKK4/7 and JNK. 32 Stimulus l GTP GDP GDP .‘f‘i’m'fit Rho (f R1100" :] W‘t‘ Effector-:1 ..aa 1 RhoGDP ' Response GDI Fig. 5. The regulation of GTPase. Rho family small GTPases act as biological switches by cycling between an inactive GDP-bound state and an active GTP-bound state. Only the active GTPases can interact with effector proteins to mediate cellular responses. The activity of GTPases is regulated by GEFS, GAPS, and GDIs. (Adapted from [140]). See text for details. 33 Chapter 2. Cdc42 induces activation loop phosphorylation and membrane targeting of MLK3 1. Abstract Mixed-lineage kinase 3 (MLK3) functions as a MAPKKK to activate multiple MAPK pathways. Our current studies demonstrate that lack of MLK3 blocks signaling of activated Cdc42 to JNK, giving strong support for the idea that Cdc42 is a physiological activator of MLK3. We show herein that Cdc42, in a prenylation- dependent manner, targets MLK3 from a perinuclear region to membranes, including the plasma membrane. Cdc42-induced membrane targeting of MLK3 is independent of MLK3 catalytic activity, but depends upon an intact Cdc42/Rae Interactive Binding (CRIB) motif, consistent with MLK3 membrane translocation being mediated through direct binding of Cdc42. Phosphorylation of MLK3’S activation loop requires MLK3 catalytic activity and is induced by Cdc42 in a prenylation-independent manner, arguing that Cdc42 binding is sufficient for activation loop autophosphorylation of MLK3. However, membrane targeting is necessary for full activation of MLK3 and maximal signaling to JNK. We previously reported that MLK3 is autoinhibited through an interaction between its N-terminal SH3 domain and a proline-containing sequence found between the leucine zipper and the CRIB motif of MLK3. Thus we propose a model in which GTP-bound Cdc42/Rae binds MLK3, disrupts SH3-mediated autoinhibition * This Chapter has been published as J Biol Chem. 2005 Dec 30; 280(52):42984-93 34 leading to dimerization and activation loop autophosphorylation. Targeting of this partially active MLK3 to membranes likely results in additional phosphorylation events that fully activate MLK3 and its ability to maximally signal through the JNK pathway. 35 2. Introduction The activation of protein kinases and the Specification of their Signaling pathways is a highly orchestrated process that is accomplished through dynamic and reversible events including phosphorylation, molecular interactions with proteins or other effector molecules, and subcellular targeting. Altered subcellular localization may impact protein kinase activation, signaling, or both. For instance, whether a protein kinase encounters an activating protein may depend upon its subcellular localization. Alternatively, many protein kinases have multiple in viva substrates and signaling pathways, and spatiotemporal localization of protein kinases provides a mechanism by which substrate and signaling specificity can be achieved. Mixed-lineage kinase 3 (MLK3) was first characterized as a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the c-Jun N-terminal kinase (JNK) pathway through the dual phosphorylation of mitogen-activated protein kinase kinases 4/7 (MKK4/7) [59, 62]. The Drosophila MLK, called Slipper, is critical for the JNK-dependent process of dorsal closure in the fly embryo [127]. MLK3-induced JNK activation is implicated in apoptosis of neuronal cells in response to trophic factor withdrawal [120, 149-151]. MLK3 also activates JNK in Jurkat T lymphocytes [80] and MCF-7 breast cancer cells [81] in response to tumor necrosis factor alpha (TNF-a) treatment. MLK3 activates the p38 pathway through phosphorylation of MKK3/6 [63], although this activity may be dependent upon the scaffold JIP2 [77, 152]. Transforming growth factor beta (TGF-B)-induced apoptosis of hepatocytes is reportedly dependent on MLK3-induced activation of p38 [83]. MLK3 also contributes to extracellular signal- 36 regulated kinase (ERK) activation [79]; and gene silencing of MLK3 using siRNA blocks B-Raf-mediated ERK activation and proliferation [34, 35]. Deciphering how MLK3 is regulated is critical to our ultimate understanding of how MLK3 integrates different MAPK signaling pathways. Autoregulatory interactions are key to controlling MLK3 activity and signaling. MLK3’S catalytic domain is flanked by an N-terminal SH3 domain and a centrally located zipper and Cdc42/Rae interactive binding (CRIB) motif. Zipper-mediated homo-oligomerization is required for full activity of MLK3, proper substrate phosphorylation, and activation of the JNK pathway [9] , 108]. Work from our lab indicates that MLK3 is autoinhibited through an interaction between its SH3 domain and a proline-containing sequence within MLK3 [99]. Phosphorylation also contributes to the regulation of MLK3. Site-directed mutagenesis data indicate that activation loop phosphorylation of Thr277 and Ser281 is critical for MLK3 activity [84]. In addition MLK3 is reported to be negatively regulated by Akt phosphorylation [88]. Finally in viva labeling coupled with mass spectrometry has revealed multiple sites of phosphorylation of MLK3, most of which are clustered at the COOH-terminus [85], whose functions are currently under study. Cdc42 and Rac are Rho family GTPases that regulate diverse cellular processes including actin cytoskeleton remodeling, vesicular transport, endocytosis, cell cycle progression, cellular transformation, motility, and cell polarity [134-138]. Like all members of the Ras superfamily of GTPases, Cdc42 and Rac are able to associate with cellular membranes by virtue of posttranslational prenylation of the Cys of the COOH- tenninal CAAX (Cys-aliphatic-aliphatic-any amino acid) motif [145, 146]. A second 37 signal for membrane localization, found in the so-called hypervariable region immediately upstream of the CAAX motif, typically contains either palmitoylation sites [147] or a series of basic residues [148] (Fig. 2). Activated forms of the small GTPases Cdc42 and Rac interact with MLK3 in a CRIB motif-dependent manner to increase MLK3’S autophosphorylation and substrate phosphorylation activity [105-107] and to potentiate MLK3-induced activation of JNK [63, 106, 107]. However little is known about how GTPases activate MLK3. Herein we provide evidence that Cdc42 induces JNK signaling through endogenous MLK3. Furthermore we Show that activated Cdc42 translocates MLK3 to membranes and induces activation loop phosphorylation of MLK3. The data presented support a mechanism whereby Cdc42 binding is sufficient for activation loop autophosphorylation of MLK3, but that prenylation-dependent, Cdc42-induced membrane targeting of MLK3, which does not, in itself, require MLK3 activity, is required for full activation of MLK3 and signaling to JNK. This work provides important insight into the molecular mechanism by which Cdc42 activates MLK3 and its signaling pathways. 38 3. Materials and Methods 3.1. Antibodies The phospho-MLK3 (Thr277/Ser281), phospho-SEKIIMKK4 (Thr26l), phospho-MKK7 (Ser271/Thr275), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyrl82), and phospho-ERK (Thr202/Tyr204) monoclonal antibodies were purchased from Cell Signaling Technology, Inc. The phospho-c-Jun (KM-1) mouse monoclonal antibody was from Santa Cruz Biotechnology, Inc. The Flag M2 monoclonal antibody and actin mouse monoclonal antibody were purchased from Sigma-Aldrich. Other antibodies used were the MLK3 rabbit polyclonal antibody [107], and horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). 3.2. siRNA The human mlk3 siRNA sequence is derived from the sequence 5'- GGGCAGTGACGTCTGGAGTTT-3' as described previously [35]. The negative control siRNA sequence is derived from the sequence 5'-GGGCAGCGACGTGTCGAGCTT—3’. HeLa cells were plated in 6-well plates and transfected with 250 nmol of siRNA oligonucleotide per well using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 20 h, transfections using F lag-Cdc42“2 expression vector were performed; and after another 20 h, cells were lysed. 3.3. Expression Vectors and Site-Directed Mutagenesis The construction of the cytomegalovirus-based expression vectors containing the cDNA for the wildtype MLK3 (pRKS-mlk3) has been described elsewhere [58]. The 39 expression plasmid construct encoding the N-terminal Flag epitope-tagged constitutively active Cdc42 (pRKS-N-Flag.cdc42m) was kindly provided by Avi Ashkenazi (Genentech, Inc.). Variants of Cdc42Vl2 containing point mutations were constructed using the Quick Change Site-directed mutagenesis method (Stratagene) using Pfic polymerase (Invitrogen) and 15 cycles of amplification. To generate pRKS-NFlag.cdc42V’2C188S, 5’-GATGTTCATAGCAGCACAGATCTGCGGCTCTTCTTCG-3’ and its reverse complement were used as primers and pRKS-NFlag.cdc42V'2 was used as the template. To substitute Lysl83, Lysl84, Arg186, Arg187 of Cdc42 with neutral Gln residues, pRKS-NFlag.cdc42V’2 C188S, was used as a template in two successive rounds of mutagenesis using the following primers and their reverse complements: 5’-CCAGAACCGAAGAAGAGCCAGAGGTCTGTGCTGCTATGAAC-3’ for the first round; and 5’-GCCTCCAGAACCGCAGCAGCAGAGCCAGCAGTCTGTGCTATGAACGCT-3’ for the second round. The presence of the desired mutations was confirmed by DNA sequencing (MSU Genomics Technology Support Facility). 3.4. Cell Lines and Transfections Human embryonic kidney (HEK) 293 cells were cultured and transfected using the calcium phosphate method as previously described [107]. HeLa cells were cultured in high glucose Dulbecco’s modified Eagle’s media containing 8% fetal bovine serum, 2 mM glutamine, and 100 U/ml penicillin/streptomycin (Invitrogen) and were transfected using Lipofectamine 2000 (Invitrogen). 40 3.5. Subcellular Fractionation Plasma membrane-enriched fractions were generated according to Stokoe et a1. with minor modifications [153]. Cells were washed twice with ice-cold phosphate- buffered saline (PBS) and disrupted in hypotonic buffer (10 mM Tris (pH 7.5) containing 35 mM NaF, 5 mM MgC12, 1 mM EGTA, 1 mM Na4PPi, 1 mM Na3V04, 100 M B- glycerophosphate, 2 mM phenylmethylsulfonyl fluoride, and 0.15 U/ml aprotinin). The cells were homogenized with 60 strokes in a Dounce homogenizer. Unbroken cells and nuclei were removed by centrifugation at 500 x g for 5 min at 4°C. Centrifugation of the supernatant at 4°C at 16,900 x g yielded a plasma membrane-enriched pellet (Pl6.9) fraction and a soluble (816.9) fraction. The pellet fractions were resuspended in lysis buffer (50 mM HEPES (pH 7.5) containing 150 mM NaCl, 1.5 mM MgC12, 2 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM NaaPPi, 10 mM NaF, 100 uM [3- glycerophosphate, 1 mM Na3VO4, 2 mM phenylmethylsulfonyl fluoride, and 0.15 U/ml aprotinin). Protein concentrations in each fraction were determined using Bradford assays according to the manufacturer’s instructions (Bio-Rad). Either equal amounts of total protein or equal cellular equivalents from the 816.9 and P16.9 fractions were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as indicated in figure legends, and Western blotting for MLK3 and Flag-Cdc42 was performed as described below. 3.6. Cell Lysis and Immunoprecipitations Cells were harvested and lysed as described previously [9]] . Clarified lysates were incubated with antibody-bound protein A-agarose for 90 min at 4 °C. 41 lmmunoprecipitates were washed three times with lysis buffer and analyzed by Western blotting. 3.7. SDS-PAGE and Western Blot Analysis Lysates and immunoprecipitates of proteins were resolved by SDS-PAGE. Proteins were transferred to nitrocellulose membranes and Western blotted using appropriate antibodies. Western blots were developed by the chemiluminescence method. Multiple exposures of the Western blots were developed, and densitometry (ImageJ, http://rsb.info.nih.gov/ij/) of unsaturated films was used to determine relative expression levels/phosphorylation. Statistics were calculated using an unpaired Student's t test. A p value less than 0.05 was considered statistically significant. 3.8. Immunofluorescence and Confocal Microscopy HeLa cells were plated onto 6-well plates containing cover slips (1 x 105 cells per well) and transiently transfected using Lipofectamine 2000 (Invitrogen). Afier transfection for 16-18 h, the cells were rinsed twice with PBS, fixed in 2% formaldehyde (Polyscience, Inc.) for 30 min at room temperature and washed three times with PBS. The cells were permeabilized with PBS containing 0.2% Triton X-100 for 10 min at 37°C and blocked in PBS containing 10% fetal calf serum (FCS), 2% bovine serum albumin (BSA) and RNAse A (100 ug/ml) for 60 min at room temperature. After incubation with the primary antibody (5 ug/ml of the MLK3 rabbit polyclonal antibody or 3.5 ug/ml Flag M2 monoclonal antibody, in PBS containing 5% BSA) for 1 h at room temperature, cells were washed three times with PBS containing 2% FCS and incubated for l h with the appropriate Alexa Fluor 488 or Alexa Fluor 546-conjugated secondary antibody 42 (Molecular Probes). After extensive washing with PBS containing 2% FCS, the nuclei were stained with TO-PRO-3 iodide (2 uM, Molecular Probes, Inc.) and mounted onto glass slides. The fluorescently labeled cells were examined with a Zeiss LSM Pascal confocal laser scanning microscope. Three separate tracks were used for capturing fluorescent images; excitation was done using 488 nm, 543 nm and 633 nm lasers with BP 500-530 nm (F lag-Cdc42, represented in Green), BP 560-615 nm (MLK3, represented in Red) and LP 650 nm (TO-PRO-3 iodide, represented in Blue) respectively as emission filters. 3.9. Immune Complex Kinase Assays For measurement of MLK3 activity in the plasma-enriched fractions, an immune complex kinase assay of the solubilized P16.9 fractions was performed. The relative amounts of MLK3 in the P16.9 fractions from cells expressing MLK3 alone or MLK3 plus Cdc42“2 were determined by densitometry (NIH image) and adjusted to ensure equal amounts of immunoprecipitated MLK3 from the P16.9 fractions. Solubilized portions of the P16.9 fractions were incubated with 20 pl of Protein A-agarose beads pre- bound with MLK3 antibody for 90 min at 4°C. The MLK3 kinase assay was carried out as described previously [91], except that 10 pg of purified GST-MKK4 was used as substrate. The extent of GST-MKK4 phosphorylation was determined by Western blotting with an anti-phospho-MKK4 antibody. For measurement of MLK3 activity in total cellular lysates, the MLK3 precipitates were washed twice with lysis buffer and twice with kinase buffer. 8 pg of recombinant, catalytically inactive GST-MKK7 K165A [81] was used as substrate, and 43 the reaction was carried out for 20 min at room temperature. The extent of GST-MKK7 phosphorylation was determined by Western blotting with a phospho-MKK7 antibody. 4. Results 4.1. Cdc42-induced JNK Activation Requires MLK3 Previous studies have demonstrated that activated Cdc42 binds MLK3 and increases its catalytic activity [106, 107]. However, these studies have relied upon ectopically expressed MLK3. To determine whether Cdc42 regulates INK Signaling through endogenous MLK3, Cdc42-induced JNK activation was measured in HeLa cells in which MLK3 expression was abolished using RNA interference[35]. AS Shown in Fig. l, Cdc42-induced JNK activation is reduced at least 5-fold upon transfection with human-specific, but not the control siRNA, indicating that MLK3 is a major physiological target of Cdc42 in signaling to JN K. 4.2. Membrane Targeting-defective Variants of Activated Cdc42 Are Able to Interact with MLK3 The COOH-terminal CAAX motif of Cdc42 serves as a target for geranylgeranylation, thus endowing Cdc42 with the ability to associate with membranes. Therefore it is tempting to speculate that prenylated, activated Cdc42 localizes MLK3 to cellular membranes. To study the potential of Cdc42-induced membrane targeting of MLK3, variants of Cdc42 that fail to undergo prenylation and/or lack the basic secondary membrane targeting motifs were constructed as diagrammed in Fig. 2A. Specifically, the prenylation site Cys] 88 in the CAAX motif of Cdc42Vl2 was mutated to Ser (F ig.2A). In yeast, this analogous change of a sulfhydryl to a hydroxyl group prevents prenylation and rescues the lethal phenotype induced by constitutively active Cdc42V12 [154]. 45 In initial fractionation experiments, the prenylation-defective mutant of Cdc42“2 C1888 was found primarily in a soluble fraction, but a small amount was still retained in a membrane-enriched fraction (data not shown). This observation suggests that, even in the absence of prenylation, the hypervariable region of Cdc42 mediates residual membrane binding, as has been shown for K-Ras [148]. Consequently the four basic residues in the hypervariable region of Cdc42, Lysl83, Lysl84, Arg186 and Arg187 were mutated to neutral glutamine residues to generate Cdc42v'2C188S, K/R4Q (Fig. 2A). Two of these residues, Lysl83 and Lysl84, are critical for endomembrane binding of Cdc42 through y-COP [155]. This variant, Cdc42v'2Cl888, K/R4Q, was present only in the soluble fraction (Fig. 3A). As has been observed for Ras [147, 148, 156], the posttranslationally modified Cdc42 migrates more rapidly than the unprenylated mutant form in SDS-PAGE. To rule out the possibility that the introduced mutations interfere with the ability of Cdc42v'2 to interact with MLK3, coimmunoprecipitation experiments were performed. 2‘"2 associate with As shown in Fig. ZB, both prenylation-defective variants of Cdc4 MLK3 to the same extent, indicating that the geranylgeranyl group and the COOH- terminal basic residues of Cdc42 are not required for association with MLK3. 4.3. Activated Cdc42 Targets MLK3 to a Plasma Membrane-enriched Fraction To determine whether Cdc42 impacts the subcellular distribution of MLK3, biochemical fractionation experiments were performed using HEK293 cells transiently expressing MLK3 and variants of constitutively active Cdc42v'2. The distribution of MLK3 between a soluble (816.9) and a plasma membrane-enriched pellet (Pl6.9) 46 fraction was assessed by Western blotting. Data from a representative experiment are shown in Fig. 3A. Based on five independent experiments, MLK3 is distributed approximately equally between the 816.9 and P16.9 fractions when expressed alone. In contrast, upon co-expression with activated Cdc42V'2, MLK3 is found predominantly in the P16.9 fraction. However, activated Cdc42Vlz is present in both fractions, as has been reported for overexpressed RasV'2 in COS cells [153]. Coexpression of MLK3 with the membrane targeting-defective, activated GTPase, Cdc42V'2C188S, K/R4Q, yielded a fractionation pattern identical to that of MLK3 alone (Fig. 3A), indicating that activated Cdc42Vl2 alters the subcellular localization of MLK3 in a prenylation-dependent manner. To confirm that MLK3 is only activated at the plasma membrane upon targeting by Cdc42, the catalytic activity of MLK3 in the P16.9 fractions was measured in in vitra kinase assays. The P16.9 fractions were solublized in Triton X-lOO-containing buffer and equal amounts of immunoprecipitated MLK3 were subjected to in vitra kinase assays using unlabeled ATP and recombinant GST-MKK4 as substrates. Phosphorylation of the activation segment of MKK4 was monitored using a phospho-specific antibody against MKK4. Data from three independent experiments demonstrate that MLK3 activity is present in the plasma membrane-enriched fractions only in the presence of active, prenylated Cdc42 (Fig. 3B). When MLK3 is expressed alone or with the prenylation- defective Cdc42v'2Cl88S, K/R4Q, negligible MLK3 activity resides in the P16.9 fraction. Taken together, these data support the hypothesis that functional prenylation is required for Cdc42-mediated targeting and activation of MLK3 at the plasma membrane. 4.4. Activated Cdc42 and MLK3 Colocalize at the Plasma Membrane 47 To examine the subcellular localization of Cdc42Vlz and MLK3 in cells, confocal microscopy experiments were performed using HeLa cells transfected with vectors encoding MLK3 and Cdc42“2 variants. MLK3 alone displays prominant perinuclear staining along with punctate, vesicular patterns and limited diffuse cytosolic staining (Fig. 4, top panel). These data are in general agreement with reports that MLK3 localizes to the Golgi [157] and centrosome regions [124]. Cdc42Vlz has been previously shown to localize to the plasma membrane [158]. When MLK3 is coexpressed with Cdc42V'2, they clearly colocalize at the plasma membrane (Fig. 4, center panel). In contrast the prenylation-defective, activated Cdc42VIZ consistently displays cytosolic staining. In the presence of prenylation-defective Cdc42v'2, MLK3 fails to localize to the plasma membrane and largely maintains perinuclear staining (Fig. 4, bottom panel). Taken altogether, these data further support the idea that activated Cdc42“2 targets MLK3 to the plasma membrane. 4.5. Requirements of MLK3 Membrane Targeting by Activated Cdc42 To determine the requirements for Cdc42-induced membrane targeting of MLK3, variants of MLK3 were expressed alone and with activated Cdc42 in HEK293 cells and the subcellular distribution of MLK3 was examined by Western blotting of equal cellular equivalents after biochemical fractionation. As shown in Fig. 5A, both wild type MLK3 and the catalytically inactive variant, MLK3 K144R, are efficiently targeted to the plasma membrane-enriched fraction when expressed with Cdc42V‘2, indicating that MLK3 activity is not required for Cdc42-induced targeting. 4s MLK3 translocation may depend on direct physical association with Cdc42 or, alternatively, may be an indirect effect of activated Cdc42 Signaling. To discern between these possibilities, we made use of the variant MLK3 I492A,S493A in which two of the conserved residues in the CRIB motif have been changed to Ala residues. This variant, which has been shown to be defective in Cdc42Vl2 binding [107], fails to undergo Cdc42- I mediated translocation as shown in Fig. 5A. These data strongly support a mechanism whereby direct interaction with activated Cdc42 is responsible for membrane targeting of MLK3. 4.6. Cdc42 Induces Activation Loop (Auto)-Phosphorylation of MLK3 Within the catalytic domain of protein kinases resides the so-called activation loop, whose phosphorylation often alters its conformation rendering the kinase (usually) catalytically active [7-10]. Site-directed mutagenesis data support phosphorylation of the activation loop within the kinase domain of MLK3 is critical for MLK3 activity [84]. Activation loop phosphorylation of MLK3 was assessed by Western blotting of cellular lysates with a phosphospecific antibody directed against activation loop-phosphorylated MLK3 (pThr277/pSer28l). As shown in Fig. 5B, wildtype MLK3 lacks activation loop phosphorylation, which is markedly enhanced upon coexpression with activated Cdc42. The kinase-defective variant of MLK3 is effectively targeted by Cdc42 to the membrane fraction (Fig. 5A) but fails to undergo activation loop phosphorylation (Fig. SB), indicating that activation loop phosphorylation is due, at least in part, to autophosphorylation. 49 If direct association with Cdc42 is required for MLK3 translocation, as our data above indicate, and if translocation is needed for MLK3 activation, it is reasonable to predict that the CRIB mutant of MLK3 would not be activated by Cdc42. In accord with this hypothesis, MLK3 I492A,S493A exhibits very . minimal activation loop phosphorylation whether expressed with or without activated Cdc42 (Fig. SB). As shown in Fig. SB, activation loop phosphorylation, to a first approximation, mirrors MLK3- induced JNK activation, as judged by Western blotting of phospho-JNK in cellular lysates. 4.7. Cdc42 Induces Activation Loop Phosphorylation Independent of Membrane Targeting Catalytic activity and GTPase binding are required for Cdc42-induced activation loop phosphorylation of MLK3. As expected, the activation loop phosphorylated MLK3 resides in the membrane fraction (Fig. 6A). One reasonable hypothesis is that Cdc42- induced membrane targeting iS critical for MLK3 activation loop phosphorylation. However we find that both the prenylation-defective and -competent versions of Cdc42 induce a comparable increase in activation loop phosphorylation of MLK3, indicating that the Cdc42-mediated increase in activation loop phosphorylation of MLK3 does not depend upon membrane targeting (Fig. 63). Taken altogether, these data imply that Cdc42 activates MLK3, at least in part, by inducing activation loop (auto)phosphorylation. Furthermore, this function of Cdc42 is independent of its ability to associate with cellular membranes and likely ensues from the physical association of the kinase with the small GTPase. 50 4.8. Membrane Targeting Contributes to Full Activation of MLK3 Both prenylation-competent and prenylation-defective variants of Cdc42VIZ promote activation loop phosphorylation of MLK3 to the same extent. However, activation loop phosphorylation may not be suflicient for full activation of MLK3. To determine whether membrane targeting contributes to the activation of MLK3 by Cdc42, in vitra MLK3 immune complex catalytic activity assays were performed from HeLa cells expressing MLK3 and Cdc42“2 variants. Recombinant, catalytically inactive GST- MKK7 was used as a substrate; and the extent of phosphorylation of the activation segment of MKK7 was measured using a phospho-specific antibody directed against MKK7. Control experiments were performed to confirm that a linear relationship exists between the amount of phospho-GST-MKK7 loaded and the resulting signal quantitated using Image] software (data not Shown). AS demonstrated previously, coexpression of MLK3 with activated Cdc42",l2 enhances the in vitra catalytic activity of MLK3 (Fig. 7). We have consistently observed that coexpression with Cdc42 increases the protein levels of MLK3 and thus it is necessary to correct the MLK3 activity for the amount of protein present. Based on three independent experiments using HeLa cells and normalizing for the amount of immunoprecipitated MLK3, MLK3 catalytic activity is increased 5.5-fold upon 2‘"2 over that of MLK3 alone, whereas coexpression with prenylation-competent Cdc4 the prenylation-defective Cdc42V'2C1888, K/R4Q induces only a 2.5-fold activation of MLK3. These data indicate membrane targeting is required for the full activation of MLK3 induced by Cdc42. 51 Prior work from our lab identified Ser555 and Ser556 of MLK3 as Sites that incorporate radiolabeled phosphate in viva upon coexpression of MLK3 with activated Cdc42 [85], leading uS to hypothesize that these might be membrane targeting-dependent, activating phosphorylation sites. However, an MLK3 variant in which these phosphorylation sites have been substituted with alanine was fully activated by Cdc42- induced membrane targeting (Y. Du and K. Schachter, data not shown), suggesting that other phosphorylation events/posttranslational modifications are responsible for the enhanced activation of MLK3 in response to membrane targeting. 4.9. Enhanced MLK3 Signaling to JNK Requires Cdc42-induced Membrane Targeting Since full activation of MLK3 requires Cdc42-induced membrane targeting one might expect membrane targeting to affect MLK3 signaling. One of the best-described functions of MLK3 is as a MAPKKK that activates the JNK pathway ([62]. In addition, activated Cdc42 potentiates MLK3-induced JNK activation [107]. To determine the impact of membrane targeting of MLK3 by Cdc42 on JNK activation, MLK3-induced .lN K activation in cells was assessed using either the phospho-specific antibodies directed against activated JNK (pThrl83/pTyr185) and/or c-Jun (pSer63). MLK3 alone induces basal phosphorylation JNK and c-Jun that is markedly enhanced upon coexpression with prenylation-competent Cdc42VIZ in HeLa cells (Fig. 8A). In contrast, despite its full ability to induce activation loop phosphorylation of MLK3, prenylation-defective Cdc42v'2C188S, K/R4Q only weakly potentiated MLK3 activation of JNK and c-Jun in cells. 52 Additional experiments show that prenylated Cdc42 alone, or MLK3 alone, only weakly activate JNK, whereas expression of the two together results in synergistic JNK activation (Fig. 8B, fourth panel), as judged by phospho-JNK Western blotting. Furthermore, only when MLK3 has been activated by prenylated Cdc42, are there Significant levels of activated MKK7 in cells, as measured by immunoblotting of cellular lysates with a phospho-MKK7 antibody (Fig. 83, third panel). From these data we conclude that functional targeting to the plasma membrane contributes to MLK3—induced activation of JNK by Cdc42, most likely reflecting the increased catalytic activity of membrane-targeted MLK3. 53 5. Discussion In this study we investigated the mechanism by which the small GTPase Cdc42 activates MLK3. Depletion of cellular MLK3 using RNA interference prevents Cdc42-induced JNK activation implicating MLK3 as a physiological target of Cdc42. Previous work from our lab showed that GTP-bound Cdc42 associates with and activates MLK3 in a CRIB motif-dependent manner [107]. In order to assess the contribution of Cdc42-induced membrane targeting to MLK3 activation and signaling, a membrane- targeting defective variant of Cdc42VIZ was engineered. This membrane targeting- defective counterpart has no detectable membrane association yet retains the ability to associate with MLK3 in coimmunoprecipitation assays. In these studies, biochemical fractionation experiments, along with confocal microscopy, indicate that prenylation- competent, but not prenylation-defective, activated Cdc42 targets MLK3 from a perinuclear region to heavy membranes, including the plasma membrane. These results are consistent with the idea that Cdc42 and MLK3 signal at the plasma membrane. Recently a biosensor approach revealed that active Cdc42 is present not only at the cell periphery, but also within the cell body near the cell periphery and at the trans- Golgi apparatus [159]. Given recent data regarding Ras Signaling on endosomes as well as on the plasma membrane [160-163] and considering the ability of both Cdc42 [159, 164] and MLK3 to localize to endomembrane structures [157], it would not at all be surprising if, under certain conditions, Cdc42 may activate MLK3 on these cellular membranes. 54 Phosphorylation within the so-called “activation loop” promotes the active conformation of many protein kinases. Substitution of phosphomimetic Asp residues for Thr277 and Ser281 within the activation loop results in active MLK3 [84]. Interestingly, using a phospho-Specific antibody, we found that GTP-bound Cdc42 potently induces activation loop phosphorylation of wildtype MLK3. However, Cdc42-induced membrane targeting is not required for activation loop phosphorylation since both 2Vl2 promote activation prenylation-competent and prenylation-defective variants of Cdc4 loop phosphorylation of MLK3 to the same extent. Furthermore, we show that Cdc42- induced activation loop phosphorylation requires MLK3 activity. These data imply that physical association with activated Cdc42 causes activation loop (auto)phosphorylation of MLK3. It is possible that Cdc42 activates MLK3 indirectly through one of the MAPKKK kinases, such as the p21-activated kinase (PAK), which is known to be activated by Cdc42 and Rac [165-168]. However our finding that a GTPase binding-defective version of MLK3 cannot be activated by Cdc42 strongly argues for Cdc42 as a direct effector of MLK3. The N-terminal region of PAK harbors a CRIB motif-containing p21-binding domain (PBD) which overlaps with an autoinhibitory domain [165-167]. The binding of the active, small GTPase disrupts this autoinhibitory conformation resulting in autophosphorylation of the activation loop and activation of PAK [165, 166]. Our lab has previously reported that MLK3 is autoinhibited through association of its N-terminal SH3 domain with a sequence containing Pro469 which, based at least on primary sequence, is Situated in close proximity between the zipper domain (amino acids 400-462) and CRIB 55 motif (amino acids 498-514) [99]. Thus, although they utilize different structural domains/elements, PAK and MLK3 appear to share a common theme of GTPase- mediated disruption of autoinhibition. In the case of PAK, crystal structures reveal a trans autoinhibited dimeric form [168]; and experimental evidence indicates that activated Cdc42 dissociates the autoinhibited PAK dimer leading to cis autophosphorylation. In contrast, zipper-mediated dimerization/oligomerization is critical for MLK3 substrate phosphorylation and signaling [91, 108]. While the precise oligomerization state of neither the autoinhibited nor the activated form of MLK3 has been experimentally determined, it has been suggested, based upon co- immunoprecipitation of differentially tagged versions of MLK3, that Cdc42 induces dimerization/oligomerization of MLK3 [93, 108]. From the results discussed above, it might be concluded that physical association with Cdc42 is sufficient for MLK3 activation. However, in vitra kinase assays for MLK3 activity revealed that maximal activation of MLK3 requires Cdc42-induced membrane targeting. The inference is that activation loop (auto)phosphorylation is required, but insufficient, for full activation of MLK3, and is consistent with the idea that membrane- targeting dependent phosphorylation events, are necessary for the full activation of MLK3 and its Signaling to JNK. It is also conceivable that enhanced accessibility of cellular substrates to MLK3 might contribute to its Signaling to JNK. The process of Ras-induced Raf activation shares some similar features with Cdc42-induced MLK3 activation. GTP-bound Ras binds and translocates c-Raf to the plasma membrane [27, 153, 169-171] and endomembranes [160-163]. Full activation of c-Raf requires activation loop phosphorylation [172] as well as membrane-dependent 56 phosphorylation, reportedly by PAK [173, 174] and Src [175-177]. However, while prenylation-defective, activated Cdc42 induces activation loop phosphorylation and partial activation of MLK3, prenylation-defective variants of Ras fail to activate c-Raf [156, 176]. A prenylation-defective variant of Ras has been shown to bind Raf [156], but binding-induced activation loop phosphorylation has not been examined. The protein kinase(s) responsible for the membrane targeting-dependent phosphorylation of MLK3 are as yet unidentified, but two MAPKKKKS, germinal center kinase (GCK) [178] and hematopoietic progenitor protein kinase 1 (I-IPKI) [179], which have been implicated in MLK3 activation, emerge as candidate MLK3 kinases. While HPKI is restricted to cells of hematopoietic lineage, both MLK3 and GCK are widely expressed in cell and tissue types [58, 180]. Interestingly it has been shown that I-IPK] can be recruited to lipid rafts [181] and GCK is found at Golgi and plasma membranes [182]. One of the major functions ascribed to MLK3 is as a MAPKKK for activation of the JNK pathway. Our finding that the prenylation-defective, activated Cdc42 variant fails to fully potentiate MLK3-induced JNK activation supports the idea that plasma membrane localization of MLK3 is an important facet of the mechanism by which Cdc42 activates MLK3-induced INK Signaling. We propose that activated Cdc42 associates with autoinhibited MLK3 through its CRIB motif, disrupting SH3-mediated autoinhibition, inducing zipper-mediated dimerization of MLK3 and subsequent (auto)phosphorylation within the activation loop of MLK3. This localization- independent, allosteric activation and activation loop phosphorylation accounts for about half of the full activation of MLK3. AS a consequence of Cdc42-induced membrane 57 targeting, MLK3 undergoes additional phosphorylation events that are required for its full activation. 58 < '8 '3 3 n X Frag-ede42v12 - + + new MLK3WB ®JNKwe -" " In a. m Flag-Cdc42 we ~11..- Actin WB Fig. 1. Cdc42-induced JNK activation requires MLK3. HeLa cells were transfected with human MLK3 siRNA or control siRNA followed by F lag-Cdc42“2 transfections, 20 h later. Cell were lysed and cellular lysates were resolved by SDS-PAGE and Western blotted using the indicated antibodies. The data shown are representative of three independent experiments. 59 H-Ras GPGQMSQKCVLS K-Ras KKKKSKTKCVIM Rac1 PVKKRKRKCLLL RhoA RRGKKKSGCLVL Cdc42 PEPKKSRRCVLL Cdc42V12C1888 l l :1". Cdc42“2 C1883, KIR4Q GO 008 MLK3 - + + + + ctrc42V12 - - + 01883333 FIag-Cdc421Pl .— - - MLK3 we .1“- MLK3 we Lysate E .i’ Flag-Cdc42WB Fig. 2. Construction of prenylation-defective variants of Cdc42V12 and the ability of Cdc42Vlz variants to associate with MLK3. A. Alignment of the COOH-termini of small GTPases and Cdc42V12 mutants. The twelve COOH-terminal amino acids of H- Ras, K-Ras, Rac1, RhoA and Cdc42 are aligned. The CAAX motif is italicized. Basic residues are shown in bold and palmitoylation Sites are underlined. The mutations in the engineered Cdc42V12 variants are shown with the LyS/Arg to Gln and/or Cys to Ser changes indicated by bald letters. B, Association of Cdc42VIZ variants with MLK3. Flag-Cdc42v'2 variants were immunoprecipitated from total cellular lysates using the Flag antibody and associated MLK3 was detected by immunoblotting with an MLK3 antibody. (Barbara C. Bock, 2000) 60 MLK3 + + + Cdc42V12 - 'l' C1888,KIR4Q 16.98PSPSP ii i.“ MLK3WB fig - Flag-Cdc42 we MLK3 - + + 61383 Cdc42V12 ' " + [mug MLK3 up w,” . a .._..... @esr-MKK4 we 9 C» . MLK3 we -- Flag-Cdc42 we Fig. 3. Effect of Cdc42": variants on the subcellular distribution of MLK3 and MLK3 activity in plasma membrane-enriched fractions. HEK293 cells were transiently transfected with expression vectors containing cDNAs indicated above each V12 2 figure. A, Subcellular distribution of MLK3 upon coexpression with Cdc4 variants. 2Vlz variants were disrupted Cells expressing MLK3 in the presence and absence of Cdc4 by Dounce homogenization in hypotonic buffer. The postnuclear fractions were centrifuged at 16,900 x g to yield soluble ($16.9) fractions and pellet (Pl6.9) fractions. Equal amounts of total protein were loaded in each lane. Western blots used to determine the distribution of MLK3 and Flag-Cdc42, using the MLK3 or Flag antibody, are Shown in the tap and lower panels, respectively. The data shown is representative of five independent experiments. B. Catalytic activity of MLK3 in P16.9 fractions. An in vitra 61 assay of MLK3 activity using equal amounts of MLK3 immunoprecipitated from solubilized P16.9 fractions was performed using GST-MKK4 as a substrate. Phosphorylation of GST-MKK4 was assessed using an antibody that recognizes phosphorylated Thr26l of MKK4. (Barbara C. Bock, 2000) 62 Phase Contrast MLK3 Flag-Cdc42 Merge .5 + + . 5‘ Fig. 4. Subcellular localization of MLK3. HeLa cells, transiently expressing MLK3 SM'IW zLAZ'OPO'BEH +911“ ovum ‘seaio manna-631:1 + 2x11" 2v12 variants, were fixed and MLK3 was stained using with or without F lag-tagged Cdc4 a rabbit MLK3 antibody and a secondary antibody conjugated with Alexa Fluor 546. Flag-tagged Cdc42V12 variants were detected by mouse monoclonal Flag antibody and a secondary antibody conjugated with Alexa Fluor 488. The nuclei were stained with T0- PRO-3 iodide. Using confocal laser scanning microscopy, more than 50 cells were examined for each transfection. Representative images are shown with F lag-Cdc42, MLK3, and nuclei represented in green, red and blue, respectively. Bar = 10 um. 63 MLK3 WT K144i! I492A,S493A Flag-Cdcnwz _- _ ._ __ _- _+ 16.98PS+P3.+P8P SPSP -- _ _ - - - — “-I- MLK3 W3 Flag-Cdc42 we B 1492A MLK3 WT K144i! 8493A Cdc42“: ' __'._'_ ' " I * - , __ @utxswe .. as a... -... a... MLK3WB *- -~- r.- r1 . @JNKWB Fig. 5. Requirements of MLK3 membrane targeting by activated Cdc42 and activation loop phosphorylation of MLK3. A, Requirements of MLK3 membrane targeting by activated Cdc42. HEK 293 cells were transiently transfected with expression vectors containing cDNAs as indicated. Subcellular fractionation experiments were performed as described in Fig. 3. The samples for all the fractions‘were loaded as equal cellular equivalents. The distribution of MLK3 and Flag-Cdc42 were shown by Western blotting using the MLK3 or Flag antibody respectively. B. Cdc42-induces activation loop (auto)phosphorylation of MLK3. HeLa cells were transiently transfected with expression vectors containing cDNAs for MLK3 variants with or without F lag- Cdc42V'2, as indicated. Equal amounts of total protein from cellular lysates were resolved by SDS-PAGE and Western blotted using the indicated antibodies. The data shown are representative of three independent experiments. (The experiments in panel A were performed in collaboration with Karen Schachter.) 64 A MLK3 + + Cdc42V12 - + 16.9 S P S P - ® MLK3 we -"'-' 2 MLK3 We - Flag-Cdc42 we MLK3 WT WT WT KD C1883 Cdc42V12 - + mm - ”tea ' i M - MLK3 we a a gr; ®T277I®8281 MLK3 we a.» "" Flag-Cdc42 WB Fig. 6. Cdc42 induces activation loop phosphorylation of MLK3 independent of membrane targeting. A. Distribution of the activation loop phosphorylated MLK3. HEK 293 cells were transiently transfected with expression vectors for MLK3, with or without F lag-Cdc42V'2. Biochemical fractionation experiments were performed as described in Fig. 3. The distribution of MLK3, F lag-Cdc42 were examined by Western blotting. The activation loop phosphorylation of MLK3 in these different fractions was determined by Western blotting using a specific phospho-MLK3 antibody. B. The effect of Cdc42VIZ variants on activation loop phosphorylation of MLK3. HEK293 cells transiently expressing wildtype (WT) MLK3 and Cdc42V12 variants, or MLK3 K144A (KD), were lysed and equal amounts of total protein were resolved by SDS-PAGE. Western blots of cellular lysates using MLK3 and phospho-MLK3 antibodies are shown in the tap and middle panels, respectively. The data Shown are representative of three independent experiments. 65 MLK3 ' +++ ems Cdc42“2 ' ' "' KIR4Q MLK3 11>] .— ®GST-MKK7 we - - ‘ MLK3 WB ._... -” MLK3 we L sate y ’ Frag-Cdc42 we -.—-- Acfln WB B a: x 3' 5. . to o 6- ‘9? c— 5- '; P: 034‘ " a. 2 a 3' o o E; 2‘ 'U Q. ‘5 1‘ "' c MLK3 - + «r- + Cdc42‘m . _ + crass KIR4O Fig. 7. MLK3 activity induced by Cdc42VIZ variants. HeLa cells were transfected with or without Cde42‘“2 variants as indicated. A, in vitra immune complex kinase assay of MLK3 using recombinant GST-MKK7 K165A as a substrate. The top panel shows phosphorylation of GST-MKK7 K165A as determined by Western blotting using a phospho-Specific antibody against MKK7; the second panel shows a Western blot of the immunoprecipitated MLK3 from the in vitra kinase assays; the third, fourth and fifth panels are Western blots indicating the levels in cellular lysates of MLK3, Flag- Cdc42Vlz variants and, as a loading control, actin. B, Quantitation of MLK3 activity assay. Phosphorylation of GST-MKK7 was quantitated by densitometry and 66 normalized to levels of immunoprecipitated MLK3 as described under “Experimental Procedures”. The means 3: SE. for fold-increase in phosphorylation of GST-MKK7 K165A from three independent experiments are shown. 67 A MLK3 -+++ C1388 -- MLK3 we I"! g. @MLKS We H . §®JNK we I. - ii ®c-Jun W8 .- Flag-Cdc42 WB -..- Actln 11113 B MLK3 ...+++ C1888 C1888 C C4 V1 ' ' d 2 2 ' + KIR4Q ' + KIR‘Q a- MLK3 we —- ‘— @MLK3 we ...... ®MKK7 we : : @JNK we v a Flag-Cdc42 we - ,w -.u.--“ Actin WB Fig. 8. Effect of Cdc42V'2 variants on MLK3-induced JNK signaling. HeLa cells were transfected with expression vectors as indicated. Equal amounts of protein from cellular lysates were resolved by SDS-PAGE and Western blotted using the indicated antibodies. The data shown are representative of three independent experiments. 68 Chapter 3. Regulation of mixed-lineage kinase 3 by guanine nucleotide exchange factor, Vav 1. Abstract Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates multiple MAPK pathways. Whereas the downstream Signaling components that are regulated by MLK3 have been largely defined, less is known about the upstream regulators of MLK3. Data presented in Chapter 2 demonstrated that the small GTPase Cdc42 is a physiological regulator of MLK3. In this study the role of Vav, one of the guanine nucleotide exchange factors that activates Cdc42, in the regulation of MLK3 was examined. Herein, we report that the expression of Vav increases MLK3 activity as judged by MLK3 autophosphorylation and substrate phosphorylation. Furthermore, Vav associates with MLK3 via at least two distinct Sites within MLK3. The MLK family inhibitor, CEP-11004 dramatically inhibits Vav-induced JNK activation. However, Silencing of the MLK3 gene by siRNA has no effect on activation of JNK-induced by expressed Vav. These data suggest Vav may signal through multiple MLKs to activate JNK. 69 2. Introduction The immediate downstream Signaling components of MLK3 have been largely defined, but the identification of the upstream activators of MLK3 is incomplete. Multiple lines of evidence point to a role for both Rae and Cdc42 as activators of MLK3. For instance Slpr, the Drasaphila MLK homolog, is critical for Rac signaling to JNK during dorsal closure in fly embryogenesis [127]. Activated forms of the small GTPases, Cdc42 and Rac, can bind to MLK3 through the CRIB motif and increase MLK3 catalytic activity [63, 105, 107, 183]. The data in Chapter 2 demonstrates that binding of activated Cdc42 promotes activation loop phosphorylation of MLK3 and partial activation of MLK3. Furthermore, activated Cdc42, in a prenylation-dependent fashion, targets MLK3 to the plasma membrane; and this prenylation-dependent membrane targeting is required for full activation of MLK3 and its signaling to INK [184]. Guanine nucleotide exchange factors (GEF S) activate GTPases by stimulating the exchange of the bound GDP for GTP [100, 101]. Only in their active, GTP-bound state, are GTPases capable of interacting with downstream effector proteins. Since MLK3 is activated by Cdc42 and Rac, the GEFS that activate Cdc42 and Rac might also promote MLK3 signaling. There are multiple GEFS that can activate Cdc42 and/or Rae. Vav was determined to be one of the most potent activators of MLK3 in an MLK3 activity assay, where several different GEFS were coexpressed with MLK3 (M. Chao 2002, unpublished data). The Vav proteins are GEFS for Rho-family GTPases. In mammals, three isoforms of Vav are Vav ] , Vav2 and Vav3. Whereas Vav] is predominantly expressed in the 70 haemopoietic system, Vav2 and Vav3 are more ubiquitously expressed [185-188]. The spectrum of GTPase targets for different Vav proteins is diverse. It has been reported that Vav] is primarily a GEF for Rac1, Rac2, RhoA and RhoG; that Vav2 acts on RhoA, RhoB and RhoG; and that Vav3 activates RhoA, RhoG and, modestly, Racl [186-191]. However, others have reported that Vavl may also activate Cdc42 and that Vav2 may serve as a GEF for Rae] and Cdc42[188, 190, 192-194]. In addition, activation of Rac1 is absent in Vavf” thymocytes and in Vav3'l' DT40 B cells [195, 196]. Activation of Cdc42 also appears to be defective in Vav1'/' T cells [197]. However, structural analysis revealed that the features that are important for the interaction with Vav, and proper Vav- induced GDP/GTP exchange are present in Rae] and RhoA, but not in Cdc42 [198]. The classical Rho family GEFS, including Vav proteins, contain a pleckstrin homology (PH) domain immediately followed by a Dbl homology (DI-I) domain. The DH domain is essential for GEF activity, whereas the PH domain has been suggested to directly affect the catalytic activity of the DH domain and to target the GEF to its appropriate intracellular locations [191, 198, 199]. Besides the tandem DH and PH domains, all Vavs also contain a number of other domains and motifs, which can contribute to protein-protein interactions [19], 198, 199] (Fig. 1). The NHz-terminus of Vav proteins contain a calponin homology (CH) domain which inhibits GEF activity, perhaps through an interaction with the cysteine-rich C1 domain[199]. The acidic region (Ac) contains at least three tyrosine phosphorylation sites [200]. Among them, Tyr] 74 has been shown to bind to the DH domain, thus inhibiting its GEF activity by keeping Vav] in an autoinhibited conformation [20]]. Phosphorylation of Tyr] 74 results in the disruption of its intramolecular interaction with the DH domain, promoting the active 71 conformation of Vav [201]. The autoinhibition of Vav may be regulated by Tyr] 74-DH interaction in collaboration with CH-Cl interaction. The COOH-terminus of Vav has two SH3 domains flanking a Single SH2 domain. Because of these multiple protein interaction domains, Vavs might also serve as an adaptor protein to facilitate the assembly of Signaling complexes and to regulate signaling cascades in a GEF activity independent manner [188, 198]. Results from mice with targeted disruption of single or multiple Vav isoforms have suggested that they play important roles in T-cell development and T-cell antigen receptor (TCR) signaling[188, 202]. Whereas disruption of Vav] results in decreased numbers of thymocytes and peripheral T cells in mice, mice deficient in either Vav2 or Vav3 alone appear to have normal T-cell development[202]. However, there is a further Significant decrease in the numbers of thymocytes and peripheral T cells in Vavl/2/3 triple knockout mice, suggesting the different Vav isoforms have redundant functions. Further analysis reveals that Vav 1 -null cells have decreased TCR Signal-induced proliferation and cytokine secretion[l 88, 202]. Vav proteins plays important roles in antigen receptor-induced cytoskeletal reorganization, and in activation of stress-activated protein kinases and several important transcription factors, such as the nuclear factor of activated T cells (N F-AT) and NF -1