lllllllllllllllllll llllllll \ 3 1293 0090\0 3 75 This is to certify that the thesis entitled A couparison of tyrosine phosphorylation pattern in normal and transfer-ed human fibroblasts. presented by Chin-Huei'Pan has been accepted towards fulfillment of the requirements for _.n.s;_degree in aimibjgiogy and Public Health gig/fl /’k (W Major professor Date May 14, 1991 0'7639 MS U is an Affirmative Action/Equal Opportunity Institution r a LIBRARY Michigan State University ‘ ,J ' a“ . ' PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE I’ll J MSU lo An Affirmative Action/Equal Opportunity Institution chna-pt A COMPARISON OF TYROSINE PHOSPHORYLATION PATTERN IN NORMAL AND TRANSFORMED HUMAN FIBROBLASTS by Chin-Huei Pan A THESIS Submitted to Michigan State University in partiai fuifiliment of.the requirements for the degree of MASTER OF SCIENCE Department of Microbioiogy and Public Heaith 1991 A camarsou or mosruE PHOSPHORYLATION PATTERN IN MI. All) women Him FIBRIBLASTS By Chin-Huei Pan This study was undertaken to determine whether there was a change in the pattern of tyrosine phosphorylated proteins between normal human fibroblasts and their derivatives. The analysis was carried out by using a phosphotyrosine-specific antibody with a Western blotting analysis. I found that the tyrosine pattern changes in the process of transformation. The data from an infinite life span but nontumorigenic cell lineage generated in our laboratory ( MSU-1.0, MSU-1.1 and MSU-1.1 V0) offer good evidence for tyrosine phosphorylation being involved in the multi-stepped cellular transformation. The pattern of tyrosine phosphorylation was identical in N-, K-, H-ras transformed cell strains, suggesting that the growth control pathway is the same regardless of the kind of res oncogene used to transform the cells. These spontaneous transformed cell strains share a very similar pattern except for an enhanced 72kDa phosphoprotein in one strain. v-fes-transformed MSU-1.1 V0 cells showed a more enhanced and different tyrosine phosphorylation pattern than the MSU-1.1 V0 cells from which they were derived. One of these hyperphosphorylated proteins was identified as the pllov'fes-encoded protein. The data showed a good ii iii correlation between the fes protein expression level and the tyrosine phosphorylation level. I also analyzed five human fibrosarcoma-derived cell line-s. Four of them showed similar but not identical patterns, suggesting they each utilize a similar growth control pathway. However, HT1080 and VTszT cell lines showed a similar pattern after vanadate treatment, suggesting they may utilize the same growth control pathway. These experiments demonstrate that the tyrosine phosphorylation pattern of human fibroblasts changes in the process of transformation. I found that several proteins with unique molecular weight showed enhanced phosphorylation at discrete steps in the process of transformation. With this information, it is possible to select apprOpriate monoclonal antibodies to identify these phosphorylated proteins, which may provide further insight into the tumorigenesis process in fibroblasts. To my mother, Tsung-wei Ho and my late father, Chi-Du Pan and my grandfather, Kuang-Che Ho iv ACKNOHLEDGEHENTS I would like to thank my graduate advisor and research director, Dr. J. Justin McCormick, for sharing with me his broad knowledge of cancer, and his many ideas and perspectives that have shaped this thesis. I also owed a special thanks to Dr. Veronica M. Maher for her direction. I want to thank the other member of my graduate committee, Dr. Jonathan Fratkin, for his advice and invaluable time he gave to my work. I also want to thank to numerous individuals whose constant encouragement, help and friendship supported me. They include Lonnie Milam, Rhey-Hwa Chen, Chia-Miao Mah, Yi-Ching Wang and Dave Reinhold. I also would like to express my gratitude to my parents, whose guidance and love have been constant throughout this endeavor. TABLE OF CONTENTS .Page LIST OF TABLES ....................................................... vi LIST OF FIGURES ...................... -: ............................... vii INTRODUCTION .......................................................... 1 CHAPTER 1. LITERATURE REVIEWS A. Role of receptor with tyrosine kinase activity (RTK) in cells. 1. ,Perspectives and summary ............................ . ...... 6 2. Structural characteristics and classification .............. 7 a. Prototypic structure ...... _; ........... , .................. 7 b. Four families of RTKs..... ............ ’ .................. 7 3. Molecular mechanism of tyrosine kinase activity ............ 10 a. Enzymology and phosphorylation .......................... 10 b. Functional consequences of receptor autophosphorylation.11 4. Substrates of RTKs in multiple pathways of signal transduction ............................................... 11 B. Role of protein tyrosine kinases (PTK) in tumorigenesis ....... 14 1. Two kinds of protein tyrosine kinases ...................... 14 a. Oncogene encoded PTKs and their cellular homologs ....... 14 b. Growth factor receptors ................................. 18 2. Oncogenic potential of protein tyrosine kinases ............ 20 C. The molecular genetics of cancer on the basis of oncogene theory ........................................................ 22 1. A proto-oncogene can be oncogenic in many ways ............. 22 a. Chromosome rearrangement ................................ 24 b. Deletion or point mutation in coding sequence ........... 24 c. Activation by gene amplification ........................ 25 References ....................................................... 27 CHAPTER II. A comparison of the pattern of phosphotyrosine-containing proteins in normal human fibroblasts, nontumorigenic but infinite life span cell lineage, and tumor-derived cell strains. Abstract ......................................................... 38 Introduction ..................................................... 39 vi Materials and methods Cells and culture conditions .................................. 42 Preparation of cell lysates ................................... 43 Sodium orthovanadate treatment ................................ 43 Analysis of v-fes protein by immunoprecipitation .............. 43 Western blotting .............................................. 45 Results The pattern of phosphotyrosine-containing proteins in the MSU-I cell linage ......................................... 48 The pattern of phosphotyrosine-containing proteins in ras transformed cell strains .................................. 51 The pattern of phosphotyrosine-containing proteins in spontaneous transformed cell strains .......................... 51 The pattern of phosphotyrosine-containing proteins in human fibrosarcoma-derived cell lines ......................... 54 Discussion ...................................................... 63 References ...................................................... 67 CHAPTER III. Enhancement of tyrosine phosphorylation in v-fes transformed human fibroblasts ........................... 70 Abstract ........................................................ 71 Introduction .................................................... 72 Materials and methods Cells and culture conditions .................................. 75 Preparation of cell lysates ................................... 75 Analysis of v-fes protein by immunoprecipitation .............. 76 Western blotting .............................................. 76 Results Enhances phosphotyrosine-containing proteins in v-fes transformed cells ............................................. 79 Identification of the p110kDa v-fes encoded protein ........... 82 Correlation of v—fes expression level and tyrosine phosphorylation level ......................................... 82 Sodium orthovanadate enhances the detection of cellular phosphotyrosine-containing proteins ........................... 88 Discussion ...................................................... 91 References ...................................................... 94 vii LIST OF TABLES CHAPTER I page 1. Mammalian protein tyrosine kinases ........................ 15 2. Oncogenes originally identified through their presence in transforming retroviruses ................................. 23 CHAPTER II 1. Characteristics of the MSU-l cell lineage ................. 44 2. ras transformed cell strains .............................. 46 3. Carcinogen treated cell strains ........................... 47 CHAPTER III 1. v-fes transfected cell strains ............................ 78 viii LIST OF FIGURES CHAPTER I page 1. Prototypic structure of RTKs ............................. 8 2. Chematic representatives of RTKs ......................... 9 CHAPTER II 1. The pattern of phosphotyrosine-containing proteins in the MSU-l cell lineage .................................. 50 2. The pattern of phosphotyrosine-containing proteins in the ras oncogene transformed cell strains ............... 53 3. The pattern of phosphotyrosine-containing proteins in the spontaneous tumorigenic MSU-1.1 cell strains ........ 56 4. The pattern of phosphotyrosine-containing proteins in the human fibrosarcoma-derived cell lines ............... 58 5. Immunoprecipitation of MCI cells with anti-v—fes antibodies .............................................. 62 CHAPTER III 1.Enhanced expression of phosphotyrosine-containing proteins in v-fes transfected cells ...................... 81 2.1dentification of the p110 v-fes protein in v-fes transformed cells ........................................ 84 3.The correlation of p110 v-fes protein expression level and tyrosine phosphorylation level ....................... 87 4.Sodium orthovanadate treatment of cells enhances the detection of cellular phosphotyrosine-containing proteins ................................................. 90 ix ABBREVIATIONS CSF Colony stimulating factor EGF Epidermal growth factor FGF Fibroblast growth factor PDGF Platelet-derived growth factor PTK Protein tyrosine kinase RTK Receptor with tyrosine kinase activity SER Serine TGF Transforming growth factor THE Threonine TYR Tyrosine Introduction According to present theory, a cancer cell results when a normal cell acquires a number of specific changes in dominant activation of proto-oncogenes and/or down-regulation of tumor suppressor genes. One common phenotype is the ability of cancer cells to proliferate in vitro in the absence or reduced levels of growth factors. These explanations are offered for the phenomena: 1) growth factor(s) are expressed which are not normally expressed; 2) increased number of normal growth factor receptors or altered growth factor receptors are synthesized; 3) various elements in the secondary messenger pathways of growth control are activated. One such pathway involves the phosphorylation of tyrosine residues on specific proteins by protein tyrosine kinases. Initially, it appeared that one could classify the protein tyrosine kinases into two groups: the growth factor receptors, and oncogenes that had integrated into retroviruses. The first group can be classified into four families (EGF, insulin, PDGF, and FGF families of receptor kinase) based upon distinct structural characteristics and sequence similarity ( for review, see Ullrich and Schlessenger, 1990). Some proto-oncogene products are altered growth factor receptor-like proteins with tyrosine kinase activity. A good example is the erbB oncoprotein, which is homologous to the EGF receptor with a large deletion in the extracellular binding domain ( Ullrich et al., 1984). Binding the appropriate growth 2 factor to a specific receptor causes a rapid phosphorylation on tyrosine residues of the receptor proteins themselves, as well as a number of cellular proteins. The second group includes the proto-oncogenes, such as c-src, c-fes etc., that have been identified as oncogenes because of their captures by retroviruses ( for review, see Hunter and Cooper, 1985). To date, several substrates of protein tyrosine kinases have been found and their biological importance has been identified. The 145 kD phospholipase c-r is phosphorylated on tyrosine residues by EGF and PDGF stimulation in vitro and in living cells (Margolis et al., 1989; Meisenhelder ’et al., 1989; Wahl et al., 1989). The stimulation of phospholipase c-r is the first step in the signal transduction pathway. Many studies also have shown that the 120kD ras GTPase-activating protein(GAP) is phosphorylated on tyrosine by the PDGF receptor in Balb/3T3 cells in response to PDGF (Kaplan et al., 1990). The GAP protein is believed to react directly with the cellular ras p21 protein and enhances the GTPase activity of the protein (Trahey and McCormick, 1987; Vogel et al., 1988; McCormick, 1990). Some other substrates of viral- oncogene proteins possessing tyrosine kinase activity have been identified, such as vinculin (Antler et al., 1985), talin (DeClue et al., 1987) and calpactin (Redke et al., 1980; Erikson et al., 1980), which are part of the cellular submembranous cytoskeleton in cells transformed by Rous sarcoma virus. Also, there are a number of phosphorylated substrates which have been found by different laboratories, but their functions remain unknown. However, it is postulated that these tyrosine— phosphorylated proteins play a role in generating a mitogenic signal which 3 can control cellular growth and differentiation and harbor a latent oncogenic potential, which when activated, results in the generation of abnormal growth signals and oncogenesis. The goal of this thesis is to investigate whether the phosphorylation of new tyrosine kinase substrate(s) or increased phosphorylation level of the same substrate(s) correlate with the degree of transformation. Since'our laboratory has generated a series of clonally derived cell strains, in which each cell strain contains a somewhat more transformed phenotype than the parental cell strain, this is an ideal test system. Studies utilizing transformed cell strains in this thesis include an infinite life span ,but nontumorigenic, MSU-l cell lineage, ras oncogene transformed cell strains, fes oncogene transformed cell strains, and spontaneous transformed cell strains. I also analyzed five human f ibrosarcoma-derived cell lines in order to examine whether their tyrosine phosphorylation pattern is similar to the transformed cell strains we generated in vitro. Chapter I of the thesis reviews the literature that provide the concept of our understanding about the importance of tyrosine kinases and their substrates in oncogenesis. Chapter II describes my work showing that the tyrosine phosphorylation pattern changes in the process of transformation. Chapter III describes my work showing that the v-fes transformed cells contained enhanced and distinct tyrosine phosphorylation pattern. One of the hyperphosphorylated proteins was identified to be the p110"'f°s encoded protein. The data also showed that the fes protein expression level correlated with the tyrosine phosphorylation level. References Antler, A.M., Greenberg, M.E., Feldman, G.M., and Hanafusa, H. (1985) Increased phosphorylation of tyrosine in vinculin does not occur upon transformation by some avian sarcoma viruses. Mol. Cell Biol. 5 263-267 DeClue, J.E., and Martin, G.S. (1987) Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cell. Mol. Cell Biol. 1 371-378 Erikson, E., and Erikson, R.L. (1980) Identification of cellular protein substrate phosphorylated by the avian sarcoma virus-transforming gene product. Cell 21 829-836 Hunter, T., and Cooper, J.A. (1985) Protein tyrosine kinases. Ann. Rev. Biochem. 54 897-930 Kaplan, D.R., Morrison, D.K., Wong, G. McCormick, F., and Williams, L.T. (1990) PDGF G-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signalling complex. Cell 51 125-133 Margolis, 8., Rhee, S.G., Ziberstein, A., Felder, S., Mervic, M., Lyall, R., Levitzki, A., Ullrich, A., Ziberstein, A., and Schlessinger, J. (1989) EGF induces tyrosine phosphorylation of phospholipase c-II: a potential mechanism for EGF receptor signalling. Cell 51 1101-1107 McCormick, F. (1990) The world according to GAP. Oncogene 5 1281-1283 Meisenhelder, J., Suh, P.G., Rhee, 5.6., and Hunter, T. (1989) Phospholipase c-r is a substrate for PDGF and EGF receptor protein- tyrosine kinse in vivo and in vitro. Cell 51 1109-1122 Redke, K., Gilmore, T., and Martin, G.S. (1980) Transformation by Rous sarcoma.virus induced in vitro progression from premalignant to neoplastic transformation of human diploid cell. In Vitro 15 821-828 Trahey, M., and McCormick, F. (1987) A cyroplasmic protein stimulates normal n-ras p21 GTPase, but does not affect oncogenic mutants. Science 255 542-545 Ullrich, A., Coussen, L., Waterfield, M.D., Hayflick, J.S., Dull, T.J., Gray, A., Tam, A.W., Lee, J., Yarden, Y., Liberman, T.A., Schlessinger, J., Downward, J., Mayes, E.L.V., Whittle, N., and Seburg, P.H., (1984) Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309 418-425 Ullrich, A., and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 51 203-212 Vogel, U.S., Dixon, R.A., Schaber, M.D., Diehl, R.E., Marshall, M.S., Scolnick, E.M., Sigal, 1.5., and Gibbs, J.8. (1988) Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature 355 90-93 4 5 Wahl, M.I., Olashaw, N.E., Nishibe, S., Rhee, S.G., Pledger, W.J., and Carpenter, G. (1989) PDGF induces rapid and sustained tyrosine phosphorylation on phospholipase c-r in quiescent 8AL8/c 3T3 cells. Mol. Cell Biol. 9 2934-2943 Chapter One Literature Review A. Role of receptors with tyrosine kinase 1.Perspectives G Sun-any A large number of growth factors present in serum stimulate a cellular interaction with a family of cell-surface receptors that have intrinsic, protein-tyrosine kinase activity. These receptors have an extracellular ligand binding domain that is linked to a cytoplasmic catalytic domain. Binding of the growth factors to their receptors transduces signals that play a key role in cellular growth regulation. So far, little is known about the possible cascade(s) that receptors with tyrosine kinase activity (RTK) trigger. An understanding of the relationship between protein phosphorylation and cellular growth regulations is important. Two areas of research have already elucidated some aspects of the RTK function. Firstly, several cloned cDNA sequences of RTK have been isolated , and they provide the information on the primary structure of a number of RTKs, and comparative analysis provides information about receptor domain function. The availability of cloned cDNAs for RTKs opens the way for understanding the biochemical mechanisms of growth signal generation. A second breakthrough has come from the realization that some oncogenes encode products that are altered RTKs, which provides valuable insights into the mechanisms of normal growth factor receptors, cellular transformation, and tumorigenesis. Further, a combination of genetic and biochemical approaches will bring a fuller understanding of the mitogenic responses in both normal cells and transformed cells. 6 7 2.Structural characteristics and classification a. Prototypic structure Growth factor receptors with tyrosine kinase activity all have a similar molecular topology (Figure 1). All have a large glycosylated, extracellular ligand binding domain, a single hydrophobic transmembrane domain, and a cytoplasmic catalytic domain that has the protein tyrosine kinase activity (Hanks et al., 1988; Yarden & Ullrich, 1988; Schiessinger, 1988; Williams, 1989). Unlike the muscarinic receptor (Kubo et al., 1986), rhodopsin receptor (Ovchimikov, 1982; Nathans and Hognoss, 1983; Zuker er al., 1985), and Bradrenergic receptor (Yarden et al., 1986; Dixon et al., 1986), each of which possess seven membrane-spanning hydrophobic domains, RTK has the ligand binding domain and the tyrosine kinase domain separated by the plasma membrane. Therefore, receptor activation due to extracellular ligand binding must be transmitted across the membrane into activation of intracellular domain function. b. Four families of RTK 0n the basis of distinct structural characteristics and sequence similarity, RTKs can be classified into four families (Figure 2.) 1. Family I: This family includes the receptors for EGF (Ullrich et al., 1984) and its close relatives, the HER 2/neu, HER 3/c-erb-3 (Kraus et al., 1989) and X mrk (Withbrodt et al., 1989). The characteristic structure of this family includes two cysteine-rich repeat sequences in the extracellular domain. 2. Family 11: In contrast to monomeric Family I RTKs, Family II DC MAINS N Ugcnd 86::st Binding ficnsnenmx , .agamaeerz"with the BCA protein assay kit (Pierce Chemical, Rockford, IL.). Witness. One hundred micromoles of sodium orthovanadate was added to the plates for 16 hours prior to harvest. Preparation of the lysates was described before. f - r in immun r i i n Lysates containing equal amount of protein (150ug) were immunoprecipitated with a rat anti-v-fes monoclonal antibody (Oncogene Inc., Manhasset, NY, Antibody Ab-l) that reacts specifically with the v- fes encoded sequence common to the translational products of the Snyder- Theiler and Gardner strains of feline sarcoma viruses. Protein-A agarose (Oncogene Inc.) coated with goat anti-rat IgG (Oncogene Inc.) was added, and the mixture was shaken for 30 minutes at.49C. The agarose fraction was washed twice with RIPA buffer, once with 50mM Tris pH7.5 containing 1M MgCl, and once again with RIPA buffer. NaDodSO,-PAGE sample buffer was 44 Table 1. Characteristics of the MSU-I cell lineage. ' Growth factor Cell Karyotype Immortality independence Tumorigenicity LG-l Diploid - - - MSU-1.0 Diploid + - - MSU-1.1 45+ 2 marker + + - chromosomes MSU-1.1 V0 45+ 2 + +++ +/- MSU-1.1 ras 45+ 2 + +++ ++ MSU-1.1 Car. 45+ 2 + +++ ++ MSU-1.1 Spon.45+2 + +++ ++ 45 added, and the samples were boiled for 5 minutes , then centrifuged, and electrophoresed in 8% NaDOdSO,-polyacrylamide gels. mum Equal amounts of protein (150ug) were loaded onto 8% NaDodSO,- polyacrylamide gel, and the proteins were seperated by electrophoresis as described by Laemmli (Laemmli, 1970). Transfer of proteins to Immobilon-P paper (Millipore Inc, Bedford, MA) was done in the presence of 25mM Tris, 192m glycine and 0.375% NaDodSO, using a Trans-Blot Electrophoretic Transfer Cell (Biorad, Richmond, Ca). Filters were blocked for 36 hours at room temperature using 3% powdered non-fat dry milk in TBS (20mM Tris; SOOmN NaCl pH7.5) with 0.2% sodium azide. After rinsing in TBS briefly, the filters were incubated overnight in 1% nfilk-TBS supplemented with 0.05% Tween 20 and lug/ml mouse anti-Phosphotyrosine monoclonal antibody PY 20 (ICN Inc. Costa Mesa, Ca.). The filters were washed four times with TBS containing 0.1% Tween 20 (washing buffer), then incubated with goat anti-mouse IgG antibody (Cappel Inc, Durham, NC) for 90 minutes. After rinsing the filters five times with the washing buffer, they were reacted with 10uCi [‘“Il Protein-A (ICN Inc.) for 2 hours. The filters were then washed five times with buffer for ten minutes each time, and exposed to Kodak X-RP films at -80°C for 36-48 hours using intensifying screens. The molecular weights of proteins were estimated by running myosin (200 kDa), phosphorylase 8 (97 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa) on each gel. 46 Table 2. ras Transformed Cell Strains. Oncogene Cell name Tumor Type mutated H-ras 2MT malignant 3MT malignant mutated N-ras 3T malignant 8T malignant v-k-ras 17828T malignant 17820T malignant 47 Table 3. Spontaneous Transformants of MSU-1.1 (A).Cell strains derived after carcinogen treatment Carcinogen Cell name Selection Tumor Type iiié """"""" GEES} """""" EQELQ'QEEQ; """ iiiigiéit ENU $0532AC4MT/T1 Morphology Malignant BPDE CSV020.St1 Soft agarose Malignant (B). Spontaneous transformed cell strains Cell name Selection Tumor type L46I.1T Soft agarose Malignant L4BI.1T Soft agarose Malignant L451.8 Morphology Malignant Results 1‘ -. - r . ’l' '1' i . -- on t ”In! or- -i in il‘ M - -ll Dom Our laboratory has developed a lineage of clonally derived cell strains from normal human foreskin fibroblasts, in which each cell strain expresses a somewhat more transformed phenotype than the cell strain from which it was derived (Table 1). In the preliminary studies, we found that there is no difference in the tyrosine phosphorylation pattern when all the cell strains were grown in the 10% serum and serum free media (Ryan et al., 1987). Therefore, in the following experiments, we grow the cells in the medium supplemented with 10% serum as described in the Materials and Methods. The phosphotyrosine-containing proteins of these cell strains were analyzed by the Western blotting technique using a specific antibody against phosphotyrosine (Figure 1). Normal fibroblasts and MSU-1.0 cells both have the growth factor dependent phenotype and showed an identical phosphorylation pattern. MSU-1.1 which is partially growth factor independent, showed a similar phosphorylation pattern , but expressed a 170k0a and a 140k0a phosphoprotein at a higher level then LG-1 and MSU-1.0 does. The spontaneous growth factor independent variant of MSU-1.1, MSU- 1.1 V0, contained two novel phosphoproteins at 180k0a and 160k0a that were not observed in LG-I, MSU-1.0 or MSU-1.1. The 170k0a and 140k0a phosphoproteins are absent in MSU-1.1 VO suggesting that the 180k0a and 160k0a are variants of these proteins. Sodium orthovanadate is an effective inhibitor of phosphotyrosine phosphatases (Leis and Kaplan, 1982; Swarup et al., 1982). Cells incubated 4B 49 Figure 1. The pattern of phosphotyrosine-containing proteins in the MSU-I cell lineage. Total proteins from cells grown in the absence of sodium orthovanadate (A) and from cells preincubated with' 100um sodium orthovanadate 13 hours prior to lysate preparation (8) were seperated by 8% SOS/PAGE gel electrophoresis and analyzed by Western blotting technique using an antiphosphotyrosine antibody (PY20) as described in the Materials and Methods. Arrows indicate phosphoproteins that are unique to the cell strains. Molecular weight of markers are given in kilodaltons on the side. The filter of panel A was exposed to a film for 36 hours, and the filter of panel 8 was exposed for 48 hours using intensifying screens. 50 (A) w is -—170 #160 «—140 43- Fig. 1 51 in the presence of the compound accumulate phosphotyrosine-containing proteins. Figure 18 shows that preincubation the MSU-I cell lineage with the compound for 12 hours results in a different extent of tyrosine phosphorylation in the various icell strains. The normal human fibroblasts ( LG-1) exhibit greater enhancement of tyrosine phosphoproteins than MSU- 1.0, MSU-1.1 or MSU-1.1 V0 does after vanadate treatment, suggesting the concentration of sodium orthovanadate is not optimal to inhibit tyrosine phosphatase acting in these strains. Perhaps these strains have more phosphatase activity. Ti‘ -. - n of . . . . . in-- on tinin- -r- -in n . r.i arm-c -ll strains The pattern of phosphotyrosinc-containing proteins of six MSU-1.1 cells strains produced by the transfection of the cells with a plasmid carrying either H-ras, N-ras, K-ras (Table 2), was also analyzed by Western blotting. No differences in tyrosine phosphorylation were observed between the proteins from the ras transformed cell strains and from the LG-I normal fibroblasts from which they were ultimately derived, except that the p210k0a protein was not detected in all the ras transformed cells (Figure 2, panel A). Cells incubated in the presence of sodium orthovanadate showed an enhanced level of phosphotyrosine- containing proteins, and a similar phosphotyrosine pattern, except the H- ras transformed cell strain (2MT) phosphorylated the p110kDa protein at a lower level than others, also the amount of p84k0a phosphoprotein was reduced in a N-ras transformed cell strain (8T) (Figure 2, panel 8). 52 Figure 2. The pattern of phosphotyrosine-containing proteins of ras oncogene transformed cell strains. Total proteins from cells grown in the absence of sodium orthovanadate (A) and from cells grown in the presence of the compound (8) were analyzed by Western blotting as described in the Materials and Methods. In panel A, lane 1,2 contained proteins from two H- ras transfected MSU-1.1 cell strains, 2MT and 3MT; lane 3,4 contained proteins from two N-ras MSU-1.1 cell strains, 3T and 8T; lane 5,6 contained two K-ras MSU-1.1 cell strains, 17828T and 17820T; and lane 7 contained proteins from normal human fibroblast. The filter of panel A was exposed to a film for 48 hours. Panel B shows one H-ras transfected MSU- 1.1 cell straine, 2MT (lane 1); two N-ras transfected MSU-1.1 cell strains , 3T and 8T(lane 2,3 ); two K-ras transfected MSU-1.1 cell strains, 17828T and 17820T (lane 4); and normal human fibroblasts, LG-l (lane 5) that were incubated with vanadate prior to lysate preparation. The filter of panel B was exposed for 16 hours using intensifying screens. 53 (A) (B) 43— Fig.2 54 I‘ -. - I . . . uh- r- in-- on .inina -r-t-in in non an‘CU um-r- derixed_cell_st:ains Three spontaneous morphologically transformed clones, L46I.5T, L4BI.1T and L451.B, were selected in a soft agarose assay, and all the strains generated malignant tumors when injected into nude mice. The cell lysates of these three tumor-derived cell strains were also examined by Western blot analysis using the anti-phosphotyrosine antibody described before. Three tunor-derived carcinogen treated MSU-1.1 cell strains (Table 3), L551.3T, S0532AC4MT/T1 and CSVO.20/ST1, weretalso analyzed. The phosphoprotein pattern of these six cell strains and the cell strain they were derived from (MUS-1.1) is very similar (Figure 3A), with the exception that the L451.B cell strain expresses a 72k0a phosphoprotein at a higher level of phosphorylation than the other cell strains (Figure 3A, lane 3). We further examined the expression level of the specific phosphoprotein in a series of L451 cell strains (Figure 3B). The morphologically transformed pretumor cells selected from a cloning assay, L451, do not contain the pp72k0a phosphoprotein or a pp125k0a protein. When the pretunor cells were injected into a nude mouse, one trilobed tumor was generated. Each lobe was cultured independently and designated as L451.A1, L451.A2 and L451.A3. These three cell strains also do not contain the phosphorylated 72kda protein, and they express the 125k0a protein at a different level than L451.B. The data suggest that different spontaneous changes involving the phosphorylation of these proteins on tyrosine residues occurred in each independent cell strain during tumorigenesis. 55 Figure 3. The pattern of phosphotyrosine-containing proteins in spontaneous tumorigenic MSU-1.1 cell strains. (A) Total cellular proteins were analyzed by the Western blot technique using an antiphosphotyrosine antibody described in the Materials and Methods. Lanes 1-6 contained the spontaneous transformed tumor-derived cells from L461, L481, L451.B, L551.3T, $0532AC4MT/T1 and CSV020.ST1 respectively. (B) Cells grown from a trilobe tumor produced by the spontaneous transformant L451 were analyzed for phosphotyrosine content as described previously and compared to other tumor-derived cells (L451.B) from the same spontaneous transformants. Lane 1 contains L451.A1 proteins, Lane 2 containes L451.A2 proteins, lane 3 contains L451.A1 proteins, lane 4 contains L451 proteins and lane 5 contains L451.8 proteins. The film of panel A was exposed for 18 hours, and the film of panel 8 was exposed for 48 hours with intensifying screens. . D K 5 2 1 P L ._p72KDI Fig.3 57 Figure 4. The pattern of phosphotyrosine-containing proteins in human fibrosarcoma-derived cell lines, SHAC, HT1080, 8387, NCI, and a spontaneous transformed cell line, VIP:FT. Total proteins from sodium orthovanadate untreated cell lines (A) and from sodium orthovanadate treated cell lines (8) were analyzed by Western blotting as described in the Materials and Methods. Panel A is NCI (lane 1), 8387 (lane 2), VIP:FT (lane 3), HT1080 (lane 4) and~SHAC (lane 5). The lysate from the normal human fibroblast, LG-1, was also run on the gel (lane 6). Panel B is HT1080 (lane 1), VIP:TF (lane 2), SHAC (lane 3), 8387 (lane 4) and NCI (lane 5). Arrows indicate unique phosphoproteins in these cell lines. The filter of panel A. was exposed to a film for 48 hours. The filter of panel 8 containing lanes 1, 2 was exposed for 6 hours and the film containing lanes 3,4,5 was exposed for 16 hours using intensifying screens. 58 (B) (A) ~140 Fig.4 59 ° .. - I . '1' oh- . -- un . in; pro - n in hu an rr- r 01A- Wines To determine whether the fibroblasts transformed in vitro were similar to cells transformed in vivo (VIP:FT is an exception), we examined the phosphotyrosine pattern of five human f ibrosarcoma-derived cell lines, HT1080, 8387, SHAC and NCI, and VIP:FT ( a spontaneous transformed human fibroblast reported to generate fibrosarcomas in nude mice) , were analyzed by Western blotting using the antibody against phosphotyrosines (figure 4A). Although the pattern of phosphoproteins was very similar to that of normal fibroblasts (LG-1), unique enhanced tyrosine kinase substrates were clearly detected in each of the cell lines, except 8387 cell strain. The VIP:FT cell line showed a unique band at 72k0a. NCI cells showed a unique band at 62k0a. No extra phosphoprotein bands were detected in the SHAC and HT1080 cell strains, however, SHAC, NCI, HT1080 and VIP:FT all expressed the p120k0a, p110kDa and 84kDa phosphoproteins at levels at least 2 fold higher than the normal fibroblasts. The normal fibroblasts and all the fibrosarcoma cell lines contain phosphorylated proteins of 120k0a, 110k0a, 80k0a, 71k0a, 60k0a, 50k0a, 46k0a and 43k0a. The pattern of the phosphotyrosine-containing proteins within the 8387 cell line and normal fibroblast cell line is alrrost identical, including a lower phosphotyrosine content of p120k0a and p110kDa bands, and a p210k0a phosphoprotein band which is absent from the other fibrosarcoma cell lines. Figure 4B shows that incubation of the cells with sodium orthovanadate enhanced the phosphorylation of the substrates of tyrosine kinase, especially the proteins over 120 kilodalton. The effect of vanadate on the various phosphoproteins differs. The pattern of 60 phosphotyrosine-containing proteins is very similar for VIP:FT and HT1080 and quite different from that of 8387, NCI and SHAC cells. Both the VIP:FT and HT1080 cell Strains contairia novel phosphoprotein, pp140k0a, that was not seen in the untreated cells. VIP:FT treated with vanadate also expressed the pp84kDa and pp72k0a at higher levels than the other fibrosarcoma cell lines. , The molecular weight of the v-fes protein is known to be 110k0a by immunoprecipitation using anti-v-fes antibody (data not shown). Since the NCI cell strain strongly expressed a p110kDa phosphoprotein at the strongest expression level and the anti-v-fes antibody is reported able to recognize human c-fes protein product (Yu et al., 1987), we decided to test the possibility that the p110kDa in the NCI cell line was indeed the c-fes protein. We harvested a v-fes transformed cell line and NCI cell line, prepared cell lysates, and then immunoprecipitated the lysates with an anti-v-fes antibody. The immunoprecipitated lysates were then electrophoresed on SOS-PAGE gel along with the nonprecipitated NCI cell lysates. Proteins were transferred to Immobilon-P paper and analyzed by Western blotting using anti-phosphotyrosine antibody. The result (Figure 5) showed that the p110kDa protein in the NCI cell line *was not immunoprecipitated by the v-fes antibody indicating that the protein is not the c-fes oncogene product. 61 Figure 5. Immunoprecipitation of NCI cells with anti-v-fes antibody. The lysate of a v-fes transfected cell strain was inmunoprecipitaed with anti- v-fes antibody as a positive control (lane 1 ). NCI cells were lysed in RIPA buffer and immunoprecipitated with an anti-v-fes antibody (lane 2), or lysed in Laemmli sample buffer without immunoprecipitaion (lane 3), Total proteins and immunoprecipitates were seperated by 8% SOS/PAGE gel electrophoresis and analyzed by Western blotting using the antiphosphotyrosine antibody as described in the Materials and Methods. The arrow indicates the p110K0a“‘“’protein. The filter was exposed to a film for 48 hours using intensifying screens. 62 Fig. 5 Discussion Western blotting technique using an anti-phosphotyrosine antibody allowed us to detect the difference>of tyrosine phosphorylation pattern in various cell stains. More or less phosphorylated proteins of specific molecular weight were observed. The explanation for the differences could be due to the differences in phosphatase activity, tyrosine kinase activity, amount of substrate proteins, growth rate or other factors. Hyperphosphorylated phosphotyrosine-containing proteins were detected in some transformed cells, such as the p72k0a protein of the VIP:FT cell line, and the p110kDa and p120k0a proteins of the HT1080, NCI, SHAC and VIP:FT cell lines. There areitwo possibilities that would explain the difference in the pattern of tyrosine phosphoproteins. First, growth factor or growth factor receptor overexpression has been reported in many tumor cell lines and transformed cell strains. For example, the A431 cell line derived from a human squamous carcinoma has an increased number of EGF receptors (Cohen et al., 1982). In addition, the production of PDGF and its receptor has been reported in the U-2 OS cell line derived from an osteosarcoma (Betshdtz et al., 1984). The activation of'a proto-oncogene which encodes a growth factor receptor-like protein could also explain the increased level observed in tyrosine phosphoryaltion. A good example of this is the overexpression of p185"‘5“°"’""u ( an EGF receptor-like protein) in human mammary and ovarian carcinomas. The HER-2/neu proto-oncogene is amplified 25-30% in breast cancers and the extent of expression is associated witha patient’s prognosis, strongly suggesting a critical role for the EGF receptor-likeityrosine kinase in tumor progression (Slamon et 63 64 al., 1989). Other investigations have suggested that the autocrine stimulation of overexpressed growth factor receptors may be essential for cellular transformation (Velu et al., 1987; Riedel et al., 1988). The overexpression of either growth factor(s) or growth factor receptor(s) may lead to the constitutive activation of tyrosine kinase(s) which may lead to the unregulated phosphorylation of specific substrates on tyrosine. A second possibility is that a proto-oncogene encoding a tyrosine kinase is activated by a point mutation, gene amplification or gene translocation‘which leads to increased tyrosine phosphorylation activity. However, so far, little evidence has shown that the activation of these genes play a role in human cancer. Reports have shown that tissue and cell lines established from turrors of neuroectodermal origin express high levels of c-src with high kinase activity (Brugge et al., 1985; Bolen et al., 1985). In addition, human fibroblasts transfected with a plasmid carrying the v-fes oncogene , which belongs to this group of oncogenes, generate invasive, fast growing tumors in athymic mice and show enhanced phosphotyrosine-containing protein expression compared to control cells (McCormick et al., unpublished data). These data suggest that the human c- fes gene involves in the generation of human tumors of mesenchymal origin from an activated c-fes. In this paper, we compared the pattern of phosphotyrosine-containing proteins in some human fibrosarcoma-derived cell lines. Each cell line showed a slightly different pattern. This suggests that there is some difference in the production of different growth factors or/and growth factor receptors, or an internal gene change of a proto-oncogene with tyrosine kinase activity are involved in the tumorigenicity of these 65 fibrosarcomas. When the cells were incubated in the presence of sodium orthovanadate, enhanced expression of phosphotyrosine-containing proteins was detected. The phosphotyrosine pattern for the VIP:FT and HT1080 was very similar but differed from that of other cell lines, suggesting that VIP:FT and HT1080 cell lines utilize the same or very similar growth pathway. The transformation of human cells is considered to be a multi- stepped process ( for review, see Barret and Fletcher, 1987). In this paper, the data from MSU-l cell lineage strongly support this theory. The pattern of the phosphotyrosine-containing proteins correlates with the transformed phenotype of each cell strain, and also with the degree of growth factor independence. Both the growth factor dependent normal human fibroblasts and MSU-1.0 cells showed a similar pattern. The partial growth factor dependent MSU-1.1 cells showed a similar pattern but exhibited a greater degree of phosphorylation of two proteins. 0n the other hand, the completely growth factor independent MSU-1.1 V0 cells contained two unique phosphoproteins. The p180k0a protein of MSU-1.1 V0 cells could be the PDGF receptor, since a high level of PDGF production has been found to be the characteristic of this cell strain. Similar evidence has also shown that tumor-derived cell lines from carcinogen transformed Syrian hamster embryo cells contained several novel tyrosine phosphoproteins in addition to those found in non-tumorigenic transformed cell strains and normal parental cells (Kanner et al., 1989). Numerous cellular substrates of receptors with tyrosine kinase activity have been identified (for review, see Ullrich and Schlessinger, 1990). Because of the limited sensitivity of the technique, we could see 66 only the proteins containing the 15-20 most highly phosphorylated tyrosines. Having seen this pattern, we could in the future detect specific phosphorylated proteins such as GAP by immunoprecipitation of the cell lysate with anti-GAP antibody followed by the immunoblot analysis with an anti-phosphotyrosine antibody (Ellis et al., 1990; Kaplan et al., 1990). The phosphotyrosine-containing proteins identified thus far have been found to play a role in cellular transformation. Many of these include receptors with tyrosine kinase activity. Most of these proteins are either proto-oncogene products, components in the secondary messenger pathways or factors that regulate thetactivity'of proto-oncogene products. The identification‘and characterization of these proteins may shed light on the mechanism of growth control pathways as well as provide an understanding of oncogenesis. Reference Antler, A.M., Greenberg, M.E., Feldman, G.M., and Hanafusa, H. 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Science 235 542-545 Tucker, R.F., Volkenent, M.E., Branum, E.L., and Moses, H.L. (1983) Comparison of intra- and extracellular transforming growth factors from nontransformed and chemically transformed mouse embryo cells. Cancer Res. 53 1581-1586 Ullrich, A., Coussen, L., Hayflick, J.S., Dull, T.J., Gray, A., Tam, A.W., Lee, J., Yarden, Y., Liberman, T.A., Schlessinger, J., Downward, J., Mayes, E.L.V., Whittle, N., Waterfield, M.D., and Seeburg, P.H. (1984) Human epidermal growth factor receptor c0NA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cell. Nature 359 418-425 Ullrich, A., and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 51 203-212 69 Velu, T.J., Beguinot, L., Vass, W.C., Willingham, M.C., Merlino, G.T., Pastan, I., and Lowy, D.R. (1987) Epidermal growth factor-dependent transformation by a human EGF receptor proto-oncogene. Science 231 1408- 410 Vogel, U.S., Dixon, R.A., Schaber, M.D., Diehl, R.E., Marshall, M.S., Scolnick, E.M., Sigal, I.S., and Gibbs, J.B. (1988) Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature 335 90-93 Yu, G., and Glazer, R.I. (1987) Purification and characterization of pm“ and p“"" related tyrosine protein kinase activities in differentiated HL- 60 leukemia cells. J. Biol. Chem. 252 17543-17548 Wahl, M.I., Olashaw, N.E., Nishibe, S., Rhee, S.G., Pledger, W.J., and Carpenter, G. (1989) PDGF induces rapid and sustained tyrosine phosphorylation of phospholipase c-r in quiescent Balb/c 3T3 cells. Mol. Cell. Biol. 9 2934-2943 CHAPTER III Enhance-ent of tyrosine phosphorylation in v-fes transfer-ed huean fibroblasts Chin-Huei Pan, Veronica M. Maher and J. Justin McCormick Carcinogenesis Laboratory, Fee Hall Department of Microbiology and Public Health Michigan State University, East Lansing, MI 48824-1316 70 Abstract Using the Western blotting analysis and a monoclonal antibody against phosphotyrosine, we found that two human fibroblast cell strains derived from tumors arising after injection of a transformed cell strain that had been transfected with a plasmid carrying provirus of the Gardner- .Arnstein strain of feline sarcoma virus (v-fes oncogene), express specific phosphotyrosine-containing proteins of molecular weight at 150k0a, 110k0a, BBkDa, 80kDa and 68kDa at higher levels. The pp110k0a in the v-fes transfected cell strains was shown to be the v-fes encoded protein by immunoprecipitation with anti-v-fes antibody. Data also showed that the expression level of the v-fes protein correlated with its tyrosine phosphorylation level. As the v-fes expression increased, the amount of phosphotyrosine-containing proteins present in the cells also increased. Untransfected control cells incubated in the presence of an inhibitor of . phosphotyrosine phosphatases, sodium orthovanadate, resulted in an enhanced phosphorylation of the same substrates of v-fes tyrosine kinase. These studies suggest that the more intense tyrosine phosphorylation in the fes expressing cells, which correlates with high expression of the v- fes protein is involved in the malignant transformation of the v-fes transfected cells. 71 Introduction The 130k0a transforming protein of Fujinami avian sarcoma virus , fps, is a tyrosine kinase that can undergo autophosphorylation on tyrosine and serine residues (Weinmaster et al., 1984; Weinmaster et al., 1985). The chicken c-fps gene is homologous to the cat c-fes gene based upon the very high degree of sequence homology and other evidence (Shibuya and Hanafusa, 1984). C-fes is the cellular homologue of the transforming gene of several independently derived, acutely transforming feline retroviruses: Gardner-Arnstein (GA), Hardy-Zuckerman 1 (H2 1) and Snyder- Theilner(ST) sarcoma virus ( for review, see Bishop and Varmus, 1982). The protooncogene product of the mammalian c-fes (NCP92) is also a cellular tyrosine kinase that can undergo autophosphorylation (Feldman et al., 1985:1987). It has been shown that c-fes expression is restricted to hematopoietic cells of the myeloid lineage. The highest levels of NCP92 were found in tissue macrophages and in boneimarrow cells (Samarat et al., 1985). This suggests that the protein may play a role in normal hematopoietic differentiation. NCP92 has been detected in the primary human myeloid leukemic cells of granulocytic and monocytic origin (Feldman et al., 1985), suggesting an important role for the c-fes protein in the mechanisms that regulate cell differentiation and proliferation. It has been shown that transfection with a plasmid containing the provirus of the Gardner-Arnstein strain of Feline sarcoma virus can 72 73 transform feline (Haynes et al., 1988) , rodent(Sodroski et al., 1984) and human fibroblasts (J.J. McCormick, unpublished data). Overexpression of human c-fes in NIH3T3 cell line can cause cellular transformation when a retroviral vector is used (Feldman et al., 1990). All the fps/fes containing retroviruses induce fibrosarcomas and myxosarcomas in experimental animals (Hanafusa et al., 1980). Since the feline c-fes and the human c-fes share 94% homology of the amino acid sequence (Roebroek et al., 1985), we postulated that the feline v-fes oncogene could act in the same manner as the activated human c-fes in vivo, and that the c-fes may be the transforming protein in some human tumors. To examine the process of malignant transformation, McCormick and his colleagues have developed a lineage of human fibroblasts ( MSU-l) in which each successive strain expresses a somewhat more transformed phenotype than the strain itiwas derived from. In this paper, we have used a spontaneous variant of the MSU-1.1 strain that is partially growth factor independent and non-tumorigenic, NSU-1.1 V0, as the recipient cell of the v-fes oncogene. The MSU-1.1 V0 cell strain is totally growth factor independent, partially' anchorage independent, and does not generate malignant tumors in athymic mice. In this paper, we show that transformation of the MSU-1.1 V0 cell strain in culture with v-fes results in cells which can produce malignant tumors in athymic mice. We detected several enhanced phosphotyrosine-containing proteins, pp150k0a, pp110k0a, ppBBkDa, pp80k0a and pp68k0a, in the v-fes transfected cells by Western blotting analysis using an anti-phosphotyrosine antibody. The pp110k0a phosphoprotein was shown to be the v-fes product by immunoprecipitation 74 assay. We also correlated the expression level of the v-fes protein with the tyrosine phosphorylation level. Materials and Methods ur i n A spontaneous variant of the infinite life span, nontumorigenic MSU- 1.1 has been selected using a low calcium, serum-free media to select for growth factor independent variants. This strain is designated MSU-1.1 V0 which is completely growth factor independent, forms small colonies in agarose and generates low grade malignant tumors in athymic mice. MSU-1.1 V0 cells were transfected with a plasmid carrying the provirus of the Gardner-Arstein strain of feline sarcoma virus. Independent clones were selected in soft agarose based on their ability to form large colonies (>300um). Each clone was then grown and 1x107 cells were injected into Balb/c athymic mice to determine the tumorigenic potential of each isolated clone. Two such cell strains (VO-fes-I and VO-fes-Z) generated progressively growing, invasive, spindle cell sarcomas in three weeks. Other three v-fes transfected cell strains (VO-fes-3, VO-fes-4, and V0- fes-5) were also studied in this paper, and their tumorigenicities were tested later (table 1). All the cells were maintained in H-McM medium, supplemented with 5% fetal bovine serum, 5% supplemented calf serum (Hyclone Inc., Logan, UT) , penicillin, streptomycin, and hydrocortisone at 37°C in water saturated incubator with 5% C02. Ereparatioo_o£_cell_lxsates To prepare samples for Western blot analysis, monolayers of cells in P100 culture dishes were washed twice with 10 ml of PBS buffer ( 15mM sodium phosphate pH7.5; 150mM NaCl), and the buffer was aspirated 75 76 completely. Two hundred microliters of protein sample buffer (10% glycerol; 0.06M Tris-Hcl pH6.8; 2% sodium dodecyl sulfate; 5% mercaptoethanol; 0.005% Bromophenol Blue) supplemented with 0.2mM sodium orhtovanadate (Aldrich Chemical Co., Milwaukee, WI), 2uM phenylmethylsulfonyl fluoride (8M8 Co., Indianapolis, IN) and 5ul/ml A- Protinin (Sigma Co., ST. Louis, MO) was applied to the plates. The viscous cell lysate was scraped from the plates by using a rubber policeman. The lysate was immediately boiled 10 minutes , sonicated thirty seconds, and stored at -80° C prior to use. Parallel plates of cells were lysed in RIPA buffer (0.1M Tris-HCl pH7.5; 0.15M NaCl; 1% Deoxycholic acid; 1% Triton x- 100; 5mM EDTA pH8.0; 0.1% NaDodSO‘ ). Total protein concentration was measured in the RIPA buffer lysate with the BCA protein assay kit. (Pierce Chemical, Rockford, IL.) Analysjs 5f v-fe; prgtein by immungpregipitatign Equal amounts of protein lysate (150ug) were immunoprecipitated with a rat anti-v-fes monoclonal antibody (Oncogene Inc., Manhasset, NY, Antibody Ab-l) that reacts specifically with the v-fes encoded sequence common to the translational products of the Snyder-Theiler and Gardner strains of feline sarcoma viruses. Protein-A agarose (Oncogene Inc.) coated with goat anti-rat IgG (Oncogene Inc.) was added, and the mixture was shaken for 30 minutes at 4°C. The agarose fraction was washed twice with RIPA buffer, once with 50mM Tris pH7.5 containing 1M MgClz and one more time with RIPA buffer. NaDodSO‘-PAGE sample buffer was added, and the samples were boiled for 5 minutes , then centrifuged, and electrophoresed in 8% NaDOdSO‘-polyacrylamide gels. 77 n l in Equal amounts of protein (150ug) were loaded onto 8% NaDodSO‘- polyacrylamide gel, and the proteins were separated by electrophoresis as described by Laemmli (Laemmli, 1970). Transfer of proteins to Immobilon-P paper (Millipore Inc, Bedford, MA) was done in the presence of 25mM Tris, 192m glycine and 0.375% NaDodSO‘ using a Trans-Blot Electrophoretic Transfer Cell (Biorad, Richmond, Ca). Filters were blocked for 36 hours at room temperature using 3% powdered non-fat dry milk in TBS (20mM Tris; 500mM NaCl pH7.5) with 0.2% sodium azide. After rinsing in TBS briefly, the filters were incubated overnight in 1% milk-TBS supplemented with 0.05% Tween 20 and lug/ml mouse anti-Phosphotyrosine monoclonal antibody, PY20 (ICN Inc. Costa Mesa, Ca.). The filters were washed four times with TBS containing 0.1% Tween 20 (washing buffer), then incubated with goat anti-mouse IgG antibody (Cappel Inc, Durham, NC) for 90 minutes. After rinsing the filters five times with the washing buffer, they were reacted with 10uCi [“51] Protein-A (ICN Inc.) for 2 hours. The filters were then washed five times with extensive amount of washing buffer for 10 minutes each time, and exposed to Kodak X-RP films at -80°C for 36-48 hours using intensifying screens. The molecular weights of proteins were estimated by myosin (200k0a), phosphorylase 8 (97k0a), bovine serum albumin (68k0a), and ovalbumin (43k0a) on each gel. '78 Table. 1 v-fes Transfected Cell Strains. Cell Strain Diameter of Colony fes Protein Tumorigenesis in Agarose Expression VO-fes-I 500um +++ + VO-fes-Z 500um +++ + V0-fes-3 280um - - VO-fes-4 440um + + VO-fes-S 9000m +++ + Results 1 -r -- oi- rho . i -- onttini . pro -in in v-f- ran f-rm-a -ll Two v-fes tumor derived cell strains, VO-fes-I and VO-fes-Z, were grown and immunoprecipitation of 35S labelled cellular proteins showed that the tumor cells expressed the v-fes oncogene product at a high level. The phosphotyrosinc-containing proteins of these two tumor-derived cell lines were detected by Western blotting using a specific antibody against phosphotyrosine. Both cell lines show identical pattern of tyrosine phosphorylation (data not shown). In contrast to MSU-1.1 VO cells, prominent tyrosine kinase substrates were clearly detected in the v-fes transfected cell lines (Figure 1). The molecular weight of these phosphoproteins shown on the blot are 150k0a, 110k0a and 88kda. Two other proteins, ppBOkda and pp68kda, also could be detected in the MSU-1.1 V0 cells from which it derived, but the expression level was much lower than that of v-fes transformed tumor-derived cells (Figure 1). If the x-ray film was exposed after a longer period of time, 10-15 bands representing phosphotyrosine-containing proteins were observed in both the normal human fibroblasts and the v-fes transformed cells. This suggests that the normal and transformed cells share many of the same phosphorylated proteins. i v-f r in Among the novel tyrosine kinase substrates in the v-fes transformed tumor-derived cell line, the pp110k0a protein had the strongest expression level and its molecular weight was very close to that of the v-fes protein. To test the possibility that the p110kDa was indeed the v-fes 79 80 Figure 1. Enhanced expression of phosphotyrosine-containing proteins in v- fes transformed cells. Proteins lysates were run on 8% SOS/PAGE gels, transferred to Immobilon-P paper and Western blotting analysis performed using an anti-phosphotyrosine antibody as described in the Material and Methods. Arrows indicate five phosphoproteins showing enhanced phosphotyrosine content in the v-fes transformed cells. Lane 1 contains lysate from control MSU-1.1 V0 cells, lane 2 contains the V-fes transfected MSU-1.1 V0 cells lysate. The molecular weights of protein standards are shown in kilodaltons on the side. The film was exposed for 18 hours using intensifying screens. 2oo— , 97— . O I Figure 1 43- 82 protein, we harvested the v-fes transformed tumor-derived cells, prepared lysates, and then immunoprecipitated the lysates with antibody against v- fes. The immunoprecipitated lysates were then subjected to SOS-PAGE gel electrophoresis along with the nonprecipitated cell lysates. Proteins were transferred to Immobilon-P paper and analyzed by Western blotting using phosphotyrosine antibody (Figure 2, lane 1). The results show that the pp110k0a protein in the tumor-derived cells is immunoprecipitated by the v-fes antibody, indicating that the protein is the v-fes oncogene product. Furthermore, since the v-fes protein can be autophosphorylated on tyrosine residues, we also immunopreicipitated the lysates with the antiphosphotyrosine antibody in order to amplify the concentration of the phosphotyrosine-containing proteins in the samples. The lysates were then run on a gel, transferred and analyzed by Western blotting as described before. A protein with an apparent molecular weight of 110K0a was detected (Figure 2, lane 4). . - a i- -f v-f- - rr- i-n l-v-l ;n- . in- oh- uh-r lg i- l-v-l Since the v-fes protein is a tyrosine kinase, and has the ability to phosphorylate other proteins in vi vo, we examined the relationship between the v-fes expression level and the tyrosine phosphorylation level. Three independent v-fes transfected agarose isolated cell strains which exhibited different v-fes protein expression levels( VO-fes-3, V0-fes-4, VO-fes-5 Table 1) were studied. Cells were metabolically labelled with [35S] methionine and cysteine, lysates were immunoprecipitated with the anti-v-fes antibody and analyzed using the SOS-PAGE electrophoresis and autoradiography (Data not shown).The pp110"’f°5 of VO-fes-3 cells is . .ii.“cih.‘hat— 83 Figure 2. Identification of the p110"’fes protein in v-fes transformed cells. Lane 1 contains the non-immunoprecipitated lysates of v-fes transformed cells. Cells were lysed in RIPA buffer for immunoprecipitation with anti-v-fes antibody (lane 2) or anti-phosphotyrosine antibody (lane 4), and the proteins were separated by 8% SOS/PAGE gel electrophoresis and analyzed by Western blotting using the anti-phosphotyrosine antibody as described before. Lane 3 contains the lysate of the VO-fes-I cells which was not precipitated with any specific antibody but with agarose-protein A as a negative control. The arrow indicates the p110"'fes protein. The film was exposed for 36 hours using intensifying screens. 84 Fig. 2 85 expressed at undetectable levels compared to the untransfected MSU-1.1 V0. VO-fes-4 cells expressed the protein pp110"'fes at levels above control levels and VO-fes-5 cells expressed the protein at levels 3-5 fold higher than VO-fes-4 cells (Table I). We then measured the tyrosine kinase activity in the cell strains using nonprecipiated cell lysates and v-fes antibody precipitated cell lysates. The cell lysates were run out on a gel and Western analysis performed using the antiphosphotyrosine antibody. Figure 3 shows that the amount of p110“es phosphotyrosine in VO-fes-S cells is similar to the level in the v-fes-I cells tested before and at least two fold higher than that of V0-fes-4 cells. In addition, the level of phosphorylation of the 150k0a, 88k0a, and thei68k0a proteins in VO-fes- 5 is higher than that of V0-fes-4. The phosphoprotein content of these proteins in V0-fes-3 is as same as that of the MUS-1.1 V0 cell strain, suggesting that the VO-fes-3 cells do not express the transfected v-fes oncogene. This data shows a good correlation between the expression level of v-fes and the v-fes tyrosine phosphorylation level within the cell. As the v-fes expression increases, the amount of phosphotyrosine present in the cell also increases. orig e gov: .r: - ' ht. ‘ h‘ r‘ ‘ ls sf ‘1 Tgr the r 0 Te ‘- contaiu1n9_nrctelns Sodium orthovanadate is an effective inhibitor of phosphotyrosine phosphatases ( Leis and Kaplan, 1982; Swarup et al., 1982). Cells incubated in the presence of the compound accumulate phosphotyrosine in proteins. The use of the inhibitor allows one to study substrates of cellular tyrosine kinases which may not be detectable under other 86 M“ protein expression level and Figure 3. The correlation of p110 tyrosine phosphorylation level. (A) Proteins lysate from nontransfected control MSU-1.1 V0 cells (lane 1), V0-fes-3 cells (lane 2), V0-fes-4 cells (lane 3), V0-fes-5 cells(lane 4) and VO-fes-I cells (lane15) weretanalyzed by Western blotting using the anti-phosphotyrosine antibody as described before. (8) V-fes transfected cells , VO-fes-3 (lane 1), VO-fes-4 (lane 2), VO-fes-S (lane 3) and VO-fes-l (lane 4), were lysed in the RIPA buffer, immunoprecipited with the anti-v-fes antibody, and then analyzed by Western blotting using the tanti-phosphotyrosine antibody. Arrows indicate the p110v'f” protein. The 90 kDa protein observed in all the cell stains immunoprecipitated with anti-v-fes antibody represent a non- specifically bound protein and demonstrates that the proteins were loaded equally on the gel. The blot of panel A was exposed to a film for 16 hours, and the blot of panel 8 was exposed to a film for 36 hours using intensifying screens. 87 (A) (B) 200- 1 2 3 4 .. .9 ‘ —pllOKDe 1 511993th 97— I 68— 43— FIg. 3 88 conditions because the phosphotyrosine turnover is too rapid in the cells. Total cellular protein from the untreated MSU-1.1 V0 (Figure 4, lane 1), a v-fes transformed tumor derived cell line (Figure 4, lane 2) and MSU-1.1 V0 treated with 100uM Sodium orthovanadate for 18 hours prior to lysis (Figure 4, lane 3) were analyzed by Western blotting with antiphosphotyrosine antibody. The preincubation of MSU-1.1 V0 cells with vanadate increased the phosphotyrosine content of all the substrates observed in the fes tumor cells with the exception of the pp110"""'es . Vanadate also enhanced the level of phosphotyrosine in three proteins not seen in the v-fes transformed cells. The molecular weights of those proteins were 180k0a, 120k0a, and 115k0a. 89 Figure 4. Sodium orthovanadate treatment of cells enhances the detection of cellular phosphotyrosine-containing proteins. Protein lysates from control MSU-1.1 V0 cells (line 1), VO-fes-I cells (line 2), MSU-1.1 V0 cells incubated with 100uM sodium orthovanadate for 18 hours (line 3) were analyzed by Western blotting using the anti-phosphotyrosine antibody as described before. Arrows indicate the enhanced phosphotyrosine-containing proteins which were observed in the v-fes transformed cell strains. Arrows with asterisk indicate novel phosphoproteins unique to vanadate treated MSU-1.1 VO cells. The film was exposed for 16 hours using intensifying screens . 90 200- ‘ iflg.4 Discussion A variety of tyrosine kinase substrates are known in cells transformed by the src oncogene, and few substrates have been identified in the cells transformed by fes or fps. Since in cells transformed by the src oncogene of Rous sarcoma virus, some of the substrates of p60"""c tyrosine kinase were also phosphorylated by other viral tyrosine kinase encoded by the oncogenes, such as v-fps (Kamps and Sefton, 1988), it is possible that some of the substrates of the fes encoded tyrosine kinase are among those identified substrates of src oncogene. These phosphoproteins include the cytoskeleton protein vinculin (Antler et al., 1985), talin (Declue et al., 1987), the fibronectin receptor complex (Hirst et al., 1986). Calpactin, also referred to as the pp36k0a protein that shows actin and phospholipid binding properties, is known to be phosphorylated by src tyrosine kinase activity (Radke et al., 1980; Erikson and Erikson, 1980). There is little evidence suggesting a functional connection between the phosphorylation of these cytoskeleton proteins and the malignant transformation of the src-transformed cells. However data showed that in src-transformed cells, foCal contacts were fewer and smaller, the cytoskeleton became disordered, and the cells rounded up and were generally less adhesive (David-Pferty and Singer, 1980). Other tyrosine'kinase substrates include calmodulin (Fukami et al., 1986), microtubule associated protein (Akiyama et al., 1986), protolytic enzymes( Cooper et al., 1983) and a PI kinase (Cohen et al., 1990). In this paper, we observed that five proteins, which are expressed at high levels, were phosphorylated on tyrosine in the v-fes transformed 91 92 human fibroblasts. On the basis of this similarity in molecular weight, the 150k0a phosphoprotein detected by us in the fes expressing cells may be the phosphorylated fibronectin receptor complex reported for the src- transformed cells (Hirst et al., 1986). The identification of these phosphoproteins may shed light on the correlation between the malignant transformation of v-fes oncogene and its tyrosine kinase activity. Incubation of cells with sodium orthovanadate can increase the steady-state level of phosphotyrosine in the substrates of cellular protein tyrosine kinases. We found that the three specific substrates of pp110Mes can also be found as phosphorylated on tyrosines in the untransfected normal cells when treated with vanadate, indicating that the pattern of tyrosine phosphorylation in the fes transfected cells and the control cells are qualitatively the same, but more intense in the fes- expressing cells. These results are consistent with the findings by Kamps and Sefton and others (Kamps and Sefton , 1988 ) who showed that all of 0"'src contain significant amounts of the phosphorylated the substrates of p6 tyrosines in normal NIH 3T3 cells treated with vanadate. These data also suggested that the viral protein tyrosine kinases‘ induce cellular transformation through the intervention in normal cellular regulatory pathways that are controlled in normal cells by tyrosine protein phosphorylation, rather than through the phosphorylation of proteins that are not natural substrates of cellular tyrosine kinase. Additionally, the data shown in this paper suggests that the 'tumorigenesis of v-fes transfected cells involves a greater amount of tyrosine phosphorylation on specific proteins. Several investigations have shown that the expression of the c- 93 fps/fes gene product in human cells is restricted to cells of the monocyte, macrophage and granulocyte lineages of both normal and tumor origin (Feldman et al., 1985). However, the v-fes of Feline Sarcoma Virus can transform NIH3T3 mouse fibroblast cells (Sodroski et al., 1984) and human fibroblasts (Milo et al., 1980), so that the resulting cells generate tumors in nude mice. 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