Sep 17, 1985 - protein of 60,000 daltons which possesses intrinsic protein kinase activity specific for tyrosine residues (1, 7, 9, 16; for a review, see reference ...
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1986, p. 735-738 0270-7306/86/020735-04$02.00/0 Copyright © 1986, American Society for Microbiology
Vol. 6, No. 2
Novel Serine Phosphorylation of pp6Oc-src in Intact Cells after Tumor Promoter Treatment LARRY E. GENTRY,* KAREN E. CHAFFIN, MOHAMMED SHOYAB, AND A. F. PURCHIO ONCOGEN, Seattle, Washington 98121 Received 17 September 1985/Accepted 21 November 1985
Treatment of normal cells with the tumor promoters 12-0-tetradecanoylphorbol-13-acetate and mezerein results in increased phosphorylation of pp60csrc. Two-dimensional tryptic phosphopeptide analysis of partial V8 protease fragments indicated that this phosphorylation takes place on a serine residue which lies within the amino-terminal 18 kilodaltons of pp60csrc and represents the major phosphorylation site following tumor promoter treatment. Untreated cells exhibited a low but detectable level of phosphorylation at this serine residue. The significance of these results with respect to the phosphoregulation of pp60c.S as well as tumor promotion is discussed.
The virally encoded oncogenic protein pp6Ov-src has been the most widely studied of the transforming proteins. It is a protein of 60,000 daltons which possesses intrinsic protein kinase activity specific for tyrosine residues (1, 7, 9, 16; for a review, see reference 15). Normal cells contain an analog of pp60v-src termed pp60c-src (6, 30). pp6Ov-src and pp6Ocsrc are phosphoproteins containing both serine and tyrosine phosphorylation sites. In both cases, the major phosphorylated serine site is located in the amino-terminal region of the proteins at serine 17 (12). Phosphorylation of this site is thought to occur in a cAMP-dependent fashion (8). The major phosphotyrosine resides within the carboxy-terminal half of the pp6Osrc molecule (8, 23, 29). For pp6Ovsrc, tyrosine 416 has been identified as the major tyrosine phosphoacceptor site (23, 29). Several minor phosphorylation sites have also been observed in the amino-terminal half of the pp65rc proteins; however, their precise locations have not been determined (5, 10, 25). Since phosphorylation-dephosphorylation has been shown to be a powerful means of regulating protein function, it is reasonable to assume that these phosphorylation events are important regulatory signals for the pp60Src proteins. Understanding the effect of the phosphorylation of these tyrosine kinases may provide insight into their function in both cellular growth and differentiation. In a previous report, we demonstrated that tumor promoters induce hyperphosphorylation of pp6Ov-src at a new serine site located within the amino-terminal 18,000 daltons of the pp60v-src molecule (24). We were therefore interested to determine whether the phosphorylation state of the cellular form of the src gene product, pp60csrc, was also affected by phorbol ester treatment. To test this, we used a normal mink lung cell line which earlier had been shown to be responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) (27, 28). Nearly confluent cultures of cells were labeled for 4 h with 1 mCi Of 32p, per ml in phosphate-free Dulbecco modified Eagle medium containing 2.5% fetal bovine serum and 2.5% bovine serum and subsequently treated for 20 min with either dimethyl sulfoxide (DMSO; 0.2% [vol/vol] final concentration) or TPA (100 ng/ml; 0.2% DMSO). The pp60csrc molecules were then immunoprecipitated as previously described (20) with monoclonal antibody 327 (a generous gift of *
J. Brugge). An autoradiogram of sodium dodecyl sulfate-gel profile of pp60c-src immunoprecipitates from 32P-labeled cells normalized for equivalent amounts of protein is shown in Fig. 1A. pp6Oc-src molecules from TPA-treated cells showed an increase in phosphorylation compared with DMSOtreated control cells. Cerenkov quantitation indicated a greater than 80% increase in phosphorylation following TPA treatment. In contrast, the specific activity of total cellular phosphoproteins did not change during the brief TPA treatments (data not shown). Figure 1B shows a limited V8 protease analysis of pp60c-src immunoprecipitated from cells treated with TPA or control cells treated with DMSO. Partial digests were performed with 50 ng of V8 protease as described previously (3, 8). Vl and V2 indicate the amino- and carboxy-terminal fragments, respectively, of the src protein (8). Fragments V3 and V4 are derived from Vl and represent the amino-terminal 18 and 16 kilodaltons of the protein (8). A comparison of lane 1 (DMSO treated) with lane 2 (TPA treated) demonstrates that the increased phosphorylation occurred predominately within the amino-terminal fragments Vl, V3, and V4. Phosphoamino acid analysis revealed that the increased phosphorylation in pp60c-src occurred at a serine residue, as reported earlier for pp6Ov-src (24). Comparative two-dimensional tryptic peptide maps are shown in Fig. 2. The V3 plus V4 protease fragments of pp60csrc were isolated from preparative V8 protease maps, digested with trypsin, and fractionated in two dimensions as previously described (8). The peptide map of pp60csrc from control cells displayed primarily one major phosphopeptide (panel A). The peptide map of the V3 plus V4 fragments from TPA-treated cells, however, revealed the dramatic appearance of another phosphopeptide migrating as a negatively charged species (panel B). Close examination of the peptide map from untreated pp60c-src indicated that a similar phosphopeptide exists, but at a much reduced level. The TPA-induced phosphorylation site of pp60csrc was similar in its electrophoretic mobility to that observed for pp60v-src (24). To test the relatedness of these tryptic phosphopeptides, we performed two-dimensional tryptic peptide mapping. The results are shown in Fig. 2. Panels B and C show the tryptic phosphopeptide maps of the V3 plus V4 fragments derived from pp6Oc-src of mink lung cells and pp60v-src of Schmidt-Ruppin D-transformed BALB/c 3T3
Corresponding author. 735
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NOTES
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FIG. 1. TPA treatment of normal mink lung cells. Panel A, immunoprecipitation analysis of pp6o0-src from detergent extracts of 32Pi-labeled cells. Immunoprecipitates were prepared from equivalent amounts of detergent-solubilized proteins and analyzed by electrophoresis on 10% polyacrylamide gels. Lane 1, immunoprecipitates from control cells treated with DMSO. Lane 2, immunoprecipitates from cells treated with TPA. Panel B, one-dimensional V8 protease mapping of isolated pp6.0c-src from control DMSO- (lane 1) or TPA-treated cells (lane 2). Protease digestions were performed with 50 ng of V8 enzyme. The numbers on the left indicate molecular size in kilodaltons.
cells, respectively. A mix of trypic phosphopeptide fragments (panel D) demonstrates that the TPA-induced phosphopeptides comigrate (peptides 2 and b). These experiments provide evidence that the major serine site phosphorylated as a result of tumor promoter treatment within the viral and cellular forms of pp6Osrc are identical. We also tested the effects of 4ot-phorbol-12,13-didecanoate (4a-PDD) and mezerein on the phosphorylation of pp6Ocsrc. The phorbol derivative 4a-PDD is biologically inactive, whereas mezerein is classified as a second-stage tumor promoter (22). Cells were labeled as described earlier and treated for 20 min with 100 ng of 4a-PDD or mezerein per ml. The pp60c-src molecules were then immunoprecipitated, and the entire molecule was used for two-dimensional tryptic phosphopeptide analysis (Fig. 3). For comparison, the results of TPA treatment are shown in Fig. 3, panel A. As expected, TPA treatment resulted in the appearance of only one additional heavily phosphorylated tryptic peptide of pp60csrc corresponding to the serine phosphorylation site. Mezerein (panel C) produced the same effect. The biologically inactive phorbol analog 4ot-PDD (panel B), on the other hand, failed to elicit phosphorylation of this serine site. One potential mediator of tumor promoter-induced phosphorylation events is the calcium- and phospholipiddependent enzyme, protein kinase C (for a review, see reference 22). This enzyme represents the major receptor for
tumor promoters and exhibits serine- and threonine-specific protein kinase activity (2, 19, 21, 22, 26). Both TPA and mezerein have been shown to bind and activate protein kinase C (2, 19, 21, 22, 26). Our results showing the specific serine phosphorylation of pp6Ocsrc in intact cells following treatment with TPA or mezerein indicate that protein kinase C may alter the phosphorylation state of the pp60csr' molecules. The similar membrane locations of the active form of protein kinase C (18) and pp6Ocsrc (11) point to the possibility of a direct interaction between these two kinases. While this paper was in review, Gould et al. (14) reported similar findings for the phosphorylation of pp60csrc. In addition, those authors provided evidence that protein kinase C may be responsible for this tumor promoterinduced phosphorylation event. What role does this phosphorylation have on the function of pp609csrc? Recent evidence has suggested that protein kinase C may be important in the phosphorylation of EGF receptor, a normal cellular tyrosine kinase (4, 13, 17). Tumor promoter treatment of cells causes enhanced serine and threonine phosphorylation of this growth factor receptor, modulating both its EGF-binding and its tyrosine kinase activities (4, 13, 17). One might expect, then, a similar regulatory role for the phosphorylation of pp60c"sr. We have compared the kinase activities of pp60csrc in immunoprecipitates prepared from control and TPA-treated cells and, to date, have observed no significant differences in the Km or Vm,,x of the enzyme with angiotension or casein as a substrate (data not shown). However, in vitro assays may not provide us with a complete picture of the importance of this phosphorylation event. The real significance of tumor promoter-induced hyperB
A
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FIG. 2. Two-dimensional tryptic phosphopeptide analysis. The V3 plus V4 protease fragments of pp6Oc-src and pp6O-src were digested with trypsin and fractionated in two dimensions. Panel A, map derived from pp6oc-src of control cells. Panel B, map derived from pp60J-src of TPA-treated cells. Panel C, map derived from pp6Ov-src of TPA-treated Schmidt-Ruppin-transformed cells. Panel D, mix of B and C. Phosphopeptides are identified by numbers or lowercase letters.
VOL. 6, 1986
NOTES B
A
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-4chromatography FIG. 3. Effects of 4a-PDD and mezerein on the phosphorylation of pp6Ocsrc in mink lung cells. Two-dimensional tryptic maps were performed on the entire pp60cs'c molecule. Panel A, tryptic phosphopeptide map of pp6Oc-rc from cells treated with TPA. Panel B, tryptic phosphopeptide map of pp60cs'c from cells treated with 4a-PDD. Panel C, tryptic phosphopeptide map of pp6Oc-src from cells treated with mezerein. The tumor promoter-induced phosphopeptide is identified by the letter b.
phosphorylation of pp6Oc-Src with regard to the phosphoregulation of pp60c-src or its involvement in tumor promotion and modulation of cellular function is not clear at present. Phosphorylation of pp60csrc at this additional serine moiety may, within the confines of the intact cell, regulate its tyrosine kinase activity or confer new substrate specificity. Such changes could result in the phosphorylation of cellular proteins involved in the regulation of growth, signal transduction, and differentiation. We are especially grateful to Joan Brugge for her generous gift of monoclonal antibody 327. We thank Tony Hunter for communication of results prior to publication. We also thank Anne Little, Deborah Stephens, Cyndy Becker, Toni M. Lenhardt, and Bonnie Kirk for typing the manuscript. LITERATURE CITED 1. Brugge, J. S., and R. L. Erikson. 1977. Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature (London) 269:346-348. 2. Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, V. Kikkawa, and Y. Nishizuka. 1982. Direct activation of Ca2+-activated, phospholipid-dependent protein kinase by tumor promoting phorbol esters. J. Biol. Chem. 257:7847-7851. 3. Cleveland, D. W., S. G. Fischer, M. W. Kirschner, and U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102-1106. 4. Cochet, C., G. N. Gill, J. Meisenhelder, J. A. Cooper, and T. Hunter. 1984. C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factorstimulated tyrosine protein kinase activity. J. Biol. Chem. 259:2553-2558. 5. Coliett, M. S., S. K. Belzer, and A. F. Purchio. 1984. Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates. Mol. Cell. Biol. 4:1213-1220. 6. Coliett, M. S., J. S. Brugge, and R. L. Erikson. 1978. Characterization of a normal cell protein related to avian sarcoma virus transforming gene product. Cell 15:1363-1370. 7. Collett, M. S., and R. L. Erikson. 1978. Protein kinase activity associated with avian sarcoma virus src gene product. Proc. Natl. Acad. Sci. USA 75:2021-2024. 8. Collett, M. S., E. Erikson, and R. L. Erikson. 1979. Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation. J. Virol. 29:770-781. 9. Collett, M. S., A. F. Purchio, and R. L. Erikson. 1980. Avian sarcoma virus-transforming protein, pp6Gsrc, shows protein
10. 11.
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19. 20. 21.
22.
23.
kinase activity specific for tyrosine. Nature (London) 285: 167-169. Collett, M. S., S. K. Wells, and A. F. Purchio. 1983. Physical modification of purified Rous sarcoma virus pp6Ov-src protein after incubation with ATP/Mg2+. Virology 128:285-297. Courtneidge, S. A., A. D. Levinson, and J. M. Bishop. 1980. The protein encoded by the transforming gene of avian sarcoma virus (pp6Osrs) and a homologous protein in normal cells (pp60Proto-Src) are associated with the plasma membrane. Proc. Natl. Acad. Sci. USA 77:3783-3787. Cross, F. R., and H. Hanafusa. 1983. Local mutagenesis of Rous sarcoma virus: the major sites of tyrosine and serine phosphorylation of pp6Osrc are dispensable for transformation. Cell 34:597-607. Davis, R. J., and M. P. Czech. 1985. Tumor promoting phorbol diesters cause the phosphorylation of epidermal growth factor receptors in normal human fibroblasts at threonine-654. Proc. Natl. Acad. Sci. USA 82:1974-1978. Gould, K. L., J. R. Woodgett, J. A. Cooper, J. E. Buss, D. Shalloway, and T. Hunter. 1985. Protein kinase C phosphorylates pp60src at a novel site. Cell 42:849-857. Hunter, T., and J. A. Cooper. 1985. Protein-tyrosine kinases. Annu. Rev. Biochem. 54:897-930. Hunter, T., and B. M. Sefton. 1980. The transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Natl. Acad. Sci. USA 77:1311-1315. Iwashita, S., and F. Fox. 1984. Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells. J. Biol. Chem. 259:2559-2567. Kraft, A. S., W. B. Anderson, H. L. Cooper, and J. J. Sando. 1982. Decrease in cytosolic calcium/phospholipid-dependent protein kinase activity following phorbol ester treatment of EL4 thymoma cells. J. Biol. Chem. 257:13193-13196. Leach, K. L., M. L. James, and P. M. Blumberg. 1983. Characterization of a specific phorbol ester aporeceptor in mouse brain cytosol. Proc. Natl. Acad. Sci. USA 80:4208-4212. Lipsich, L. A., A. J. Lewis, and J. S. Brugge. 1983. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J. Virol. 48:352-360. Niedel, J. E., L. S. Kuhn, and G. R. Vandenbark. 1983. Phorbol diester receptor copurifies with protein kinase C. Proc. Natl. Acad. Sci. USA 80:36-40. Nishizuka, Y. 1984. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature (London) 308:693-698. Patschinsky, T., T. Hunter, F. S. Esch, J. A. Cooper, and B. M.
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NOTES Sefton. 1982. Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation. Proc. Natl. Acad. Sci. USA 79:973-977. Purchio, A. F., M. Shoyab, and L. E. Gentry. 1985. TPA treatment of RSV transformed cells causes site-specific increased phosphorylation of pp60-src. Science 229:1393-1395. Purchio, A. F., S. K. Wells, and M. S. Collett. 1983. Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp6Ov-srC protein after incubation with ATP plus Mg2 . Mol. Cell. Biol. 3:1589-1597. Shoyab, M., and R. Boaze, Jr. 1984. Isolation and characterization specific receptor for biologically active phorbol and ingenol esters. Arch. Biochem. Biophys. 1:197-205. Shoyab, M., J. E. DeLarco, and G. J. Todaro. 1979. Biologically
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active phorbol esters specifically alter affinity of epidermal growth factor membrane receptors. Nature (London) 279: 387-391. 28. Shoyab, M., and G. J. Todaro. 1980. Specific high affinity cell membrane receptors for biologically active phorbol and ingenol esters. Nature (London) 288:451-455. 29. Smart, J. E., H. Oppermann, A. P. Czernilofsky, A. F. Purchio, R. L. Erikson, and J. M. Bishop. 1981. Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp6O-src) and its normal cellular homologue (pp6Oc-sc). Proc. Natl. Acad. Sci. USA 78:6013-6017. 30. Takeya, T., and H. Hanafusa. 1983. Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism ofgenerating the transforming virus. Cell 32: 881-890.
