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. 1993 Sep;13(9):5290–5300. doi: 10.1128/mcb.13.9.5290

Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae.

S M Murphy 1, M Bergman 1, D O Morgan 1
PMCID: PMC360223  PMID: 7689149

Abstract

The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bergman M., Mustelin T., Oetken C., Partanen J., Flint N. A., Amrein K. E., Autero M., Burn P., Alitalo K. The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity. EMBO J. 1992 Aug;11(8):2919–2924. doi: 10.1002/j.1460-2075.1992.tb05361.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cartwright C. A., Eckhart W., Simon S., Kaplan P. L. Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain. Cell. 1987 Apr 10;49(1):83–91. doi: 10.1016/0092-8674(87)90758-6. [DOI] [PubMed] [Google Scholar]
  3. Cicchetti P., Mayer B. J., Thiel G., Baltimore D. Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho. Science. 1992 Aug 7;257(5071):803–806. doi: 10.1126/science.1379745. [DOI] [PubMed] [Google Scholar]
  4. Clark E. A., Brugge J. S. Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets. Mol Cell Biol. 1993 Mar;13(3):1863–1871. doi: 10.1128/mcb.13.3.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cooper J. A., Gould K. L., Cartwright C. A., Hunter T. Tyr527 is phosphorylated in pp60c-src: implications for regulation. Science. 1986 Mar 21;231(4744):1431–1434. doi: 10.1126/science.2420005. [DOI] [PubMed] [Google Scholar]
  6. Cooper J. A., King C. S. Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src. Mol Cell Biol. 1986 Dec;6(12):4467–4477. doi: 10.1128/mcb.6.12.4467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cooper J. A., Runge K. Avian pp60c-src is more active when expressed in yeast than in vertebrate fibroblasts. Oncogene Res. 1987 Sep-Oct;1(4):297–310. [PubMed] [Google Scholar]
  8. Czernilofsky A. P., Levinson A. D., Varmus H. E., Bishop J. M., Tischer E., Goodman H. Corrections to the nucleotide sequence of the src gene of Rous sarcoma virus. Nature. 1983 Feb 24;301(5902):736–738. doi: 10.1038/301736b0. [DOI] [PubMed] [Google Scholar]
  9. Fukui Y., O'Brien M. C., Hanafusa H. Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix. Mol Cell Biol. 1991 Mar;11(3):1207–1213. doi: 10.1128/mcb.11.3.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gould K. L., Hunter T. Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity. Mol Cell Biol. 1988 Aug;8(8):3345–3356. doi: 10.1128/mcb.8.8.3345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grandori C., Hanafusa H. p60c-src is complexed with a cellular protein in subcellular compartments involved in exocytosis. J Cell Biol. 1988 Dec;107(6 Pt 1):2125–2135. doi: 10.1083/jcb.107.6.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hirai H., Varmus H. E. Mutations in src homology regions 2 and 3 of activated chicken c-src that result in preferential transformation of mouse or chicken cells. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8592–8596. doi: 10.1073/pnas.87.21.8592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hirai H., Varmus H. E. Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src. Mol Cell Biol. 1990 Apr;10(4):1307–1318. doi: 10.1128/mcb.10.4.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaplan J. M., Varmus H. E., Bishop J. M. The src protein contains multiple domains for specific attachment to membranes. Mol Cell Biol. 1990 Mar;10(3):1000–1009. doi: 10.1128/mcb.10.3.1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kaplan K. B., Swedlow J. R., Varmus H. E., Morgan D. O. Association of p60c-src with endosomal membranes in mammalian fibroblasts. J Cell Biol. 1992 Jul;118(2):321–333. doi: 10.1083/jcb.118.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kato J. Y., Takeya T., Grandori C., Iba H., Levy J. B., Hanafusa H. Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein. Mol Cell Biol. 1986 Dec;6(12):4155–4160. doi: 10.1128/mcb.6.12.4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kemp B. E., Pearson R. B. Design and use of peptide substrates for protein kinases. Methods Enzymol. 1991;200:121–134. doi: 10.1016/0076-6879(91)00134-i. [DOI] [PubMed] [Google Scholar]
  18. Kmiecik T. E., Johnson P. J., Shalloway D. Regulation by the autophosphorylation site in overexpressed pp60c-src. Mol Cell Biol. 1988 Oct;8(10):4541–4546. doi: 10.1128/mcb.8.10.4541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kmiecik T. E., Shalloway D. Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell. 1987 Apr 10;49(1):65–73. doi: 10.1016/0092-8674(87)90756-2. [DOI] [PubMed] [Google Scholar]
  20. Kornbluth S., Jove R., Hanafusa H. Characterization of avian and viral p60src proteins expressed in yeast. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4455–4459. doi: 10.1073/pnas.84.13.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Linstedt A. D., Vetter M. L., Bishop J. M., Kelly R. B. Specific association of the proto-oncogene product pp60c-src with an intracellular organelle, the PC12 synaptic vesicle. J Cell Biol. 1992 Jun;117(5):1077–1084. doi: 10.1083/jcb.117.5.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lowe C., Yoneda T., Boyce B. F., Chen H., Mundy G. R., Soriano P. Osteopetrosis in Src-deficient mice is due to an autonomous defect of osteoclasts. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4485–4489. doi: 10.1073/pnas.90.10.4485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. MacAuley A., Okada M., Nada S., Nakagawa H., Cooper J. A. Phosphorylation of Src mutants at Tyr 527 in fibroblasts does not correlate with in vitro phosphorylation by CSK. Oncogene. 1993 Jan;8(1):117–124. [PubMed] [Google Scholar]
  25. Matsuda M., Mayer B. J., Fukui Y., Hanafusa H. Binding of transforming protein, P47gag-crk, to a broad range of phosphotyrosine-containing proteins. Science. 1990 Jun 22;248(4962):1537–1539. doi: 10.1126/science.1694307. [DOI] [PubMed] [Google Scholar]
  26. Morgan D. O., Kaplan J. M., Bishop J. M., Varmus H. E. Production of p60c-src by baculovirus expression and immunoaffinity purification. Methods Enzymol. 1991;200:645–660. doi: 10.1016/0076-6879(91)00177-x. [DOI] [PubMed] [Google Scholar]
  27. Musacchio A., Gibson T., Lehto V. P., Saraste M. SH3--an abundant protein domain in search of a function. FEBS Lett. 1992 Jul 27;307(1):55–61. doi: 10.1016/0014-5793(92)80901-r. [DOI] [PubMed] [Google Scholar]
  28. Nada S., Okada M., MacAuley A., Cooper J. A., Nakagawa H. Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. Nature. 1991 May 2;351(6321):69–72. doi: 10.1038/351069a0. [DOI] [PubMed] [Google Scholar]
  29. Okada M., Nada S., Yamanashi Y., Yamamoto T., Nakagawa H. CSK: a protein-tyrosine kinase involved in regulation of src family kinases. J Biol Chem. 1991 Dec 25;266(36):24249–24252. [PubMed] [Google Scholar]
  30. Okada M., Nakagawa H. A protein tyrosine kinase involved in regulation of pp60c-src function. J Biol Chem. 1989 Dec 15;264(35):20886–20893. [PubMed] [Google Scholar]
  31. Parsons S. J., Creutz C. E. p60c-src activity detected in the chromaffin granule membrane. Biochem Biophys Res Commun. 1986 Jan 29;134(2):736–742. doi: 10.1016/s0006-291x(86)80482-x. [DOI] [PubMed] [Google Scholar]
  32. Parsons S. J., McCarley D. J., Ely C. M., Benjamin D. C., Parsons J. T. Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin. J Virol. 1984 Aug;51(2):272–282. doi: 10.1128/jvi.51.2.272-282.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Partanen J., Armstrong E., Bergman M., Mäkelä T. P., Hirvonen H., Huebner K., Alitalo K. cyl encodes a putative cytoplasmic tyrosine kinase lacking the conserved tyrosine autophosphorylation site (Y416src). Oncogene. 1991 Nov;6(11):2013–2018. [PubMed] [Google Scholar]
  34. Pawson T., Gish G. D. SH2 and SH3 domains: from structure to function. Cell. 1992 Oct 30;71(3):359–362. doi: 10.1016/0092-8674(92)90504-6. [DOI] [PubMed] [Google Scholar]
  35. Piwnica-Worms H., Saunders K. B., Roberts T. M., Smith A. E., Cheng S. H. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell. 1987 Apr 10;49(1):75–82. doi: 10.1016/0092-8674(87)90757-4. [DOI] [PubMed] [Google Scholar]
  36. Resh M. D. Specific and saturable binding of pp60v-src to plasma membranes: evidence for a myristyl-src receptor. Cell. 1989 Jul 28;58(2):281–286. doi: 10.1016/0092-8674(89)90842-8. [DOI] [PubMed] [Google Scholar]
  37. Reynolds A. B., Vila J., Lansing T. J., Potts W. M., Weber M. J., Parsons J. T. Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus. EMBO J. 1987 Aug;6(8):2359–2364. doi: 10.1002/j.1460-2075.1987.tb02512.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Roussel R. R., Brodeur S. R., Shalloway D., Laudano A. P. Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10696–10700. doi: 10.1073/pnas.88.23.10696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rubin G. M. Preparation of RNA and ribosomes from yeast. Methods Cell Biol. 1975;12:45–64. doi: 10.1016/s0091-679x(08)60951-6. [DOI] [PubMed] [Google Scholar]
  40. Schild D., Brake A. J., Kiefer M. C., Young D., Barr P. J. Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2916–2920. doi: 10.1073/pnas.87.8.2916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schuh S. M., Brugge J. S. Investigation of factors that influence phosphorylation of pp60c-src on tyrosine 527. Mol Cell Biol. 1988 Jun;8(6):2465–2471. doi: 10.1128/mcb.8.6.2465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Seidel-Dugan C., Meyer B. E., Thomas S. M., Brugge J. S. Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src. Mol Cell Biol. 1992 Apr;12(4):1835–1845. doi: 10.1128/mcb.12.4.1835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  44. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Soriano P., Montgomery C., Geske R., Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693–702. doi: 10.1016/0092-8674(91)90499-o. [DOI] [PubMed] [Google Scholar]
  46. Taylor S. J., Shalloway D. The cell cycle and c-Src. Curr Opin Genet Dev. 1993 Feb;3(1):26–34. doi: 10.1016/s0959-437x(05)80337-5. [DOI] [PubMed] [Google Scholar]
  47. Thomas J. E., Soriano P., Brugge J. S. Phosphorylation of c-Src on tyrosine 527 by another protein tyrosine kinase. Science. 1991 Oct 25;254(5031):568–571. doi: 10.1126/science.1719633. [DOI] [PubMed] [Google Scholar]
  48. Waksman G., Kominos D., Robertson S. C., Pant N., Baltimore D., Birge R. B., Cowburn D., Hanafusa H., Mayer B. J., Overduin M. Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides. Nature. 1992 Aug 20;358(6388):646–653. doi: 10.1038/358646a0. [DOI] [PubMed] [Google Scholar]
  49. Wong S., Reynolds A. B., Papkoff J. Platelet activation leads to increased c-src kinase activity and association of c-src with an 85-kDa tyrosine phosphoprotein. Oncogene. 1992 Dec;7(12):2407–2415. [PubMed] [Google Scholar]
  50. Yu H., Rosen M. K., Shin T. B., Seidel-Dugan C., Brugge J. S., Schreiber S. L. Solution structure of the SH3 domain of Src and identification of its ligand-binding site. Science. 1992 Dec 4;258(5088):1665–1668. doi: 10.1126/science.1280858. [DOI] [PubMed] [Google Scholar]

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