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. 1993 Aug 15;90(16):7696–7700. doi: 10.1073/pnas.90.16.7696

The Src family tyrosine kinases are required for platelet-derived growth factor-mediated signal transduction in NIH 3T3 cells.

G M Twamley-Stein 1, R Pepperkok 1, W Ansorge 1, S A Courtneidge 1
PMCID: PMC47209  PMID: 8356071

Abstract

Three members of the Src family of protein tyrosine kinases Src, Fyn, and Yes associate with the activated platelet-derived growth factor (PDGF) receptor in vivo. This interaction requires the Src homology 2 (SH2) domain of the Src family member and causes activation of the intrinsic activity of the Src family kinases. We microinjected cells with DNA encoding catalytically inactive forms of the Src and Fyn proteins and examined their effects on PDGF-mediated signaling in vivo. Kinase-inactive Src and Fyn inhibited PDGF-stimulated entry of cells into S phase, whereas kinase-active forms of the proteins had no inhibitory effects. An intact SH2 domain was required for inhibition. Furthermore, when kinase-inactive Fyn was comicroinjected with a plasmid expressing activated Ras, the cells could enter S phase, indicating that the expression of kinase-inactive Fyn did not damage cell viability. Injection of an antibody specific for Src, Fyn, and Yes also reduced signal transduction through the PDGF receptor but only when injected within 8 hr of PDGF stimulation. Together these results indicate that the ubiquitously expressed Src family members are required for PDGF-induced mitogenic signaling.

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Selected References

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

  1. Alemà S., Casalbore P., Agostini E., Tatò F. Differentiation of PC12 phaeochromocytoma cells induced by v-src oncogene. Nature. 1985 Aug 8;316(6028):557–559. doi: 10.1038/316557a0. [DOI] [PubMed] [Google Scholar]
  2. Ansorge W., Pepperkok R. Performance of an automated system for capillary microinjection into living cells. J Biochem Biophys Methods. 1988 Aug;16(4):283–292. doi: 10.1016/0165-022x(88)90062-0. [DOI] [PubMed] [Google Scholar]
  3. Bolen J. B., Rowley R. B., Spana C., Tsygankov A. Y. The Src family of tyrosine protein kinases in hemopoietic signal transduction. FASEB J. 1992 Dec;6(15):3403–3409. doi: 10.1096/fasebj.6.15.1281458. [DOI] [PubMed] [Google Scholar]
  4. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  5. Courtneidge S. A., Dhand R., Pilat D., Twamley G. M., Waterfield M. D., Roussel M. F. Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor. EMBO J. 1993 Mar;12(3):943–950. doi: 10.1002/j.1460-2075.1993.tb05735.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Courtneidge S. A., Smith A. E. The complex of polyoma virus middle-T antigen and pp60c-src. EMBO J. 1984 Mar;3(3):585–591. doi: 10.1002/j.1460-2075.1984.tb01852.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fantl W. J., Escobedo J. A., Martin G. A., Turck C. W., del Rosario M., McCormick F., Williams L. T. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell. 1992 May 1;69(3):413–423. doi: 10.1016/0092-8674(92)90444-h. [DOI] [PubMed] [Google Scholar]
  8. Hatakeyama M., Kono T., Kobayashi N., Kawahara A., Levin S. D., Perlmutter R. M., Taniguchi T. Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association. Science. 1991 Jun 14;252(5012):1523–1528. doi: 10.1126/science.2047859. [DOI] [PubMed] [Google Scholar]
  9. Heldin C. H., Westermark B. Signal transduction by the receptors for platelet-derived growth factor. J Cell Sci. 1990 Jun;96(Pt 2):193–196. doi: 10.1242/jcs.96.2.193. [DOI] [PubMed] [Google Scholar]
  10. Kaplan D. R., Morrison D. K., Wong G., McCormick F., Williams L. T. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990 Apr 6;61(1):125–133. doi: 10.1016/0092-8674(90)90220-9. [DOI] [PubMed] [Google Scholar]
  11. Kaplan D. R., Whitman M., Schaffhausen B., Pallas D. C., White M., Cantley L., Roberts T. M. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987 Sep 25;50(7):1021–1029. doi: 10.1016/0092-8674(87)90168-1. [DOI] [PubMed] [Google Scholar]
  12. Kashishian A., Kazlauskas A., Cooper J. A. Phosphorylation sites in the PDGF receptor with different specificities for binding GAP and PI3 kinase in vivo. EMBO J. 1992 Apr;11(4):1373–1382. doi: 10.1002/j.1460-2075.1992.tb05182.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kazlauskas A., Cooper J. A. Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins. Cell. 1989 Sep 22;58(6):1121–1133. doi: 10.1016/0092-8674(89)90510-2. [DOI] [PubMed] [Google Scholar]
  14. Kazlauskas A., Ellis C., Pawson T., Cooper J. A. Binding of GAP to activated PDGF receptors. Science. 1990 Mar 30;247(4950):1578–1581. doi: 10.1126/science.2157284. [DOI] [PubMed] [Google Scholar]
  15. Kazlauskas A., Kashishian A., Cooper J. A., Valius M. GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit. Mol Cell Biol. 1992 Jun;12(6):2534–2544. doi: 10.1128/mcb.12.6.2534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kremer N. E., D'Arcangelo G., Thomas S. M., DeMarco M., Brugge J. S., Halegoua S. Signal transduction by nerve growth factor and fibroblast growth factor in PC12 cells requires a sequence of src and ras actions. J Cell Biol. 1991 Nov;115(3):809–819. doi: 10.1083/jcb.115.3.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kypta R. M., Goldberg Y., Ulug E. T., Courtneidge S. A. Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell. 1990 Aug 10;62(3):481–492. doi: 10.1016/0092-8674(90)90013-5. [DOI] [PubMed] [Google Scholar]
  18. Kypta R. M., Hemming A., Courtneidge S. A. Identification and characterization of p59fyn (a src-like protein tyrosine kinase) in normal and polyoma virus transformed cells. EMBO J. 1988 Dec 1;7(12):3837–3844. doi: 10.1002/j.1460-2075.1988.tb03269.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lowenstein E. J., Daly R. J., Batzer A. G., Li W., Margolis B., Lammers R., Ullrich A., Skolnik E. Y., Bar-Sagi D., Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. doi: 10.1016/0092-8674(92)90167-b. [DOI] [PubMed] [Google Scholar]
  20. Luttrell D. K., Luttrell L. M., Parsons S. J. Augmented mitogenic responsiveness to epidermal growth factor in murine fibroblasts that overexpress pp60c-src. Mol Cell Biol. 1988 Jan;8(1):497–501. doi: 10.1128/mcb.8.1.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Margolis B., Rhee S. G., Felder S., Mervic M., Lyall R., Levitzki A., Ullrich A., Zilberstein A., Schlessinger J. EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling. Cell. 1989 Jun 30;57(7):1101–1107. doi: 10.1016/0092-8674(89)90047-0. [DOI] [PubMed] [Google Scholar]
  22. Mayer B. J., Jackson P. K., Baltimore D. The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):627–631. doi: 10.1073/pnas.88.2.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Meisenhelder J., Suh P. G., Rhee S. G., Hunter T. Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell. 1989 Jun 30;57(7):1109–1122. doi: 10.1016/0092-8674(89)90048-2. [DOI] [PubMed] [Google Scholar]
  24. Molloy C. J., Bottaro D. P., Fleming T. P., Marshall M. S., Gibbs J. B., Aaronson S. A. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature. 1989 Dec 7;342(6250):711–714. doi: 10.1038/342711a0. [DOI] [PubMed] [Google Scholar]
  25. Mori S., Rönnstrand L., Yokote K., Engström A., Courtneidge S. A., Claesson-Welsh L., Heldin C. H. Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases. EMBO J. 1993 Jun;12(6):2257–2264. doi: 10.1002/j.1460-2075.1993.tb05879.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mulcahy L. S., Smith M. R., Stacey D. W. Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature. 1985 Jan 17;313(5999):241–243. doi: 10.1038/313241a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Rönnstrand L., Mori S., Arridsson A. K., Eriksson A., Wernstedt C., Hellman U., Claesson-Welsh L., Heldin C. H. Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma. EMBO J. 1992 Nov;11(11):3911–3919. doi: 10.1002/j.1460-2075.1992.tb05484.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Smith M. R., DeGudicibus S. J., Stacey D. W. Requirement for c-ras proteins during viral oncogene transformation. Nature. 1986 Apr 10;320(6062):540–543. doi: 10.1038/320540a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Songyang Z., Shoelson S. E., Chaudhuri M., Gish G., Pawson T., Haser W. G., King F., Roberts T., Ratnofsky S., Lechleider R. J. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. doi: 10.1016/0092-8674(93)90404-e. [DOI] [PubMed] [Google Scholar]
  31. Sorrentino V., Pepperkok R., Davis R. L., Ansorge W., Philipson L. Cell proliferation inhibited by MyoD1 independently of myogenic differentiation. Nature. 1990 Jun 28;345(6278):813–815. doi: 10.1038/345813a0. [DOI] [PubMed] [Google Scholar]
  32. Superti-Furga G., Fumagalli S., Koegl M., Courtneidge S. A., Draetta G. Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src. EMBO J. 1993 Jul;12(7):2625–2634. doi: 10.1002/j.1460-2075.1993.tb05923.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Twamley G. M., Kypta R. M., Hall B., Courtneidge S. A. Association of Fyn with the activated platelet-derived growth factor receptor: requirements for binding and phosphorylation. Oncogene. 1992 Oct;7(10):1893–1901. [PubMed] [Google Scholar]
  34. Wahl M. I., Nishibe S., Suh P. G., Rhee S. G., Carpenter G. Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalization and extracellular calcium. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1568–1572. doi: 10.1073/pnas.86.5.1568. [DOI] [PMC free article] [PubMed] [Google Scholar]

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