Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 Sep;12(9):3903–3909. doi: 10.1128/mcb.12.9.3903

Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

C J Molloy 1, T P Fleming 1, D P Bottaro 1, A Cuadrado 1, S A Aaronson 1
PMCID: PMC360268  PMID: 1508192

Abstract

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.

Full text

PDF
3903

Images in this article

3905Image
on p.3905
3906Image
on p.3906
3907Image
on p.3907
3907Image
on p.3907

Selected References

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

  1. Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [PubMed] [Google Scholar]
  2. Alonso T., Morgan R. O., Marvizon J. C., Zarbl H., Santos E. Malignant transformation by ras and other oncogenes produces common alterations in inositol phospholipid signaling pathways. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4271–4275. doi: 10.1073/pnas.85.12.4271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  4. Benjamin C. W., Connor J. A., Tarpley W. G., Gorman R. R. NIH-3T3 cells transformed by the EJ-ras oncogene exhibit reduced platelet-derived growth factor-mediated Ca2+ mobilization. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4345–4349. doi: 10.1073/pnas.85.12.4345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brott B. K., Decker S., Shafer J., Gibbs J. B., Jove R. GTPase-activating protein interactions with the viral and cellular Src kinases. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):755–759. doi: 10.1073/pnas.88.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Calés C., Hancock J. F., Marshall C. J., Hall A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature. 1988 Apr 7;332(6164):548–551. doi: 10.1038/332548a0. [DOI] [PubMed] [Google Scholar]
  7. Cuadrado A., Molloy C. J. Overexpression of phospholipase C-gamma in NIH 3T3 fibroblasts results in increased phosphatidylinositol hydrolysis in response to platelet-derived growth factor and basic fibroblast growth factor. Mol Cell Biol. 1990 Nov;10(11):6069–6072. doi: 10.1128/mcb.10.11.6069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Di Marco E., Pierce J. H., Fleming T. P., Kraus M. H., Molloy C. J., Aaronson S. A., Di Fiore P. P. Autocrine interaction between TGF alpha and the EGF-receptor: quantitative requirements for induction of the malignant phenotype. Oncogene. 1989 Jul;4(7):831–838. [PubMed] [Google Scholar]
  9. Downward J., Graves J. D., Warne P. H., Rayter S., Cantrell D. A. Stimulation of p21ras upon T-cell activation. Nature. 1990 Aug 23;346(6286):719–723. doi: 10.1038/346719a0. [DOI] [PubMed] [Google Scholar]
  10. Ellis C., Moran M., McCormick F., Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990 Jan 25;343(6256):377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  11. Fazioli F., Kim U. H., Rhee S. G., Molloy C. J., Segatto O., Di Fiore P. P. The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency. Mol Cell Biol. 1991 Apr;11(4):2040–2048. doi: 10.1128/mcb.11.4.2040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Field J., Broek D., Kataoka T., Wigler M. Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jun;7(6):2128–2133. doi: 10.1128/mcb.7.6.2128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Finzi E., Fleming T., Segatto O., Pennington C. Y., Bringman T. S., Derynck R., Aaronson S. A. The human transforming growth factor type alpha coding sequence is not a direct-acting oncogene when overexpressed in NIH 3T3 cells. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3733–3737. doi: 10.1073/pnas.84.11.3733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gibbs J. B., Marshall M. S., Scolnick E. M., Dixon R. A., Vogel U. S. Modulation of guanine nucleotides bound to Ras in NIH3T3 cells by oncogenes, growth factors, and the GTPase activating protein (GAP). J Biol Chem. 1990 Nov 25;265(33):20437–20442. [PubMed] [Google Scholar]
  15. Gibbs J. B., Schaber M. D., Marshall M. S., Scolnick E. M., Sigal I. S. Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells. J Biol Chem. 1987 Aug 5;262(22):10426–10429. [PubMed] [Google Scholar]
  16. 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]
  17. Hall A. ras and GAP--who's controlling whom? Cell. 1990 Jun 15;61(6):921–923. doi: 10.1016/0092-8674(90)90054-i. [DOI] [PubMed] [Google Scholar]
  18. Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Margolis B., Zilberstein A., Franks C., Felder S., Kremer S., Ullrich A., Rhee S. G., Skorecki K., Schlessinger J. Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis. Science. 1990 May 4;248(4955):607–610. doi: 10.1126/science.2333512. [DOI] [PubMed] [Google Scholar]
  25. Marshall M. S., Gibbs J. B., Scolnick E. M., Sigal I. S. Regulatory function of the Saccharomyces cerevisiae RAS C-terminus. Mol Cell Biol. 1987 Jul;7(7):2309–2315. doi: 10.1128/mcb.7.7.2309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsui T., Heidaran M., Miki T., Popescu N., La Rochelle W., Kraus M., Pierce J., Aaronson S. Isolation of a novel receptor cDNA establishes the existence of two PDGF receptor genes. Science. 1989 Feb 10;243(4892):800–804. doi: 10.1126/science.2536956. [DOI] [PubMed] [Google Scholar]
  27. McCormick F. The world according to GAP. Oncogene. 1990 Sep;5(9):1281–1283. [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Moran M. F., Polakis P., McCormick F., Pawson T., Ellis C. Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein. Mol Cell Biol. 1991 Apr;11(4):1804–1812. doi: 10.1128/mcb.11.4.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nishizuka Y. Perspectives on the role of protein kinase C in stimulus-response coupling. J Natl Cancer Inst. 1986 Mar;76(3):363–370. [PubMed] [Google Scholar]
  32. Rake J. B., Quiñones M. A., Faller D. V. Inhibition of platelet-derived growth factor-mediated signal transduction by transforming ras. Suppression of receptor autophosphorylation. J Biol Chem. 1991 Mar 15;266(8):5348–5352. [PubMed] [Google Scholar]
  33. Satoh T., Endo M., Nakafuku M., Nakamura S., Kaziro Y. Platelet-derived growth factor stimulates formation of active p21ras.GTP complex in Swiss mouse 3T3 cells. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5993–5997. doi: 10.1073/pnas.87.15.5993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Satoh T., Nakamura S., Kaziro Y. Induction of neurite formation in PC12 cells by microinjection of proto-oncogenic Ha-ras protein preincubated with guanosine-5'-O-(3-thiotriphosphate). Mol Cell Biol. 1987 Dec;7(12):4553–4556. doi: 10.1128/mcb.7.12.4553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stacey D. W., Kung H. F. Transformation of NIH 3T3 cells by microinjection of Ha-ras p21 protein. Nature. 1984 Aug 9;310(5977):508–511. doi: 10.1038/310508a0. [DOI] [PubMed] [Google Scholar]
  36. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  37. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  38. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  39. Wahl M. I., Olashaw N. E., Nishibe S., Rhee S. G., Pledger W. J., Carpenter G. Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells. Mol Cell Biol. 1989 Jul;9(7):2934–2943. doi: 10.1128/mcb.9.7.2934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Williams L. T. Signal transduction by the platelet-derived growth factor receptor. Science. 1989 Mar 24;243(4898):1564–1570. doi: 10.1126/science.2538922. [DOI] [PubMed] [Google Scholar]
  41. Zhang K., DeClue J. E., Vass W. C., Papageorge A. G., McCormick F., Lowy D. R. Suppression of c-ras transformation by GTPase-activating protein. Nature. 1990 Aug 23;346(6286):754–756. doi: 10.1038/346754a0. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES