Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Apr;11(4):1804–1812. doi: 10.1128/mcb.11.4.1804

Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein.

M F Moran 1, P Polakis 1, F McCormick 1, T Pawson 1, C Ellis 1
PMCID: PMC359849  PMID: 2005883

Abstract

The p21ras GTPase-activating protein (GAP) down-regulates p21ras by stimulating its intrinsic GTPase activity. GAP is found predominantly as a monomer in the cytosol of normal cells. However, in cells expressing an activated cytoplasmic protein-tyrosine kinase, p60v-src, or stimulated with epidermal growth factor, GAP becomes phosphorylated on tyrosine and serine and forms distinct complexes with two phosphoproteins of 62 and 190 kDa (p62 and p190). In v-src-transformed Rat-2 cells, a minor fraction of GAP associates with the highly tyrosine phosphorylated p62 to form a complex that is localized at the plasma membrane and in the cytosol. In contrast, the majority of GAP enters a distinct complex with p190 that is exclusively cytosolic and contains predominantly phosphoserine. Epidermal growth factor stimulation also induces a marked conversion of monomeric GAP to higher-molecular-weight species in rat fibroblasts. The GAP-p190 complex is dependent on phosphorylation and shows reduced GAP activity. These results indicate that protein-tyrosine kinases induce GAP to form multiple heteromeric complexes, which are strong candidates for regulators or targets of p21ras.

Full text

PDF
1804

Images in this article

1806Image
on p.1806
1807Image
on p.1807
1808Image
on p.1808
1808Image
on p.1808
1809Image
on p.1809
1809Image
on p.1809

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. Anderson D., Koch C. A., Grey L., Ellis C., Moran M. F., Pawson T. Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors. Science. 1990 Nov 16;250(4983):979–982. doi: 10.1126/science.2173144. [DOI] [PubMed] [Google Scholar]
  3. Brooks-Wilson A. R., Ball E., Pawson T. The myristylation signal of p60v-src functionally complements the N-terminal fps-specific region of P130gag-fps. Mol Cell Biol. 1989 May;9(5):2214–2219. doi: 10.1128/mcb.9.5.2214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Downward J., Riehl R., Wu L., Weinberg R. A. Identification of a nucleotide exchange-promoting activity for p21ras. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5998–6002. doi: 10.1073/pnas.87.15.5998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Kamps M. P., Sefton B. M. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 1988 Apr;2(4):305–315. [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. 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]
  12. Koch C. A., Moran M., Sadowski I., Pawson T. The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function. Mol Cell Biol. 1989 Oct;9(10):4131–4140. doi: 10.1128/mcb.9.10.4131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Krengel U., Schlichting I., Scherer A., Schumann R., Frech M., John J., Kabsch W., Pai E. F., Wittinghofer A. Three-dimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules. Cell. 1990 Aug 10;62(3):539–548. doi: 10.1016/0092-8674(90)90018-a. [DOI] [PubMed] [Google Scholar]
  14. Letwin K., Yee S. P., Pawson T. Novel protein-tyrosine kinase cDNAs related to fps/fes and eph cloned using anti-phosphotyrosine antibody. Oncogene. 1988 Dec;3(6):621–627. [PubMed] [Google Scholar]
  15. Luo K., Hurley T. R., Sefton B. M. Transfer of proteins to membranes facilitates both cyanogen bromide cleavage and two-dimensional proteolytic mapping. Oncogene. 1990 Jun;5(6):921–923. [PubMed] [Google Scholar]
  16. Margolis B., Li N., Koch A., Mohammadi M., Hurwitz D. R., Zilberstein A., Ullrich A., Pawson T., Schlessinger J. The tyrosine phosphorylated carboxyterminus of the EGF receptor is a binding site for GAP and PLC-gamma. EMBO J. 1990 Dec;9(13):4375–4380. doi: 10.1002/j.1460-2075.1990.tb07887.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marshall M. S., Hill W. S., Ng A. S., Vogel U. S., Schaber M. D., Scolnick E. M., Dixon R. A., Sigal I. S., Gibbs J. B. A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity. EMBO J. 1989 Apr;8(4):1105–1110. doi: 10.1002/j.1460-2075.1989.tb03480.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Moran M. F., Koch C. A., Anderson D., Ellis C., England L., Martin G. S., Pawson T. Src homology region 2 domains direct protein-protein interactions in signal transduction. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8622–8626. doi: 10.1073/pnas.87.21.8622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morrison D. K., Kaplan D. R., Escobedo J. A., Rapp U. R., Roberts T. M., Williams L. T. Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF beta-receptor. Cell. 1989 Aug 25;58(4):649–657. doi: 10.1016/0092-8674(89)90100-1. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pawson T. Non-catalytic domains of cytoplasmic protein-tyrosine kinases: regulatory elements in signal transduction. Oncogene. 1988 Nov;3(5):491–495. [PubMed] [Google Scholar]
  24. Polakis P. G., Rubinfeld B., Evans T., McCormick F. Purification of a plasma membrane-associated GTPase-activating protein specific for rap1/Krev-1 from HL60 cells. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):239–243. doi: 10.1073/pnas.88.1.239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reedijk M., Liu X. Q., Pawson T. Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor. Mol Cell Biol. 1990 Nov;10(11):5601–5608. doi: 10.1128/mcb.10.11.5601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rossomando A. J., Payne D. M., Weber M. J., Sturgill T. W. Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6940–6943. doi: 10.1073/pnas.86.18.6940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Satoh T., Endo M., Nakafuku M., Akiyama T., Yamamoto T., Kaziro Y. Accumulation of p21ras.GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7926–7929. doi: 10.1073/pnas.87.20.7926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  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. Tanaka K., Nakafuku M., Satoh T., Marshall M. S., Gibbs J. B., Matsumoto K., Kaziro Y., Toh-e A. S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein. Cell. 1990 Mar 9;60(5):803–807. doi: 10.1016/0092-8674(90)90094-u. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Trahey M., Wong G., Halenbeck R., Rubinfeld B., Martin G. A., Ladner M., Long C. M., Crosier W. J., Watt K., Koths K. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. doi: 10.1126/science.3201259. [DOI] [PubMed] [Google Scholar]
  33. Weinmaster G., Zoller M. J., Smith M., Hinze E., Pawson T. Mutagenesis of Fujinami sarcoma virus: evidence that tyrosine phosphorylation of P130gag-fps modulates its biological activity. Cell. 1984 Jun;37(2):559–568. doi: 10.1016/0092-8674(84)90386-6. [DOI] [PubMed] [Google Scholar]
  34. West M., Kung H. F., Kamata T. A novel membrane factor stimulates guanine nucleotide exchange reaction of ras proteins. FEBS Lett. 1990 Jan 1;259(2):245–248. doi: 10.1016/0014-5793(90)80019-f. [DOI] [PubMed] [Google Scholar]
  35. Wolfman A., Macara I. G. A cytosolic protein catalyzes the release of GDP from p21ras. Science. 1990 Apr 6;248(4951):67–69. doi: 10.1126/science.2181667. [DOI] [PubMed] [Google Scholar]
  36. Xu G. F., O'Connell P., Viskochil D., Cawthon R., Robertson M., Culver M., Dunn D., Stevens J., Gesteland R., White R. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell. 1990 Aug 10;62(3):599–608. doi: 10.1016/0092-8674(90)90024-9. [DOI] [PubMed] [Google Scholar]

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

RESOURCES