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. 1996 Jun;16(6):2561–2569. doi: 10.1128/mcb.16.6.2561

A Ras-GTPase-activating protein SH3-domain-binding protein.

F Parker 1, F Maurier 1, I Delumeau 1, M Duchesne 1, D Faucher 1, L Debussche 1, A Dugue 1, F Schweighoffer 1, B Tocque 1
PMCID: PMC231246  PMID: 8649363

Abstract

We report the purification of a Ras-GTPase-activating protein (GAP)-binding protein, G3BP, a ubiquitously expressed cytosolic 68-kDa protein that coimmunoprecipitates with GAP. G3BP physically associates with the SH3 domain of GAP, which previously had been shown to be essential for Ras signaling. The G3BP cDNA revealed that G3BP is a novel 466-amino-acid protein that shares several features with heterogeneous nuclear RNA-binding proteins, including ribonucleoprotein (RNP) motifs RNP1 and RNP2, an RG-rich domain, and acidic sequences. Recombinant G3BP binds effectively to the GAP SH3 domain G3BP coimmunoprecipitates with GAP only when cells are in a proliferating state, suggesting a recruitment of a GAP-G3BP complex when Ras is in its activated conformation.

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Selected References

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  1. Booker G. W., Gout I., Downing A. K., Driscoll P. C., Boyd J., Waterfield M. D., Campbell I. D. Solution structure and ligand-binding site of the SH3 domain of the p85 alpha subunit of phosphatidylinositol 3-kinase. Cell. 1993 May 21;73(4):813–822. doi: 10.1016/0092-8674(93)90259-s. [DOI] [PubMed] [Google Scholar]
  2. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  3. Bustelo X. R., Suen K. L., Michael W. M., Dreyfuss G., Barbacid M. Association of the vav proto-oncogene product with poly(rC)-specific RNA-binding proteins. Mol Cell Biol. 1995 Mar;15(3):1324–1332. doi: 10.1128/mcb.15.3.1324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cai H., Szeberényi J., Cooper G. M. Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells. Mol Cell Biol. 1990 Oct;10(10):5314–5323. doi: 10.1128/mcb.10.10.5314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chenevert J., Corrado K., Bender A., Pringle J., Herskowitz I. A yeast gene (BEM1) necessary for cell polarization whose product contains two SH3 domains. Nature. 1992 Mar 5;356(6364):77–79. doi: 10.1038/356077a0. [DOI] [PubMed] [Google Scholar]
  6. Clark G. J., Quilliam L. A., Hisaka M. M., Der C. J. Differential antagonism of Ras biological activity by catalytic and Src homology domains of Ras GTPase activation protein. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4887–4891. doi: 10.1073/pnas.90.11.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [PubMed] [Google Scholar]
  8. Drubin D. G., Mulholland J., Zhu Z. M., Botstein D. Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I. Nature. 1990 Jan 18;343(6255):288–290. doi: 10.1038/343288a0. [DOI] [PubMed] [Google Scholar]
  9. Duchesne M., Schweighoffer F., Parker F., Clerc F., Frobert Y., Thang M. N., Tocqué B. Identification of the SH3 domain of GAP as an essential sequence for Ras-GAP-mediated signaling. Science. 1993 Jan 22;259(5094):525–528. doi: 10.1126/science.7678707. [DOI] [PubMed] [Google Scholar]
  10. Egan S. E., Giddings B. W., Brooks M. W., Buday L., Sizeland A. M., Weinberg R. A. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature. 1993 May 6;363(6424):45–51. doi: 10.1038/363045a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Flynn D. C., Leu T. H., Reynolds A. B., Parsons J. T. Identification and sequence analysis of cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate. Mol Cell Biol. 1993 Dec;13(12):7892–7900. doi: 10.1128/mcb.13.12.7892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fumagalli S., Totty N. F., Hsuan J. J., Courtneidge S. A. A target for Src in mitosis. Nature. 1994 Apr 28;368(6474):871–874. doi: 10.1038/368871a0. [DOI] [PubMed] [Google Scholar]
  14. Gout I., Dhand R., Hiles I. D., Fry M. J., Panayotou G., Das P., Truong O., Totty N. F., Hsuan J., Booker G. W. The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell. 1993 Oct 8;75(1):25–36. [PubMed] [Google Scholar]
  15. Hobert O., Jallal B., Schlessinger J., Ullrich A. Novel signaling pathway suggested by SH3 domain-mediated p95vav/heterogeneous ribonucleoprotein K interaction. J Biol Chem. 1994 Aug 12;269(32):20225–20228. [PubMed] [Google Scholar]
  16. Jung G., Korn E. D., Hammer J. A., 3rd The heavy chain of Acanthamoeba myosin IB is a fusion of myosin-like and non-myosin-like sequences. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6720–6724. doi: 10.1073/pnas.84.19.6720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kenan D. J., Query C. C., Keene J. D. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. doi: 10.1016/0968-0004(91)90088-d. [DOI] [PubMed] [Google Scholar]
  18. Khosravi-Far R., Der C. J. The Ras signal transduction pathway. Cancer Metastasis Rev. 1994 Mar;13(1):67–89. doi: 10.1007/BF00690419. [DOI] [PubMed] [Google Scholar]
  19. Kolch W., Heidecker G., Lloyd P., Rapp U. R. Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature. 1991 Jan 31;349(6308):426–428. doi: 10.1038/349426a0. [DOI] [PubMed] [Google Scholar]
  20. Lazaris-Karatzas A., Smith M. R., Frederickson R. M., Jaramillo M. L., Liu Y. L., Kung H. F., Sonenberg N. Ras mediates translation initiation factor 4E-induced malignant transformation. Genes Dev. 1992 Sep;6(9):1631–1642. doi: 10.1101/gad.6.9.1631. [DOI] [PubMed] [Google Scholar]
  21. Leto T. L., Lomax K. J., Volpp B. D., Nunoi H., Sechler J. M., Nauseef W. M., Clark R. A., Gallin J. I., Malech H. L. Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src. Science. 1990 May 11;248(4956):727–730. doi: 10.1126/science.1692159. [DOI] [PubMed] [Google Scholar]
  22. Li N., Batzer A., Daly R., Yajnik V., Skolnik E., Chardin P., Bar-Sagi D., Margolis B., Schlessinger J. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature. 1993 May 6;363(6424):85–88. doi: 10.1038/363085a0. [DOI] [PubMed] [Google Scholar]
  23. Lim W. A., Richards F. M., Fox R. O. Structural determinants of peptide-binding orientation and of sequence specificity in SH3 domains. Nature. 1994 Nov 24;372(6504):375–379. doi: 10.1038/372375a0. [DOI] [PubMed] [Google Scholar]
  24. Liu X., Marengere L. E., Koch C. A., Pawson T. The v-Src SH3 domain binds phosphatidylinositol 3'-kinase. Mol Cell Biol. 1993 Sep;13(9):5225–5232. doi: 10.1128/mcb.13.9.5225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lock P., Fumagalli S., Polakis P., McCormick F., Courtneidge S. A. The human p62 cDNA encodes Sam68 and not the RasGAP-associated p62 protein. Cell. 1996 Jan 12;84(1):23–24. doi: 10.1016/s0092-8674(00)80989-7. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Martin G. A., Yatani A., Clark R., Conroy L., Polakis P., Brown A. M., McCormick F. GAP domains responsible for ras p21-dependent inhibition of muscarinic atrial K+ channel currents. Science. 1992 Jan 10;255(5041):192–194. doi: 10.1126/science.1553544. [DOI] [PubMed] [Google Scholar]
  29. Matunis M. J., Michael W. M., Dreyfuss G. Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein. Mol Cell Biol. 1992 Jan;12(1):164–171. doi: 10.1128/mcb.12.1.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mayeda A., Munroe S. H., Cáceres J. F., Krainer A. R. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J. 1994 Nov 15;13(22):5483–5495. doi: 10.1002/j.1460-2075.1994.tb06883.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mayer B. J., Hamaguchi M., Hanafusa H. A novel viral oncogene with structural similarity to phospholipase C. Nature. 1988 Mar 17;332(6161):272–275. doi: 10.1038/332272a0. [DOI] [PubMed] [Google Scholar]
  32. McGlade J., Brunkhorst B., Anderson D., Mbamalu G., Settleman J., Dedhar S., Rozakis-Adcock M., Chen L. B., Pawson T. The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion. EMBO J. 1993 Aug;12(8):3073–3081. doi: 10.1002/j.1460-2075.1993.tb05976.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mollat P., Zhang G. Y., Frobert Y., Zhang Y. H., Fournier A., Grassi J., Thang M. N. Non-neutralizing monoclonal antibodies against Ras GTPase-activating protein: production, characterization and use in an enzyme immunometric assay. Biotechnology (N Y) 1992 Oct;10(10):1151–1156. doi: 10.1038/nbt1092-1151. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. Otsu M., Hiles I., Gout I., Fry M. J., Ruiz-Larrea F., Panayotou G., Thompson A., Dhand R., Hsuan J., Totty N. Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. Cell. 1991 Apr 5;65(1):91–104. doi: 10.1016/0092-8674(91)90411-q. [DOI] [PubMed] [Google Scholar]
  38. Pleiman C. M., Hertz W. M., Cambier J. C. Activation of phosphatidylinositol-3' kinase by Src-family kinase SH3 binding to the p85 subunit. Science. 1994 Mar 18;263(5153):1609–1612. doi: 10.1126/science.8128248. [DOI] [PubMed] [Google Scholar]
  39. Prasad K. V., Kapeller R., Janssen O., Repke H., Duke-Cohan J. S., Cantley L. C., Rudd C. E. Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase. Mol Cell Biol. 1993 Dec;13(12):7708–7717. doi: 10.1128/mcb.13.12.7708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pronk G. J., Bos J. L. The role of p21ras in receptor tyrosine kinase signalling. Biochim Biophys Acta. 1994 Dec 30;1198(2-3):131–147. doi: 10.1016/0304-419x(94)90010-8. [DOI] [PubMed] [Google Scholar]
  41. Pronk G. J., de Vries-Smits A. M., Ellis C., Bos J. L. Complex formation between the p21ras GTPase-activating protein and phosphoproteins p62 and p190 is independent of p21ras signalling. Oncogene. 1993 Oct;8(10):2773–2780. [PubMed] [Google Scholar]
  42. Qiu R. G., Chen J., Kirn D., McCormick F., Symons M. An essential role for Rac in Ras transformation. Nature. 1995 Mar 30;374(6521):457–459. doi: 10.1038/374457a0. [DOI] [PubMed] [Google Scholar]
  43. Ren R., Mayer B. J., Cicchetti P., Baltimore D. Identification of a ten-amino acid proline-rich SH3 binding site. Science. 1993 Feb 19;259(5098):1157–1161. doi: 10.1126/science.8438166. [DOI] [PubMed] [Google Scholar]
  44. Rodaway A. R., Teahan C. G., Casimir C. M., Segal A. W., Bentley D. L. Characterization of the 47-kilodalton autosomal chronic granulomatous disease protein: tissue-specific expression and transcriptional control by retinoic acid. Mol Cell Biol. 1990 Oct;10(10):5388–5396. doi: 10.1128/mcb.10.10.5388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rozakis-Adcock M., Fernley R., Wade J., Pawson T., Bowtell D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 1993 May 6;363(6424):83–85. doi: 10.1038/363083a0. [DOI] [PubMed] [Google Scholar]
  46. Saksela K., Cheng G., Baltimore D. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J. 1995 Feb 1;14(3):484–491. doi: 10.1002/j.1460-2075.1995.tb07024.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schweighoffer F., Barlat I., Chevallier-Multon M. C., Tocque B. Implication of GAP in Ras-dependent transactivation of a polyoma enhancer sequence. Science. 1992 May 8;256(5058):825–827. doi: 10.1126/science.1317056. [DOI] [PubMed] [Google Scholar]
  48. Settleman J., Albright C. F., Foster L. C., Weinberg R. A. Association between GTPase activators for Rho and Ras families. Nature. 1992 Sep 10;359(6391):153–154. doi: 10.1038/359153a0. [DOI] [PubMed] [Google Scholar]
  49. Settleman J., Narasimhan V., Foster L. C., Weinberg R. A. Molecular cloning of cDNAs encoding the GAP-associated protein p190: implications for a signaling pathway from ras to the nucleus. Cell. 1992 May 1;69(3):539–549. doi: 10.1016/0092-8674(92)90454-k. [DOI] [PubMed] [Google Scholar]
  50. Seuwen K., Lagarde A., Pouysségur J. Deregulation of hamster fibroblast proliferation by mutated ras oncogenes is not mediated by constitutive activation of phosphoinositide-specific phospholipase C. EMBO J. 1988 Jan;7(1):161–168. doi: 10.1002/j.1460-2075.1988.tb02796.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Siomi H., Choi M., Siomi M. C., Nussbaum R. L., Dreyfuss G. Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome. Cell. 1994 Apr 8;77(1):33–39. doi: 10.1016/0092-8674(94)90232-1. [DOI] [PubMed] [Google Scholar]
  52. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  53. Stahl M. L., Ferenz C. R., Kelleher K. L., Kriz R. W., Knopf J. L. Sequence similarity of phospholipase C with the non-catalytic region of src. Nature. 1988 Mar 17;332(6161):269–272. doi: 10.1038/332269a0. [DOI] [PubMed] [Google Scholar]
  54. Szeberényi J., Cai H., Cooper G. M. Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells. Mol Cell Biol. 1990 Oct;10(10):5324–5332. doi: 10.1128/mcb.10.10.5324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Taylor S. J., Shalloway D. An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis. Nature. 1994 Apr 28;368(6474):867–871. doi: 10.1038/368867a0. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. Wasenius V. M., Saraste M., Salvén P., Erämaa M., Holm L., Lehto V. P. Primary structure of the brain alpha-spectrin. J Cell Biol. 1989 Jan;108(1):79–93. doi: 10.1083/jcb.108.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Weng Z., Rickles R. J., Feng S., Richard S., Shaw A. S., Schreiber S. L., Brugge J. S. Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions. Mol Cell Biol. 1995 Oct;15(10):5627–5634. doi: 10.1128/mcb.15.10.5627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Weng Z., Thomas S. M., Rickles R. J., Taylor J. A., Brauer A. W., Seidel-Dugan C., Michael W. M., Dreyfuss G., Brugge J. S. Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains. Mol Cell Biol. 1994 Jul;14(7):4509–4521. doi: 10.1128/mcb.14.7.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. White M. A., Nicolette C., Minden A., Polverino A., Van Aelst L., Karin M., Wigler M. H. Multiple Ras functions can contribute to mammalian cell transformation. Cell. 1995 Feb 24;80(4):533–541. doi: 10.1016/0092-8674(95)90507-3. [DOI] [PubMed] [Google Scholar]
  61. Wu H., Reynolds A. B., Kanner S. B., Vines R. R., Parsons J. T. Identification and characterization of a novel cytoskeleton-associated pp60src substrate. Mol Cell Biol. 1991 Oct;11(10):5113–5124. doi: 10.1128/mcb.11.10.5113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yang Y. S., Garbay C., Duchesne M., Cornille F., Jullian N., Fromage N., Tocque B., Roques B. P. Solution structure of GAP SH3 domain by 1H NMR and spatial arrangement of essential Ras signaling-involved sequence. EMBO J. 1994 Mar 15;13(6):1270–1279. doi: 10.1002/j.1460-2075.1994.tb06379.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Yatani A., Okabe K., Polakis P., Halenbeck R., McCormick F., Brown A. M. ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K+ channels. Cell. 1990 Jun 1;61(5):769–776. doi: 10.1016/0092-8674(90)90187-j. [DOI] [PubMed] [Google Scholar]
  64. Yu H., Chen J. K., Feng S., Dalgarno D. C., Brauer A. W., Schreiber S. L. Structural basis for the binding of proline-rich peptides to SH3 domains. Cell. 1994 Mar 11;76(5):933–945. doi: 10.1016/0092-8674(94)90367-0. [DOI] [PubMed] [Google Scholar]
  65. 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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