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. 1994 Oct;14(10):6715–6726. doi: 10.1128/mcb.14.10.6715

Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation.

A K Arvidsson 1, E Rupp 1, E Nånberg 1, J Downward 1, L Rönnstrand 1, S Wennström 1, J Schlessinger 1, C H Heldin 1, L Claesson-Welsh 1
PMCID: PMC359202  PMID: 7935391

Abstract

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.

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Selected References

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

  1. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrews D. M., Kitchin J., Seale P. W. Solid-phase synthesis of a range of O-phosphorylated peptides by post-assembly phosphitylation and oxidation. Int J Pept Protein Res. 1991 Nov;38(5):469–475. doi: 10.1111/j.1399-3011.1991.tb01528.x. [DOI] [PubMed] [Google Scholar]
  3. Arvidsson A. K., Heldin C. H., Claesson-Welsh L. Transduction of circular membrane ruffling by the platelet-derived growth factor beta-receptor is dependent on its kinase insert. Cell Growth Differ. 1992 Dec;3(12):881–887. [PubMed] [Google Scholar]
  4. Basu T. N., Gutmann D. H., Fletcher J. A., Glover T. W., Collins F. S., Downward J. Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients. Nature. 1992 Apr 23;356(6371):713–715. doi: 10.1038/356713a0. [DOI] [PubMed] [Google Scholar]
  5. Boguski M. S., McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. doi: 10.1038/366643a0. [DOI] [PubMed] [Google Scholar]
  6. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  7. Brady S. T. Motor neurons and neurofilaments in sickness and in health. Cell. 1993 Apr 9;73(1):1–3. doi: 10.1016/0092-8674(93)90151-f. [DOI] [PubMed] [Google Scholar]
  8. Burgering B. M., de Vries-Smits A. M., Medema R. H., van Weeren P. C., Tertoolen L. G., Bos J. L. Epidermal growth factor induces phosphorylation of extracellular signal-regulated kinase 2 via multiple pathways. Mol Cell Biol. 1993 Dec;13(12):7248–7256. doi: 10.1128/mcb.13.12.7248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Claesson-Welsh L., Eriksson A., Morén A., Severinsson L., Ek B., Ostman A., Betsholtz C., Heldin C. H. cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules. Mol Cell Biol. 1988 Aug;8(8):3476–3486. doi: 10.1128/mcb.8.8.3476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Claesson-Welsh L., Hammacher A., Westermark B., Heldin C. H., Nistér M. Identification and structural analysis of the A type receptor for platelet-derived growth factor. Similarities with the B type receptor. J Biol Chem. 1989 Jan 25;264(3):1742–1747. [PubMed] [Google Scholar]
  11. Claesson-Welsh L. Signal transduction by the PDGF receptors. Prog Growth Factor Res. 1994;5(1):37–54. doi: 10.1016/0955-2235(94)90016-7. [DOI] [PubMed] [Google Scholar]
  12. Clark S. G., Stern M. J., Horvitz H. R. C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature. 1992 Mar 26;356(6367):340–344. doi: 10.1038/356340a0. [DOI] [PubMed] [Google Scholar]
  13. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Fukui Y., Hanafusa H. Phosphatidylinositol kinase activity associates with viral p60src protein. Mol Cell Biol. 1989 Apr;9(4):1651–1658. doi: 10.1128/mcb.9.4.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gale N. W., Kaplan S., Lowenstein E. J., Schlessinger J., Bar-Sagi D. Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras. Nature. 1993 May 6;363(6424):88–92. doi: 10.1038/363088a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Gotoh N., Tojo A., Muroya K., Hashimoto Y., Hattori S., Nakamura S., Takenawa T., Yazaki Y., Shibuya M. Epidermal growth factor-receptor mutant lacking the autophosphorylation sites induces phosphorylation of Shc protein and Shc-Grb2/ASH association and retains mitogenic activity. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):167–171. doi: 10.1073/pnas.91.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heldin C. H., Westermark B. Platelet-derived growth factor: mechanism of action and possible in vivo function. Cell Regul. 1990 Jul;1(8):555–566. doi: 10.1091/mbc.1.8.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Kazlauskas A., Feng G. S., Pawson T., Valius M. The 64-kDa protein that associates with the platelet-derived growth factor receptor beta subunit via Tyr-1009 is the SH2-containing phosphotyrosine phosphatase Syp. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6939–6943. doi: 10.1073/pnas.90.15.6939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lechleider R. J., Sugimoto S., Bennett A. M., Kashishian A. S., Cooper J. A., Shoelson S. E., Walsh C. T., Neel B. G. Activation of the SH2-containing phosphotyrosine phosphatase SH-PTP2 by its binding site, phosphotyrosine 1009, on the human platelet-derived growth factor receptor. J Biol Chem. 1993 Oct 15;268(29):21478–21481. [PubMed] [Google Scholar]
  25. 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]
  26. Li W., Nishimura R., Kashishian A., Batzer A. G., Kim W. J., Cooper J. A., Schlessinger J. A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase. Mol Cell Biol. 1994 Jan;14(1):509–517. doi: 10.1128/mcb.14.1.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Matuoka K., Shibasaki F., Shibata M., Takenawa T. Ash/Grb-2, a SH2/SH3-containing protein, couples to signaling for mitogenesis and cytoskeletal reorganization by EGF and PDGF. EMBO J. 1993 Sep;12(9):3467–3473. doi: 10.1002/j.1460-2075.1993.tb06021.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Medema R. H., Bos J. L. The role of p21ras in receptor tyrosine kinase signaling. Crit Rev Oncog. 1993;4(6):615–661. [PubMed] [Google Scholar]
  30. Miyazono K., Hellman U., Wernstedt C., Heldin C. H. Latent high molecular weight complex of transforming growth factor beta 1. Purification from human platelets and structural characterization. J Biol Chem. 1988 May 5;263(13):6407–6415. [PubMed] [Google Scholar]
  31. Mori S., Heldin C. H., Claesson-Welsh L. Ligand-induced ubiquitination of the platelet-derived growth factor beta-receptor plays a negative regulatory role in its mitogenic signaling. J Biol Chem. 1993 Jan 5;268(1):577–583. [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Nånberg E., Westermark B. Platelet-derived growth factor increases the turnover of GTP/GDP on ras in permeabilized fibroblasts. J Biol Chem. 1993 Aug 25;268(24):18187–18194. [PubMed] [Google Scholar]
  35. Olivier J. P., Raabe T., Henkemeyer M., Dickson B., Mbamalu G., Margolis B., Schlessinger J., Hafen E., Pawson T. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos. Cell. 1993 Apr 9;73(1):179–191. doi: 10.1016/0092-8674(93)90170-u. [DOI] [PubMed] [Google Scholar]
  36. Pelicci G., Lanfrancone L., Grignani F., McGlade J., Cavallo F., Forni G., Nicoletti I., Grignani F., Pawson T., Pelicci P. G. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell. 1992 Jul 10;70(1):93–104. doi: 10.1016/0092-8674(92)90536-l. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Rozakis-Adcock M., McGlade J., Mbamalu G., Pelicci G., Daly R., Li W., Batzer A., Thomas S., Brugge J., Pelicci P. G. Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature. 1992 Dec 17;360(6405):689–692. doi: 10.1038/360689a0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Rönnstrand L., Terracio L., Claesson-Welsh L., Heldin C. H., Rubin K. Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor. J Biol Chem. 1988 Jul 25;263(21):10429–10435. [PubMed] [Google Scholar]
  41. 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]
  42. Satoh T., Fantl W. J., Escobedo J. A., Williams L. T., Kaziro Y. Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types. Mol Cell Biol. 1993 Jun;13(6):3706–3713. doi: 10.1128/mcb.13.6.3706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schlessinger J., Ullrich A. Growth factor signaling by receptor tyrosine kinases. Neuron. 1992 Sep;9(3):383–391. doi: 10.1016/0896-6273(92)90177-f. [DOI] [PubMed] [Google Scholar]
  44. Segatto O., Pelicci G., Giuli S., Digiesi G., Di Fiore P. P., McGlade J., Pawson T., Pelicci P. G. Shc products are substrates of erbB-2 kinase. Oncogene. 1993 Aug;8(8):2105–2112. [PubMed] [Google Scholar]
  45. Simon M. A., Dodson G. S., Rubin G. M. An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell. 1993 Apr 9;73(1):169–177. doi: 10.1016/0092-8674(93)90169-q. [DOI] [PubMed] [Google Scholar]
  46. Skolnik E. Y., Lee C. H., Batzer A., Vicentini L. M., Zhou M., Daly R., Myers M. J., Jr, Backer J. M., Ullrich A., White M. F. The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. EMBO J. 1993 May;12(5):1929–1936. doi: 10.1002/j.1460-2075.1993.tb05842.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. Valius M., Kazlauskas A. Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell. 1993 Apr 23;73(2):321–334. doi: 10.1016/0092-8674(93)90232-f. [DOI] [PubMed] [Google Scholar]
  50. Weima S. M., van Rooijen M. A., Mummery C. L., Feyen A., de Laat S. W., van Zoelen E. J. Identification of the type-B receptor for platelet-derived growth factor in human embryonal carcinoma cells. Exp Cell Res. 1990 Feb;186(2):324–331. doi: 10.1016/0014-4827(90)90312-x. [DOI] [PubMed] [Google Scholar]
  51. Wennström S., Siegbahn A., Yokote K., Arvidsson A. K., Heldin C. H., Mori S., Claesson-Welsh L. Membrane ruffling and chemotaxis transduced by the PDGF beta-receptor require the binding site for phosphatidylinositol 3' kinase. Oncogene. 1994 Feb;9(2):651–660. [PubMed] [Google Scholar]
  52. Westermark B., Siegbahn A., Heldin C. H., Claesson-Welsh L. B-type receptor for platelet-derived growth factor mediates a chemotactic response by means of ligand-induced activation of the receptor protein-tyrosine kinase. Proc Natl Acad Sci U S A. 1990 Jan;87(1):128–132. doi: 10.1073/pnas.87.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yokote K., Mori S., Hansen K., McGlade J., Pawson T., Heldin C. H., Claesson-Welsh L. Direct interaction between Shc and the platelet-derived growth factor beta-receptor. J Biol Chem. 1994 May 27;269(21):15337–15343. [PubMed] [Google Scholar]
  54. van der Geer P., Hunter T. Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts. EMBO J. 1993 Dec 15;12(13):5161–5172. doi: 10.1002/j.1460-2075.1993.tb06211.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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