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. 1996 Oct 1;15(19):5299–5313.

Mutation of a Src phosphorylation site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis.

K Hansen 1, M Johnell 1, A Siegbahn 1, C Rorsman 1, U Engström 1, C Wernstedt 1, C H Heldin 1, L Rönnstrand 1
PMCID: PMC452274  PMID: 8895575

Abstract

Ligand induced activation of the beta-receptor for platelet-derived growth factor (PDGF) leads to activation of Src family tyrosine kinases. We have explored the possibility that the receptor itself is a substrate for Src. We show that Tyr934 in the kinase domain of the PDGF receptor is phosphorylated by Src. Cell lines expressing a beta-receptor mutant, in which Tyr934 was replaced with a phenyalanine residue, showed reduced mitogenic signaling in response to PDGF-BB. In contrast, the mutant receptor mediated increased signals for chemotaxis and actin reorganization. Whereas the motility responses of cells expressing wild-type beta-receptors were attenuated by inhibition of phosphatidylinositol 3'-kinase, those of cells expressing the mutant receptor were only slightly influenced. In contrast, PDGF-BB-induced chemotaxis of the cells with the mutant receptor was attenuated by inhibition of protein kinase C, whereas the chemotaxis of cells expressing the wild-type beta-receptor was less affected. Moreover, the PDGF-BB-stimulated tyrosine phosphorylation of phospholipase C-gamma was increased in the mutant receptor cells compared with wild-type receptor cells. In conclusion, the characteristics of the Y934F mutant suggest that the phosphorylation of Tyr934 by Src negatively modulates a signal transduction pathway leading to motility responses which involves phospholipase C-gamma, and shifts the response to increased mitogenicity.

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Selected References

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

  1. Akimoto K., Takahashi R., Moriya S., Nishioka N., Takayanagi J., Kimura K., Fukui Y., Osada S. i., Mizuno K., Hirai S. i. EGF or PDGF receptors activate atypical PKClambda through phosphatidylinositol 3-kinase. EMBO J. 1996 Feb 15;15(4):788–798. [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. Blume-Jensen P., Wernstedt C., Heldin C. H., Rönnstrand L. Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact cells. J Biol Chem. 1995 Jun 9;270(23):14192–14200. doi: 10.1074/jbc.270.23.14192. [DOI] [PubMed] [Google Scholar]
  4. Bokoch G. M. Chemoattractant signaling and leukocyte activation. Blood. 1995 Sep 1;86(5):1649–1660. [PubMed] [Google Scholar]
  5. Boonen G. J., de Koster B. M., VanSteveninck J., Elferink J. G. Neutrophil chemotaxis induced by the diacylglycerol kinase inhibitor R59022. Biochim Biophys Acta. 1993 Jul 28;1178(1):97–102. doi: 10.1016/0167-4889(93)90114-5. [DOI] [PubMed] [Google Scholar]
  6. Calalb M. B., Polte T. R., Hanks S. K. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol. 1995 Feb;15(2):954–963. doi: 10.1128/mcb.15.2.954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Claesson-Welsh L. Platelet-derived growth factor receptor signals. J Biol Chem. 1994 Dec 23;269(51):32023–32026. [PubMed] [Google Scholar]
  10. 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]
  11. Dekker L. V., Parker P. J. Protein kinase C--a question of specificity. Trends Biochem Sci. 1994 Feb;19(2):73–77. doi: 10.1016/0968-0004(94)90038-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Gilbert S. H., Perry K., Fay F. S. Mediation of chemoattractant-induced changes in [Ca2+]i and cell shape, polarity, and locomotion by InsP3, DAG, and protein kinase C in newt eosinophils. J Cell Biol. 1994 Oct;127(2):489–503. doi: 10.1083/jcb.127.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hagihara H., Nomoto A., Mutoh S., Yamaguchi I., Ono T. Role of inflammatory responses in initiation of atherosclerosis: effects of anti-inflammatory drugs on cuff-induced leukocyte accumulation and intimal thickening of rabbit carotid artery. Atherosclerosis. 1991 Nov;91(1-2):107–116. doi: 10.1016/0021-9150(91)90192-6. [DOI] [PubMed] [Google Scholar]
  15. Hawkins P. T., Eguinoa A., Qiu R. G., Stokoe D., Cooke F. T., Walters R., Wennström S., Claesson-Welsh L., Evans T., Symons M. PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curr Biol. 1995 Apr 1;5(4):393–403. doi: 10.1016/s0960-9822(95)00080-7. [DOI] [PubMed] [Google Scholar]
  16. Heldin C. H. Dimerization of cell surface receptors in signal transduction. Cell. 1995 Jan 27;80(2):213–223. doi: 10.1016/0092-8674(95)90404-2. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hyers T. M., Tricomi S. M., Liao J. J. Inhibition of some human neutrophil functions by the cyclooxygenase inhibitor ketorolac tromethamine. J Leukoc Biol. 1992 May;51(5):490–495. doi: 10.1002/jlb.51.5.490. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kundra V., Escobedo J. A., Kazlauskas A., Kim H. K., Rhee S. G., Williams L. T., Zetter B. R. Regulation of chemotaxis by the platelet-derived growth factor receptor-beta. Nature. 1994 Feb 3;367(6462):474–476. doi: 10.1038/367474a0. [DOI] [PubMed] [Google Scholar]
  21. Kundra V., Soker S., Zetter B. R. Excess early signaling activity inhibits cellular chemotaxis toward PDGF-BB. Oncogene. 1994 May;9(5):1429–1435. [PubMed] [Google Scholar]
  22. Lee S. B., Rhee S. G. Significance of PIP2 hydrolysis and regulation of phospholipase C isozymes. Curr Opin Cell Biol. 1995 Apr;7(2):183–189. doi: 10.1016/0955-0674(95)80026-3. [DOI] [PubMed] [Google Scholar]
  23. Lee Y. H., Kim H. S., Pai J. K., Ryu S. H., Suh P. G. Activation of phospholipase D induced by platelet-derived growth factor is dependent upon the level of phospholipase C-gamma 1. J Biol Chem. 1994 Oct 28;269(43):26842–26847. [PubMed] [Google Scholar]
  24. Liao F., Shin H. S., Rhee S. G. In vitro tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2 by src-family protein tyrosine kinases. Biochem Biophys Res Commun. 1993 Mar 31;191(3):1028–1033. doi: 10.1006/bbrc.1993.1320. [DOI] [PubMed] [Google Scholar]
  25. Mellstroöm K., Höglund A. S., Nistér M., Heldin C. H., Westermark B., Lindberg U. The effect of platelet-derived growth factor on morphology and motility of human glial cells. J Muscle Res Cell Motil. 1983 Oct;4(5):589–609. doi: 10.1007/BF00712117. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Moriya S., Kazlauskas A., Akimoto K., Hirai S., Mizuno K., Takenawa T., Fukui Y., Watanabe Y., Ozaki S., Ohno S. Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):151–155. doi: 10.1073/pnas.93.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nakanishi H., Brewer K. A., Exton J. H. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1993 Jan 5;268(1):13–16. [PubMed] [Google Scholar]
  29. Nakanishi O., Shibasaki F., Hidaka M., Homma Y., Takenawa T. Phospholipase C-gamma 1 associates with viral and cellular src kinases. J Biol Chem. 1993 May 25;268(15):10754–10759. [PubMed] [Google Scholar]
  30. Nomoto A., Mutoh S., Hagihara H., Yamaguchi I. Smooth muscle cell migration induced by inflammatory cell products and its inhibition by a potent calcium antagonist, nilvadipine. Atherosclerosis. 1988 Aug;72(2-3):213–219. doi: 10.1016/0021-9150(88)90083-4. [DOI] [PubMed] [Google Scholar]
  31. Obermeier A., Tinhofer I., Grunicke H. H., Ullrich A. Transforming potentials of epidermal growth factor and nerve growth factor receptors inversely correlate with their phospholipase C gamma affinity and signal activation. EMBO J. 1996 Jan 2;15(1):73–82. [PMC free article] [PubMed] [Google Scholar]
  32. Pawson T. Protein modules and signalling networks. Nature. 1995 Feb 16;373(6515):573–580. doi: 10.1038/373573a0. [DOI] [PubMed] [Google Scholar]
  33. Peterson J. E., Jelinek T., Kaleko M., Siddle K., Weber M. J. c phosphorylation and activation of the IGF-I receptor in src-transformed cells. J Biol Chem. 1994 Nov 4;269(44):27315–27321. [PubMed] [Google Scholar]
  34. 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]
  35. Schneider C., Newman R. A., Sutherland D. R., Asser U., Greaves M. F. A one-step purification of membrane proteins using a high efficiency immunomatrix. J Biol Chem. 1982 Sep 25;257(18):10766–10769. [PubMed] [Google Scholar]
  36. Siegbahn A., Hammacher A., Westermark B., Heldin C. H. Differential effects of the various isoforms of platelet-derived growth factor on chemotaxis of fibroblasts, monocytes, and granulocytes. J Clin Invest. 1990 Mar;85(3):916–920. doi: 10.1172/JCI114519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Songyang Z., Carraway K. L., 3rd, Eck M. J., Harrison S. C., Feldman R. A., Mohammadi M., Schlessinger J., Hubbard S. R., Smith D. P., Eng C. Catalytic specificity of protein-tyrosine kinases is critical for selective signalling. Nature. 1995 Feb 9;373(6514):536–539. doi: 10.1038/373536a0. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Stover D. R., Becker M., Liebetanz J., Lydon N. B. Src phosphorylation of the epidermal growth factor receptor at novel sites mediates receptor interaction with Src and P85 alpha. J Biol Chem. 1995 Jun 30;270(26):15591–15597. doi: 10.1074/jbc.270.26.15591. [DOI] [PubMed] [Google Scholar]
  40. Toker A., Meyer M., Reddy K. K., Falck J. R., Aneja R., Aneja S., Parra A., Burns D. J., Ballas L. M., Cantley L. C. Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. J Biol Chem. 1994 Dec 23;269(51):32358–32367. [PubMed] [Google Scholar]
  41. 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]
  42. Wasilenko W. J., Payne D. M., Fitzgerald D. L., Weber M. J. Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene. Mol Cell Biol. 1991 Jan;11(1):309–321. doi: 10.1128/mcb.11.1.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Wright T. M., Hoffman R. D., Nishijima J., Jakoi L., Snyderman R., Shin H. S. Leukocyte chemoattraction by 1,2-diacylglycerol. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1869–1873. doi: 10.1073/pnas.85.6.1869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yeo E. J., Kazlauskas A., Exton J. H. Activation of phospholipase C-gamma is necessary for stimulation of phospholipase D by platelet-derived growth factor. J Biol Chem. 1994 Nov 11;269(45):27823–27826. [PubMed] [Google Scholar]

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