Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1992 Nov;11(11):3911–3919. doi: 10.1002/j.1460-2075.1992.tb05484.x

Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma.

L Rönnstrand 1, S Mori 1, A K Arridsson 1, A Eriksson 1, C Wernstedt 1, U Hellman 1, L Claesson-Welsh 1, C H Heldin 1
PMCID: PMC556901  PMID: 1396585

Abstract

Two novel sites of autophosphorylation were localized to the C-terminal tail of the PDGF beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) compared to the wild type PDGF beta-receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC-gamma could be detected. These data show that tyrosine phosphorylation of PLC-gamma is dependent on autophosphorylation of the PDGF beta-receptor at Tyr1009 and Tyr1021.

Full text

PDF
3919

Images in this article

3912Image
on p.3912
3913Image
on p.3913
3914Image
on p.3914
3914Image
on p.3914
3915Image
on p.3915
3917Image
on p.3917

Selected References

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

  1. Arteaga C. L., Johnson M. D., Todderud G., Coffey R. J., Carpenter G., Page D. L. Elevated content of the tyrosine kinase substrate phospholipase C-gamma 1 in primary human breast carcinomas. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10435–10439. doi: 10.1073/pnas.88.23.10435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auger K. R., Serunian L. A., Soltoff S. P., Libby P., Cantley L. C. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989 Apr 7;57(1):167–175. doi: 10.1016/0092-8674(89)90182-7. [DOI] [PubMed] [Google Scholar]
  3. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolton A. E., Hunter W. M. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J. 1973 Jul;133(3):529–539. doi: 10.1042/bj1330529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  6. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [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., Eriksson A., Westermark B., Heldin C. H. cDNA cloning and expression of the human A-type platelet-derived growth factor (PDGF) receptor establishes structural similarity to the B-type PDGF receptor. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4917–4921. doi: 10.1073/pnas.86.13.4917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Coughlin S. R., Escobedo J. A., Williams L. T. Role of phosphatidylinositol kinase in PDGF receptor signal transduction. Science. 1989 Mar 3;243(4895):1191–1194. doi: 10.1126/science.2466336. [DOI] [PubMed] [Google Scholar]
  11. Courtneidge S. A., Kypta R. M., Cooper J. A., Kazlauskas A. Platelet-derived growth factor receptor sequences important for binding of src family tyrosine kinases. Cell Growth Differ. 1991 Oct;2(10):483–486. [PubMed] [Google Scholar]
  12. Downward J., Yarden Y., Mayes E., Scrace G., Totty N., Stockwell P., Ullrich A., Schlessinger J., Waterfield M. D. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature. 1984 Feb 9;307(5951):521–527. doi: 10.1038/307521a0. [DOI] [PubMed] [Google Scholar]
  13. Escobedo J. A., Kaplan D. R., Kavanaugh W. M., Turck C. W., Williams L. T. A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine. Mol Cell Biol. 1991 Feb;11(2):1125–1132. doi: 10.1128/mcb.11.2.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Hill T. D., Dean N. M., Mordan L. J., Lau A. F., Kanemitsu M. Y., Boynton A. L. PDGF-induced activation of phospholipase C is not required for induction of DNA synthesis. Science. 1990 Jun 29;248(4963):1660–1663. doi: 10.1126/science.2163545. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Kazlauskas A., Cooper J. A. Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins. Cell. 1989 Sep 22;58(6):1121–1133. doi: 10.1016/0092-8674(89)90510-2. [DOI] [PubMed] [Google Scholar]
  20. Kazlauskas A., Durden D. L., Cooper J. A. Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit. Cell Regul. 1991 Jun;2(6):413–425. doi: 10.1091/mbc.2.6.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Kim H. K., Kim J. W., Zilberstein A., Margolis B., Kim J. G., Schlessinger J., Rhee S. G. PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254. Cell. 1991 May 3;65(3):435–441. doi: 10.1016/0092-8674(91)90461-7. [DOI] [PubMed] [Google Scholar]
  24. Kim J. W., Sim S. S., Kim U. H., Nishibe S., Wahl M. I., Carpenter G., Rhee S. G. Tyrosine residues in bovine phospholipase C-gamma phosphorylated by the epidermal growth factor receptor in vitro. J Biol Chem. 1990 Mar 5;265(7):3940–3943. [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Laudano A. P., Buchanan J. M. Phosphorylation of tyrosine in the carboxyl-terminal tryptic peptide of pp60c-src. Proc Natl Acad Sci U S A. 1986 Feb;83(4):892–896. doi: 10.1073/pnas.83.4.892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Margolis B. Proteins with SH2 domains: transducers in the tyrosine kinase signaling pathway. Cell Growth Differ. 1992 Jan;3(1):73–80. [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. McKeehan W. L., Hamilton W. G., Ham R. G. Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2023–2027. doi: 10.1073/pnas.73.6.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Miyazono K., Okabe T., Urabe A., Takaku F., Heldin C. H. Purification and properties of an endothelial cell growth factor from human platelets. J Biol Chem. 1987 Mar 25;262(9):4098–4103. [PubMed] [Google Scholar]
  35. Mohammadi M., Honegger A. M., Rotin D., Fischer R., Bellot F., Li W., Dionne C. A., Jaye M., Rubinstein M., Schlessinger J. A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1. Mol Cell Biol. 1991 Oct;11(10):5068–5078. doi: 10.1128/mcb.11.10.5068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Mori S., Claesson-Welsh L., Heldin C. H. Identification of a hydrophobic region in the carboxyl terminus of the platelet-derived growth factor beta-receptor which is important for ligand-mediated endocytosis. J Biol Chem. 1991 Nov 5;266(31):21158–21164. [PubMed] [Google Scholar]
  38. Mori S., Heldin C. H., Claesson-Welsh L. Ligand-induced polyubiquitination of the platelet-derived growth factor beta-receptor. J Biol Chem. 1992 Mar 25;267(9):6429–6434. [PubMed] [Google Scholar]
  39. Nishibe S., Wahl M. I., Hernández-Sotomayor S. M., Tonks N. K., Rhee S. G., Carpenter G. Increase of the catalytic activity of phospholipase C-gamma 1 by tyrosine phosphorylation. Science. 1990 Nov 30;250(4985):1253–1256. doi: 10.1126/science.1700866. [DOI] [PubMed] [Google Scholar]
  40. Ostman A., Bäckström G., Fong N., Betsholtz C., Wernstedt C., Hellman U., Westermark B., Valenzuela P., Heldin C. H. Expression of three recombinant homodimeric isoforms of PDGF in Saccharomyces cerevisiae: evidence for difference in receptor binding and functional activities. Growth Factors. 1989;1(3):271–281. doi: 10.3109/08977198908998003. [DOI] [PubMed] [Google Scholar]
  41. Pantazis P., Bonner W. M. Quantitative determination of histone modification. H2A acetylation and phosphorylation. J Biol Chem. 1981 May 10;256(9):4669–4675. [PubMed] [Google Scholar]
  42. Rhee S. G., Suh P. G., Ryu S. H., Lee S. Y. Studies of inositol phospholipid-specific phospholipase C. Science. 1989 May 5;244(4904):546–550. doi: 10.1126/science.2541501. [DOI] [PubMed] [Google Scholar]
  43. Rotin D., Margolis B., Mohammadi M., Daly R. J., Daum G., Li N., Fischer E. H., Burgess W. H., Ullrich A., Schlessinger J. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma. EMBO J. 1992 Feb;11(2):559–567. doi: 10.1002/j.1460-2075.1992.tb05087.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rönnstrand L., Beckmann M. P., Faulders B., Ostman A., Ek B., Heldin C. H. Purification of the receptor for platelet-derived growth factor from porcine uterus. J Biol Chem. 1987 Mar 5;262(7):2929–2932. [PubMed] [Google Scholar]
  45. Vega Q. C., Cochet C., Filhol O., Chang C. P., Rhee S. G., Gill G. N. A site of tyrosine phosphorylation in the C terminus of the epidermal growth factor receptor is required to activate phospholipase C. Mol Cell Biol. 1992 Jan;12(1):128–135. doi: 10.1128/mcb.12.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wahl M. I., Nishibe S., Kim J. W., Kim H., Rhee S. G., Carpenter G. Identification of two epidermal growth factor-sensitive tyrosine phosphorylation sites of phospholipase C-gamma in intact HSC-1 cells. J Biol Chem. 1990 Mar 5;265(7):3944–3948. [PubMed] [Google Scholar]
  47. Wahl M. I., Nishibe S., Suh P. G., Rhee S. G., Carpenter G. Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalization and extracellular calcium. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1568–1572. doi: 10.1073/pnas.86.5.1568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wahl M., Carpenter G. Selective phospholipase C activation. Bioessays. 1991 Mar;13(3):107–113. doi: 10.1002/bies.950130303. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. 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]
  51. Yarden Y., Escobedo J. A., Kuang W. J., Yang-Feng T. L., Daniel T. O., Tremble P. M., Chen E. Y., Ando M. E., Harkins R. N., Francke U. Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors. Nature. 1986 Sep 18;323(6085):226–232. doi: 10.1038/323226a0. [DOI] [PubMed] [Google Scholar]
  52. Yu J. C., Heidaran M. A., Pierce J. H., Gutkind J. S., Lombardi D., Ruggiero M., Aaronson S. A. Tyrosine mutations within the alpha platelet-derived growth factor receptor kinase insert domain abrogate receptor-associated phosphatidylinositol-3 kinase activity without affecting mitogenic or chemotactic signal transduction. Mol Cell Biol. 1991 Jul;11(7):3780–3785. doi: 10.1128/mcb.11.7.3780. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES