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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Jun;82(12):4080–4084. doi: 10.1073/pnas.82.12.4080

Platelet-derived growth factor mimics phorbol diester action on epidermal growth factor receptor phosphorylation at threonine-654.

R J Davis, M P Czech
PMCID: PMC397938  PMID: 2987962

Abstract

Addition of platelet-derived growth factor (PDGF) to quiescent WI-38 human fetal lung fibroblasts mimics the effect of tumor-promoting phorbol diesters to inhibit the high-affinity binding of 125I-labeled epidermal growth factor (125I-EGF). PDGF, like phorbol diesters, was found to increase the phosphorylation state of EGF receptors immunoprecipitated from intact fibroblasts that were labeled to equilibrium with [32P]phosphate. Phosphoamino acid analysis of the EGF receptors indicated that both PDGF and phorbol diesters increased the level of [32P]phosphoserine and [32P]phosphothreonine. Phosphopeptide mapping of the EGF receptor demonstrated that PDGF increased the phosphorylation of several sites and induced the phosphorylation of a site that was not observed to be phosphorylated on EGF receptors isolated from control cells. This latter phosphorylation site on the EGF receptor was identified as threonine-654, previously shown to be phosphorylated in response to phorbol diesters in intact cells or by purified protein kinase C in vitro. Further, it was observed that PDGF mimicked the action of phorbol diesters to inhibit the EGF-dependent tyrosine phosphorylation of the EGF receptor in [32P]phosphate-labeled fibroblasts. These results are consistent with the hypothesis that increases in diacylglycerol and Ca2+ levels caused by addition of PDGF to fibroblasts activate protein kinase C and that this kinase, at least in part, mediates the effect of PDGF on the phosphorylation of the EGF receptor. The data further suggest that protein kinase C may play an important role in the regulation of cellular metabolism and proliferation by PDGF.

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Selected References

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

  1. Ashendel C. L., Staller J. M., Boutwell R. K. Protein kinase activity associated with a phorbol ester receptor purified from mouse brain. Cancer Res. 1983 Sep;43(9):4333–4337. [PubMed] [Google Scholar]
  2. Assoian R. K., Grotendorst G. R., Miller D. M., Sporn M. B. Cellular transformation by coordinated action of three peptide growth factors from human platelets. 1984 Jun 28-Jul 4Nature. 309(5971):804–806. doi: 10.1038/309804a0. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J., Heslop J. P., Irvine R. F., Brown K. D. Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor. Biochem J. 1984 Aug 15;222(1):195–201. doi: 10.1042/bj2220195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bowen-Pope D. F., Dicorleto P. E., Ross R. Interactions between the receptors for platelet-derived growth factor and epidermal growth factor. J Cell Biol. 1983 Mar;96(3):679–683. doi: 10.1083/jcb.96.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown K. D., Dicker P., Rozengurt E. Inhibition of epidermal growth factor binding to surface receptors by tumor promotors. Biochem Biophys Res Commun. 1979 Feb 28;86(4):1037–1043. doi: 10.1016/0006-291x(79)90221-3. [DOI] [PubMed] [Google Scholar]
  6. Carpenter G., King L., Jr, Cohen S. Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor. J Biol Chem. 1979 Jun 10;254(11):4884–4891. [PubMed] [Google Scholar]
  7. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
  8. Cochet C., Gill G. N., Meisenhelder J., Cooper J. A., Hunter T. C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Feb 25;259(4):2553–2558. [PubMed] [Google Scholar]
  9. Cochran B. H., Zullo J., Verma I. M., Stiles C. D. Expression of the c-fos gene and of an fos-related gene is stimulated by platelet-derived growth factor. Science. 1984 Nov 30;226(4678):1080–1082. doi: 10.1126/science.6093261. [DOI] [PubMed] [Google Scholar]
  10. Collins M. K., Sinnett-Smith J. W., Rozengurt E. Platelet-derived growth factor treatment decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells. J Biol Chem. 1983 Oct 10;258(19):11689–11693. [PubMed] [Google Scholar]
  11. Cooper J. A., Bowen-Pope D. F., Raines E., Ross R., Hunter T. Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins. Cell. 1982 Nov;31(1):263–273. doi: 10.1016/0092-8674(82)90426-3. [DOI] [PubMed] [Google Scholar]
  12. Davis R. J., Czech M. P. Tumor-promoting phorbol diesters cause the phosphorylation of epidermal growth factor receptors in normal human fibroblasts at threonine-654. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1974–1978. doi: 10.1073/pnas.82.7.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Davis R. J., Czech M. P. Tumor-promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor. J Biol Chem. 1984 Jul 10;259(13):8545–8549. [PubMed] [Google Scholar]
  14. Davis R. J., Ganong B. R., Bell R. M., Czech M. P. sn-1,2-Dioctanoylglycerol. A cell-permeable diacylglycerol that mimics phorbol diester action on the epidermal growth factor receptor and mitogenesis. J Biol Chem. 1985 Feb 10;260(3):1562–1566. [PubMed] [Google Scholar]
  15. Dicker P., Rozengurt E. Stimulation of DNA synthesis by tumour promoter and pure mitogenic factors. Nature. 1978 Dec 14;276(5689):723–726. doi: 10.1038/276723a0. [DOI] [PubMed] [Google Scholar]
  16. Doolittle R. F., Hunkapiller M. W., Hood L. E., Devare S. G., Robbins K. C., Aaronson S. A., Antoniades H. N. Simian sarcoma virus onc gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor. Science. 1983 Jul 15;221(4607):275–277. doi: 10.1126/science.6304883. [DOI] [PubMed] [Google Scholar]
  17. Downward J., Parker P., Waterfield M. D. Autophosphorylation sites on the epidermal growth factor receptor. Nature. 1984 Oct 4;311(5985):483–485. doi: 10.1038/311483a0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Ek B., Heldin C. H. Characterization of a tyrosine-specific kinase activity in human fibroblast membranes stimulated by platelet-derived growth factor. J Biol Chem. 1982 Sep 10;257(17):10486–10492. [PubMed] [Google Scholar]
  20. Ek B., Heldin C. H. Use of an antiserum against phosphotyrosine for the identification of phosphorylated components in human fibroblasts stimulated by platelet-derived growth factor. J Biol Chem. 1984 Sep 10;259(17):11145–11152. [PubMed] [Google Scholar]
  21. Ek B., Westermark B., Wasteson A., Heldin C. H. Stimulation of tyrosine-specific phosphorylation by platelet-derived growth factor. Nature. 1982 Feb 4;295(5848):419–420. doi: 10.1038/295419a0. [DOI] [PubMed] [Google Scholar]
  22. Frackelton A. R., Jr, Tremble P. M., Williams L. T. Evidence for the platelet-derived growth factor-stimulated tyrosine phosphorylation of the platelet-derived growth factor receptor in vivo. Immunopurification using a monoclonal antibody to phosphotyrosine. J Biol Chem. 1984 Jun 25;259(12):7909–7915. [PubMed] [Google Scholar]
  23. Frantz C. N., Stiles C. D., Scher C. D. The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate enhances the proliferative response of Balb/c-3T3 cells to hormonal growth factors. J Cell Physiol. 1979 Sep;100(3):413–424. doi: 10.1002/jcp.1041000305. [DOI] [PubMed] [Google Scholar]
  24. Friedman B., Frackelton A. R., Jr, Ross A. H., Connors J. M., Fujiki H., Sugimura T., Rosner M. R. Tumor promoters block tyrosine-specific phosphorylation of the epidermal growth factor receptor. Proc Natl Acad Sci U S A. 1984 May;81(10):3034–3038. doi: 10.1073/pnas.81.10.3034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Glenn K., Bowen-Pope D. F., Ross R. Platelet-derived growth factor. III. Identification of a platelet-derived growth factor receptor by affinity labeling. J Biol Chem. 1982 May 10;257(9):5172–5176. [PubMed] [Google Scholar]
  26. Habenicht A. J., Glomset J. A., King W. C., Nist C., Mitchell C. D., Ross R. Early changes in phosphatidylinositol and arachidonic acid metabolism in quiescent swiss 3T3 cells stimulated to divide by platelet-derived growth factor. J Biol Chem. 1981 Dec 10;256(23):12329–12335. [PubMed] [Google Scholar]
  27. Heldin C. H., Wasteson A., Westermark B. Interaction of platelet-derived growth factor with its fibroblast receptor. Demonstration of ligand degradation and receptor modulation. J Biol Chem. 1982 Apr 25;257(8):4216–4221. [PubMed] [Google Scholar]
  28. Hunter T., Ling N., Cooper J. A. Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature. 1984 Oct 4;311(5985):480–483. doi: 10.1038/311480a0. [DOI] [PubMed] [Google Scholar]
  29. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Iwashita S., Fox C. F. Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells. J Biol Chem. 1984 Feb 25;259(4):2559–2567. [PubMed] [Google Scholar]
  31. Johnsson A., Heldin C. H., Wasteson A., Westermark B., Deuel T. F., Huang J. S., Seeburg P. H., Gray A., Ullrich A., Scrace G. The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor. EMBO J. 1984 May;3(5):921–928. doi: 10.1002/j.1460-2075.1984.tb01908.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Johnsson A., Heldin C. H., Westermark B., Wasteson A. Platelet-derived growth factor: identification of constituent polypeptide chains. Biochem Biophys Res Commun. 1982 Jan 15;104(1):66–74. doi: 10.1016/0006-291x(82)91941-6. [DOI] [PubMed] [Google Scholar]
  33. Kelly K., Cochran B. H., Stiles C. D., Leder P. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell. 1983 Dec;35(3 Pt 2):603–610. doi: 10.1016/0092-8674(83)90092-2. [DOI] [PubMed] [Google Scholar]
  34. Kikkawa U., Takai Y., Tanaka Y., Miyake R., Nishizuka Y. Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters. J Biol Chem. 1983 Oct 10;258(19):11442–11445. [PubMed] [Google Scholar]
  35. Lee L. S., Weinstein I. B. Mechanism of tumor promoter inhibition of cellular binding of epidermal growth factor. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5168–5172. doi: 10.1073/pnas.76.10.5168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Leof E. B., Olashaw N. E., Pledger W. J., O'Keefe E. J. Cyclic AMP potentiates down regulation of epidermal growth factor receptors by platelet-derived growth factor. Biochem Biophys Res Commun. 1982 Nov 16;109(1):83–91. doi: 10.1016/0006-291x(82)91569-8. [DOI] [PubMed] [Google Scholar]
  37. Matrisian L. M., Larsen B. R., Finch J. S., Magun B. E. Further purification of epidermal growth factor by high-performance liquid chromatography. Anal Biochem. 1982 Sep 15;125(2):339–351. doi: 10.1016/0003-2697(82)90015-x. [DOI] [PubMed] [Google Scholar]
  38. McCaffrey P. G., Friedman B., Rosner M. R. Diacylglycerol modulates binding and phosphorylation of the epidermal growth factor receptor. J Biol Chem. 1984 Oct 25;259(20):12502–12507. [PubMed] [Google Scholar]
  39. Moolenaar W. H., Tertoolen L. G., de Laat S. W. Growth factors immediately raise cytoplasmic free Ca2+ in human fibroblasts. J Biol Chem. 1984 Jul 10;259(13):8066–8069. [PubMed] [Google Scholar]
  40. Niedel J. E., Kuhn L. J., Vandenbark G. R. Phorbol diester receptor copurifies with protein kinase C. Proc Natl Acad Sci U S A. 1983 Jan;80(1):36–40. doi: 10.1073/pnas.80.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nishimura J., Huang J. S., Deuel T. F. Platelet-derived growth factor stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3 cell membranes. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4303–4307. doi: 10.1073/pnas.79.14.4303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pessin J. E., Gitomer W., Oka Y., Oppenheimer C. L., Czech M. P. beta-Adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes. J Biol Chem. 1983 Jun 25;258(12):7386–7394. [PubMed] [Google Scholar]
  43. Pike L. J., Bowen-Pope D. F., Ross R., Krebs E. G. Characterization of platelet-derived growth factor-stimulated phosphorylation in cell membranes. J Biol Chem. 1983 Aug 10;258(15):9383–9390. [PubMed] [Google Scholar]
  44. Pike L. J., Gallis B., Casnellie J. E., Bornstein P., Krebs E. G. Epidermal growth factor stimulates the phosphorylation of synthetic tyrosine-containing peptides by A431 cell membranes. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1443–1447. doi: 10.1073/pnas.79.5.1443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Savage C. R., Jr, Cohen S. Epidermal growth factor and a new derivative. Rapid isolation procedures and biological and chemical characterization. J Biol Chem. 1972 Dec 10;247(23):7609–7611. [PubMed] [Google Scholar]
  46. Scher C. D., Shepard R. C., Antoniades H. N., Stiles C. D. Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle. Biochim Biophys Acta. 1979 Aug 10;560(2):217–241. doi: 10.1016/0304-419x(79)90020-9. [DOI] [PubMed] [Google Scholar]
  47. Sharkey N. A., Leach K. L., Blumberg P. M. Competitive inhibition by diacylglycerol of specific phorbol ester binding. Proc Natl Acad Sci U S A. 1984 Jan;81(2):607–610. doi: 10.1073/pnas.81.2.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shoyab M., De Larco J. E., Todaro G. J. Biologically active phorbol esters specifically alter affinity of epidermal growth factor membrane receptors. Nature. 1979 May 31;279(5712):387–391. doi: 10.1038/279387a0. [DOI] [PubMed] [Google Scholar]
  49. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
  50. Ushiro H., Cohen S. Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A-431 cell membranes. J Biol Chem. 1980 Sep 25;255(18):8363–8365. [PubMed] [Google Scholar]
  51. Waterfield M. D., Scrace G. T., Whittle N., Stroobant P., Johnsson A., Wasteson A., Westermark B., Heldin C. H., Huang J. S., Deuel T. F. Platelet-derived growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus. Nature. 1983 Jul 7;304(5921):35–39. doi: 10.1038/304035a0. [DOI] [PubMed] [Google Scholar]
  52. Wharton W., Leof E., Pledger W. J., O'Keefe E. J. Modulation of the epidermal growth factor receptor by platelet-derived growth factor and choleragen: effects on mitogenesis. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5567–5571. doi: 10.1073/pnas.79.18.5567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wrann M., Fox C. F., Ross R. Modulation of epidermal growth factor receptors on 3T3 cells by platelet-derived growth factor. Science. 1980 Dec 19;210(4476):1363–1365. doi: 10.1126/science.6254158. [DOI] [PubMed] [Google Scholar]

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