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. 1987 Apr;6(4):921–926. doi: 10.1002/j.1460-2075.1987.tb04839.x

Embryonal carcinoma-derived growth factor activates protein kinase C in vivo and in vitro.

L C Mahadevan, A Aitken, J Heath, J G Foulkes
PMCID: PMC553483  PMID: 3595564

Abstract

We have recently reported that a polypeptide mitogen, the embryonal carcinoma-derived growth factor (ECDGF), induces phosphorylation of the epidermal growth factor (EGF) receptor in intact C3H 10T 1/2 mouse fibroblasts with concomittant loss of high affinity EGF binding sites. This phenomenon appears to be mediated through an activation of protein kinase C. Several groups have described an acidic 80,000 dalton protein substrate of protein kinase C. In this paper, we demonstrate that the addition of ECDGF or the phorbol ester TPA to intact C3H 10T 1/2 cells results in the enhanced phosphorylation of this 80 kd protein in vivo. Furthermore, this response is demonstrable in vitro. Thus the addition of ECDGF, the phorbol ester TPA, protein kinase C or phosphoinositidase C to crude membranes prepared from C3H 10T 1/2 cells resulted in the enhanced phosphorylation of this protein. Data obtained by phosphopeptide mapping of the 80 kd protein show that the ECDGF-induced activation of protein kinase C in our membrane preparations is comparable with that obtained in vivo. The availability of an in vitro system in which this response is preserved should now allow a detailed biochemical analysis of the steps between binding of a mitogen to its receptor and the activation of protein kinase C.

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Selected References

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

  1. Aitken A., Bilham T., Cohen P., Aswad D., Greengard P. A specific substrate from rabbit cerebellum for guanosine-3':5'-monophosphate-dependent protein kinase. III. Amino acid sequences at the two phosphorylation sites. J Biol Chem. 1981 Apr 10;256(7):3501–3506. [PubMed] [Google Scholar]
  2. Allan D., Michell R. H. A calcium-activated polyphosphoinositide phosphodiesterase in the plasma membrane of human and rabbit erythrocytes. Biochim Biophys Acta. 1978 Apr 4;508(2):277–286. doi: 10.1016/0005-2736(78)90330-9. [DOI] [PubMed] [Google Scholar]
  3. Blackshear P. J., Wen L., Glynn B. P., Witters L. A. Protein kinase C-stimulated phosphorylation in vitro of a Mr 80,000 protein phosphorylated in response to phorbol esters and growth factors in intact fibroblasts. Distinction from protein kinase C and prominence in brain. J Biol Chem. 1986 Jan 25;261(3):1459–1469. [PubMed] [Google Scholar]
  4. Blackshear P. J., Witters L. A., Girard P. R., Kuo J. F., Quamo S. N. Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways. J Biol Chem. 1985 Oct 25;260(24):13304–13315. [PubMed] [Google Scholar]
  5. Cockcroft S., Gomperts B. D. Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase. Nature. 1985 Apr 11;314(6011):534–536. doi: 10.1038/314534a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Downes C. P., Michell R. H. The polyphosphoinositide phosphodiesterase of erythrocyte membranes. Biochem J. 1981 Jul 15;198(1):133–140. doi: 10.1042/bj1980133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Garrels J. I. Quantitative two-dimensional gel electrophoresis of proteins. Methods Enzymol. 1983;100:411–423. doi: 10.1016/0076-6879(83)00070-1. [DOI] [PubMed] [Google Scholar]
  10. Harrington C. A., Eichberg J. Norepinephrine causes alpha 1-adrenergic receptor-mediated decrease of phosphatidylinositol in isolated rat liver plasma membranes supplemented with cytosol. J Biol Chem. 1983 Feb 25;258(4):2087–2090. [PubMed] [Google Scholar]
  11. Heath J. K., Isacke C. M. PC13 embryonal carcinoma-derived growth factor. EMBO J. 1984 Dec 1;3(12):2957–2962. doi: 10.1002/j.1460-2075.1984.tb02240.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heath J. K., Mahadevan L., Foulkes J. G. The role of EGF receptor transmodulation in embryonal carcinoma-derived growth factor-induced mitogenesis. EMBO J. 1986 Aug;5(8):1809–1814. doi: 10.1002/j.1460-2075.1986.tb04430.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kasuga M., Zick Y., Blithe D. L., Crettaz M., Kahn C. R. Insulin stimulates tyrosine phosphorylation of the insulin receptor in a cell-free system. Nature. 1982 Aug 12;298(5875):667–669. doi: 10.1038/298667a0. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Litosch I., Wallis C., Fain J. N. 5-Hydroxytryptamine stimulates inositol phosphate production in a cell-free system from blowfly salivary glands. Evidence for a role of GTP in coupling receptor activation to phosphoinositide breakdown. J Biol Chem. 1985 May 10;260(9):5464–5471. [PubMed] [Google Scholar]
  16. Low M. G. Phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. Methods Enzymol. 1981;71(Pt 100):741–746. doi: 10.1016/0076-6879(81)71087-5. [DOI] [PubMed] [Google Scholar]
  17. Lucas D. O., Bajjalieh S. M., Kowalchyk J. A., Martin T. F. Direct stimulation by thyrotropin-releasing hormone (TRH) of polyphosphoinositide hydrolysis in GH3 cell membranes by a guanine nucleotide-modulated mechanism. Biochem Biophys Res Commun. 1985 Oct 30;132(2):721–728. doi: 10.1016/0006-291x(85)91192-1. [DOI] [PubMed] [Google Scholar]
  18. Marche P., Koutouzov S., Meyer P. Metabolism of phosphoinositides in the rat erythrocyte membrane. A reappraisal of the effect of magnesium on the 32P incorporation into polyphosphoinositides. Biochim Biophys Acta. 1982 Mar 12;710(3):332–340. doi: 10.1016/0005-2760(82)90116-3. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  21. Rodriguez-Pena A., Rozengurt E. Phosphorylation of an acidic mol. wt. 80 000 cellular protein in a cell-free system and intact Swiss 3T3 cells: a specific marker of protein kinase C activity. EMBO J. 1986 Jan;5(1):77–83. doi: 10.1002/j.1460-2075.1986.tb04180.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rozengurt E., Collins M. Molecular aspects of growth factor action: receptors and intracellular signals. J Pathol. 1983 Nov;141(3):309–331. doi: 10.1002/path.1711410310. [DOI] [PubMed] [Google Scholar]
  23. Rozengurt E., Rodriguez-Pena M., Smith K. A. Phorbol esters, phospholipase C, and growth factors rapidly stimulate the phosphorylation of a Mr 80,000 protein in intact quiescent 3T3 cells. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7244–7248. doi: 10.1073/pnas.80.23.7244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sherr C. J., Rettenmier C. W., Sacca R., Roussel M. F., Look A. T., Stanley E. R. The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Cell. 1985 Jul;41(3):665–676. doi: 10.1016/s0092-8674(85)80047-7. [DOI] [PubMed] [Google Scholar]
  25. Smith C. D., Lane B. C., Kusaka I., Verghese M. W., Snyderman R. Chemoattractant receptor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in human polymorphonuclear leukocyte membranes. Requirement for a guanine nucleotide regulatory protein. J Biol Chem. 1985 May 25;260(10):5875–5878. [PubMed] [Google Scholar]
  26. Thom D., Powell A. J., Lloyd C. W., Rees D. A. Rapid isolation of plasma membranes in high yield from cultured fibroblasts. Biochem J. 1977 Nov 15;168(2):187–194. doi: 10.1042/bj1680187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tsuda T., Kaibuchi K., Kawahara Y., Fukuzaki H., Takai Y. Induction of protein kinase C activation and Ca2+ mobilization by fibroblast growth factor in Swiss 3T3 cells. FEBS Lett. 1985 Oct 28;191(2):205–210. doi: 10.1016/0014-5793(85)80009-0. [DOI] [PubMed] [Google Scholar]
  28. 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]

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