Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1988 Oct;62(10):3649–3654. doi: 10.1128/jvi.62.10.3649-3654.1988

Mutation of a protein kinase C phosphorylation site in the erbB protein of avian erythroblastosis virus.

S J Decker 1, B Dorai 1, S Russell 1
PMCID: PMC253506  PMID: 2901498

Abstract

Tumor promoter-stimulated phosphorylation of threonine 98 of the erbB protein of avian erythroblastosis virus (AEV) correlates with inhibition of erbB-dependent mitogenesis. To more clearly define the role of phosphorylation of this residue in regulation of the activity of the erbB protein, we have constructed erbB mutations which encode alanine (Ala-98), tyrosine (Tyr-98), or serine (Ser-98) at position 98. The biosynthesis and stability of the three mutant proteins were similar to those of the wild-type erbB protein, and all three retained the ability to transform chicken embryo fibroblasts. Treatment of transformed CEF with 12-tetradecanoylphorbol-13-acetate (TPA) stimulated incorporation of 32Pi into wild-type and mutant erbB proteins and resulted in a slight decrease in the electrophoretic mobilities of all the erbB proteins. Tryptic maps of erbB phosphopeptides showed no endogenous or TPA-stimulated phosphorylation of alanine 98 or tyrosine 98 in cells transformed by the Ala-98 and Tyr-98 mutants. Analysis of tryptic phosphopeptides by high-pressure liquid chromatography revealed that TPA treatment of cells stimulated phosphorylation of other sites of the erbB protein in addition to threonine 98. A high endogenous level of phosphorylation of serine 98 of the Ser-98 mutant protein was found, and TPA treatment of cells did not result in further phosphorylation of this residue. Cells transformed by wild-type and mutant AEV were equally sensitive to TPA-dependent inhibition of growth in soft agar and TPA-dependent inhibition of [3H]thymidine incorporation. TPA treatment inhibited tyrosine phosphorylation to a similar extent in cells transformed by wild-type or Ala-98 AEV. These data indicate that phosphorylation of threonine 98 of the erbB protein is not responsible for TPA-dependent inhibition of growth of AEV-transformed cells or TPA-induced inhibition of erbB-dependent tyrosine phosphorylation. TPA-stimulated phosphorylation of the erbB protein at other sites may mediate these effects. The data also show that subtle changes in a phosphorylation site (i.e., changing threonine to serine) can drastically alter recognition by protein kinases.

Full text

PDF
3649

Images in this article

3650Image
on p.3650
3651Image
on p.3651
3651Image
on p.3651

Selected References

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

  1. Bramson H. N., Kaiser E. T., Mildvan A. S. Mechanistic studies of cAMP-dependent protein kinase action. CRC Crit Rev Biochem. 1984;15(2):93–124. doi: 10.3109/10409238409102298. [DOI] [PubMed] [Google Scholar]
  2. Cassel D., Pike L. J., Grant G. A., Krebs E. G., Glaser L. Interaction of epidermal growth factor-dependent protein kinase with endogenous membrane proteins and soluble peptide substrate. J Biol Chem. 1983 Mar 10;258(5):2945–2950. [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. Decker S. J. Effects of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on metabolism of the epidermal growth factor receptor in normal human fibroblasts. Mol Cell Biol. 1984 Sep;4(9):1718–1724. doi: 10.1128/mcb.4.9.1718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Decker S. J. Phosphorylation of the erbB gene product from an avian erythroblastosis virus-transformed chick fibroblast cell line. J Biol Chem. 1985 Feb 25;260(4):2003–2006. [PubMed] [Google Scholar]
  8. 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]
  9. Edelman A. M., Blumenthal D. K., Krebs E. G. Protein serine/threonine kinases. Annu Rev Biochem. 1987;56:567–613. doi: 10.1146/annurev.bi.56.070187.003031. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Frykberg L., Palmieri S., Beug H., Graf T., Hayman M. J., Vennström B. Transforming capacities of avian erythroblastosis virus mutants deleted in the erbA or erbB oncogenes. Cell. 1983 Jan;32(1):227–238. doi: 10.1016/0092-8674(83)90513-5. [DOI] [PubMed] [Google Scholar]
  12. Glass D. B., Krebs E. G. Protein phosphorylation catalyzed by cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Annu Rev Pharmacol Toxicol. 1980;20:363–388. doi: 10.1146/annurev.pa.20.040180.002051. [DOI] [PubMed] [Google Scholar]
  13. Graf T., Royer-Pokora B., Schubert G. E., Beug H. Evidence for the multiple oncogenic potential of cloned leukemia virus: in vitro and in vitro studies with avian erythroblastosis virus. Virology. 1976 Jun;71(2):423–433. doi: 10.1016/0042-6822(76)90370-6. [DOI] [PubMed] [Google Scholar]
  14. Gullick W. J., Downward J., Waterfield M. D. Antibodies to the autophosphorylation sites of the epidermal growth factor receptor protein-tyrosine kinase as probes of structure and function. EMBO J. 1985 Nov;4(11):2869–2877. doi: 10.1002/j.1460-2075.1985.tb04016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hashimoto E., Takeda M., Nishizuka Y., Hamana K., Iwai K. Studies on the sites in histones phosphorylated by adenosine 3':5'-monophosphate-dependent and guanosine 3':5'-monophosphate-dependent protein kinases. J Biol Chem. 1976 Oct 25;251(20):6287–6293. [PubMed] [Google Scholar]
  16. Hunter T., Cooper J. A. Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells. Cell. 1981 Jun;24(3):741–752. doi: 10.1016/0092-8674(81)90100-8. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Kanba S., Kanba K. S., Richelson E. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibits muscarinic (M1) receptor-mediated inositol phosphate release and cyclic GMP formation in murine neuroblastoma cells (clone N1E-115). Eur J Pharmacol. 1986 Jun 5;125(1):155–156. doi: 10.1016/0014-2999(86)90096-8. [DOI] [PubMed] [Google Scholar]
  20. Kelleher D. J., Pessin J. E., Ruoho A. E., Johnson G. L. Phorbol ester induces desensitization of adenylate cyclase and phosphorylation of the beta-adrenergic receptor in turkey erythrocytes. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4316–4320. doi: 10.1073/pnas.81.14.4316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lee L. S., Weinstein I. B. Tumor-promoting phorbol esters inhibit binding of epidermal growth factor to cellular receptors. Science. 1978 Oct 20;202(4365):313–315. doi: 10.1126/science.308698. [DOI] [PubMed] [Google Scholar]
  22. Leeb-Lundberg L. M., Cotecchia S., Lomasney J. W., DeBernardis J. F., Lefkowitz R. J., Caron M. G. Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5651–5655. doi: 10.1073/pnas.82.17.5651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lin C. R., Chen W. S., Lazar C. S., Carpenter C. D., Gill G. N., Evans R. M., Rosenfeld M. G. Protein kinase C phosphorylation at Thr 654 of the unoccupied EGF receptor and EGF binding regulate functional receptor loss by independent mechanisms. Cell. 1986 Mar 28;44(6):839–848. doi: 10.1016/0092-8674(86)90006-1. [DOI] [PubMed] [Google Scholar]
  24. Moolenaar W. H., Aerts R. J., Tertoolen L. G., de Laat S. W. The epidermal growth factor-induced calcium signal in A431 cells. J Biol Chem. 1986 Jan 5;261(1):279–284. [PubMed] [Google Scholar]
  25. Neckameyer W. S., Wang L. H. Molecular cloning and characterization of avian sarcoma virus UR2 and comparison of its transforming sequence with those of other avian sarcoma viruses. J Virol. 1984 Jun;50(3):914–921. doi: 10.1128/jvi.50.3.914-921.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pike L. J., Eakes A. T. Epidermal growth factor stimulates the production of phosphatidylinositol monophosphate and the breakdown of polyphosphoinositides in A431 cells. J Biol Chem. 1987 Feb 5;262(4):1644–1651. [PubMed] [Google Scholar]
  27. Whiteley B., Cassel D., Zhuang Y. X., Glaser L. Tumor promoter phorbol 12-myristate 13-acetate inhibits mitogen-stimulated Na+/H+ exchange in human epidermoid carcinoma A431 cells. J Cell Biol. 1984 Sep;99(3):1162–1166. doi: 10.1083/jcb.99.3.1162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yamamoto T., Hihara H., Nishida T., Kawai S., Toyoshima K. A new avian erythroblastosis virus, AEV-H, carries erbB gene responsible for the induction of both erythroblastosis and sarcomas. Cell. 1983 Aug;34(1):225–232. doi: 10.1016/0092-8674(83)90153-8. [DOI] [PubMed] [Google Scholar]
  29. Yamamoto T., Nishida T., Miyajima N., Kawai S., Ooi T., Toyoshima K. The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell. 1983 Nov;35(1):71–78. doi: 10.1016/0092-8674(83)90209-x. [DOI] [PubMed] [Google Scholar]
  30. Zavoico G. B., Halenda S. P., Sha'afi R. I., Feinstein M. B. Phorbol myristate acetate inhibits thrombin-stimulated Ca2+ mobilization and phosphatidylinositol 4,5-bisphosphate hydrolysis in human platelets. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3859–3862. doi: 10.1073/pnas.82.11.3859. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES