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. 1992 Jun 15;284(Pt 3):705–710. doi: 10.1042/bj2840705

Identification of a protein transiently phosphorylated by activators of endothelial cell function as the heat-shock protein HSP27. A possible role for protein kinase C.

L Santell 1, N S Bartfeld 1, E G Levin 1
PMCID: PMC1132595  PMID: 1622389

Abstract

Several agonists of endothelial cell function (thrombin, histamine, dioctanoylglycerol, phorbol 12-myristate 13-acetate, interleukin-1) have previously been shown to enhance the level of phosphorylation of an undefined 29,000-M(r) protein (P29). Comparison of this protein with other phosphoproteins suggested that it may be related to the mammalian heat-shock protein HSP27. Immunoprecipitation and immunoblot analysis with antibodies specific for human HSP27 demonstrated that P29 was immunochemically identical with HSP27. Further characterization of agonist-induced phosphorylation of HSP27 indicated that phosphorylation occurred exclusively on serine residues, and phosphopeptide analysis of tryptic- and chymotryptic-cleavage products demonstrated that the phosphopeptides generated were identical for each agonist and okadaic acid. Down-regulation of protein kinase C-alpha by prolonged treatment with phorbol esters eliminated the ability of phorbol 12-myristate 13-acetate, dioctanoylglycerol, thrombin and histamine to phosphorylate HSP27 above background levels and deceased interleukin-1-stimulated HSP27 phosphorylation by 60%. These data suggest that the various agonists employed stimulate HSP27 phosphorylation through similar mechanisms and that protein kinase C is probably involved.

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Selected References

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

  1. Arrigo A. P., Pauli D. Characterization of HSP27 and three immunologically related polypeptides during Drosophila development. Exp Cell Res. 1988 Mar;175(1):169–183. doi: 10.1016/0014-4827(88)90264-9. [DOI] [PubMed] [Google Scholar]
  2. Arrigo A. P. Tumor necrosis factor induces the rapid phosphorylation of the mammalian heat shock protein hsp28. Mol Cell Biol. 1990 Mar;10(3):1276–1280. doi: 10.1128/mcb.10.3.1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arrigo A. P., Welch W. J. Characterization and purification of the small 28,000-dalton mammalian heat shock protein. J Biol Chem. 1987 Nov 15;262(32):15359–15369. [PubMed] [Google Scholar]
  4. Brock T. A., Capasso E. A. Thrombin and histamine activate phospholipase C in human endothelial cells via a phorbol ester-sensitive pathway. J Cell Physiol. 1988 Jul;136(1):54–62. doi: 10.1002/jcp.1041360107. [DOI] [PubMed] [Google Scholar]
  5. Buxbaum J. D., Dudai Y. A quantitative model for the kinetics of cAMP-dependent protein kinase (type II) activity. Long-term activation of the kinase and its possible relevance to learning and memory. J Biol Chem. 1989 Jun 5;264(16):9344–9351. [PubMed] [Google Scholar]
  6. Chrétien P., Landry J. Enhanced constitutive expression of the 27-kDa heat shock proteins in heat-resistant variants from Chinese hamster cells. J Cell Physiol. 1988 Oct;137(1):157–166. doi: 10.1002/jcp.1041370119. [DOI] [PubMed] [Google Scholar]
  7. Cooper J. A., Gould K. L., Cartwright C. A., Hunter T. Tyr527 is phosphorylated in pp60c-src: implications for regulation. Science. 1986 Mar 21;231(4744):1431–1434. doi: 10.1126/science.2420005. [DOI] [PubMed] [Google Scholar]
  8. Darbon J. M., Tournier J. F., Tauber J. P., Bayard F. Possible role of protein phosphorylation in the mitogenic effect of high density lipoproteins on cultured vascular endothelial cells. J Biol Chem. 1986 Jun 15;261(17):8002–8008. [PubMed] [Google Scholar]
  9. Feuerstein N., Cooper H. L. Rapid phosphorylation-dephosphorylation of specific proteins induced by phorbol ester in HL-60 cells. Further characterization of the phosphorylation of 17-kilodalton and 27-kilodalton proteins in myeloid leukemic cells and human monocytes. J Biol Chem. 1984 Mar 10;259(5):2782–2788. [PubMed] [Google Scholar]
  10. Feuerstein N., Cooper H. L. Rapid protein phosphorylation induced by phorbol ester in HL-60 cells. Unique alkali-stable phosphorylation of a 17,000-dalton protein detected by two-dimensional gel electrophoresis. J Biol Chem. 1983 Sep 10;258(17):10786–10793. [PubMed] [Google Scholar]
  11. Kim Y. J., Shuman J., Sette M., Przybyla A. Arsenate induces stress proteins in cultured rat myoblasts. J Cell Biol. 1983 Feb;96(2):393–400. doi: 10.1083/jcb.96.2.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kwiatkowski A. P., King M. M. Autophosphorylation of the type II calmodulin-dependent protein kinase is essential for formation of a proteolytic fragment with catalytic activity. Implications for long-term synaptic potentiation. Biochemistry. 1989 Jun 27;28(13):5380–5385. doi: 10.1021/bi00439a010. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Landry J., Chrétien P., Lambert H., Hickey E., Weber L. A. Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells. J Cell Biol. 1989 Jul;109(1):7–15. doi: 10.1083/jcb.109.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Leach K. L., Powers E. A., McGuire J. C., Dong L., Kiley S. C., Jaken S. Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity. J Biol Chem. 1988 Sep 15;263(26):13223–13230. [PubMed] [Google Scholar]
  16. Leach K. L., Ruff V. A., Wright T. M., Pessin M. S., Raben D. M. Dissociation of protein kinase C activation and sn-1,2-diacylglycerol formation. Comparison of phosphatidylinositol- and phosphatidylcholine-derived diglycerides in alpha-thrombin-stimulated fibroblasts. J Biol Chem. 1991 Feb 15;266(5):3215–3221. [PubMed] [Google Scholar]
  17. Levin E. G., Marotti K. R., Santell L. Protein kinase C and the stimulation of tissue plasminogen activator release from human endothelial cells. Dependence on the elevation of messenger RNA. J Biol Chem. 1989 Sep 25;264(27):16030–16036. [PubMed] [Google Scholar]
  18. Levin E. G., Marzec U., Anderson J., Harker L. A. Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells. J Clin Invest. 1984 Dec;74(6):1988–1995. doi: 10.1172/JCI111620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Levin E. G., Santell L. Phosphorylation of an Mr = 29,000 protein by IL-1 is susceptible to partial down-regulation after endothelial cell activation. J Immunol. 1991 Jun 1;146(11):3772–3778. [PubMed] [Google Scholar]
  20. Levin E. G., Santell L. Thrombin- and histamine-induced signal transduction in human endothelial cells. Stimulation and agonist-dependent desensitization of protein phosphorylation. J Biol Chem. 1991 Jan 5;266(1):174–181. [PubMed] [Google Scholar]
  21. Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Regazzi R., Eppenberger U., Fabbro D. The 27,000 daltons stress proteins are phosphorylated by protein kinase C during the tumor promoter-mediated growth inhibition of human mammary carcinoma cells. Biochem Biophys Res Commun. 1988 Apr 15;152(1):62–68. doi: 10.1016/s0006-291x(88)80680-6. [DOI] [PubMed] [Google Scholar]
  24. Robaye B., Hepburn A., Lecocq R., Fiers W., Boeynaems J. M., Dumont J. E. Tumor necrosis factor-alpha induces the phosphorylation of 28kDa stress proteins in endothelial cells: possible role in protection against cytotoxicity? Biochem Biophys Res Commun. 1989 Aug 30;163(1):301–308. doi: 10.1016/0006-291x(89)92135-9. [DOI] [PubMed] [Google Scholar]
  25. Strulovici B., Daniel-Issakani S., Baxter G., Knopf J., Sultzman L., Cherwinski H., Nestor J., Jr, Webb D. R., Ransom J. Distinct mechanisms of regulation of protein kinase C epsilon by hormones and phorbol diesters. J Biol Chem. 1991 Jan 5;266(1):168–173. [PubMed] [Google Scholar]
  26. Welch W. J. Phorbol ester, calcium ionophore, or serum added to quiescent rat embryo fibroblast cells all result in the elevated phosphorylation of two 28,000-dalton mammalian stress proteins. J Biol Chem. 1985 Mar 10;260(5):3058–3062. [PubMed] [Google Scholar]

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