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

• Published in: Biochemical journal 1992-06, Vol.284 (3), p.705-710
• Main Authors: SANTELL, L, BARTFELD, N. S, LEVIN, E. G
• Format: Article
• Language: English
• Subjects: Autoradiography; Biological and medical sciences; Blotting, Western; Cell physiology; Cells, Cultured; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Fundamental and applied biological sciences. Psychology; Heat-Shock Proteins - isolation & purification; Heat-Shock Proteins - metabolism; Humans; Molecular and cellular biology; Peptide Mapping; Phosphates - metabolism; Phosphopeptides - analysis; Phosphoproteins - isolation & purification; Phosphoproteins - metabolism; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C - metabolism; Responses to growth factors, tumor promotors, other factors; Tetradecanoylphorbol Acetate - pharmacology; Trypsin; Umbilical Veins
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2840705

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.