Cell stress-induced phosphorylation of ATF2 and c-Jun transcription factors in rat ventricular myocytes.
Ventricular myocytes are exposed to various pathologically important cell stresses in vivo. In vitro, extreme stresses (sorbitol-induced hyperosmotic shock in the presence or absence of okadaic acid, and anisomycin) were applied to ventricular myocytes cultured from neonatal rat hearts to induce a robust activation of the 46 and 54 kDa stress-activated protein kinases (SAPKs). These activities were increased in nuclear extracts of cells in the absence of any net import of SAPK protein. Phosphorylation of ATF2 and c-Jun was increased as shown by the appearance of reduced-mobility species on SDS/PAGE, which were sensitive to treatment with protein phosphatase 2A. Hyperosmotic shock and anisomycin had no effect on the abundance of ATF2. In contrast, cell stresses induced a greater than 10-fold increase in total c-Jun immunoreactivity detected on Western blots with antibody to c-Jun (KM-1). Cycloheximide did not inhibit this increase, which we conclude represents phosphorylation of c-Jun. This conclusion was supported by use of a c-Jun(phospho-Ser-73) antibody. Immunostaining of cells also showed increases in nuclear phospho-c-Jun in response to hyperosmotic stress. Severe stress (hyperosmotic shock+okadaic acid for 2 h) induced proteins (migrating at approx. 51 and 57 kDa) that cross-reacted strongly with KM-1 antibodies in both the nucleus and the cytosol. These may represent forms of c-Jun that had undergone further modification. These studies show that stresses induce phosphorylation of transcription factors in ventricular myocytes and we suggest that this response may be pathologically relevant.
Clerk
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Division of Cardiac Medicine, Imperial College School of Medicine at N.H.L.I., Dovehouse Street, London SW3 6LY, U.K.
Sugden
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D051698:Activating Transcription Factor 2; D000818:Animals; D000831:Animals, Newborn; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D006352:Heart Ventricles; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors / Q000378:metabolism*
D051698:Activating Transcription Factor 2; C497107:Atf2 protein, rat; D017362:Cyclic AMP Response Element-Binding Protein; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases