CELL STRESS-INDUCED PHOSPHORYLATION OF ATF2 AND C-JUN TRANSCRIPTION FACTORS IN RAT VENTRICULAR MYOCYTES

Ventricular myocytes are exposed to various pathologically important c ell stresses in vivo. In vitro, extreme stresses (sorbitol-induced hyp erosmotic shock in the presence or absence of okadaic acid, and anisom ycin) were applied to ventricular myocytes cultured from neonatal rat hearts to induce a robust activation of the 46 and 54 kDa stress-activ ated protein kinases (SAPKs). These activities were increased in nucle ar 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 appea rance of reduced-mobility species on SDS/PAGE, which were sensitive to treatment with protein phosphatase 2A. Hyperosmotic shock and anisomy cin had no effect on the abundance of ATF2. In contrast, cell stresses induced a greater than 10-fold increase in total c-Jun immunoreactivi ty detected on Western blots with antibody to c-Jun (KM-1). Cyclohexim ide did not inhibit this increase, which we conclude represents phosph orylation of c-Jun. This conclusion was supported by use of a c-Jun(ph ospho-Ser-73) antibody. Immunostaining of cells also showed increases in nuclear phospho-c-Jun in response to hyperosmotic stress. Severe st ress (hyperosmotic shock + okadaic acid for 2 h) induced proteins (mig rating at approx. 51 and 57 kDa) that cross-reacted strongly with KM-1 antibodies in both the nucleus and the cytosol. These may represent f orms of c-Jun that had undergone further modification. These studies s how that stresses induce phosphorylation of transcription factors in v entricular myocytes and we suggest that this response may be pathologi cally relevant.