STIMULATION OF MULTIPLE MITOGEN-ACTIVATED PROTEIN-KINASE SUB-FAMILIESBY OXIDATIVE STRESS AND PHOSPHORYLATION OF THE SMALL HEAT-SHOCK-PROTEIN, HSP25 27, IN NEONATAL VENTRICULAR MYOCYTES/

We investigated the activation of three subfamilies of mitogen-activat ed protein kinases (MAPKs), namely the stress-activated protein kinase s/c-Jun N-terminal kinases (SAPKs/JNKs), the extracellularly responsiv e kinases (ERKs) and p38-MAPK, by oxidative stress as exemplified by H 2O2 in primary cultures of neonatal rat ventricular myocytes. The 46 a nd 54 kDa species of SAPKs/JNKs were activated 5- and 10-fold, respect ively, by 0.1 mM H2O2 (the maximally effective concentration). Maximal activation occurred at 15-30 min, but was still detectable after 2 h. Both ERK1 and ERK2 were activated 16-fold by 0.1 mM H2O2 with a simil ar time course to the SAPKs/JNKs, and this was comparable with their a ctivation by 1 mu M PMA, the most powerful activator of ERKs that we h ave so far identified in these cells. The activation of ERKs by H2O2 w as inhibited by PD98059, which inhibits the activation of MAPK (or ERK ) kinases, and by the protein kinase C (PKC) inhibitor, GF109203X. ERK activation was also inhibited by downregulation of PMA-sensitive PKC isoforms. p38-MAPK was activated by 0.1 mM H2O2 as shown by an increas e in its phosphorylation. However, maximal phosphorylation (activation ) was more rapid (< 5 min) than for the SAPKs/JNKs or the ERKs. We stu died the downstream consequences of p38-MAPK activation by examining a ctivation of MAPK-activated protein kinase 2 (MAPKAPK2) and phosphoryl ation of the MAPKAPK2 substrate, the small heat shock protein HSP25/27 . As with p38-MAPK, MAPKAPK2 was rapidly activated (maximal within 5 m in) by 0.1 mM H2O2. This activation was abolished by 10 mu M SB203580, a selective inhibitor of certain p38-MAPK isoforms. The phosphorylati on of HSP25/27 rapidly followed activation of MAPKAPK2 and was also in hibited by SB203580. Phosphorylation of HSP25/27 was associated with a decrease in its aggregation state. These data indicate that oxidative stress is a powerful activator of all three MAPK subfamilies in neona tal rat ventricular myocytes. Activation of all three MAPKs has been a ssociated with the development of the hypertrophic phenotype. However, stimulation of p38-MAPK and the consequent phosphorylation of HSP25/2 7 may also be important in cardioprotection.