PAK2 is cleaved and activated during hyperosmotic shock-induced apoptosis via a caspase-dependent mechanism: Evidence for the involvement of oxidative stress

Hyperosmotic shock elicits a stress response in mammalian cells and can lea d to apoptotic cell death. In the present study, we report that hyperosmoti c shock can induce activation of a 36 kDa kinase detected by an in-gel kina se assay in several cell types, including mouse Balb/c 3T3 fibroblasts, and human Hep 3B and A431 cells. This 36 kDa kinase can be recognized by an an tibody against the C-terminal region of a family of p21(Cdc42/Rac)-activate d kinases (PAKs) on immunoblot. Further studies with this antibody and a PA K2-specific antibody against the N-terminal region of PAK2 demonstrate that hyperosmotic shock can induce cleavage of PAK2 to generate a 36 kDa C-term inal catalytic fragment in cells. The cleavage and activation of PAK2 was f ound to be closely associated with both DNA fragmentation and activation of an ICE/CED-3 family cysteine protease termed caspase-3 in hyperosmotically shocked cells. Furthermore, pretreating the cells with two caspase inhibit ors (Ac-DEVD-cho and Ac-YVAD-cmk) could inhibit both cleavage/activation of PAK2 and DNA fragmentation induced by hyperosmotic shock. Moreover, all th ese hyperosmotic shock-induced changes (i.e., activation of caspase-3, clea vage/activation of PAK2, and DNA fragmentation) in cells could be blocked b y antioxidants such as ascorbic acid (vitamine C), alpha-tocopherol (vitami ne E), dithiothreitol, beta-mercaptoethanol, and glutathione. Taken togethe r, our results show that PAK2 is cleaved and activated via a caspase-depend ent mechanism during hyperosmotic shock-induced apoptosis and suggest the i nvolvement of antioxidant-preventable oxidative stress in inducing this pro cess. J. Cell. Physiol. 178:397-408, 1999. (C) 1999 Wiley-Liss, Inc.