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 lead to apoptotic cell death. In the present study, we report that hyperosmotic shock can induce activation of a 36 kDa kinase detected by an in-gel kinase 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 antibody against the C-terminal region of a family of p21Cdc42/Rac-activated kinases (PAKs) on immunoblot. Further studies with this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 demonstrate that hyperosmotic shock can induce cleavage of PAK2 to generate a 36 kDa C-terminal catalytic fragment in cells. The cleavage and activation of PAK2 was found 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 inhibitors (Ac-DEVD-cho and Ac-YVAD-cmk) could inhibit both cleavage/activation of PAK2 and DNA fragmentation induced by hyperosmotic shock. Moreover, all these hyperosmotic shock-induced changes (i.e., activation of caspase-3, cleavage/activation of PAK2, and DNA fragmentation) in cells could be blocked by antioxidants such as ascorbic acid (vitamine C), alpha-tocopherol (vitamine E), dithiothreitol, beta-mercaptoethanol, and glutathione. Taken together, our results show that PAK2 is cleaved and activated via a caspase-dependent mechanism during hyperosmotic shock-induced apoptosis and suggest the involvement of antioxidant-preventable oxidative stress in inducing this process.
Chan
W H
WH
Department of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Yu
J S
JS
Yang
S D
SD
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects; D006528:Carcinoma, Hepatocellular; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D004789:Enzyme Activation; D006801:Humans; D006982:Hypertonic Solutions; D008113:Liver Neoplasms; D051379:Mice; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014466:Ultraviolet Rays; D054462:p21-Activated Kinases