Chan W H, Yu J S, Yang S D
Department of Life Sciences, National Tsing Hua University, Taiwan, ROC.
J Protein Chem. 1998 Jul;17(5):485-94. doi: 10.1023/a:1022578820147.
Heat shock induces a stress response in mammalian cells and can also lead to apoptotic cell death. Here we report that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be drastically activated in several cell types by heat shock. Immunoblot analysis revealed that this 36-kDa MBP kinase can be recognized by an antibody against the C-terminal region of a family of p21Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as tools, we further demonstrated that heat shock can induce cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment in mouse Balb/c 3T3 and human Hep 3B cells. The kinetic profile of appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in these cells induced by heat shock. In addition, the heat shock-induced 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 heat shock-treated Hep 3B cells. Moreover, blockage of the activation of caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors of caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could substantially diminish the extent of heat shock-induced cleavage/activation of PAK2. Overall, our results point out that PAK2 is cleaved and activated during the heat shock-induced apoptotic cell death process and suggest that caspase-3 is involved in this process.
热休克可诱导哺乳动物细胞产生应激反应，也可导致细胞凋亡性死亡。在此我们报告，通过凝胶内激酶分析检测到的一种36 kDa的髓鞘碱性蛋白（MBP）激酶，在几种细胞类型中可被热休克强烈激活。免疫印迹分析显示，这种36 kDa的MBP激酶可被一种针对p21Cdc42/Rac激活激酶（PAKs）家族C末端区域的抗体识别。通过使用该抗体以及一种针对PAK2 N末端区域的PAK2特异性抗体作为工具，我们进一步证明，在小鼠Balb/c 3T3细胞和人Hep 3B细胞中，热休克可诱导PAK2裂解，产生一个36 kDa的C末端催化片段。PAK2的36 kDa C末端催化片段出现的动力学曲线与这些细胞中热休克诱导的36 kDa MBP激酶的激活完全匹配。此外，在热休克处理的Hep 3B细胞中，现已发现热休克诱导的PAK2裂解和激活与DNA片段化以及一种名为caspase-3的ICE/CED-3家族半胱氨酸蛋白酶的激活密切相关。此外，用两种caspase特异性四肽抑制剂（Ac-DEVD-cho和Ac-YVAD-cmk）预处理细胞来阻断caspase-3的激活，可显著降低热休克诱导的PAK2裂解/激活程度。总体而言，我们的结果指出，PAK2在热休克诱导的细胞凋亡性死亡过程中发生裂解并被激活，提示caspase-3参与了这一过程。
