Abstract
Autophosphorylation-dependent protein kinase (auto-kinase) was found in pig brain and liver based on its unique autophosphorylation/activation property (Yang et al., 1987a, b). In this thesis, the kinase purified from pig liver was partially sequenced. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21Cdc42/Rac-activated protein kinase 2 (PAK2) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of PAK by immunoblot analysis. The results in Section Ⅰ demonstrate that auto-kinase is a member of the PAK family. To investigate the possible in vivo functional role of auto-kinase, the effects of environmental stresses (including heat shock, hyperosmotic shock, and UV irradiation) on PAK2 activities were studied in Sections Ⅱ. A 36-kDa myelin basic protein (MBP) kinase activity detected by an in-gel kinase assay could be drastically increased in several cell types by environmental stresses. Immunoblot analysis revealed that this 36-kDa MBP kinase could be recognized by an antibody against the C-terminal region of PAK. By using this antibody as a probe, it was demonstrated that environmental stresses could induce cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment (activated auto-kinase). The kinetic profile of increasing amounts of the 36-kDa activated auto-kinase matched exactly with the activation of the 36-kDa MBP kinase activity in these cells induced by environmental stresses. The results in Section Ⅱ demonstrate that cleavage of PAK2 to generate activated auto-kinase is a common signaling event when cells are subjected to environmental stresses. Further studies reveal that the environmental stresses-induced cleavage of PAK2 to generate activated auto-kinase is closely associated with both DNA fragmentation and activation of an ICE/CED-3 family cysteine protease termed caspase-3. 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 environmental stresses-induced cleavage of PAK2 to generate activated auto-kinase. In addition, all the biochemical changes (i.e. activation of caspase-3, and cleavage of PAK2 to generate activated auto-kinase) in hyperosmotically shocked cells could be blocked by antioxidants such as ascorbic acid (vitamine C), a-tocopherol (vitamine E), dithiothreitol, b-mercaptoethanol and glutathione. Taken together, the results in Section Ⅲ demonstrate that PAK2 is cleaved to generate activated auto-kinase via a caspase-dependent mechanism during environmental stresses-induced cell responses and suggest an involvement of oxidative stress in inducing this process. In searching for compounds that can modulate the environmental stresses-induced biochemical changes, genistein (an isoflavone possessing inhibitory activity against protein tyrosine kinases (PTKs) and topoisomerase) was found to have profound effects on this process. All the environmental stresses-induced biochemical changes including caspase-3 activation and cleavage of PAK2 to generate activated auto-kinase could be blocked by genistein. By using UV irradiation as a stress trigger, it was found that azide, a free radical scavenger, could also effectively block the UV-induced cell responses. On the other hand, two typical inhibitors for PTKs, tyrphostin A47 and herbimycin A had no effect on UV-induced cell responses. Similar observations could also be observed in heat and hyperosmotic shock-induced cell responses. The results suggest that the inhibitory effect of genistein on environmental stresses-induced cell responses is not dependent on its property as a PTK inhibitor. Flow cytometric analysis using the cell permeant dye DCF-DA as an indicator of the generation of ROS revealed that environmental stresses including heat shock, hyperosmotic shock and UV irradiation could increase the extent of the intracellular oxidative stress. Furthermore, the UV-induced oxidative stress in cells could be prevented by genistein. The result implies that genistein may act as an antioxidant to scavenge reactive oxygen species generated in cells upon environmental stresses. Taken together, the results in Section Ⅲ demonstrate that oxidative stress is involved in the caspase-mediated cleavage of PAK2 to generate activated auto-kinase in environmental stress-induced cell responses and that genistein is a potent inhibitor for this process. To further study the mechanism linking oxidative stress to caspase-3/PAK2 activation in environmental stresses, photodynamic treatment (PDT) was used as another model in this thesis since PDT can directly induce intracellular oxidative stress. The results show that caspase-3 activation and cleavage of PAK2 to generate activated auto-kinase also occurred in mammalian cells treated with PDT. To further elucidate the missing link between singlet oxygen generation and caspase-3 activation, the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway was studied in PDT-treated A431 cell. The results show that PDT could induce a two-stage activation of the JNK in A431 cell. Studies using scavengers for various ROS and inhibitors for caspases revealed that the early-stage JNK activation is singlet oxygen-dependent whereas the late-stage JNK activation is dependent on the singlet oxygen-mediated caspase activation. Experiments using JNK and PAK2 antisense oligonucleotides further show that during PDT-induced cell responses in A431 cell, the early JNK activation is required for caspase activation and the late JNK activation is regulated by the caspase-mediated cleavage/activation of PAK2. Collectively, a signaling cascade model for PDT-induced cell responses in A431 cell can be obtained from the results of Section Ⅳ, which involves singlet oxygen, JNK, caspase, and PAK2/auto-kinase. In conclusion, the results presented in this thesis demonstrate that generation of reactive oxygen species, cleavage/activation of caspase-3, cleavage of PAK2 to generate activated auto-kinase, and activation of JNK may represent the common sequential signal transducing events in cell responses to environmental stresses and photodynamic treatment.