Study on the molecular mechanism of proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis

• Main Authors: TANG, TSWEN-KEI, 唐存愷
• Other Authors: WEN-CHANG CHANG
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/40282586821606340803

博士 === 國立臺灣大學 === 生化科學研究所 === 86 === An UV irradiation-induced A431 cell apoptosis system in combination with an in-gel kinase assay was employed to investigate the possible kinase candidate that might directly involve in the apoptotic-signaling cascade. A 36-kDa myelin basic protein(MBP)kinase was found.It could be activated during the early stages of UV irradiation-induced apoptosis and its activation accompanied DNA fragmentation in A431 cells.Immunoblot anslysis revealed that this 36-kDa kinase cound be recognized by an anti-PAK autibody against the C- terminal regions of a a family of p21Cdc42/Rac-activated kinases(PAKs). Using an anti- PAK antibody and a specific anti-PAK2 antibody against the N-terminal region of PAK2 as studying tool, we demonstrated that UV irradiation caused the cleavage of PAK2 to produce a 30-kDa N-terminal kinase fragment and a 36-kDa C-terminal fragment that became activated in A431 cells. Meanwhile, the caspase-3(cysteinyl aspartate-directed protease-3), but not caspase-1 and caspase-2, was activated in UV-induced apoptotic A431 cells induced by UV irradiation, when the activation of caspase-3 was blocked by the specific tetrapeptidic caspase-3 inhibitors (Ac-DEVD-cho and Ac-YVAD-cmk). These rusults suggested that the cleavage of PAK2 was possibly causedby the activated caspase-3. To further study the cleavage and activation of PAK2, the role of intracellular ReactiveOxygen Species (ROS) in the apoptotic A431 cells was investiged.The intracellar ROS was generated by exposure of A431 cells preloaded with the oxidation-senstitive dye 2',7'-dichlorofluorescin diacetate (DCF-DA) to UV irradiation. The flow cytometric analysis was used to detect the production of ROS. Curcumin(50 μM),a free-radical scavenger, was found to be an effective inhibitor to block the cleavage/activation of PAK2 and the activation of caspase-3 during the early stage of UV irradiation-induced apoptosis in A431 cells. The protective effect of curcumin was significantly attenuated by sodium nitroprusside(SNP), a NO tenerator. The dexamethasone, an inhibitor of inducible NO synthase, could also effectively inhibit the cleavage of PAK2 and the activation of caspase-3 in the UV-induced apoptotic A431 cells. Therefore, we proposed that the intracellular NO, which was produced by UV irradiation inductions,might be a mediator of caspase-3 activation during apoptosis.