Exposure of mammalian cells to ultraviolet (UV) light elicits a cellular response and can also lead to apoptotic cell death. In this report, we show that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be dramatically activated during the early stages of UV irradiation-triggered apoptosis of A431 cells. Immunoblot analysis revealed that this 36-kDa MBP kinase could be recognized by an antibody against the C-terminal regions 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 studying tools, we further demonstrated that UV irradiation caused cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment and a 30-kDa N-terminal fragment in A431 cells. The appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in A431 cells upon UV irradiation. In addition, UV irradiation also led to activation of CPP32/caspase-3, but not ICH-1L/caspase-2 and ICE/caspase-1, in A431 cells and the kinetics of activation of CPP32/caspase-3 appeared to correlate well with that of DNA fragmentation and of cleavage/activation of PAK2, respectively. Moreover, blockage of activation of CPP32/caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors for caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could significantly attenuate the extent of cleavage/activation of PAK2 induced by UV irradiation. Collectively, the results demonstrate that cleavage and activation of PAK2 can be induced during the early stages of UV irradiation-triggered apoptosis and indicate the involvement of CPP32/caspase-3 in this process.
HYS-32 is a novel derivative of combretastatin-A4 (CA-4) previously shown to induce microtubule coiling in rat primary astrocytes. In this study, we further investigated the signaling mechanism and EB1, a microtubule-associated end binding protein, involved in HYS-32-induced microtubule catastrophes. Confocal microscopy with double immunofluorescence staining revealed that EB1 accumulates at the growing microtubule plus ends, where they exhibit a bright comet-like staining pattern in control astrocytes. HYS-32 induced microtubule catastrophes in both a dose- and time-dependent manner and dramatically increased the distances between microtubule tips and the cell border. Treatment of HYS-32 (5 μM) eliminated EB1 localization at the microtubule plus ends and resulted in an extensive redistribution of EB1 to the microtubule lattice without affecting the β-tubulin or EB1 protein expression. Time-lapse experiments with immunoprecipitation further displayed that the association between EB-1 and β-tubulin was significantly decreased following a short-term treatment (2 h), but gradually increased in a prolonged treatment (6-24 h) with HYS-32. Further, HYS-32 treatment induced GSK3β phosphorylation at Y216 and S9, where the ratio of GSK3β-pY216 to GSK3β-pS9 was first elevated followed by a decrease over time. Co-treatment of astrocytes with HYS-32 and GSK3β inhibitor SB415286 attenuated the HYS-32-induced microtubule catastrophes and partially prevented EB1 dissociation from the plus end of microtubules. Furthermore, co-treatment with PI3K inhibitor LY294002 inhibited HYS-32-induced GSK3β-pS9 and partially restored EB1 distribution from the microtubule lattice to plus ends. Together these findings suggest that HYS-32 induces microtubule catastrophes by preventing EB1 from targeting to microtubule plus ends through the GSK3β signaling pathway.
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