Findings from this study also
Findings from this study also indicate that DAPK regulated BimEL expression during OGD as DAPK shRNA transfection inhibited OGD-induced increase of BimEL. A previous study shows that Bim is found in ER compartment in stressed 6 his tag but not in normal cells, indicating translocation of Bim during ER stress (Morishima et al., 2004). To detect whether the increased BimEL expression was associated with ER stress activation during OGD exposure, BimEL localization was examined. As expected, BimEL translocated to ER following OGD treatment, which suggests that BimEL was indeed involved in the ER stress during OGD. These data thus suggest that DAPK caused ER stress by up-regulating BimEL expression during OGD treatment. ERK1/2 is a member of the mitogen-activated protein kinase (MAPK) family and ERK1/2 can be activated by MEK through a cascade of upstream kinases (Wang et al., 2011). Once activated, ERK1/2 phosphorylates its substrates, leading to a number of cell processes including cell proliferation and cell survival. One of its substrates is BimEL. Phosphorylation of BimEL by ERK1/2 results in the ubiquitination and degradation of BimEL (Weston et al., 2003, Leung et al., 2008). Since a previous study reveals an interaction between DAPK and ERK1/2 in 293T cells (Chen et al., 2005), we hypothesized that DAPK regulated BimEL through ERK1/2 during OGD. Data from the present study showed that phospho-ERK1/2 decreased following the OGD treatment, and DAPK shRNA transfection prevented OGD-induced decrease of phospho-ERK1/2, indicating that DAPK inhibited ERK1/2 activation during OGD. However, IP result revealed no direct interaction between DAPK and ERK1/2 (data not shown). Although phospho-ERK1/2 decreased following OGD treatment, there was increased BimEL associated with phospho-ERK1/2. Consistent with these results, treatment of cells with OGD also resulted in increased phospho-BimEL level. These findings suggest that phospho-ERK1/2 phosphorylated BimEL which was then designated for degradation, and DAPK inhibited phospho-ERK1/2 expression during OGD. JNK1/2 is a member of MAPK family and studies have shown that JNK1/2 activation promotes neuronal apoptosis (Xia et al., 1995, Le-Niculescu et al., 1999). A previous study has revealed that DAPK physically interacts with protein kinase D (PKD) in response to oxidative stress, and PKD in turn activates JNK. Knockdown of either DAPK or PKD attenuates JNK activation (Eisenberg-Lerner and Kimchi, 2007). To study if JNK was involved in the DAPK–BimEL signaling pathway during OGD treatment, we first examined how DAPK affected JNK1/2 expression. Knockdown of DAPK caused a marked reduction of phospho-JNK1/2, the active form of this enzyme, during OGD exposure, whereas no direct interaction was found between DAPK and JNK1/2 (data not shown). Whether PKD was involved in the DAPK–JNK1/2 signaling pathway during OGD treatment still needs to be investigated. Subsequently, effect of JNK1/2 on BimEL expression was studied. Inhibition of JNK1/2 with a specific inhibitor, SP600125, dramatically prevented BimEL increase during OGD exposure, suggesting that JNK1/2 played a critical role in BimEL up-regulation. These results are consistent with previous observations that JNK induces Bim expression following the treatment with UV irradiation (Ley et al., 2003). Interestingly, although JNK1/2 up-regulates Bim expression in cells treated with OGD or UV irradiation, the signaling pathways are differentially regulated in these two conditions. In the UV irradiation study, JNK up-regulates Bim through ERK activation. In the OGD condition, ERK activation was not affected with the presence of JNK inhibitor, suggesting ERK1/2 was not involved in the JNK1/2–BimEL signaling pathway (Wang et al., 2011). Collectively, these data indicate DAPK activated JNK1/2 which then increased BimEL expression during OGD. Although JNK1/2 inhibitor resulted in much lower BimEL expression, cells with the inhibitor still showed slightly increased BimEL expression following OGD treatment, suggesting some other signaling pathways might also be responsible for the BimEL increase during OGD.