Phosphorylation of Ser45 and Ser59 of αB-crystallin and p38/extracellular regulated kinase activity determine αB-crystallin-mediated protection of rat brain astrocytes from C2-ceramide- and staurosporine-induced cell death

We previously demonstrated that alpha B-crystallin and protease-activated receptor (PAR) are involved in protection of astrocytes against C2-ceramide- and staurosporine-induced cell death [Li et al. (2009) J. Neurochem. 110, 1433-1444]. Here, we further investigated the mechanism of cytoprotection by alpha B-crystallin. Our current data revealed that after down-regulation of alpha B-crystallin by siRNA, cell death caused by C2-ceramide and staurosporine is increased. Furthermore, we investigated the mechanism of cytoprotection of astrocytes by intracellular alpha B-crystallin. Application of specific inhibitors of p38 and extracellular regulated kinase (ERK) abrogates the protection of astrocytes by over-expression of alpha B-crystallin. Thus, p38 and ERK contribute to protective processes by alpha B-crystallin. To reveal the molecular mechanism of alpha B-crystallin-mediated cytoprotection, we mimicked phosphorylation or unphosphorylation of alpha B-crystallin. In these experiments, we found that the phosphorylation of alpha B-crystallin at Ser45 and Ser59 is required for protection. Ser19 phosphorylation of alpha B-crystallin does not contribute to protection. Moreover, we detected that PAR-2 activation increases the phosphorylation level of alpha B-crystallin at Ser59, but does not affect the expression level of alpha B-crystallin. Thus, endogenous alpha B-crystallin has protective capacity employing a mechanism, which involves regulation of the phosphorylation status of alpha B-crystallin and p38 and ERK activity. Moreover, we report that PAR-2 activation evokes the phosphorylation of alpha B-crystallin to increase astrocytes survival.