The phosphorylation of tyrosine 332 is necessary for the caspase 3-dependent cleavage of PKCδ and the regulation of cell apoptosis

Protein kinase C δ (PKCδ plays a major role in the regulation of cell apoptosis and survival. PKCδ is cleaved by caspase 3 to generate a constitutively active catalytic domain that mediates both its apoptotic and anti-apoptotic effects. The caspase cleavage site of PKCδ in the hinge region is flanked by the two tyrosine residues, Y311 and Y332. Here, we examined the role of the phosphorylation of tyrosines 311 and 332 in the cleavage and apoptotic function of PKCδ using the apoptotic stimuli, TRAIL and cisplatin. Tyrosine 332 was constitutively phosphorylated in the A172 and HeLa cells and was further phosphorylated by TRAIL and cisplatin. This phosphorylation was inhibited by the Src inhibitors, PP2 and SU6656, and by silencing of Src. Treatment of the A172 and HeLa cells with TRAIL induced cleavage of the WT PKCδ and of the PKCδY311F mutant, whereas a lower level of cleavage was observed in the PKCδY332F mutant. Similarly, a smaller degree of cleavage of the PKCδY332 mutant was observed in LNZ308 cells treated with cisplatin. Mutation of Y332F affected the apoptotic function of PKCδ; overexpression of the PKCδY332 mutant increased the apoptotic effect of TRAIL, whereas it decreased the apoptotic effect of cisplatin. Inhibition of Src decreased the cleavage of PKCδ and modified the apoptotic responses of the cells to TRAIL and cisplatin, similar to effect of the PKCδY332F mutant. These results demonstrate that the phosphorylation of tyrosine 332 by Src modulates the cleavage of PKCδ and the sensitivity of glioma cells to TRAIL and cisplatin.