Mechanism of inhibition of sequestration of protein kinase C α/βII by ceramide -: Roles of ceramide-activated protein phosphatases and phosphorylation/dephosphorylation of protein kinase C α/βII on threonine 638/641

Sustained activation of protein kinase C (PKC) isoenzymes alpha and beta II leads to their translocation to a perinuclear region and to the formation of the pericentrion, a PKC-dependent subset of recycling endosomes. In MCF-7 human breast cancer cells, the action of the PKC activator 4 beta-phorbol-12-myristate-13-acetate (PMA) evokes ceramide formation, which in turn prevents PKC alpha/beta II translocation to the pericentrion. In this study we investigated the mechanisms by which ceramide negatively regulates this translocation of PKC alpha/beta II. Upon PMA treatment, HEK-293 cells displayed dual phosphorylation of PKC alpha/beta II at carboxyl-terminal sites (Thr638/641 and Ser-657/660), whereas in MCF-7 cells PKC alpha/beta II were phosphorylated at Ser-657/660 but not Thr-638/641. Inhibition of ceramide synthesis by fumonisin B1 overcame the defect in PKC phosphorylation and restored translocation of PKC alpha/beta II to the pericentrion. To determine the involvement of ceramide-activated protein phosphatases in PKC regulation, we employed small interference RNA to silence individual Ser/Thr protein phosphatases. Knockdown of isoforms alpha or beta of the catalytic subunits of protein phosphatase 1 not only increased phosphorylation of PKC alpha/beta II at Thr-638/641 but also restored PKC alpha/beta II translocation to the pericentrion. Mutagenesis approaches in HEK-293 cells revealed that mutation of either Thr-641 or Ser-660 to Ala in PKC beta II abolished sequestration of PKC, implying the indispensable roles of phosphorylation of PKC alpha/beta II at those sites for their translocation to the pericentrion. Reciprocally, a point mutation of Thr-641 to Glu, which mimics phosphorylation, in PKC beta II overcame the inhibitory effects of ceramide on PKC translocation in PMA-stimulated MCF-7 cells. Therefore, the results demonstrate a novel role for carboxyl-terminal phosphorylation of PKC alpha/beta II in the translocation of PKC to the pericentrion, and they disclose specific regulation of PKC autophosphorylation by ceramide through the activation of specific isoforms of protein phosphatase 1.