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

被引:31
|
作者
Kitatani, Kazuyuki [1 ]
Idkowiak-Baldys, Jolanta [1 ]
Hannun, Yusuf A. [1 ]
机构
[1] Med Univ S Carolina, Dept Biochem & Mol Biol, Charleston, SC 29425 USA
关键词
D O I
10.1074/jbc.M609162200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:20647 / 20656
页数:10
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