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J. Biol. Chem., Vol. 282, Issue 28, 20647-20656, July 13, 2007

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Mechanism of Inhibition of Sequestration of Protein Kinase C /II by Ceramide

ROLES OF CERAMIDE-ACTIVATED PROTEIN PHOSPHATASES AND PHOSPHORYLATION/DEPHOSPHORYLATION OF PROTEIN KINASE C /II ON THREONINE 638/641^*

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, 29425

Sustained activation of protein kinase C (PKC) isoenzymes and 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-phorbol-12-myristate-13-acetate (PMA) evokes ceramide formation, which in turn prevents PKC/II translocation to the pericentrion. In this study we investigated the mechanisms by which ceramide negatively regulates this translocation of PKC/II. Upon PMA treatment, HEK-293 cells displayed dual phosphorylation of PKC/II at carboxyl-terminal sites (Thr-638/641 and Ser-657/660), whereas in MCF-7 cells PKC/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/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 or of the catalytic subunits of protein phosphatase 1 not only increased phosphorylation of PKC/II at Thr-638/641 but also restored PKCII translocation to the pericentrion. Mutagenesis approaches in HEK-293 cells revealed that mutation of either Thr-641 or Ser-660 to Ala in PKCII abolished sequestration of PKC, implying the indispensable roles of phosphorylation of PKC/II at those sites for their translocation to the pericentrion. Reciprocally, a point mutation of Thr-641 to Glu, which mimics phosphorylation, in PKCII 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/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.

Received for publication, September 27, 2006 , and in revised form, May 14, 2007.

^* This work was supported in part by National Institutes of Health Grant HL 43707 (to Y. A. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave., P. O. Box 250509, Charleston, SC 29425. Tel.: 843-792-4321; Fax: 843-792-4322; E-mail: hannun{at}musc.edu.

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