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

Kazuyuki Kitatani, Jolanta Idkowiak-Baldys, Yusuf A. Hannun

Research output: Contribution to journalArticlepeer-review

31 Citations (Scopus)

Abstract

Sustained activation of protein kinase C (PKC) isoenzymes α and βII 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 4β-phorbol-12-myristate-13-acetate (PMA) evokes ceramide formation, which in turn prevents PKCα/βII translocation to the pericentrion. In this study we investigated the mechanisms by which ceramide negatively regulates this translocation of PKCα/βII. Upon PMA treatment, HEK-293 cells displayed dual phosphorylation of PKCα/βII at carboxyl-terminal sites (Thr-638/641 and Ser-657/660), whereas in MCF-7 cells PKCα/βII 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 PKCα/βII 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 α or β of the catalytic subunits of protein phosphatase 1 not only increased phosphorylation of PKCα/βII at Thr-638/641 but also restored PKCβII translocation to the pericentrion. Mutagenesis approaches in HEK-293 cells revealed that mutation of either Thr-641 or Ser-660 to Ala in PKCβII abolished sequestration of PKC, implying the indispensable roles of phosphorylation of PKCα/βII at those sites for their translocation to the pericentrion. Reciprocally, a point mutation of Thr-641 to Glu, which mimics phosphorylation, in PKCβII 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 PKCα/βII 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.

Original languageEnglish
Pages (from-to)20647-20656
Number of pages10
JournalJournal of Biological Chemistry
Volume282
Issue number28
DOIs
Publication statusPublished - 2007 Jul 13

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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