title: Mechanism of inhibition of sequestration of protein kinase C alpha/betaII by ceramide. Roles of ceramide-activated protein phosphatases and phosphorylation/dephosphorylation of protein kinase C alpha/betaII on threonine 638/641.
abstract: Sustained activation of protein kinase C (PKC) isoenzymes alpha and betaII 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 4beta-phorbol-12-myristate-13-acetate (PMA) evokes ceramide formation, which in turn prevents PKCalpha/betaII translocation to the pericentrion. In this study we investigated the mechanisms by which ceramide negatively regulates this translocation of PKCalpha/betaII. Upon PMA treatment, HEK-293 cells displayed dual phosphorylation of PKCalpha/betaII at carboxyl-terminal sites (Thr-638/641 and Ser-657/660), whereas in MCF-7 cells PKCalpha/betaII 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 PKCalpha/betaII 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 PKCalpha/betaII at Thr-638/641 but also restored PKCbetaII translocation to the pericentrion. Mutagenesis approaches in HEK-293 cells revealed that mutation of either Thr-641 or Ser-660 to Ala in PKCbetaII abolished sequestration of PKC, implying the indispensable roles of phosphorylation of PKCalpha/betaII at those sites for their translocation to the pericentrion. Reciprocally, a point mutation of Thr-641 to Glu, which mimics phosphorylation, in PKCbetaII 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 PKCalpha/betaII 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kitatani
authors.forename: Jolanta
authors.initials: J
authors.lastname: Idkowiak-Baldys
authors.forename: Yusuf A
authors.initials: YA
authors.lastname: Hannun
mesh_terms: D019943:Amino Acid Substitution; D002273:Carcinogens / Q000494:pharmacology; D020134:Catalytic Domain / Q000502:physiology; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D037341:Fumonisins / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D020125:Mutation, Missense; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054645:Protein Phosphatase 1; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000235:genetics / Q000378:metabolism
chemical_list: D002273:Carcinogens; D002518:Ceramides; D004791:Enzyme Inhibitors; D037341:Fumonisins; D007527:Isoenzymes; D013912:Threonine; C056933:fumonisin B1; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D013755:Tetradecanoylphorbol Acetate