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.
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.
Kitatani
Kazuyuki
K
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA.
Idkowiak-Baldys
Jolanta
J
Hannun
Yusuf A
YA
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
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