Mechanism of Inhibition of Sequestration of Protein Kinase C α/βII by Ceramide
2007; Elsevier BV; Volume: 282; Issue: 28 Linguagem: Inglês
10.1074/jbc.m609162200
ISSN1083-351X
AutoresKazuyuki Kitatani, Jolanta Idkowiak‐Baldys, Yusuf A. Hannun,
Tópico(s)Cellular transport and secretion
ResumoSustained 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. 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. Protein kinase C (PKC) 3The abbreviations used are: PKC, protein kinase C; cPKC, conventional PKC; C6-ceramide, N-hexanoyl-d-erythro-sphingosine; FB1, fumonisin B1; FBS, fetal bovine serum; GFP, green fluorescent protein; PBS, phosphate-buffered saline; PLD, phospholipase D; PMA, 4β-phorbol 12-myristate 13-acetate; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; siRNA, small interference RNA. is a family of several kinases that play key roles in signal transduction with multiple isoforms that are divided into three groups: conventional PKC (cPKC) (α, βI, βII, γ), novel PKC (δ,∊,η,θ), and atypical PKC (ζ, λ(τ)) (1Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4232) Google Scholar, 2Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (837) Google Scholar). A major mechanism for activation of cPKCs and novel PKCs involves acute and reversible translocation to the plasma membrane of these isoenzymes in response to either diacylglycerol or its analog 12-myristate-13-acetate (PMA), and this occurs within 30 s to a few minutes after stimulation. Recently, we and others (3Idkowiak-Baldys J. Becker K.P. Kitatani K. Hannun Y.A. J. Biol. Chem. 2006; 281: 22321-22331Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 4Becker K.P. Hannun Y.A. J. Biol. Chem. 2003; 278: 52747-52754Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 5Becker K.P. Hannun Y.A. J. Biol. Chem. 2004; 279: 28251-28256Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 6Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 7Hu T. Exton J.H. J. Biol. Chem. 2004; 279: 35702-35708Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) reported that, upon sustained stimulation of PKC (within 30-60 min), two of the cPKCs, PKCα and PKCβII, are sequestered into a subset of recycling endosomes, termed the "pericentrion," which becomes associated with the Rab11-positive compartment. The formation of the pericentrion regulates endocytosis and results in sequestration of several recycling components, including membrane lipids (3Idkowiak-Baldys J. Becker K.P. Kitatani K. Hannun Y.A. J. Biol. Chem. 2006; 281: 22321-22331Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). It is also important in the regulation of phospholipase D (PLD) activity and may dictate the site of PLD activation (7Hu T. Exton J.H. J. Biol. Chem. 2004; 279: 35702-35708Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). This novel translocation of PKC is regulated by both PLD (5Becker K.P. Hannun Y.A. J. Biol. Chem. 2004; 279: 28251-28256Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and ceramide (6Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). PLD (8Exton J.H. Biochim. Biophys. Acta. 1999; 1439: 121-133Crossref PubMed Scopus (337) Google Scholar, 9Frohman M.A. Sung T.C. Morris A.J. Biochim. Biophys. Acta. 1999; 1439: 175-186Crossref PubMed Scopus (277) Google Scholar, 10Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (924) Google Scholar) cleaves glycerophospholipids to form phosphatidic acid, which has been implicated in regulation of intracellular vesicular trafficking (11Jenkins G.M. Frohman M.A. Cell. Mol. Life Sci. 2005; 62: 2305-2316Crossref PubMed Scopus (395) Google Scholar). PLD activity is required for PMA-induced translocation of PKCβII to the pericentrion but not to the plasma membrane (4Becker K.P. Hannun Y.A. J. Biol. Chem. 2003; 278: 52747-52754Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In contrast, ceramide is involved in a negative feedback pathway that prevents translocation of PKC to the pericentrion (6Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), but the detailed mechanism remains unclear. Ceramide (12Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1500) Google Scholar, 13Ogretmen B. Hannun Y.A. Nat. Rev. Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1012) Google Scholar, 14Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (731) Google Scholar, 15Levade T. Auge N. Veldman R.J. Cuvillier O. Negre-Salvayre A. Salvayre R. Circ. Res. 2001; 89: 957-968Crossref PubMed Scopus (147) Google Scholar, 16Merrill Jr., A.H. Schmelz E.M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol. 1997; 142: 208-225Crossref PubMed Scopus (566) Google Scholar) is a key bioactive sphingolipid whose levels increase with exposure to various stimuli, including Fas ligands, chemotherapeutic drugs, tumor necrosis factor-α, and heat stress. Multiple lines of evidence implicate the generated ceramide in mediating/regulating cellular responses, including inflammatory responses (17Kitatani K. Akiba S. Sato T. Cell. Signal. 2004; 16: 967-974Crossref PubMed Scopus (20) Google Scholar, 18Chen C.C. Sun Y.T. Chen J.J. Chang Y.J. Mol. Pharmacol. 2001; 59: 493-500Crossref PubMed Scopus (121) Google Scholar), senescence (19Venable M.E. Lee J.Y. Smyth M.J. Bielawska A. Obeid L.M. J. Biol. Chem. 1995; 270: 30701-30708Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar), cell cycle arrest (20Dbaibo G.S. Pushkareva M.Y. Jayadev S. Schwarz J.K. Horowitz J.M. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1347-1351Crossref PubMed Scopus (203) Google Scholar), and apoptosis (21Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (786) Google Scholar, 22Garzotto M. White-Jones M. Jiang Y. Ehleiter D. Liao W.C. Haimovitz-Friedman A. Fuks Z. Kolesnick R. Cancer Res. 1998; 58: 2260-2264PubMed Google Scholar). The generation of ceramide in response to those stimuli involves one or more of several pathways including de novo synthesis and activation of acid or neutral sphingomyelinases (12Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1500) Google Scholar, 13Ogretmen B. Hannun Y.A. Nat. Rev. Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1012) Google Scholar, 14Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (731) Google Scholar, 15Levade T. Auge N. Veldman R.J. Cuvillier O. Negre-Salvayre A. Salvayre R. Circ. Res. 2001; 89: 957-968Crossref PubMed Scopus (147) Google Scholar, 16Merrill Jr., A.H. Schmelz E.M. Dillehay D.L. Spiegel S. Shayman J.A. Schroeder J.J. Riley R.T. Voss K.A. Wang E. Toxicol. Appl. Pharmacol. 1997; 142: 208-225Crossref PubMed Scopus (566) Google Scholar). In recent studies (6Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 23Kitatani K. Idkowiak-Baldys J. Bielawski J. Taha T.A. Jenkins R.W. Senkal C.E. Ogretmen B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2006; 281: 36793-36802Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) we found that in MCF-7 cells PMA induced the generation of ceramide from the salvage of free sphingoid bases, formed in turn from the breakdown of complex sphingolipids. Activation of this salvage pathway was implicated in preventing translocation of PKC to the pericentrion in response to PMA. The ability of ceramide to inhibit translocation of PKC to the pericentrion suggested that ceramide interfered with a key step involved in mediating the effects of PMA on PKC. Therefore, it became important to define the mechanism by which ceramide exerted its effects on PKC. Several enzymes have been shown to be activated in vitro by ceramide and have, thus, emerged as candidate transducers of ceramide action. These ceramide mediators include kinase suppressor of Ras (24Zhang Y. Yao B. Delikat S. Bayoumy S. Lin X.H. Basu S. McGinley M. Chan-Hui P.Y. Lichenstein H. Kolesnick R. Cell. 1997; 89: 63-72Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), cathepsin D (25Heinrich M. Wickel M. Schneider-Brachert W. Sandberg C. Gahr J. Schwandner R. Weber T. Saftig P. Peters C. Brunner J. Kronke M. Schutze S. EMBO J. 1999; 18: 5252-5263Crossref PubMed Scopus (306) Google Scholar), PKCζ (26Bourbon N.A. Yun J. Kester M. J. Biol. Chem. 2000; 275: 35617-35623Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), and ceramide-activated protein phosphatases, which include protein phosphatase 1 (PP1) (27Chalfant C.E. Kishikawa K. Mumby M.C. Kamibayashi C. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 20313-20317Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 28Chalfant C.E. Ogretmen B. Galadari S. Kroesen B.J. Pettus B.J. Hannun Y.A. J. Biol. Chem. 2001; 276: 44848-44855Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and protein phosphatase 2A (PP2A) (29Chalfant C.E. Szulc Z. Roddy P. Bielawska A. Hannun Y.A. J. Lipid Res. 2004; 45: 496-506Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 30Mora A. Sabio G. Risco A.M. Cuenda A. Alonso J.C. Soler G. Centeno F. Cell. Signal. 2002; 14: 557-562Crossref PubMed Scopus (94) Google Scholar, 31Dobrowsky R.T. Kamibayashi C. Mumby M.C. Hannun Y.A. J. Biol. Chem. 1993; 268: 15523-15530Abstract Full Text PDF PubMed Google Scholar, 32Kowluru A. Metz S.A. FEBS Lett. 1997; 418: 179-182Crossref PubMed Scopus (50) Google Scholar, 33Galadari S. Hago A. Patel M. Exp. Mol. Med. 2001; 33: 240-244Crossref PubMed Scopus (8) Google Scholar). Moreover, multiple studies have shown that ceramide signaling results in dephosphorylation of key proteins such as the retinoblastoma proteins (20Dbaibo G.S. Pushkareva M.Y. Jayadev S. Schwarz J.K. Horowitz J.M. Obeid L.M. Hannun Y.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1347-1351Crossref PubMed Scopus (203) Google Scholar), Bcl-2 (34Ruvolo P.P. Deng X. Ito T. Carr B.K. May W.S. J. Biol. Chem. 1999; 274: 20296-20300Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), PKCα (35Lee J.Y. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1996; 271: 13169-13174Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), Akt (36Schubert K.M. Scheid M.P. Duronio V. J. Biol. Chem. 2000; 275: 13330-13335Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), p38 (23Kitatani K. Idkowiak-Baldys J. Bielawski J. Taha T.A. Jenkins R.W. Senkal C.E. Ogretmen B. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2006; 281: 36793-36802Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 37Kitatani K. Akiba S. Hayama M. Sato T. Arch. Biochem. Biophys. 2001; 395: 208-214Crossref PubMed Scopus (29) Google Scholar), and SR proteins (38Jenkins G.M. Cowart L.A. Signorelli P. Pettus B.J. Chalfant C.E. Hannun Y.A. J. Biol. Chem. 2002; 277: 42572-42578Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and these substrates in turn mediate specific downstream functions of ceramide. Moreover, exogenous ceramide has been shown to influence both PLD (39Venable M.E. Bielawska A. Obeid L.M. J. Biol. Chem. 1996; 271: 24800-24805Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and PKC (35Lee J.Y. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1996; 271: 13169-13174Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 40Nakamura Y. Nakashima S. Ojio K. Banno Y. Miyata H. Nozawa Y. J. Immunol. 1996; 156: 256-262PubMed Google Scholar) itself, raising the possibility that ceramide generated from the salvage pathway could target either or both of these to inhibit PKC translocation to the pericentrion. Indeed, several studies have shown that short-chain ceramides inhibit PLD both in vitro (39Venable M.E. Bielawska A. Obeid L.M. J. Biol. Chem. 1996; 271: 24800-24805Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and in cells (40Nakamura Y. Nakashima S. Ojio K. Banno Y. Miyata H. Nozawa Y. J. Immunol. 1996; 156: 256-262PubMed Google Scholar, 41Suchard S.J. Hinkovska-Galcheva V. Mansfield P.J. Boxer L.A. Shayman J.A. Blood. 1997; 89: 2139-2147Crossref PubMed Google Scholar). Exogenous ceramide has also been shown to induce dephosphorylation of PKCα on several phosphorylation sites including Thr-497 or Ser-657 (42Lee J.Y. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2000; 275: 29290-29298Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Given these considerations, it became important to determine the mechanisms by which PKCs translocate to the pericentrion and the mechanisms by which ceramide interferes with this process. In this study we employed HEK-293 and MCF-7 cells and investigated the mechanisms by which ceramide evokes a negative feedback pathway for cPKC translocation to the pericentrion. The results demonstrate a novel role for carboxyl-terminal phosphorylation sites (autophosphorylation sites) of cPKCs (43Flint A.J. Paladini R.D. Koshland Jr., D.E. Science. 1990; 249: 408-411Crossref PubMed Scopus (109) Google Scholar, 44Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 45Dutil E.M. Keranen L.M. DePaoli-Roach A.A. Newton A.C. J. Biol. Chem. 1994; 269: 29359-29362Abstract Full Text PDF PubMed Google Scholar) in the translocation of PKC to the pericentrion, and they disclose very specific effects of the salvage-generated ceramide on regulation of the phosphorylation of these sites through activation of members of the PP1 family of protein phosphatases. Materials—Fumonisin B1 (FB1) was purchased from Alexis Corp. (Carlsbad, CA). Rabbit polyclonal antibody specific for phosphorylated PKCα/βII at Thr-638/641 (#9375) or rabbit polyclonal antibody specific for phosphorylated conventional/novel PKCs at a carboxyl-terminal residue homologous to Ser-660 of PKCβII (#9371) were from Cell Signaling Technology. Phosphatidylbutanol and N-hexanoyl-d-erythro-sphingosine (C6-ceramide) were from Avanti Polar Lipids Inc. (Alabaster, AL). Enhanced chemiluminescence kit was from Amersham Biosciences. Rabbit polyclonal antibody for Rab11 was from Zymed Laboratories Inc. (South San Francisco, CA). AlexaFluor488- or AlexaFluor555-conjugated anti-IgG antibodies and AlexaFluor555-transferrin were purchased from Molecular Probes. PMA, okadaic acid, and tautomycin were from Calbiochem. CD59 antibody was a generous gift from Dr. Stephen Tomlinson (Medical University of South Carolina, Charleston, SC). pcDNA3.1-mCherry was generated by Dr. Guangwei Du (SUNY, Stony Brook, NY) using pRSET-BmCherry, which was a gift from Dr. Roger Y. Tsien (University of California San Diego, CA). Other reagents were obtained from Sigma. Cell Culture—HEK-293 cells were maintained in minimal essential media supplemented with 10% (v/v) fetal bovine serum (FBS). MCF-7 cells were grown in RPMI 1640 cells supplemented with l-glutamine and 10% (v/v) FBS. Cells were maintained at less than 80% confluence under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37 °C). Plasmids—Expression vectors encoding green fluorescent protein (GFP)-PKCα, GFP-PKCβII, and four-point mutants GFP-PKCβII-T641A, GFP-PKCβII-S660A, GFP-PKCβII-T641E, and GFP-PKCβII-S660E have previously been described (4Becker K.P. Hannun Y.A. J. Biol. Chem. 2003; 278: 52747-52754Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 46Feng X. Hannun Y.A. J. Biol. Chem. 1998; 273: 26870-26874Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). PKCβII was subcloned into XhoI and BamHI sites of pcDNA3.1-mCherry to generate mCherry-PKCβII. Confocal Microcopy—Cells growing on glass coverslips were fixed for 10 min at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS) and washed with PBS. Confocal laser microscopy was performed using an LSM510 microscope (Carl Zeiss, NY). Cells were counted as translocated to the pericentrion if GFP-PKCs was observed in the perinuclear region, and GFP-PKCs translocation was assessed by blinded quantification of confocal microscopy pictures (3 fields for each counted for an approximate sampling of 50-100 cells for each determination). Indirect Immunofluorescence—Transfected cells with GFP-tagged PKCs were fixed for 10 min at room temperature with 4% formaldehyde in PBS and washed with PBS. Cells were treated for 10 min with 0.1% Triton X-100, washed with PBS, and blocked for 1 h with PBS containing 2% human serum. The primary antibodies specific for Rab11 or CD59 were diluted in PBS containing 2% human serum and incubated for 90 min at room temperature or overnight at 4 °C. Samples were washed with PBS, and AlexaFluor488- or AlexaFluor555-conjugated anti-IgG antibodies were applied for 1 h at room temperature in PBS containing 2% human serum. Confocal laser microscopy was performed using an LSM510 microscope (Carl Zeiss, NY). Palmitate Labeling—For chase experiments, MCF-7 cells were labeled (24-30 h) in 35-mm dishes with 2 μCi/ml [3H]palmitate in RPMI 1640 supplemented with 10% FBS. Cells were then washed 3 times with PBS, and RPMI 1640 supplemented with 10% FBS was added. Cells were pretreated with 0.4% 1-butanol for 10 min followed by stimulation. The culture media were removed, and the cells were washed rapidly three times with ice-cold PBS. Total cellular lipids were extracted by the modified method of Bligh and Dyer (65Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar). Lipids were separated by thin-layer chromatography on a silica gel G plate with ethyl acetate/iso-octane/acetic acid (9:5:2, v/v/v) as the developing system (6Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The plates were sprayed with EN3HANCE Spray (PerkinElmer Life Sciences) to amplify the tritium signal and then exposed for autoradiography for 24 h. The area corresponding to phosphatidylbutanol was scraped off, and the radioactivity was measured by liquid scintillation counting. Western Blotting—Cells were washed three times with PBS supplemented with Halt™ phosphatase inhibitor mixture (Pierce) and then lysed using Laemmli buffer. The protein samples (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred to nitrocellulose membranes, blocked with PBS, 0.1% Tween 20 containing 5% nonfat dried milk, washed with PBS-Tween, and incubated with rabbit polyclonal antibody for phospho-PKCα/βII (1:1000) or rabbit polyclonal antibody for PKCα (1:2000) in 0.1% Tween 20 containing 5% nonfat dried milk. The blots were washed with PBS-Tween and incubated with secondary antibody conjugated with horseradish peroxidase in PBS-Tween containing 5% nonfat dried milk. Detection was performed using enhanced chemiluminescence reagent. Transfection with Small Interference RNA (siRNA)—Cells (2 × 105 cells/60-mm dish) were transfected with double-stranded siRNAs for individual isoforms of PP1 catalytic subunit (PP1c) or PP2A catalytic subunit (PP2Ac) using Oligofectamine (Invitrogen) according to the manufacturer's instructions. After 48 h transfection reagents were washed out, and cells were stimulated with PMA in RPMI1640 supplemented with 10% FBS. Specific siRNAs for PP2Ac-β (sc-36301) and PP1c-β (sc-36295) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The other sequences of siRNAs for scrambled RNA, PP1c-α, and PP1c-γ were AATTCTCCGAACGTGTCACGT, AAGCACGACTTGGACCTCATC, and AAGAGGCAGTTGGTCACTCTG, respectively. Effects of Ceramide on PLD Activation and GFP-PKCα Translocation in PMA-stimulated MCF-7 Cells—PLD activation has been shown to be required for the translocation of PKCα and/or PKCβII to the pericentrion in HEK-293 cells or COS-7 cells (5Becker K.P. Hannun Y.A. J. Biol. Chem. 2004; 279: 28251-28256Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 7Hu T. Exton J.H. J. Biol. Chem. 2004; 279: 35702-35708Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In a previous study we showed that PKCβII failed to translocate to the pericentrion in MCF-7 (6Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). This inhibition was mediated by ceramide generated from the salvage pathway. Because ceramide was previously shown to inhibit PLD activation (39Venable M.E. Bielawska A. Obeid L.M. J. Biol. Chem. 1996; 271: 24800-24805Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 40Nakamura Y. Nakashima S. Ojio K. Banno Y. Miyata H. Nozawa Y. J. Immunol. 1996; 156: 256-262PubMed Google Scholar, 41Suchard S.J. Hinkovska-Galcheva V. Mansfield P.J. Boxer L.A. Shayman J.A. Blood. 1997; 89: 2139-2147Crossref PubMed Google Scholar), we wondered whether the effects of ceramide on PKC in MCF-7 cells are due to inhibition of PLD. MCF-7 cells labeled with [3H]palmitate were stimulated with PMA for 1 h in the presence of 0.4% 1-butanol, and then phosphatidylbutanol levels were determined as a measure of PLD activity. As shown in Fig. 1A, generation of phosphatidylbutanol was stimulated after PMA treatment. This stimulation was not affected by FB1. Under these conditions, FB1 inhibited ceramide formation and restored the translocation of GFP-PKCβII (6Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). FB1 treatment also resulted in GFP-PKCα sequestration and colocalization with the Rab11-positive subset of the recycling compartment (Fig. 1B). These results show that 1) ceramide generated from the salvage pathway does not inhibit PLD and 2) the effects of ceramide on PKC translocation to the pericentrion are not due to inhibition of PLD. PKCα/βII Phosphorylation upon PMA Treatment in MCF-7 and HEK-293 Cells—Because activation of PLD by PMA appears to be a direct result of the action of PMA and the interaction of PKC with PLD, the above results suggested that the site of ceramide action is independent of PLD activation and may be at the level of PKC itself. The association of PKCα/βII with membranes has been shown to be modulated by specific phosphorylation of residues Thr-638/641 or Ser-657/660 that have been identified as autophosphorylation sites (Thr-641 in PKCβII corresponds to Thr-638 in PKCα, whereas Ser-660 in PKCβII corresponds to Ser-657 in PKCα) (44Keranen L.M. Dutil E.M. Newton A.C. Curr. Biol. 1995; 5: 1394-1403Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 45Dutil E.M. Keranen L.M. DePaoli-Roach A.A. Newton A.C. J. Biol. Chem. 1994; 269: 29359-29362Abstract Full Text PDF PubMed Google Scholar, 47Bornancin F. Parker P.J. Curr. Biol. 1996; 6: 1114-1123Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 48Borner C. Filipuzzi I. Wartmann M. Eppenberger U. Fabbro D. J. Biol. Chem. 1989; 264: 13902-13909Abstract Full Text PDF PubMed Google Scholar). We, therefore, wondered if the translocation of PKCα/βII to the pericentrion might be modulated by phosphorylation. The effects of PMA on the phosphorylation status of PKCα/βII were evaluated in both MCF-7 cells and HEK-293 cells, since PMA induces PKC translocation to the pericentrion in HEK-293 cells but not in MCF-7 cells (3Idkowiak-Baldys J. Becker K.P. Kitatani K. Hannun Y.A. J. Biol. Chem. 2006; 281: 22321-22331Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 4Becker K.P. Hannun Y.A. J. Biol. Chem. 2003; 278: 52747-52754Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 5Becker K.P. Hannun Y.A. J. Biol. Chem. 2004; 279: 28251-28256Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 6Becker K.P. Kitatani K. Idkowiak-Baldys J. Bielawski J. Hannun Y.A. J. Biol. Chem. 2005; 280: 2606-2612Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Those two cell lines were treated with 100 nm PMA, and then acute phosphorylation of PKCα/βII at both Thr-638/641 and Ser-657/660 was assessed using two distinct antibodies targeting those sites; one antibody against Thr-638/641 is specific for PKCα/βII, whereas the second antibody against Ser-657/660 detects phosphorylation on both conventional and novel PKC. Using the last antibody, we were able to detect phosphorylation of overexpressed GFP-PKCα and GFP-PKCβII after PMA treatment in both cell lines (data not shown). This immunoreactivity does not negate phosphorylation of other PKC isoforms but clearly indicated phosphorylation of PKCα/βII at Ser-657/660 in those cell lines. PMA treatment of HEK-293 and MCF-7 over the indicated time course induced acute phosphorylation of PKCα/βII at both Thr-638/641 and Ser-657/660 in HEK-293 cells. By contrast, PKCα/βII phosphorylation at Thr-638/641 was not seen in PMA-stimulated MCF-7 cells (Fig. 2A). Because PKCβ was not detected in MCF-7 (49Ways D.K. Kukoly C.A. deVente J. Hooker J.L. Bryant W.O. Posekany K.J. Fletcher D.J. Cook P.P. Parker P.J. J. Clin. Investig. 1995; 95: 1906-1915Crossref PubMed Scopus (269) Google Scholar, 50Shanmugam M. Krett N.L. Maizels E.T. Cutler Jr., R.E. Peters C.A. Smith L.M. O'Brien M.L. Park-Sarge O.K. Rosen S.T. Hunzicker-Dunn M. Mol. Cell. Endocrinol. 1999; 148: 109-118Crossref PubMed Scopus (44) Google Scholar), the antibody against phosphorylated PKCα/βII at Thr-638/641 is suggested mostly to detect phosphorylated PKCα at Thr-638 in this cell line. During the time course of these studies, PKCα levels in both
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