Ceramide-induced Apoptosis by Translocation, Phosphorylation, and Activation of Protein Kinase Cδ in the Golgi Complex

Abstract

Protein kinase C (PKC), a Ca2+/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCδ in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCδ-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCδ expression using small interfering RNA. Ceramide induced the translocation of PKCδ to the Golgi complex and the concomitant activation of PKCδ via phosphorylation of Tyr311 and Tyr332 in the hinge region of the enzyme. Unphosphorylatable PKCδ (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCδ lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCδ to the Golgi complex and that PKCδ is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCδ by small interfering RNA to study the significance of phosphorylation of Tyr311 and Tyr332 in PKCδ for ceramide-induced apoptosis and found that phosphorylation of Tyr311 and Tyr332 is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCδ, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event. Protein kinase C (PKC), a Ca2+/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCδ in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCδ-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCδ expression using small interfering RNA. Ceramide induced the translocation of PKCδ to the Golgi complex and the concomitant activation of PKCδ via phosphorylation of Tyr311 and Tyr332 in the hinge region of the enzyme. Unphosphorylatable PKCδ (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCδ lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCδ to the Golgi complex and that PKCδ is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCδ by small interfering RNA to study the significance of phosphorylation of Tyr311 and Tyr332 in PKCδ for ceramide-induced apoptosis and found that phosphorylation of Tyr311 and Tyr332 is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCδ, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event. The protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; PTP, proteintyrosine phosphatase; GFP, green fluorescent protein; siRNA, small interfering RNA. family is a group of phospholipid-dependent serine/threonine protein kinases consisting of at least 10 subtypes that can be classified into three subgroups, classical, novel, and atypical (1Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3732) Google Scholar, 2Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4242) Google Scholar, 3Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2376) Google Scholar). The classical PKCs (α, βI, βII, and γ), which have a Ca2+-binding region (C2 region) and two cysteine-rich regions, are activated by Ca2+, phosphatidylserine and diacylglycerol, or phorbol esters (4Ono Y. Fujii T. Igarashi K. Kuno T. Tanaka C. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4868-4871Crossref PubMed Scopus (411) Google Scholar). The novel PKCs (δ, ϵ, η, and θ), which lack the C2 region, are activated by phosphatidylserine and diacylglycerol or phorbol esters without Ca2+ (5Osada S. Mizuno K. Saido T.-C. Suzuki K. Kuroki T. Ohno S. Mol. Cell. Biol. 1992; 12: 3930-3938Crossref PubMed Google Scholar). The atypical PKCs (ζ and ι/λ), which lack the C2 region and have only one cysteine-rich region, are dependent on phosphatidylserine, but are not affected by diacylglycerol, phorbol esters, or Ca2+ (6Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103Crossref PubMed Scopus (448) Google Scholar). There is increasing evidence showing that PKC is involved in apoptosis (7Gutcher I. Webb P.R. Anderson N.G. Cell. Mol. Life Sci. 2003; 60: 1061-1070Crossref PubMed Scopus (178) Google Scholar). Among multiple subtypes of PKC, PKCδ has been implicated as a pro-apoptotic kinase, mostly by acting as a target of caspase-3 (8Blass M. Kronfeld I. Kazimirsky G. Blumberg P.M. Brodie C. Mol. Cell. Biol. 2002; 22: 182-195Crossref PubMed Scopus (174) Google Scholar), whereas PKCα and PKCϵ inhibit apoptosis by phosphorylating Bcl-2 or by increasing the expression of Bcl-2, respectively (9Gubina E. Rinaudo M.S. Szallasi Z. Blumberg P.M. Mufson R.A. Blood. 1998; 91: 823-829Crossref PubMed Google Scholar, 10Ruvolo P.P. Deng X. Carr B.K. May W.S. J. Biol. Chem. 1998; 273: 25436-25442Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). Apoptotic stimuli such as DNA-damaging agents (11Emoto Y. Manome Y. Meinhardt G. Kisaki H. Kharbanda S. Robertson M. Ghayur T. Wong W. Kamen R. Weichselbaum R. EMBO J. 1995; 14: 6148-6156Crossref PubMed Scopus (652) Google Scholar), Fas ligand (12Mizuno K. Noda K. Araki T. Imaoka T. Kobayashi Y. Akita Y. Shimonaka M. Kishi S. Ohno S. Eur. J. Biochem. 1997; 250: 7-18Crossref PubMed Scopus (119) Google Scholar), and etoposide (8Blass M. Kronfeld I. Kazimirsky G. Blumberg P.M. Brodie C. Mol. Cell. 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Masters S.C. Fu H. Hakomori S. J. Biol. Chem. 2003; 278: 41557-41565Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In addition, it has been reported that PKCδ is essential for the apoptosis of keratinocytes and LaNCaP cells in response to 12-phorbol 13-myristate T. J. M. Kuroki T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. Blumberg P.M. Mol. Cell. Biol. 1999; PubMed Google Scholar). Ceramide by of has as implicated in various cellular responses, including cell 1998; PubMed Scopus Google Scholar, Science. PubMed Scopus Google Scholar, S. EMBO J. 1995; 14: PubMed Scopus Google Scholar, P.P. Y. M. J. 1996; PubMed Scopus Google Scholar, Science. 1996; 274: PubMed Scopus Google Scholar). The regulation of PKC by ceramide has been but the are ceramide has been to activate PKCα or to inhibit PKCδ in cells in A. D. J. 1998; PubMed Scopus Google Scholar). has also been reported that ceramide the translocation of PKCδ and PKCϵ the to the H. T. 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Biol. 1998; PubMed Google Scholar). of PKCδ Protein in HeLa cells the and in essential and into HeLa cells using to the of cells and for at least The was of and 10 The of was a at a at of the of translocation was as a of at for in the Golgi complex, or was using the PKCδ in the the in the was used as of by The in region is as the in the or Golgi complex by the in the in a region of the Golgi or was in at least of These and to a of translocation. of and by cells or ceramide a and in for The cells then and for The of and was the fluorescent cells various of PKCδ of 10 10 and and at The cells in of of 10 10 10 and and 10 at and the was used at for the of the was or for at and then protein for at for at and three without Ca2+ or the in of without Ca2+ or and used for phosphorylation or studies as to and The or for at The or using of was by the of into as H. E. H. T. H. Y. K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. Y. Fujii M. H. Kikkawa U. Mol. Cell. Biol. 1998; PubMed Google Scholar). In the kinase of or HeLa cells or of as the without PKC such as phosphatidylserine or The 10 of and The was for 10 at RNA and PKCδ of PKCδ as T. T. Y. Biochem. 2002; PubMed Scopus Google Scholar). of small interfering RNA expression of was of of various in essential for HeLa cells and and for The cells then of in and the of cells showing apoptotic in was a two or to be of the cells and of PKCδ in the Apoptotic of the in HeLa cells in response to a of in of the HeLa but not the of nuclear the of the apoptotic of involvement of PKCδ in ceramide-induced apoptosis was examined using the PKCδ-specific inhibitor rottlerin and knockdown of PKCδ by inhibited the apoptotic of the of apoptotic cells to without the of cell apoptosis. of endogenous PKCδ in HeLa cells using PKCδ T. T. Y. Biochem. 2002; PubMed Scopus Google Scholar) inhibited the apoptotic response to in HeLa cells Furthermore, the of M. M. J. T. Kuroki T. M. 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The and phosphorylation by ceramide as whereas of Tyr311 or Tyr332 the ceramide-induced and phosphorylation was in the to phosphorylation or demonstrated that both Tyr311 and Tyr332 ceramide Ceramide-induced tyrosine phosphorylation of PKCδ was blocked by and of family kinases study the phosphorylation of Tyr311 and Tyr332 and the kinase of PKCδ, we the kinase of PKCδ and its in HeLa cells and without ceramide in by the kinase of PKCδ and the and but the kinase of the and not of Tyr311 or Tyr332 the ceramide-induced phosphorylation of Tyr311 and Tyr332 be essential for PKCδ and of PKCδ in the Golgi have demonstrated the ceramide-induced activation of PKCδ concomitant its translocation to the Golgi complex T. S. Y. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar). the tyrosine phosphorylation of PKCδ is for translocation of PKCδ to the Golgi complex, we the ceramide-induced translocation of PKCδ, and in HeLa to PKCδ, the also to the Golgi complex in response to ceramide We that the tyrosine phosphorylation and activation of PKCδ are not necessary for the translocation of PKCδ by ceramide tyrosine phosphorylation is not necessary for the translocation of PKCδ to the Golgi complex, it is that PKCδ is its accumulation in the Golgi PKCδ in the Golgi complex or is we the tyrosine phosphorylation in HeLa cells for using for in the region and was also found on the as in the region but the green of in the did not the of that PKCδ is in the However, was also in the region in HeLa cells a was also in HeLa cells not it is that the tyrosine phosphorylation of endogenous PKCδ was by endogenous and PKCδ, we utilized the which the expression of endogenous PKCδ, but not PKCδ In HeLa cells PKCδ, ceramide-induced accumulation of in the region was only in cells on the in The of in the region in cells that PKCδ in the Golgi complex by ceramide is at Tyr311 and it was the tyrosine phosphorylation of PKCδ or translocation to the Golgi has been reported that the tyrosine phosphorylation and activation of PKCδ without its S. Y. Fujii M. H. Kikkawa U. Mol. Cell. Biol. 1998; PubMed Google Scholar). PKCδ is we a PKCδ lacking the C1B domain which could be by in the but failed to translocate to the Golgi complex ceramide in did not translocate induced the tyrosine phosphorylation and kinase activation of and PKCδ to a was activated by and These ceramide PKCδ in the Golgi complex via its C1B domain and then was and activated in the compartment. of PKCδ to the Golgi complex of the enzyme the Golgi complex, but not its to the Golgi T. S. Y. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar). The of of PKCδ be by the tyrosine phosphorylation of We examined the of the inhibitor on the activation and accumulation of PKCδ by in of the of PKCδ in the Golgi complex and that in the Furthermore, the of PKCδ by to of PKCδ for Apoptotic in HeLa the functional apoptosis and PKCδ targeting to the Golgi complex by its tyrosine we ceramide-induced apoptotic in HeLa cells PKCδ or its that HeLa cells or a apoptotic response to to that of cells However, endogenous PKCδ was in the Golgi complex, as demonstrated in and cause apoptosis that is of of We examined the of or PKCδ on the apoptotic response to in HeLa cells that for knockdown of endogenous The of PKCδ on ceramide-induced apoptosis was by of In could not the apoptotic induced by ceramide These that the phosphorylation of Tyr311 and Tyr332 in PKCδ is necessary for the apoptotic of We the phosphorylation of Tyr311 and Tyr332 in PKCδ is for apoptosis or the targeting and of PKCδ to the Golgi complex is necessary for the apoptotic of was and activated in the without translocation and that tyrosine phosphorylation and activation of PKCδ in the did not induce cell of tyrosine phosphorylation of PKCδ on apoptosis in HeLa of on the kinase and tyrosine phosphorylation of in the kinase of in HeLa cells by kinase was HeLa cells and for The kinase of was by kinase are as a of the kinase The tyrosine phosphorylation of was in HeLa cells HeLa cells and for the tyrosine phosphorylation of by was by of on the translocation of in HeLa failed to induce the translocation of However, 10 induced the translocation of 10 of on apoptosis in HeLa HeLa cells 10 or or for was by PKC the of PKC has been in and the of PKC subtype in various has been K. Y. J. Biol. 1997; PubMed Scopus Google Scholar, Y. K. K. J. Biol. 1998; PubMed Scopus Google Scholar). of PKC on PKC subtypes Y. K. K. J. Biol. 1998; PubMed Scopus Google Scholar), and stimuli and PKC translocation to the specific are necessary for the and phosphorylation of in the S. Y. H. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the translocation of PKC in cellular S. Y. Fujii M. H. Kikkawa U. Mol. Cell. Biol. 1998; PubMed Google Scholar), this that the targeting of PKC subtypes in cell We have demonstrated that ceramide by of via by translocation of PKCδ the to the Golgi complex and its activation by tyrosine phosphorylation T. S. Y. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar). In this study, we the importance of PKCδ and its tyrosine phosphorylation in the of apoptosis in HeLa cells of PKCδ via tyrosine phosphorylation has been reported by and are for the tyrosine by various stimuli (8Blass M. Kronfeld I. Kazimirsky G. Blumberg P.M. Brodie C. Mol. Cell. Biol. 2002; 22: 182-195Crossref PubMed Scopus (174) Google Scholar, W. M. J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, I. Kazimirsky G. Blumberg P.M. Brodie C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. Tanaka M. Y. H. Y. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). of tyrosine that two tyrosine and in the hinge region of PKCδ are the tyrosine phosphorylation of PKCδ in HeLa whereas of or did not the phosphorylation. is that phosphorylation of both Tyr311 and Tyr332 is necessary for the activation of The is in the by H. E. H. T. H. Y. K. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) that phosphorylation of Tyr311 is a critical in the of active PKCδ in response to in However, the the tyrosine phosphorylation of PKCδ and its activation is and it is that tyrosine are at Ceramide has been to activate tyrosine including Y. C. 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Full Text Full Text PDF PubMed Scopus Google Scholar, Szallasi Z. U. Blumberg P.M. J. 1998; PubMed Scopus Google Scholar). The tyrosine phosphorylation of PKCδ by ceramide was inhibited by of family suggesting that a tyrosine kinase in the family PKCδ in response to the of family kinases in the Golgi complex and the phosphorylation of specific by the kinases have been reported U. P. R. Eur. J. Biol. 2002; PubMed Scopus Google Scholar, M. M. J. Biol. 1997; PubMed Scopus Google Scholar, H. H. Biochem. PubMed Scopus Google Scholar), it is that tyrosine kinase such as PKCδ in the Golgi complex in HeLa cells ceramide The using and the PKCδ inhibitor that activation of PKCδ is for ceramide-induced apoptosis. has been that the apoptotic of PKCδ is the cleavage of PKCδ in the hinge region, by the of the constitutively active catalytic of the catalytic domain of PKCδ by caspase-3 has been reported in cells DNA-damaging agents (11Emoto Y. Manome Y. Meinhardt G. Kisaki H. Kharbanda S. Robertson M. Ghayur T. Wong W. Kamen R. Weichselbaum R. EMBO J. 1995; 14: 6148-6156Crossref PubMed Scopus (652) Google Scholar), Fas ligand (12Mizuno K. Noda K. Araki T. Imaoka T. Kobayashi Y. Akita Y. Shimonaka M. Kishi S. Ohno S. Eur. J. Biochem. 1997; 250: 7-18Crossref PubMed Scopus (119) Google Scholar), and etoposide (8Blass M. Kronfeld I. Kazimirsky G. Blumberg P.M. Brodie C. Mol. Cell. Biol. 2002; 22: 182-195Crossref PubMed Scopus (174) Google Scholar, 13Reyland M.E. Anderson S.M. Matassa A.A. Barzen K.A. Quissell D.O. J. Biol. Chem. 1999; 274: 19115-19123Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 14DeVries T.A. Neville M.C. Reyland M.E. EMBO J. 2002; 21: 6050-6060Crossref PubMed Scopus (208) Google Scholar). However, of PKCδ was in HeLa cells ceramide by T. S. Y. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar). In addition, the active of caspase-3 was in cell and the inhibitor failed to inhibit the ceramide-induced apoptosis in HeLa cells These suggest that the caspase-3 not to be involved in this ceramide-induced apoptotic mechanism by PKCδ in HeLa such as in this is that the caspase-3 cleavage in PKCδ is the two tyrosine phosphorylation Tyr311 and is that tyrosine phosphorylation the caspase-3 cleavage the but PKCδ without the mechanism of the activation is has been demonstrated that the C1B domain is for the translocation of PKCϵ to the Golgi complex induced by and ceramide K. Y. M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of the C1B domain of PKCϵ its translocation to the Golgi PKCϵ a C1B domain PKCδ also ceramide-induced translocation to the Golgi complex K. Y. M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), this that PKCδ lacking the C1B domain translocate to the Golgi complex in response to is supposed to be activated by phosphorylation of Tyr311 and which are in the the we have in to PKCδ translocation of PKCδ to the Golgi complex is indispensable for the apoptotic of studies T. S. Y. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar) demonstrated that ceramide the of of PKCδ the Golgi Ceramide translocates PKCδ by increasing its the Golgi complex in the is that of both the and accumulation of that PKCδ or the Golgi complex is by tyrosine phosphorylation. However, as accumulation of PKCδ in the Golgi complex was of tyrosine phosphorylation this that of PKCδ the Golgi complex is by of PKCδ by The small in PKCδ ceramide be to the that of the PKCδ in the was and of the PKC was activated by ceramide both activation and of PKCδ only in the Golgi The species-specific knockdown of PKCδ utilized in this study is a for the of of PKCδ failed to on ceramide-induced apoptosis is to the that a of endogenous PKCδ is in HeLa cells to induce the significance of the tyrosine phosphorylation of PKCδ for ceramide-induced it was necessary to the of endogenous However, knockdown of endogenous PKCδ only that endogenous PKCδ is for the of apoptosis. of endogenous PKCδ was by PKCδ, which is to demonstrated that Tyr311 and Tyr332 are in the Golgi complex ceramide and then induce apoptosis. In this we the importance of the PKCδ targeting mechanism and in the of apoptosis in HeLa cells We have demonstrated that ceramide the translocation of PKCδ to the Golgi complex and its activation via phosphorylation of Tyr311 and Tyr332 by kinases and that this activation in the of apoptosis. of PKCδ is for apoptotic and the targeting of PKCδ to the Golgi complex is essential in ceramide-induced apoptosis. T. S. Y. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar), the activation of such as and in the translocation and activation of PKCδ, suggesting that apoptosis via PKCδ-specific targeting to the Golgi complex the target of PKCδ in this mechanism is not the suggest that the targeting mechanism of PKCδ via tyrosine phosphorylation and translocation to the Golgi is indispensable for ceramide-induced apoptosis.