Journal of Biological Chemistry · 2006 · 55 citations · 49 references
Life SciencesCell BiologyMolecular BiologyApoptosisCell SignalingSignal TransductionApoptosis In CancerApoptosis RegulationApoptotic Signal TransductionApoptotic SignalingBcl-2 Family ProteinsCytochrome C ReleaseAnti-apoptotic ProteinsApoptosis Signaling PathwaysApoptosis Signaling RegulationApoptotic ResponseCytochrome C-mediated Caspase ActivationMitochondrial Apoptotic SignalingMitochondrial PathwaysPro-apoptotic Bax Activation
Proteolytic cleavage and subsequent activation of protein kinase C (PKC) δ is required for apoptosis induced by a variety of genotoxic agent, including UV radiation. In addition, overexpression of the constitutively active PKCδ catalytic fragment (PKCδ-cat) is sufficient to trigger Bax activation, cytochrome c release, and apoptosis. While PKCδ is a key apoptotic effector, the downstream target(s) responsible for the mitochondrial apoptotic cascade are not known. We found that expression of the active PKCδ-cat in HaCaT cells triggers a reduction in the anti-apoptotic protein Mcl-1, similar to UV radiation. The down-regulation of Mcl-1 induced by PKCδ-cat was not at the mRNA level but was due to decreased protein half-life. Overexpression of Mcl-1 protected HaCaT cells from both UV and PKCδ-cat-induced apoptosis and blocked the release of cytochrome c from the mitochondria, indicating that Mcl-1 down-regulation was required for apoptosis signaling. Indeed, down-regulation of Mcl-1 with siRNA slightly increased the basal apoptotic rate of HaCaT cells and dramatically sensitized them to UV or PKCδ-cat-induced apoptosis. HaCaT cells with down-regulated Mcl-1 had higher activated Bax protein, as measured by Bax cross-linking, indicating that Mcl-1 down-regulation is sufficient for Bax activation. Finally, recombinant PKCδ could phosphorylate Mcl-1 in vitro, identifying Mcl-1 as a direct target for PKCδ. Overall our results identify Mcl-1 as an important target for PKCδ-cat that can mediate its pro-apoptotic effects on mitochondria to amplify the apoptotic signaling induced by a wide range of apoptotic stimuli. Proteolytic cleavage and subsequent activation of protein kinase C (PKC) δ is required for apoptosis induced by a variety of genotoxic agent, including UV radiation. In addition, overexpression of the constitutively active PKCδ catalytic fragment (PKCδ-cat) is sufficient to trigger Bax activation, cytochrome c release, and apoptosis. While PKCδ is a key apoptotic effector, the downstream target(s) responsible for the mitochondrial apoptotic cascade are not known. We found that expression of the active PKCδ-cat in HaCaT cells triggers a reduction in the anti-apoptotic protein Mcl-1, similar to UV radiation. The down-regulation of Mcl-1 induced by PKCδ-cat was not at the mRNA level but was due to decreased protein half-life. Overexpression of Mcl-1 protected HaCaT cells from both UV and PKCδ-cat-induced apoptosis and blocked the release of cytochrome c from the mitochondria, indicating that Mcl-1 down-regulation was required for apoptosis signaling. Indeed, down-regulation of Mcl-1 with siRNA slightly increased the basal apoptotic rate of HaCaT cells and dramatically sensitized them to UV or PKCδ-cat-induced apoptosis. HaCaT cells with down-regulated Mcl-1 had higher activated Bax protein, as measured by Bax cross-linking, indicating that Mcl-1 down-regulation is sufficient for Bax activation. Finally, recombinant PKCδ could phosphorylate Mcl-1 in vitro, identifying Mcl-1 as a direct target for PKCδ. Overall our results identify Mcl-1 as an important target for PKCδ-cat that can mediate its pro-apoptotic effects on mitochondria to amplify the apoptotic signaling induced by a wide range of apoptotic stimuli. The induction of apoptosis in keratinocytes by UV radiation is an important protective mechanism from sunlight-induced skin tumors (1Grossman D. Kim P.J. Blanc-Brude O.P. Brash D.E. Tognin S. Marchisio P.C. Altieri D.C. J. Clin. Invest. 2001; 108: 991-999Crossref PubMed Scopus (197) Google Scholar). UV induces keratinocyte apoptosis primarily via the intrinsic or mitochondrial death effector pathway (2Sitailo L.A. Tibudan S.S. Denning M.F. J. Biol. Chem. 2002; 277: 19346-19352Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 3Takasawa R. Tanuma S. Apoptosis. 2003; 8: 291-299Crossref PubMed Scopus (21) Google Scholar). The UV mitochondrial apoptotic signaling pathway requires the early loss of Mcl-1, which allows the release of cytochrome c and other pro-apoptotic factors from the mitochondria. Release of these pro-apoptotic mediators triggers the activation of caspase-9 and ultimately the activation of effector caspases such as caspase-3 (2Sitailo L.A. Tibudan S.S. Denning M.F. J. Biol. Chem. 2002; 277: 19346-19352Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar). While caspase-3 cleaves a wide range of protein substrates, the widely expressed death substrate PKCδ 2The abbreviations used are: PKC, protein kinase C; Tam, 4-hydroxytamoxifen; PKCδ-cat, PKCδ catalytic fragment; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; ER, estrogen receptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Z, benzyloxycarbonyl. is critical as inhibition of PKCδ cleavage and/or activation protects from progressive caspase activation and apoptosis induced by a variety of stimuli, indicating positive feedback regulation between PKCδ cleavage and caspase activation (5Denning M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 6DeVries T.A. Neville M.C. Reyland M.E. EMBO J. 2002; 21: 6050-6060Crossref PubMed Scopus (209) Google Scholar, 7Fujii T. Garcia-Bermejo M.L. Bernabo J.L. Caamano J. Ohba M. Kuroki T. Li Yuspa S.H. 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Med. 1996; 184: 2399-2404Crossref PubMed Scopus (450) Google Scholar, 12Bharti A. Kraeft S.K. Gounder M. Pandey P. Jin S. Yuan Z.M. Lees-Miller S.P. Weichselbaum R. Weaver D. Chen L.B. Kufe D. Kharbanda S. Mol. Cell. Biol. 1998; 18: 6719-6728Crossref PubMed Scopus (202) Google Scholar, 13Sitailo L.A. Tibudan S.S. Denning M.F. J. Invest. Dermatol. 2004; 123: 434-443Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). PKCδ-cat has been localized to the mitochondria and the nucleus (6DeVries T.A. Neville M.C. Reyland M.E. EMBO J. 2002; 21: 6050-6060Crossref PubMed Scopus (209) Google Scholar, 8Denning M.F. Wang Y. Tibudan S. Nickoloff B.J. Qin J.Z. Cell Death Diff. 2002; 9: 40-52Crossref PubMed Scopus (171) Google Scholar) and induces apoptosis involving Bax activation, cytochrome c release, and caspase activation (8Denning M.F. Wang Y. Tibudan S. Nickoloff B.J. Qin J.Z. Cell Death Diff. 2002; 9: 40-52Crossref PubMed Scopus (171) Google Scholar, 13Sitailo L.A. Tibudan S.S. Denning M.F. J. Invest. Dermatol. 2004; 123: 434-443Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Although several PKCδ targets have been identified, including p73β (14Ren J. Datta R. Shioya H. Li Y. Oki E. Biedermann V. Bharti A. Kufe D. J. Biol. Chem. 2002; 277: 33758-33765Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), DNA-PK (12Bharti A. Kraeft S.K. Gounder M. Pandey P. Jin S. Yuan Z.M. Lees-Miller S.P. Weichselbaum R. Weaver D. Chen L.B. Kufe D. Kharbanda S. Mol. Cell. Biol. 1998; 18: 6719-6728Crossref PubMed Scopus (202) Google Scholar), Rad9 (15Yoshida K. Wang H.G. Miki Y. Kufe D. EMBO J. 2003; 22: 1431-1441Crossref PubMed Scopus (134) Google Scholar), phospholipid scramblases 1 and 3 (16Liu J. Chen J. Dai Q. Lee R.M. Cancer Res. 2003; 63: 1153-1156PubMed Google Scholar, 17Frasch S.C. Henson P.M. Kailey J.M. Richter D.A. Janes M.S. Fadok V.A. Bratton D.L. J. Biol. Chem. 2000; 275: 23065-23073Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 18He Y. Liu J. Durrant D. Yang H.S. Sweatman T. Lothstein L. Lee R.M. Cancer Res. 2005; 65: 10016-10023Crossref PubMed Scopus (21) Google Scholar), p38δ-ERK1/2 (19Efimova T. Broome A.M. Eckert R.L. Mol. Cell. Biol. 2004; 24: 8167-8183Crossref PubMed Scopus (68) Google Scholar), and nuclear lamin B (20Cross T. Griffiths G. Deacon E. Sallis R. Gough M. Watters D. Lord J.M. Oncogene. 2000; 19: 2331-2337Crossref PubMed Scopus (199) Google Scholar), it is unclear how these substrates can directly initiate the mitochondrial apoptotic cascade. During UV-induced apoptosis, the loss of Mcl-1 is required for the mitochondrial translocation of Bax, release of cytochrome c, and caspase activation (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar). The anti-apoptotic activity of Mcl-1 is due to its ability to bind and sequester pro-apoptotic Bcl-2 family proteins such as Bak and Bax (21Zhou P. Qian L. Kozopas K.M. Craig R.W. Blood. 1997; 89: 630-643Crossref PubMed Google Scholar, 22Cuconati A. Mukherjee C. Perez D. White E. Genes Dev. 2003; 17: 2922-2932Crossref PubMed Scopus Google Scholar, Chen L. G. A. E. J.M. Huang D.C. Genes Dev. 2005; 19: PubMed Scopus Google Scholar). Mcl-1 Bak and/or Bax are to and mediate in the mitochondria, the release of cytochrome c A. M.C. S. Cancer Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In the cell types to Mcl-1 has a and its are apoptosis at the and level D. M. J. G. J.M. 2003; PubMed Scopus Google Scholar). In the UV-induced loss of Mcl-1 is at the mRNA and in Mcl-1 protein was in cells to (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar, J.Z. P. J. L.A. Denning M.F. Nickoloff B.J. J. Cell 2004; PubMed Scopus Google Scholar). identify PKCδ targets that can initiate the mitochondrial apoptotic have on the Bcl-2 family of these proteins are by We found that PKCδ-cat triggers the and activation of Bax L.A. Tibudan S.S. Denning M.F. J. Invest. Dermatol. 2004; 123: 434-443Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In that expression of the active PKCδ-cat increased and of Mcl-1, and have Mcl-1 as a direct target by can the ability of PKCδ-cat to trigger mitochondrial apoptosis and identify Mcl-1 as a key in the positive feedback the apoptotic cascade of a wide variety of genotoxic Cell and keratinocyte cell HaCaT P. D. J. A. J. Cell Biol. PubMed Scopus Google Scholar), by Cancer was in with with UV from a of with the with by at for 1 at as (2Sitailo L.A. Tibudan S.S. Denning M.F. J. Biol. Chem. 2002; 277: 19346-19352Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 8Denning M.F. Wang Y. Tibudan S. Nickoloff B.J. Qin J.Z. Cell Death Diff. 2002; 9: 40-52Crossref PubMed Scopus (171) Google Scholar). siRNA cells with and with 1 for at 3 for the catalytic fragment of PKCδ was expressed from an as an estrogen protein that is activated by the cells with L.A. Tibudan S.S. Denning M.F. J. Invest. Dermatol. 2004; 123: 434-443Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The protein has basal PKCδ catalytic fragment and is used to it In the catalytic fragment of PKCδ (PKCδ-cat) in was used of the protein (8Denning M.F. Wang Y. Tibudan S. Nickoloff B.J. Qin J.Z. Cell Death Diff. 2002; 9: 40-52Crossref PubMed Scopus (171) Google Scholar). The higher expression the can in the of and cell apoptosis is L.A. Tibudan S.S. Denning M.F. J. Invest. Dermatol. 2004; 123: 434-443Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Mcl-1 was by W. Lee Cancer Y. Mol. Cancer 2003; Google Scholar) and was the of the (8Denning M.F. Wang Y. Tibudan S. Nickoloff B.J. Qin J.Z. Cell Death Diff. 2002; 9: 40-52Crossref PubMed Scopus (171) Google Scholar, 1996; PubMed Scopus Google Scholar). of the Bcl-2 was L.A. Tibudan S.S. Denning M.F. J. Invest. Dermatol. 2004; 123: 434-443Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). a the was in cells with from P. by and with 1 1996; PubMed Scopus Google Scholar). siRNA the to target the Mcl-1 J. L.A. A. H. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) was the of siRNA was which a target with to in the of the was by in cells as for was by as by the The cells on a to the Cell and cell by and cells in CHAPS, and for cytochrome c release by the with and them in 1 1 was to and the cells for at The cells by at for at and the by to with and with or on an used for Mcl-1 at at cytochrome c cell at and at Mcl-1 was in of on the PKCδ activated with or the active PKCδ-cat to or from cells and in the of and for at the active PKCδ-cat, recombinant PKCδ was with recombinant active caspase-3 as (8Denning M.F. Wang Y. Tibudan S. Nickoloff B.J. Qin J.Z. Cell Death Diff. 2002; 9: 40-52Crossref PubMed Scopus (171) Google Scholar). the kinase the with and in proteins on and to for by and of Mcl-1 was by cells with for to protein and cell to Mcl-1 by Mcl-1 protein by and Mcl-1 for that the of Mcl-1 in the and in was and the from the was from HaCaT cells The was used in the with the as in the of PKCδ-cat Mcl-1 is an anti-apoptotic Bcl-2 family protein that can bind to and sequester pro-apoptotic proteins such as Bax and Bak (21Zhou P. Qian L. Kozopas K.M. Craig R.W. Blood. 1997; 89: 630-643Crossref PubMed Google Scholar, 22Cuconati A. Mukherjee C. Perez D. White E. Genes Dev. 2003; 17: 2922-2932Crossref PubMed Scopus Google Scholar, Chen L. G. A. E. J.M. Huang D.C. Genes Dev. 2005; 19: PubMed Scopus Google Scholar). apoptosis induced by both PKCδ-cat and UV the activation of Bax L.A. Tibudan S.S. Denning M.F. J. Invest. Dermatol. 2004; 123: 434-443Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), Mcl-1 protein in HaCaT cells induced to apoptosis by PKCδ-cat expression or UV induce the activity of PKCδ-cat, HaCaT cells with a a protein protein has basal PKCδ activity that can activated by the cells with L.A. Tibudan S.S. Denning M.F. J. Invest. Dermatol. 2004; 123: 434-443Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). a cells with an and with The in that Mcl-1 to of in cells with the and Mcl-1 was of the was activated with with UV a reduction in Mcl-1 which was UV a similar down-regulation of Mcl-1 by and UV a Mcl-1 that primarily the of Mcl-1, Mcl-1 was to of in cells with Tam, with UV Mcl-1 to of Mcl-1 by PKCδ-cat to mRNA protein and mRNA are to have a and Mcl-1 can at the level of and protein (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar, 22Cuconati A. Mukherjee C. Perez D. White E. Genes Dev. 2003; 17: 2922-2932Crossref PubMed Scopus Google Scholar, D. M. J. G. J.M. 2003; PubMed Scopus Google Scholar). how PKCδ-cat Mcl-1 protein measured Mcl-1 protein by HaCaT cells with the protein for and Mcl-1 by in and Mcl-1 protein had a of in cells and activation of with the to UV the of Mcl-1 protein to but the kinase not Mcl-1 a measured the of Mcl-1 in cells which are to have Mcl-1 of that is not by UV radiation (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar). that the of Mcl-1 in cells is and is not decreased by indicating that cell for the regulation and of We Mcl-1 mRNA to PKCδ-cat or UV a which could to the Mcl-1 protein The in 3 that not an in Mcl-1 mRNA The mRNA of other Bcl-2 family and not UV a in mRNA for Bcl-2 family these results that PKCδ-cat triggers loss of Mcl-1 by the of Mcl-1 by the reduction in Mcl-1 by PKCδ-cat is required for apoptosis Mcl-1 in HaCaT cells by and apoptosis by PKCδ-cat or cells to UV radiation. the overexpression of the Mcl-1 in HaCaT In a was that in to the J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), the of Mcl-1 requires that PKCδ-cat expression or UV induced cell death and the overexpression of Mcl-1 protected from the cell that Mcl-1 expression protected from PKCδ-cat and UV induced apoptosis and as measured by of PKCδ-cat, not trigger apoptosis, that kinase activity is required for PKCδ-cat to induce apoptosis. The release of cytochrome c from the mitochondria is a key in the activation of caspase-9 and is regulation by anti-apoptotic Bcl-2 family proteins such as Mcl-1 (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar). Mcl-1 expression can the release of cytochrome c by PKCδ-cat L.A. Tibudan S.S. Denning M.F. J. Invest. Dermatol. 2004; 123: 434-443Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), cytochrome c by in the release of cytochrome c the and could blocked by Mcl-1 Mcl-1 expression blocked UV-induced release of cytochrome c from the mitochondria. that the caspase not cytochrome c release by PKCδ-cat, indicating that cytochrome c release is an these results that the loss of Mcl-1 by PKCδ-cat the release of cytochrome c and subsequent apoptotic signaling. of Mcl-1 with siRNA HaCaT to the reduction in Mcl-1 by PKCδ-cat is sufficient to trigger apoptosis, HaCaT cells with a a siRNA Mcl-1 and apoptosis. that the Mcl-1 siRNA Mcl-1 protein to of with an reduction to of 3 that level of inhibition is similar to the reduction in Mcl-1 by of In of apoptosis by that Mcl-1 siRNA a small but in apoptosis a in HaCaT cells the catalytic fragment of PKCδ or to UV the Mcl-1 siRNA a in apoptosis that was Mcl-1 can bind and sequester the pro-apoptotic protein Bax (21Zhou P. Qian L. Kozopas K.M. Craig R.W. Blood. 1997; 89: 630-643Crossref PubMed Google Scholar), and the induction of apoptosis by the catalytic fragment of PKCδ or UV the and of Bax L.A. Tibudan S.S. Denning M.F. J. Invest. Dermatol. 2004; 123: 434-443Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We down-regulation of Mcl-1 with siRNA was sufficient to The Bax in that the Mcl-1 siRNA induced the of Bax and increased the of Bax of the PKCδ-cat and UV increased the Bax and Bax and the Mcl-1 siRNA the and of these results that a reduction in Mcl-1 can dramatically cells to apoptotic stimuli. PKCδ our results that the catalytic fragment of PKCδ of Mcl-1, and of Mcl-1 by a variety of has been to its and activity A.M. Craig R.W. Oncogene. 2004; PubMed Scopus Google Scholar, S. K. T. Y. M. J. A. H. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (202) Google Scholar, A.M. Craig R.W. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), PKCδ can directly phosphorylate Mcl-1, or Bcl-2 as a was from with recombinant PKCδ in a kinase and the proteins by and that PKCδ a protein at in the from cell and in to the Mcl-1 in of Bcl-2 or Mcl-1 in the of PKCδ was indicating that the of Mcl-1 was and due to PKCδ. results with PKCδ-cat by caspase-3 cleavage not at with the Mcl-1 was not by PKCδ. results that Mcl-1 is a direct substrate for PKCδ is a widely expressed death substrate activated by caspase-3 cleavage in cells to apoptosis by a variety of In have Mcl-1, an anti-apoptotic Bcl-2 family as an important target for apoptosis by expression of PKCδ-cat the down-regulation of Mcl-1 by Mcl-1 protein and the loss of Mcl-1 was required for PKCδ-cat-induced cytochrome c release and apoptosis The loss of Mcl-1 is in UV-induced apoptosis (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar), and that the cleavage and activation of PKCδ required for the down-regulation of Mcl-1 by UV radiation A.M. Denning M.F. Cell Death Differ. 2005; 12: 224-232Crossref PubMed Scopus (59) Google Scholar). these by that PKCδ-cat is sufficient to Mcl-1 down-regulation and the down-regulation of Mcl-1 by The expression of Mcl-1 is types of regulation at both the and and to and apoptotic D. M. J. G. J.M. 2003; PubMed Scopus Google Scholar, R. Y. L. Oncogene. 2002; 21: PubMed Scopus Google Scholar, M. C. P. S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of PKCδ-cat in Mcl-1 UV loss of Mcl-1 with the apoptotic of HaCaT cells to UV radiation PKCδ-cat and UV decreased the of Mcl-1 protein and but not PKCδ-cat, Mcl-1 mRNA In UV has been to trigger the loss of Mcl-1 in cells by its and had of Mcl-1 protein (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar). our Mcl-1 that UV not Mcl-1 in cells indicating cell regulation for Mcl-1 the apoptotic regulation of Mcl-1 in HaCaT a keratinocyte cell is to UV skin as these cells UV in and the cells by UV radiation P. D. J. A. J. Cell Biol. PubMed Scopus Google Scholar, T.A. R. P. J. PubMed Scopus Google Scholar). In UV a reduction in Mcl-1 mRNA and protein with our results in HaCaT cells in 1 and 3 J.Z. P. J. L.A. Denning M.F. Nickoloff B.J. J. Cell 2004; PubMed Scopus Google Scholar). Mcl-1 is by and factors R. Y. Oncogene. 2002; 21: PubMed Scopus Google Scholar). The down-regulation of an anti-apoptotic protein such as Mcl-1 is not sufficient to induce apoptosis as including HaCaT Bcl-2 family (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar, V. Qin J.Z. Denning M.F. D. Nickoloff B.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Allen A. M.J. D. J. Dermatol. 2003; Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Mcl-1 and Mcl-1 has a for cells J.L. S. K. Genes Dev. 2000; PubMed Google Scholar, H. H. S. K. 2005; PubMed Scopus Google Scholar). a a siRNA Mcl-1, to Mcl-1 a level with that by PKCδ-cat 1 and We found a small but in basal apoptosis and Bax the down-regulation of Mcl-1 with siRNA dramatically sensitized HaCaT cells to PKCδ-cat and UV-induced Bax activation and apoptosis due to Mcl-1 loss by the of PKCδ-cat and Mcl-1 siRNA or the of apoptosis by reduction of Mcl-1 by siRNA not trigger apoptosis to the as the active which Mcl-1 our results that by PKCδ-cat with Mcl-1 loss to trigger apoptosis. In addition, reduction of Mcl-1 by siRNA in other cell types was not sufficient to induce of apoptosis or cell death (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar, 22Cuconati A. Mukherjee C. Perez D. White E. Genes Dev. 2003; 17: 2922-2932Crossref PubMed Scopus Google Scholar). We that Mcl-1 is directly by PKCδ-cat in kinase activity was required for increased of Mcl-1 and apoptosis is a mechanism for Mcl-1 for Furthermore, both PKCδ-cat and Mcl-1 are localized to the mitochondria, the active kinase and its substrate at the (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar, 8Denning M.F. Wang Y. Tibudan S. Nickoloff B.J. Qin J.Z. Cell Death Diff. 2002; 9: 40-52Crossref PubMed Scopus (171) Google Scholar). Mcl-1 can by several protein induced at a extracellular signal-regulated kinase in the of Mcl-1, and or a protein induced an of Mcl-1 A.M. Craig R.W. Oncogene. 2004; PubMed Scopus Google Scholar, A.M. Craig R.W. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, M. L. A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Mcl-1 was at and by kinase in to and was with of Mcl-1 and apoptosis S. K. T. Y. M. J. A. H. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Mcl-1 was by at in to or inhibition to increased and C. E. Mol. Cell. 21: Full Text Full Text PDF PubMed Scopus Google Scholar). Mcl-1 can by the and is to cleavage by caspases and B (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar, M. L. A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, C. E. Mol. Cell. 21: Full Text Full Text PDF PubMed Scopus Google Scholar, J. L.A. H. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The is and sufficient for Mcl-1 and Q. Gao W. Du F. Wang X. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). We found that the increased Mcl-1 protein not a for the pathway in Mcl-1 in HaCaT During apoptosis induced by UV and other stimuli, PKCδ cleavage and activation are and caspase activation, Mcl-1 loss is an early required for caspase activation (4Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (526) Google Scholar, M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 8Denning M.F. Wang Y. Tibudan S. Nickoloff B.J. Qin J.Z. Cell Death Diff. 2002; 9: 40-52Crossref PubMed Scopus (171) Google Scholar, 11Ghayur T. Hugunin M. Talanian R.V. Ratnofsky S. Quinlan C. Emoto Y. Pandey P. Datta R. Huang Y. Kharbanda S. Allen H. Kamen R. Wong W. Kufe D. J. Exp. Med. 1996; 184: 2399-2404Crossref PubMed Scopus (450) Google Scholar, K. K. T. T. Y. Y. M. S. S. J. 1997; PubMed Scopus Google Scholar, Y. Y. G. H. Kharbanda S. M. T. Wong Kamen R. Weichselbaum R. Kufe D. EMBO J. PubMed Scopus Google Scholar). it for PKCδ-cat to mediate the Mcl-1 down-regulation that activation, cytochrome c release, and caspase activation. the ability of PKCδ-cat to phosphorylate and the of Mcl-1 can PKCδ cleavage and activation are required for a apoptotic as a positive feedback between PKCδ cleavage and activation, Mcl-1 and caspase activation. Mcl-1 is Bcl-2 family in that it is to a variety of factors or J. G. J. Cell Biol. 2005; PubMed Scopus Google Scholar) and is required for early and in the and of cells J.L. S. K. Genes Dev. 2000; PubMed Google Scholar, H. H. S. K. 2005; PubMed Scopus Google Scholar). Mcl-1 has in cell indicating it has K. D. Liu J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The and cell of Mcl-1 are with and required to from signaling including the PKCδ apoptotic pathway our results identify Mcl-1 as a key in the positive feedback by PKCδ-cat in cells to apoptosis. We of the Cancer for with J. Nickoloff and Qin for We W. Lee Cancer for the Mcl-1
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