Inactivation of the Inhibitory κB Protein Kinase/Nuclear Factor κB Pathway by Par-4 Expression Potentiates Tumor Necrosis Factor α-induced Apoptosis
1999; Elsevier BV; Volume: 274; Issue: 28 Linguagem: Inglês
10.1074/jbc.274.28.19606
ISSN1083-351X
AutoresMarı́a T. Diaz-Meco, Marı́a-José Lallena, Alicia Monjas, Sonia Frutos, Jorge Moscat,
Tópico(s)Immune Response and Inflammation
ResumoPar-4 is a novel protein identified in cells undergoing apoptosis. The ability of Par-4 to promote apoptotic cell death is dependent on the binding and inactivation of the atypical protein kinases C (PKCs). This subfamily of kinases has been reported to control nuclear factor κB (NF-κB) through the regulation of the IκB kinase activity. NF-κB activation by tumor necrosis factor α (TNFα) provides a survival signal that impairs the TNFα-induced apoptotic response. We show here that expression of Par-4 inhibits the TNFα-induced nuclear translocation of p65 as well as the κB-dependent promoter activity. Interestingly, Par-4 expression blocks inhibitory κB protein (IκB) kinase activity, which leads to the inhibition of IκB phosphorylation and degradation, in a manner that is dependent on its ability to inhibit λ/ιPKC. Of potential functional relevance, the expression of Par-4 allows TNFα to induce apoptosis in NIH-3T3 cells. In addition, the down-regulation of Par-4 levels by oncogenic Ras sensitizes cells to TNFα-induced NF-κB activation. Par-4 is a novel protein identified in cells undergoing apoptosis. The ability of Par-4 to promote apoptotic cell death is dependent on the binding and inactivation of the atypical protein kinases C (PKCs). This subfamily of kinases has been reported to control nuclear factor κB (NF-κB) through the regulation of the IκB kinase activity. NF-κB activation by tumor necrosis factor α (TNFα) provides a survival signal that impairs the TNFα-induced apoptotic response. We show here that expression of Par-4 inhibits the TNFα-induced nuclear translocation of p65 as well as the κB-dependent promoter activity. Interestingly, Par-4 expression blocks inhibitory κB protein (IκB) kinase activity, which leads to the inhibition of IκB phosphorylation and degradation, in a manner that is dependent on its ability to inhibit λ/ιPKC. Of potential functional relevance, the expression of Par-4 allows TNFα to induce apoptosis in NIH-3T3 cells. In addition, the down-regulation of Par-4 levels by oncogenic Ras sensitizes cells to TNFα-induced NF-κB activation. protein kinase C atypical protein kinase C cytomegalovirus dithiothreitol extracellular signal regulated kinase Fas-associated death domain protein fetal calf serum FADD-like interleukin-1β-converting enzyme glutathioneS-transferase hemagglutinin inhibitor of apoptosis inhibitory κB protein IκB kinase tumor necrosis factor receptor-associated factor interleukin-1 polyacrylamide gel electrophoresis phosphate-buffered saline prostate-apoptosis-response 4 phenylmethylsulfonyl fluoride nuclear factor κB nuclear localization signal tumor necrosis factor α The atypical protein kinase C (PKC)1 subfamily of isozymes (aPKCs) has recently been the focus of considerable attention. It is composed of two members, ζPKC and λ/ιPKC (1Nishizuka Y. 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Boghaert E. Gillis D. Liu G. Nair P. Monning S. Collini P. Mattson M.P. Sukhatme V.P. Zimmer S.G. Wood D.P. McRoberts J.W. Shi Y. Rangnekar V.M. Mol. Cell. Biol. 1997; 17: 3823-3832Crossref PubMed Scopus (180) Google Scholar), as well as that Par-4 may be a mediator of neuronal apoptosis (28Guo Q. Fu W. Xie J. Luo H. Sells S.F. Geddes J.W. Bondada V. Rangnekar V.M. Mattson M.P. Nature Gen. 1998; 4: 957-962Google Scholar). Therefore, the study of the mechanisms of action of Par-4 and the atypical PKCs may be of great importance for the understanding of the signaling events involved in programmed cell death.Because the atypical PKCs regulate ERK (see above) and this kinase has been shown to be important in cell survival (3Berra E. Municio M.M. Sanz L. Frutos S. Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 1997; 17: 4346-4354Crossref PubMed Scopus (159) Google Scholar, 19Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar, 20Gardner A.M. Johnson G.L. J. Biol. Chem. 1996; 271: 14560-14566Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 21Parrizas M. Saltiel A.R. LeRoith D. J. Biol. Chem. 1997; 272: 154-161Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 22Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1332Crossref PubMed Scopus (5027) Google Scholar), the mechanisms whereby Par-4 induces apoptosis may at least in part involve this pathway. However, this may not be the only way for Par-4 to induce apoptosis. Thus, NF-κB that is a target of the atypical PKCs (4Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 11Sontag E. Sontag J.M. Garcia A. EMBO. J. 1997; 16: 5662-5671Crossref PubMed Scopus (177) Google Scholar,13Bjorkoy G. Overvatn A. Dı́az-Meco M.T. Moscat J. Johansen T. J. Biol. Chem. 1995; 270: 21299-21306Crossref PubMed Scopus (73) Google Scholar, 14Diaz-Meco M.T. Berra E. Municio M.M. Sanz L. Lozano J. Dominguez I. Diaz-Golpe V. Lain de Lera M.T. Alcamı́ J. Payá C.V. Arenzana-Seisdedos F. Virelizier J.L. Moscat J. Mol. Cell. Biol. 1993; 13: 4770-4775Crossref PubMed Google Scholar, 15Dominguez I. Sanz L. Arenzana-Seisdedos F. Diaz-Meco M.T. Virelizier J.L. Moscat J. Mol. Cell. Biol. 1993; 13: 1290-1295Crossref PubMed Google Scholar, 16Folgueira L. McElhinny J.A. Bren G.D. MacMorran W.S. Diaz-Meco M.T. Moscat J. Payá C.V. J. Virol. 1996; 70: 223-231Crossref PubMed Google Scholar, 17Lozano J. Berra E. Municio M.M. Diaz-Meco M.T. Dominguez I. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 19200-19202Abstract Full Text PDF PubMed Google Scholar) is a recognized anti-apoptotic molecule (18Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2925) Google Scholar, 23Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2440) Google Scholar, 24Wang C.-Y. Mayo M.W. Baldwin A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2499) Google Scholar, 25Wu M. Lee H. Bellas R.E. Schauer S.L. Arsura M. Katz D. Fitzgerald M.J. Rothstein T.L. Sherr D.H. Sonenshein G.E. EMBO J. 1996; 15: 4682-4690Crossref PubMed Scopus (553) Google Scholar). Therefore, it is conceivable that the impairment of the NF-κB pathway through the inhibition of the atypical PKCs may contribute to the pro-apoptotic actions of Par-4. TNFα, although it shares some components of the Fas signaling cascade, is an interesting example of the activation by a single cytokine of two different pathways with opposite effects. Thus, TNFα triggers the caspase-8 route to apoptosis and simultaneously activates NF-κB, which is an important survival signal (for a recent review, see Ref. 29Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar, and references therein). Therefore, TNFα induces apoptosis if the NF-κB pathway is inactivated (18Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2925) Google Scholar, 23Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2440) Google Scholar, 24Wang C.-Y. Mayo M.W. Baldwin A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2499) Google Scholar, 25Wu M. Lee H. Bellas R.E. Schauer S.L. Arsura M. Katz D. Fitzgerald M.J. Rothstein T.L. Sherr D.H. Sonenshein G.E. EMBO J. 1996; 15: 4682-4690Crossref PubMed Scopus (553) Google Scholar). The most classical form of NF-κB is a heterodimer of p50 and p65 (Rel A) (30Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4581) Google Scholar, 31Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5542) Google Scholar, 32Thanos D. 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Recently, two IκB kinases (IKKα and IKKβ) have been identified that phosphorylate residues 32 and 36 of IκBα, and whose activity is potently stimulated by TNF and IL-1 (36DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1896) Google Scholar, 37Mercurio F. Zhu B. Murray W. Shevchenko A. Bennett B.L. Li J.W. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1839) Google Scholar, 38Réginer C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 39Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1062) Google Scholar, 40Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1570) Google Scholar). Results from this laboratory demonstrated that the atypical PKCs are critical regulators of the IKK activity, which offers a mechanistic explanation to the proposed role of these PKCs on NF-κB activation (41Lallena M.J. Diaz-Meco M.T. Bren G. Payá C.V. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar).The studies reported here address the possibility that Par-4 potentiates TNF-induced apoptosis by inhibiting NF-κB through the blockade of the aPKC-IKK signaling cascade.DISCUSSIONThe molecules involved in signaling events controlling cell death and survival are important targets for novel therapies in cancer and inflammatory diseases. The recent discovery that the product ofpar-4, a gene induced in cells undergoing apoptosis (26Sells S.F. Wood D.P. Joshi-Barve S.S. Muthukumar S. Jacob R.J. Crist S.A. Humphreys S. Rangnekar V.M. Cell Growth & Diff. 1994; 5: 457-466PubMed Google Scholar), binds to and inhibits the atypical subfamily of PKCs (3Berra E. Municio M.M. Sanz L. Frutos S. Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 1997; 17: 4346-4354Crossref PubMed Scopus (159) Google Scholar, 43Diaz-Meco M.T. Municio M.M. Sanchez P. Lozano J. Moscat J. Mol. Cell. Biol. 1996; 16: 105-114Crossref PubMed Google Scholar), suggests that these kinases play important roles in the control of cell survival. Actually, the overexpression of wild-type ζPKC or λ/ιPKC (3Berra E. Municio M.M. Sanz L. Frutos S. Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 1997; 17: 4346-4354Crossref PubMed Scopus (159) Google Scholar, 6Murray N.R. Fields A.P. J. Biol. Chem. 1997; 272: 27521-27524Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 43Diaz-Meco M.T. Municio M.M. Sanchez P. Lozano J. Moscat J. Mol. Cell. Biol. 1996; 16: 105-114Crossref PubMed Google Scholar) protects cells from apoptosis induced by different genotoxic and stress insults. Therefore, the investigation of the mechanisms whereby the atypical PKCs regulate cell function is of potential great interest. In this regard, the ability of the atypical PKCs to regulate ERK (3Berra E. Municio M.M. Sanz L. Frutos S. Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 1997; 17: 4346-4354Crossref PubMed Scopus (159) Google Scholar, 46Berra E. Diaz-Meco M.T. Moscat J. J. Biol. Chem. 1998; 273: 10792-10797Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) seems important from the point of view of programmed cell death because the basal ERK activity has been shown to be critical for cell survival at least in some cell systems (22Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1332Crossref PubMed Scopus (5027) Google Scholar, 47Canman C.E. Kastan M.B. Nature. 1996; 384: 213-214Crossref PubMed Scopus (173) Google Scholar). Therefore, one of the mechanisms whereby the inhibition of the atypical PKCs by Par-4 induces apoptosis could at least in part involve the reduction of the ERK activity (3Berra E. Municio M.M. Sanz L. Frutos S. Diaz-Meco M.T. Moscat J. Mol. Cell. Biol. 1997; 17: 4346-4354Crossref PubMed Scopus (159) Google Scholar, 46Berra E. Diaz-Meco M.T. Moscat J. J. Biol. Chem. 1998; 273: 10792-10797Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). This pathway is antagonized by serum survival factors acting through the phosphoinositide 3-kinase/Akt cascade (46Berra E. Diaz-Meco M.T. Moscat J. J. Biol. Chem. 1998; 273: 10792-10797Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), unveiling an interesting cross-talk between different signaling events, which explains why Par-4 induces apoptosis more efficiently in cell cultures maintained under low serum conditions.There are other apoptotic pathways that are activated in a more direct way such as those of the Fas system (46Berra E. Diaz-Meco M.T. Moscat J. J. Biol. Chem. 1998; 273: 10792-10797Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Briefly, Fas binds to the adapter molecule FADD, which interacts with caspase-8 (also known as FLICE/MACH) triggering the whole caspase cascade (46Berra E. Diaz-Meco M.T. Moscat J. J. Biol. Chem. 1998; 273: 10792-10797Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Apoptosis induced by TNFα, although it shares some components of the Fas signaling cascade, is an interesting example of the activation by a single cytokine of two different pathways with opposite effects (30Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4581) Google Scholar, 31Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5542) Google Scholar, 32Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1216) Google Scholar). Thus, TNFα triggers the caspase-8 route to apoptosis and simultaneously activates NF-κB through independent although highly interconnected elements (29Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5112) Google Scholar). NF-κB is an important survival signal, and TNFα induces apoptosis if the NF-κB pathway is inactivated (30Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4581) Google Scholar, 31Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5542) Google Scholar, 32Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1216) Google Scholar). Because the atypical PKCs contribute to the activation of NF-κB by regulating the IKK enzymatic activity, in the study reported here, we sought to investigate if Par-4 may provide a signal that inactivates NF-κB, allowing TNFα to induce apoptosis. We show in this study that expression of Par-4 blocks NF-κB and IKK activation in a manner that is dependent on the inhibition of the atypical PKCs. These are very interesting observations because they provide independent evidence, other than those obtained with dominant negative mutants (11Sontag E. Sontag J.M. Garcia A. EMBO. J. 1997; 16: 5662-5671Crossref PubMed Scopus (177) Google Scholar, 13Bjorkoy G. Overvatn A. Dı́az-Meco M.T. Moscat J. Johansen T. J. Biol. Chem. 1995; 270: 21299-21306Crossref PubMed Scopus (73) Google Scholar, 14Diaz-Meco M.T. Berra E. Municio M.M. Sanz L. Lozano J. Dominguez I. Diaz-Golpe V. Lain de Lera M.T. Alcamı́ J. Payá C.V. Arenzana-Seisdedos F. Virelizier J.L. Moscat J. Mol. Cell. Biol. 1993; 13: 4770-4775Crossref PubMed Google Scholar, 17Lozano J. Berra E. Municio M.M. Diaz-Meco M.T. Dominguez I. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 19200-19202Abstract Full Text PDF PubMed Google Scholar, 43Diaz-Meco M.T. Municio M.M. Sanchez P. Lozano J. Moscat J. Mol. Cell. Biol. 1996; 16: 105-114Crossref PubMed Google Scholar), pseudosubstrate peptide inhibitors (15Dominguez I. Sanz L. Arenzana-Seisdedos F. Diaz-Meco M.T. Virelizier J.L. Moscat J. Mol. Cell. Biol. 1993; 13: 1290-1295Crossref PubMed Google Scholar), or antisense oligonucleotides (16Folgueira L. McElhinny J.A. Bren G.D. MacMorran W.S. Diaz-Meco M.T. Moscat J. Payá C.V. J. Virol. 1996; 70: 223-231Crossref PubMed Google Scholar), that the atypical PKCs are critically involved in the control of NF-κB and IKK activation (41Lallena M.J. Diaz-Meco M.T. Bren G. Payá C.V. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). Also of potential functional relevance is our finding that expression of Par-4, which by itself is a relatively weak inducer of apoptosis in the presence of high serum concentrations, allows TNFα to promote cell death, most probably by inhibiting NF-κB. Collectively, these results set an scenario in which the inhibition of the atypical PKCs by Par-4 induces apoptosis in resting cells by reducing ERK activity if the serum/phosphoinositide 3-kinase/Akt system is simultaneously inactivated (46Berra E. Diaz-Meco M.T. Moscat J. J. Biol. Chem. 1998; 273: 10792-10797Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). However, if the induction of apoptosis is an active process, as in the case of TNFα signaling, Par-4 contributes to cell death by inactivating the stimulation of the NF-κB survival branch. This suggests that potential inhibitors of the atypical PKCs could be used as sensitizers in cancer therapy. How NF-κB promotes cell survival is a matter of recent interest. Thus, Baldwin and co-workers (48Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2562) Google Scholar) have demonstrated that the induction of TRAF1, TRAF2, cIAP1, and cIAP2 may account for the anti-apoptotic effects of TNFα signaling. In addition, other molecules such as IEX-1L (49Wu M.X. Ao Z. Prasad K.V. Wu R. Schlossman S.F. Science. 1998; 281: 998-1001Crossref PubMed Google Scholar) have also been demonstrated to mediate NF-κB-induced protection. Future work in our laboratory will address the impact that Par-4 and the atypical PKCs have on the expression of these survival genes.A critical question that arises from this study is the pathophysiological implications of these findings. We have evidence that tumor transformation by the Ras oncogene provokes a profound depletion of Par-4 mRNA and protein levels (see above). This may be of potential functional relevance from the point of view of tumor progression because it may constitute a mechanism whereby cancer cells are able to survive and proliferate more efficiently than the normal cells. We demonstrate here that the down-regulation of Par-4 by oncogenic Ras has a measurable impact on TNFα signaling toward NF-κB activation. Thus, cells expressing oncogenic Ras display a much more efficient nuclear translocation of p65 in response to TNFα than control cells. This also seems to be dependent on the down-regulation of Par-4, because its ectopic expression dramatically inhibits Ras synergistic actions on NF-κB. Together, all these results will be in good agreement with a role of Par-4 during tumorigenic transformation. In this regard, recent data from Rangnekar and co-workers (50Cook J. Krishnan S. Ananth S. 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