JNK Phosphorylation and Activation of BAD Couples the Stress-activated Signaling Pathway to the Cell Death Machinery

Abstract

The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in mediating apoptosis in the developing and mature organism. The JNK signaling pathway is thought to induce apoptosis via transcription-dependent and transcription-independent mechanisms that remain to be elucidated. In this study, we report a novel mechanism by which the JNK signaling pathway directly activates a component of the cell death machinery. We have found that JNK catalyzes the phosphorylation of the BH3-only protein BAD at the distinct site of serine 128 in vitro. Activation of the JNK signaling pathway induces the BAD serine 128 phosphorylation in vivo, including in primary granule neurons of the developing rat cerebellum. The JNK-induced BAD serine 128 phosphorylation promotes the apoptotic effect of BAD in primary neurons by antagonizing the ability of growth factors to inhibit BAD-mediated apoptosis. These findings indicate that BAD is a novel substrate of JNK that links the stress-activated signaling pathway to the cell death machinery. The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in mediating apoptosis in the developing and mature organism. The JNK signaling pathway is thought to induce apoptosis via transcription-dependent and transcription-independent mechanisms that remain to be elucidated. In this study, we report a novel mechanism by which the JNK signaling pathway directly activates a component of the cell death machinery. We have found that JNK catalyzes the phosphorylation of the BH3-only protein BAD at the distinct site of serine 128 in vitro. Activation of the JNK signaling pathway induces the BAD serine 128 phosphorylation in vivo, including in primary granule neurons of the developing rat cerebellum. The JNK-induced BAD serine 128 phosphorylation promotes the apoptotic effect of BAD in primary neurons by antagonizing the ability of growth factors to inhibit BAD-mediated apoptosis. These findings indicate that BAD is a novel substrate of JNK that links the stress-activated signaling pathway to the cell death machinery. Regulation of cell death is critical to the normal development and homeostasis of multicellular organisms. In the developing nervous system, neurons are produced in excess, and naturally occurring neuronal cell death is thus critical in ensuring that neurons form the appropriate connections (1Lewin G.R. Barde Y.-A. Annu. Rev. Neurosci. 1996; 19: 289-317Google Scholar). In the mature nervous system, abnormally occurring neuronal cell death contributes to the pathogenesis of a variety of diseases including stroke, epilepsy, and neurodegenerative diseases (2Choi D.W. Curr. Opin. Neurobiol. 1996; 6: 667-672Google Scholar, 3Lowenstein D.H. Shimosaka S., So, Y.T. Simon R.P. Epilepsy Res. 1991; 10: 49-54Google Scholar, 4Cotman C.W. Neurobiol. Aging. 1998; 19: S29-S32Google Scholar). Not surprisingly, the mechanisms that underlie neuronal cell death have been the subject of intense interest. Studies of cell death in a variety of organisms have revealed that members of the Bcl-2 family of proteins act as gatekeepers of the cell death machinery (5Thompson C.B. Science. 1995; : 267Google Scholar, 6Reed J.C. Adv. Pharmacol. 1997; 41: 501Google Scholar, 7Chao D.T. Korsmeyer S.J. Annu. Rev. Immunol. 1998; 16: 395Google Scholar). Under conditions in which cells are destined to undergo programmed cell death (apoptosis), the proapoptotic multidomain Bcl-2 proteins, including Bax, induce the release of cytochromec from mitochondria leading to the activation of a caspase cascade that executes the cell death program (8Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Google Scholar, 9Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Google Scholar). The prosurvival multidomain Bcl-2 proteins, including Bcl-2 and Bcl-xl, interact with and inhibit the proapoptotic multidomain Bcl-2 proteins (10Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Google Scholar). The BH3-only subfamily of Bcl-2 proteins appears to play an important role in regulating the function of the multidomain Bcl-2 protein in response to signals from the cell surface or the cytoskeleton (11Huang D.C. Strasser A. Cell. 2000; 103: 839-842Google Scholar). The BH3-only protein BAD promotes cell death by interacting with and inhibiting the prosurvival multidomain Bcl-2 proteins (10Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Google Scholar). Survival factors suppress BAD-mediated apoptosis by inducing the phosphorylation of BAD at serine 136, serine 112, or both, culminating in the interaction and sequestration of phosphorylated BAD by members of the 14-3-3 family of proteins (12Zha J. Harada H. Yang E. Jocket J. Korsmeyer S.J. Cell. 1996; 87: 619-628Google Scholar). The protein kinases Akt, p21-activated kinase 1, and p70S6 kinase appear to mediate survival factor-induced phosphorylation of BAD serine 136 (13del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Google Scholar, 14Datta S.R. Dudek H. Tao X. Masters S., Fu, H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Google Scholar, 15Blume-Jensen P. Janknecht R. Hunter T. Curr. Biol. 1998; 8: 779-782Google Scholar, 16Schurmann A. Mooney A.F. Sanders L.C. Sells M.A. Wang H.G. Reed J.C. Bokoch G.M. Mol. Cell. Biol. 2000; 20: 453-461Google Scholar, 17Harada H. Andersen J.S. Mann M. Terada N. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9666-9670Google Scholar). On the other hand, the protein kinases Rsk, protein kinase A, and p21-activated kinase 1 are thought to mediate survival factor-induced phosphorylation of BAD serine 112 (16Schurmann A. Mooney A.F. Sanders L.C. Sells M.A. Wang H.G. Reed J.C. Bokoch G.M. Mol. Cell. Biol. 2000; 20: 453-461Google Scholar, 18Bonni A. Brunet A. West A. Datta S.R. Takasu M. Greenberg M.E. Science. 1999; 286: 1358-1362Google Scholar, 19Tan Y. Ruan H. Demeter M.R. Comb M.J. J. Biol. Chem. 1999; 274: 34859-34867Google Scholar, 20Shimamura A. Ballif B.A. Richards S.A. Blenis J. Curr. Biol. 2000; 10: 127-135Google Scholar, 21Harada H. Becknell B. Wilm M. Mann M. Huang L.J. Taylor S.S. Scott J.D. Korsmeyer S.J. Mol. Cell. 1999; 3: 413-422Google Scholar). Recently, we reported that BAD is also the target of an apoptotic signaling pathway (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). We found that the protein kinase Cdc2, previously established as controlling the transition of proliferating cells through mitosis, mediates apoptosis of newly generated postmitotic granule neurons of the developing rat cerebellum (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). Cdc2 triggers the phosphorylation of BAD at the novel site of serine 128, which lies near the growth factor-induced site of phosphorylation of serine 136 (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). The Cdc2-induced phosphorylation of BAD at serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins and thereby activates the apoptotic effect of BAD (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). A major question that is raised by these findings is whether BAD, in particular BAD serine 128, is the target of other protein kinases that propagate apoptotic signals. The c-Jun N-terminal kinase (JNK) 1The abbreviations used are: JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-related kinase kinase; MEKK1, MEK kinase 1; MKK3, MAPK kinase 3; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; IGF1, insulin-like growth factor 1; LC, liquid chromatography; MS, mass spectrometry; P6, postnatal day 6 rats; DIV, days in vitro . represents a group of mitogen-activated protein kinases (MAPKs) that mediate stress-induced cellular responses including programmed cell death (23Davis R.J. Cell. 2000; 103: 239-252Google Scholar). JNK is thought to induce apoptosis via transcription-dependent and transcription-independent mechanisms that remain incompletely understood. Studies of JNK-induced neuronal apoptosis suggest that JNK-induced phosphorylation of the transcription factor c-Jun and the consequent expression of c-Jun-induced genes mediate JNK-induced apoptosis (24Estus S. Zaks W.J. Freeman R.S. Gruda M. Bravo R. Johnson Jr., E.M. J. Cell Biol. 1994; 127: 1717-1727Google Scholar, 25Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1334Google Scholar, 26Watson A. Eilers A. Lallemand D. Kyriakis J. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 751-762Google Scholar, 27Yang D.D. Kuan C.Y. Whitmarsh A.J. Rincon M. Zheng T.S. Davis R.J. Rakic P. Flavell R.A. Nature. 1997; 389: 865-870Google Scholar, 28Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Google Scholar, 29Le-Niculescu H. Bonfoco E. Kasuya Y. Claret F.X. Green D.R. Karin M. Mol. Cell. Biol. 1999; 19: 751-763Google Scholar). In addition to transcription-dependent mechanisms, JNK promotes cell death by directly regulating the cell death machinery (30Tournier C. Hess P. Yang D.D., Xu, J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Google Scholar). JNK has been reported to catalyze the phosphorylation of Bcl-2 (31Fan M. Goodwin M., Vu, T. Brantley-Finley C. Gaarde W.A. Chambers T.C. J. Biol. Chem. 2000; 275: 29980-29985Google Scholar, 32Maundrell K. Antonsson B. Magnenat E. Camps M. Muda M. Chabert C. Gillieron C. Boschert U. Vial-Knecht E. Martinou J.C. Arkinstall S. J. Biol. Chem. 1997; 272: 25238-25242Google Scholar, 33Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Google Scholar, 34Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Google Scholar, 35Srivastava R.K., Mi, Q.-S. Hardwick J.M. Longo D.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3775-3780Google Scholar). The JNK-induced phosphorylation of Bcl-2 appears to suppress the prosurvival function of Bcl-2 (33Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Google Scholar, 35Srivastava R.K., Mi, Q.-S. Hardwick J.M. Longo D.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3775-3780Google Scholar). However, under certain circumstances, the phosphorylation of Bcl-2 at the site of the JNK-induced phosphorylation of Bcl-2 has been suggested to be required for the prosurvival function of Bcl-2 (34Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Google Scholar). In this study, we have characterized a novel mechanism by which JNK directly activates the cell death machinery in neurons. We have found that JNK catalyzes the phosphorylation of the BH3-only protein BADin vitro at the distinct site of serine 128. Activation of the JNK signaling pathway in vivo was found to induce the serine 128 phosphorylation of BAD, including endogenous BAD in primary cerebellar granule neurons. We also found that JNK signaling pathway-induced phosphorylation of BAD at serine 128 promotes the apoptotic effect of BAD in granule neurons by opposing growth factor-inhibition of BAD-mediated apoptosis. These findings define a new mechanism by which the stress-activated signaling pathway triggers apoptosis. In addition, together with our recent characterization of the Cdc2-BAD apoptotic pathway (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar), our results suggest that BAD serine 128 represents a common link for apoptosis-inducing protein kinases with the cell death machinery. The BAD plasmids (pCDNA3-BAD wild type and pCDNA3-BADS128A) (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar), activated MEKK1 (36See R.H. Calvo D. Shi Y. Kawa H. Luke M.P. Yuan Z. J. Biol. Chem. 2001; 276: 16310-16317Google Scholar), and activated MKK3 (Stratagene) have been described. The N-20 BAD (Santa Cruz Biotechnology), phospho112-BAD (New England Biolabs), phospho136-BAD (New England Biolabs), anti-hemagglutinin (Santa-Cruz Biotechnology), phospho-JNK (New England Biolabs), and phospho-p38MAPK (New England Biolabs) antibodies were purchased. The phospho128-BAD antibody was generated as described (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). The JNK inhibitor II (SP600125) and the p38MAPK inhibitor (SB203580) were from In vitro kinase were as described in the for the protein kinase In the in A, the kinase of Biotechnology), 1 of substrate and of in MOPS, 1 and was at for In the in 1 and was (New England Biolabs) was used at 1 and protein kinase A was used at induces the phosphorylation of BAD at serine 128 A, BAD was to an in vitro kinase with or and with the phospho128-BAD antibody or the N-20 BAD antibody The is a of BAD was to an in vitro kinase with or protein kinase A and with the phospho128-BAD the N-20 BAD the phospho112-BAD or the phospho136-BAD The is a of that was phosphorylated by JNK was with in were by liquid as described H. S.A. Cell. 2001; Scholar). were by and by an mass as described H. S.A. Cell. 2001; Scholar). were as described (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). proteins were by and to were for 1 in a and the were with primary antibody of for 1 at by the appropriate antibody at for were by cells were in were the day and cells to the The of was to of with 6 of in of to at for and 1 of was to the The was to the cells for cells were to In the in which were the and were used at and and were at the that was to were from granule neurons were as described (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google and a the in from postnatal day 6 that were in for or days were with of BAD or and of activated MEKK1 or the together with of a The was granule neurons in a for and were to and with were of and in in the or of for were and to a antibody to Cell survival and death were in neurons the of the and the as by the Cell were in a We the mechanism by which JNK directly the cell death machinery via the BH3-only protein In a recent (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google we found that Cdc2 directly the cell to the cell death machinery by inducing the phosphorylation of BAD at the novel site of serine 128. JNK, Cdc2, is a and serine 128 to the type of that be phosphorylated by JNK, we the that JNK the stress-induced signaling to the cell death machinery via the phosphorylation of BAD at serine 128. We whether JNK BAD in vitro. In an in vitro kinase we found that JNK the phosphorylation of a protein 1 whether JNK induces the phosphorylation of BAD at serine 128, we that was phosphorylated by JNK to mass and the BAD was from the with and to were that the of of the serine 128 to revealed that serine 128 this is modified by a group 1 These results indicate that JNK induces the phosphorylation of BAD at serine 128 in vitro. the that JNK BAD at serine 128, we a antibody that we raised to BAD BAD is phosphorylated at serine 128 (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). BAD protein was to an in vitro kinase with JNK or Cdc2 and with the phospho128-BAD or the N-20 BAD antibody that BAD of phosphorylation In these we found that JNK the phosphorylation of BAD at serine 128 as as Cdc2 We also whether JNK induces the phosphorylation of BAD at the of serine 112 and serine 136, that are phosphorylated by kinases survival signals (10Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Google Scholar). kinase kinase phosphorylated BADin vitro at serine 112 and serine 136 at serine 128 JNK the phosphorylation of BAD at serine 128 to BAD at serine 112 or serine 136 these results that JNK BAD at serine vitro. We whether activation of the JNK signaling pathway induces the phosphorylation of BAD at serine 128 We BAD or together with an expression the activated form of the kinase MEKK1 in the cell MEKK1 is the MAPK kinase kinase that activates the JNK signaling day we cell to the phospho128-BAD or the N-20 BAD We found that the expression of activated MEKK1 together with BAD the phosphorylation of BAD at serine 128 in cells The phospho128-BAD was in the phospho128-BAD antibody to a BAD in which serine 128 was with that was with activated MEKK1 in these cells these results suggest that activation of the JNK pathway triggers the phosphorylation of BAD serine 128 in We the mechanism by which MEKK1 induces the phosphorylation of BAD serine 128 in MEKK1 has been reported to induce protein kinase that in the activation of the JNK and p38MAPK (23Davis R.J. Cell. 2000; 103: 239-252Google Scholar). In activated MEKK1 induce the phosphorylation of JNK and p38MAPK that the activation of these protein kinases as by of of cells antibodies that the phosphorylated of p38MAPK or JNK the of p38MAPK and JNK in the phosphorylation of BAD serine 128, we the ability of a form of the p38MAPK MKK3 to induce the phosphorylation of BAD at serine 128. The expression of activated MKK3 in cells the phosphorylation of p38MAPK JNK We found that activated MKK3 to induce the BAD serine 128 phosphorylation that p38MAPK mediate BAD serine 128 In other we the ability of activated MEKK1 to induce BAD phosphorylation in cells that were with the p38MAPK inhibitor or the JNK inhibitor D.T. Xu, J.C. A. S. Y. S.S. D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: Scholar). We found that the p38MAPK inhibitor to the ability of activated MEKK1 to induce the BAD 128 phosphorylation the JNK inhibitor the BAD serine 128 phosphorylation these suggest that JNK mediates the ability of stress-induced signals to induce the phosphorylation of BAD at serine 128. whether the JNK signaling pathway induces the phosphorylation of endogenous BAD in vivo, we whether a to induce the JNK signaling pathway in cells (23Davis R.J. Cell. 2000; 103: 239-252Google Scholar), induces the phosphorylation of BAD at serine 128 in We found that of cells to the phosphorylation of endogenous BAD at serine 128 of JNK by the inhibitor phosphorylation of BAD at 128 These results suggest that JNK mediates the phosphorylation of BAD 128 in whether the JNK signaling pathway induces the phosphorylation of BAD that is we the phosphorylation of endogenous BAD in cerebellar granule neurons that were to We found that induces the phosphorylation of endogenous BAD at serine 128 in cerebellar granule neurons We also the effect of activated MEKK1 the phosphorylation of endogenous BAD in cerebellar granule neurons. We of cerebellar granule neurons that were with the activated MEKK1 expression or the expression We found that a of granule neurons with the phospho128-BAD However, phospho128-BAD was in a of granule neurons activated MEKK1 These results suggest that activation of the JNK signaling pathway induces the phosphorylation of endogenous BAD at serine 128 in granule neurons. the effect of the JNK-induced phosphorylation of BAD at serine 128 the function of BAD, we BAD or together with activated MEKK1 in primary cerebellar granule neurons. granule neurons were of survival factors or with a of that activates the for The expression of BAD apoptosis of cerebellar granule and activation BAD-mediated apoptosis The expression of activated MEKK1 together with BAD BAD-mediated apoptosis in survival granule cell However, activation to inhibit the apoptotic effect of BAD BAD was with activated MEKK1 These results suggest that activated MEKK1 induces a that growth factor of We the role of phosphorylation of BAD at serine 128 in of BAD-mediated apoptosis. We the effect of activated MEKK1 the apoptotic effect of a BAD in which serine 128 was with In to the ability of activated MEKK1 to the apoptotic effect of wild type BAD in granule activated MEKK1 to the ability of to inhibit the apoptotic effect of our results suggest that activation of the JNK signaling pathway induces the phosphorylation of BAD at serine 128 and thereby promotes the apoptotic effect of BAD by antagonizing growth factor-inhibition of In this study, we have characterized a novel mechanism by which the JNK signaling pathway promotes neuronal apoptosis. We have found that JNK catalyzes the phosphorylation of the BH3-only protein BAD at the distinct site of serine 128 in vitro. Activation of the JNK signaling pathway the phosphorylation of BAD serine vivo, including in primary cerebellar granule neurons. The JNK-induced phosphorylation of BAD serine 128 triggers the apoptotic effect of BAD by opposing growth factor-inhibition of BAD-mediated apoptosis. findings define a mechanism that directly links the JNK signaling pathway to the cell death machinery. We have BAD serine 128 as a target of Cdc2-induced apoptosis in cerebellar granule neurons (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). The that the JNK signaling pathway also induces BAD-mediated apoptosis via the phosphorylation of BAD serine 128 that BAD serine 128 represents a common target that links kinases apoptotic signals with the cell death machinery. Cdc2 induces the phosphorylation of BAD serine 128 in newly generated granule neurons that are from postnatal day 6 and in vitro for days (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). we found that activation of the JNK signaling pathway induces BAD-mediated apoptosis in mature granule neurons These the that the developing cerebellar granule kinases target BAD serine 128 at distinct of granule The of BAD serine 128 as a site phosphorylated by JNK represents the report of a BH3-only protein that is by a of the stress-activated protein kinase be important in the to whether the JNK signaling pathway other members of the BH3-only family of A link JNK and the cell death machinery has been suggested by of JNK-induced phosphorylation of Bcl-2 (31Fan M. Goodwin M., Vu, T. Brantley-Finley C. Gaarde W.A. Chambers T.C. J. Biol. Chem. 2000; 275: 29980-29985Google Scholar, 32Maundrell K. Antonsson B. Magnenat E. Camps M. Muda M. Chabert C. Gillieron C. Boschert U. Vial-Knecht E. Martinou J.C. Arkinstall S. J. Biol. Chem. 1997; 272: 25238-25242Google Scholar, 33Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Google Scholar, 34Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Google Scholar, 35Srivastava R.K., Mi, Q.-S. Hardwick J.M. Longo D.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3775-3780Google Scholar). The JNK-induced phosphorylation of Bcl-2 appears to suppress the prosurvival function of Bcl-2 (33Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Google Scholar, 35Srivastava R.K., Mi, Q.-S. Hardwick J.M. Longo D.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3775-3780Google Scholar). In to the effect of phosphorylation Bcl-2 family proteins the function of these proteins the serine 128 phosphorylation of BAD, whether is by Cdc2 or by JNK, activates the apoptotic function of We have found that the JNK-induced serine 128 phosphorylation of BAD promotes the apoptotic effect of BAD by antagonizing growth factor-inhibition of BAD-mediated apoptosis. The serine 128 phosphorylation of BAD growth factor of BAD by inhibiting the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins (22Konishi Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). The that JNK-induced phosphorylation of BAD at serine 128 growth factor of BAD the that BAD is a of for survival and apoptotic signals. kinases including Akt, Rsk, p21-activated kinase 1, p70S6 and protein kinase A induce the phosphorylation of BAD at serine 112 or serine 136 leading to the sequestration of BAD with 14-3-3 proteins (12Zha J. Harada H. Yang E. Jocket J. Korsmeyer S.J. Cell. 1996; 87: 619-628Google Scholar, Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Google Scholar, 14Datta S.R. Dudek H. Tao X. Masters S., Fu, H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Google Scholar, 15Blume-Jensen P. Janknecht R. Hunter T. Curr. Biol. 1998; 8: 779-782Google Scholar, 16Schurmann A. Mooney A.F. Sanders L.C. Sells M.A. Wang H.G. Reed J.C. Bokoch G.M. Mol. Cell. Biol. 2000; 20: 453-461Google Scholar, 17Harada H. Andersen J.S. Mann M. Terada N. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9666-9670Google Scholar, 18Bonni A. Brunet A. West A. Datta S.R. Takasu M. Greenberg M.E. Science. 1999; 286: 1358-1362Google Scholar, 19Tan Y. Ruan H. Demeter M.R. Comb M.J. J. Biol. Chem. 1999; 274: 34859-34867Google Scholar, 20Shimamura A. Ballif B.A. Richards S.A. Blenis J. Curr. Biol. 2000; 10: 127-135Google Scholar, 21Harada H. Becknell B. Wilm M. Mann M. Huang L.J. Taylor S.S. Scott J.D. Korsmeyer S.J. Mol. Cell. 1999; 3: 413-422Google Scholar), the apoptosis-inducing kinases JNK and Cdc2 induce the phosphorylation of BAD at serine 128 and Y. Lehtinen M. Donovan N. Bonni A. Mol. Cell. 2002; 9: 1005-1016Google Scholar). BAD as a protein that prosurvival and proapoptotic signals with the effect to the of the cell to or undergo apoptosis. JNK-induced apoptosis is thought to play an important role in the development of the nervous C.Y. Yang D.D. D.R. Davis R.J. Rakic P. Flavell R.A. 1999; Scholar, K. K. L. Karin M. Wagner E.F. Dev. 1999; Scholar). In be important to whether the JNK-induced phosphorylation of BAD plays a role in neuronal cell death normal apoptosis is also thought to to neuronal cell to including that in epilepsy, and neurodegenerative diseases of the (2Choi D.W. Curr. Opin. Neurobiol. 1996; 6: 667-672Google Scholar, 3Lowenstein D.H. Shimosaka S., So, Y.T. Simon R.P. Epilepsy Res. 1991; 10: 49-54Google Scholar, 4Cotman C.W. Neurobiol. Aging. 1998; 19: S29-S32Google Scholar). be to whether JNK-induced BAD serine 128 phosphorylation contributes to neuronal cell in these We S. for of BAD by mass and Y. Shi for the activated MEKK1