Regulation of Bad Phosphorylation and Association with Bcl-xL by the MAPK/Erk Kinase

Phosphorylation of the Bcl-2 family protein Bad may represent an important bridge between survival signaling by growth factor receptors and the prevention of apoptosis. Bad phosphorylation was examined following cytokine stimulation, which revealed phosphorylation on a critical residue, serine 112, in a MEK-dependent manner. Furthermore, Bad phosphorylation also increased on several sites distinct from serine 112 but could not be detected on serine 136, previously thought to be a protein kinase B/Akt-targeted residue. Serine 112 phosphorylation was shown to be absolutely required for dissociation of Bad from Bcl-xL. These results demonstrate for the first time in mammalian cells the involvement of the Ras-MAPK pathway in the phosphorylation of Bad and the regulation of its function. Phosphorylation of the Bcl-2 family protein Bad may represent an important bridge between survival signaling by growth factor receptors and the prevention of apoptosis. Bad phosphorylation was examined following cytokine stimulation, which revealed phosphorylation on a critical residue, serine 112, in a MEK-dependent manner. Furthermore, Bad phosphorylation also increased on several sites distinct from serine 112 but could not be detected on serine 136, previously thought to be a protein kinase B/Akt-targeted residue. Serine 112 phosphorylation was shown to be absolutely required for dissociation of Bad from Bcl-xL. These results demonstrate for the first time in mammalian cells the involvement of the Ras-MAPK pathway in the phosphorylation of Bad and the regulation of its function. cysteine-aspartate specific proteases Bcl-xL-associated death inducer extracellular regulated kinase granulocyte-macrophage colony-stimulating factor interleukin mitogen-activated protein kinase MAPK/Erk kinase protein kinase B phosphatidylinositol 3′-OH kinase polyacrylamide gel electrophoresis cAMP-dependent protein kinase Apoptosis is a universal phenomenon whereby a damaged cell, a virally infected cell, or a cell that is no longer receiving a specific extracellular survival signal destroys itself. The process of apoptosis involves various discreet levels, ultimately leading to the activation of cysteine-aspartate specific proteases (caspases)1 (1Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6159) Google Scholar). An intensely studied family of proteins involved upstream of caspase activation share homology with Bcl-2, one of the first apoptosis regulating proteins identified as an oncogene. These proteins may function as a checkpoint for life and death decisions (2Chao D.T. Korsmeyer S.J. Annu. Rev. Immunol. 1998; 16: 395-419Crossref PubMed Scopus (1517) Google Scholar). Cytokines prevent the onset of apoptosis and caspase activation by activating both protein and lipid kinase cascades, which may converge on the Bcl-2 family. In this way, the cytoprotective actions of these signaling pathways may involve the up-regulation of death antagonists, as well as the post-translational modification of Bcl-2 family proteins, which alters their role in propagating the apoptotic signal. Cytokine receptors of the hemopoietic superfamily activate a number of well studied signaling pathways following tyrosine kinase activation, including p21ras and PI3K and their downstream targets. PI3K-generated 3′-phosphoinositides mediate activation of a family of phospholipid-dependent kinases, which includes PDK1 (3Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar) and an incompletely characterized PDK2 (4Andjelkovic M. Maira S.M. Cron P. Parker P.J. Hemmings B.A. Mol. Cell. Biol. 1999; 19: 5061-5072Crossref PubMed Scopus (101) Google Scholar, 5Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 6Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. . Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (947) Google Scholar). PDK1 is an important activation loop kinase and is responsible for the phosphorylation of PKB/c-Akt, p70 S6 kinase, and protein kinase Cδ (reviewed in Ref. 7Belham C. Wu S. Avruch J. Curr. Biol. 1999; 9: 93-96Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Ras-activated Raf operates in a parallel pathway, by phosphorylating and activating downstream targets including the dual specificity kinase MAPK/Erk kinase (MEK; also termed MKK)-1 and 2 (8Crews C.M. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8205-8209Crossref PubMed Scopus (152) Google Scholar, 9Gomez N. Cohen P. Nature. 1991; 353: 170-173Crossref PubMed Scopus (419) Google Scholar, 10Kyriakis J.M. App H. Zhang X.F. Banerjee P. Brautigan D.L. Rapp U.R. Avruch J. Nature. 1992; 358: 417-421Crossref PubMed Scopus (977) Google Scholar, 11Zheng C.F. Guan K.L. J. Biol. Chem. 1993; 268: 11435-11439Abstract Full Text PDF PubMed Google Scholar). The ability of Ras to induce transformation is believed in part to involve sustained activation of Raf, MEK, and MAPK activity (12Thomas S.M. DeMarco M. D'Arcangelo G. Halegoua S. Brugge J.S. Cell. 1992; 68: 1031-1040Abstract Full Text PDF PubMed Scopus (503) Google Scholar, 13Leevers S.J. Marshall C.J. EMBO J. 1992; 11: 569-574Crossref PubMed Scopus (383) Google Scholar, 14Lange-Carter C.A. Johnson G.L. Science. 1994; 265: 1458-1461Crossref PubMed Scopus (296) Google Scholar). MEK has a very narrow substrate specificity, restricted to the threonine and tyrosine residues of p44erk1 and p42erk2. Phosphorylation by MEK of this family of MAPKs dramatically increases their activity, resulting in phosphorylation of further downstream targets including p90rsk (15Wood K.W. Sarnecki C. Roberts T.M. Blenis J. Cell. 1992; 68: 1041-1050Abstract Full Text PDF PubMed Scopus (662) Google Scholar), as well as translocation to the nucleus where they phosphorylate transcription factors involved in immediate/early gene expression (reviewed in Refs. 16Su B. Karin M. Curr. Opin. Immunol. 1996; 8: 402-411Crossref PubMed Scopus (719) Google Scholar and 17Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1163) Google Scholar). Two recently developed inhibitors of MEK1, PD98059 (18Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2593) Google Scholar) and MEK1/2, U0126 (19Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2751) Google Scholar), have been extensively characterized, and shown to be highly selective in their inhibition of the MAPK pathway. One Bcl-2 family member shown recently to be a target of cytokine-stimulated signaling is Bad (20Yang E. Zha J. Jockel J. Boise L.H. Thompson C.B. Korsmeyer S.J. Cell. 1995; 80: 285-291Abstract Full Text PDF PubMed Scopus (1894) Google Scholar). Induction of Bad phosphorylation on multiple serine residues influences its subcellular distribution, from an association with Bcl-xL at the mitochondria, to a cytosolic location, associated with 14-3-3 (21Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2254) Google Scholar). The association of Bad with Bcl-xL is mediated through dimerization of conserved BH3 domains (22Zha J. Harada H. Osipov K. Jockel J. Waksman G. Korsmeyer S.J. J. Biol. Chem. 1997; 272: 24101-24104Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 23Kelekar A. Chang B.S. Harlan J.E Fesik S.W. Thompson C.B. Mol. Cell. Biol. 1997; 17: 7040-7046Crossref PubMed Scopus (270) Google Scholar), characteristic for other BH3 domain containing proteins. Phosphorylation of residues in proximity to the BH3 domain of Bad may alter the affinity of Bad for Bcl-xL, promoting dissociation. This may relieve Bcl-xL of some negative influence, allowing protection of cells from apoptosis. The specific residues on Bad phosphorylated in response to survival factors are serine 112 and serine 136 (21Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2254) Google Scholar). Mutation of either of these residues to alanine potentiates death following transient transfection with Bad, suggesting that both are critical in the disruption of Bad-Bcl-xL heterodimers. Dephosphorylation of Bad by specific phosphatases, such as calcineurin, reverses the cycle back to an unphosphorylated, death-promoting agonist during Ca+2-induced apoptosis (24Wang H.G. Pathan N. Ethell I.M. Krajewski S. Yamaguchi Y. Shibasaki F. McKeon F. Bobo T. Franke T.F. Reed J.C. Science. 1999; 284: 339-343Crossref PubMed Scopus (963) Google Scholar). The phosphorylation of Bad occurs by unknown, cytokine-stimulated pathways. Besides cytokines, many other receptors for growth and survival factors can activate pathways leading to phosphorylation of Bad, including the receptors for epidermal growth factor, platelet-derived growth factor, insulin-like growth factor-1, and Stem cell factor (21Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2254) Google Scholar, 25del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1986) Google Scholar, 26Datta S.R. Dudek H. Tao X. Masters S. Fu H.A. Gotoh Y. Greenburg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4946) Google Scholar, 27Scheid M.P. Duronio V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7439-7444Crossref PubMed Scopus (276) Google Scholar). In this respect, oncogenes involved in the signal transduction of each of these receptors may bypass the requirement for extracellular stimuli to maintain protection from apoptosis, in part by increasing Bad phosphorylation. Therefore, the detailing of specific signaling pathways involved in the regulation of Bad is critical in our understanding of oncogenesis. In our previous report, we showed that the mobility shift of Bad, indicative of phosphorylation, could be blocked by a MEK inhibitor, PD 98059 (27Scheid M.P. Duronio V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7439-7444Crossref PubMed Scopus (276) Google Scholar). This lead us to perform a careful analysis of Bad phosphorylation following MEK inhibition. Here we show that Bad is targeted on serine 112 by a MEK-dependent pathway, whereas phosphorylation of two other residues occurs in a MEK-independent manner. Serine 112 phosphorylation was found to be required for dissociation of Bad from Bcl-xL. These results demonstrate for the first time the involvement of a Ras-controlled signaling pathway leading to the phosphorylation and inactivation of a pro-apoptotic Bcl-2 family member in mammalian cells. This ultimately may lead to better to the apoptosis during such as to Bad and Bcl-xL from and and Bad and from PD98059 was from U0126 was from and from was a from James of and at and was at These of shown previously to induce increases in tyrosine phosphorylation. containing and a from of from or cells at and in a in with and as a of of cytokine by in in which of the was with but cells and in for at to in the the mobility shift of Bad, or cells various with and on for and to 2 of was and the at Bad with of protein at for with and in by for in a polyacrylamide gel with a and to blocked with for and with from or from at was detected with and to the These and to Bad with Bcl-xL, with the following In these 2 of for at and for an with of protein at for and by as cells of cytokine as in and in with with at for 2 Bad was from as on a gel with an of and and Bad was detected by and by either from the gel by or by a Bad from various and as was from the gel and with in at by and the was to and with of and in of electrophoresis was on at for The in the in and by In two with was but a was also these with analysis was by the and with of to for The was and the with of was on of and at for detected by In each of the a of a of and was also that was by the phosphorylation of Bad in we first Bad from cell with a kinase a of Two of the with containing phosphorylated and cells with with cells an in phosphorylation of two residues distinct from either or as well as the of a to the with we to phosphorylation following results cells with or Stem cell factor not The including the one containing and to which serine phosphorylation previous have shown that Bad phosphorylation by the hemopoietic and is MEK activation (27Scheid M.P. Duronio V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7439-7444Crossref PubMed Scopus (276) Google Scholar). This of the mobility shift of Bad on a phenomenon to phosphorylated proteins. further the phosphorylation of Bad downstream of cytokine Bad from cells was examined following with the MEK PD 98059 with this the ability of or to induce Bad phosphorylation with but not phosphorylation. further for MEK as an upstream of Bad phosphorylation, a of MEK was of cells with U0126 (19Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2751) Google Scholar) also the phosphorylation of Bad by also the of with to activate Bad can be phosphorylated in by (21Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2254) Google Scholar). have previously shown that a in in as well as promoting phosphorylation on in a M.P. P.R. Duronio V. 1999; PubMed Google Scholar). these increased Bad phosphorylation by with In to stimulation, inhibition of MEK not the in Bad phosphorylation by MEK-dependent Bad phosphorylation was also in cell 2 In these both PD 98059 and U0126 the phosphorylation of Bad, which the in phosphorylation of p42erk2. which sites of phosphorylation downstream of with and with by with Bad was by and by In the of MEK activity, the phosphorylation was whereas the other two not The has one phosphorylation and a and to demonstrate that was MEK-dependent phosphorylation, we a with PD 98059 or U0126 and with revealed a in Bad to B was on this from the and that the a not these results that the phosphorylated downstream of MEK was and that MEK activation by is for the role of MEK in Bad phosphorylation, we MEK-dependent phosphorylation of was important for Bad-Bcl-xL apoptosis, Bad may a role in promoting the death signal by Bcl-xL (21Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2254) Google Scholar, 23Kelekar A. Chang B.S. Harlan J.E Fesik S.W. Thompson C.B. Mol. Cell. Biol. 1997; 17: 7040-7046Crossref PubMed Scopus (270) Google Scholar). In our a of Bad with Bcl-xL from cells that been of cytokine for the of these two proteins during the of apoptosis with for or in an and of Bad-Bcl-xL with dissociation. phosphorylation was for this cells with for in the of the activation of MEK Bad-Bcl-xL association to to cells In as well as hemopoietic factor, which has been shown previously to activate (27Scheid M.P. Duronio V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7439-7444Crossref PubMed Scopus (276) Google Scholar) but not Ras or MAPK Duronio V. J.S. J. Immunol. 1992; Google Scholar). with a requirement for activation, was to dissociation between Bad and Bcl-xL In each of these we examined the activation of both MAPK and and in a MEK-independent with our that these activate (27Scheid M.P. 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