p90RSK Blocks Bad-mediated Cell Death via a Protein Kinase C-dependent Pathway

Although activation of protein kinase C (PKC) is known to promote cell survival and protect against cell death, the PKC targets and pathways that serve this function have remained elusive. Here we demonstrate that two potent activators of PKC, 12-O-tetradecanoylphorbol-13-acetate and bryostatin, both stimulate phosphorylation of Bad at Ser112, a site known to regulate apoptotic cell death by interleukin-3. PKC inhibitors but not PI 3-kinase/Akt inhibitors block 12-O-tetradecanoylphorbol-13-acetate-stimulated Bad phosphorylation. PKC isoforms tested in vitro were unable to phosphorylate Bad at Ser112, suggesting that PKC acts indirectly to activate a downstream Bad kinase. p90RSK and family members RSK-2 and RSK-3 are activated by phorbol ester and phosphorylate Bad at Ser112 both in vitro andin vivo. p90RSK stimulates binding of Bad to 14-3-3 and blocks Bad-mediated cell death in a Ser112-dependent manner. These findings suggest that p90RSK can function in a PKC-dependent pathway to promote cell survival via phosphorylation and inactivation of Bad-mediated cell death. Although activation of protein kinase C (PKC) is known to promote cell survival and protect against cell death, the PKC targets and pathways that serve this function have remained elusive. Here we demonstrate that two potent activators of PKC, 12-O-tetradecanoylphorbol-13-acetate and bryostatin, both stimulate phosphorylation of Bad at Ser112, a site known to regulate apoptotic cell death by interleukin-3. PKC inhibitors but not PI 3-kinase/Akt inhibitors block 12-O-tetradecanoylphorbol-13-acetate-stimulated Bad phosphorylation. PKC isoforms tested in vitro were unable to phosphorylate Bad at Ser112, suggesting that PKC acts indirectly to activate a downstream Bad kinase. p90RSK and family members RSK-2 and RSK-3 are activated by phorbol ester and phosphorylate Bad at Ser112 both in vitro andin vivo. p90RSK stimulates binding of Bad to 14-3-3 and blocks Bad-mediated cell death in a Ser112-dependent manner. These findings suggest that p90RSK can function in a PKC-dependent pathway to promote cell survival via phosphorylation and inactivation of Bad-mediated cell death. interleukin 12-O-tetradecanoylphorbol-13-acetate protein kinase C cAMP-response element-binding protein mitogen-activated protein mitogen-activated protein kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase glutathione S-transferase maltose-binding protein 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside terminal deoxynucleotidyl transferase-mediated x-dUTP nick end labeling Survival factors prevent cells from undergoing cell death or apoptosis by inhibiting the execution of the cell death program. Recently, the convergence of signaling pathways activated by IL-31 and other survival factors with the cell death machinery has been traced to their ability to stimulate phosphatidylinositol (PI) 3-kinase (1Yao R. Cooper G.M. 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Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the of p90RSK in regulating cell survival has not been Here we demonstrate that of and protein kinase C cells against Bad-mediated cell death and also stimulates phosphorylation of Bad at of PKC block Bad suggesting that PKC phosphorylate in we have been unable to in vitro phosphorylation of Bad by and PKC suggesting that PKC a downstream Bad kinase. demonstrate that TPA p90RSK in a PKC-dependent and that in vitro p90RSK can phosphorylate Bad at of p90RSK in cells stimulates Bad phosphorylation at Furthermore, treatment of cells with TPA or overexpression of p90RSK Bad-mediated cell death. These suggest that the of phorbol ester and PKC cell survival act via a pathway a phosphorylation of Bad at suggest that the pathway may in regulating cell death at the Bcl-xL checkpoint via phosphorylation and inactivation of and cell were from Phosphorylation Bad and and Bad were from was from and were from protein kinase and protein kinase A were from and was by and PKC inhibitors and and were from bryostatin was from was from and in cell death the was from was from cells were in with cells were in with was by of the of Bad from a and sites of of the the of the S. S. Res. PubMed Scopus Google Scholar). and were by from and from a protein and were by and was by and and were by and were by with to of was by both the and the of protein the were by to with the was a of the were at and of cells was as Y. J. A. S. P. M.J. J. 1996; PubMed Scopus Google Scholar) with of as in the of was at with cells were and in and were with TPA the indicated to were with from cells with Bad and with or TPA and in cell were at were with cell and with and were with were by cells in of from cells were by and to were with in at with at with to at and the p90RSK was by of p90RSK from cell cells were with inhibitors and with TPA were in cell from cells were with and with protein A at by were with cell and with kinase were in of kinase with and of protein or and Bad and at Kinase were with and Bad phosphorylation was by and Bad from to were with a to the of were by at or were with the Ser112 to Ser136 to or the both Ser112 and Ser136 were to were by with and the protein the as by J. T.C. J. Protein Sci. 1998; PubMed Scopus Google Scholar). the were away from by binding to cells were with or cells were and with with in and with in cells were with terminal deoxynucleotidyl and at with with with and with and cells were in at with cells were with in cells were a Phosphorylation A.J. Chen J. G. J. P. 1991; PubMed Scopus Google Scholar) against Ser112 and Ser136 of Bad were as were shown to be the by and by and protein not site a of was by to the C of Bad or Bad at Ser112, Ser136, or both sites and J. T.C. J. Protein Sci. 1998; PubMed Scopus Google Scholar). shown in Ser112 protein at Ser112 or Ser112 and Ser136, whereas Ser136 protein at Ser136 or Ser112 and These demonstrate a of both to their in vivo. cells were with or or were and against Bad Ser112 or Bad of cells with TPA the phosphorylation of at of Ser112 to the of Ser136 to not block Ser112 the site of the Ser112 and were at as Bad other agents that also stimulate phosphorylation of Bad at Ser112 the Bad Ser112 of cells with TPA or growth factor the of phosphorylation at and growth factor also phosphorylation at Ser112 TPA a phosphorylation of of that remained treatment of PKC, as well as TPA Bad Ser112 phosphorylation in cells phorbol are known to activate and PKC isoforms as well as other pathways the kinase we inhibitors to the of pathways Bad phosphorylation at of cells with or two PI 3-kinase wortmannin and Bad phosphorylation. of and the or Bad phosphorylation at Ser112 the ability of PKC inhibitors to block Bad phosphorylation A and (also known as or is a potent of and Bad phosphorylation at Furthermore, phosphorylation at Ser112 to suggesting Bad phosphorylation. of cells with a potent of and also Bad phosphorylation. treatment with or Ser112 phosphorylation of were has been to inhibit PKC isoforms and but to have the isoforms and G. H. G. H. C. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). potent by and the of to block signaling to Bad suggest a the PKC to the protein kinase phosphorylating Bad at both TPA and bryostatin induce Bad phosphorylation and PKC inhibitors block Bad we tested the ability of PKC isoforms to phosphorylate Bad at and PKC isoforms were tested their ability to phosphorylate the PKC isoforms were able to phosphorylate and of the isoforms tested were unable to phosphorylate Bad at not that PKC may activate a downstream protein kinase that in Bad at of p90RSK was this protein kinase is activated by TPA downstream of PKC R. P. A. J. 1993; PubMed Scopus Google Scholar). of p90RSK in vitro is to be with the in which Bad Ser112 is E. J. Mol. Cell. Biol. PubMed Scopus Google Scholar). phosphorylate Bad at Ser112, p90RSK was from or cells a kinase were and as and Bad phosphorylation was by a Ser112 TPA p90RSK kinase that Bad and the Bad but not the and were also in a kinase protein as and Bad phosphorylation was by Ser112 and Ser136 shown in TPA stimulates the ability of p90RSK to phosphorylate at Ser112 but not at These suggest that in Bad at Ser112 but not at that p90RSK was downstream of PKC a pathway to Bad phosphorylation at Ser112, p90RSK a of sensitivity to that Bad we the sensitivity of p90RSK kinase to or by cells with a TPA cell were p90RSK was and kinase was as Bad Ser112 phosphorylation with or with and in or p90RSK in with their Bad Ser112 phosphorylation A with the that these inhibitors block p90RSK p90RSK was from cell and were in the of and of of the PKC inhibitors or in vitro at Kinase were as shown in a the of and to block p90RSK in p90RSK in vitro with that these are not inhibiting p90RSK the ability of p90RSK to stimulate Bad phosphorylation at Ser112 by cells were with and or and cells were in the a and with TPA were by Ser112 of p90RSK Bad phosphorylation at Ser112 in the of TPA suggesting that p90RSK is at in the of TPA Bad Ser112 phosphorylation was by TPA in cells with p90RSK of Bad Ser112 to but not Ser136 to blocks phosphorylation. the of other family members Bad Ser112 phosphorylation. RSK-2 and RSK-3 were from or TPA cells and kinase were as and Bad phosphorylation was by Ser112 shown in TPA stimulates the ability of both RSK-2 and RSK-3 to phosphorylate Bad at Ser112 in a of kinase. the of we by cell and RSK-3 were and RSK-3 not the ability of RSK-2 and RSK-3 to stimulate Bad phosphorylation in we cells with and overexpression of RSK-2 or RSK-3 Bad Ser112 phosphorylation that was enhanced TPA treatment Phosphorylation of Bad at Ser112 and Ser136 has been to 14-3-3 Bad to that block (5Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2257) Google Scholar). treatment of cells with TPA or overexpression of p90RSK stimulate binding of Bad to 14-3-3 were from cells or cells with and or with TPA cell were with or of with 14-3-3 was by Ser112 or Bad shown in both TPA and p90RSK stimulate binding of to 14-3-3 or PKC and p90RSK function in a pathway that Bad-mediated cell death, activation of the pathway block Bad-mediated cell death via phosphorylation and of 14-3-3 this we and cells with Bad and a as a to of with and with in with Bad in a in the of cells and in the of cells and in the of cells is with apoptotic death of a of the cells Bad the of death by we cell death the of cells from in S.D. were by from shown in of cells with Bad by in the death of a of cells Bad-mediated cell death was by treatment with TPA p90RSK also block Bad-mediated cell death, we p90RSK with Bad of cells with Bad or by in cell death, as by the or by of cells as Bad-mediated cell death was by with p90RSK Similarly, with cell death mediated by the Bad phosphorylation sites p90RSK we and with induce the death of a of and cell death by Bad can be by with However, cell death by Bad or was not by suggesting that phosphorylation of Bad at Ser112 is p90RSK to function as a death Although a of growth and survival factors activate PKC, and PKC activation is known to promote cell the PKC cell survival are largely unknown. in this a the activation of PKC cell PKC activation by TPA is shown to in the phosphorylation of Bad at Ser112, and this is with of Bad-mediated cell death. PKC does not to phosphorylate Bad at Ser112 in we by that the protein acts downstream of PKC and in can phosphorylate Bad at p90RSK Bad at Ser112 in the of PKC inhibitors Bad phosphorylation are with their p90RSK and phosphorylation in vivo. overexpression of p90RSK in cells stimulates Bad Ser112 phosphorylation and blocks cell death. these findings a pathway the activation of PKC and p90RSK and phosphorylation of Bad at may function to Bad-mediated cell death and promote cell by Zha (5Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2257) Google Scholar) Bad Ser112 and Ser136 as two sites that by IL-3 were able to block the of have a survival pathway to Bad phosphorylation that PI activation of by phosphorylation of Bad at Ser136 (12Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4957) Google Scholar, 13del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1989) Google Scholar, Bellacosa A. S.G. Tsichlis P.N. Hay N. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar, P. R. T. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). Datta that Akt activation in the phosphorylation of Bad at Ser136 but not pathway by Datta by the PI 3-kinase inhibitors wortmannin and LY294002 and the phosphorylation of Bad Ser136 (12Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4957) Google Scholar). pathway we have is not by PI 3-kinase to Bad phosphorylation at Ser112, and does not involve TPA does not stimulate Akt in the cells we have not pathway a from the PI 3-kinase/Akt to Bad phosphorylation and cell that pathways Bad to cell Survival factors such as IL-3 also in activation of protein kinase A. protein kinase A has recently been as a Bad kinase phosphorylating Bad at Ser112 (14Harada H. Becknell B. Wilm M. Mann M. Huang L.J. Taylor S.S. Scott J.D. Korsmeyer S.J. Mol. Cell. 1999; 3: 413-422Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar). a can Bad at both Ser112 and Ser136 and promote apoptosis in a (15Wang 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 (967) Google Scholar). have a pathway cell survival in to and insulin-like growth factor-1 (7Scheid M.P. Duronio V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7439-7444Crossref PubMed Scopus (276) Google Scholar, G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (966) Google Scholar), suggesting the existence of survival pathway from PKC to the activation of p90RSK is PKC has been to activate the kinase at and R. Y. C. H. Science. 1998; PubMed Scopus Google Scholar, Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar), and can phosphorylate p90RSK at in vitro and in N. J. P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, R. P. A. J. 1993; PubMed Scopus Google Scholar), to that PKC p90RSK via the However, of with a D.R. A. P. A.R. J. Biol. 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