Bcl-2 Phosphorylation by p38 MAPK

Abstract

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser87 and Thr56 as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38α knock-out mice (p38α-/- MEF), whereas they occur within 12 h of serum withdrawal in p38α+/+ MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress. The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser87 and Thr56 as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38α knock-out mice (p38α-/- MEF), whereas they occur within 12 h of serum withdrawal in p38α+/+ MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress. Bcl-2 family proteins play differential roles in the regulation of mitochondria-mediated apoptosis, by either promoting or inhibiting the release of apoptogenic molecules from mitochondria to the cytosol (1Breckenridge D.G. Xue D. Curr. Opin. Cell Biol. 2004; 16: 647-652Crossref PubMed Scopus (234) Google Scholar, 2Marzo I. Brenner C. Kroemer G. Biomed. Pharmacother. 1998; 52: 248-251Crossref PubMed Scopus (71) Google Scholar). Bcl-2 family proteins modulate the mitochondrial permeability through interaction with adenine nucleotide translocator, voltage-dependent anion channel, by ADP/ATP exchange, or by interfering with oxidative phosphorylation during apoptosis (2Marzo I. Brenner C. Kroemer G. Biomed. Pharmacother. 1998; 52: 248-251Crossref PubMed Scopus (71) Google Scholar). The three-dimensional structure of Bcl-2 proteins suggests that they are also capable of forming channels in mitochondrial membranes (3Minn A.J. Velez P. Schendel S.L. Liang H. Muchmore S.W. Fesik S.W. Fill M. Thompson C.B. Nature. 1997; 385: 353-357Crossref PubMed Scopus (727) Google Scholar, 4Muchmore S.W. Sattler M. Liang H. Meadows R.P. Harlan J.E. Yoon H.S. Nettesheim D. Chang B.S. Thompson C.B. Wong S.L. Ng S.L. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1298) Google Scholar, 5Schendel S.L. Montal M. Reed J.C. Cell Death. Differ. 1998; 5: 372-380Crossref PubMed Scopus (277) Google Scholar). Recent evidence indicates that the antiapoptotic functions of Bcl-2 can be regulated by post-translational modifications, including phosphorylation (6Blagosklonny M.V. Leukemia. 2001; 15: 869-874Crossref PubMed Scopus (135) Google Scholar), dimerization (7Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1228) Google Scholar), and/or proteolytic cleavage (8Cheng E.H. Kirsch D.G. Clem R.J. Ravi R. Kastan M.B. Bedi A. Ueno K. Hardwick J.M. Science. 1997; 278: 1966-1968Crossref PubMed Scopus (1015) Google Scholar). There is still controversy over the significance of Bcl-2 phosphorylation, and two opposing models were initially proposed, the “taxol-induced” and the “interleukin-3-induced” Bcl-2 phosphorylation (6Blagosklonny M.V. Leukemia. 2001; 15: 869-874Crossref PubMed Scopus (135) Google Scholar). In the first model, Bcl-2 phosphorylation induces a strong inhibition of the antiapoptotic potential and is related to hyperphosphorylation of several kinases, including Raf-1 kinase, but is independent of protein kinase C (9Blagosklonny M.V. Schulte T. Nguyen P. Trepel J. Neckers L.M. Cancer Res. 1996; 56: 1851-1854PubMed Google Scholar). In the second model, stimulation with growth factors, such as interleukin-3, induces Bcl-2 phosphorylation, which increases the antiapoptotic potential of the protein (10Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar), whereas, in contrast, interleukin-3 deprivation inhibits Bcl-2-phosphorylation and induces cell death. However, subsequent reports clearly show that hyperphosphorylated Bcl-2 cannot be considered a hallmark of the growth/survival mechanism of action and that triggering specific tyrosine kinase receptors decreases Bcl-2 phosphorylation still inhibiting cell death (6Blagosklonny M.V. Leukemia. 2001; 15: 869-874Crossref PubMed Scopus (135) Google Scholar). For example, binding of insulin receptor substrate proteins to Bcl-2, triggered by insulin/insulin-receptor interaction, inhibits its phosphorylation and has been proposed as one of the survival mechanisms induced by insulin (11Ueno H. Kondo E. Yamamoto-Honda R. Tobe K. Nakamoto T. Sasaki K. Mitani K. Furusaka A. Tanaka T. Tsujimoto Y. Kadowaki T. Hirai H. Mol. Biol. Cell. 2000; 11: 735-746Crossref PubMed Scopus (51) Google Scholar). In memory B lymphocytes, as well as in continuos B lymphoblastoid cell lines, nerve growth factor (NGF) 3The abbreviations used are: NGF, nerve growth factor; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEF, mouse embryo fibroblast(s); MDCK, Madin-Darby canine kidney; GST, glutathione S-transferase; PBS, phosphate-buffered saline; MALDI, matrix-assisted laser desorptionionization;TOF,time-of-flight;CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; rp38, recombinant p38; WT, wild type; MS, mass spectrometry; ERK, extracellular signal-regulated kinase; PARP, poly-(ADP-ribose) polymerase. inhibits apoptosis by preventing Bcl-2 phosphorylation (12Rosini P. De Chiara G. Bonini P. Lucibello M. Marcocci M.E. Garaci E. Cozzolino F. Torcia M. J. Biol. Chem. 2004; 279: 14016-14023Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 13Rosini P. De Chiara G. Lucibello M. Garaci E. Cozzolino F. Torcia M. Biochem. Biophys. Res. Commun. 2000; 278: 753-759Crossref PubMed Scopus (45) Google Scholar, 14Torcia M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). are not and the of Bcl-2 in cellular able to or Bcl-2 is that such the action of Bcl-2 kinases, which residues of the protein. In Bcl-2 phosphorylation in one of the serine and threonine residues located in a nonstructured loop that links the BH3 and BH4 Hardwick J.M. S. A. PubMed Scopus Google Scholar), and kinases and been to modulate such phosphorylation J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. T. C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (277) Google Scholar, Y. S. J. Y. M. H. Y. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, K. B. E. M. M. C. C. E. J.C. S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). as a potential Bcl-2 kinase, and has been to Bcl-2 phosphorylation and K. B. E. M. M. C. C. E. J.C. S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). However, reports activation is Bcl-2 phosphorylation, kinases, such as are A. Y. J. De P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). during of activation and Bcl-2 phosphorylation has been H.S. H. P. T. J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). p38 MAPK is in several including triggered by and Bcl-2 phosphorylation (12Rosini P. De Chiara G. Bonini P. Lucibello M. Marcocci M.E. Garaci E. Cozzolino F. Torcia M. J. Biol. Chem. 2004; 279: 14016-14023Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 13Rosini P. De Chiara G. Lucibello M. Garaci E. Cozzolino F. Torcia M. Biochem. Biophys. Res. Commun. 2000; 278: 753-759Crossref PubMed Scopus (45) Google Scholar, 14Torcia M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. E. Y. T. H. Mol. Cell PubMed Scopus Google Scholar, S. T. Cell Res. 2004; PubMed Scopus Google Scholar). these Bcl-2 hyperphosphorylation induced by p38 MAPK always to proapoptotic as by the release of cytochrome c in cellular and in M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In to JNK, Bcl-2 phosphorylation is with p38 MAPK activation and is by a direct interaction Bcl-2 and the activated in the mitochondrial in cytochrome c release and caspase apoptosis, triggered a p38 MAPK not protein synthesis and can be by p38 the these the that several kinases can specific Bcl-2 residues and the to In this study, we by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser87 and Thr56 as the Bcl-2 residues phosphorylated by p38 of these residues with inhibits cytochrome c release from mitochondria the of activated p38 also that of the p38α MAPK apoptosis induced by serum withdrawal in embryonic fibroblasts and that Bcl-2 phosphorylation induced by p38 MAPK plays a key role in the early induction of apoptosis following serum Cell fibroblasts from p38α and p38α were by of K. T. R. M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). were in with and cell were by and in with c were from MAPK, and mouse Bcl-2 were from p38 MAPK were from and the p38 MAPK from recombinant p38 and were from recombinant and its and were as N-terminal protein using in protein by cell in phosphate-buffered with and The cell with to and with and The to a and using a with the were with glutathione in with and of Bcl-2 as M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The Bcl-2 by a using a and the in as Bcl-2 were in the as proteins in and Bcl-2 were in as N-terminal protein in as and were in using as the and the and of Bcl-2, in which and to the phosphorylated threonine and serine of Bcl-2, The as Y. S. A. K. E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). kinase by in the or of activated p38 MAPK in kinase and and Both and were and were from and to with as A. M. M. Chem. 1996; PubMed Scopus Google Scholar, M. A. T. S. L. T. M. Nature. 1996; PubMed Scopus Google Scholar). were from and by mass For mass were in and using of the were with the laser to with a of c by were in phosphate-buffered and in of mitochondrial were through cell and were a The and the the mitochondrial and with or of E. or or with the obtained by the with and of p38 MAPK, with or and the phosphorylation in the of p38 mitochondria were also used as were and the and proteins were by to a The with cytochrome c to by a with The by of p38α+/+ and under conditions of serum deprivation and of in the of as were and a The were with the by with The by of Bcl-2 phosphorylation a kinase by of recombinant p38 or and of recombinant in kinase in the or of In of the kinase by of recombinant or or and of recombinant p38 in kinase p38 Bcl-2 proteins were used as were by a and with and The by the of recombinant of or recombinant were with of in kinase in the of of were and p38α+/+ and were in a of h were with and in serum and 12 the were in PBS, in with and with in with serum in to the were with c in serum h in a The were in and with the in PBS, and by a to a with a p38 MAPK of Thr56 and Ser87 Bcl-2 of c from and of protein kinases, including the MAPK family been to Bcl-2 phosphorylation J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. T. C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (277) Google Scholar, Y. S. J. Y. M. H. Y. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, K. B. E. M. M. C. C. E. J.C. S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). p38 MAPK to Bcl-2 phosphorylation and cell death in memory B withdrawal of survival factor M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). with Bcl-2 in which and with we that and Bcl-2 from apoptosis induced by p38 MAPK these as of the kinase M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In to evidence that p38 phosphorylation of the Ser87 and Thr56 residues of Bcl-2 inhibits the antiapoptotic of the we the Bcl-2 in recombinant and mitochondria from MDCK, a canine cell that not Bcl-2 A. B. J. 1996; 15: PubMed Scopus Google Scholar). as M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), were with recombinant Bcl-2 with recombinant Bcl-2 of the loop the and or Bcl-2 wild as and of recombinant proteins in the activated p38 MAPK to the and the release of cytochrome c as a of Bcl-2 antiapoptotic h of p38 MAPK induced the release of cytochrome c from Bcl-2 and the by a of the J.C. S. S. A. PubMed Scopus Google Scholar). contrast, the p38 MAPK-induced release of cytochrome c not from mitochondria the Bcl-2 and from Bcl-2 or Bcl-2 with that from Bcl-2 the Bcl-2 mitochondria were to p38 MAPK p38 MAPK to release of c from mitochondria not to Bcl-2, that the MAPK interaction related to the as M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the the that the of Thr56 and Ser87 residues with inhibits the c release induced by p38 MAPK action p38 MAPK Bcl-2 in Thr56 and Ser87 and a direct of the Bcl-2 residues phosphorylated by activated p38 MAPK, we a mass spectrometry of of Bcl-2 phosphorylation by p38 MAPK in kinase by recombinant Bcl-2 with in the or of activated p38 The were and to phosphorylated or Bcl-2 protein were and to were from and mass by in by the of the two and two to as and were from of Bcl-2 in a we B the residues and a not the of the phosphorylated by p38 The used as Both the mass to the of the and its were in the p38 to mass of mass of the from Bcl-2 and p38 Bcl-2 Both a to which the Thr56 as the one within the MAPK mass of the from p38 Bcl-2 a to phosphorylated that the the Thr56 of we the phosphorylation of the B to the B mass of mass of the from Bcl-2 and p38 Bcl-2 a to B the mass of from p38 Bcl-2 a to the B that the one the and within the MAPK However, to the or that one the of the were were in not Thus, and B were in the mass the of these in as with the is with the by the the of the and B of not using and For the mass to the of the phosphorylation the as of p38 MAPK action the Thr56 and one the residues and and 14Torcia M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Thr56 and Ser87 residues of Bcl-2 as the of the to direct evidence of phosphorylation, we used a or of the protein were by with the and the and of Bcl-2 obtained by in kinase with recombinant p38 MAPK were by and with as the the proteins p38 MAPK but not the However, a phosphorylated Bcl-2 also using Furthermore, able to phosphorylation of Bcl-2 that they are of p38 MAPK in kinase with activated as a direct phosphorylation of the Thr56 or Ser87 residues of However, activated and p38 MAPK were able to substrate In to the of the and to the residues of p38 MAPK a kinase by recombinant Bcl-2 or recombinant Bcl-2 as a recombinant with p38 that the the protein and the but not Bcl-2 protein p38 MAPK the the Bcl-2 and Bcl-2 but not the the not with protein not to p38 that the with and with the obtained with the Thr56 and Ser87 as the Bcl-2 residues phosphorylated by p38 The were used to in a in which activation of p38 MAPK induced by the in Bcl-2 phosphorylation, with Ser87 and Thr56 the M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that and a of phosphorylated Bcl-2 the that obtained with recombinant to the of the phosphorylated Bcl-2, as by A. S. J. Google Scholar). phosphorylation of residues by Thr56 and Ser87 as the of p38 MAPK action also in this cellular of Bcl-2 and specific to that the Thr56 but also the Ser87 Bcl-2 residues are specific of p38 MAPK of p38 MAPK-induced Bcl-2 in Cell of by clearly show that p38 MAPK phosphorylation of the Thr56 and Ser87 Bcl-2 residues the antiapoptotic of the protein. In to the of this in a cellular we obtained embryonic fibroblasts from p38α wild mice or knock-out (p38α-/- mice K. T. R. M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google and induced apoptosis by serum Cell from p38α+/+ or MEF, in the or of were and activation of p38 MAPK and phosphorylation of Bcl-2 were by techniques with specific that of activated p38 MAPK h serum in p38α+/+ MEF, but not in with p38 MAPK that the in MEF, is in p38α+/+ p38α+/+ and were also in the or of the p38 MAPK 12 the of a of Bcl-2 in the cell with specific antibodies, the of Bcl-2 The h of serum withdrawal in p38α+/+ and by In contrast, phosphorylated Bcl-2 protein not in of that activation of p38 MAPK is Bcl-2 phosphorylation induced by serum in to the in a second of we activated p38 MAPK cytochrome c release from mitochondria serum of p38α+/+ or were in and in and 12 were with with with c and by c a mitochondrial in p38α+/+ and of cytochrome c in a of p38α+/+ but not in the of p38α+/+ with of cytochrome c and of to contrast, the mitochondrial of cytochrome c not in Furthermore, a serum mitochondrial structure the as T. J. Cell Mol. PubMed Scopus Google in p38α+/+ MEF, but not in not we p38α+/+ and under conditions of serum deprivation in the or of the specific and cleavage as a of cleavage is in p38α+/+ and is by In contrast, cleavage not the in MEF, that and activation of p38 MAPK play a role in apoptosis induced by serum that fibroblasts are to apoptosis induced by A. S. E. A. C. M. Mol. Biol. Cell. 2004; 15: PubMed Scopus Google Scholar). The apoptosis with of the mitochondrial proapoptotic protein Bax and of the receptor as well as with MAPK A. S. E. A. C. M. Mol. Biol. Cell. 2004; 15: PubMed Scopus Google Scholar). In to the of proteins in the in we p38α+/+ and in the of serum and Bax and by that Bax and not in either p38α+/+ or MEF, from to h serum that in the early of serum cytochrome c release from mitochondria is not by synthesis of proapoptotic proteins but is the of p38 Bcl-2 However, h serum a in as well as in Bax protein in p38α+/+ not that are in the events that in this model, following serum of the mechanisms protein is phosphorylation, event that to a of the with or decrease in its of the binding The of the Bcl-2 family of proteins a and regulation by of protein is J.C. Res. 1996; PubMed Scopus Google phosphorylation events modulate by triggering or inhibiting the Furthermore, Bcl-2 be the of kinases, and are the and of the phosphorylated Thus, is not Bcl-2, phosphorylation is either as or its (6Blagosklonny M.V. 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Chem. 2001; Full Text Full Text PDF PubMed Scopus Google under these p38 MAPK the kinase Bcl-2 In the study, we by mass spectrometry and techniques with specific the two residues Ser87 and Thr56 as phosphorylated by p38 mass of Bcl-2 to to that p38 MAPK two residues within the loop the and of of the phosphorylation by not to the of the two identified Bcl-2 the of specific antibodies, with Bcl-2 and to Thr56 and Ser87 as Bcl-2 of p38 MAPK are with that of B cell with the Bcl-2 and the to In this we clearly that activated p38 MAPK is able to release of cytochrome c from mitochondria of the canine cell the of recombinant Bcl-2, the of MAPK interaction in triggering the In this the Bcl-2 as well as the or Bcl-2 recombinant to cytochrome c release by activated p38 MAPK, as with Bcl-2 evidence suggests that p38 MAPK-induced phosphorylation of the Ser87 and Thr56 residues of Bcl-2 able to decrease the antiapoptotic potential of the protein. 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