BAD Ser-155 Phosphorylation Regulates BAD/Bcl-XL Interaction and Cell Survival

Abstract

The BH3 domain of BAD mediates its death-promoting activities via heterodimerization to the Bcl-XL family of death regulators. Growth and survival factors inhibit the death-promoting activity of BAD by stimulating phosphorylation at multiple sites including Ser-112 and Ser-136. Phosphorylation at these sites promotes binding of BAD to 14-3-3 proteins, sequestering BAD away from the mitochondrial membrane where it dimerizes with Bcl-XL to exert its killing effects. We report here that the phosphorylation of BAD at Ser-155 within the BH3 domain is a second phosphorylation-dependent mechanism that inhibits the death-promoting activity of BAD. Protein kinase A, RSK1, and survival factor signaling stimulate phosphorylation of BAD at Ser-155, blocking the binding of BAD to Bcl-XL. RSK1 phosphorylates BAD at both Ser-112 and Ser-155 and rescues BAD-mediated cell death in a manner dependent upon phosphorylation at both sites. The BH3 domain of BAD mediates its death-promoting activities via heterodimerization to the Bcl-XL family of death regulators. Growth and survival factors inhibit the death-promoting activity of BAD by stimulating phosphorylation at multiple sites including Ser-112 and Ser-136. Phosphorylation at these sites promotes binding of BAD to 14-3-3 proteins, sequestering BAD away from the mitochondrial membrane where it dimerizes with Bcl-XL to exert its killing effects. We report here that the phosphorylation of BAD at Ser-155 within the BH3 domain is a second phosphorylation-dependent mechanism that inhibits the death-promoting activity of BAD. Protein kinase A, RSK1, and survival factor signaling stimulate phosphorylation of BAD at Ser-155, blocking the binding of BAD to Bcl-XL. RSK1 phosphorylates BAD at both Ser-112 and Ser-155 and rescues BAD-mediated cell death in a manner dependent upon phosphorylation at both sites. mitogen-activated protein kinase protein kinase A 12-O-tetradecanoylphorbol-13-acetate 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside glutathione S-transferase human embryonic kidney wild type 3-isobutyl-1-methylxanthine maltose-binding protein BAD is one member of a group of “BH3 domain only” proteins that appears to transduce death signals from the cytoplasm to the mitochondrial membrane and induce apoptosis (1Yang E. Zha J. Jockel J. Boise L.H. Thompson C.B. Korsmeyer S.J. Cell. 1995; 80: 285-291Abstract Full Text PDF PubMed Scopus (1897) Google Scholar, 2Zha 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, 3Korsmeyer S.J. Cancer Res. 1999; 59 (suppl.): 1693s-1700sGoogle Scholar). Intracellular survival pathways can inhibit cell death by phosphorylating BAD on several different sites blocking death-promoting activity. Studies by Zha et al. (2Zha 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) first identified BAD Ser-112 and Ser-136 as two sites that when phosphorylated in response to interleukin-3 blocked the cytotoxic effects of BAD. Survival factors such as interleukin-3 activate a phosphatidylinositol 3-kinase-dependent pathway involving the activation of Akt followed by the phosphorylation of BAD at Ser-136 (4del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1989) Google Scholar, 5Datta 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, 6Blume-Jensen P. Janknecht R. Hunter T. Curr. Biol. 1998; 8: 779-782Abstract Full Text Full Text PDF PubMed Google Scholar). Interleukin-3 signaling can also activate protein kinase C-, MAPK-,1 and RSK1-dependent pathways that stimulate the phosphorylation of BAD at Ser-112 (7Tan Y. Ruan H. Demeter M.R. Comb M.J. J. Biol. Chem. 1999; 274: 34859-34867Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 8Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1682) Google Scholar, 9Shimamura A. Ballif B.A. Richards S.A. Blenis J. Curr. Biol. 2000; 10: 127-135Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 10Fang X., Yu, S. Eder A. Mao M. Bast Jr., R.C. Boyd D. Mills G.B. Oncogene. 1999; 18: 6635-6640Crossref PubMed Scopus (237) Google Scholar, 11Scheid M.P. Schubert K.M. Duronio V. J. Biol. Chem. 1999; 274: 31108-31113Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Interleukin-3 can also increase cAMP levels resulting in activation of PKA. Membrane-based PKA has been identified as a BAD kinase that phosphorylates BAD at Ser-112 (12Harada 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). Calcineurin, a calcium-dependent phosphatase, promotes apoptosis by dephosphorylating BAD at both Ser-112 and Ser-136 (13Wang 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). Phosphorylation at Ser-112 and Ser-136 creates binding sites for the phospho-specific binding of 14-3-3 proteins, which retain BAD in the cytoplasm and prevent cytotoxic interactions with Bcl-XL at the mitochondrial membrane (2Zha 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, 5Datta 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, 7Tan Y. Ruan H. Demeter M.R. Comb M.J. J. Biol. Chem. 1999; 274: 34859-34867Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Although the precise mechanism these molecules use to induce cell death is still unknown, deletion analysis of BAD has demonstrated the importance of the BH3 domain in mediating both its heterodimerization with Bcl-XL and its death-promoting activity (14Zha 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 (267) Google Scholar). Site-specific mutagenesis of the closely related BH3 domain of BAX has revealed the importance of the hydrophobic surface of the BH3 α-helical face for dimerization and cell death activity (15Wang K. Gross A. Waksman G. Korsmeyer S.J. Mol. Cell. Biol. 1998; 18: 6083-6089Crossref PubMed Scopus (189) Google Scholar). BAX is thought to interact with Bcl-XL at the mitochondrial membrane by fitting into an α-helical groove. The binding of Bcl-XL by the BH3 domain may cause Bcl-XL to release Apaf1 or regulate other Bcl-XL activities resulting in a caspase 9-initiated cascade of proteolysis and induction of apoptosis (16Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3268) Google Scholar, 17Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1263) Google Scholar). In the present study, we demonstrate that BAD can be phosphorylated at Ser-155, which is located within the middle of the hydrophobic amphipathic face of the BH3 α helix. Phosphorylation at Ser-155, similar to phosphorylation at Ser-112 and Ser-136, inhibits BAD-induced death-promoting activity but does so in a different manner. Phosphorylation at Ser-155 does not induce 14-3-3 binding and cytoplasmic sequestration but instead directly blocks BH3-dependent dimerization with Bcl-XL. Forskolin, 3-isobutyl-1-methylxanthine (IBMX), TPA, isoproterenol, fetal calf serum, and mammalian cell culture medium were obtained from Sigma. Epidermal growth factor and insulin-like growth factor I were from Life Technologies, Inc. Phosphorylation state-specific anti-BAD(Ser-155) antibody, control anti-BAD antibodies, and the chemiluminescent Western detection kit were obtained from Cell Signaling Technology. Bcl-XL-specific antibodies were purchased from Santa Cruz. RSK1-specific monoclonal antibody was from Transduction Laboratories. Protein kinase A, calmodulin-dependent protein kinase, casein kinase I, casein kinase II, and ERK2 were purchased from New England Biolabs. Protein kinase C was purchased from Calbiochem, and the X-gal staining kit was from Invitrogen. The QuickChange site-directed mutagenesis kit was obtained from Stratagene, and the FuGENE 6 transfection reagent was from Roche Molecular Biochemicals. The GST·BAD mammalian expression vector was constructed by fusion of the complete coding sequence of BAD that has been amplified from a mouse brain cDNA library (CLONTECH) and cloned into theBamHI/NotI sites of the eukaryotic expression vector pEBG. pEBG expresses an amino-terminal GST fusion of the cloned gene under the control of the strong constitutively active human EF-1α promoter (18Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar). GST·BAD S112A/S136A mammalian expression vectors were constructed as described previously (7Tan Y. Ruan H. Demeter M.R. Comb M.J. J. Biol. Chem. 1999; 274: 34859-34867Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). GST·BAD S155A and S155E mammalian expression vectors were constructed by site-directed mutagenesis using the QuickChange site-directed mutagenesis kit. pMT2·RSK1 was provided by Joseph Avruch and pMT2·RSK2 and RSK3 were provided by Christian Bjorbaek. The pEAK 10·Bcl-XL vector was constructed by fusion of the complete coding sequence of Bcl-XL amplified from pCMV·Bcl-XL (provided by Stanley Korsmeyer) and cloned into the EcoRI/NotI site of the pEAK10 vector. MBP·Bcl-XL vector was constructed by fusion of the complete coding sequence of Bcl-XL into the EcoRI/NotI of pMalC2 vector. The anti-phospho-BAD(Ser-155) antibodies were generated by immunizing rabbits with synthetic phosphopeptides covalently coupled to keyhole limpet hemocyanin. The presence of phospho-specific immunoreactivity was detected by the enzyme-linked immunosorbent assay using both phosphorylated and nonphosphorylated peptides. After purification of IgG using protein A-agarose, phosphopeptide-specific antibodies were purified by first passing the antibodies over immobilized nonphosphorylated peptide to remove antibodies that are reactive to nonphosphorylated epitopes. The nonabsorbed fraction was then passed over a column of immobilized phosphopeptide. After extensive washing, the retained immunoglobulins were eluted at low pH, rapidly neutralized, dialyzed, and concentrated. 2 μg of GST·BAD protein were incubated with different kinases in 50 μl of kinase buffer (25 mm Tris, pH 7.4, 5 mm β-glycerol phosphate, 2 mm dithiothreitol, 0.1 mmNa3VO4, 10 mmMgCl2) and 200 μm ATP for 30 min at 30 °C. Kinase reactions were terminated with SDS sample buffer, and BAD phosphorylation was detected by immunoblotting. HEK293 cells were maintained in modified minimum Eagle's medium supplemented with 10% (v/v) horse serum. Transient transfection of HEK293 cells was performed as described previously (19Tan Y. Rouse J. Zhang A. Cariati S. Cohen P. Comb M.J. EMBO J. 1996; 15: 4629-4642Crossref PubMed Scopus (568) Google Scholar) with 2 μg of GST·BAD, GST·BAD S112A/S136A, or GST·BAD S155A and 10 μg of the catalytic subunit of PKA or 10 μg of pMT2·RSK1, RSK2, or RSK3 in 10-cm2 plates. The total amount of transfected DNA was maintained at 20 μg with pEGB. Following calcium phosphate transfections, cells were glycerol-shocked and incubated for 24 h in medium containing 10% horse serum and for 18 h without serum. Cells were treated with regulators for the indicated times. For the survival assay, HEK293 cells were transfected with 0.5 μg of Rous sarcoma virus β-galactosidase, 1 μg of GST·BAD or BAD mutants, and 0.5 μg of pMT2·RSK1 using FuGENE 6 transfection reagent. 24 h after transfection, cells were grown in medium without serum for 18 h and then fixed for X-gal staining. Cell extracts were prepared by lysing 5 × 105 cells in 100 μl of SDS sample buffer (2% SDS, 10% glycerol, 80 mm Tris, pH 6.8, 0.15 m2-mercaptoethanol, 0.02% bromphenol blue). Extracts from 105 cells (20 μl) were fractionated by 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blots were blocked with 5% nonfat dry milk in TBST (10 mm Tris, pH 7.5, 150 mm NaCl, 0.05% Tween 20) for 1 h at room temperature, incubated with primary antibody overnight at 4 °C, incubated with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature, and visualized using the chemiluminescent Western detection kit. HEK293 cells were transfected with plasmids encoding pEAK10·Bcl-XL, wild-type GST·BAD, or GST·BAD containing S155A or S155E mutants as indicated. After 24 h, the cells were deprived of serum and 18 h later were harvested in cell buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm 1 mm mm 1 mmNa3VO4, 1 1 Cell extracts were incubated with Bcl-XL antibody overnight at 4 and protein for 2 were with cell buffer and with and proteins were eluted with SDS sample buffer for Western or MBP·Bcl-XL proteins to were with extracts prepared from HEK293 cells transfected with plasmids encoding wild-type BAD mutants, or treated with or without and in cell Cell extracts were incubated overnight at 4 °C, were with cell buffer and with and proteins were eluted with SDS sample buffer for Western in Ser-155 within the BH3 of BAD Ser-155 is in a protein kinase A that may be phosphorylated by protein the that phosphorylation at the of the BAD BH3 we generated a phosphorylation state-specific antibody J. G. J. P. Scopus Google Scholar) Ser-155 of BAD as described under The antibody was to be for the phosphorylated by enzyme-linked immunosorbent assay not and by Western using phosphorylated and nonphosphorylated GST·BAD proteins 2 We antibody to the of several different protein kinases to BAD at Ser-155 in with a GST·BAD fusion protein as demonstrated in A, of the protein kinases protein kinase A was to BAD at to in activation of protein kinase A using stimulate phosphorylation of BAD at HEK293 cells were transfected with plasmids encoding wild-type GST·BAD or GST·BAD S112A/S136A or S155A Cell extracts were prepared and using phospho-specific antibodies BAD Ser-155 and BAD Ser-112 as as phosphorylation anti-BAD antibodies 2 the phosphorylation of GST·BAD at both Ser-155 and The site of the anti-phospho-BAD(Ser-155) and antibodies was using the BAD of Ser-155 or Ser-112 to immunoreactivity with the phospho-specific Although the is to 14-3-3 (2Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2257) Google it was phosphorylated at Ser-155 in response to that Ser-155 can be phosphorylated in the of phosphorylation at Ser-112 and Ser-136. in 2 a and phosphorylation of BAD at of HEK293 cells with the catalytic subunit of protein kinase A also the phosphorylation of BAD at Ser-155 2 phosphorylates BAD at Ser-155 in and in A, GST·BAD was phosphorylated using the indicated kinases and using anti-phospho-BAD(Ser-155) casein kinase protein kinase calmodulin-dependent protein kinase BAD Ser-155 HEK293 cells were transfected with plasmids encoding GST·BAD or S112A/S136A or S155A mutants as indicated. After 24 h, the cells were deprived of serum and 18 h later were treated with 30 μm and 0.5 mm for 30 min to as indicated. of BAD phosphorylation at HEK293 cells were transfected as described and treated with 30 and 0.5 mm for the indicated to PKA BAD Ser-155 HEK293 cells were transfected with plasmids encoding GST·BAD and the catalytic subunit of PKA as described cell extracts were and was using anti-phospho-BAD(Ser-155) antibody or anti-BAD antibody We also other that stimulate phosphorylation of BAD at with TPA, isoproterenol, or insulin-like growth factor the increase in phosphorylation at Ser-155 rapidly a phosphorylation of BAD that was within 5 min of and h after RSK1 in these cells (7Tan Y. Ruan H. Demeter M.R. Comb M.J. J. Biol. Chem. 1999; 274: 34859-34867Abstract Full Text Full Text PDF PubMed Scopus (219) Google we RSK1 phosphorylates BAD at RSK1 was from or HEK293 cells using a RSK1-specific monoclonal kinase were performed using wild-type and GST·BAD fusion proteins as and BAD phosphorylation was detected by using anti-phospho-BAD(Ser-155) antibody RSK1 phosphorylated BAD at Ser-155, and activity was after with RSK1 also BAD at Ser-155 in HEK293 cells were with plasmids encoding RSK1 and of RSK1 BAD phosphorylation at Ser-155 in the of and Ser-155 phosphorylation was by in cells transfected with RSK1 4 of two closely related and also in of BAD phosphorylation at Ser-155 4 and RSK1 both stimulate phosphorylation of BAD at binding of and sequestration (2Zha 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, 7Tan Y. Ruan H. Demeter M.R. Comb M.J. J. Biol. Chem. 1999; 274: 34859-34867Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). in BAD with 14-3-3 was also phosphorylated at and RSK1 both the amount of BAD with BAD phosphorylation at and in A, RSK1 phosphorylates BAD at Ser-155 in Cell extracts prepared from HEK293 cells with or without 200 were using a RSK1-specific antibody were incubated in a kinase using GST·BAD or the S155A as BAD phosphorylation was detected by using anti-phospho-BAD(Ser-155) and BAD Ser-155 phosphorylation in HEK293 cells were transfected with plasmids encoding GST·BAD or GST·BAD S155A and RSK1, or RSK3 as indicated. After 24 h, cells were deprived of serum and 18 h later were treated with for 30 min as indicated to cell extracts were prepared and using anti-phospho-BAD(Ser-155) or anti-BAD antibody BAD 14-3-3 HEK293 cells were transfected with plasmids encoding wild-type GST·BAD and RSK1 and were treated with as described in the to Cell extracts were incubated with or to as indicated. After extensive washing, proteins to the were eluted using SDS sample was using anti-phospho-BAD(Ser-155) Ser-155 in the of the BH3 domain to an in BAD heterodimerization with cell death we the of Ser-155 phosphorylation and on heterodimerization with Bcl-XL. BAD and BAD S155E from were phosphorylated in with the catalytic subunit of and its to to Bcl-XL was by on immobilized MBP·Bcl-XL in 5 A, PKA binding of wild-type BAD to Bcl-XL but on the binding of BAD S155A to Bcl-XL. with the of phosphorylation at Ser-155, we also that BAD S155E blocked binding to Bcl-XL both in and in 5 in 5 levels of BAD and BAD S155A were with Bcl-XL BAD S155E was We also using immobilized Bcl-XL to BAD from extracts prepared from HEK293 cells transfected with BAD or in BAD and S155A were retained on the Bcl-XL BAD S155E was these that phosphorylation of Ser-155 or of Ser-155 to blocks binding of BAD to Bcl-XL. in BAD and BAD S155A were cell death In HEK293 cells 24 h after transfection with BAD or BAD was survival of transfected of BAD with RSK1 can BAD-mediated cell BAD S155A or BAD S112A/S136A and survival BAD S155E cell death when with or BAD. of phosphorylation sites the cytotoxic with by that survival signals in at two to inhibit the activity of first by phosphorylating BAD at Ser-112 and Ser-136 to stimulate binding of 14-3-3 proteins and sequestering BAD from the mitochondrial membrane (2Zha 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) and second by phosphorylation of BAD at Ser-155, which directly inhibits BH3 and with Bcl-XL. Phosphorylation of BAD at Ser-155 is to a phosphate directly the BH3 hydrophobic face and the Bcl-XL hydrophobic (14Zha 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 (267) Google J.M. D. Korsmeyer S.J. D. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). is with the effects of BAD Ser-155 as as involving mutagenesis of BAD and BAX BH3 (14Zha 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 (267) Google Scholar, K. Gross A. Waksman G. Korsmeyer S.J. Mol. Cell. Biol. 1998; 18: 6083-6089Crossref PubMed Scopus (189) Google Scholar). The BH3 Ser-155 is similar to BAX where mutagenesis has the importance of BAD Ser-155 in mediating BAX heterodimerization (15Wang K. Gross A. Waksman G. Korsmeyer S.J. Mol. Cell. Biol. 1998; 18: 6083-6089Crossref PubMed Scopus (189) Google Scholar). here for BAD BAX blocked heterodimerization and BAX death-promoting activity (15Wang K. Gross A. Waksman G. Korsmeyer S.J. Mol. Cell. Biol. 1998; 18: 6083-6089Crossref PubMed Scopus (189) Google Scholar). of the of BH3 domain K. Korsmeyer S.J. Genes Dev. 1996; 10: PubMed Scopus Google Scholar, H. H. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, X. H. C. X. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google has that the BH3 hydrophobic face is within the and is by caspase the death-promoting activity J.M. D. Korsmeyer S.J. D. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. H. J. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. G. 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Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google complete sequestration may activation of both that phosphorylates BAD at both Ser-112 and Ser-155 and rescues BAD-mediated cell death in a manner dependent upon phosphorylation at both sites. is to Ser-155 phosphorylation a to of cell death or is to BAD-induced death-promoting activity. the protein kinases of the family are of the cascade that are by different growth and survival factors A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1682) Google Scholar, 9Shimamura A. Ballif B.A. Richards S.A. Blenis J. Curr. Biol. 2000; 10: 127-135Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, J. S. A. PubMed Scopus Google Scholar, Science. 1999; 286: PubMed Scopus Google Ser-155 may to be a of multiple survival We for Joseph Avruch for pMT2·RSK1, Christian for pMT2·RSK2 and and for the fusion proteins, and for GST·BAD and Stanley Korsmeyer for and BAD Ser-112 and Ser-136 and pCMV·Bcl-XL We also and for and and for with