Growth Factors Inactivate the Cell Death Promoter BAD by Phosphorylation of Its BH3 Domain on Ser155
2000; Elsevier BV; Volume: 275; Issue: 32 Linguagem: Inglês
10.1074/jbc.m002526200
ISSN1083-351X
AutoresXiaomai Zhou, Yimao Liu, Gillian Payne, Robert J. Lutz, Thomas W. Chittenden,
Tópico(s)Signaling Pathways in Disease
ResumoThe Bcl-2 family protein BAD promotes apoptosis by binding through its BH3 domain to Bcl-xL and related cell death suppressors. When BAD is phosphorylated on either Ser112 or Ser136, it forms a complex with 14-3-3 in the cytosol and no longer interacts with Bcl-xLat the mitochondria. Here we show that phosphorylation of a distinct site Ser155, which is at the center of the BAD BH3 domain, directly suppressed the pro-apoptotic function of BAD by eliminating its affinity for Bcl-xL. Protein kinase A functioned as a BAD Ser155 kinase both in vitro and in cells. BAD Ser155 was found to be a major site of phosphorylation induced following stimulation by growth factors and prevented by protein kinase A inhibitors but not by inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Growth factors inhibited BAD-induced apoptosis in both a Ser112/Ser136- and a Ser155-dependent fashion. Thus, growth factors engage an anti-apoptotic signaling pathway that inactivates BAD by direct modification of its BH3 cell death effector domain. The Bcl-2 family protein BAD promotes apoptosis by binding through its BH3 domain to Bcl-xL and related cell death suppressors. When BAD is phosphorylated on either Ser112 or Ser136, it forms a complex with 14-3-3 in the cytosol and no longer interacts with Bcl-xLat the mitochondria. Here we show that phosphorylation of a distinct site Ser155, which is at the center of the BAD BH3 domain, directly suppressed the pro-apoptotic function of BAD by eliminating its affinity for Bcl-xL. Protein kinase A functioned as a BAD Ser155 kinase both in vitro and in cells. BAD Ser155 was found to be a major site of phosphorylation induced following stimulation by growth factors and prevented by protein kinase A inhibitors but not by inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Growth factors inhibited BAD-induced apoptosis in both a Ser112/Ser136- and a Ser155-dependent fashion. Thus, growth factors engage an anti-apoptotic signaling pathway that inactivates BAD by direct modification of its BH3 cell death effector domain. Bcl-2 homology domain platelet-derived growth factor epidermal growth factor glutathione S-transferase cAMP-dependent protein kinase A polymerase chain reaction hemagglutinin PKA inhibitor phosphatidylinositol Bcl-2 family proteins are important regulators of apoptosis that function during development and other physiological processes but also contribute to pathological conditions associated with inappropriate cell survival such as cancer (1Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3254) Google Scholar, 2Strasser A. Huang D.C. Vaux D.L. Biochim. Biophys. Acta. 1997; 1333: F151-F178PubMed Google Scholar). This still expanding family contains both pro- and anti-apoptotic members characterized by the presence of one or more Bcl-2 homology (BH)1 domains. Of these domains, BH3 has proven to be a key element in pro-apoptotic Bcl-2 homologs that mediate both protein binding and cell death functions (1Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3254) Google Scholar,3Chittenden T. Flemington C. Houghton A.B. Ebb R.G. Gallo G.J. Elangovan B. Chinnadurai G. Lutz R.J. EMBO J. 1995; 14: 5589-5596Crossref PubMed Scopus (438) Google Scholar). In Caenorhabditis elegans, the BH3-containing protein EGL-1 functions at the most proximal point in a pathway required for all programmed cell death, and in mammalian cells BH3 proteins transduce signals from cell surface receptors to a central cell death pathway regulated by Bcl-2 (4Conradt B. Horvitz H.R. Cell. 1998; 93: 519-529Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 5Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3790) Google Scholar, 6Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3080) Google Scholar). BAD is a "BH3-only" pro-apoptotic Bcl-2 family member whose function is regulated by phosphorylation in response to survival factors such as nerve growth factor, insulin-like growth factor-1, and interleukin-3 (7Yang 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, 8Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2255) Google Scholar, 9Datta 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 (4946) Google Scholar). In its unphosphorylated state, BAD forms heterodimers with anti-apoptotic Bcl-2 homologs and promotes cell death. These activities are inhibited by phosphorylation of BAD on either of two serine residues, Ser112 or Ser136 (8Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2255) Google Scholar). The serine/threonine kinase Akt that is activated by growth factors through a PI 3-kinase-dependent mechanism phosphorylates BAD on Ser136 (9Datta 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 (4946) Google Scholar, 10Peso L.D. González-Garcı́a M. Page C. Herera R. Nuñez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1987) Google Scholar). The ribosomal S6 kinases and a mitochondria-localized cAMP-dependent protein kinase (PKA) have been reported to phosphorylate BAD on Ser112 (11Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1680) Google Scholar, 12Harada H. Becknell B. Wilm M. Mann M. Huang L.J.-S. 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) following stimulation by growth factors and interleukin-3, respectively. When BAD is phosphorylated on these sites, it is sequestered in the cytosol in a complex with 14-3-3 and fails to interact with Bcl-xL at mitochondrial membranes (8Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2255) Google Scholar). The present study provides genetic, biochemical, and biological evidence that growth factor-induced phosphorylation on the novel site Ser155, which is within the functionally critical BH3 domain, directly blocks BAD binding to Bcl-xL and may represent another major regulatory mechanism of BAD inactivation. A cDNA for murine BAD was obtained by reverse transcriptase-PCR using mRNA isolated from FL5.12 cells and the following PCR primers: 5′-GCCTCCAGGATCCAAGATGGGAACC-3′ and 5′-GGAGCGGGTAGAATTCCGGGATG-3′. When this cDNA, which encodes the 204-amino acid BAD protein (7Yang 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), was cloned into pcDNA3 (Invitrogen) and was expressed in cells, the protein product was significantly larger than the endogenous BAD protein (approximately 30 kDa versus 23 kDa, respectively) detected with an anti-BAD antibody (C-20, Santa Cruz Biotechnology, Inc.). A methionine residue at position 43 of the mouse sequence corresponds to the first methionine residue of the human BAD. We generated a cDNA for the shorter form of murine BAD by using an upstream PCR primer surrounding the second methionine residue (5′-TGGAGACCAGGATCCCAGAGTAGCT-3′) and the same downstream primer as above. The expressed construct co-migrated with the endogenous BAD of FL5.12 cells, suggesting that the shorter form of BAD (162 amino acids) is the major translation product of BAD in these cells. Serine residues 112, 136, 134, and 155 were substituted with alanine, and serine 155 was also substituted with aspartic acid using PCR-mediated mutagenesis in the context of the shorter form of BAD. (Amino acid numbering was not changed to be consistent with previous conventions; serine positions are actually 70, 94, 92, and 113, respectively.) The nucleotide sequence of BAD and its mutants was confirmed by DNA sequencing. The cDNA of BAD and its mutants were cloned into a pcDNA3-HA vector, which introduces the HA epitope at the amino terminus of BAD coding sequences. The cDNA encoding the human protein kinase inhibitor for cAMP-dependent protein kinase (PKI) was isolated from total mRNA of HeLa cells by reverse transcriptase-PCR and was inserted in frame into pcDNA3-HA. Rat-1, COS-7, and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfections were carried out with the Superfect reagent from Qiagen or the GenePortor transfection reagent from Gene Therapy Systems. For generation of cell lysates, cells were washed with ice-cold phosphate-buffered saline and lysed in radioimmune precipitation buffer (50 mm HEPES, pH 7.5, 1% deoxycholate acid, 1% Nonidet P-40, 0.1% SDS, 150 mmNaCl, 1 mm Na3(VO)4, 1 mm NaO3P7, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 20 μg/ml aprotinin). Lysates were cleared by centrifugation at 16,000 ×g for 15 min at 4 °C. For Western blot analysis, protein samples were electrophoresed on 16 or 18% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Blots were blocked in Western wash buffer (40 mm Tris, pH 8.0, 150 mm NaCl, 0.2% Nonidet P-40) with 5% bovine serum albumin for 1 h at room temperature. Blots were incubated with primary antibody diluted in Western wash buffer with 3% bovine serum albumin at room temperature for 1–2 h and then were washed and incubated with secondary horseradish peroxidase-coupled antibody diluted in Western wash buffer with 1.5% bovine serum albumin and washed extensively. Proteins were detected by the ECL method according to the manufacturer's instructions (Amersham Pharmacia Biotech). Anti-HA antibody (3F10) was from Roche Molecular Biochemicals. Anti-Akt (sc1618) was from Santa Cruz Biotechnology, Inc., anti-p473-Akt (9271S) was from New England BioLabs, and anti-activated EGF receptor (E12120) was from Transduction Laboratories. DNA encoding BAD and various mutants was excised from pcDNA3 as a BamHI-EcoRI fragment and inserted into the BamHI-EcoRI cloning sites of pGEX-2T (Amersham Pharmacia Biotech). GST fusion proteins were expressed in Escherichia coli strain DH5α. Cells (500 ml) were grown to an A 595 of 0.7–0.9 at 37 °C and induced with 2 mm isopropyl β-d-thiogalactopyranoside at 30 °C overnight. Cells were collected by centrifugation and suspended in 20 ml of HBS (10 mm HEPES, pH 7.5, 3.4 mm EDTA, 150 mm NaCl) plus 1% (v/v) Triton X-100 and 10 mmβ-mercaptoethanol. Cells were lysed by two passages through a Microfluidizer M110S (Microfluidics), and cell debris was removed by centrifugation for 30 min at 20,000 × g. A 20% ammonium sulfate precipitation was performed on the cell lysate to remove aggregated protein, and GST·BAD was purified from the supernatant by glutathione-agarose chromatography. The fractions containing GST·BAD were pooled and concentrated using a Centriprep 3 (Amicon), and the protein concentration was determined by the Bradford assay (Bio-Rad). In vitro kinase assays were carried out in 30-μl volumes containing 10 mm HEPES, pH 7.5, 100 mm NaCl, 12 mm MgCl2, 1 mm dithiothreitol, 15 μCi of [γ-32P]ATP (6000 Ci/mmol, NEN Life Science Products), 11 units of protein kinase A (catalytic subunit from bovine heart, Calbiochem), and 1 μg of purified GST·BAD. Reactions were incubated for 30 min at 30 °C and terminated by the addition of SDS-polyacrylamide gel electrophoresis sample buffer. The anti-phospho-Ser155-BAD antibody was produced at Bio-Synthesis Inc. (Lewisville, TX). A phosphopeptide of the sequence NH2-GCQRYGRELRRMpSDESVDSF-COOH where pS is phosphoserine was synthesized at >70% purity, conjugated to keyhole limpet hemocyanin, and injected into rabbits. Immune serum (10 weeks) reacted with Ser155-phosphorylated BAD in Western blot analysis (1:500 dilution). This antibody reacts exclusively with the Ser155-phosphorylated form of BAD. The antibody recognized the phosphorylated (upper) band of BAD S112A/S136A from transfected cell lysates, did not react with the lower unphosphorylated band (see Fig. 2 C), and failed to recognize either Ser112- or Ser136-phosphorylated BAD (data not shown). HeLa cells were plated in 12-well plates at 5 × 104 cells/well 1 day prior to transfection. Cells were transfected with BAD (or BAD mutants) and β-galactosidase expression plasmids in a ratio of 4:1 (0.6 μg:0.15 μg) using the Superfect transfection reagent (Qiagen). Twenty-four hours after transfection, cells were cultured in serum-free medium or in serum-free medium plus EGF for an additional 12 h. β-Galactosidase activity was measured in extracts using a fluorogenic substrate (MUG, Bio-Rad FluorAceTM β-galactosidase reporter assay, 170–3150). The loss of β-galactosidase activity in these assays reflects apoptosis and elimination of the transfected cells, and the β-galactosidase reductions were reversed by the addition of a broad spectrum caspase inhibitor, Z-VAD-fluoromethylketone (where Z is benzyloxycarbonyl, data not shown). Immunlon 2 (Dynatech) microtiter plates were coated with 5 μg/ml neutravidin (50 μl/well, Pierce) in sodium bicarbonate buffer, pH 9.0, overnight at 4 °C. All remaining steps were conducted at room temperature. Plates were washed two times with phosphate-buffered saline containing 0.1% Tween 20 (wash buffer) and blocked for 1 h with 0.2 ml/well 1% normal goat serum in phosphate-buffered saline. Following two additional washes, 1.25 μg/ml Bak BH3 19-mer peptide (residues 71–89) that was biotinylated at the amino terminus was added to the wells in 50 μl of 10 mm HEPES buffer, pH 7.2, containing 150 mm KCl, 5 mm MgCl2, 1 mm EGTA, and 0.2% Nonidet P-40 (Nonidet P-40 buffer). After 30 min, the wells were washed twice with wash buffer, and GST·Bcl-xL (0.25 μm in 50 μl of Nonidet P-40 buffer) was added in the absence or presence of BAD or Bak BH3 peptides. Following a 1-h incubation, the plates were washed twice with wash buffer, and the amount of bound GST·Bcl-xL was determined by enzyme-linked immunosorbent assay using an anti-GST primary antibody and a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Jackson) with ABTS (Zymed Laboratories Inc.) as substrate. Five washes were conducted following each 1-h antibody incubation. GST·Bcl-xL fusion protein was produced in E. coli by a similar procedure that was described for the production of GST·BAD (see above). Immunoblot analysis of a BAD mutant that was unable to be phosphorylated on Ser112 and Ser136 revealed that BAD was phosphorylated on a third site. BAD S112A/S136A expressed in COS-7 cells or in stably transfected MCF-7 cells (data not shown) migrated as a doublet on a large dimension 16% SDS-polyacrylamide gel (Fig. 1 A). Phosphatase treatment of the transfected cell lysate completely eliminated the upper band of this doublet, confirming the existence of a phosphorylation site on BAD distinct from both Ser112 and Ser136. The functional significance was examined by testing the impact of this phosphorylation on the ability of BAD to dimerize with Bcl-xL. GST·Bcl-xL bound only to the faster migrating form of BAD in lysate from BAD S112A/S136A-transfected cells (Fig. 1 A), indicating that phosphorylation on the site distinct from Ser112 and Ser136 prevents interaction with Bcl-xL. To localize the novel phosphorylation site in BAD, selected serine residues in addition to Ser112 and Ser136 were substituted with alanine. Alanine substitution of Ser155but not Ser134 eliminated the slower migrating band of the doublet when expressed in COS-7 cells (BAD S112A/S136A/S155A; Fig.1 B) suggesting that Ser155 could be a third site of phosphorylation on BAD. A single Ser155 to alanine mutation significantly reduced the overall extent of BAD phosphorylation in transfected HeLa cells and eliminated the slowest migrating phosphorylated form of BAD observed following stimulation with serum or EGF (Fig. 1 C). Phosphorylation on Ser155 was confirmed directly by demonstrating reactivity of BAD to a phospho-Ser155-specific antibody (see below). Interestingly, Ser155 is located at the center of the BH3 domain of BAD, the structural element that mediates BAD/Bcl-xL dimerization (13Kelekar 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, 14Ottilie S. Diaz J.L. Horne W. Chang J. Wang Y. Wilson G. Chang S. Weeks S. Fritz L.C. Oltersdorf T. J. Biol. Chem. 1997; 272: 30866-30872Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 15Zha 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). The sequence surrounding Ser155, LRRM SD, matches the consensus phosphorylation site for mammalian PKA,X RR X S X(16Kemp B.E. Pearson R.B. Trends Biochem. Sci. 1990; 15: 342-346Abstract Full Text PDF PubMed Scopus (807) Google Scholar). To test whether PKA can phosphorylate BAD on Ser155 in vitro, purified PKA was incubated with GST fusion proteins of BAD or different BAD mutants in the presence of [γ-32P]ATP. PKA did phosphorylate wild-type GST·BAD (Fig. 2 A) although not in the presence of a PKI fragment that is a specific PKA inhibitor (17Day R.N. Walder J.A. Maurer R.A. J. Biol. Chem. 1989; 264: 431-436Abstract Full Text PDF PubMed Google Scholar) (data not shown). Alanine substitution of Ser112 and Ser136, either alone or in combination, did not substantially reduce the level of BAD phosphorylation by PKA in vitro (Fig. 2 A). In contrast, mutation of Ser155 dramatically reduced (S155A single mutant) or completely abolished (S112A/S136A/S155A triple mutant) phosphorylation (Fig. 2 A) indicating that Ser155 is the major phosphorylation site of PKA in vitro. To test whether PKA may function as a Ser155 kinase of BADin vivo, we examined whether known activators of PKA would stimulate BAD Ser155 phosphorylation in cells. Forskolin, which stimulates PKA through the activation of adenylate cyclase, rapidly induced a sustained gel mobility shift of BAD S112A/S136A in transfected HeLa cells that was sensitive to phosphatase treatment (Fig. 2 B). In contrast, forskolin had no effect on the BAD S112A/S136A/S155A mutant, indicating that Ser155 is the likely site of phosphorylation by a cAMP-dependent kinasein vivo (Fig. 2 B). An anti-phospho-Ser155-specific BAD antibody (see "Experimental Procedures") reacted with the phosphorylated bands of wild-type BAD and BAD S112A/S136A induced by forskolin in transfected HeLa cells (Fig. 2 C, upper panels). This antibody is specific for phospho-Ser155-BAD; it failed to react with BAD S155A or the BAD triple mutant although the expression levels of BAD and the BAD mutants were at similar levels (Fig. 2 C, lower panels). These results provide direct evidence for BAD phosphorylation on Ser155. Because adenylate cyclase is a target of activated G protein-coupled receptors (18Linder M.E. Gilman A.G. Sci. Am. 1992; : 56-65PubMed Google Scholar), we also examined whether BAD Ser155 phosphorylation would respond to ligands of G protein-coupled receptors. Of the ligands tested in HeLa cells,l-epinephrine rapidly induced a gel mobility shift of BAD S112A/S136A (Fig. 2 D) that was blocked by mutation of BAD Ser155 to alanine or by the co-expression of PKI (17Day R.N. Walder J.A. Maurer R.A. J. Biol. Chem. 1989; 264: 431-436Abstract Full Text PDF PubMed Google Scholar) (data not shown). Together, these results demonstrate that BAD Ser155 can be phosphorylated in cells either by PKA directly or through a PKA-dependent pathway. Growth factors suppress the pro-apoptotic function of BAD in part through the activation of Akt, which functions as a BAD Ser136 kinase (9Datta 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 (4946) Google Scholar, 10Peso L.D. González-Garcı́a M. Page C. Herera R. Nuñez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1987) Google Scholar). We examined whether growth factor stimulation would also induce Ser155 phosphorylation of BAD. EGF stimulated the phosphorylation of BAD on Ser155 in transfected HeLa cells, which was blocked by co-expression of PKI (Fig.3 A). To test whether phosphorylation of Ser155 is likewise dependent on the PI 3-kinase/Akt pathway, we treated transfected HeLa cells with wortmannin, an inhibitor of PI 3-kinase, and analyzed BAD Ser155 phosphorylation following EGF stimulation. Wortmannin treatment prevented the activation of endogenous Akt by PI 3-kinase following EGF stimulation but did not impair EGF-induced phosphorylation of BAD on Ser155 (Fig. 3 B). In parallel assays, BAD Ser155 phosphorylation was prevented by the addition of the PKA inhibitor H89 without affecting the phosphorylation of Akt. The EGF receptor kinase inhibitor AG1478 blocked the phosphorylation of both Akt and BAD Ser155. These results demonstrate that EGF stimulates the phosphorylation of BAD Ser155 through a PKA-dependent mechanism that is distinct from the PI 3-kinase/Akt pathway. To examine whether endogenous BAD is phosphorylated on Ser155 in response to growth factors, serum-starved Rat-1 fibroblasts were stimulated with PDGF, endogenous BAD was immunoprecipitated, and Ser155 phosphorylation was analyzed by Western blot with the anti-phospho-Ser155 antibody. PDGF induced the phosphorylation of endogenous BAD on Ser155, which was significantly reduced by pretreatment of cells with the PKA inhibitor H89 but not the PI 3-kinase inhibitor wortmannin (Fig. 3,C and D). The pro-apoptotic function of BAD requires its ability to heterodimerize with Bcl-xL and related proteins (13Kelekar 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, 14Ottilie S. Diaz J.L. Horne W. Chang J. Wang Y. Wilson G. Chang S. Weeks S. Fritz L.C. Oltersdorf T. J. Biol. Chem. 1997; 272: 30866-30872Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 15Zha 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). Thus, the phosphorylation of Ser155 in response to growth/survival factors (Fig. 3) and its apparent inhibitory effect on binding to Bcl-xL (Fig. 1 A) suggest that it may suppress the pro-apoptotic activity of BAD. In keeping with this possibility, mutation of BAD Ser155 to a residue that cannot be phosphorylated enhanced the pro-apoptotic function of BAD in transient transfection assays. BAD S155A showed a modest but highly reproducible enhancement of toxicity compared with wild-type BAD, and the S112A/S136A/S155A triple mutant consistently exhibited greater toxicity than the S112A/S136A double mutant (Fig.4 A, open bars). Western blot analysis demonstrated that the enhanced toxicity of these mutants could not be attributed to elevated levels of protein expression in these assays (Fig. 4 A, inset). EGF stimulation, which induces BAD Ser155 phosphorylation, suppressed the pro-apoptotic function of BAD in these assays. Mutation of Ser155 to alanine alone did not eliminate the protective effect of EGF consistent with the ability of EGF to inactivate BAD through the phosphorylation of Ser112 and/or Ser136. However, the pro-apoptotic activity of a BAD S112A/S136A double mutant was also suppressed by EGF stimulation revealing the ability of EGF to inactivate BAD through a distinct mechanism. Our data indicate that this mechanism involves Ser155 because mutation of Ser155 to alanine in the context of S112A/S136A completely inhibited the anti-apoptotic effect of EGF stimulation (S112A/S136A/S155A triple mutant, Fig.4 A, AAA, filled bars). The β-galactosidase reporter assay was valid as a measurement of apoptosis because BAD-induced reduction of β-galactosidase activity was reversed by the treatment of cells with Z-VAD, a broad spectrum caspase inhibitor (data not shown). These results indicate that the anti-apoptotic effects of EGF can be mediated through Ser155 phosphorylation independently of Ser112/Ser136 phosphorylation and are consistent with the distinct EGF-activated signaling pathways that lead to the phosphorylation of BAD on Ser136 and on Ser155 (Fig. 3, B and D). To further evaluate the impact of Ser155 phosphorylation on the pro-apoptotic activity of BAD, we substituted Ser155with aspartic acid (S155D) to mimic the negatively charged phospho-Ser155 residue. Upon expression in cells, BAD S155D showed no pro-apoptotic activity compared with wild-type BAD (Fig.4 B). Consistent with this result, the BAD S155D mutant did not associate with co-expressed Bcl-xL in a co-immunoprecipitation experiment although wild-type BAD and BAD S155A did (data not shown). These results further support the hypothesis that phosphorylation of Ser155 leads to the functional inactivation of BAD in cells. Ser155 within a PKA consensus site is evolutionarily conserved at the center of the BH3 domain of BAD whereas BH3 domains of other known pro-apoptotic Bcl-2 homologs contain glycine at this position (Fig.5 A). NMR studies have revealed that BH3 forms an α-helical structure, which binds to a hydrophobic cleft on the surface of Bcl-xL (19Sattler M. Liang H. Nettesheim D. Meadows R.P. Harlan J.E. Eberstadt M. Yoon H.S. Shuker S.B. Chang B.S. Minn A.J. Thompson C.B. Fesik S.W. Science. 1997; 275: 983-986Crossref PubMed Scopus (1291) Google Scholar). We tested whether the addition of a phosphate group on BAD Ser155 interferes with this interaction by measuring the affinity of synthetic BAD BH3 peptides for Bcl-xL in an in vitro competition binding assay. A BH3 peptide encompassing BAD residues 143–168 bound to Bcl-xL with an affinity similar to a Bak BH3 control peptide. However, the affinity of a matched peptide that was phosphorylated on Ser155 was reduced by greater than 100-fold (Fig. 5 B). Moreover, in the context of full-length BAD produced by in vitro translation phosphorylation by PKA induced a gel mobility shift of wild-type (but not S155A mutant) BAD and blocked the ability of BAD to bind to Bcl-xL (Fig.5 C). These results demonstrate that phosphorylation on Ser155 is sufficient to directly inactivate the heterodimerization function of BH3 and provide a biochemical mechanism for how the pro-apoptotic function of BAD is suppressed by Ser155 phosphorylation. Mutational studies have provided strong evidence that BAD promotes apoptosis by dimerizing with Bcl-xL and related proteins (13Kelekar 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), emphasizing the importance of understanding how phosphorylation regulates this interaction. Our findings suggest that phosphorylation on different sites within BAD have mechanistically distinct consequences; Ser155 phosphorylation directly prevents heterodimerization by abolishing the affinity of BAD BH3 for Bcl-xL whereas phosphorylation on Ser112 and Ser136 that are located outside the BH3 domain may inhibit dimerization with Bcl-xL indirectly. In particular, phosphorylation on Ser112 or Ser136, but not Ser155, generates a consensus binding site for the cytosolic protein 14-3-3 that may bind and alter the subcellular distribution of BAD to prevent interaction with Bcl-xL at mitochondrial membranes (8Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2255) Google Scholar). Phosphorylation of BAD provides an important link between extracellular survival factors and the intrinsic cell death pathway regulated by Bcl-2. The prevailing model for anti-apoptotic signaling by growth/survival factors emphasizes a pathway involving the PI 3-kinase-dependent activation of Akt, phosphorylation of BAD on Ser136, and subsequent dissociation from Bcl-xL. Our findings identify a distinct anti-apoptotic pathway that leads to the biochemical inactivation of the BAD BH3 domain through a PKA-dependent but PI 3-kinase/Akt-independent mechanism. PKA-mediated phosphorylation of BAD Ser155 may explain, at least in part, the anti-apoptotic effects of PKA activation that have been observed in some settings (20Berridge M.V. Tan A.S. Hilton C.J. Exp. Hematol. 1993; 21: 269-276PubMed Google Scholar, 21Parvathenani L.K. Buescher E.S. Chacon-Cruz E. Beebe S.J. J. Biol. Chem. 1998; 273: 6736-6743Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22Fladmark K.E. Gjertsen B.T. Doskeland S.O. Vintermyr O.K. Biochem. Biophys. Res. Commun. 1997; 232: 20-25Crossref PubMed Scopus (58) Google Scholar) and PI 3-kinase-independent survival signals triggered by growth factors (23Kulik G. Weber M.J. Mol. Cell. Biol. 1998; 18: 6711-6718Crossref PubMed Scopus (230) Google Scholar, 24Burtscher I. Compagni A. Lamm G.M. Christofori G. Cancer Res. 1999; 59: 3923-3926PubMed Google Scholar). Precisely how growth factors activate PKA in cells is not clear although there have been reports showing that PDGF activates PKA by stimulating its release from cell membrane (25deBlaquiere J. Walker F. Michelangeli V.P. Fabri L. Burgess A.W. J. Biol. Chem. 1994; 269: 4812-4818Abstract Full Text PDF PubMed Google Scholar) and that EGF activates PKA via Grb-2-mediated recruitment of PKA to the EGF receptor (26Tortora G. Pepe S. Bianco C. Damiano V. Ruggiero A. Baldassarre G. Corbo C. Cho-Chung Y.S. Bianco A.R. Ciardiello F. Int. J. Cancer. 1994; 59: 712-716Crossref PubMed Scopus (24) Google Scholar). Mitochondrial membrane-localized PKA was identified as an interleukin-3-induced kinase of BAD Ser112 (12Harada H. Becknell B. Wilm M. Mann M. Huang L.J.-S. 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). This is a paradox because our results indicate that Ser112 is a poor substrate for PKA relative to Ser155 both in vitro and in cells. It is possible that Ser155 may prove to be an important target for the mitochondria-localized PKA because the BAD substrate used in that study was a mutant lacking Ser155 (12Harada H. Becknell B. Wilm M. Mann M. Huang L.J.-S. 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). The convergence of multiple growth factor-stimulated pathways on BAD underscores the important role of this BH3 protein in anti-apoptotic signaling by cell surface receptors. It has been generally proposed that BAD kinases modify the unphosphorylated form of BAD bound to Bcl-xL at mitochondrial membranes, thereby releasing free active Bcl-xL. Both PKA and Akt, however, are unable to phosphorylate BAD in vitro on residues Ser155and Ser136, respectively, when BAD is in a pre-existing complex with Bcl-xL. 2X.-M. Zhou and R. J. Lutz, unpublished observations. Although substrate accessibility may be different in cells, this finding suggests that PKA and Akt may inactivate BAD not by dissociating existing BAD·Bcl-xL complexes but rather by preventing its accumulation in a dephosphorylated "active" state. The relative significance of PKA- and Akt-mediated pathways to the inactivation of BAD in different settings remains to be determined. In tumor cell lines, our preliminary results indicate that basal BAD Ser155 phosphorylation is higher than in non-transformed cells and is more refractory to dephosphorylation upon growth factor deprivation. 3X.-M. Zhou and Y. Liu, unpublished observations. This raises the possibility that the inactivation of BAD BH3 through Ser155phosphorylation makes an important contribution to the suppression of apoptosis in cancer cells. We thank our colleagues at Apoptosis Technology, Inc. and BioChem Pharma, Inc. for numerous reagents and helpful discussions.
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