Phosphorylation of the Pro-apoptotic Protein BIK

Abstract

BIK is a pro-apoptotic BCL-2 family member and is the founding member of a subfamily of pro-apoptotic proteins known as “BH3-alone” proteins. Ectopic expression of BIK induces apoptosis in variety of mammalian cells. BIK complexes with various anti-apoptotic BCL-2 family proteins such as adenovirus E1B-19K and BCL-2 via the BH3 domain. However, the heterodimerization activity of BIK alone is insufficient for its apoptotic activity. Previous studies have shown that phosphorylation regulates the functional activity of both anti-apoptotic and pro-apoptotic members of the BCL-2 family. Here, we have examined phosphorylation of BIK and its effect on the apoptotic activity of BIK. We show that BIK exists as a phosphoprotein and is phosphorylated at residues 33 (threonine) and 35 (serine). Mutation of the phosphorylation sites, in which the Thr and Ser residues were changed to alanine residues, reduced the apoptotic activity of BIK without significantly affecting its ability to heterodimerize with BCL-2. Our results suggest that phosphorylation of BIK is required for eliciting efficient apoptotic activity. Partial purification of the protein kinase from HeLa cell cytoplasmic extracts suggest that BIK may be phosphorylated by a casein kinase II-related enzyme. BIK is a pro-apoptotic BCL-2 family member and is the founding member of a subfamily of pro-apoptotic proteins known as “BH3-alone” proteins. Ectopic expression of BIK induces apoptosis in variety of mammalian cells. BIK complexes with various anti-apoptotic BCL-2 family proteins such as adenovirus E1B-19K and BCL-2 via the BH3 domain. However, the heterodimerization activity of BIK alone is insufficient for its apoptotic activity. Previous studies have shown that phosphorylation regulates the functional activity of both anti-apoptotic and pro-apoptotic members of the BCL-2 family. Here, we have examined phosphorylation of BIK and its effect on the apoptotic activity of BIK. We show that BIK exists as a phosphoprotein and is phosphorylated at residues 33 (threonine) and 35 (serine). Mutation of the phosphorylation sites, in which the Thr and Ser residues were changed to alanine residues, reduced the apoptotic activity of BIK without significantly affecting its ability to heterodimerize with BCL-2. Our results suggest that phosphorylation of BIK is required for eliciting efficient apoptotic activity. Partial purification of the protein kinase from HeLa cell cytoplasmic extracts suggest that BIK may be phosphorylated by a casein kinase II-related enzyme. BCL-2 homology domain dithiothreitol hemagglutinin protein kinase C amino acid(s) baby rat kidney polyacrylamide gel electrophoresis wild type phosphate-buffered saline 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside casein kinase II Apoptosis is a physiological process of cell death shared by all multicellular organisms and is critical for removing unwanted cells during development. This process is essential for the maintenance of normal tissue homeostasis. Cell death or survival is dependent on the receipt of continuous signals from the extracellular environment. These signals are then transduced through the cell surface to intracellular molecules that regulate apoptotic cell death. A number of such regulators have been identified. Among them, the BCL-2 family proteins play an important role in both induction and suppression of apoptosis (reviewed in Ref. 1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4755) Google Scholar). The anti-apoptotic members include the cellular proteins such as BCL-2, BCL-xL, MCL-1, BFL1, and BCL-w and viral proteins such as Epstein-Barr virus BHRF1 and adenovirus E1B-19K proteins. These cellular and viral proteins suppress apoptosis induced by diverse stimuli. Certain pro-apoptotic proteins such as BAX and BAK, despite sharing extensive homology with BCL-2, induce apoptosis when overexpressed. Most other pro-apoptotic proteins such as BAD, BID, BIK, BIM, BNIP3, BNIP1, HRK, and NOXA share only a single domain (BH3)1 with BCL-2 and are hence designated “BH3-alone” proteins; the human BIK protein is the founding member of this family. In Caenorhabditis elegans, a BH3-alone protein Egl-1 has been shown to be essential for developmentally programmed death of somatic cells (2Conradt B. Horvitz H.R. Cell. 1998; 93: 519-529Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). An interesting feature of the BCL-2 family of proteins is the ability of the pro-apoptotic members to heterodimerize with the anti-apoptotic members (reviewed in Ref. 1Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4755) Google Scholar). This suggests that one of the mechanisms by which anti-apoptotic members of the BCL-2 family suppress apoptosis may be through heterodimerization with pro-apoptotic members. However, at least in the case of BIK, heterodimerization with anti-apoptotic proteins such as BCL-2 and BCL-xL is insufficient for induction of apoptosis (3Elangovan B. Chinnadurai G. J. Biol. Chem. 1997; 272: 24494-24498Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), suggesting that other factors may also influence the apoptotic activity of BIK. Certain apoptotic stimuli appear to modulate the expression and activity of the BCL-2 family proteins at the level of transcription as well as by post-translational modifications. For example, expression of the BH3-alone pro-apoptotic gene Noxa is activated during p53-mediated apoptosis (4Odo E. Ohki R. Murasawa H. Nemoto J. Shibue T. Yamashita T. Tokino T. Taniguchi T. Tanaka N. Science. 2000; 288: 1053-1058Crossref PubMed Scopus (1678) Google Scholar). Hypoxic conditions have shown to activate the expression of another pro-apoptotic protein BNIP3 (5Bruick R.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9082-9087Crossref PubMed Scopus (655) Google Scholar). The BH3-alone protein BIM (6O'Connor L. Strasser A. O'Reilly L.A. Hausmann G. Adams J.M. Cory S. Huang D.C. EMBO J. 1998; 17: 384-395Crossref PubMed Scopus (943) Google Scholar) has been shown to be essential for cytokine withdrawal-induced apoptosis in hemopoietic cells (7Bouillet P. Metcalf D. Huang D.C. Tarlinton D.M. Kay T.W. Kontgen F. Adams J.M. Science. 1999; 286: 1735-1738Crossref PubMed Scopus (1285) Google Scholar). BIM is normally sequestered to the microtubules in an inactive form and is released during apoptosis (8Puthalakath H. Huang D.C. O'Reilly L.A. King S.M. Strasser A. Mol. Cell. 1999; 3: 287-296Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). During apoptosis mediated by the death receptors such as CD95, the BH3-alone protein BID is proteolytically cleaved by caspase-8 to a pro-apoptotic form (tBID) from an inactive form (9Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3050) Google Scholar, 10Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3756) Google Scholar). Post-translational modifications such as phosphorylation also play important roles in regulating the activity of both anti-apoptotic and pro-apoptotic BCL-2 family proteins. BCL-2 contains several potential serine/threonine phosphorylation sites within the “variable region,” which is located between the BH3 and BH4 domains. Phosphorylation of a serine residue (Ser-70) within this region of BCL-2 has been shown to be required for its full anti-apoptotic activity (11Ito T. Deng X. Boyd C. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Deletion of the variable region of BCL-2 has been shown to relieve a novel proliferation restraining activity of BCL-2 (12Uhlman E.J. D'Sa-Eipper C. Chinnadurai G. Cancer Res. 1996; 56: 2506-2509PubMed Google Scholar). Both BCL-2 and BCL-xL have been shown to be phosphorylated in cells following treatment with microtubule disrupting agents (13Poruchynsky M.S. Wang E.E. Rudin C.M. Blagosklonny M.V. Fojo T. Cancer Res. 1998; 58: 3331-3338PubMed Google Scholar). Taken together, it can be concluded that the activities of BCL-2 and BCL-xL can be influenced by phosphorylation. Similarly, phosphorylation of the pro-apoptotic protein BAD on Ser-112, Ser-136, and Ser-155, following interleukin-3 treatment, has been shown to prevent its heterodimerization with BCL-2 and BCL-xL, thus rendering it inactive (14Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2233) Google Scholar, 15Harada H. Becknell B. Wilm M. Mann M. Huang L.J. Taylor S.S. Scott J.D. Mol. Cell. 1999; 3: 413-422Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 16Peso L.D. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1974) Google Scholar, 17Zhou X.M. Liu Y. Payne G. Lutz R.J. Chittenden T. J. Biol. Chem. 2000; 275: 25046-25051Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 18Tan Y. Demeter M.R. Ruan H. Comb M.J. J. Biol. Chem. 2000; 275: 25868-25869Google Scholar). Several protein kinases have been shown to phosphorylate the BCL-2 family members. The pro-apoptotic protein BAD is a target for phosphorylation by the survival promoting protein kinase Akt (19Datta 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 (4895) Google Scholar) and mitochondrial protein kinase A (15Harada H. Becknell B. Wilm M. Mann M. Huang L.J. Taylor S.S. Scott J.D. Mol. Cell. 1999; 3: 413-422Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). Similarly, phosphorylation of non-BCL-2 members also modulates apoptosis. Phosphorylation of pro-caspase 9 by Akt appears to inhibit activation of this caspase by proteolysis (20Cardone M.H. Roy N. Stennicke H.R. Salveson G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2704) Google Scholar). Exposure to DNA damaging agents that leads to p53-mediated apoptosis also induces phosphorylation of p53 at Ser-392 by casein kinase II (CKII) (21Blaydes J.P. Hupp T.R. Oncogene. 1998; 17: 1045-1052Crossref PubMed Scopus (94) Google Scholar). In this article, we report that the pro-apoptotic protein BIK is a phosphoprotein and that phosphorylation is required for the full apoptotic activity of BIK. The HA-tagged BIK expression plasmid (pcDNA3HA-BIK) has been described (22Boyd J.M. Gallo G. Elangovan B. Houghton B. Malstrom S. Avery B.J. Ebb R.G. Subramanian T. Chittenden C. Lutz R.J. Chinnadurai G. Oncogene. 1995; 11: 1921-1928PubMed Google Scholar). Plasmids pET21b-BIK and mutant BIK were constructed by cloning polymerase chain reaction-amplified BIK cDNA encoding aa 1–134 in expression vector pET21b (Novagen) in frame with the His6 tag at the C terminus. All BIK mutants were constructed by polymerase chain reaction according to a method described by Taylor et al. (23Taylor J.W. Ott J. Eckstein F. Nucleic Acids Res. 1985; 13: 8765-8785Crossref PubMed Scopus (563) Google Scholar). His-tagged wt and mutant BIK proteins were expressed in Escherichia coli strain BL21 and purified over Ni2+-NTA-agarose resin (Qiagen). One liter of bacterial culture was centrifuged and resuspended in 10 ml of sonication buffer (50 mm Tris-Cl, pH 7.5, 50 mm NaCl, 0.5 mm DTT, 1% Nonidet P-40). The cell suspension was sonicated four times for 1 min each. The lysates were centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was diluted with sonication buffer without Nonidet P-40 to adjust the Nonidet P-40 concentration to 0.5%. The extracts were loaded onto the Ni2+-NTA columns equilibrated with sonication buffer containing 0.5% Nonidet P-40. The proteins were eluted with a linear gradient of 0–0.3 m imidazole. The wt and mutant (T33A/S35A) BIK proteins were expressed in BSC40 cells using vaccinia/T7 RNA polymerase vector system (24Ausubel F.M. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1992Google Scholar). Briefly, cells were transfected with 10 μg of pcDNA3-based plasmids expressing either HA-tagged wt or mutant (T33A/S35A) BIK and infected with 10 pfu/cell vaccinia virus vTF7–3. The cells were labeled either in phosphate-free DMEM with 100 μCi/ml [32P]orthophosphoric acid (PerkinElmer Life Sciences) or in methionine-free DMEM with [35S]methionine-cysteine mixture (PerkinElmer Life Sciences) for 4 h. Eighteen to 20 h after infection, cells were lysed with 50 mm Tris, pH 7.4, 150 mmNaCl, 0.1% SDS, 1% Nonidet P-40 with the protease inhibitors leupeptin and aprotinin. Cell lysates were subjected to immunoprecipitation using HA monoclonal antibody 12CA5 (Roche Molecular Biochemicals), and the immunoprecipitates were resolved by 12% SDS-PAGE. The proteins were visualized by autoradiography. wt BIK amino or mutant BIK (T33A/S35A) proteins were phosphorylated in in mm pH mm DTT, 10 mm 0.5 mm 50 of by of HeLa cell cytoplasmic or purified HeLa cell The mixture was at 30 for 30 min following of by with the BIK protein was by and examined by autoradiography. BIK was phosphorylated in using HeLa cell The phosphorylated protein was by and the gel containing phosphorylated BIK was and with and 50 mm The protein was with 100 μg of in 50 for h at °C. The were in at for h and were to the which were onto a and by electrophoresis at for h with pH buffer T. P. M. T. Cell. Full Text PDF PubMed Scopus Google Scholar). The of were by and autoradiography. HeLa cells in were transfected with μg of HA wt BIK or mutant BIK (T33A/S35A) expressed from the with μg of the plasmid by the The cells were at h after and with Briefly, the cells were with and with 0.5% for the cells with for were with ml of mm mm mm 1 in at The cells were examined and cells were as and cells. were using baby rat kidney cells from as described T. M. R.J. Chinnadurai G. Oncogene. Google Scholar). The of this to the activity of pro-apoptotic proteins has been described T. Chinnadurai G. W.S. in Molecular and New Scholar). HeLa cells × were from tissue culture with The cell was in buffer A pH 7.5, 0.1% Nonidet 0.5 and mm lysed at 4 for 30 and centrifuged at 12,000 × The supernatant was loaded on to a The was with 10 ml of buffer A and eluted with a continuous gradient were on A 12% containing purified BIK was μg of purified HeLa cell was with by The proteins were and to functional activity. The gel was in 100 ml of a containing 50 mm Tris-Cl, pH and for 1 h to The were with 50 mm Tris-Cl, pH and mm for 1 h. The proteins were then in a containing 50 mm Tris-Cl, pH and m for 1 h. The concentration of was then reduced from m to thus of proteins. In gel phosphorylation was by the in 10 ml of kinase buffer mm pH mm DTT, 10 mm 0.5 mm 50 and μCi/ml (PerkinElmer Life Sciences) for 1 h. The were times with ml of acid and 1% and subjected to the human BIK protein expressed in mammalian cells was examined by it as a of one of the may the phosphorylated form of BIK, we labeled BIK either with [35S]methionine-cysteine mixture or with [32P]orthophosphoric For this HA-tagged BIK was expressed in BSC40 cells using the vaccinia virus expression system (24Ausubel F.M. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1992Google Scholar). The proteins were with the HA antibody and by 1 The BIK protein that was labeled with as a as in the case of of BIK labeled with that the may be the phosphorylated form of BIK 1 BIK is also we labeled cells either with or BIK with a BIK antibody and by 1 This also that BIK is These results that BIK is a the phosphorylation of BIK we BIK be phosphorylated in For this a His-tagged of BIK that the was expressed in purified using Ni2+-NTA-agarose and as the for phosphorylation. The BIK protein that was with a cytoplasmic from HeLa cells was phosphorylated BIK without the was phosphorylated The cytoplasmic was also through a and the various were to ability to phosphorylate the These results that the kinase activity that phosphorylated BIK the phosphorylated amino of BIK, we acid of in phosphorylated BIK. For this were and to phosphorylate BIK. The acid that Ser and Thr residues were phosphorylated and phosphorylation of was results were also using in phosphorylated BIK the phosphorylation we by all and residues within the acid region of BIK to alanine residues The various mutants were with the HA and expressed in cells. The protein extracts were by and with the HA All the mutant proteins mutant were expressed as proteins. The various mutant mutant were expressed as suggesting that are in phosphorylation in the other mutant only the suggesting that this mutant may be in phosphorylation. that mutant is in we of cells transfected with wt BIK or mutant with of the proteins labeled with that both wt and mutant proteins were expressed at was of in the mutant protein These results suggest that mutant is in phosphorylation conditions the wt BIK protein is phosphorylated The was also to phosphorylation of purified BIK These results suggest that residues of BIK are required for phosphorylation. which of the residues the phosphorylation of BIK, we constructed single amino acid mutants of BIK in which either or was to an residue and The mutant proteins were labeled with and and Both mutants and were to be in suggesting that both residues are required for phosphorylation of BIK. However, results between the that both residues may be phosphorylated in a or only one is phosphorylated and the other residue a the acid that both Thr and Ser residues are it is that both and may be target for mutants and BIK mutants were by A number of are in I. that expressed both the and of BIK were for and the mutant that expressed only the form was for phosphorylation. in a BIK mutants were by A number of are in I. that expressed both the and of BIK were for and the mutant that expressed only the form was for phosphorylation. the role of phosphorylation on BIK we the apoptotic activities of wt BIK and mutant in a apoptosis HeLa cells were transfected with plasmids expressing wt BIK or mutant with a plasmid that coli In cells transfected with the vector of the cells expressed the apoptotic at all DNA examined in cells transfected with wt BIK or mutant BIK, apoptosis as a of DNA concentration 4 In cells transfected with wt BIK, of the transfected cells on the DNA In cells transfected with the mutant BIK, was significantly cell death at all DNA suggesting that the the apoptotic activity of BIK. In to the apoptosis the of wt BIK and the mutant BIK were also examined in a cell survival T. Chinnadurai G. W.S. in Molecular and New Scholar). This survival is on the that of a pro-apoptotic gene with the and in cells the of In cells transfected with the and wt BIK, of was reduced as with cells transfected with the and the vector 4 In cells transfected with the and the was in the number of with cells expressing wt BIK, suggesting that the mutant induced efficient cell death. Both the apoptosis 4 and the cell survival 4 suggest that phosphorylation of BIK is required for the of efficient cell death activity. We also examined the effect of phosphorylation of BIK on its ability to heterodimerize with the cellular protein BCL-2. For this cells were transfected with HA-tagged wt BIK or mutant (T33A/S35A) BIK or vector and BCL-2. extracts were either with the HA antibody or with the BCL-2 The immunoprecipitates were subjected to using the BCL-2 antibody This that the mutant with BCL-2 at to that of wt BIK, suggesting that phosphorylation of BIK at residues and significantly influence its for BCL-2. In we have also that the mutant an an with wt BIK the of the protein kinase for phosphorylation of BIK, we the amino acid of BIK for the phosphorylation sites for known protein that BIK contains four sites and sites of the sites and to the phosphorylation sites by of BIK, the that may be a kinase for phosphorylation of BIK. be that of other potential and phosphorylation sites phosphorylation of BIK the of the protein we the of the in the HeLa cell cytoplasmic by the in gel kinase using BIK protein as the This that was phosphorylated by proteins of and is interesting to that of of proteins and are to the of the that the HeLa cell cytoplasmic the and and of protein kinase We also the effect of purified on phosphorylation of BIK. shown in wt BIK was phosphorylated by the mutant was in phosphorylation. These results the that may be the kinase for phosphorylation of BIK. We then the effect of a on phosphorylation of by purified and HeLa cell BIK phosphorylation by in a However, phosphorylation of BIK by HeLa cell extracts was significantly by the extracts Taken together, the that a kinase to to may be the kinase for phosphorylation of BIK. We have that the human pro-apoptotic protein BIK is a phosphoprotein and that it is phosphorylated on phosphorylation sites We have that phosphorylation is required for efficient cell death. This is of the role of phosphorylation on another BH3-alone pro-apoptotic protein In the case of BAD, survival factors induce phosphorylation via kinases Akt and protein kinase A (19Datta 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 (4895) Google Scholar) and influence its apoptotic activity. The by which phosphorylation a role in the apoptotic activity of BIK is has been that BH3 proteins apoptosis by with BCL-2 family proteins Ref. K. Science. 1997; 275: PubMed Scopus (552) Google Scholar). In the case of BAD, phosphorylation of a Ser residue within the BH3 domain its ability to with BCL-2 X.M. Liu Y. Payne G. Lutz R.J. Chittenden T. J. Biol. Chem. 2000; 275: 25046-25051Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, S.R. A. L. A. Greenberg M.E. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), which may at least of BAD activity. Here, we have that the phosphorylation mutant of BIK with BCL-2 at to that of wt BIK, despite its apoptotic activity. We have also results in studies between the mutant (T33A/S35A) and BCL-xL These to (3Elangovan B. Chinnadurai G. J. Biol. Chem. 1997; 272: 24494-24498Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) that heterodimerization of BIK with cellular proteins is alone insufficient for its pro-apoptotic activity. is that phosphorylation of BIK may play role in with other potential cellular In case of pro-apoptotic proteins and BID, the BH3 domain in to apoptotic stimuli (9Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3050) Google Scholar, 10Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3756) Google Scholar, M. H. D. M. Science. 1997; 275: PubMed Scopus Google Scholar). In the case of BID, protein appears to the BH3 domain. is that phosphorylation of BIK that the BH3 domain. Our results suggest that BIK may be a target for phosphorylation by a kinase to kinases have shown to phosphorylate apoptosis such as p53 and N. A. E. M. 1999; PubMed Scopus Google Scholar, S.R. Nucleic Acids Res. 1996; PubMed Scopus Google Scholar, T.R. Cell. Full Text PDF PubMed Scopus Google Scholar, K. M.H. M. S. H. J.M. B. Cell Mol. Biol. Res. Google Scholar), BIK be the BCL-2 family protein target for apoptosis has been shown to be by expression of the Akt N. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). appears that the effect of Akt on BIK activity may be as Akt the activity of a number of other pro-apoptotic stimuli. it has been that Akt may inhibit BAD and apoptosis by expression of BCL-xL R.G. M. M. J. 2000; PubMed Scopus Google Scholar). BIK is expressed in several human and appears to be expressed at in and S. Chinnadurai G. 2000; PubMed Scopus Google Scholar). is that the activity of BIK may be activated by phosphorylation in to apoptotic stimuli in various human The effect of various apoptotic stimuli on phosphorylation of BIK to be