Identification of the Major Phosphorylation Site in Bcl-xL Induced by Microtubule Inhibitors and Analysis of Its Functional Significance

Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 → Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 → Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Co-expression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax. Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 → Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 → Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Co-expression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax. Mitochondrial or intrinsic apoptosis is regulated by the Bcl-2 family of proteins which exhibit either pro- or anti-apoptotic properties (for review, see Refs. 1Youle R.J. Strasser A. Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3503) Google Scholar, 2Danial N.N. Clin. Cancer Res. 2007; 13: 7254-7263Crossref PubMed Scopus (346) Google Scholar, 3Letai A.G. Nat. Rev. Cancer. 2008; 8: 121-132Crossref PubMed Scopus (461) Google Scholar). The BH3-only pro-apoptotic members act as essential initiators of intrinsic apoptosis, whereas the multidomain pro-apoptotic Bax and Bak act as essential mediators of mitochondrial membrane permeability. Bcl-2 proteins function in a hierarchical fashion, and different models have been proposed to explain their complex interrelationships (1Youle R.J. Strasser A. Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3503) Google Scholar, 2Danial N.N. Clin. Cancer Res. 2007; 13: 7254-7263Crossref PubMed Scopus (346) Google Scholar, 3Letai A.G. Nat. Rev. Cancer. 2008; 8: 121-132Crossref PubMed Scopus (461) Google Scholar). The activator-sensitizer model proposes that BH3-only proteins comprise two subsets, the activators and the sensitizers. Activators, which include Bim and Bid, once activated themselves, in turn directly activate Bax and Bak. Sensitizers such as Bad and Bik, on the other hand, appear to act by neutralizing anti-apoptotic Bcl-2 family members and do so by displacing activator proteins bound to anti-apoptotic Bcl-2 proteins or by precluding anti-apoptotic Bcl-2 proteins from sequestering activator BH3-only proteins. In the indirect model Bax and Bak are bound to anti-apoptotic Bcl-2 proteins and become liberated and activated as a consequence of displacement by BH3-only proteins. Microtubule inhibitors are a class of compounds that bind to tubulin or microtubules and suppress microtubule dynamics (for review, see Refs. 4Mollinedo F. Gajate C. Apoptosis. 2003; 8: 413-450Crossref PubMed Scopus (419) Google Scholar, 5Rowinsky E.K. Donehower R.C. The Chemotherapy Source Book. 2nd Ed. Williams and Wilkins, Baltimore, MD2015Google Scholar, 6Jordan M.A. Wilson L. Nat. Rev. Cancer. 2004; 4: 253-265Crossref PubMed Scopus (3498) Google Scholar). Treatment of cells with these agents leads to prolonged activation of the mitotic spindle checkpoint, mitotic arrest, and eventually cell death, providing a rationale for their use as antitumor agents. The first microtubule inhibitor utilized for cancer chemotherapy was the vinca alkaloid vinblastine from the plant Catharanthus roseus, which was put into clinical practice to treat pediatric hematological malignancies in the mid-1950s. Currently, vinblastine is used in adjuvant and combination chemotherapy for several tumor types including breast, testicular, Hodgkin lymphoma, non-Hodgkin lymphoma, and Kaposi sarcoma. Another class of microtubule inhibitors is the taxanes with paclitaxel (Taxol, Food and Drug Administration approval in 1995), becoming a highly successful anticancer agent. Although microtubule inhibitors have played a key role in cancer chemotherapy for many decades, a molecular explanation of how cells die in response to mitotic checkpoint activation has proven difficult to establish. One of the complicating factors is that microtubule inhibitors activate many different signaling pathways (4Mollinedo F. Gajate C. Apoptosis. 2003; 8: 413-450Crossref PubMed Scopus (419) Google Scholar, 7Bhalla K.N. Oncogene. 2003; 22: 9075-9086Crossref PubMed Scopus (393) Google Scholar). However, one highly consistent observation is the phosphorylation of anti-apoptotic Bcl-2 proteins including Bcl-2 (8Haldar S. Jena N. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4507-4511Crossref PubMed Scopus (746) Google Scholar) and Bcl-xL (9Poruchynsky M.S. Wang E.E. Rudin C.M. Blagosklonny M.V. Fojo T. Cancer Res. 1998; 58: 3331-3338PubMed Google Scholar). Although Bcl-2 appears to undergo different types of phosphorylation involved in both cell survival and cell death, extensive multisite phosphorylation occurs in response to microtubule inhibitors (for review, see Ref. 10Ruvolo P.P. Deng X. May W.S. Leukemia. 2001; 15: 515-522Crossref PubMed Scopus (375) Google Scholar). Expression of phospho-defective forms of Bcl-2 renders some cell types more resistant to microtubule inhibitors, suggesting that phosphorylation antagonizes Bcl-2 anti-apoptotic function (11Srivastava R.K. Mi Q. Hardwick M.J. Longo D.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3775-3780Crossref PubMed Scopus (333) Google Scholar, 12Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Crossref PubMed Scopus (917) Google Scholar), whereas another study suggested that drug-induced multisite phosphorylation promotes Bcl-2 anti-apoptosis function through protein stabilization (13Brichese L. Barboule N. Heliez C. Valette A. Exp. Cell Res. 2002; 278: 101-111Crossref PubMed Scopus (42) Google Scholar). Bcl-xL phosphorylation also occurs in response to microtubule inhibitors (9Poruchynsky M.S. Wang E.E. Rudin C.M. Blagosklonny M.V. Fojo T. Cancer Res. 1998; 58: 3331-3338PubMed Google Scholar) and other apoptotic stimuli including ionizing radiation (14Kharbanda S. Saxena S. Yoshida K. Pandey P. Kaneki M. Wang Q. Cheng K. Chen Y. Campbell A. Sudha T. Yuan Z. Narula J. Weichselbaum R. Nalin C. Kufe D. J. Biol. Chem. 2000; 275: 322-327Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). In light of information that, among many apoptotic regulatory proteins, Bcl-xL expression correlates uniquely with drug resistance (15Amundson S.A. Myers T.G. Scudiero D. Kitada S. Reed J.C. Fornace Jr., A.J. Cancer Res. 2000; 60: 6101-6110PubMed Google Scholar), it is particularly important to understand mechanisms that govern Bcl-xL function. However, information on the functional significance and enzymology of Bcl-xL phosphorylation is controversial. For example, whereas several reports suggest that phosphorylation is catalyzed by c-Jun NH2-terminal kinase (JNK), 3The abbreviations used are:JNKc-Jun NH2-terminal kinaseGAPDHglyceraldehyde-3-phosphate dehydrogenaseHAhemagglutininMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideMSmass spectrometryMALDImatrix-assisted laser desorption ionizationVBLvinblastineTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 3The abbreviations used are:JNKc-Jun NH2-terminal kinaseGAPDHglyceraldehyde-3-phosphate dehydrogenaseHAhemagglutininMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideMSmass spectrometryMALDImatrix-assisted laser desorption ionizationVBLvinblastineTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine in one study phosphorylation was reported to occur on serine (serine 62) (16Basu A. Haldar S. FEBS Lett. 2003; 538: 41-47Crossref PubMed Scopus (156) Google Scholar), and in another study phosphorylation was reported to occur on threonine residues (threonines 47 and 115) (14Kharbanda S. Saxena S. Yoshida K. Pandey P. Kaneki M. Wang Q. Cheng K. Chen Y. Campbell A. Sudha T. Yuan Z. Narula J. Weichselbaum R. Nalin C. Kufe D. J. Biol. Chem. 2000; 275: 322-327Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). These apparent discrepancies appear to stem from the fact that potential sites of phosphorylation have been inferred based in particular on the assumption that JNK is the responsible kinase rather than directly determined. However, studies implicating JNK in microtubule inhibitor-induced Bcl-xL/Bcl-2 phosphorylation (12Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Crossref PubMed Scopus (917) Google Scholar, 14Kharbanda S. Saxena S. Yoshida K. Pandey P. Kaneki M. Wang Q. Cheng K. Chen Y. Campbell A. Sudha T. Yuan Z. Narula J. Weichselbaum R. Nalin C. Kufe D. J. Biol. Chem. 2000; 275: 322-327Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 16Basu A. Haldar S. FEBS Lett. 2003; 538: 41-47Crossref PubMed Scopus (156) Google Scholar) have been complicated by the fact that JNK also plays a key role in cell cycle progression. In a carefully designed study using synchronized cells examined over an extended time-course, we showed that when cells are treated with both a JNK inhibitor and a microtubule inhibitor, the JNK inhibitor slows the normal progression to mitotic arrest, and events such as Bcl-xL/Bcl-2 phosphorylation are delayed by many hours (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar). in the context of JNK microtubule inhibitor-induced Bcl-xL/Bcl-2 phosphorylation occurs to a normal (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar). the on Bcl-xL/Bcl-2 function of of putative JNK sites are by apparent of the kinase c-Jun NH2-terminal kinase mass spectrometry laser desorption vinblastine c-Jun NH2-terminal kinase mass spectrometry laser desorption vinblastine In this study we the direct biochemical of a major site of phosphorylation in Bcl-xL induced by vinblastine and other microtubule an of the site of phosphorylation and is to the that when sites are inferred indirect Site-directed mutagenesis showed that cells expressing a phospho-defective Bcl-xL mutant are highly that phosphorylation is a key regulatory for anti-apoptotic function. of wild-type or phospho-defective Bcl-xL resistance to Bax induced cell death, whereas a phospho-mimic Bcl-xL mutant failed to cells from Bax, in part to reduced Bax These studies provide into the role of Bcl-xL phosphorylation induced by microtubule was from Cell for or for Bax and were from Cell and Bax was from was from was from was from an complex and were from was from was from and and vinblastine were from Cell and cell was in at and in with and overexpressing or were by cells with Bcl-xL with in and in as M. Lyle L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of Cell and cell were and as (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar). and mitochondrial were using the from were with for and with for were with Bcl-xL for at a of and with and with for at a of were performed at cells were with for at and with in for protein and were using and laser of a Cell were in and the vinblastine was was for by of the and was at are to and treated cells were to a of for of apoptosis using the cell In this are by at of Bcl-xL was performed using H. P. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) with cells were in of and inhibitors on at for The was with to the and to the was for were and the was and the was as M. Lyle L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and in of and were to and which was performed with of Bcl-xL was using the same that of protein, from were used for and the of and were the were was performed in of and with for at were in a were by using in and in of the of Bcl-xL and Bax by was performed as M. Lyle L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were and was by with was into two of which was with of and the other with of The were in for and analysis Chem. 2004; PubMed Scopus Google Scholar, A.J. M. J. Res. 4: PubMed Scopus Google Scholar). mass was of with a mass a the from the with the mass were to mass analysis with a mass single the and information was used to identify Bcl-xL with the A.J. M. J. Res. 4: PubMed Scopus Google Scholar). The combination of from and for of Bcl-xL. to residues of Bcl-xL Ser-62 with and (for was by of cell (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar) and of in and of and were for at were by the of to and in and of were to were in with to a for and an was using a using Site-directed Bcl-xL in as a The and were using the mutagenesis to the The of were by of or wild-type phospho-mimic or phospho-defective mutant were used to cells using to the were performed using the of or cell were to the level of protein by cell cells were and and cells were and by and Bax of Bax activation was performed as M. Lyle L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of as a cell a mitotic by apoptotic cell in response to with vinblastine and is and to (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar). it an model for the molecular mitotic and apoptosis and for the role of Bcl-xL phosphorylation these on from that Bcl-xL is a protein, we cells overexpressing Bcl-xL to the of protein for purification that was mitochondrial and also in the as for the protein (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar) that these Bcl-xL is see was mitochondrial was confirmed by was used as a and it is that it was more to in cells cells In was in cells in cells vinblastine studies were performed to the properties of cell survival and apoptosis that cells overexpressing were and vinblastine was to the of cells of survival was for cells treated with vinblastine these the of cells to cells to this the are consistent with cells to resistant to cell was by of which confirmed that vinblastine caused mitotic of Bcl-xL expression level of Bcl-xL. cells were and or treated with for and using of protein were performed with and or and Bcl-xL were on a and with The and are as are light and from the cells are resistant to or cells were to cell with the of vinblastine for The are the or cells were or treated with and to apoptosis as on the at The are Bcl-xL from it was important to for Treatment of cells with vinblastine a in such in was by However, by to protein and the of vinblastine to a in was These with of suggest that the of of the protein, the for of was to were and many were we as a purification was by different of and of Bcl-xL in the be The was as was from the by and to and performed with Bcl-xL showed at with the from cells a compared with the from cells at and were also as these were also in performed in the of Bcl-xL and light and from the were and to mass analysis as was Bcl-xL vinblastine a mass of of Bcl-xL or was of the for the of these One showed single phosphorylation vinblastine analysis of this revealed the of Chem. 2004; PubMed Scopus Google Scholar) and or which phosphorylation to Ser-62 and the other or the threonine Chem. 2004; PubMed Scopus Google Scholar). analysis of the of the site of phosphorylation to Ser-62 showed of consistent with a molecular mass than and is one of two sites in Bcl-xL to be F. S.J. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The other in Bcl-xL to F. S.J. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), also showed Ser-62 by a the ability of Ser-62 in the of Bcl-xL to as a site of phosphorylation vinblastine a and Bcl-xL residues was and utilized as a as in was with an from vinblastine treated no phosphorylation was when an from cells was were when were from cells treated with or of Ser-62 Bcl-xL was to and several were that the mutant protein at a level with In to wild-type failed to undergo a in response to vinblastine also cells overexpressing Bcl-xL with another site in the of Bcl-xL reported to be by JNK (14Kharbanda S. Saxena S. Yoshida K. Pandey P. Kaneki M. Wang Q. Cheng K. Chen Y. Campbell A. Sudha T. Yuan Z. Narula J. Weichselbaum R. Nalin C. Kufe D. J. Biol. Chem. 2000; 275: 322-327Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar), to The was in suggesting that the of phosphorylation of was to of the site and was a consequence of whereas both wild-type Bcl-xL and vinblastine with a failed to do so and suggested that Ser-62 is the major site of serine microtubule inhibitors were including paclitaxel and and these failed to phosphorylation of whereas both wild-type and were Ser-62 is the major site in response to microtubule inhibitors in of Ser-62 were to of Ser-62 the apoptotic response to Treatment of cells with vinblastine induced apoptosis, whereas two expressing were with than that of the level of apoptosis the same cells expressing exhibited the same of apoptotic as cells expressing wild-type that the apoptotic response of was to phosphorylation and to was as an apoptotic and vinblastine was to in cells expressing wild-type Bcl-xL In Bax activation was in cells expressing phospho-defective wild-type Bcl-xL whereas Bax expression was In cell vinblastine a of suggesting that cells expressing undergo mitotic the apoptotic response is to a in a to mitotic Bcl-xL with have suggested that Bcl-xL directly with Bax and pro-apoptotic function (1Youle R.J. Strasser A. Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3503) Google Scholar, 2Danial N.N. Clin. Cancer Res. 2007; 13: 7254-7263Crossref PubMed Scopus (346) Google Scholar, 3Letai A.G. Nat. Rev. Cancer. 2008; 8: 121-132Crossref PubMed Scopus (461) Google Scholar, 4Mollinedo F. Gajate C. Apoptosis. 2003; 8: 413-450Crossref PubMed Scopus (419) Google Scholar). One is that phosphorylation of Bcl-xL with Bax. this cells expressing wild-type Bcl-xL and were or treated with and cell were to Bcl-xL by for Bcl-xL and Bax. in the ability of wild-type Bcl-xL to bind Bax was vinblastine whereas retained the ability to bind Bax vinblastine These suggest that phosphorylation of Bcl-xL to Bax. Functional with and the function of Bcl-xL a phospho-mimic mutant was also cells were transiently with a or or and were and for Bcl-xL. in expression were In expression of the proteins was as the of cells was was with of the Bcl-xL cell were and for Bax, and was performed which showed expression However, it was from of the cells that in the of performed with Bax or with Bax, a of the cells were and the in cells are in and of from is in of cell effects on Bax were performed. in wild-type and were to bind Bax, a significantly reduced of Bax with These suggest that the reduced ability of to the adverse effects of Bax expression be to a reduced capacity to Bax. Bcl-xL is a key anti-apoptotic protein that characteristically phosphorylation in response to microtubule inhibitors (9Poruchynsky M.S. Wang E.E. Rudin C.M. Blagosklonny M.V. Fojo T. Cancer Res. 1998; 58: 3331-3338PubMed Google Scholar). designed to the role of phosphorylation have on sites by JNK, which a on the of the Jr., Nat. Rev. Mol. Cell Biol. 2007; 8: PubMed Scopus Google Scholar). are such sites in and One suggested Ser-62 to be the site of JNK phosphorylation (16Basu A. Haldar S. FEBS Lett. 2003; 538: 41-47Crossref PubMed Scopus (156) Google Scholar), and another suggested the two to be by JNK (14Kharbanda S. Saxena S. Yoshida K. Pandey P. Kaneki M. Wang Q. Cheng K. Chen Y. Campbell A. Sudha T. Yuan Z. Narula J. Weichselbaum R. Nalin C. Kufe D. J. Biol. Chem. 2000; 275: 322-327Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). However, we have that JNK is responsible for microtubule inhibitor induced Bcl-xL phosphorylation (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar). these we a to directly identify phosphorylation sites in Bcl-xL. this we of in mass spectrometry which of residues Chem. 2004; PubMed Scopus Google Scholar, J. M. C. 2002; PubMed Scopus Google Scholar). of of Bcl-xL from and cells a major site of phosphorylation at which occurs in the of the (1Youle R.J. Strasser A. Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3503) Google Scholar, 2Danial N.N. Clin. Cancer Res. 2007; 13: 7254-7263Crossref PubMed Scopus (346) Google Scholar, 3Letai A.G. Nat. Rev. Cancer. 2008; 8: 121-132Crossref PubMed Scopus (461) Google Scholar). the by the mass spectrometry was no of major phosphorylation at other In vinblastine and studies have that Bcl-xL also and T. C. it that in the of the and it appears that Ser-62 is the single major site with which multisite phosphorylation in response to microtubule inhibitors P.P. Deng X. May W.S. Leukemia. 2001; 15: 515-522Crossref PubMed Scopus (375) Google Scholar). Ser-62 is by a in the Bcl-xL suggesting that the kinase responsible is However, this fact is with to the of the Bcl-xL kinase over of phosphorylation sites are or Jr., Nat. Rev. Mol. Cell Biol. 2007; 8: PubMed Scopus Google Scholar), the of The protein are protein and protein and these have been as suggesting that JNK is responsible for Bcl-xL phosphorylation are controversial. In JNK inhibitor the in effects on JNK were reported (16Basu A. Haldar S. FEBS Lett. 2003; 538: 41-47Crossref PubMed Scopus (156) Google Scholar). In to c-Jun delayed Bcl-xL phosphorylation phosphorylation at (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar, L. Lyle J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). these we are the kinase using the to be an for a kinase to that the kinase is from T. and T. C. in expressing phospho-defective Bcl-xL were resistant to vinblastine compared with cells expressing of wild-type cells expressing Bcl-xL a at another potential an apoptotic response as cells expressing wild-type Bcl-xL. is a that effects of and the key role of Ser-62 in Bcl-xL anti-apoptotic function. Although the suggest that phosphorylation antagonizes Bcl-xL anti-apoptotic do the we showed by that Bcl-xL and Bax in cells vinblastine M. Lyle L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the we have of the of the phospho-defective mutant to that phosphorylation the ability of wild-type Bcl-xL to bind Bax was vinblastine whereas phospho-defective Bcl-xL retained the ability to bind Bax vinblastine In Bax is and Bcl-xL is both in the and mitochondrial (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar), with Bax to the vinblastine M. Lyle L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it appear that a key role for Bcl-xL in this is to Bax, and Bcl-xL Bax is to to the of phospho-defective and forms of Bcl-xL for the that Bcl-xL phosphorylation promotes apoptosis through a release of Bax. Co-expression of either wild-type or cells from cell of failed to provide the same level of in part through a reduced capacity to Bax Currently, more studies of cells expressing these are to the role of Bcl-xL phosphorylation in of pro-apoptotic Bcl-2 family analysis of Ser-62 revealed that was Another residues from also exhibited of These residues were in Bcl-xL from both and of which are by a in of proteins Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar), occurs PubMed Scopus Google Scholar). we used to Bcl-xL from it is that this and the of these residues in cells analysis biochemical that and are residues in Bcl-xL. studies have that antagonizes Bcl-xL anti-apoptotic function F. S.J. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, R. D. Biol. 2007; PubMed Scopus Google Scholar). is of that occurs in the same as phosphorylation is to that phosphorylation and both of which Bcl-xL anti-apoptotic functional occurs in response to F. S.J. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, R. D. Biol. 2007; PubMed Scopus Google Scholar) and phosphorylation in response to microtubule (9Poruchynsky M.S. Wang E.E. Rudin C.M. Blagosklonny M.V. Fojo T. Cancer Res. 1998; 58: 3331-3338PubMed Google Scholar), these act rather than in to Bcl-xL. Bcl-xL or Bcl-2 phosphorylation occurs apoptosis, and it has been that phosphorylation However, more studies using synchronized cells have that microtubule inhibitor-induced phosphorylation is mitotic and to apoptosis be first (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar). several reports have that apoptosis occurs when become (17Du L. Lyle Oncogene. PubMed Scopus Google Scholar, Y. C. R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, L. K. Cancer Res. Google Scholar). Although these suggest that phosphorylation apoptosis, by no the that phosphorylation is a for these be with using phospho-defective which that phosphorylation anti-apoptotic One is that apoptosis in response to microtubule inhibitors is a with phosphorylation of a that is an phospho-defective the first also be studies are on the molecular events the of phosphorylation and the of apoptosis to the of this Lyle for with