Reversible Phosphorylation of Bcl2 following Interleukin 3 or Bryostatin 1 Is Mediated by Direct Interaction with Protein Phosphatase 2A

Abstract

Interleukin 3 (IL-3) stimulates the net growth of murine factor-dependent NSF/N1.H7 and FDC-P1/ER myeloid cells by stimulating proliferation and suppressing apoptosis. Recently, we discovered that Bcl2 is phosphorylated at an evolutionarily conserved serine residue (Ser70) after treatment with the survival agonists IL-3 or bryostatin 1, a potent activator of protein kinase (Ito, T., Deng, X., Carr, B., and May, W. S. (1997)J. Biol. Chem.272, 11671–11673). In addition, an intact Ser70 was found to be required for Bcl2's ability to suppress apoptosis after IL-3 withdrawal or toxic chemotherapy. We now show that phosphorylation of Bcl2 occurs rapidly after the addition of agonist to IL-3-deprived cells and can be reversed by the action of an okadaic acid (OA)-sensitive phosphatase. A role for protein phosphatase (PP) 2A as the Bcl2 regulatory phosphatase is supported by several observations: 1) dephosphorylation of Bcl2 is blocked by OA, a potent PP1 and PP2A inhibitor; 2) intracellular PP2A, but not PP1, co-localizes with Bcl2; 3) the purified PP2Ac catalytic subunit directly dephosphorylates Bcl2 in vitro in an OA-sensitive manner; 4) the purified PP2Ac catalytic subunit preferentially dephosphorylates Bcl2 in vitro compared with PP1 and PP2B; 5) reciprocal immunoprecipitation studies indicate a direct interaction between PP2A and hemagglutinin (HA)-Bcl2; and 6) treatment of factor-deprived cells with bryostatin 1 dramatically increases the association between PP2A and Bcl2. Increased association between Bcl2 and PP2A occurs 15 min after agonist stimulation when Bcl2 phosphorylation has peaked and immediately before dephosphorylation. An agonist-induced increased association of PP2A and Bcl2 fails to occur in cells expressing the inactive, phosphorylation-negative S70A Bcl2 mutant, which indicates that an intact Ser70 site is necessary and sufficient for the interaction to occur. Functional phosphorylation of Bcl2 at Ser70 is proposed to be a dynamic process regulated by the sequential action of an agonist-activated Bcl2 kinase and PP2A. Interleukin 3 (IL-3) stimulates the net growth of murine factor-dependent NSF/N1.H7 and FDC-P1/ER myeloid cells by stimulating proliferation and suppressing apoptosis. Recently, we discovered that Bcl2 is phosphorylated at an evolutionarily conserved serine residue (Ser70) after treatment with the survival agonists IL-3 or bryostatin 1, a potent activator of protein kinase (Ito, T., Deng, X., Carr, B., and May, W. S. (1997)J. Biol. Chem.272, 11671–11673). In addition, an intact Ser70 was found to be required for Bcl2's ability to suppress apoptosis after IL-3 withdrawal or toxic chemotherapy. We now show that phosphorylation of Bcl2 occurs rapidly after the addition of agonist to IL-3-deprived cells and can be reversed by the action of an okadaic acid (OA)-sensitive phosphatase. A role for protein phosphatase (PP) 2A as the Bcl2 regulatory phosphatase is supported by several observations: 1) dephosphorylation of Bcl2 is blocked by OA, a potent PP1 and PP2A inhibitor; 2) intracellular PP2A, but not PP1, co-localizes with Bcl2; 3) the purified PP2Ac catalytic subunit directly dephosphorylates Bcl2 in vitro in an OA-sensitive manner; 4) the purified PP2Ac catalytic subunit preferentially dephosphorylates Bcl2 in vitro compared with PP1 and PP2B; 5) reciprocal immunoprecipitation studies indicate a direct interaction between PP2A and hemagglutinin (HA)-Bcl2; and 6) treatment of factor-deprived cells with bryostatin 1 dramatically increases the association between PP2A and Bcl2. Increased association between Bcl2 and PP2A occurs 15 min after agonist stimulation when Bcl2 phosphorylation has peaked and immediately before dephosphorylation. An agonist-induced increased association of PP2A and Bcl2 fails to occur in cells expressing the inactive, phosphorylation-negative S70A Bcl2 mutant, which indicates that an intact Ser70 site is necessary and sufficient for the interaction to occur. Functional phosphorylation of Bcl2 at Ser70 is proposed to be a dynamic process regulated by the sequential action of an agonist-activated Bcl2 kinase and PP2A. interleukin 3 bryostatin 1 okadaic acid protein phosphatase protein kinase C endoplasmic reticulum polyacrylamide gel electrophoresis wild type. Multipotential hematopoietic growth factors such as interleukin 3 (IL-3)1 mediate net cell growth by stimulating proliferation and suppressing apoptosis (1Williams G.T. Smith C.A. Spooncer E. Dexter T.M. Taylor D.R. Nature. 1990; 343: 76-79Crossref PubMed Scopus (871) Google Scholar). Many of the molecular details concerning the mechanisms regulating signaling components necessary for cell proliferation have been determined. For example, the activation of non-receptor protein tyrosine kinases such as the Janus-activated kinases and the coupling to signals generated by cytoplasmic signal transducers and activators of transcription and/or the RAS/RAF-1/mitogen-activated protein kinase cascade have been demonstrated (2Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar). However, relatively little is known about the signaling mechanism(s) involved in factor-induced suppression of apoptosis. The proto-oncogene Bcl2 is the prototype member of a critical new gene family that functions to suppress apoptosis in a variety of cell systems including factor-dependent lymphohematopoietic and neural cells (3Vaux D.L. Cory S. Adams T.M. Nature. 1988; 335: 440-442Crossref PubMed Scopus (2738) Google Scholar, 4Yang E. Korsmeyer S.J. Blood. 1996; 88: 386-401Crossref PubMed Google Scholar). Whereas Bcl2 is localized in the endoplasmic reticulum and nuclear envelope, current evidence indicates that expression in the outer mitochondrial membrane is required for its potent antiapoptosis function (5Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Need J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar). A compelling model predicts that Bcl2 functions by preventing the release of potent mitochondrial activators of the cytosolic death effector caspases that mediate the intracellular proteolysis that is characteristic of apoptosis (6Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar). Several mechanisms have been proposed to explain the apoptosis-suppressing activity of Bcl2 (7Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5878) Google Scholar). Bcl2 can heterodimerize with a proapoptotic binding partner, BAX, to neutralize its death effector properties. The binding of Bcl2 to the mitochondrial apoptosis-activating factor (Apaf-1) and/or the blockade of cytochrome C release from the mitochondria may prevent the activation of the two death proteases, caspases 9 and 3 (8Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmed M. Alnermi E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6259) Google Scholar, 9Zou H. Henzel W.J. Lin A. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar). Bcl2 also acts by modulating/slowing the collapse of the mitochondrial transmembrane potential that occurs during apoptosis (6Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar), apparently by inhibiting the transition in mitochondrial membrane permeability (10Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1268) Google Scholar). Whereas any one or a combination of these potential functions of Bcl2 may operate to suppress apoptosis, the mechanism(s) by which Bcl2 may be regulated to preserve mitochondrial integrity has not been identified. Recently, we discovered that Bcl2 is post-translationally modified by phosphorylation on an evolutionarily conserved serine site (serine 70), and that IL-3 induces and increases phosphorylation at this site (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar,12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). Similar results have also been reported for nerve growth factor-induced phosphorylation of Bcl2, which is associated with the suppression of apoptosis in rat PC12W pheochromocytoma cells (13Horiuchi M. Hayashida W. Kambe T. Yamada T. Dzau V.J. J. Biol. Chem. 1997; 272: 19022-19026Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Site-specific mutational analysis of Bcl2 revealed that an intact serine 70 is required for phosphorylation as well as maximal and potent suppression of apoptosis (12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). Thus, phosphorylation represents at least one important post-receptor signaling mechanism that may positively regulate the antiapoptotic function of Bcl2, at least in factor-dependent cells. However, others have reported an inability to easily detect Bcl2 phosphorylation under continuous growth conditions (14Gajewski T. Thompson C.B. Cell. 1996; 87: 589-592Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), suggesting a possible role for a Bcl2 phosphatase in reversing phosphorylation. Reversible phosphorylation is a well-understood mechanism for the rapid regulation of critical cell functions including metabolism, signal transduction, cell division, growth, and memory (15Cohen P. Klumpp S. Schelling D.L. FEBS Lett. 1989; 250: 601-606Crossref PubMed Scopus Google Scholar, M. Cancer Biol. PubMed Scopus Google Scholar). IL-3 signaling is a dynamic process that results in signal by the rapid of critical post-receptor components such as Janus-activated kinase and protein kinase (2Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar). Whereas protein kinase C an IL-3 post-receptor kinase (2Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar), can directly Bcl2 in vitro and in (1Williams G.T. Smith C.A. Spooncer E. Dexter T.M. Taylor D.R. Nature. 1990; 343: 76-79Crossref PubMed Scopus (871) Google Scholar, W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar, Deng X. Carr May W.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), a regulatory Bcl2 phosphatase that the action of an agonist-activated Bcl2 kinase has not been identified. to a protein phosphatase is involved in regulating Bcl2 phosphorylation in cells and to the mechanism(s) involved in regulating Bcl2 dephosphorylation. Bcl2 and and as (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar). murine IL-3 was by of 1 was the of G. The purified catalytic subunit of PP2A was as J. Biol. Chem. Full Text PDF PubMed Google Scholar). and PP2Ac from and the was from PP1, PP2A, and from The was from from NSF/N1.H7 cells the of H. S. D. Exp. 1990; Google Scholar). for expression of of wild mutant, or Bcl2 and as (12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). FDC-P1/ER cells expressing of wild Bcl2 and as (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar, M. N. May W.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with acid or as (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar). in of IL-3 for and in and/or for 1 before the addition of IL-3 or bryostatin 1 (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar). or as in the or the cells and in and immunoprecipitation was as (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar). The to gel to a and to for the at as (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar). The membrane was also for analysis to Bcl2 or the by as (12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). For Bcl2, or Bcl2, analysis and on cell in of was as (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar). with acid and with bryostatin 1 for 15 Bcl2 was as and the in and in of phosphatase PP1 and or and 1 PP2Ac or of PP1, PP2A, or and the for the at as K. J. 1994; PubMed Scopus Google Scholar, K. J. 1993; PubMed Scopus Google Scholar). The was by the addition of and the was for min before as The activity of PP2A, PP1, and was by the phosphatase the of Bcl2 and PP2A was as J.C. Cell. 1996; 87: Full Text Full Text PDF PubMed Scopus Google Scholar). cells in 1 and for by a and at the The was at to the membrane that the The was at to the membrane that the and endoplasmic reticulum The the cytosolic The purified by a as S.M. May W.S. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). of protein from to and by as IL-3 and may apoptosis in myeloid NSF/N1.H7 and FDC-P1/ER cells by the serine phosphorylation of Bcl2 (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar). However, little is known about the mechanism(s) of regulation of Bcl2 phosphorylation and a Bcl2 protein phosphatase also be studies to the and of Bcl2 phosphorylation after agonist addition to factor-deprived cells. The cells with acid and for with a of IL-3 or agonists to the survival (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar). the cells and in Bcl2 was and as (12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). IL-3 and the phosphorylation of Bcl2, which maximal by 15 or min in NSF/N1.H7 or FDC-P1/ER or was found to be rapidly to a by the of expression of Bcl2 not during agonist the of phosphorylation is to from the sequential action of a Bcl2 kinase and phosphatase. the PP1 and/or PP2A protein may be the cells with okadaic acid a potent PP1 and PP2A treatment for min in the of the dephosphorylation of Bcl2 as and in a of Bcl2 1 However, the was compared with that after agonist addition 1 and Bcl2 at S70A Bcl2 was to be phosphorylated from or phosphorylation that the site was that an kinase may have been by the treatment with OA, by inhibiting a a Bcl2 PP2A is the protein phosphatase in cells E. K. J. N. G. Cell. Biol. 1997; PubMed Scopus Google Scholar). PP2A activity is reported to be by by PP1, an of 1 compared with an for PP1 P. 1990; Full Text PDF PubMed Scopus Google Scholar, P. Klumpp S. Schelling D.L. FEBS Lett. 1989; 250: PubMed Scopus Google Scholar), and PP2Ac can Bcl2 in vitro in an OA-sensitive 3 has been in cell that relatively of can a of PP2A with little or on PP1 B. P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). a relatively of was in the the results with but not a role for PP2A or a phosphatase in Bcl2 in intact myeloid cells. studies to which purified protein phosphatase directly and Bcl2 in Bcl2 was as a for the of PP1, PP2A, or for the that purified PP2A rapidly and Bcl2 3 with and The indicate that PP2A is PP1 or and indicate that phosphorylated Bcl2 is a potential for PP2A. Bcl2 is an membrane protein on the outer mitochondrial membrane (6Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4422) Google Scholar, D. G. Milliman Korsmeyer S.J. Nature. 1990; PubMed Scopus Google Scholar), and nuclear membrane M. Korsmeyer S.J. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). a suppression of apoptosis by Bcl2 is to mitochondrial W. A. B. J. 1996; PubMed Scopus Google Scholar, Susin S.A. N. B. Kroemer G. FEBS Lett. 1996; PubMed Scopus Google Scholar), we that a Bcl2 phosphatase may with Bcl2. studies as under indicate that Bcl2 and the and catalytic of the PP2A G. 1993; PubMed Scopus Google in the membrane which mitochondrial as well as in the and nuclear membrane PP1 is not in the or nuclear membrane has been reported to be a protein E. T. Nature. 1997; PubMed Scopus Google Scholar), this protein phosphatase was found to be localized in the cytoplasmic and of which little Bcl2. of associated with the mitochondrial membrane of as by 4) as reported T. Cell. Full Text PDF PubMed Scopus Google Scholar, T. T. H. H. M. J. 1997; PubMed Scopus Google Scholar). a role for PP2A as a Bcl2 phosphatase in at least in murine factor-dependent myeloid cells. to an OA-sensitive phosphatase may directly with Bcl2 to about dephosphorylation after agonist reciprocal immunoprecipitation with or was NSF/N1.H7 cells that as under The cells with and with for a at which Bcl2 phosphorylation is maximal 1 or PP2Ac was and by that the and a that was by analysis as the catalytic subunit of PP2A The membrane was with (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google to as a is known to heterodimerize with Bcl2 under conditions Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5878) Google Scholar). be with an but not when the PP2A was suggesting that the PP2A may the interaction under these conditions the that PP2Ac is with Bcl2 a a Bcl2 was with and that Bcl2 was also by the PP2Ac the of the interaction a was with cell to for an interaction between PP1 and Bcl2. results indicate that and but not and PP1, can be from of cells a and PP1 is as by the of cell not these that PP2A but apparently not PP1 may directly to Bcl2 and regulate phosphorylation. PP2A was also found to be associated with Bcl2, after IL-3 for at In this the addition of a rapid and in the association of Bcl2 and PP2A after 15 a by which Bcl2 phosphorylation has maximal results for two myeloid cell these indicate that the interaction is not to a cell a the that the association results from an of treatment induces an increased association of PP2Ac with Bcl2 but not with S70A cells expressing Bcl2 or cells expressing Bcl2 or the of function S70A Bcl2 protein of IL-3 for by the addition of bryostatin for to and of the association between PP2Ac and Bcl2, as by and PP2Ac and by with PP2Ac or Bcl2 as under Increased PP2A association may Bcl2. agonist-induced Bcl2 phosphorylation occurs on Ser70 (12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar), a was to an intact Ser70 phosphorylation site is required for the in association after agonist-induced Bcl2 phosphorylation. expressing or Bcl2 or the phosphorylation-negative S70A Bcl2 and of IL-3 for as (12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). was to Bcl2 and any in the interaction between Bcl2 and PP2A was by immunoprecipitation after of the cells. indicate that and Bcl2, but not the phosphorylation-negative S70A Bcl2 mutant, to a in association between Bcl2 and PP2Ac Increased association between Bcl2 and PP2A, not occur Bcl2 phosphorylation is maximal the increased association Bcl2 suggesting that phosphorylated Ser70 may be required for any in association of PP2Ac and Bcl2. However, the treatment of cells with to Bcl2 phosphatase activity not with agonist-induced association of not these indicate that the increased association is of PP2Ac We Bcl2 phosphorylation by IL-3 or the survival agonist bryostatin 1 is a or dynamic indicate that Bcl2 phosphorylation is a dynamic process that can be regulated by the of a Bcl2 kinase (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar, T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google and a Bcl2 phosphatase 1 and Bcl2 phosphorylation occurs rapidly after the addition of IL-3 or bryostatin and maximal by Bcl2 an but dephosphorylation to a of phosphorylation by which indicates the of a Bcl2 phosphatase. we that can Bcl2 in and that the survival agonists or bryostatin 1 can phosphorylation at the Ser70 suggesting a role for (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar, Deng X. Carr May W.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We have now discovered that phosphorylation of Bcl2 may occur the action of PP2A. Whereas agonist-induced Bcl2 phosphorylation is after 15 an of Bcl2 phosphorylation can be when the potent phosphatase is 1, suggesting a role for an OA-sensitive phosphatase. PP2A but not PP1 is with Bcl2 in mitochondria Bcl2 is known to function W. A. B. J. 1996; PubMed Scopus Google and Susin S.A. N. B. Kroemer G. FEBS Lett. 1996; PubMed Scopus Google Scholar), the of Bcl2 phosphorylation may be directly regulated by the action of a Bcl2 such as and a of PP2A. with a direct PP2A but not PP1 or was found to be at vitro 3 PP2A is associated with Bcl2, when cells have been of IL-3 for a by which Bcl2 phosphorylation is 1 the interaction between Bcl2 and PP2A under conditions may for the of Bcl2 phosphorylation after IL-3 Bcl2 in its ability to suppress apoptosis (12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google and at least one mechanism by which factor withdrawal may apoptosis. However, agonist is a rapid in Bcl2 phosphorylation is which is by a in the association of Bcl2 and PP2A that phosphorylation on mechanism to function to regulate the of agonist-induced Bcl2 phosphorylation and for the but of phosphorylation under growth conditions However, the toxic can of Bcl2 at Ser70 1 at least in that dynamic Bcl2 phosphorylation may be important for cell phosphorylation may with Bcl2 function as S. N. S. A. PubMed Scopus Google Scholar, Korsmeyer S.J. M. D.L. Cell. Biol. PubMed Scopus Google Scholar). a of cells to not a Bcl2 phosphatase but also critical that Bcl2 is not sufficient for cell survival (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar, T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). in of a necessary role for the dynamic regulation of Bcl2 phosphorylation in has been found that nerve growth factor-induced Bcl2 phosphorylation can be reversed by binding to rat PC12W pheochromocytoma cells that on nerve growth factor for survival (13Horiuchi M. Hayashida W. Kambe T. Yamada T. Dzau V.J. J. Biol. Chem. 1997; 272: 19022-19026Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). was discovered to apoptosis in the of nerve growth apparently by a protein kinase phosphatase M. Hayashida W. Kambe T. Yamada T. Dzau V.J. J. Biol. Chem. 1997; 272: 19022-19026Abstract Full Text Full Text PDF PubMed Scopus (266) Google that may also Bcl2 dephosphorylation directly or these results that reversing growth factor-induced Bcl2 phosphorylation and/or with dynamic Bcl2 phosphorylation may have a on cell The activity and/or of a phosphatase the of Bcl2 phosphorylation and an intact Ser70 phosphorylation but not PP2Ac to be required The interaction between PP2A and Bcl2 may from an increased of phosphorylated Ser70 or a potential in Bcl2 from phosphorylation. the the increased association between Bcl2 and PP2A may function to the dynamic of Bcl2 phosphorylation and the of phosphorylation that is required under growth The results also the potential of an Bcl2 kinase in addition to IL-3 or (11May W.S. Tyler P.G. Ito T. Armstrong D.K. Qatsha K.A. Davidson N.E. J. Biol. Chem. 1994; 269: 26865-26870Abstract Full Text PDF PubMed Google Scholar, T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). Bcl2 can when is in the of agonist 1 which that the of OA-sensitive phosphatase activity may a protein kinase to Bcl2 However, the of this potential Bcl2 kinase is not Whereas PP2A has been associated with the regulation of cell M. Cancer Biol. PubMed Scopus Google Scholar, G. 1993; PubMed Scopus Google Scholar), is known about mitochondrial PP2A may apoptosis. results a mechanism for regulation of Bcl2 that PP2A. The also the evidence for a of PP2A that is associated with the mitochondrial an interaction with Bcl2. PP2A by direct protein interaction may PP2A function and an important role in Bcl2 phosphorylation and In of this in which PP2A function the dephosphorylation of the protein E. G. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the phosphatase has also been reported to directly with and be by Bcl2 in cells E. T. Nature. 1997; PubMed Scopus Google Scholar). interaction apparently the nuclear of and However, is evidence that is involved in the regulation of Bcl2 phosphorylation and is not to at the and is not with Bcl2 after the agonist treatment of a role for in Bcl2 phosphorylation at least in factor-dependent myeloid cells. the binding of PP2A or to Bcl2 may have and the potential of Bcl2 in with and regulating components involved in apoptosis. has been reported that phosphorylation of Bcl2 is not in cells (14Gajewski T. Thompson C.B. Cell. 1996; 87: 589-592Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). that a to detect Bcl2 phosphorylation in cells may at least in the increased activity of a phosphatase or the of a mitochondrial Bcl2 kinase Deng X. Carr May W.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, a of Bcl2 phosphorylation in cells may be to detect also inhibiting an associated Bcl2 phosphatase In agonist-induced phosphorylation of Bcl2 at Ser70 can now be in dynamic with the of phosphorylation regulated by the action of a Bcl2 kinase and PP2A. to of the mechanisms the regulation of Bcl2 which is required for and potent Bcl2 function (12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). this regulatory mechanism a potential for