Novel Role for JNK as a Stress-activated Bcl2 Kinase
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m100279200
ISSN1083-351X
AutoresXingming Deng, Lei Xiao, Wenhua Lang, Fengqin Gao, Peter P. Ruvolo, W. Stratford May,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoInterleukin (IL)-3-induced Bcl2 phosphorylation at Ser70 may be required for its full and potent antiapoptotic activity. However, in the absence of IL-3, increased expression of Bcl2 can also prolong cell survival. To determine how Bcl2 may be functionally phosphorylated following IL-3 withdrawal, astress-activated Bcl2 kinase (SAK) was sought. Results indicate that anisomycin, a potent activator of the stress kinase JNK/SAPK, can induce Bcl2 phosphorylation at Ser70 and that JNK1 can be latently activated following IL-3 withdrawal to mediate Bcl2 phosphorylation. JNK1 directly phosphorylates Bcl2 in vitro, co-localizes with Bcl2, and collaborates with Bcl-2 to mediate prolonged cell survival in the absence of IL-3 or following various stress applications. Dominant-negative (DN)-JNK1 can block both anisomycin and latent IL-3 withdrawal-induced Bcl2 phosphorylation (>90%) and potently enhances cell death. Furthermore, low dose okadaic acid (OA), a potent protein phosphatase 1 and 2A inhibitor, can activate the mitogen-activated protein kinases JNK1 and ERK1/2, but not p38 kinase, to induce Bcl2 phosphorylation and prolong cell survival in factor-deprived cells. Since PD98059, a specific MEK inhibitor, can only partially inhibit OA-induced Bcl2 phosphorylation but completely blocks OA-induced Bcl2 phosphorylation in cells expressing DN-JNK1, this supports the conclusion that OA may stimulate Bcl2 phosphorylation via a mechanism involving both JNK1 and ERK1/2. Collectively, these findings indicate a novel role for JNK1 as a SAK and may explain, at least in part, how functional phosphorylation of Bc12 can occur in the absence of growth factor. Interleukin (IL)-3-induced Bcl2 phosphorylation at Ser70 may be required for its full and potent antiapoptotic activity. However, in the absence of IL-3, increased expression of Bcl2 can also prolong cell survival. To determine how Bcl2 may be functionally phosphorylated following IL-3 withdrawal, astress-activated Bcl2 kinase (SAK) was sought. Results indicate that anisomycin, a potent activator of the stress kinase JNK/SAPK, can induce Bcl2 phosphorylation at Ser70 and that JNK1 can be latently activated following IL-3 withdrawal to mediate Bcl2 phosphorylation. JNK1 directly phosphorylates Bcl2 in vitro, co-localizes with Bcl2, and collaborates with Bcl-2 to mediate prolonged cell survival in the absence of IL-3 or following various stress applications. Dominant-negative (DN)-JNK1 can block both anisomycin and latent IL-3 withdrawal-induced Bcl2 phosphorylation (>90%) and potently enhances cell death. Furthermore, low dose okadaic acid (OA), a potent protein phosphatase 1 and 2A inhibitor, can activate the mitogen-activated protein kinases JNK1 and ERK1/2, but not p38 kinase, to induce Bcl2 phosphorylation and prolong cell survival in factor-deprived cells. Since PD98059, a specific MEK inhibitor, can only partially inhibit OA-induced Bcl2 phosphorylation but completely blocks OA-induced Bcl2 phosphorylation in cells expressing DN-JNK1, this supports the conclusion that OA may stimulate Bcl2 phosphorylation via a mechanism involving both JNK1 and ERK1/2. Collectively, these findings indicate a novel role for JNK1 as a SAK and may explain, at least in part, how functional phosphorylation of Bc12 can occur in the absence of growth factor. interleukin-3 mitogen-activated protein kinase C-Jun N-terminal protein kinase stress-activated Bcl2kinase dominant negative JNK extracellular signal-regulated kinase okadaic acid Bryostatin-1 anisomycin protein phosphatase 2A staurosporine PD98059 protein kinase C polyacrylamide gel electrophoresis fluorescence-activated cell sorter Apoptosis is a highly organized, physiologic mechanism of destroying injured and abnormal cells. Apoptosis is implicated in the regulation of development, differentiation, and homeostasis (1Dixon S.C. Soriano B.J. Lush R.M. Borner M.M. Figg W.D. Ann. Pharmacother. 1997; 31: 76-82Crossref PubMed Scopus (78) Google Scholar).Bcl2 is the first identified survival gene involved in the control of apoptosis (2Gajewski T.F. Thompson C.B. Cell. 1996; 87: 589-592Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Greater than 15 Bcl2 family members, representing both anti-apoptotic and pro-apoptotic members, have been identified to date in mammalian cells and viruses (3Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4780) Google Scholar). Results have suggested potential mechanism(s) by which Bcl2 functions including: (a) heterodimerization with its proapoptotic partner, Bax (4Oltvai Z.N. Milliman C.L. Korsmeyer S.J. 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May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar). However, Bcl2 prolongs cell survival under various stress applications including growth factor withdrawal. Thus, if phosphorylation is required for Bcl2s full and potent antiapoptotic function, how might Bcl2 phosphorylation occur under these conditions? To experimentally address this question, we hypothesized the existence of astress-activated Bcl2 kinase (SAK).The c-Jun N-terminal kinases (JNKs) are classic stress-activated protein kinases (15Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1705) Google Scholar). JNKs are potently and preferentially activated following various cell stress applications including UV irradiation (16Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1374) Google Scholar), heat and osmotic shock (16Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1374) Google Scholar), treatment with a protein synthesis inhibitor (17Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2408) Google Scholar), inflammatory cytokines (17Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2408) Google Scholar), growth factor withdrawal (18Le-Niculescu H. Bonfoco E. Kasuya Y. Claret F.X. Green D.R. Karin M. Mol. Cell. Biol. 1999; 19: 751-763Crossref PubMed Scopus (439) Google Scholar), and treatment with chemotherapy drugs including paclitaxel (19Srivastava R.K. Mi Q.S. Hardwick J.M. Longo D.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3775-3780Crossref PubMed Scopus (332) Google Scholar,20Yamamoto K. Ichijo H. Korsmeyer S. Mol. Cell. Biol. 1999; 19: 8469-8478Crossref PubMed Scopus (909) Google Scholar), adriamycin, vinblastine, and etoposide (21Osborn M.T. Chambers T.C. J. Biol. Chem. 1996; 271: 30950-30955Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). JNK is also activated in response to cell stress induced by certain DNA-damaging agents including 1-β-d-arabinofuranosylcytosine (22), cis-platinum, and mitomycin C (22Kharbanda S. Ren R. Pandey P. Shafman T.D. Feller S.M. Weichselbaum R.R. Kufe D.W. Nature. 1995; 376: 785-788Crossref PubMed Scopus (458) Google Scholar). Recently, JNK has been implicated in the regulation of apoptosis (23Derijard B. Hibi M. Wu I. Barrett T. Su Bing Deng Tiliang Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2949) Google Scholar, 24Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3593) Google Scholar). However, the mechanism(s) and functional role for JNK in the regulation of apoptosis are not clear (24Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3593) Google Scholar). On the one hand, reports suggest that activation of JNK can be associated with induction of apoptosis. For example, it was found that nerve growth factor withdrawal from post-mitotic rat PC12 pheochromocytoma cells resulted in JNK activation in association with apoptosis (18Le-Niculescu H. Bonfoco E. Kasuya Y. Claret F.X. Green D.R. Karin M. Mol. Cell. Biol. 1999; 19: 751-763Crossref PubMed Scopus (439) Google Scholar, 25Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5027) Google Scholar). JNK is also activated following other stress applications including irradiation, tumor necrosis factor-α, or Fas-ligand treatment (FasL; 26, 27). In addition, in both sympathetic neurons and PC12 cells, expression of a dominant-negative mutant of c-Jun, or microinjection of c-Jun neutralizing antibodies, are able to inhibit apoptosis (28Estus S. Zaks W.J. Freeman R.S. Gruda M. Bravo R. Johnson E.M. J. Cell Biol. 1994; 127: 1717-1727Crossref PubMed Scopus (786) Google Scholar, 29Ham J. Babij C. Whitfield J. Pfarr C.M. Lallemand D. Yaniv M. Rubin L.L. Neuron. 1995; 14: 927-939Abstract Full Text PDF PubMed Scopus (757) Google Scholar). These data also suggest that phosphorylation of c-Jun by JNK is closely associated with apoptosis. However, these studies did not indicate a potential mechanism by which JNK activation and/or c-Jun phosphorylation might trigger apoptosis. Interestingly, it has recently been proposed that the JNK/c-Jun/FasL signaling pathway may play a role in induction of neuronal cell death in response to various stresses via up-regulation of FasL expression (18Le-Niculescu H. Bonfoco E. Kasuya Y. Claret F.X. Green D.R. Karin M. Mol. Cell. Biol. 1999; 19: 751-763Crossref PubMed Scopus (439) Google Scholar). On the other hand, evidence has been reported that JNK activation is involved in cell survival. Epidermal growth factor or phorbol ester treatment of cells induces activation of JNK in association with cell survival (23Derijard B. Hibi M. Wu I. Barrett T. Su Bing Deng Tiliang Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2949) Google Scholar). Lymphocytes deficient in upstream activation of JNK by JNKK1/SEK1 are reported to be more sensitive to Fas-mediated apoptosis (30Nishina H. Fishcher K.D. Radvanyi L. Shahinian A. Hakem R. Rubies E.A. Bernstein A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; 385: 350-353Crossref PubMed Scopus (308) Google Scholar), suggesting that JNK activation is required for prolonged cell survival following a stress event. In support of this notion, lymphocytes from TRAF2−/− mice, which are defective in JNK activation, are highly sensitive to tumor necrosis factor-induced apoptosis (31Yeh W. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. Pompa J, L. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar). Even stronger evidence in favor of this hypothesis derives from the analysis of compound mutant JNK1−/−JNK2−/− embryos that exhibit increased apoptosis within the developing forebrain (32Kuan C.Y. Yang D.D. Samanta Roy D.R. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar, 33Sabapathy K. Jochum W. Hochedlinger K. Chang L. Karin M. Wager E.F. Mech. Dev. 1999; 89: 115-124Crossref PubMed Scopus (300) Google Scholar). Moreover, it has been reported that integrin-mediated survival signaling can be mediated by the JNK pathway (34Almeida E.A. Iiic D. Han Q. Hauck C.R. Jin F. Kawakatsu H. Schlaepfer D.D. Damsky C.H. J. Cell Biol. 2000; 149: 741-754Crossref PubMed Scopus (335) Google Scholar). Therefore, the JNKs may potentially play a dual role (i.e. both positive or negative) in regulating apoptosis. Our findings indicate that JNK1 can serve as both a SAK and an IL-3/agonist-activated Bcl2 kinase involved in Bcl2 regulation. Thus, JNK1, as an SAK, may be important for prolonged survival following stress stimuli that otherwise would lead to rapid cell death.DISCUSSIONSince phosphorylation of Bcl2 may be required for its full and potent antiapoptotic activity (12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar), a SAK was postulated to help explain why WT but not the non-phosphorylated S70A Bcl2 mutant could prolong cell survival following IL-3 withdrawal or treatment with some stress applications. Evidence reported here suggests that JNK1 is a SAK. First, stress stimuli such as IL-3 withdrawal or OA treatment were found to activate JNK1 either latently or rapidly, respectively, in close association with Bcl2 phosphorylation and prolonged cell survival as compared with cells expressing S70A Bcl2. Second, DN-JNK1 was able to block Bcl2 phosphorylation and prolong survival following these stress applications. Third, JNK1 was found to co-localize with Bcl2 in the mitochondria and could directly phosphorylate Bcl2 at Ser70 in vitro. Collectively, these findings provide strong evidence that JNK1 is a physiologic SAK. IL-3 withdrawal is a potent physiological stress that leads to apoptosis but can be delayed if WT Bcl2 is expressed. Our findings demonstrate that IL-3 withdrawal can result in the latent activation of JNK1 (up to 3-fold) in close association with Bcl2 phosphorylation at Ser70. In contrast, the other MAPKs, ERKs1/2 and p38, are not latently activated or are inactivated upon IL-3 withdrawal, indicating that MAPKs other than JNK1 are probably not involved in latent Bcl2 phosphorylation. These findings may explain, at least in part, how Bc12 can prolong cell survival during stress.Recent reports from our laboratory demonstrated that OA can induce Bcl2 phosphorylation at Ser70, suggesting a role for an OA-sensitive Bcl2 phosphatase in regulating phosphorylation (35Deng X. Ito T. Carr B. Mumby M. May W.S. J. Biol. Chem. 1998; 273: 34157-34163Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Results reported here mechanistically extend these findings and indicate that OA-induced Bcl2 phosphorylation may occur as a result of the combined activation of the ERKs1/2 and JNK1 by a mechanism that involves the inhibition of a PP2A-like phosphatase activity since the combination of DN-JNK1 and PD98059 can completely block OA-induced Bcl2 phosphorylation. Also, whether the OA-activated death/survival pendulum sways one way or the other may depend on the concentration(s) of OA used. Results indicate that at low concentrations of OA (i.e. 10 nm), which can selectively inhibit PP2A but not PP1 (35Deng X. Ito T. Carr B. Mumby M. May W.S. J. Biol. Chem. 1998; 273: 34157-34163Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), survival dramatically increases in cells expressing WT but not the non-phosphorylatable S70A Bcl2 following IL-3 withdrawal (Fig. 1). These findings further support and extend our previous findings that prolonged cell survival is dependent upon an intact Ser70 Bcl-2 phosphorylation site (12Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar).The findings indicate that while anisomycin treatment of cells does not prolong survival in the absence of IL-3 (Fig. 6 D), anisomycin also does not significantly increase the rate of cell death under these conditions (Fig. 6 D), even though anisomycin is reported to be toxic to cells by inhibiting protein synthesis (39Shu J. Hitomi M. Sacey D. Oncogene. 1996; 13: 2421-2430PubMed Google Scholar). This may potentially be explained since anisomycin provides a potential survival signal by activating JNK1 and resulting in Bcl2 phosphorylation (Fig. 6 C). However, any survival advantage gained by inducing Bc12 phosphorylation may, under certain circumstances, be opposed by a toxic effect such as the inhibition of protein synthesis with the outcome being neutral with respect to survival.While a precise role for JNK1 in the regulation of programmed cell death is not clear (31Yeh W. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. Pompa J, L. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar), some reports have concluded that activation of JNK induces apoptosis (18Le-Niculescu H. Bonfoco E. Kasuya Y. Claret F.X. Green D.R. Karin M. Mol. Cell. Biol. 1999; 19: 751-763Crossref PubMed Scopus (439) Google Scholar, 25Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5027) Google Scholar, 26Chen Y. Wang X. Templeton D. Davis R.J. Tan T-H. J. Biol. Chem. 1996; 271: 31929-31936Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 27Verheeij M. Bose R. Lin X Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1710) Google Scholar, 28Estus S. Zaks W.J. Freeman R.S. Gruda M. Bravo R. Johnson E.M. J. Cell Biol. 1994; 127: 1717-1727Crossref PubMed Scopus (786) Google Scholar, 29Ham J. Babij C. Whitfield J. Pfarr C.M. Lallemand D. Yaniv M. Rubin L.L. Neuron. 1995; 14: 927-939Abstract Full Text PDF PubMed Scopus (757) Google Scholar). By contrast, other reports indicate that JNK activation is required for cell survival (23Derijard B. Hibi M. Wu I. Barrett T. Su Bing Deng Tiliang Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2949) Google Scholar, 30Nishina H. Fishcher K.D. Radvanyi L. Shahinian A. Hakem R. Rubies E.A. Bernstein A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; 385: 350-353Crossref PubMed Scopus (308) Google Scholar, 31Yeh W. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. Pompa J, L. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 32Kuan C.Y. Yang D.D. Samanta Roy D.R. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar, 45Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 191-194Crossref Scopus (280) Google Scholar). Such discrepancies in the published literature may be due, at least in part, to specific cell type differences. Our findings clearly indicate that activated JNK1 can cooperate with WT Bcl2 to prolong cell survival in factor-dependent cells following stress applications that would otherwise lead to rapid cell death. These findings are also consistent with other reports that JNK1 can phosphorylate Bcl2 (19; 20). Thus, DN-JNK1 not only can block stress-induced Bcl2 phosphorylation but can also promote a more rapid cell death following such applications (Fig. 6 D). These data strongly suggest that the functional role of JNK1 as a SAK is to prolong cell survival following stress which, under some circumstances, may represent only an agonal attempt to maintain the antiapoptotic function of Bc12.Interestingly, our findings indicate that JNK1 is not only latently activated following IL-3 withdrawal but also can be rapidly activated following the addition of the survival agonists IL-3 or Bryo. These findings support the notion that JNK1 may play a dual role in both IL-3-mediated survival and stress-activated signaling relative to cell survival (Fig. 8). The dynamics of IL-3-induced JNK1 activation and Bcl2 phosphorylation is rapid (15–30 min) and reversible (Fig. 8) (35Deng X. Ito T. Carr B. Mumby M. May W.S. J. Biol. Chem. 1998; 273: 34157-34163Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), while that for activation of JNK1 and Bcl2 phosphorylation is latent, being observed only between 6 and 12 h following IL-3 withdrawal (Fig. 2, A and B). Any differential mechanism(s) for early versus latent activation of JNK1 are not yet clear, and further studies will be required. The rapid activation of JNK1 may result from PKC activation that occurs following IL-3 addition to cells because bryostatin-1, a potent PKC activator and survival stimulator in factor-dependent cells (8May 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), can also rapidly activate cellular JNK1 (Fig. 8). On the other hand, latent activation of JNK1, which may account for prolonged cell survival following stress, does not appear to involve PKC or other MAPKs, including the ERKs or p38 kinase (Fig. 2). Presently, how JNK1 is latently activated remains to be determined. Interestingly, only the WT, phosphorylatable Bcl-2 but not the non-phosphorylatable S70A Bcl2 can prolong cell survival following stress, clearly indicating a requirement for an intact Ser70 site for Bcl2s potent antiapoptotic function following stress. Bryostatin-1 can stimulate JNK1 activity, suggesting that PKC may somehow act as an upstream activator of JNK1, at least in IL-3 dependent cells. Interestingly, since DN-JNK1 can only partially inhibit IL-3-induced Bcl2 phosphorylation, this provides further strong evidence that JNK1 may also play a role in agonist-induced Bcl2 phosphorylation. Since the activity of JNK1/SAPK has been reported to be inhibited by the potent PKC inhibitor, staurosporine (46Yue T.L. Wang C. Romnic A.M. Kikly K. Keller P. DeWolf W.E. Hart T.K. Thomas H.C. Storer B. Gu J.L. Wang X. Feuerstein G.Z. J. Mol. Cell Cardiol. 1998; 30: 495-507Abstract Full Text PDF PubMed Scopus (152) Google Scholar), this would help to explain why the combination of staurosporine and PD98059 can completely inhibit IL-3-induced Bcl2 phosphorylation (14Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar).Multiple kinases have been proposed to mediate the phosphorylation of Bcl2 or Bcl-XL following various stimuli (20Yamamoto K. Ichijo H. Korsmeyer S. Mol. Cell. Biol. 1999; 19: 8469-8478Crossref PubMed Scopus (909) Google Scholar). However, the precise functional role(s) of Bcl2 phosphorylation is not completely clear. By contrast to our (8May 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. 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Inactivation by paclitaxel is thought to occur when Bcl2 is phosphorylated at two other residues (Thr69 and Ser87) in addition to Ser70 (i.e.multiple-site phosphorylation) (20Yamamoto K. Ichijo H. Korsmeyer S. Mol. Cell. Biol. 1999; 19: 8469-8478Crossref PubMed Scopus (909) Google Scholar). Therefore, it is possible that multiple-site phosphorylation may differ functionally from Ser70 mono-site phosphorylation to regulate Bcl2s antiapoptotic function, perhaps by inducing an additional conformational change. While further work will be required to explain the mechanism, it should be noted that paclitaxel can also act directly on mitochondria isolated from human cancer cells to mediate the release of cytochrome c and activate caspases that initiate apoptosis in a mechanism apparently independent of Bcl2 (52Andre N. Braguer D. Brasseur G. Goncalves A. Lemesle D. Guise S. Jordan M.A. Guise S. Jordan M.A. Briand C. Cancer Res. 2000; 60: 5349-5353PubMed Google Scholar). Thus the role of multisite Bcl2 phosphorylation in paclitaxel-induced death is mechanistically unclear.Our findings indicate that JNK1-induced phosphorylation of Bcl2 may be required to facilitate prolonged survival following stress conditions that ultimately lead to cell death if not reversed. The mechanism(s) by which phosphorylation can enhance Bcl2s function is not yet clear but, at least in part, phosphorylation appears to maintain a stable association between Bcl2 and its proapoptotic partner, Bax (14Deng X. Ruvolo P. Carr B.K. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1578-1583Crossref PubMed Scopus (221) Google Scholar). In addition, phosphorylation of Bcl2 has been recently reported to prevent degradation and thereby prolong the expression and presumably the antiapoptotic function of Bcl2 (49Dimmeler S. Breitschopf K. Haendeler J. Zeiher A.M. J. Exp. Med. 1999; 189: 1815-1822Crossref PubMed Scopus (284) Google Scholar). Thus, inhibition of Bcl2 degradation, either by suppressing a ubiquitin-dependent proteasomal pathway or by mimicking continuous phosphorylation of the putative phosphorylation sites in Bcl2, was able to confer cell resistance to apoptosis (49Dimmeler S. Breitschopf K. Haendeler J. Zeiher A.M. J. Exp. Med. 1999; 189: 1815-1822Crossref PubMed Scopus (284) Google Scholar). With respect to a functional role for stress-induced Bcl2 phosphorylation by JNK1, our findings suggest a novel mechanism by which Bcl2 may protect cells from apoptosis following various stress applications. Thus, JNK1 may play an important role in regulating apoptosis through phosphorylation of Bcl2 (Fig.9). These findings would help to explain the apparent paradox between JNK activation and cell survival or death. Furthermore, these findings suggest that a novel antineoplastic strategy may be to inactivate Bcl2 by inhibiting the SAK, JNK1. Apoptosis is a highly organized, physiologic mechanism of destroying injured and abnormal cells. Apoptosis is implicated in the regulation of development, differentiation, and homeostasis (1Dixon S.C. Soriano B.J. Lush R.M. Borner M.M. Figg W.D. Ann. Pharmacother. 1997; 31: 76-82Crossref PubMed Scopus (78) Google Scholar).Bcl2 is the first identified survival gene involved in the control of apoptosis (2Gajewski T.F. Thompson C.B. Cell. 1996; 87: 589-592Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Greater than 15 Bcl2 family members, representing both anti-apoptotic and pro-apoptotic members, have been identified to date in mammalian cells and viruses (3Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4780) Google Scholar). Results have suggested potential mechanism(s) by which Bcl2 functions including: (a) heterodimerization with its proapoptotic partner, Bax (4Oltvai Z.N. Milliman C.L. Korsmeyer S.J. 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