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Bcl-xL Blocks Activation of Related Adhesion Focal Tyrosine Kinase/Proline-rich Tyrosine Kinase 2 and Stress-activated Protein Kinase/c-Jun N-terminal Protein Kinase in the Cellular Response to Methylmethane Sulfonate

1999; Elsevier BV; Volume: 274; Issue: 13 Linguagem: Inglês

10.1074/jbc.274.13.8618

ISSN

1083-351X

Autores

Pramod S. Pandey, Shalom Avraham, Andrew E. Place, Vijay Kumar, Pradip K. Majumder, Keding Cheng, Atsuko Nakazawa, Satya Saxena, Surender Kharbanda,

Tópico(s)

Cytokine Signaling Pathways and Interactions

Resumo

The stress-activated protein kinase/c-Jun N-terminal protein kinase (JNK) is induced in response to ionizing radiation and other DNA-damaging agents. Recent studies indicate that activation of JNK is necessary for induction of apoptosis in response to diverse agents. Here we demonstrate that methylmethane sulfonate (MMS)-induced activation of JNK is inhibited by overexpression of the anti-apoptotic protein Bcl-xL, but not by caspase inhibitors CrmA and p35. By contrast, UV-induced JNK activity is insensitive to Bcl-xL. The results demonstrate that treatment with MMS is associated with an increase in tyrosine phosphorylation of related adhesion focal tyrosine kinase (RAFTK)/proline-rich tyrosine kinase 2 (PYK2), an upstream effector of JNK and that this phosphorylation is inhibited by overexpression of Bcl-xL. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced JNK activation. The results indicate that inhibition of RAFTK phosphorylation by MMS in Bcl-xL cells is attributed to an increase in tyrosine phosphatase activity in these cells. Hence, treatment of Bcl-xL cells with sodium vanadate, a tyrosine phosphatase inhibitor, restores MMS-induced activation of RAFTK and JNK. These findings indicate that RAFTK-dependent induction of JNK in response to MMS is sensitive to Bcl-xL, but not to CrmA and p35, by a mechanism that inhibits tyrosine phosphorylation and thereby activation of RAFTK. Taken together, these findings support a novel role for Bcl-xL that is independent of the caspase cascade. The stress-activated protein kinase/c-Jun N-terminal protein kinase (JNK) is induced in response to ionizing radiation and other DNA-damaging agents. Recent studies indicate that activation of JNK is necessary for induction of apoptosis in response to diverse agents. Here we demonstrate that methylmethane sulfonate (MMS)-induced activation of JNK is inhibited by overexpression of the anti-apoptotic protein Bcl-xL, but not by caspase inhibitors CrmA and p35. By contrast, UV-induced JNK activity is insensitive to Bcl-xL. The results demonstrate that treatment with MMS is associated with an increase in tyrosine phosphorylation of related adhesion focal tyrosine kinase (RAFTK)/proline-rich tyrosine kinase 2 (PYK2), an upstream effector of JNK and that this phosphorylation is inhibited by overexpression of Bcl-xL. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced JNK activation. The results indicate that inhibition of RAFTK phosphorylation by MMS in Bcl-xL cells is attributed to an increase in tyrosine phosphatase activity in these cells. Hence, treatment of Bcl-xL cells with sodium vanadate, a tyrosine phosphatase inhibitor, restores MMS-induced activation of RAFTK and JNK. These findings indicate that RAFTK-dependent induction of JNK in response to MMS is sensitive to Bcl-xL, but not to CrmA and p35, by a mechanism that inhibits tyrosine phosphorylation and thereby activation of RAFTK. Taken together, these findings support a novel role for Bcl-xL that is independent of the caspase cascade. The cellular response to certain stress inducers includes cell cycle arrest and, in certain cases, lethality. However, the intracellular signals that control the induction of these events are mainly unclear. Whereas p53 has been implicated in promoting apoptosis induced by ionizing radiation (IR) 1The abbreviations used are:IR, ionizing radiation; SV, sodium vanadate; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal protein kinase; PARP, poly(ADP-ribose) polymerase; proline-rich tyrosine kinase, RAFTK, related adhesion focal tyrosine kinase; MMS, methylmethane sulfonate; PKCδ, protein kinase C δ; TNF, tumor necrosis factor; GST, glutathioneS-transferase; ICE, interleukin-converting enzyme; PYK2, proline-rich tyrosine kinase.1The abbreviations used are:IR, ionizing radiation; SV, sodium vanadate; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal protein kinase; PARP, poly(ADP-ribose) polymerase; proline-rich tyrosine kinase, RAFTK, related adhesion focal tyrosine kinase; MMS, methylmethane sulfonate; PKCδ, protein kinase C δ; TNF, tumor necrosis factor; GST, glutathioneS-transferase; ICE, interleukin-converting enzyme; PYK2, proline-rich tyrosine kinase. exposure (1Lowe S.W. 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More direct evidence for involvement of interleukin-converting enzyme (ICE)-like proteases in apoptosis comes from studies utilizing the cowpox virus protein CrmA (10Ray C.A. Black R.A. Kronheim S.R. Greenstreet T.A. Sleath P.R. Salvesen G.S. Pickup D.J. Cell. 1992; 69: 597-604Abstract Full Text PDF PubMed Scopus (886) Google Scholar) and the baculovirus protein p35 (11Bump N.J. Hackett M. Hugunin M. Seshagiri S. Brady K. Chen P. Ferenz C. Franklin S. Ghayur T. Li P. Licari P. Mankovich J. Shi L. Greenberg A.H. Miller L.K. Wong W.W. Science. 1995; 269: 1885-1888Crossref PubMed Scopus (600) Google Scholar), which are direct inhibitors of at least certain members of the caspase family. Methylmethane sulfonate (MMS) is a monofunctional alkylating agent and is a potent inducer of cellular stress leading to chromosomal aberrations, point mutations, and cell killing (12Beranek D.T. Weis C.C. Swenson D.H. 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The stress-activated protein kinase (SAPK) (also known as c-Jun N-terminal kinase (JNK)) is induced by tumor necrosis factor (TNF), anisomycin, and interleukin-1 (16Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2941) Google Scholar, 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 (2403) Google Scholar). JNK is also activated by UV light, osmotic shock, IR, and a variety of other DNA damaging agents (18Gupta S. Campbell D. Dérijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1333) Google Scholar, 19Derijard B. Raingeaud J. Barrett T. Wu I.-H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1400) Google Scholar, 20Kharbanda S. Ren R. Pandey P. Shafman T. Feller S. Weichselbaum R. Kufe D. 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By contrast, other studies have shown that TNF-induced apoptosis is independent of JNK activation (32Liu Z.G. Hsu H.L. Goeddel D.V. Karin M. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1778) Google Scholar). Therefore, the role of JNK in mediating apoptosis may be inducer- and cell type-specific. Activation of the related adhesion focal tyrosine kinase (RAFTK) has been shown to be upstream to JNK in the response to TNF or UV in certain cell types (33Tokiwa G. Dikic I. Lev S. Schlessinger J. Science. 1996; 273: 792-794Crossref PubMed Scopus (285) Google Scholar). RAFTK (also known as PYK2 and Ca2+-dependent tyrosine kinase (CADTK)) is involved in signaling upstream to the extracellular-regulated kinase (ERK) pathway (34Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Llowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1242) Google Scholar, 35Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. 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Thus, RAFTK may function as an intermediate that links various calcium signals with both short and long term responses in neuronal cells (34Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Llowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1242) Google Scholar,36Yu H. Li X. Marchetto G.S. Dy R. Hunter D. Calvo B. Dawson T.L. Wilm M. Anderegg R.J. Graves L.M. Earp S.H. J. Biol. Chem. 1996; 271: 29993-29998Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). In this study, we demonstrate that MMS-induced activation of JNK in U-937 cells is blocked by overexpressing Bcl-xL and not CrmA or p35. By contrast, Bcl-xL had no apparent effect on UV-induced JNK activity. We also demonstrate that, in contrast to UV, MMS-induced activation of JNK is RAFTK-dependent and that tyrosine phosphorylation of RAFTK is sensitive to Bcl-xL. Moreover, treatment of U-937/Bcl-xL cells with sodium vanadate (SV) restores MMS-induced tyrosine phosphorylation of RAFTK and activation of JNK. Human U-937/neo, U-937/Bcl-xL, U-937/CrmA, and U-937/p35 cells (37Datta R. Manome Y. Taneja N. Boise L.H. Weischlebaum R. Wong W.W. Kufe D. Cell Growth and Differentiation. 1995; 6: 363-370PubMed Google Scholar, 38Datta R. Kojima H. Banach D. Bump N.J. Talanian R.V. Alnemri E.S. Weischlebaum R. Wong W.W. Kufe D. J. Biol. Chem. 1997; 272: 1965-1969Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine. PC12 cells were grown in DMEM containing 10% HI horse serum, 5% heat-inactivated-fetal bovine serum, and antibiotics. Cells (1 × 106/flask) were seeded 24 h before treating with 1 mm MMS (Sigma) with or without 200 μm SV (Sigma). Cells were also treated with 40 J/m2 UV (UV StratalinkerTM 1800, Stratagene). Cells were treated with 1 mm MMS with or without 200 μm SV. The initial number of cells seeded was 1 × 105/ml. After indicated times, the numbers of live cells was determined by trypan blue exclusion. Cells were washed with phosphate-buffered saline and lysed in 1 ml of lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1 mm SV, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and 10 μg/ml of leupeptin and aprotinin) as described (21Kharbanda S. Pandey P. Ren R. Mayer B. Zon L. Kufe D. J. Biol. Chem. 1995; 270: 30278-30281Crossref PubMed Scopus (127) Google Scholar). Lysates were incubated with anti-JNK antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4 °C and then for 45 min after addition of protein A-Sepharose. The immune complexes were washed three times with lysis buffer and once with kinase buffer and resuspended in kinase buffer containing [γ-32P]ATP (6000 Ci/mmol; NEN Life Science Products) and GST-Jun (1–102) (39Saleem A. Yuan Z.M. Taneja N. Rubin E. Kufe D. Kharbanda S. J. Immunol. 1995; 154: 4150-4156PubMed Google Scholar). The reactions were incubated for 15 min at 30 °C and terminated by the addition of SDS sample buffer. The proteins were analyzed by 10% SDS-polyacrylamide gel electrophoresis and autoradiography. Cell lysates were separated by electrophoresis in SDS-polyacrylamide gels and then transferred to nitrocellulose paper. Immunoblot analyses were performed using anti-Bcl-x (40Boise L.H. Gonzaiz-Garcia M. Postema C.E. Ding L. Lindsten T. Turka L.A. Mao X. Nunez G. Thompson C.B. Cell. 1993; 74: 597-608Abstract Full Text PDF PubMed Scopus (2908) Google Scholar), anti-PKCδ (Santa Cruz) or anti-Tyr(P) (4G10, Upstate Biotechnology, Lake Placid, NY) antibodies. The antigen-antibody complexes were visualized by chemiluminescence (ECL, Amersham Pharmacia Biotech). Preparation of lysates and immunoblotting for PARP were performed as described using the C-2–10 anti-PARP monoclonal antibody (41Desnoyers S. Shah G.M. Brochu G. Poirier G.G. Ann. Biochem. 1994; 218: 470-473Crossref Scopus (30) Google Scholar). After washing with phosphate-buffered saline/Tween, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech) for anti-PARP antibody. Cells were washed with phosphate-buffered saline and lysed in 1 ml of lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1 mm SV, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and 10 μg/ml leupeptin and aprotinin) as described (42Kharbanda S. Pandey P. Jin S. Satoshi I. Bharti A. Yuan Z.M. Weichselbaum R. Weaver D. Kufe D. Nature. 1997; 386: 732-735Crossref PubMed Scopus (237) Google Scholar). Lysates were incubated with anti-RAFTK antibody (43Avraham S. London R. Fu Y. Ota S. Hiregowdara D. Li J. Jiang S. Pasztor L.M. White R.A. Groopman J.E. Avraham H. J. Biol. Chem. 1995; 270: 27742-27751Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) for 1 h at 4 °C and then for 45 min after addition of protein A-Sepharose. The immune complexes were analyzed by immunoblotting with anti-Tyr(P) (4G10, Upstate Biotechnology) or anti-RAFTK antibodies. PC12 cells were transiently transfected with vector or Flag-RAFTK (K-M) with pEBG-SAPK using LipofectAMINE (Life Technologies, Inc.). After transfection, cells were treated with MMS, and total cell lysates were subjected to incubation with GST protein adsorbed on GSH beads. The protein precipitates were analyzed by GST-Jun immune complex kinase assays as described above. Total cell lysates were also analyzed by immunoblotting with anti-GST-SAPK. U-937 or U-937/Bcl-xL cells were treated with 200 μmSV and harvested after 3 h. Cells were lysed in phosphate-free 50 mm Tris-HCl, pH 7.5, buffer using a sonicator. The PTPase activity in whole cell lysate was measured by a nonradioactive photometric enzyme immunoassay kit (Boehringer Mannheim). The reactions were performed directly in the wells of a microtiter plate with biotin-labeled synthetic tyrosine-phosphorylated peptide substrate (100 mm) bound to the streptavidin matrix. Following the addition of vanadate (500 μm) to quench the reactions, the fractions of unmetabolized substrate in the reactions were determined immunochemically using peroxidase conjugated anti-phosphotyrosine antibodies. The development of color was monitored at 405 nm in a microtiter plate reader. To determine the involvement of Bcl-xL in MMS- and UV-induced apoptosis, we assessed cleavage of the 116-kDa PARP protein to an 85-kDa fragment in response to these agents. As expected, MMS-treatment of U-937 cells resulted in PARP cleavage (Fig.1 A). Similar findings were obtained when U-937 cells were treated with UV, and there was no detectable cleavage of PARP when U-937/Bcl-xL cells were treated with MMS or UV (Fig. 1 A). Previous studies have shown that PKCδ undergoes caspase-3-mediated proteolytic cleavage in an apoptotic pathway induced in response to diverse forms of stress (7Emoto Y. Manome G. Meinhardt G. Kisaki H. Kharbanda S. Robertson M. Ghayur T. Wong W.W. Kamen R. Weichselbaum R. Kufe D. EMBO J. 1995; 14: 6148-6156Crossref PubMed Scopus (652) Google Scholar, 44Ghayur T. Hugunin M. Talanian R.V. Ratnofsky S. Quinlan C. Emoto Y. Pandey P. Datta R. Kharbanda S. Allen H. Kamen R. Wong W. Kufe D. J. Exp. Med. 1996; 184: 2399-2404Crossref PubMed Scopus (446) Google Scholar). To further determine the activation of caspase-3 by MMS or UV, cell lysates from MMS- or UV-treated U-937 and U-937/Bcl-xLcells were analyzed by immunoblotting with PKCδ. Similar to PARP cleavage, the results demonstrate that MMS or UV-treatment of U-937 cells results in PKCδ cleavage (Fig. 1 B). Moreover, there was no detectable cleavage of PKCδ when U-937/Bcl-xLcells were treated with MMS or UV (Fig. 1 B). We also compared the proliferation of U-937 and U-937/Bcl-xL cells in the presence of varying concentrations of MMS. At lower MMS concentrations (25–50 μm), there was little if any effect of MMS on proliferation of either cell type. At higher concentrations of MMS (1 mm), in contrast to U-937/Bcl-xL, approximately 50% of U-937 cells were dead (based on trypan blue exclusion) by 12–16 h. Moreover, more than 75% U-937 cells were dead when treated with 1 mm MMS for 24–36 h. These findings indicate that overexpression of Bcl-xL significantly blocks MMS-induced cell death. Moreover, overexpression of Bcl-xL also blocked MMS- and UV-induced internucleosomal DNA fragmentation, a hallmark of apoptosis (data not shown). Taken together, these findings indicated that overexpression of Bcl-xL is associated with inhibition of apoptosis in the response to MMS and UV. Whereas JNK activation has been associated with induction of apoptosis, we assayed anti-JNK immunoprecipitates for phosphorylation of GST-Jun. The results demonstrate increased JNK activity and no change in JNK protein levels in wild type cells treated with MMS or UV (Fig. 1, C andD, and data not shown). Importantly, MMS-induced JNK activity was completely inhibited in cells overexpressing Bcl-xL (Fig. 1 C). By contrast, Bcl-xL had no detectable effect on UV-induced JNK activity (Fig. 1 D). To rule out a clonal effect, three additional, independently selected clones of U-937/Bcl-xL cells (clones 2, 9, and 10) that express high levels of this protein (data not shown) were treated with MMS. Analysis of anti-JNK immunoprecipitates from these clones also demonstrated inhibition of JNK activation by Bcl-xL (Fig. 1 E). Thus, whereas both MMS- and UV-induced apoptosis is inhibited in U-937/Bcl-xL cells, only MMS-induced JNK activity is blocked by Bcl-xL. These findings suggest that Bcl-xL functions upstream to the activation of JNK by MMS, but not UV. Previous studies have shown that Bcl-xL functions upstream to activation of caspase 3 (7Emoto Y. Manome G. Meinhardt G. Kisaki H. Kharbanda S. Robertson M. Ghayur T. Wong W.W. Kamen R. Weichselbaum R. Kufe D. EMBO J. 1995; 14: 6148-6156Crossref PubMed Scopus (652) Google Scholar, 44Ghayur T. Hugunin M. Talanian R.V. Ratnofsky S. Quinlan C. Emoto Y. Pandey P. Datta R. Kharbanda S. Allen H. Kamen R. Wong W. Kufe D. J. Exp. Med. 1996; 184: 2399-2404Crossref PubMed Scopus (446) Google Scholar). Because MMS- and UV-induced PARP cleavage is inhibited in U-937/Bcl-xL cells, we asked whether this event is also sensitive to CrmA or p35. There was no detectable cleavage of PARP in MMS-treated U-937/CrmA or U-937/p35 cells (Fig. 2 A). Similar results were obtained when cell lysates were analyzed for cleavage of PKCδ (data not shown). However, the finding that p35, but not CrmA, blocks UV-induced cleavage of PARP (Fig. 2 B) indicated that UV and MMS activate caspases by different mechanisms. Analysis of U-937/CrmA and U-937/p35 cells for MMS- or UV-induced JNK activity demonstrated that, in contrast to Bcl-xL, overexpression of CrmA or p35 in U-937 cells has no detectable effect on activation of JNK in response to these agents (Fig. 2 C). The inhibition of JNK by MMS in U-937/Bcl-xL, but not in U-937/CrmA or U-937/p35 cells, suggested that activation of JNK by MMS is upstream to caspase activation. To further assess this issue, U-937 cells treated with MMS for various times were analyzed for activation of JNK and cleavage of procaspase-3. The results demonstrate that whereas JNK is activated by MMS at 1 h, cleavage of procaspase-3 is detected only after 4–6 h (Fig. 2 D). Taken together, these findings indicate that in U-937 cells treated with MMS, Bcl-xLfunctions upstream of JNK activation by a mechanism either upstream from or independent of caspase inhibition. In this context, previous studies have shown that caspases act both upstream and downstream to JNK activation depending upon cell type and inducer (45Cahill M. Peter M. Kischkel F. Chinnaiyan A. Dixit V. Krammer P. Nordheim A. Oncogene. 1996; 13: 2087-2096PubMed Google Scholar, 46Frisch S. Vuori K. Kelaita D. Sicks S. J. Cell Biol. 1996; 135: 1377-1382Crossref PubMed Scopus (225) Google Scholar, 47Park D.S. Stefanis L. Yan I.Y. Farinelli S.E. Greene L.A. J. Biol. Chem. 1996; 271: 21898-21905Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Previous studies have shown that activation of JNK by TNF, MMS, or UV is induced by c-Abl-independent mechanisms (20Kharbanda S. Ren R. Pandey P. Shafman T. Feller S. Weichselbaum R. Kufe D. Nature. 1995; 376: 785-788Crossref PubMed Scopus (456) Google Scholar, 22Kharbanda S. Saleem A. Shafman T. Emoto Y. Taneja N. Rubin E. Weichselbaum R. Woodgett J. Avruvh J. Kyriakis J. Kufe D. J. Biol. Chem. 1995; 270: 18871-18874Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 29Pandey P. Raingeaud J. Kaneki M. Weichselbaum R. Davis R. Kufe D. Kharbanda S. J. Biol. Chem. 1996; 271: 23775-23779Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In this context, other studies have shown that the RAFTK tyrosine kinase plays a key role as an upstream regulator of the JNK pathway in response to UV in PC12 cells (33Tokiwa G. Dikic I. Lev S. Schlessinger J. Science. 1996; 273: 792-794Crossref PubMed Scopus (285) Google Scholar). RAFTK is activated by phosphorylation on tyrosine (33Tokiwa G. Dikic I. Lev S. Schlessinger J. Science. 1996; 273: 792-794Crossref PubMed Scopus (285) Google Scholar). Treatment of U-937 cells with MMS, but not UV, resulted in increased tyrosine phosphorylation of RAFTK (Fig.3 A). There was also no detectable tyrosine phosphorylation of RAFTK following treatment with agents, such as cis-platinum and IR, that activate c-Abl (data not shown). Whereas Bcl-xL blocks MMS-induced JNK activation and MMS induces tyrosine phosphorylation of RAFTK, we asked whether Bcl-xL functions upstream to RAFTK. The results demonstrate that Bcl-xL blocks MMS-induced tyrosine phosphorylation of RAFTK (Fig. 3 B). By contrast, inhibition of caspases by overexpressing CrmA or p35 had no effect on RAFTK tyrosine phosphorylation (data not shown). RAFTK is also expressed in PC12 neuroblastoma cells (34Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Llowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1242) Google Scholar, 43Avraham S. London R. Fu Y. Ota S. Hiregowdara D. Li J. Jiang S. Pasztor L.M. White R.A. Groopman J.E. 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The results demonstrate that treatment of PC12 cells expressing RAFTK K-M mutant, but not the empty vector, with MMS is associated with a significant inhibition of JNK activity (Fig. 4 C). Other studies have shown that the protein-tyrosine kinase c-Src functions upstream to JNK in response to MMS (48Liu Z.G. Baskaran R. Lea-Chou E.T. Wood L. Chen Y. Karin M. Wang J.Y.J. Nature. 1996; 384: 273-276Crossref PubMed Scopus (346) Google Scholar). Because c-Src is activated by a RAFTK-dependent mechanism (35Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar), these results together indicate that RAFTK acts as an upstream activator of the JNK signaling pathway in the cellular response to MMS.Figure 4RAFTK-dependent activation of JNK by MMS. A, PC12 cells were treated with 1 mm MMS for 30 min. Total cell lysates were subjected to immunoprecipitation with anti-RAFTK and analyzed by immunoblotting with anti-Tyr(P) antibody. B, total cell lysates from control and MMS-treated PC12 cells were subjected to immunoprecipitation with anti-JNK antibody. The protein precipitates were analyzed by in vitro immune complex kinase assays as described. C,PC12 cells were transiently transfected with vector or Flag-RAFTK K-M. The cells were also cotransfected with pEBG-SAPK. Fourty-eight hours after transfection, cells were treated with 1 mm MMS and harvested after 30 min. Cell lysates were incubated with GST, and the protein precipitates were analyzed by in vitro immune complex kinase assays (top panel). GST-protein precipitates were also analyzed by immunoblotting with anti-GST-SAPK (middle panel). The fold activation in JNK activity is expressed as the mean ± S.D. of three independent experiments (bottom panel).View Large Image Figure ViewerDownload (PPT) Tyrosine phosphorylation of proteins has been implicated as playing critical roles in regulating cell death and survival. Previous studies have shown that treatment of certain cell types with tyrosine phosphatase inhibitor, SV, is associated with the regulation of apoptosis (49Yang C. Chang J. Gorospe M. Passaniti A. Cell Growth Differ. 1996; 7: 161-171PubMed Google Scholar, 50Yousefi S. Green D.R. Blaser K. Simon H.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10868-10872Crossref PubMed Scopus (215) Google Scholar). Because the results of the present study demonstrate that overexpression of Bcl-xL inhibits tyrosine phosphorylation of RAFTK, we sought to determine whether a tyrosine phosphatase is involved in this regulation. To compare the overall tyrosine phosphatase activity in U-937 and U-937/Bcl-xLcells, we measured the background tyrosine phosphorylation of proteins in these cell types and total tyrosine phosphatase activity. U-937 and U-937/Bcl-xL cells treated with SV were also assayed for total tyrosine phosphatase activity. The results demonstrate that overexpression of Bcl-xL in U-937 cells is associated with an increase (approximately 3–4-fold) in total tyrosine phosphatase activity, and as expected, treatment with SV inhibited this activity to basal levels (Fig. 5 A). In concert with the increase in phosphatase activity, background tyrosine phosphorylation of proteins in U-937/Bcl-xL cells is significantly lower than that in U-937 cells (Fig. 5 B). To determine the role of a tyrosine phosphatase in the regulation of RAFTK activity by MMS, cells were treated with SV with or without MMS, and anti-RAFTK immunoprecipitates were analyzed by immunoblotting with anti-Tyr(P). The results demonstrate that treatment with SV increases MMS-induced tyrosine phosphorylation of RAFTK in U-937 cells (Fig.5 C). Importantly, treatment of U-937/Bcl-xLcells with SV restores tyrosine phosphorylation of RAFTK in response to MMS (Fig. 5 D). Taken together, these findings indicate that MMS-induced tyrosine phosphorylation and activation of RAFTK is regulated by a tyrosine phosphatase in U-937 cells. The 4-fold higher basal activity of tyrosine phosphatases in U-937/Bcl-xLcells contributes to inhibition of MMS-induced tyrosine phosphorylation of RAFTK. To determine whether treatment of cells with SV affects MMS-induced JNK activity, U-937 and U-937/Bcl-xL cells were treated with SV with or without MMS, and anti-JNK immunoprecipitates were analyzed by immune complex JNK kinase assays. The results demonstrate that treatment with SV significantly increases MMS-induced JNK activity in U-937 cells (Fig. 6 A). More importantly, treatment of U-937/Bcl-xL cells with SV restored JNK activation in response to MMS (Fig. 6 B). In this context, a recent study has suggested that activation of JNK by MMS and UV is mediated by distinct upstream modulators and that the MMS-response may be regulated by a tyrosine phosphatase (15Wilhelm D. Bender K. Knebel A. Angel P. Mol. Cell. Biol. 1997; 17: 4792-4800Crossref PubMed Scopus (224) Google Scholar). Moreover, activation of RAFTK by MMS is regulated by a tyrosine phosphatase and functions upstream to the activation of caspases. Taken together, the findings of our study indicate that MMS induces tyrosine phosphorylation of RAFTK and contributes directly to the activation of JNK. To determine whether inhibition of phosphatase activity by SV also affects MMS-induced cell death, U-937 and U-937/Bcl-xLcells were treated with SV with or without MMS, and cell death was assessed by trypan blue exclusion. The results demonstrate that treatment with SV increases MMS-induced cell death in U-937 cells (not shown). More importantly, SV treatment of U-937/Bcl-xLcells, which are resistant to MMS, was associated with approximately a 30% increase in cell death in response to MMS (Fig. 6 C). Taken together, these findings suggest that inhibition of phosphatase activity by SV potentiates cell death in response to MMS. Control of apoptosis and progression of cell cycle are closely linked processes, acting to preserve homeostasis and developmental morphogenesis. Proteins that regulate apoptosis, such as Bcl-2, have also been implicated in control of the cell (51Carmeliet P. Dor Y. Herbert J.M. Fukumura D. Brusselmans K. Dewerchin M. Neeman M. Bono F. Abramovitch R. Maxwell P. Koch C.J. Ratcliffe P. Moons L. Jain R.K. Collen D. Keshet E. Nature. 1998; 394: 485-490Crossref PubMed Scopus (2179) Google Scholar, 52Yin D.X. Schimke R.T. Proc. Natl. Acad. Sci. U. S. 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The present findings demonstrate that in contrast to CrmA or p35, Bcl-xL blocks MMS-induced activation of RAFTK, JNK, and induction of apoptosis. Thus, the RAFTK → JNK pathway is upstream to caspase activation in the cascade of MMS-induced apoptosis. We thank Drs. John Kyriakis, Joseph Avruch, Ajay Rana, Jim Woodgett, and Leonard Zon for providing various JNK cDNA constructs and anti-GST SAPK antibody and Dr. Hawa Avraham for critical reading of the manuscript. We thank Rebecca Farber for excellent technical assistance.

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