Catalase Activity Is Regulated by c-Abl and Arg in the Oxidative Stress Response
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m301292200
ISSN1083-351X
AutoresCheng Cao, Yumei Leng, Donald Küfe,
Tópico(s)Fungal Plant Pathogen Control
ResumoThe Abl family of mammalian non-receptor tyrosine kinases includes c-Abl and Arg. Recent studies have demonstrated that c-Abl and Arg are activated in the response of cells to oxidative stress. This work demonstrates that catalase, a major effector of the cellular defense against H2O2, interacts with c-Abl and Arg. The results show that H2O2 induced binding of c-Abl and Arg to catalase. The SH3 domains of c-Abl and Arg bound directly to catalase at a P293FNP site. c-Abl and Arg phosphorylated catalase at Tyr231 and Tyr386 in vitro and in the response of cells to H2O2. The functional significance of the interaction is supported by the demonstration that cells deficient in both c-Abl and Arg exhibit substantial increases in H2O2 levels. In addition, c-abl –/– arg –/– cells exhibited a marked increase in H2O2-induced apoptosis compared with that found in the absence of either kinase. These findings indicate that c-Abl and Arg regulate catalase and that this signaling pathway is of importance to apoptosis in the oxidative stress response. The Abl family of mammalian non-receptor tyrosine kinases includes c-Abl and Arg. Recent studies have demonstrated that c-Abl and Arg are activated in the response of cells to oxidative stress. This work demonstrates that catalase, a major effector of the cellular defense against H2O2, interacts with c-Abl and Arg. The results show that H2O2 induced binding of c-Abl and Arg to catalase. The SH3 domains of c-Abl and Arg bound directly to catalase at a P293FNP site. c-Abl and Arg phosphorylated catalase at Tyr231 and Tyr386 in vitro and in the response of cells to H2O2. The functional significance of the interaction is supported by the demonstration that cells deficient in both c-Abl and Arg exhibit substantial increases in H2O2 levels. In addition, c-abl –/– arg –/– cells exhibited a marked increase in H2O2-induced apoptosis compared with that found in the absence of either kinase. These findings indicate that c-Abl and Arg regulate catalase and that this signaling pathway is of importance to apoptosis in the oxidative stress response. Normal aerobic metabolism is associated with the production of reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; SH, Src homology; MEFs, mouse embryo fibroblasts; GST, glutathione S-transferase; BSO, l-buthionine sulfoxime; DCF-DA, 2′,7′-dichlorofluorescein diacetate. including superoxides, hydrogen peroxide (H2O2), hydroxyl radicals, and nitric oxide. A substantial amount of oxygen reduced by the mitochondrial respiratory chain is converted to superoxide and then to H2O2 by the mitochondrial superoxide dismutase (1Fridovich I. J. Biol. Chem. 1997; 272: 18515-18517Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar). H2O2 is readily diffusible across cell membranes and functions as a signaling molecule in diverse cellular events. Mitogenic signals induced by certain growth factors and activated Ras are mediated by H2O2 production (2Sundaresan M. Yu Z.-X. Ferrans V. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2322) Google Scholar, 3Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1441) Google Scholar). H2O2 activates c-Jun, c-Fos, and NF-κB and thereby regulates gene transcription (4Devary Y. Gottlieb R.A. Smeal T. Karin M. Cell. 1992; 71: 1081-1091Abstract Full Text PDF PubMed Scopus (796) Google Scholar, 5Schreck R. Rieber P. Baeuerle P. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3430) Google Scholar). In addition, the extracellular signal-regulated protein kinase, c-Jun N-terminal kinases, p70S6K, and p90rsk are activated by H2O2 signaling (2Sundaresan M. Yu Z.-X. Ferrans V. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2322) Google Scholar, 6Lo Y. Wong J. Cruz T. J. Biol. Chem. 1996; 271: 15703-15707Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 7Bai J. Rodriguez A. Melendez J. Cederbaum A. J. Biol. Chem. 1999; 274: 26217-26224Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The generation of H2O2 by normal cellular metabolism is also associated with damage to DNA, proteins, and lipids (8Croteau D. Bohr V. J. Biol. Chem. 1997; 272: 25409-25412Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 9Berlett S. Stadtman E. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2809) Google Scholar) and the induction of apoptosis (10Jacobson M.D. Trends. Biochem. Sci. 1996; 21: 83-86Abstract Full Text PDF PubMed Scopus (730) Google Scholar, 11Manna S.K. Zhang H.J. Yan T. Oberley L.W. Aggarwal B.B. J. Biol. Chem. 1998; 273: 13245-13254Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). Although few insights are available regarding the mechanisms responsible for ROS-induced cell death, H2O2 activates topoisomerase II-mediated cleavage of chromosomal DNA and thereby apoptosis (12Li T. Chen A. Yu C. Mao Y. Wang H. Liu L. Genes Dev. 1999; 13: 1553-1560Crossref PubMed Scopus (149) Google Scholar). The p66shc adaptor protein (13Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1479) Google Scholar) and the p85 subunit of phosphatidylinositol 3-kinase (14Yin Y. Terauchi Y. Solomon G. Aizawa S. Rangarajan P. Yazaki Y. Kadowaki T. Barrett J. Nature. 1998; 391: 707-710Crossref PubMed Scopus (151) Google Scholar) have been implicated in the apoptotic response to H2O2. Other studies have indicated that p53-induced apoptosis is mediated by ROS (12Li T. Chen A. Yu C. Mao Y. Wang H. Liu L. Genes Dev. 1999; 13: 1553-1560Crossref PubMed Scopus (149) Google Scholar, 15Johnson T. Yu Z.-X. Ferrans V. Lowenstein R. Finkel T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11848-11852Crossref PubMed Scopus (525) Google Scholar, 16Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2246) Google Scholar) and that H2O2-induced apoptosis is p53-dependent (13Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1479) Google Scholar, 14Yin Y. Terauchi Y. Solomon G. Aizawa S. Rangarajan P. Yazaki Y. Kadowaki T. Barrett J. Nature. 1998; 391: 707-710Crossref PubMed Scopus (151) Google Scholar). The predominant enzymatic mechanisms that regulate intracellular H2O2 levels are mediated by catalase and glutathione peroxidase. The tetrameric catalase converts H2O2 to H2O and O2 in peroxisomes (17Amstad P. Peskin A. Shah G. Mirault M.E. Moret R. Zbinden I. Cerutti P. Biochemistry. 1991; 30: 9305-9313Crossref PubMed Scopus (266) Google Scholar). With the exception of rat myocardial cells, catalase is not detectable in mitochondria (18Radi R. Turrens J.F. Chang L.Y. Bush K.M. Crapo J.D. Freeman B.A. J. Biol. Chem. 1991; 266: 22028-22034Abstract Full Text PDF PubMed Google Scholar). Glutathione peroxidase converts H2O2 to H2O in a reaction that oxidizes GSH to its disulfide form (GSSG). In turn, GSH is regenerated from GSSG by glutathione reductase. Regulation of H2O2 by the glutathione redox cycle is mediated in the cytosol and mitochondria. Little is known about the regulation of catalase or glutathione peroxidase, particularly the effects of post-translational modifications on their activities. The widely expressed mammalian c-Abl and Arg non-receptor tyrosine kinases (19Goff S.P. Gilboa E. Witte O.N. Baltimore D. Cell. 1980; 22: 777-785Abstract Full Text PDF PubMed Scopus (280) Google Scholar, 20Kruh G.D. King C.R. Kraus M.H. Popescu N.C. Amsbaugh S.C. McBride W.O. Aaronson S.A. Science. 1986; 234: 1545-1548Crossref PubMed Scopus (73) Google Scholar, 21Kruh G.D. Perego R. Miki T. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5802-5806Crossref PubMed Scopus (136) Google Scholar) have been implicated in cellular responses to oxidative and other types of stress (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 (463) Google Scholar, 23Sun X. Majumder P. Shioya H. Wu F. Kumar S. Weichselbaum R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 17237-17240Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 24Cao C. Ren X. Kharbanda S. Koleske A.J. Prasad K. Kufe D. J. Biol. Chem. 2001; 276: 11465-11468Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 25Ito Y. Pandey P. Mishra N. Kumar S. Narula N. Kharbanda S. Saxena S. Kufe D. Mol. Cell. Biol. 2001; 21: 6233-6242Crossref PubMed Scopus (118) Google Scholar). The N-terminal SH3, SH2, and kinase domains of c-Abl and Arg share ∼90% identity. The C-terminal regions of c-Abl and Arg share 29% identity and are distinguished from the other non-receptor tyrosine kinases by the presence of globular and filamentous actin-binding domains (26Van Etten R.A. Jackson P.K. Baltimore D. Sanders M.C. Matsuddaira P.T. Janmey P.A. J. Cell Biol. 1994; 124: 325-340Crossref PubMed Scopus (237) Google Scholar). c-Abl differs from Arg by the presence of a nuclear localization signal (26Van Etten R.A. Jackson P.K. Baltimore D. Sanders M.C. Matsuddaira P.T. Janmey P.A. J. Cell Biol. 1994; 124: 325-340Crossref PubMed Scopus (237) Google Scholar) and DNA-binding sequences (27Kipreos E.T. Wang J.Y.J. Science. 1992; 256: 382-385Crossref PubMed Scopus (177) Google Scholar) in the C-terminal region. c-Abl also differs from Arg by localization to the nucleus and extranuclear organelles, whereas Arg is expressed predominantly in the cytoplasm (28Wang B. Kruh G.D. Oncogene. 1996; 13: 193-197PubMed Google Scholar). The cytoplasmic form of c-Abl is activated in the response of cells to H2O2 by a mechanism dependent on protein kinase Cδ (23Sun X. Majumder P. Shioya H. Wu F. Kumar S. Weichselbaum R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 17237-17240Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 29Sun X. Wu F. Datta R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 7470-7473Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). c-Abl is targeted to mitochondria in H2O2-treated cells and induces loss of the mitochondrial transmembrane potential (30Kumar S. Bharti A. Mishra N. Kharbanda S. Saxena S. Kufe D. J. Biol. Chem. 2001; 276: 17281-17285Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Mitochondrial targeting of c-Abl in response to oxidative stress is also associated with cytochrome c release and induction of apoptosis (23Sun X. Majumder P. Shioya H. Wu F. Kumar S. Weichselbaum R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 17237-17240Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In concert with these findings, c-Abl-deficient cells exhibit resistance to H2O2-induced cell death (23Sun X. Majumder P. Shioya H. Wu F. Kumar S. Weichselbaum R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 17237-17240Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 30Kumar S. Bharti A. Mishra N. Kharbanda S. Saxena S. Kufe D. J. Biol. Chem. 2001; 276: 17281-17285Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Other studies have shown that Arg is activated by oxidative stress and that this response involves Arg-mediated phosphorylation of the pro-apoptotic Siva-1 protein (24Cao C. Ren X. Kharbanda S. Koleske A.J. Prasad K. Kufe D. J. Biol. Chem. 2001; 276: 11465-11468Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). H2O2-induced apoptosis is attenuated in Arg-deficient cells, and this defect is corrected by reconstituting Arg expression (24Cao C. Ren X. Kharbanda S. Koleske A.J. Prasad K. Kufe D. J. Biol. Chem. 2001; 276: 11465-11468Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Moreover, recent findings indicate that ROS induce c-Abl-Arg heterodimers and that both c-Abl and Arg are necessary as effectors in the apoptotic response to oxidative stress (31Cao C. Leng Y. Kufe D. J. Biol. Chem. 2003; 278: 12961-12967Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This study demonstrates that c-Abl and Arg interact with catalase in the response of cells to oxidative stress. The functional significance of these findings is supported by the demonstration that cells deficient in both c-Abl and Arg exhibit elevated intracellular H2O2 levels and a pronounced apoptotic response to oxidative stress. Cell Culture—293 cells, MCF-7, MCF-7/c-Abl(K-R) (32Yuan Z. Huang Y. Ishiko T. Kharbanda S. Weichselbaum R. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1437-1440Crossref PubMed Scopus (178) Google Scholar), and MCF-7/Arg(K-R) (24Cao C. Ren X. Kharbanda S. Koleske A.J. Prasad K. Kufe D. J. Biol. Chem. 2001; 276: 11465-11468Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) cells and established lines from wild-type, c-abl –/–, arg –/–, and c-abl –/– arg –/– mouse embryo fibroblasts (MEFs) were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. U-937 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mml-glutamine, and antibiotics. Cells were treated with H2O2 (Sigma) and STI571 (Novartis, Basel, Switzerland). Transient transfections were performed with LipofectAMINE (Invitrogen). Vectors—FLAG-tagged c-Abl, Arg, and catalase and their mutants were expressed by cloning into the pcDNA3.1-based FLAG vector. His/Express-tagged constructs were prepared by cloning into pcDNA4HisMAX, which contains N-terminal His and Express tags (Invitrogen). Myc-tagged c-Abl and catalase vectors were prepared by cloning into pCMV-Myc (Clontech). Glutathione S-transferase (GST) fusion proteins were generated by expression of pGEX4T2-based vectors in Escherichia coli BL21(DE3). Immunoprecipitation and Immunoblot Analysis—Cell lysates were prepared in lysis buffer (50 mm Tris-HCl (pH 7.5), 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 10 mm sodium fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin A) containing 1% Nonidet P-40. Soluble protein was subjected to immunoprecipitation with anti-c-Abl (K-12, Santa Cruz Biotechnology), anti-catalase (Sigma), anti-Myc (Santa Cruz Biotechnology), anti-FLAG (M5, Sigma), or anti-Express (Invitrogen) antibody. Immunoblot analysis was performed with anti-catalase (Calbiochem), anti-c-Abl (sc-2411, Santa Cruz Biotechnology), anti-Arg (24Cao C. Ren X. Kharbanda S. Koleske A.J. Prasad K. Kufe D. J. Biol. Chem. 2001; 276: 11465-11468Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), anti-Myc, anti-FLAG, anti-Express, or anti-Tyr(P) (4G10, Upstate Biotechnology, Inc.) antibody. The antigen-antibody complexes were visualized by chemiluminescence (ECL, Amersham Biosciences). Binding Assays—Cell lysates were incubated with 5 μg of GST or GST fusion proteins bound to glutathione beads. After incubation for 2 h at 4 °C, the adsorbates were washed with lysis buffer and then subjected to immunoblot analysis. An aliquot of the total lysate (2%, v/v) was included as a control. For direct binding assays, purified GST fusion proteins were incubated with in vitro translated 35S-labeled catalase. The adsorbates were analyzed by SDS-PAGE and autoradiography. Kinase Assays—Purified human catalase (2 μg; Sigma) or GST-catalase (2 μg) was incubated with GST-c-Abl (prepared in Sf9 cells) or GST-Arg (prepared in E. coli) in kinase buffer (20 mm HEPES (pH 7.5), 75 mm KCl, 10 mm MgCl2, and 10 mm MnCl2) containing 2.5 μCi of [γ-32P]ATP for 30 min at 30 °C. The reaction products were analyzed by SDS-PAGE and autoradiography. Assessment of Catalase Activity—For the substrate solution, 0.1 ml of 30% H2O2 was added to 50 ml of 0.05 m phosphate buffer (pH 7.0). The A 240 of the solution was adjusted to between 0.550 and 0.520. The reactions were initiated by adding 0.1 ml of the enzyme solution to 2.9 ml of the substrate solution in a silica cuvette (1-cm light path) at 25 °C. The time required for the A 240 to decrease from 0.450 to 0.400 corresponds to the decomposition of 3.45 μmol of H2O2 in the 3-ml assay (Sigma catalog). FLAG-catalase or the FLAG-catalase(Y-F) mutants were expressed in 293 cells with c-Abl, c-Abl(K-R), Arg, or Arg(K-R). Anti-FLAG immunoprecipitates were assayed for catalase activity and by immunoblotting for catalase protein. Densitometric scanning of the signals was compared with that obtained with dilutions of crystalline catalase (Sigma). Measurement of Intracellular H 2 O 2 Levels—Cells were treated with 0.3 mml-buthionine sulfoxime (BSO; Sigma) for 16 h and were then incubated with 5 μm 2′,7′-dichlorofluorescein diacetate (DCF-DA; Sigma) for 30 min at 37 °C in the dark. The cells were then incubated in the absence and presence of 0.5 mm H2O2 for 30 min at 37 °C. The fluorescence of oxidized DCF was measured at an excitation wavelength of 480 nm and an emission wavelength of 525 nm by flow cytometry (BD Biosciences). The geometric mean obtained from 104 cells was used to calculate the relative H2O2 concentration. Apoptosis Assays—DNA content was assessed by staining ethanol-fixed and citrate buffer-permeabilized cells with propidium iodide and monitoring by FACScan (BD Biosciences). The numbers of cells with sub-G1 DNA were determined with a MODFIT LT program (24Cao C. Ren X. Kharbanda S. Koleske A.J. Prasad K. Kufe D. J. Biol. Chem. 2001; 276: 11465-11468Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). c-Abl and Arg Associate with Catalase—To determine whether c-Abl associates with catalase in the cellular response to oxidative stress, lysates from H2O2-treated MCF-7 cells were subjected to immunoprecipitation with anti-c-Abl antibody. Immunoblot analysis of the precipitates with anti-catalase antibody demonstrated a low level of constitutive c-Abl binding to catalase (Fig. 1A). However, treatment with 0.25 mm H2O2 resulted in increased formation of c-Abl-catalase complexes (Fig. 1A). Similar findings were obtained when cells were treated with 0.5 and 1.0 mm H2O2 (Fig. 1A). However, exposures to 2.0 mm H2O2 were associated with decreased formation of c-Abl-catalase complexes (Fig. 1A). In the reciprocal experiment, analysis of anti-catalase immunoprecipitates by immunoblotting with anti-c-Abl antibody confirmed that H2O2 induced the association of c-Abl and catalase and that the extent of the interaction was dependent on the concentration of H2O2 (Fig. 1B). Similar results were obtained for an H2O2-dependent association between Arg and catalase (Fig. 1B). The demonstration that H2O2 induced binding of catalase with c-Abl and Arg in U-937 cells indicates that the interaction with catalase occurs in different cell types (Fig. 1C). To confirm binding of catalase to c-Abl and Arg, 293 cells were transfected to express Myc-tagged catalase and FLAG-tagged c-Abl or Arg. Immunoblot analysis of anti-FLAG immunoprecipitates with anti-catalase antibody demonstrated constitutive binding of catalase and c-Abl (Fig. 1D). The finding that kinase-inactive c-Abl(K-R) also associated with catalase demonstrates that binding is independent of the c-Abl kinase function (Fig. 1D). Similar results were obtained for a constitutive interaction between catalase and Arg or Arg(K-R) (Fig. 1D). These findings indicate that c-Abl and Arg associate with catalase and that the interaction is increased in response to oxidative stress. Binding of c-Abl and Arg to Catalase—To further define the association between c-Abl and catalase, lysates from MCF-7 cells were incubated with GST fusion proteins containing the c-Abl SH2 or SH3 domain. Analysis of the adsorbates by immunoblotting with anti-catalase antibody demonstrated binding of catalase to the c-Abl SH2 and SH3 domains (Fig. 2A). Similar findings were obtained for binding of catalase to Arg SH2 and Arg SH3 domain (Fig. 2A). As a control, there was no detectable binding of catalase to GST proteins containing the Grb2 SH2 or SH3 domain (Fig. 2A). To assess whether binding of c-Abl and catalase is direct, GST-c-Abl and GST-Arg fusion proteins were incubated with purified 35S-labeled FLAG-catalase. Analysis of the adsorbates by SDS-PAGE and autoradiography demonstrated binding to the c-Abl and Arg SH3 domains (Fig. 2B). By contrast, there was no detectable binding of catalase to the GST proteins containing the c-Abl or Arg SH2 domain (Fig. 2B). These results suggest that catalase is tyrosine-phosphorylated in MCF-7 cells, but not after in vitro translation. Catalase contains a potential proline-rich site (293PFNP296) for SH3 domain binding. Lysates from 293 cells expressing a FLAG-catalase(P293A) mutant were analyzed for binding to the c-Abl SH3 domain. The results demonstrate that, in contrast to wild-type catalase, binding of the c-Abl SH3 domain was abrogated with catalase(P293A) (Fig. 2C). Similar results were obtained with the Arg SH3 domain (Fig. 2C). To show that the PFNP motif is important for the interaction between c-Abl and catalase in vivo, lysates from cells expressing Myc-c-Abl and FLAG-catalase or FLAG-catalase(P293A) were subjected to immunoprecipitation with anti-Myc antibody. Analysis of the immunoprecipitates by immunoblotting with anti-FLAG antibody demonstrated binding of c-Abl to catalase, but not to catalase(P293A) (Fig. 2D). In a similar analysis, there was no detectable binding of Myc-Arg to catalase(P293A) (data not shown). These findings demonstrate that c-Abl and Arg bind directly to catalase through interactions of their SH3 domains and the catalase PFNP site. Tyrosine Phosphorylation of Catalase in Response to Oxidative Stress—To determine whether c-Abl phosphorylates catalase, recombinant c-Abl was incubated with purified catalase and [γ-32P]ATP. Analysis of the reaction products by SDS-PAGE and autoradiography demonstrated phosphorylation of catalase (Fig. 3A, left panel). To confirm these findings, similar reactions were performed in the presence of unlabeled ATP. Immunoblot analysis of the reaction products with anti-Tyr(P) antibody demonstrated that catalase was phosphorylated at tyrosine (Fig. 3A, right panel). In concert with the demonstration that ROS-induced activation of c-Abl is mediated by protein kinase Cδ (23Sun X. Majumder P. Shioya H. Wu F. Kumar S. Weichselbaum R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 17237-17240Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 29Sun X. Wu F. Datta R. Kharbanda S. Kufe D. J. Biol. Chem. 2000; 275: 7470-7473Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), the addition of H2O2 to the reaction had no apparent effect on tyrosine phosphorylation of catalase (data not shown). The results also demonstrate that catalase is a substrate for Arg (Fig. 3B). To assess c-Abl-mediated phosphorylation of catalase in vivo, lysates from cells expressing Myc-catalase were immunoprecipitated with anti-Myc antibody. Analysis of the precipitates by immunoblotting with anti-Tyr(P) antibody demonstrated phosphorylation of catalase at tyrosine (Fig. 3C). The finding that treatment of the cells with the c-Abl/Arg kinase inhibitor STI571 (33Druker B.J. Tamura S. Buchdunger E. Ohno S. Segal G.M. Fanning S. Zimmermann J. Lydon N.B. Nat. Med. 1996; 2: 561-566Crossref PubMed Scopus (3167) Google Scholar) decreased tyrosine phosphorylation of catalase provided support for a c-Abl- and/or Arg-dependent mechanism (Fig. 3C). Cells expressing Myc-catalase and c-Abl also showed phosphorylation of catalase at tyrosine (Fig. 3D). By contrast, tyrosine phosphorylation of catalase was inhibited in cells expressing c-Abl(K-R) (Fig. 3D). Catalase was also subject to tyrosine phosphorylation in cells expressing kinase-active Arg, but not kinase-inactive Arg(K-R) (Fig. 3D). To assess tyrosine phosphorylation of catalase in response to oxidative stress, anti-catalase immunoprecipitates from lysates of H2O2-treated MCF-7 cells were subjected to immunoblotting with anti-Tyr(P) antibody. The results demonstrate that tyrosine phosphorylation of catalase was increased in response to 1.0 (but not 0.25 or 2.0) mm H2O2 (Fig. 4A). By contrast, H2O2-induced increases in tyrosine phosphorylation of catalase were not found in MCF-7 cells stably expressing c-Abl(K-R) or Arg(K-R) (Fig. 4B). Moreover, pretreatment with 1 and 10 μm STI571 blocked H2O2-induced tyrosine phosphorylation of catalase (Fig. 4C). These findings collectively demonstrate that catalase is phosphorylated by a c-Abl- and/or Arg-dependent mechanism in the oxidative stress response. Catalase Is Regulated by Phosphorylation at Tyr231 and Tyr386—To define the phosphorylation sites, catalase was incubated with c-Abl and then subjected to tryptic digestion. Analysis of the fragments by high pressure liquid chromatography separation and Edman sequencing demonstrated phosphorylation at Tyr231 and Tyr386. Compared with wild-type catalase, mutation of Tyr231 to Phe was associated with a decrease in c-Abl-mediated phosphorylation (Fig. 5A). Mutation of Tyr386 to Phe was also associated with a decrease in c-Abl phosphorylation (Fig. 5A). The catalase(Y231F/Y386F) double mutant exhibited comparable decreases in c-Abl phosphorylation, but not complete abrogation (Fig. 5A). Similar results were obtained for Arg-mediated phosphorylation of the catalase mutants (data not shown). Because these findings indicate that phosphorylation of catalase by c-Abl and Arg is not restricted to Tyr231 and Tyr386, we generated mutants at the other 14 tyrosine sites. The absence of a detectable effect of these mutants on c-Abl and Arg phosphorylation indicated that Tyr231 and Tyr386 are the predominant sites (data not shown). To determine whether in vivo phosphorylation of catalase is affected by the tyrosine mutations, 293 cells were transfected to express Myc-c-Abl and FLAG-catalase or FLAG-catalase mutated at Tyr231, Tyr386, or Tyr231/Tyr386. Immunoblot analysis of anti-FLAG immunoprecipitates with anti-Tyr(P) antibody showed a substantial decrease in tyrosine phosphorylation of the catalase(Y231F) mutant (Fig. 5B). The extent of tyrosine phosphorylation was also decreased with catalase(Y386F) and the double mutant (Fig. 5B). Similar findings were obtained when Express-Arg was expressed with wild-type catalase and the Tyr-to-Phe mutants (Fig. 5C). To determine whether catalase is regulated by phosphorylation at Tyr231 and Tyr386, anti-FLAG immunoprecipitates from 293 cells expressing FLAG-catalase and c-Abl or c-Abl(K-R) were analyzed for catalase activity. The results demonstrate that, compared with wild-type c-Abl, cotransfection of c-Abl(K-R) substantially decreased catalase activity (Fig. 5D). Compared with wild-type FLAG-catalase, the activity was decreased when FLAG-catalase(Y231F) or FLAG-catalase(Y386F) was expressed with c-Abl (Fig. 5D). The demonstration that the activity of the FLAG-catalase(Y231F/Y386F) double mutant was comparable to that of the single Tyr-to-Phe mutants indicates that phosphorylation at both Tyr231 and Tyr386 is needed for stimulation of catalase activity (Fig. 5D). Moreover, the inhibitory effects of c-Abl(K-R) on catalase activity were much less pronounced with these mutants than with the wild-type enzyme (Fig. 5D). Similar results were obtained when the wild-type and mutant FLAG-catalase proteins were expressed with Arg or Arg(K-R) (data not shown). These findings indicate that (i) c-Abl and Arg activate catalase by phosphorylation at both Tyr231 and Tyr386, and (ii) expression of c-Abl(K-R) or Arg(K-R) attenuates catalase activity. c-Abl and Arg Regulate Intracellular H 2 O 2 Levels—Intracellular H2O2 levels are controlled predominantly by catalase and the glutathione redox cycle. To assess the protective effects of catalase, BSO was used to inhibit the glutathione redox cycle by depleting intracellular levels of glutathione (7Bai J. Rodriguez A. Melendez J. Cederbaum A. J. Biol. Chem. 1999; 274: 26217-26224Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). MEFs were treated with BSO and monitored for intracellular H2O2 levels by fluorescence spectrophotometry using the oxidant-sensitive dye DCF-DA. The results demonstrate that, compared with wild-type MEFs, arg –/– cells exhibited somewhat higher intracellular H2O2 levels (Fig. 6A, upper panel). Increased H2O2 levels were more apparent in c-abl –/– cells (Fig. 6A, upper panel). Importantly, even higher levels of H2O2 were found in c-abl –/– arg –/– cells (Fig. 6A, upper panel). Treatment of the wild-type MEFs with H2O2 was associated with an increase in H2O2 levels (Fig. 6A, lower panel). H2O2 treatment of c-abl –/– and arg –/– cells was also associated with higher intracellular H2O2 levels (Fig. 6A, lower panel). Moreover, the finding that the c-abl –/– arg –/– cells exhibited a striking increase in H2O2 levels supports more pronounced dysregulation of catalase than that observed in c-abl –/– or arg –/– cells (Fig. 6A, lower panel). Analysis of three experiments confirmed significant differences for H2O2 levels in control and H2O2-treated c-abl –/– arg –/– cells compared with wild-type, c-abl –/–, and arg –/– cells (Fig. 6B). In addition, analysis of two independent clones of c-abl –/– arg –/– cells indicated that the substantial differences were not due to clonal variation (Fig. 6B). To confirm the basis for dysregulation of H2O2 homeostasis, c-abl –/– arg –/– cells were transduced with a retroviral vector expressing Arg (24Cao C. Ren X. Kharbanda S. Koleske A.J. Prasad K. Kufe D. J. Biol. Chem. 2001; 276: 11465-11468Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The results demonstrate that Arg expression was associated with decreases in H2O2 l
Referência(s)