SOCS1 Inhibits Tumor Necrosis Factor-induced Activation of ASK1-JNK Inflammatory Signaling by Mediating ASK1 Degradation
2006; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m512338200
ISSN1083-351X
AutoresYun He, Wei Zhang, Rong Zhang, Haifeng Zhang, Min Wang,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoWe have previously shown that ASK1 undergoes ubiquitination and degradation in resting endothelial cells (EC) and that proinflammatory cytokine tumor necrosis factor (TNF) induces deubiquitination and stabilization, leading to ASK1 activation. However, the mechanism for the regulation of ASK1 stability is not known. In the present study, we have shown that SOCS1, a member of suppressor of cytokine signaling, induces ASK1 degradation. SOCS1 was constitutively expressed in EC and formed a labile complex with ASK1 that can be stabilized by proteasomal inhibitors. The phosphotyrosine-binding SH2 domain of SOCS1 was critical for its association with ASK1. Thus a SOCS1 mutant defective in phosphotyrosine binding failed to bind to and induce ASK1 degradation. Phosphotyrosine of ASK1 was induced in response to growth factors, and TNF induced dephosphorylation and dissociation of ASK1 from SOCS1. ASK1 with a mutation at Tyr-718 diminished the binding to SOCS1, suggesting that the phosphotyrosine-718 of ASK1 is critical for SOCS1 binding. Moreover, ASK1 expression and activity were up-regulated in SOCS1-deficient mice and derived EC, resulting in enhanced TNF-induced activation of JNK, expression of proinflammatory molecules, and apoptotic responses. We concluded that SOCS1 functions as a negative regulator in TNF-induced inflammation in EC, in part, by inducing ASK1 degradation. We have previously shown that ASK1 undergoes ubiquitination and degradation in resting endothelial cells (EC) and that proinflammatory cytokine tumor necrosis factor (TNF) induces deubiquitination and stabilization, leading to ASK1 activation. However, the mechanism for the regulation of ASK1 stability is not known. In the present study, we have shown that SOCS1, a member of suppressor of cytokine signaling, induces ASK1 degradation. SOCS1 was constitutively expressed in EC and formed a labile complex with ASK1 that can be stabilized by proteasomal inhibitors. The phosphotyrosine-binding SH2 domain of SOCS1 was critical for its association with ASK1. Thus a SOCS1 mutant defective in phosphotyrosine binding failed to bind to and induce ASK1 degradation. Phosphotyrosine of ASK1 was induced in response to growth factors, and TNF induced dephosphorylation and dissociation of ASK1 from SOCS1. ASK1 with a mutation at Tyr-718 diminished the binding to SOCS1, suggesting that the phosphotyrosine-718 of ASK1 is critical for SOCS1 binding. Moreover, ASK1 expression and activity were up-regulated in SOCS1-deficient mice and derived EC, resulting in enhanced TNF-induced activation of JNK, expression of proinflammatory molecules, and apoptotic responses. We concluded that SOCS1 functions as a negative regulator in TNF-induced inflammation in EC, in part, by inducing ASK1 degradation. The vascular cell that primarily limits the inflammatory and atherosclerotic process is the vascular endothelial cell (EC). 3The abbreviations used are: EC, endothelial cell; TNF, tumor necrosis factor; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; JAK, Janus kinase; Trx, thioredoxin; IFN, interferon; IGF, insulin-like growth factor; HUVEC, human umbilical vein EC; BAEC, bovine aortic EC; MLEC, mouse lung EC; ECGS, EC growth factors; HA, hemagglutinin; GST, glutathione S-transferase; PI, propidium iodide; SOCS, suppressor of cytokine signaling; TRAF, TNF reporter-associated factor; CIS, cytokine-inducible Src homology 2-containing protein; RT, reverse transcription; KO, knockout; WT, wild type; DN, dominant negative. Inflammatory cytokines such as tumor necrosis factor-α (TNF) induce EC dysfunction by disturbing normal homeostasis, relaxation, and survival by triggering signal transduction and gene transcription (1Madge L.A. Pober J.S. Exp. Mol. Pathol. 2001; 70: 317-325Crossref PubMed Scopus (263) Google Scholar). In addition to the NF-κB pathway, the stress-activated MAP kinases c-Jun N-terminal kinase (JNK) and p38 MAPKs have been shown to be critical for TNF-induced gene expression of proinflammatory molecules such as E-selectin and VCAM-1 (2Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, is an upstream activator of JNK/p38 MAPK cascades (3Matsuzawa A. Nishitoh H. Tobiume K. Takeda K. Ichijo H. Antioxid. Redox. Signal. 2002; 4: 415-425Crossref PubMed Scopus (205) Google Scholar). Studies from our laboratory and others have demonstrated that ASK1 functions as an effector in TNF-induced inflammation in EC (4Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Investig. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar, 5Liu Y. Min W. Circ. Res. 2002; 90: 1259-1266Crossref PubMed Scopus (314) Google Scholar, 6Yamawaki H. Pan S. Lee R.T. Berk B.C. J. Clin. Investig. 2005; 115: 733-738Crossref PubMed Scopus (206) Google Scholar). Thus ASK1 can be activated by almost all inflammatory stimuli such as TNF, interleukin-1, and reactive oxygen species. In contrast, anti-inflammatory factors such as antioxidants and shear stress inhibit ASK1 activity (4Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Investig. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar, 6Yamawaki H. Pan S. Lee R.T. Berk B.C. J. Clin. Investig. 2005; 115: 733-738Crossref PubMed Scopus (206) Google Scholar). ASK1 is a 170-kDa protein that functionally is composed of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain. The C-terminal domain of ASK1 binds to the TRAF domain of TRAF2 and TRAF6 (7Nishitoh H. Saitoh M. Mochida Y. Takeda K. Nakano H. Rothe M. Miyazono K. Ichijo H. Mol. Cell. 1998; 2: 389-395Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). We have recently shown that the association with TRAF2 followed by recruitment of AIP1 (ASK1-interacting protein-1, a Ras-GAP (GTPase-activating protein) family protein) is required for ASK1 activation by TNF (8Zhang R. Al-Lamki R. Bai L. Streb J.W. Miano J.M. Bradley J. Min W. Circ. Res. 2004; 94: 1483-1491Crossref PubMed Scopus (215) Google Scholar). On the other hand, several cellular inhibitors including thioredoxin (Trx), glutaredoxin, and 14-3-3 bind to and inhibit ASK1 activity in resting cells (8Zhang R. Al-Lamki R. Bai L. Streb J.W. Miano J.M. Bradley J. Min W. Circ. Res. 2004; 94: 1483-1491Crossref PubMed Scopus (215) Google Scholar, 9Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2092) Google Scholar, 10Zhang R. He X. Liu W. Lu M. Hsieh J.T. Min W. J. Clin. Investig. 2003; 111: 1933-1943Crossref PubMed Scopus (128) Google Scholar, 11Song J.J. Rhee J.G. Suntharalingam M. Walsh S.A. Spitz D.R. Lee Y.J. J. Biol. Chem. 2002; 277: 46566-46575Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 12Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (314) Google Scholar). Specifically, redox sensors Trx and glutaredoxin in reduced forms bind to ASK1 and block cytokine/stress-induced ASK1 activation (4Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Investig. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar, 5Liu Y. Min W. Circ. Res. 2002; 90: 1259-1266Crossref PubMed Scopus (314) Google Scholar, 9Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2092) Google Scholar). 14-3-3, a phosphoserine-binding molecule, binds to ASK1 specifically via Ser-967 of ASK1 and inhibits ASK1-induced apoptosis (4Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Investig. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar, 12Zhang L. Chen J. Fu H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8511-8515Crossref PubMed Scopus (314) Google Scholar, 13Zhang R. Luo D. Miao R. Bai L. Ge Q. Sessa W.C. Min W. Oncogene. 2005; 24: 3954-3963Crossref PubMed Scopus (150) Google Scholar). The mechanism by which ASK1 activity is regulated in EC is not fully understood. We have previously shown that TNF activates ASK1, in part, by dissociating preexisting complexes of ASK1 with 14-3-3 and Trx. In contrast, atheroprotective laminar flow inhibits TNF-induced ASK1 and JNK activation by preventing the release of ASK1 from 14-3-3 and Trx (4Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Investig. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar). Furthermore, we have shown that ASK1 is a labile protein and undergoes ubiquitination/degradation in resting EC. Although cellular inhibitors such as Trx promote ASK1 ubiquitination/degradation, proinflammatory cytokines such as TNF induce deubiquitination and stabilization of ASK1 (5Liu Y. Min W. Circ. Res. 2002; 90: 1259-1266Crossref PubMed Scopus (314) Google Scholar). Thus regulation of ASK1 stability is a critical step in ASK1 activation. However, the mechanism for ASK1 degradation is not understood. SOCS1, a member of the SOCS family of proteins, was first identified as an inhibitor of cytokine signaling. The role of SOCS1 in T cell function has been extensively studied (14Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (606) Google Scholar, 15Marine J.C. Topham D.J. McKay C. Wang D. Parganas E. Stravopodis D. Yoshimura A. Ihle J.N. Cell. 1999; 98: 609-616Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). SOCS1 deletion causes perinatal lethality with death by 2–3 weeks due to uncontrolled inflammation. Introducing an interferon-γ (IFN-γ) deficiency or introducing neutralizing antibody to IFN-γ eliminates lethality, suggesting that lymphocyte-produced IFN-γ is critical to SOCS1-associated perinatal lethality (15Marine J.C. Topham D.J. McKay C. Wang D. Parganas E. Stravopodis D. Yoshimura A. Ihle J.N. Cell. 1999; 98: 609-616Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 16Alexander W.S. Starr R. Fenner J.E. Scott C.L. Handman E. Sprigg N.S. Corbin J.E. Cornish A.L. Darwiche R. Owczarek C.M. Kay T.W. Nicola N.A. Hertzog P.J. Metcalf D. Hilton D.J. Cell. 1999; 98: 597-608Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). Thus SOCS1 functions as a feedback regulator in IFN-γ signaling. Mechanistic studies suggest that SOCS1 via its N-terminal domain binds to and inhibits the kinase activities of all members of JAK kinase family (JAK1–3 and Tyk2), kinases critical for signaling in many cytokines in immune cells (17Alexander W.S. Hilton D.J. Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (608) Google Scholar). Thus SOCS1 is generally considered as an anti-inflammatory molecule by suppressing cytokine production from T cells, macrophages, and antigen presentation from dendritic cells (17Alexander W.S. Hilton D.J. Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (608) Google Scholar). Eight SOCS family members (CIS, SOCS1–7) have been identified and are defined by a characteristic structure composed of a highly variable N-terminal region, a central SH2 domain, and a highly conserved 40–50-amino acid motif (called SOCS box) at the C terminus. SOCS1 also functions as an inhibitor in other cytokine signaling by various mechanisms. For example, SOCS1 attenuates insulin/IGF-1 signaling by binding to the insulin/IGF-1 receptors to inhibit the receptor kinase activity and by targeting insulin/IGF-1 receptor substrate-1 (IRS-1) for proteasome degradation (18Rui L. Yuan M. Frantz D. Shoelson S. White M.F. J. Biol. Chem. 2002; 12: 12Google Scholar, 19Mooney R.A. Senn J. Cameron S. Inamdar N. Boivin L.M. Shang Y. Furlanetto R.W. J. Biol. Chem. 2001; 276: 25889-25893Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Besides IFN-γ, other inflammatory cytokines such as TNF and interleukin-6 also induce SOCS1 in fat and muscle tissues and inhibit insulin/IGF-1 signaling (18Rui L. Yuan M. Frantz D. Shoelson S. White M.F. J. Biol. Chem. 2002; 12: 12Google Scholar, 19Mooney R.A. Senn J. Cameron S. Inamdar N. Boivin L.M. Shang Y. Furlanetto R.W. J. Biol. Chem. 2001; 276: 25889-25893Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Thus SOCS1 has been implicated in inflammation-induced insulin resistance. A general model has been proposed for SOCS protein-targeted protein degradation; SOCS box contains a conserved elongin BC-binding motif (BC box) and mediates interaction with elongin BC complex. In turn, the elongin complex associates with the putative ubiquitin ligase cullin-2. Signaling proteins (e.g. JAKs) associated with the N-terminal or SH2 domains of SOCS proteins could be ubiquitinated by cullin-2 and are targeted for degradation by the proteasome (20Ilangumaran S. Rottapel R. Immunol. Rev. 2003; 192: 196-211Crossref PubMed Scopus (55) Google Scholar, 21Krebs D.L. Hilton D.J. Stem Cells (Durham). 2001; 19: 378-387Crossref PubMed Scopus (661) Google Scholar). Recent data suggest that SOCS1 may also function as an inhibitor in TNF signaling. Thus SOCS1-knock-out (KO) mice or cells derived from the mice are hypersensitive to TNF (22Morita Y. Naka T. Kawazoe Y. Fujimoto M. Narazaki M. Nakagawa R. Fukuyama H. Nagata S. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5405-5410Crossref PubMed Scopus (171) Google Scholar). However, the mechanism by which SOCS1 suppresses TNF signaling is not known. In the present study, we have shown that SOCS1 via its SH2 domain binds to the phosphotyrosine residues on ASK1 to induce ASK1 degradation in an elongin complex-dependent manner. TNF induced dephosphorylation of ASK1 and dissociation of ASK1 from SOCS1, resulting in ASK1-JNK activation. Moreover, SOCS1-KO mouse tissues and derived EC showed increased ASK1 expression and enhanced TNF-induced ASK1-JNK activation, gene expression of proinflammatory molecules, as well as apoptotic responses. We concluded that SOCS1 functions as a negative regulator in TNF-induced inflammatory signaling in EC. Plasmid Construction—Expression plasmids for human SOCS1, -2, -3, -6, and CIS expression plasmids were provided by Dr. Robert Mooney (University of Rochester, NY) (19Mooney R.A. Senn J. Cameron S. Inamdar N. Boivin L.M. Shang Y. Furlanetto R.W. J. Biol. Chem. 2001; 276: 25889-25893Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar); expression plasmids for elongin B and C were provided by Dr. Paul Rothman (Columbia University, NY) (23Chen X.P. Losman J.A. Cowan S. Donahue E. Fay S. Vuong B.Q. Nawijn M.C. Capece D. Cohan V.L. Rothman P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2175-2180Crossref PubMed Scopus (163) Google Scholar). ASK1 constructs were described previously (4Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Investig. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar, 5Liu Y. Min W. Circ. Res. 2002; 90: 1259-1266Crossref PubMed Scopus (314) Google Scholar). The mutant SOCS1, SOCS3, and ASK1 were constructed by site-directed mutagenesis using QuikChange™ site-directed mutagenesis kit (Stratagene) according to the protocol of the manufacturer. Cells and Cytokines—Bovine aortic endothelial cells (BAEC) were purchased from Clonetics (San Diego, CA). Human umbilical vein EC (HUVEC) were from Boyer Center for Molecular Medicine Cell Culture Core, Yale University. Human recombinant TNF was purchased from R&D Systems (Minneapolis, MN) and used at 10 ng/ml, and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Cell Transfection—Transfection of BAEC was performed by Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen). Cells were cultured at 90% confluence in 6-well plates and were transfected with total of 4 μg of plasmid constructs as indicated. Cells were harvested at 36–48 h after transfection, and cell lysates were used for protein assays. Isolation of SOCS1-deficient Mouse Lung EC (MLEC)—SOCS1+/– and SOCS3+/– mice were from Dr. James Ihle (St. Jude Children’s Research Hospital, Memphis, TN) (15Marine J.C. Topham D.J. McKay C. Wang D. Parganas E. Stravopodis D. Yoshimura A. Ihle J.N. Cell. 1999; 98: 609-616Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 24Marine J.C. McKay C. Wang D. Topham D.J. Parganas E. Nakajima H. Pendeville H. Yasukawa H. Sasaki A. Yoshimura A. Ihle J.N. Cell. 1999; 98: 617-627Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). MLEC isolation was performed as we described (25Pan S. An P. Zhang R. He X. Yin G. Min W. Mol. Cell. Biol. 2002; 22: 7512-7523Crossref PubMed Scopus (115) Google Scholar) followed by immunoselection and immortalization modified from the protocol described by Lim et al. (26Lim Y.C. Garcia-Cardena G. Allport J.R. Zervoglos M. Connolly A.J. Gimbrone Jr., M.A. Luscinskas F.W. Am. J. Pathol. 2003; 162: 1591-1601Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). For immunoselection, 10 μl of beads (per T-75 of mouse lung cells) were washed three times with 1 ml of buffer A (phosphate-buffered saline +2% fetal bovine serum) and resuspended in 100 μl of buffer A. 10 μl (10 μg) of anti-mouse ICAM-2 or 10 μl (10 μg) of PECAM-1 were added and rocked at 4 °C for 2 h. Beads were washed three times and resuspended in 160 μl of buffer A. Confluent mouse lung cells cultured in a T-75 flask were placed at 4 °C for 5 min and incubated with the beads at 4 °C for 1 h. Cells were then washed with warm phosphate-buffered saline and treated with 3 ml of warm Trypsin/EDTA. When cells were detached, 7 ml of growth media were added. An empty 15-ml tube in the magnetic field was placed on the holder, and the cell suspension (∼10 ml) was added slowly by placing the pipette on the wall of the tube so that the cells pass through the magnetic field. Cells were incubated for 5 min, and the media were carefully aspirated. The 15-ml tube was removed from the magnetic holder, and the beads/cells were resuspended in 10 ml of media. The selected cells were plated on 0.2% gelatin-coated flasks and cultured for 3–7 days. When the cells were confluent, another round of immunoselection was repeated. Antibody Array Screening—The antibody array membranes were provided by Dr. Y. Eugene Chin (Brown University School of Medicine, Providence, RI) (27Wang Y. Wu T.R. Cai S. Welte T. Chin Y.E. Mol. Cell. Biol. 2000; 20: 4505-4512Crossref PubMed Scopus (158) Google Scholar). 100 polyclonal or monoclonal antibodies, including those against SOCS family proteins, were immobilized on polyvinylidene difluoride membranes (5 by 5 cm) at predetermined positions. The antibody array membranes were then incubated with 5% milk at room temperature for 2 h followed by incubation with cell lysates from BAEC in the presence or absence of HA-tagged ASK1. After incubation for 2 h, the membranes were washed three times with phosphate-buffered saline with 0.1% Tween 20 and blotted with horseradish peroxidase-conjugated anti-HA antibody (Roche Diagnosis) for 2 h followed by three washes and enhanced chemiluminescence (ECL) detection. Immunoprecipitation and Immunoblotting—EC (HUVEC, BAEC, or MLEC) after various treatments were washed twice with cold phosphate-buffered saline and lysed in 1.5 ml of cold lysis buffer (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.1% Triton X-100, 0.75% Brij 96, 1 mm sodium orthovanadate, 1 mm sodium fluoride, 1 mm sodium pyrophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mm phenylmethylsulfonyl fluoride, 1 mm EDTA) for 20 min on ice. Protein concentrations were determined with a Bio-Rad kit. For immunoprecipitation to analyze protein interaction in vivo, 400 μg of cell lysate supernatant were precleared by incubating with 5 μg of normal rabbit serum plus protein A/G-agarose beads on rotator at 4 °C overnight. The lysates were then incubated with 5 μg of the first protein-specific antiserum (e.g. anti-SOCS1 from Medical and Biological Laboratory) for 2 h with 50 μl of protein A/G-agarose beads. The immune complexes were collected after each immunoprecipitation by centrifugation at 14,000 × g for 10 min followed by four washes with lysis buffer. The immune complexes were subjected to SDS-PAGE followed by immunoblot (Immobilon P, Millipore, Milford, MA) with the second protein (e.g. ASK1)-specific antibody (H300, Santa Cruz Biotechnology). The chemiluminescence was detected using an ECL kit according to the instructions of the manufacturer (Amersham Biosciences). For detection of FLAG-tagged proteins (e.g. SOCS proteins), anti-FLAG M2 antibody (Sigma) was used for immunoblot. For detection of HA-tagged proteins (ASK1 and elongin B/C), anti-HA antibody (Roche Diagnostics) was used for immunoblot. ASK1 and JNK Kinase Assays—ASK1 and JNK assays were performed as described previously (4Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Investig. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar, 5Liu Y. Min W. Circ. Res. 2002; 90: 1259-1266Crossref PubMed Scopus (314) Google Scholar) using GST-MKK4 and GST-c-Jun-(1–80) fusion protein as a substrate, respectively. Briefly, a total of 400 μg of cell lysates was immunoprecipitated with 5 μg of antibody against ASK1 or JNK1 (Santa Cruz Biotechnology). The immunoprecipitates were mixed with 10 μg of GST-MKK4 or GST-c-Jun-(1–80) suspended in the kinase buffer (20 mm Hepes, pH 7.6, 20 mm MgCl2, 25 mm β-glycerophosphate, 100 μm sodium orthovanadate, 2 mm dithiothreitol, 20 μm ATP) containing 1 μl (10 μCi) of [γ-32P]ATP. The kinase assay was performed at 25 °C for 30 min. The reaction was terminated by the addition of Laemmli sample buffer, and the products were resolved by SDS-PAGE (12%) followed by protein transferring to a membrane (Immobilon P). The phosphorylated GST-MKK4 or GST-c-Jun-(1–80) was visualized by autoradiography. The membrane was further used for Western blot with anti-ASK1 or anti-JNK1. GST-SOCS1 Pull-down Assay—GST fusion protein preparation and GST pull-down assay were performed as described previously (4Liu Y. Yin G. Surapisitchat J. Berk B.C. Min W. J. Clin. Investig. 2001; 107: 917-923Crossref PubMed Scopus (94) Google Scholar, 5Liu Y. Min W. Circ. Res. 2002; 90: 1259-1266Crossref PubMed Scopus (314) Google Scholar). Briefly, GST-SOCS1 fusion proteins expressed in Escherichia coli XL-1 blue were affinity-purified on glutathione-Sepharose beads (Amersham Biosciences). 400 μg of cell lysates expressing HA-tagged ASK1 were incubated overnight at 4 °C with 10 μg of GST-SOCS1 bound to glutathione-Sepharose in the lysis buffer. The beads were washed four times with the lysis buffer before the addition of boiling Laemmli sample buffer. Bound ASK1 proteins were resolved on SDS-PAGE and detected by Western blot with anti-HA or anti-FLAG antibody. Quantitation of Apoptotic Cell—CCell killing assays were performed as described previously with a modification (5Liu Y. Min W. Circ. Res. 2002; 90: 1259-1266Crossref PubMed Scopus (314) Google Scholar, 8Zhang R. Al-Lamki R. Bai L. Streb J.W. Miano J.M. Bradley J. Min W. Circ. Res. 2004; 94: 1483-1491Crossref PubMed Scopus (215) Google Scholar, 10Zhang R. He X. Liu W. Lu M. Hsieh J.T. Min W. J. Clin. Investig. 2003; 111: 1933-1943Crossref PubMed Scopus (128) Google Scholar, 13Zhang R. Luo D. Miao R. Bai L. Ge Q. Sessa W.C. Min W. Oncogene. 2005; 24: 3954-3963Crossref PubMed Scopus (150) Google Scholar). The propidium iodide (PI) exclusion method for loss of integrity of cell membranes was used to assess viability. In brief, cells were suspended in phosphate-buffered saline containing 25 μg/ml PI for 5 min at 37 °C and then subjected to analytic flow cytometry on a FACSort (BD Biosciences) immediately after labeling. A light scatter gate was set up to eliminate cell debris from the analysis. The PI fluorescence signal was recorded on the FL3 channel and analyzed by using CellQuest software. Phosphatidylserine translocation, which precedes loss of PI exclusion in apoptotic cell death, was assessed by an annexin V-fluorescein isothiocyanate staining kit (Roche Diagnostics) following the manufacturer’s protocol. For nuclear morphology, cells were stained with 4′,6-diamidino-2-phenylindole, and apoptotic cell (nuclei condensation) were visualized under UV microscope. RNA Isolation and Quantitative Real-time RT-PCR—Total RNA was isolated from EC with a Qiagen RNeasy mini kit (Qiagen Inc., Valencia, CA) as recommended by the supplier. Total RNA was quantitated by OD at 260 using a Du-64 spectrophotometer (Beckman Instruments). Using an equal amount of total RNA (200 ng) from EC, stimulated under various conditions, mRNA was primed with random hexamers, and cDNA was synthesized from mRNA by TaqMan reverse transcription with MultiScribe reverse transcriptase (Applied Biosystems, Foster, CT) according to the manufacturer’s description. The final cDNA product was used for subsequent cDNA amplification by polymerase chain reaction. cDNA was amplified and quantitated by using SYBR Green PCR reagents from Applied Biosystems according to the manufacturer’s instructions. Briefly, the cDNA for the specific genes (E-selectin, VCAM-1, SOCS1) and 18 S rRNA were amplified by AmpliTag Gold DNA polymerase using specific primers, which were synthesized by Yale Howard Hughes Medical Institute/Keck oligonucleotide synthetic facility (Yale University School of Medicine, New Haven, CT). The cDNA for 18 S rRNA was amplified by using a specific forward primer (5′-TTC CGA TAA CGA ACG AGA CTCT-3′) and a specific reverse primer (5′-TGG CTG AAC GCC ACT TGTC-3′). The following specific forward and reverse primers were used to amplify the gene of interest: SOCS1, 5′-TCC GTT CGC ACG CCG ATT AC-3′ and 5′-TCA AAT CTG GAA GGG GAA GG-3′; E-selectin, 5′-CAT CCA ACG AAC CAA AGA CTCG-3′ and 5′-GGC ACT TGC AGG TGT AAC TATT-3′; VCAM-1, 5′-AGT TGG GGA TTC GGT TGT TCT-3′ and 5′-CCC CTC ATT CCT TAC CAC CC-3′ The PCR reaction mixture (final volume 25 μl) contained 5 μl of cDNA, 1 μl of 10 μm forward primer, 1 μl of 10 μm reverse primer, 2.5 μl of PCR 10× SYBR Green PCR buffer, 3 μl of 25 mm MgCl2,2 μl of dNTP mix (2.5 mm dATP, 2.5 mm dCTP, 2.5 mm dGTP, and 5 mm dUTP), 0.25 μl of AmpErase UNG (1 unit/μl uracil-N-glycosylase), 0.125 μl of AmpliTag Gold DNA polymerase (5 units/μl of AmpliTag Gold DNA polymerase), and 10.125 μl of H2O. The PCR reaction was performed in triplicate (3 wells of C96-well plate). The reaction was amplified with iCycler iQ multicolor real time PCR detector (Bio-Rad) for 37 cycles with melting at 94 °C for 30 s, an annealing at 58 °C for 30 s, and extension at 72 °C for 1 min in iCycler iQ PCR 96-well plates (Bio-Rad). The relative quantification values for the interest gene expression were calculated from the accurate CT, which is the PCR cycle at which an increase in reporter fluorescence from SYBR Green dye can be first detected obtained above a baseline signal. CT values for 18 S rRNA cDNA were subtracted from CT values for the interest gene cDNA for each well to calculate –CT. The triplicate –CT values for each sample were averaged. To calculate the fold induction of the interest gene mRNA in cells treated with cytokines over control cells, the averaged –CT values calculated for control cells were subtracted from –CT values calculated for cytokine-treated cells to calculate –CT. Then, the fold induction for each well was calculated by using the 2 – (–CT) formula. The fold induction value for triplicate wells was averaged, and data are presented as the mean ± S.E. of triplicate wells. SOCS1 Binds to ASK1 and Induces ASK1 Degradation—To understand the mechanism of ASK1 degradation, we performed antibody arrays using HA-ASK1 as bait (see “Experimental Procedures”). Results showed that ASK1 interacted with several members of the SOCS protein family that have been implicated in protein degradation. This result prompted us to reason that SOCS family protein may target ASK1 to degradation machinery. We first confirmed the interaction of ASK1 with SOCS proteins by co-immunoprecipitation assays. ASK1 was co-transfected with FLAG-tagged SOCS1, -2, -3, -6, or CIS into bovine EC (BAEC). Expression of SOCS and ASK1 was determined by Western blot with anti-FLAG and anti-ASK1 antibody, respectively. SOCS proteins showed similar levels in expression (Fig. 1a). Interestingly, ASK1 expression was dramatically different in the presence of different SOCS proteins. Co-expression of SOCS1 and SOCS3 significantly decreased ASK1 expression (Fig. 1b). Association of ASK1 with SOCS was determined by immunoprecipitation with anti-FLAG antibody followed by Western blot with anti-ASK1 antibody. SOCS1 and SOCS3 strongly bind to ASK1 (Fig. 1c). These data suggest that SOCS1 and SOCS3 might bind to ASK1 and induce ASK1 degradation. We focused on SOCS1 for further studies to determine the role of SOCS1 in ASK1 degradation. The SH2 Domain of SOCS1 Is Critical for the Association with ASK1—To define the critical domain of SOCS1 for ASK1 binding and degradation, we generated SOCS1 truncated mutants in GST- or FLAG-tagged constructs (5Liu Y. Min W. Circ. Res. 2002; 90: 1259-1266Crossref PubMed Scopus (314) Google Scholar). SOCS1-DC contains a deletion of the C-terminal SOCS box, and SOCS1-DN contains a deletion of the N-terminal domain (Fig. 2a). Preliminary data indicated that both SOCS1-DC and SOCS1-DN bind to ASK1 in a GST pull-down assay (not shown), suggesting that the SH2 domain might be critical for ASK1 interaction. We then mutated the Arg-105 (to Lys) within the SH2 domain of SOCS1, which has been previously shown to be critical for phosphotyrosine binding (14Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref
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