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• W2000971871 abstract "Janus kinase 1/signal transducers and activators of transcription 3 (JAK1/STAT3) pathway is one of the recognized oncogenic signaling pathways that frequently overactivated in a variety of human tumors. Despite rapid progress in elucidating the molecular mechanisms of activation of JAK/STAT pathway, the processes that regulate JAK/STAT deactivation need to be further clarified. Here we demonstrate that CUE domain-containing 2 (CUEDC2) inhibits cytokine-induced phosphorylation of JAK1 and STAT3 and the subsequent STAT3 transcriptional activity. Further analysis by a yeast two-hybrid assay showed that CUEDC2 could engage in a specific interaction with a key JAK/STAT inhibitor, SOCS3 (suppressors of cytokine signaling 3). The interaction between CUEDC2 and SOCS3 is required for the inhibitory effect of CUEDC2 on JAK1 and STAT3 activity. Additionally, we found CUEDC2 functions collaboratively with SOCS3 to inhibit JAK1/STAT3 signaling by increasing SOCS3 stability via enhancing its association with Elongin C. Therefore, our findings revealed a new biological activity for CUEDC2 as the regulator of JAK1/STAT3 signaling and paved the way to a better understanding of the mechanisms by which SOCS3 has been linked to suppression of the JAK/STAT pathway. Janus kinase 1/signal transducers and activators of transcription 3 (JAK1/STAT3) pathway is one of the recognized oncogenic signaling pathways that frequently overactivated in a variety of human tumors. Despite rapid progress in elucidating the molecular mechanisms of activation of JAK/STAT pathway, the processes that regulate JAK/STAT deactivation need to be further clarified. Here we demonstrate that CUE domain-containing 2 (CUEDC2) inhibits cytokine-induced phosphorylation of JAK1 and STAT3 and the subsequent STAT3 transcriptional activity. Further analysis by a yeast two-hybrid assay showed that CUEDC2 could engage in a specific interaction with a key JAK/STAT inhibitor, SOCS3 (suppressors of cytokine signaling 3). The interaction between CUEDC2 and SOCS3 is required for the inhibitory effect of CUEDC2 on JAK1 and STAT3 activity. Additionally, we found CUEDC2 functions collaboratively with SOCS3 to inhibit JAK1/STAT3 signaling by increasing SOCS3 stability via enhancing its association with Elongin C. Therefore, our findings revealed a new biological activity for CUEDC2 as the regulator of JAK1/STAT3 signaling and paved the way to a better understanding of the mechanisms by which SOCS3 has been linked to suppression of the JAK/STAT pathway. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway was originally described as a signal-transducing pathway induced by interferons (1Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Abstract Full Text PDF PubMed Scopus (1250) Google Scholar). However, it has now been demonstrated that the JAK/STAT pathway mediates a multitude of distinct biological signals. JAK kinases are associated with cell surface receptors, such as cytokine and tyrosine kinase receptors. Binding of ligand triggers JAK-mediated phosphorylation of specific tyrosine residues in the cytoplasmic portion of the receptor. Cytoplasmic STAT proteins are recruited to the membrane by phosphorylated receptor and then phosphorylated by JAKs. Phosphorylated STATs then dimerize via their Src homology 2 (SH2) 4The abbreviations used are: SH2Src homology 2SOCSsuppressor of cytokine signalingPIASprotein inhibitor of activated statsCUEcoupling of ubiquitin conjugation to endoplasmic reticulum degradationCUEDC2CUE domain-containing 2EpoRerythropoietin receptorMEMminimum essential mediumaaamino acidsIRF-1interferon regulatory factor-1. domains, translocate to the nucleus, and transactivate target genes (2Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2526) Google Scholar, 3Levy D.E. Lee C.K. J. Clin. Invest. 2002; 109: 1143-1148Crossref PubMed Scopus (758) Google Scholar). JAK/STAT pathway regulates many cellular processes critical for hematopoiesis, immune response, and allelotaxy, including innate and adaptive immune function, development, proliferation, differentiation, apoptosis, and inflammation. Duration and degree of JAK/STAT activation are tightly controlled, and any deregulation will lead to disease, including tumor development (4Igaz P. Tóth S. Falus A. Inflamm Res. 2001; 50: 435-441Crossref PubMed Scopus (102) Google Scholar). Src homology 2 suppressor of cytokine signaling protein inhibitor of activated stats coupling of ubiquitin conjugation to endoplasmic reticulum degradation CUE domain-containing 2 erythropoietin receptor minimum essential medium amino acids interferon regulatory factor-1. The STAT protein family is composed of multiple members, termed STAT1–6. STAT3 was first described as a DNA-binding protein activated by epidermal growth factor and interleukin-6 (IL-6) capable of interacting with an enhancer element in the promoters of acute phase genes (5Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Crossref PubMed Scopus (1735) Google Scholar, 6Bowman T. Garcia R. Turkson J. Jove R. Oncogene. 2000; 19: 2474-2488Crossref PubMed Scopus (1593) Google Scholar). Later studies demonstrated that STAT3 is activated in response to several cytokines and growth factors, such as interferon (IFN) and leptin. In normal cells, STAT3 activation is transient like other STAT family members; however, in a large number of primary tumors and cancer-derived cell lines, it remains persistently activated, which may be caused by impairment of negative regulation or mutation of STAT3 itself (7Bromberg J. J. Clin. Invest. 2002; 109: 1139-1142Crossref PubMed Scopus (755) Google Scholar, 8Lesina M. Kurkowski M.U. Ludes K. Rose-John S. Treiber M. Klöppel G. Yoshimura A. Reindl W. Sipos B. Akira S. Schmid R.M. Algül H. Cancer Cell. 2011; 19: 456-469Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). Evidence indicates that constitutive activation of STAT3 may contribute to cellular transformation, induce tumor angiogenesis, and suppress anti-tumor immune responses, further enhancing tumor progression (9Yu H. Jove R. Nat. Rev. Cancer. 2004; 4: 97-105Crossref PubMed Scopus (1972) Google Scholar, 10Lee H. Deng J. Kujawski M. Yang C. Liu Y. Herrmann A. Kortylewski M. Horne D. Somlo G. Forman S. Jove R. Yu H. Nat. Med. 2010; 16: 1421-1428Crossref PubMed Scopus (316) Google Scholar). Therefore, STAT3 proteins are emerging as ideal targets for cancer therapy (9Yu H. Jove R. Nat. Rev. Cancer. 2004; 4: 97-105Crossref PubMed Scopus (1972) Google Scholar, 11Sinibaldi D. Wharton W. Turkson J. Bowman T. Pledger W.J. Jove R. Oncogene. 2000; 19: 5419-5427Crossref PubMed Scopus (264) Google Scholar, 12O'Shea J.J. Gadina M. Schreiber R.D. Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar). A large body of literature has been generated on the regulation of the JAK/STAT pathway. After well defining of the positive regulators of this signaling pathway, three main classes of proteins that negatively control the JAK/STAT pathway were concluded, including SOCS (suppressors of cytokine signaling) proteins, PIAS (protein inhibitors of activated stats) family proteins, and protein-tyrosine phosphatases (13Greenhalgh C.J. Hilton D.J. J. Leukoc. Biol. 2001; 70: 348-356PubMed Google Scholar). The SOCS family consists of eight members, including cytokine-inducible SH2 protein and SOCS-1–7. The SOCS proteins contain a central SH2 domain and a conserved carboxyl-terminal region called the SOCS box. Expression of SOCS proteins is rapidly induced by cytokine-mediated STAT activation (14Krebs D.L. Hilton D.J. J. Cell Sci. 2000; 113: 2813-2819Crossref PubMed Google Scholar) and subsequently leads to down-regulation of cytokine-induced signal transduction. Therefore, SOCS proteins act in a classic negative feedback loop to regulate JAK/STAT signal transduction. Despite the structural similarity of SOCS family members, they inhibit JAK/STAT signaling through different mechanisms. For example, cytokine-inducible SH2 protein, which was the first family member identified, was shown to compete with STAT5 for binding sites within the erythropoietin receptor (EpoR), thereby attenuating the JAK2/STAT5 signaling pathway (15Matsumoto A. Masuhara M. Mitsui K. Yokouchi M. Ohtsubo M. Misawa H. Miyajima A. Yoshimura A. Blood. 1997; 89: 3148-3154Crossref PubMed Google Scholar). SOCS1 binds to the activation loop of JAKs via its SH2 domain and inhibits JAK kinase activity (16Yasukawa 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). The detailed mechanism by which SOCS3 functions to inhibit JAK/STAT signaling has not been well characterized. Some studies have reported that SOCS3 could also bind to and directly inhibit JAKs (17Sasaki A. Yasukawa H. Suzuki A. Kamizono S. Syoda T. Kinjyo I. Sasaki M. Johnston J.A. Yoshimura A. Genes Cells. 1999; 4: 339-351Crossref PubMed Scopus (311) Google Scholar), whereas other reports suggest that it is necessary for SOCS3 to associate with the activated cytokine receptors to attenuate JAK/STAT signaling (18Nicholson S.E. De Souza D. Fabri L.J. Corbin J. Willson T.A. Zhang J.G. Silva A. Asimakis M. Farley A. Nash A.D. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6493-6498Crossref PubMed Scopus (398) Google Scholar, 19Sasaki A. Yasukawa H. Shouda T. Kitamura T. Dikic I. Yoshimura A. J. Biol. Chem. 2000; 275: 29338-29347Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 20Bjorbak C. Lavery H.J. Bates S.H. Olson R.K. Davis S.M. Flier J.S. Myers Jr., M.G. J. Biol. Chem. 2000; 275: 40649-40657Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 21Hörtner M. Nielsch U. Mayr L.M. Johnston J.A. Heinrich P.C. Haan S. J. Immunol. 2002; 169: 1219-1227Crossref PubMed Scopus (111) Google Scholar, 22Hörtner M. Nielsch U. Mayr L.M. Heinrich P.C. Haan S. Eur. J. Biochem. 2002; 269: 2516-2526Crossref PubMed Scopus (69) Google Scholar). CUEDC2 is a CUE domain-containing protein. Previous work from our laboratory showed that CUEDC2 interacts with the progesterone receptor and promotes progesterone-induced proteasomal degradation of the progesterone receptor (23Zhang P.J. Zhao J. Li H.Y. Man J.H. He K. Zhou T. Pan X. Li A.L. Gong W.L. Jin B.F. Xia Q. Yu M. Shen B.F. Zhang X.M. EMBO J. 2007; 26: 1831-1842Crossref PubMed Scopus (67) Google Scholar). More importantly, we found CUEDC2 is a crucial determinant of resistance to endocrine therapies in breast cancer through affecting estrogen receptor α protein stability (24Pan X. Zhou T. Tai Y.H. Wang C. Zhao J. Cao Y. Chen Y. Zhang P.J. Yu M. Zhen C. Mu R. Bai Z.F. Li H.Y. Li A.L. Liang B. Jian Z. Zhang W.N. Man J.H. Gao Y.F. Gong W.L. Wei L.X. Zhang X.M. Nat. Med. 2011; 17: 708-714Crossref PubMed Scopus (113) Google Scholar). These functions provide insight into the mechanism by which CUEDC2 regulates breast cancer cells. In this study, we demonstrate that CUEDC2 inhibits JAK1/STAT3 activation by attenuating their phosphorylation. In addition, we identify CUEDC2 as a novel SOCS3 binding partner that stabilizes SOCS3 protein, resulting in suppression of JAK1/STAT3 signaling. Therefore, our novel findings suggest that CUEDC2 cooperates with SOCS3 to suppress the JAK/STAT signaling pathway. HA-CUEDC2 full-length and truncated mutants were described as before (25Li H.Y. Liu H. Wang C.H. Zhang J.Y. Man J.H. Gao Y.F. Zhang P.J. Li W.H. Zhao J. Pan X. Zhou T. Gong W.L. Li A.L. Zhang X.M. Nat. Immunol. 2008; 9: 533-541Crossref PubMed Scopus (117) Google Scholar). To generate bacterial expression vector for GST-CUEDC2 and the mutants, the corresponding CUEDC2 cDNAs (1–287, 1–133, 1–180,133–287, and 180–287 aa) were cloned in-frame into pGEX-KG vector (Amersham Biosciences). FLAG-SOCS3 and FLAG-SOCS3 (ΔSH2) were amplified by PCR from mammary library and cloned into pCDNA3.0 vector; other FLAG-SOCS3 deletion mutants were kindly provided by Dr. Fred Schaper (26Schmitz J. Weissenbach M. Haan S. Heinrich P.C. Schaper F. J. Biol. Chem. 2000; 275: 12848-12856Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). PACT-Luc and m67-Luc were, respectively, provided by Dr. Tarik Moroy (27Rödel B. Tavassoli K. Karsunky H. Schmidt T. Bachmann M. Schaper F. Heinrich P. Shuai K. Elsässer H.P. Möröy T. EMBO J. 2000; 19: 5845-5855Crossref PubMed Scopus (117) Google Scholar) and Dr. Yong-Yun Kong (28Kwon M.C. Koo B.K. Moon J.S. Kim Y.Y. Park K.C. Kim N.S. Kwon M.Y. Kong M.P. Yoon K.J. Im S.K. Ghim J. Han Y.M. Jang S.K. Shong M. Kong Y.Y. EMBO J. 2008; 27: 642-653Crossref PubMed Scopus (56) Google Scholar). FLAG-STAT3 constructs were obtained from Dr. Darnell Jr., and GST-STAT3 was from Dr. XinMin Cao. IFN-γ activating sequence reporter was gift from Dr. Geoffrey L. Greene. JAK1 expression plasmid was kindly provided from Dr. Claude Haan. To generate Gp130 expression construct, a DNA fragment containing the intracytoplasmic domain of gp130 was amplified by PCR and inserted into PXJ40-HA vector. HEK293T cells and HeLa cells were cultured in DMEM containing 10% newborn calf serum at 37 °C in a humidified atmosphere of 5% CO2. HepG2 cells were maintained in MEM containing 10% fetal bovine serum. Cells were transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer's instruction. Luciferase assays were carried out using the luciferase kit (Promega, Madison, WI) as described by the manufacturer's instructions, and luciferase activities were determined using a Dual-Luciferase Reporter Assay System (Promega). All experiments were repeated at least three times. For immunoprecipitation experiments, cells were lysed in E1A buffer (50 mm Hepes, pH 7.6, 250 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA) containing a mixture of protease inhibitors. Immunoprecipitations were performed by incubating whole cell extracts with the indicated antibody and rocking at 4 °C for 6 h after the preincubating with protein A/G-Sepharose (Santa Cruz Biotechnology),. Immunoprecipitates were washed 3 times and resuspended in 40 μl of 1× SDS sample buffer, then resolved by SDS-PAGE. All the samples for phosphorylation assays were prepared in M2 buffer. Mouse anti-HA antibody, rabbit anti-SOCS3 (sc-9023), and anti-STAT3 (sc-7179) antibodies were purchased from Santa Cruz Biotechnology. Anti-FLAG (M2) (F3165) monoclonal antibody was from Sigma. Rabbit anti-pSTAT3 (Tyr-705) (#9131), anti-JAK1 (#3332), and anti-pJAK1 (#1022/1023) (#3331) were purchased from cell signaling technology. CUEDC2 monoclonal antibody and GAPDH and GFP polyclonal antibodies were prepared in our laboratory. GST and GST fusion proteins were expressed in DH5α and purified according to the manufacturer's instructions (GE Healthcare). FLAG-SOCS3 protein, obtained from the whole cell lysates of HEK293T cells, which were transfected with FLAG-SOCS3 and/or its mutants plasmids, were incubated with GST and GST-CUEDC2 or its truncate fusion protein bound to agarose beads in 1 ml of binding buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 2% glycerol, and 0.1% Nonidet P-40) containing a protease inhibitor mixture at 4 °C for 6 h. Beads were then washed 3 times and resuspended in 30 μl of 1× SDS-PAGE sample buffer and detected by immunoblotting. The two small interfering RNAs (siRNA) that target CUEDC2 were purchased, respectively, from Invitrogen (#1, HSS149051) and Dharmacon (#2, J-014272-20); target sequences were, respectively, 5′-CCAAGAUGAGGCAACUGGCGCUGAG-3′ (#1) and 5′-CAUCAGAGGAGAACUUCGA-3′ (#2). For control siRNA against Photinus pyralis luciferase gene (Invitrogen), the target sequence was 5′-GGAUUUCGAGUCGUCUUAAUGUAUA-3′. Relative expression of endogenous CUEDC2 was detected by anti-CUEDC2 (from our laboratory). SOCS3 was selectively suppressed by using the RNA interference method, and the siRNA used for targeting human SOCS3 were: 5′-CCAAGAACCUGCGCATCCA-3′ and 5′-TGGATGCGCAGGTTCTTGG-3′ (29Komyod W. Böhm M. Metze D. Heinrich P.C. Behrmann I. Mol. Cancer Res. 2007; 5: 271-281Crossref PubMed Scopus (44) Google Scholar). The pSUPER retro shRNA retrovirus vector expressing CUEDC2 siRNA (target sequence 5′-GAAGCTGATCCGATACATC-3′; 5′- GTACATGATGGTGGATAGC-3′) were constructed by recombinant DNA technology. The packaging cells Phoenix (from ATCC) were transfected with these combinant plasmids using a liposome-based transfection method, and virus supernatant was collected and then infected into HeLa cells. The stable integrant was selected using G418 for 2 weeks. For HepG2 cell lines stably expressing CUEDC2, CUEDC2 cDNA was inserted into pBabe-retro-puro retrovirus vector. HepG2 cells were infected with virus supernatant from Phoenix cells transfected with pBabe-GFP or pBabe-CUEDC2, and stable integrant was selected with puromycin for 2 weeks. HeLa cells stably expressing CUEDC2 were described as before (25Li H.Y. Liu H. Wang C.H. Zhang J.Y. Man J.H. Gao Y.F. Zhang P.J. Li W.H. Zhao J. Pan X. Zhou T. Gong W.L. Li A.L. Zhang X.M. Nat. Immunol. 2008; 9: 533-541Crossref PubMed Scopus (117) Google Scholar). CUEDC2 interacts with progesterone receptor and estrogen receptor, leading to the ubiquitination and proteasome-dependent degradation of these two proteins (23Zhang P.J. Zhao J. Li H.Y. Man J.H. He K. Zhou T. Pan X. Li A.L. Gong W.L. Jin B.F. Xia Q. Yu M. Shen B.F. Zhang X.M. EMBO J. 2007; 26: 1831-1842Crossref PubMed Scopus (67) Google Scholar, 24Pan X. Zhou T. Tai Y.H. Wang C. Zhao J. Cao Y. Chen Y. Zhang P.J. Yu M. Zhen C. Mu R. Bai Z.F. Li H.Y. Li A.L. Liang B. Jian Z. Zhang W.N. Man J.H. Gao Y.F. Gong W.L. Wei L.X. Zhang X.M. Nat. Med. 2011; 17: 708-714Crossref PubMed Scopus (113) Google Scholar). To gain further insight into the function of CUEDC2 and elucidate other potential roles of CUEDC2 in cytokine-mediated signal transduction, we investigated whether CUEDC2 regulates other transcription factors that are also ubiquitinated. To investigate this possibility, reporter gene assays were used to determine the effect of CUEDC2 on the transcriptional activity of various transcription factors. STAT3 activity was shown to be affected by CUEDC2 in a screening assay. 5W.-N. Zhang, L. Wang, Q. Wang, X. Luo, D.-F. Fang, Y. Chen., X. Pan, J.-H. Man, Q. Xia, B.-F. Jin, W.-H. Li, T. Li, B. Liang, L. Chen, W.-L. Gong, M. Yu, A.-L. Li, T. Zhou, and H.-Y Li, unpublished data. As shown in Fig. 1A, HEK293T cells were transfected with pACT-Luc (a luciferase reporter plasmid containing the promoter of α1-antichymotrypsin that is has two STAT3 binding sites) (27Rödel B. Tavassoli K. Karsunky H. Schmidt T. Bachmann M. Schaper F. Heinrich P. Shuai K. Elsässer H.P. Möröy T. EMBO J. 2000; 19: 5845-5855Crossref PubMed Scopus (117) Google Scholar) and an increasing dose of HA-CUEDC2 constructs. Results show that CUEDC2 clearly inhibits IFN-α-induced STAT3 transcriptional activity in a dose-dependent manner. Similar results were also obtained in HeLa cells (supplemental Fig. 1A). To further confirm the role of endogenous CUEDC2 in STAT3 transcriptional activation, CUEDC2 expression was knocked down using two different siRNA in HEK293T cells. As anticipated, IFN-α-induced STAT3 activation was higher in CUEDC2 knockdown cells compared with control cells. Western blot analysis demonstrates that both of the two CUEDC2 siRNA, but not control siRNA, specifically reduced the expression of endogenous CUEDC2 (Fig. 1B and supplemental Fig. 1B). To exclude the nonspecific effect that may be caused by the α1-antichymotrypsin reporter gene, the same experiments were performed by using other STAT3-responsive luciferase reporter constructs of GAS (IFN-γ-activating sequence) reporter and m67 reporter (a synthetic STAT3-responsive promoter) (31Leong H. Mathur P.S. Greene G.L. Breast Cancer Res. Treat. 2009; 117: 505-515Crossref PubMed Scopus (52) Google Scholar, 32Bromberg J.F. Wrzeszczynska M.H. Devgan G. Zhao Y. Pestell R.G. Albanese C. Darnell Jr., J.E. Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2517) Google Scholar), and the results showed that CUEDC2 also inhibits the expression of these two reporters in a dose-dependent manner (Fig. 1C and supplemental Fig. 1C). To test the specificity of CUEDC2 on STAT3 activation, a luciferase reporter construct (4× interferon regulatory factor-1 (IRF-1)) containing four copies of the STAT binding sequence from the IRF-1 gene were used (33Chung C.D. Liao J. Liu B. Rao X. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (809) Google Scholar). Cells cotransfected with STAT3 and (4× IRF-1) reporter showed a 200-fold increase of luciferase expression upon IFN-α treatment. In keeping with the above data, expression of CUEDC2 strongly inhibited IFN-α-induced STAT3-dependent gene expression. However, CUEDC2 had no such inhibitory effect on STAT1-mediated transcriptional activation in response to IFN-α (Fig. 1, D and E), indicating that CUEDC2 specifically inhibits STAT3 transcriptional activity. Because STAT3 is also the major mediator of IL-6 signaling, it was next investigated whether CUEDC2 plays a role in IL-6-mediated STAT3 activation. As shown in Fig. 1F, CUEDC2 expression decreases IL-6-induced STAT3 activation in HepG2 cells. Notably, our results also demonstrate that CUEDC2 can inhibit STAT3 activation triggered by forced expression of JAK1 or the intracytoplasmic domain of cell surface receptor glycoprotein 130 (gp130) (Fig. 1G). These data suggest that CUEDC2-mediated suppression of STAT3 activation is not restricted to a particular stimulation but may be a general mechanism of STAT3 regulation. Phosphorylation of STAT3 at specific residues, particularly Tyr-705, is necessary for its activation. Therefore, it was next determined whether CUEDC2 inhibits STAT3 activity through affecting its phosphorylation. To this end, either HA-CUEDC2 or control vectors were co-transfected with FLAG-STAT3 into HEK293T cells. Time course analysis of STAT3 phosphorylation demonstrates that, in response to IFN-α treatment, the amount of phosphorylated STAT3 increased substantially in the cells transfected with a control vector (HA vector); in contrast, this phosphorylation of STAT3 was markedly decreased in cells transfected with the CUEDC2 vector (HA-CUEDC2) (Fig. 2A). To analyze the phosphorylation status of endogenous STAT3, HeLa cells stably expressing FLAG-CUEDC2 (HeLa/CUEDC2) or the control vector (HeLa/Vector) were treated with or without IFN-α for the indicated times, and cell lysate was resolved by SDS-PAGE and subjected to Western blot analysis. The results show that IFN-α-induced phosphorylation of STAT3 was much less in cells expressing CUEDC2 compared with control cells. Levels of total STAT3 protein were similar in both samples (Fig. 2B). Next we examined the influence of CUEDC2 on IL-6-induced STAT3 phosphorylation. In keeping with the luciferase reporter assay results, IL-6-induced phosphorylation of STAT3 at Tyr-705 was also reduced in HepG2 cells stably expressing CUEDC2 (HepG2/CUEDC2) compared with control cells expressing GFP (HepG2/GFP) (Fig. 2C). However, inhibition of CUEDC2 by siRNA in HepG2 cells led to an elevated level of phosphorylated STAT3 (Fig. 2D). Furthermore, the impact of CUEDC2 on JAK1 phosphorylation was also investigated in HeLa cells because of its major role in STAT3 activation. The results showed that, just as STAT3, IFN-α-induced JAK1 phosphorylation was lower in cells stably expressing CUEDC2 compared with the control cells (Fig. 2E). Consistent with these results, phosphorylation levels of JAK1 and STAT3 were elevated when CUEDC2 was knocked down by shRNA(#1) in HeLa cells (Fig. 2F). To rule out the off-target effect, we tested the kinetics of JAK1 and STAT3 phosphorylation with a different CUEDC2 shRNA(#2), and the same results were also obtained (Fig. 2G). Thus, these data indicate that CUEDC2 inhibits the phosphorylation of JAK1 and STAT3 and thus attenuates STAT3 transcriptional activation.FIGURE 1CUEDC2 inhibits STAT3 transcriptional activity. A, HEK293T cells were transiently transfected in a 12-well plate with pACT luciferase reporter (200 ng), FLAG-STAT3 (200 ng), and increasing amounts of HA-CUEDC2 vectors (0, 200, and 500 ng) as indicated. 24 h after transfection, cells were stimulated with IFN-α (50 ng/ml) for an additional 6 h, and luciferase activity was measured. Renilla reporter pRL-TK (20 ng/well) vectors were used as an internal control for transfection efficiency. B, HEK 293T cells were transfected with control siRNA or the two different CUEDC2 siRNAs (20 nm) (#1 and #2). 24 h later pACT-Luc and FLAG-STAT3 plasmids were cotransfected as in A. After another 24 h, cells were treated with IFN-α for 6 h, and luciferase reporter assays were performed. C, HEK293T cells were transfected the same as in A, except the luciferase reporter gene was replaced by STAT3 responsive IFN-γ activating sequence reporter (500 ng/well). D and E, HEK293T cells were transfected with 4× IRF-1 luciferase reporter (500 ng/well) construct and STAT3 or STAT1(200 ng/well) together with or without CUEDC2 as indicated. Twenty-four hours after transfection, cells were left untreated (open columns) or treated with IFN-α (50 ng/ml) for 6 h, and luciferase activity was determined. F, HepG2 cells were transiently transfected with pACT-Luc (200 ng/well) together with increasing amounts of CUEDC2 vectors; 24 h after transfection cells were starved for 16–18 h in MEM with 0.5% serum then treated with IL-6 (100 ng/ml) for another 6 h, and luciferase activity was measured.G, JAK1 or glycoprotein 130 (gp130) expression vectors (200 ng/well) were co-transfected with pACT-Luc, FLAG-STAT3, and HA-CUEDC2 (or HA-vector) into HEK293T cells; 24 h later cells were harvested and detected for luciferase activity. All the results are the means ± S.E. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because CUEDC2 inhibits JAK1 and STAT3 phosphorylation and the subsequent STAT3 transcriptional activity, was next determined whether CUEDC2 directly interacts with JAK1 and/or STAT3. However, we found that no association was observed between CUEDC2 and either JAK1 or STAT3, although the positive controls (JAK1 interacts with SOCS1 and STAT3 interacts with PIAS3) in the same assay worked well (supplemental Fig. 2) (33Chung C.D. Liao J. Liu B. Rao X. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (809) Google Scholar, 34Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1234) Google Scholar). This indicates that CUEDC2 inhibits JAK1/STAT3 signaling by recruiting some other essential molecules. To further explore how CUEDC2 mediates JAK1 and STAT3 inhibition, it was determined whether SOCS or SHP (the SH2 domain-containing protein-tyrosine phosphatase) family members, which are key negative regulators of JAK/STAT signaling, interact with CUEDC2 in a yeast strain, AH109, by performing β-galactosidase assays. Interestingly, out of all the proteins tested, CUEDC2 only interacts with SOCS3 (Fig. 3A). The specificity of the interaction between CUEDC2 and SOCS3 was further confirmed by GST pulldown assays. Results show that substantial amounts of SOCS3 were pulled down by GST-CUEDC2 but not by GST alone (Fig. 3B, upper panel). Additionally, SOCS1, another member of the SOCS family proteins, could bind neither GST nor GST-CUEDC2 (Fig. 3B, lower panel). These results suggest that CUEDC2 specifically associates with SOCS3 in vitro. To further verify this interaction in eukaryotic cells, HA-CUEDC2 and FLAG-SOCS3 were co-expressed into HEK293T cells, and co-immunoprecipitation experiments were performed with anti-FLAG antibody and followed by Western blot analysis. Again, data show that CUEDC2 associates with SOCS3 specifically (Fig. 3C). As data from Fig. 3, A–C, demonstrate that these two proteins interact was performed with exogenous protein expression, we next examined whether CUEDC2 and SOCS3 interact under physiological conditions, To do so, cell extracts from HeLa cells were prepared, and immunoprecipitations were performed with anti-CUEDC2 antibody or mouse immunoglobulin G (IgG) control. Precipitates were resolved by SDS-PAGE followed by Western blot analysis using SOCS3 antibody. As shown in Fig. 3D, SOCS3 was detected in the immunoprecipitates obtained from cell extracts with antibody to CUEDC2 (anti-CUEDC2) but not with control IgG. Collectively, these data indicate that CUEDC2 interacts with SOCS3 in vitro and in vivo. To delineate which region of CUEDC2 was responsible for association with SOCS3, a series of GST-CUEDC2 deletion mutants was constructed (Fig. 4A), and GST pulldown assays were performed in HEK293T cells. As indicated in Fig. 4B, only wild type CUEDC2 and the carboxyl-terminal region of CUEDC2 (133–287 aa) interacted with SOCS3; none of the other CUEDC2 mutants (1–133, 1–180, and 180–287aa) was enough to bind SOCS3. Therefore, these results revealed that association of CUEDC2 and SOCS3 requires the carboxyl-terminal region including the CUE domain. To analyze the binding domains of SOCS3 in better detail, SOCS3 deletion mutants, respectively, lacking the 23 amino-terminal amino acids (SOCS3 ΔN), the amino-terminal region identified as an extension of the SH2 domain (SOCS3 ΔESS), the SH2 domain (SOCS3 ΔSH2) and the carboxyl-terminal SOCS-box (SOCS3 ΔBox) were generated (Fig. 4C). These" @default.
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• W2000971871 title "CUEDC2 (CUE Domain-containing 2) and SOCS3 (Suppressors of Cytokine Signaling 3) Cooperate to Negatively Regulate Janus Kinase 1/Signal Transducers and Activators of Transcription 3 Signaling" @default.
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