The WD Motif-Containing Protein RACK-1 Functions as a Scaffold Protein Within the Type I IFN Receptor-Signaling Complex This information is current as Anna Usacheva, Xinyong Tian, Raudel Sandoval, Debra of September 28, 2021. Salvi, David Levy and Oscar R. Colamonici J Immunol 2003; 171:2989-2994; ; doi: 10.4049/jimmunol.171.6.2989 http://www.jimmunol.org/content/171/6/2989 Downloaded from References This article cites 39 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/171/6/2989.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology The WD Motif-Containing Protein RACK-1 Functions as a Scaffold Protein Within the Type I IFN Receptor-Signaling Complex1 Anna Usacheva,2* Xinyong Tian,2* Raudel Sandoval,* Debra Salvi,* David Levy,† and Oscar R. Colamonici3* The WD repeat-containing protein receptor for activated protein kinase C (RACK)-1 has been linked to a variety of signaling systems including protein kinase C, growth factors, and IFNs. In the IFN system, RACK-1 functions as an adaptor recruiting the transcription factor STAT1 to the receptor complex. However, RACK-1 should play a broader role in type I IFN signaling because mutation of the RACK-1 binding site in the IFN-␣ receptor 2/␤ subunit of the type I IFN receptor abrogates not only STAT1, but also STAT2, activation. In this study, we demonstrate that RACK-1 serves as a scaffold protein for a multiprotein complex that Downloaded from includes the IFN-␣ receptor 2/␤-chain of the receptor, STAT1, Janus kinase 1, and tyrosine kinase 2. In vitro data further suggest that within this complex tyrosine kinase 2 is the tyrosine kinase responsible for the phosphorylation of STAT1. Finally, we provide evidence that RACK-1 may also serve as a scaffold protein in other cytokine systems such as IL-2, IL-4, and erythropoietin. The Journal of Immunology, 2003, 171: 2989–2994. ytokines and IFNs (1–3) activate kinases of the Janus to the receptor complex and no specific STAT1-docking tyrosine http://www.jimmunol.org/ kinase (Jak)4 family and transcription factors designated has been identified within the ␣ or ␤ subunits of the receptor (13, C as STAT (4–7). Although there is some promiscuity in 15, 20, 21). Moreover, the tyrosine phosphorylation of STAT1 STAT activation by different cytokines (i.e., STAT1 is activated requires the previous phosphorylation of STAT2 (22). Interest- by IFN-␣, IFN-␥, IL-6, LIF, IL-10, etc.), specific knockout mice ingly, mutation of the RACK-1 binding site of ␤L has an impact models have demonstrated that the biological role of a distinct on type I IFN signaling that goes beyond activation of STAT1 STAT is restricted to specific systems (for review see Ref. 8). For because it also impairs activation/phosphorylation of STAT2. This example, STAT1 is only specifically required for type I (IFN-␣, finding raises the question as to whether RACK-1 recruits other -␤,or-␻) and type II (IFN-␥) signaling (9, 10). signaling components to the receptor complex. In most cytokine systems, activation of STATs through tyrosine RACK-1 functions are not restricted to protein kinase C (PKC) by guest on September 28, 2021 phosphorylation requires their previous recruitment to distinct or IFN signaling because RACK-1 interacts with Src homology 2 phosphotyrosines within the receptor subunits (reviewed in Refs. (SH2)-containing proteins such as src, phospholipase C ␥, and ras- 7, 8, and 11). In the case of the type I IFNR, STAT2 is constitu- GTPase-activating protein (GAP) (23, 24), ␤ integrins (25), tively associated with the ␤L subunit (also designated as IFN-␣ PDE4D5 (26), the ␤ common subunit of the GM-CSF/IL-3/IL-5 receptor 2) in a phosphotyrosine-independent manner and has ad- receptors (27), and insulin-like growth factor (IGF) receptor (28). ditional phosphotyrosine-dependent docking sites on the ␣ and ␤L Because RACK-1 is a WD repeat-contained protein with no en- chain (12–16). Interestingly, full activation of STAT2 by type I zymatic activity it has been proposed that it functions as a scaffold IFNs requires the presence of at least two of these three docking protein that recruits specific signaling elements. For instance, scaf- sites (13). Activation of STAT1 by type I IFNs differs significantly fold proteins bring together multiple components of the mitogen- from its activation by IFN-␥ and the activation of STATs in gen- activated protein kinase signaling (29, 30). eral by other cytokines. For example, the adaptor protein receptor We sought to determine whether RACK-1, in addition to serving for activated protein kinase C (RACK)-1 (17–19) recruits STAT1 as an adaptor between the ␤L chain of the receptor and STAT1, was required for docking other components of the type I IFN re- ceptor system such as Jak1 and tyrosine kinase (Tyk)2. We also *Department of Pharmacology, University of Illinois, Chicago, IL 60612; and †De- endeavored to determine whether Jak1 and Tyk2 could be respon- partment of Pathology, New York University School of Medicine, New York, New sible for tyrosine phosphorylation of STAT1 and STAT2. Our York 10016 findings indicate that RACK-1 directly interacts with Tyk2 and Received for publication April 14, 2003. Accepted for publication July 16, 2003. Jak1, however, only Tyk2 can phosphorylate STAT1 in vitro. Fi- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance nally, we provide evidence that RACK-1 associates with other with 18 U.S.C. Section 1734 solely to indicate this fact. cytokine receptors such as erythropoietin, IL-2R␤, and IL-4R␣, 1 This work was supported by National Institutes of Health Grants CA55079 and suggesting that RACK-1 could play a role in signaling by other GM54709 (to O.R.C.). cytokines. 2 A.U. and X.T. contributed equally to this manuscript. 3 Address correspondence and reprint requests to Dr. Oscar R. Colamonici, Depart- Materials and Methods ment of Pharmacology (M/C868), University of Illinois, 835 South Wolcott Avenue, Cell lines, reagents, and antiviral assays Room E403, Chicago, IL 60612. E-mail address: [email protected]. 4 Abbreviations used in this paper: Jak, Janus kinase; RACK, receptor for activated U-266 and L-929 cells were grown in RPMI 1640 supplemented with 10% 8 protein kinase C; SH2, Src homology 2; IGF, insulin-like growth factor; Tyk, tyrosine (v/v) FBS. Human IFN-␣2 (specific activity 2.2 ϫ 10 U/mg) was a gift of kinase; PKC, protein kinase C; EPOR, erythropoietin receptor. R. Bordens (Schering-Plough, Kenilworth, NJ). The anti-phosphotyrosine Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 2990 RACK-1 AND IFN SIGNALING Ab 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY) and the anti-RACK-1 (IgM), -STAT1, and -Jak1 mAbs were purchased from BD Transduction Laboratories (Lexington, KY). The anti-RACK1 Ab (IgG) used for the experiments described (see Fig. 2C) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-STAT1 sera were kindly provided by Dr. A. Larner (Cleveland Clinic, Cleveland, OH). Immunoprecipitation and immunoblotting Cells (1 ϫ 107 cells/immunoprecipitation) were treated as indicated, and then lysed in lysis buffer (20 mM Tris-HCl, pH 6.6, containing 1% Nonidet P-40, 50 mM NaCl, 1 mM EDTA, 2.5% glycerol v/v, 1.0 mM sodium fluoride, 1.0 mM sodium orthovanadate, 1.0 mM PMSF, 0.5 ␮g/ml leu- peptin, and 5.0 ␮g/ml trypsin inhibitor) for 30 min at 4°C. Immunopre- cipitations were performed as previously described (13). Proteins were transferred to polyvinylidene difluoride membranes, immunoblotted with the indicated Abs, and developed using a chemiluminescent detection method (Pierce, Rockford, IL). GST-fusion proteins and mammalian expression construct The different GST fusion proteins encoding the cytoplasmic domain of the ␣ and ␤L subunits of the type I IFNR, IL-2R␤, IL-4␣, and GST-RACK-1 have been described previously (31, 32). GST-STAT1 corresponds to the Downloaded from full-length STAT1 sequence subcloned into pGEX-2T. For in vitro kinase assays, GST-STAT1 was eluted from the GSH-Sepharose beads using 10 mM glutathione and ϳ2–4 ␮g/assay used as substrate. The amount of GST used per pull-down was estimated from gels stained with Coomassie blue FIGURE 1. RACK-1 interacts with tyrosine-phosphorylated proteins. and compared to BSA standards. Pull-down experiments and immunoblot- U-266 (2 ϫ 107 cells/immunoprecipitation) lysates obtained from IFN-␣- ting were performed using the same procedure described above for treated (10,000 U/ml) or control cells were used for pull-down experiments immunoprecipitations. with the indicated GST fusion proteins or for immunoprecipitation with a http://www.jimmunol.org/ In vitro translation and in vitro kinase assays combination of anti-STAT1 and anti-STAT2 Abs.