The WD Motif-Containing Protein RACK-1 Functions as a Scaffold Protein Within the Type I IFN -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! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 28, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 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 STAT1 to the receptor complex. However, RACK-1 should play a broader role in type I IFN signaling because mutation of the RACK-1 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, 1, and 2. In vitro data further suggest that within this complex 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 systems such as IL-2, IL-4, and . 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 (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, ␥, and ras- 7, 8, and 11). In the case of the type I IFNR, STAT2 is constitu- GTPase-activating protein (GAP) (23, 24), ␤ (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 (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, . 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. Immunoblotting was sequentially performed with the anti-phosphotyrosine (4G10, top panel) For in vitro translation assays, proteins were produced using a T7 wheat and anti-STAT1 (lower panel) mAbs. The migration of phosphoproteins germ in vitro transcription/translation (Promega, Madison, WI) follow- ing manufacturer’s procedure. [35S]Methionine proteins were incubated pulled down by GST-RACK-1 (asterisks and arrow) and STAT1/STAT2 with the indicated GST fusion proteins overnight, washed, and analyzed by (dashes) is indicated. SDS-PAGE as described for immunoprecipitations. In vitro kinase assays were performed after immunoprecipitation with the indicated Abs as pre- viously described (32). interaction with STAT1, the association of Jak1 and RACK-1 is not affected by IFN-␤ treatment. The level of tyrosine-phospho- Results rylated Jak1 associated with RACK-1 was slightly lower in this by guest on September 28, 2021 RACK-1 interacts with Jak1 and Tyk2 experiment but consistently present. As expected, GST-␤L also We have previously reported that RACK-1 interacts with the non- pulled down Jak1 as previously reported (31). As observed in Fig. phosphorylated form of STAT1 (21). Fig. 1 confirms this finding 1, GST-RACK-1, GST-␤L, and in some experiments the anti-Jak1 because GST-RACK-1 pulls down only nonphosphorylated Ab (Fig. 2A, lane 8) pulled down a tyrosine-phosphorylated pro- STAT1 from lysates of either control or cells treated with 5000 tein with a slightly slower electrophoretic mobility that resembled U/ml of IFN␣2 (Fig. 1, lower panel, lanes 3 and 7), while the Tyk2 (Fig. 2A). Fig. 2B shows that indeed the tyrosine phosphor- anti-STAT1 Ab detects the phosphorylated (Fig. 1, upper panel, ylated and nonphosphorylated forms of Tyk2 were pulled down by lane 4) and nonphosphorylated (Fig. 1, lower panel, lanes 4 and 8) GST-RACK-1 (lanes 3 and 8). STAT1. As expected, a GST encoding the long form of the ␤ We next determined whether an interaction between endoge- subunit of the type I IFNR also pulls down the nonphosphorylated nous RACK-1 and Jak1 or Tyk2 could be detected by coimmu- form of STAT1 (Fig. 1, lower panel, lanes 2 and 6) through its noprecipitation. Fig. 2C shows that indeed the anti-Jak1 and -Tyk2 interaction with RACK-1. Interestingly, this experiment also dem- Abs (Fig. 2C, lanes 2 and 3) can coprecipitate RACK-1 indicating onstrated that GST-RACK-1 and GST-␤L pulled down tyrosine- that the interaction also occurs in vivo. These data suggest that phosphorylated proteins (Fig. 1, upper panel, asterisks) with elec- RACK-1 not only interacts with ␤L and STAT1 as previously trophoretic mobility similar to Jak1 and Tyk2 from lysates reported (21), but also with Jak1 and Tyk2. obtained from IFN-treated cells. Additionally, only GST-RACK-1 interacted with a tyrosine-phosphorylated protein with an approx- RACK-1 interacts with different cytokine receptors imate molecular mass of 100 kDa (lane 3, arrow) that resembles RACK-1 has been reported to associate with other cytokine and the electrophoretic mobility of the ␤-chain of the receptor. How- growth factor receptors such as the ␤ common chain of the GM- ever, the identity of this protein has not been confirmed due to CSF/IL-3/IL-5 receptors and IGF receptors (27, 28). Because heavy reactivity of the anti-␤L serum (raised using GST-␤LasAg) RACK-1 interacts with the ␤L chain, STAT1, Jak1, and Tyk2, we with the GST part of GST-RACK-1 (data not shown). next examined whether it could also interact with the ␣-chain of To determine whether two of the phosphoproteins associated the type I IFNR as well as other cytokine receptors. GST fusion with RACK-1 corresponded to Jak1 and Tyk2, we performed new proteins for type I IFNR ␣-chain, IL-2R␤ and ␥, IL-4R␣, and pull-down experiments using cell lysates obtained from control erythropoietin receptor (EPOR) were used to pull-down RACK-1 and IFN-␤-treated U-266 cells. Fig. 2A shows that the protein from cell lysates. Fig. 3 shows that RACK-1 interacts with the pulled down by GST-RACK-1 (Fig. 2A, middle panel, lanes 2 and ␣-chain of the type I IFNR (Fig. 3, lane 7), although the interaction 6) is recognized by an anti-Jak1 Ab and has the same electro- appears to be weaker than with the ␤-chain (Fig. 3, lane 8). Inter- phoretic mobility as the Jak1 protein immunoprecipitated with an estingly, RACK-1 also associated with the IL-2R␤ chain (Fig. 3, anti-Jak1 Ab (Fig. 2A, middle panel, lanes 4 and 8). Unlike the lane 5), a GST-IL-4R␣ that encompasses residues 283–429 (Fig. The Journal of Immunology 2991

FIGURE 3. RACK-1 interacts with distinct cytokine receptors. U-266 cell lysates were used as source of RACK-1 in pull-down experiments with the indicated GST-fusion proteins. Immunoprecipitation with an anti-RACK-1 mAb or IgM control were used as positive and negative controls, respectively. Immunoblotting was performed with anti- RACK-1 and -GST mAbs. Downloaded from

translated RACK-1 associates with GST-␤L (Fig. 4A, lane 3) but fails to interact with GST-␣ or GST control (Fig. 4A, lanes 1 and

2). These results suggest two possibilities: 1) the interactions of the http://www.jimmunol.org/ ␣-chain with RACK-1 (Fig. 4A) may not be direct but rather me- diated by other proteins within the receptor complex, or 2) the ␣-chain has a lower affinity for RACK-1 that masks the detection FIGURE 2. RACK-1 interacts with Jak1 and Tyk2. U-266 (2 ϫ 107 of the association under these experimental conditions. cells/immunoprecipitation) lysates obtained from IFN-␤-treated (10,000 We also studied the nature of the interaction between RACK-1 U/ml) or control cells were used for pull-down experiments with the in- and Jak1 and Tyk2 using a wheat germ in vitro translation system. dicated GST fusion proteins or for immunoprecipitation with anti-Jak1 (A, Fig. 4B shows that GST-RACK-1 interacts with in vitro translated Jak1), anti-Tyk2, or control normal rabbit serum (B, Tyk2 and NR, respec- Jak1 and Tyk2 (Fig. 4B, lanes 4 and 9) suggesting that the inter-

tively). Western blotting (WB) was first performed with the anti- by guest on September 28, 2021 phosphotyrosine Ab 4G10, followed by stripping of the filters and immu- action between RACK-1 and both Jaks is direct. noblotting with anti-Jak1 (A) or -Tyk2 (B) and -GST Abs. GST control is Surprisingly, the GST-␣ chain, which did not interact with not detected because it was run out of the gel. C, Cell lysates (1 mg/ RACK-1, also failed to pull-down significant amounts of Tyk2 immunoprecipitation) were immunoprecipitated with the indicated Abs or produced in wheat germ in vitro translation systems (Fig. 4B, lanes purified rabbit sera (normal rabbit serum), and Western blot analysis was 2 and 7). The association of the ␣-chain and Tyk2 was previously performed using an anti-RACK-1 IgG mAb (Santa Cruz Biotechnology). described using as sources of Tyk2 mammalian cell lysates, rabbit ␮ Approximately 200 g of total cell lysate (input) were used as positive reticulocyte in vitro translation systems and baculovirus extracts control. (33–35). To determine whether there were differences in the in- teraction between the ␣-chain and Tyk2 that depended on the sys- tem used, we compared in parallel the association between the 3, lane 3), and with the EPOR (Fig. 3, lane 14). However, two ␣-chain and Tyk2 using as sources of the kinase either cell lysate other regions of the IL-4R␣ (amino acids 209–288 and 429–561, or wheat germ or rabbit reticulocyte lysate in vitro translation sys- Fig. 3, lanes 2, and 4), IL-2R␥ (Fig. 3, lanes 6 and 11), or GST tems. Fig. 4C shows that indeed GST-␣ associates with Tyk2 pro- control (Fig. 3, lane 10) failed to interact with RACK-1. These data demonstrate that RACK-1 interacts specifically with both sub- duced by in vitro translation in rabbit reticulocyte lysates and with units of the type I IFNR and also suggest that it may play a role in Tyk2 present in cell lysates (Fig. 4C, lanes 7 and 12), but not when signaling by other cytokine receptors such IL-2, IL-4 and EPORs. the kinase is produced in a system likely to be devoid of other proteins involved in cytokine signaling such as wheat germ lysates RACK-1 interacts directly with Tyk2, Jak1, and IFN-␣R␤L, and (Fig. 4C, lane 2). These data suggest that the association of the indirectly with the ␣-chain of the type I IFNR ␣-chain of the type I IFNR with Tyk2 is mediated by unidentified The finding that RACK-1 associated with proteins that are part of proteins that are not present in wheat germ systems (see a large receptor complex (both receptor subunits, STAT1, Tyk2, Discussion). and Jak1) raised the possibility that some interactions may not be Of note, Fig. 4 demonstrates that there is a direct interaction direct but rather mediated by other components of the complex. between ␤L and Tyk2 (Fig. 4, B and C, lanes 8 and 3, respective- For example, because both Jak1 and RACK-1 associate with the ly). This suggests that there is some degree of promiscuity in the ␤L chain, GST-RACK-1 could pull-down Jak1 through the inter- Jak binding site of ␤L similar to that previously reported for the action of the kinase with ␤L. To address this question, RACK-1 gp130 family of receptors (36). Alternatively, ␤L may indepen- was produced using a wheat germ in vitro translation system, dently bind either of these two kinases as previously reported for which did not contain Jak kinases or IFNR homologs, and pull- the IL-2R␤ chain (37) (see Discussion). Altogether, these data sug- down experiments were performed with GST-RACK-1, GST-␣, gest that RACK-1 may be the central docking element in a com- and GST-␤L fusion proteins. As previously reported (21), in vitro- plex that involves Jak1, Tyk2, STAT1, and ␤L. 2992 RACK-1 AND IFN SIGNALING

not clearly improved by the addition of the phospho-STAT2 pep- tide in this experiment, although in others there was a slight in- crease (data not shown). This result demonstrates that at least in in vitro systems Tyk2, but not Jak1, can phosphorylate STAT1, rais- ing the possibility that Tyk2 may also phosphorylate STAT1 in vivo. Unfortunately, we have not succeeded in producing a suit- able GST-STAT2 to determine which kinase was responsible for STAT2 phosphorylation.

Discussion Adaptor or scaffold proteins have been shown to have an important role in signaling cascades by contributing to the assembly of spe- cific signaling complexes (29, 30). Although RACK-1 was origi- nally described as a receptor for activated PKC-␤, it has recently become clear that this scaffold protein has functions that exceed FIGURE 4. RACK-1 interacts directly with Tyk2, Jak1, and the ␤L PKC signaling. RACK-1 appears to be important in a diverse 35 chain of the type I IFNR. A,[ S]Methionine RACK1 was produced using group of signaling pathways such as src kinases, cAMP, integrins, a wheat germ T7 in vitro transcription/translation kit and used for pull- and cytokines including type I IFNs (20, 21, 25–27). In the type I Downloaded from downs with the indicated GST fusion proteins or for immunoprecipitation IFN system, RACK-1 associates with the ␤-chain of the receptor with the anti-RACK1 Ab. B, Similar experiment as in A, except that Tyk2 and Jak1 were used for in vitro translation. C, Similar experiment as in A and recruits STAT1 to the receptor complex. However, there is and B except that Tyk2 was produced by in vitro translation using either evidence supporting a broader role for RACK-1 in type I IFN wheat germ (lanes 1–5) or rabbit reticulocyte (lanes 6–10) systems. In signaling. The most compelling argument is that mutation of the lanes 11–15, U-266 cell lysates were used as a source of Tyk2 protein. region of the ␤L chain containing the RACK-1 binding site not

only abolishes STAT1 tyrosine phosphorylation, but also phos- http://www.jimmunol.org/ phorylation of STAT2. This finding suggests that RACK-1 is ei- Tyk2 phosphorylates STAT1 in vitro ther necessary for the appropriate receptor configuration that al- lows STAT2 phosphorylation by the Jaks or that RACK-1 may The finding that RACK-1 can associate with STAT1, Tyk2, and recruit other signaling proteins to the receptor complex that are Jak1, raises the question as to which kinase phosphorylates this required for STAT2 activation. We explored here the hypothesis STAT factor. To address this question, purified GST-STAT1 was that the scaffolding function of RACK-1 is not limited to the re- used as a substrate in immunocomplex in vitro kinase assays after cruitment of STAT1 but rather involves other components of the immunoprecipitation with either anti-Tyk2 or anti-Jak1 Abs. We type I IFNR complex. Our results demonstrate that in addition to

added to the reaction a STAT2 peptide containing phospho or non- by guest on September 28, 2021 the ␤L chain and STAT1, RACK-1 interacts with Tyk2, Jak1, and phosphotyrosine 691. The rationale for this approach was 2-fold: the ␣-chain of the receptor present in mammalian cell lysates. first, phosphorylation of STAT1 is preceded by phosphorylation of However, unlike the interaction between RACK-1 and STAT1, STAT2 and may require the previous binding of phosphotyrosine RACK-1 still associates with phosphorylated Jaks. 691 of STAT2 to the SH2 domain of STAT1. Second, RACK-1 The finding that the ␣ and ␤ subunits interact independently interacts with a region of STAT1 that involves part of the SH2 with the Jaks and RACK-1 raised the question as to whether domain of STAT1 and the phosphopeptide may participate in ex- RACK-1 was serving as an adaptor protein or simply as one more posing the target tyrosine of STAT1 (data not shown). Fig. 5 protein within the multiprotein complex. If RACK-1 indeed func- shows that GST-STAT1 is phosphorylated after IFN treatment tions as a scaffold protein, it must directly interact with different only in immunocomplexes that include Tyk2. Phosphorylation was components of the complex directly. Experiments performed with the receptor subunits and Tyk2 and Jak1 proteins produced in wheat germ in vitro translation system, which should be devoid of IFNR subunits and Jaks, demonstrated that RACK-1 interacts di- rectly with Tyk2 and Jak1. However, we did not detect a direct interaction between RACK-1 and the ␣-chain of the receptor using a wheat germ lysate system. This would explain that GST- RACK-1 pulled down a lower amount of the ␣-chain than ␤L from cell lysates (Fig. 3) and that we have not been able to detect RACK-1 in coimmunoprecipitations with the ␣-chain (20). Alter- natively, RACK-1 could have lower affinity for the ␣-chain than for ␤L, and the low amounts of RACK-1 produced in the wheat germ in vitro translation system are not enough to detect the FIGURE 5. STAT1 is phosphorylated by Tyk2 in vitro. U-266 cells association. 7 (1 ϫ 10 cells/lane) were treated with IFN-␤ (10,000 U/ml) for 15 min or The scaffolding function of RACK-1 within the receptor com- left untreated. Immunoprecipitations were performed with anti-Tyk2, anti- plex may explain why disruption of the interaction between Jak1 Abs or normal rabbit serum (NR) and the immunocomplexes were RACK-1 and ␤L results in deficient activation of not only STAT1, subjected to in vitro kinase assays using as a substrate GST-STAT1 in the ␤ presence of nonphosphorylated (STAT2) or phosphorylated (pY-STAT2) but also STAT2 (21). In this scenario, L, which directly and peptides. Lower panel, Immunoblotting with anti-GST to detect GST- through phosphotyrosines docks STAT2, may not make STAT2 STAT1. No differences were noted in this experiment between phosphor- available for phosphorylation by Jaks. Interestingly, specific mea- ylated and nonphosphorylated STAT2. Similar results were also obtained sles virus proteins impede IFN-␣ signaling by preventing the as- without the addition of peptide. sociation of RACK-1 with STAT1, and the activation of Jak1, The Journal of Immunology 2993 supporting the concept that RACK-1 is critical for the phosphor- 3. Leonard, W. J., and J. J. O’Shea. 1998. JAKs and STATS: biological implica- ylation of STAT1 and STAT2 by the appropriate kinases (38, 39). tions. Annu. Rev. Immunol. 16:293. 4. Darnell, J. E. J. 1997. Stats and regulation. Science 277:1630. The finding that RACK-1 associated with Jak1 and Tyk2 also 5. Schindler, S., and J. J. E. Darnell. 1995. Transcriptional responses to polypeptide raised the question as to which kinase was responsible for the ligands: the JAK-STAT pathway. Annu. Rev. Biochem. 64:621. phosphorylation of STAT1. Immunocomplex kinase assays using 6. Stahl, N., T. J. Farruggella, T. J. Boulton, Z. Zhong, J. J. E. Darnell, and G. D. Yancopoulos. 1995. Choice of Stats and other substrates specified by mod- as a substrate GST-STAT1 revealed that Tyk2, but not Jak1, was ular tyrosine-based motifs in cytokine receptors. Science 267:1349. able to phosphorylate STAT1 in vitro. Although the conditions in 7. Schindler, C. 1999. Cytokines and JAK-STAT signaling. Exp. Cell Res. 253:7. in vitro kinase assays do not always reflect the in vivo scenario, the 8. Schindler, C., and I. Strehlow. 2000. Cytokines and STAT signaling. Adv. Phar- finding that Jak1 was unable to phosphorylate STAT1 in vitro macol. 47:113. 9. Meraz, M. A., J. M. White, K. C. F. Sheehan, E. A. Bach, S. J. Rodij, A. S. Dighe, weakens a possible role of this kinase in STAT1 phosphorylation. D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Campbell, et al. 1996. Targeted Unfortunately, we have not been able to produce a form of STAT2 disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in suitable for in vitro kinase assays to determine whether Jak1 is the Jak-Stat signaling pathway. Cell 84:431. 10. Durbin, J. E., R. Hackenmiller, M. C. Simon, and D. E. Levy. 1996. Targeted responsible for the phosphorylation of this factor. However, it was disruption of the mouse Stat1 gene results in compromised innate immunity to previously reported that mouse embryonic fibroblasts null for the viral disease. Cell 84:443. ␣-chain that were stably transfected with the human ␤L chain were 11. Ihle, J. E. 1996. STATS: signal transducers and activators of transcription. Cell 84:331. able to show low levels of phosphorylation of Jak1 and STAT2, 12. Yan, H., K. Krishnan, A. Greenlund, S. Gupta, J. T. E. Lim, R. Schreiber, but not Tyk2 or STAT1 suggesting that Jak1 could be responsible C. Schindler, and J. J. Krolewski. 1996. Phosphorylated -␣ receptor 1 for STAT2 phosphorylation (13). Altogether, these results would subunit (IFNaR1) acts as a docking site for the latent form of the 113 kDa STAT2 protein. EMBO J. 15:1064. support a model in which Tyk2 and Jak1 are responsible for the Downloaded from 13. Nadeau, O. W., P. Domanski, A. Usacheva, S. Uddin, L. P. Platanias, P. Pitha, phosphorylation of STAT1 and STAT2, respectively. R. Raz, D. Levy, B. Majchrzak, E. Fish, and O. R. Colamonici. 1999. The prox- An unexpected finding was that the ␣-chain of the receptor did imal tyrosines of the cytoplasmic domain of the ␤ chain of the type I interferon receptor are essential for signal transducer and activator of transcription (Stat) 2 not show a significant association with Tyk2 produced in wheat activation. J. Biol. Chem. 274:4045. germ lysate systems. This lack of interaction is not due to folding 14. Li, X., S. Leung, I. M. Kerr, and G. S. Stark. 1997. Functional subdomains of problems of either in vitro-translated Tyk2 or bacterially produced STAT2 required for preassociation with the ␣ interferon receptor for signaling. Mol. Cell. Biol. 17:2048. GST␣ chain because the same wheat germ preparation of the ki- http://www.jimmunol.org/ 15. Wagner, T. C., S. Velichko, D. Vogel, M. R. Rani, S. Leung, R. M. Ransohoff, nase was able to interact with the ␤-chain and RACK-1 (Fig. 4, B G. R. Stark, H. D. Perez, and E. Croze. 2002. Interferon signaling is depen- and C), and the same GST␣ chain pulled down Tyk2 produced in dent on specific tyrosines located within the intracellular domain of IFNAR2c: expression of IFNAR2c tyrosine mutants in U5A cells. J. Biol. rabbit reticulocyte lysates or mammalian cells (Fig. 4C). These Chem. 277:1493. data suggest that the interaction between Tyk2 and the ␣-chain 16. Nguyen, V. P., A. Z. Saleh, A. E. Arch, H. Yan, F. Piazza, J. Kim, and requires an intermediary protein. These results do not necessarily J. J. Krolewski. 2002. STAT2 binding to the interferon-␣ receptor 2 subunit is not ␣ contradict previous reports describing the association between the required for interferon- signaling. J. Biol. Chem. 277:9713. 17. Mochly-Rosen, D., B. L. Smith, C. H. Chen, M. H. Disatnik, and D. Ron. 1995. ␣-chain and Tyk2 because those experiments were performed us- Interaction of protein kinase C with RACK1, a receptor for activated C-kinase: a ing Tyk2 expressed in mammalian cells or produced in baculovirus role in ␤ protein kinase C mediated . Biochem. Soc. Trans. systems (33–35). 23:596. by guest on September 28, 2021 18. Mochly-Rosen, D., H. Khaner, J. Lopez, and B. L. Smith. 1991. Intracellular Interestingly, our results also revealed that the ␤-chain itself receptors for activated protein kinase C: identification of a binding site for the could interact with Tyk2. A possible explanation could be that . J. Biol. Chem. 266:14866. there is some degree of flexibility between the kinase binding sites 19. Ron, D., C. H. Chen, J. Caldwell, L. Jamieson, E. Orr, and D. Mochly-Rosen. 1994. Cloning of an for protein kinase C: a homolog of the of ␤L and the conserved regions of the kinases that interact with ␤ subunit of G proteins. [Published erratum appears in 1995 Proc. Natl. Acad. cytokine receptors. This scenario would not be very different from Sci. USA 92:2016]. Proc. Natl. Acad. Sci. USA 91:839. that reported for the gp130 subunit of the IL-6R which can asso- 20. Croze, E., A. Usacheva, D. Asarnow, R. D. Minshall, H. D. Perez, and O. R. Colamonici. 2000. RACK-1, a WD motif containing protein, specifically ciate with Jak1, Tyk2, or Jak2 (36). Alternatively, the ␤L subunit associates with the human type I IFN receptor. J. Immunol. 165:5127. has the ability to interact independently with both kinases as pre- 21. Usacheva, A., R. Smith, G. E. Baida, E. Croze, and O. R. Colamonici. 2001. The viously reported for the ␤-chain of the IL-2R (37). adaptor RACK1 recruits STAT1 to the type I interferon receptor and is required for STAT activation. J. Biol. Chem. 276:22948. Finally, it was not surprising that RACK-1 also interacted with 22. Leung, S., S. A. Qureshi, I. M. Kerr, J. E. J. Darnell, and G. R. Stark. 1995. Role other cytokine receptors such as the IL-2R␤, IL-4R␣, and EPOR of STAT2 in the ␣ interferon signaling pathway. Mol. Cell. Biol. 15:1312. that it has been previously reported to participate in signaling by 23. Chang, B. Y., K. B. Conroy, E. M. Machleder, and C. A. Cartwright. 1998. RACK1, a receptor for activated C kinase and a homolog of the ␤ subunit of G other growth factor receptors such as the IGF-1 and IL-3/IL-5/ proteins, inhibits activity of src tyrosine kinases and growth of NIH 3T3 cells. GM-GSF receptors (27, 28). Therefore, RACK-1 may represent a Mol. Cell. Biol. 18:3245. modular protein whose function as a scaffold goes beyond its ini- 24. Chang, B. Y., M. Chiang, and C. A. Cartwright. 2001. The interaction of Src and ␤ RACK1 is enhanced by activation of protein kinase C and tyrosine phosphory- tial description as a receptor for activated PKC . There are many lation of RACK1. J. Biol. Chem. 276:20346. questions regarding the role of RACK-1 that have not been ad- 25. Liliental, J., and D. D. Chang. 1998. Rack1, a receptor for activated protein dressed. For instance, do all these systems absolutely require kinase C, interacts with ␤ subunit. J. Biol. Chem. 273:2379. RACK-1 for signaling? Are there other WD repeat-containing pro- 26. Yarwood, S. J., M. R. Steele, G. Scotland, M. D. Houslay, and G. B. Bolger. 1999. The RACK1 signaling scaffold protein selectively interacts with the cAMP- teins that can substitute or function redundantly and therefore, specific phosphodiesterase PDE4D5 isoform. J. Biol. Chem. 274:14909. complement the absence of RACK-1? So far, experiments using 27. Geijsen, N., M. Spaargaren, J. A. Raaijmakers, J. W. Lammers, L. Koenderman, siRNA have not produced a decrease large enough in RACK-1 and P. J. Coffer. 1999. Association of RACK1 and PKC␤ with the common ␤-chain of the IL-5/IL-3/GM-CSF receptor. Oncogene 18:5126. levels that would allow us to answer these questions (our unpub- 28. Hermanto, U., C. S. Zong, W. Li, and L. H. Wang. 2002. RACK1, an insulin-like lished observation). growth factor I (IGF-I) receptor-interacting protein, modulates IGF-I-dependent integrin signaling and promotes cell spreading and contact with . Mol. Cell. Biol. 22:2345. 29. Yasuda, J., A. J. Whitmarsh, J. Cavanagh, M. Sharma, and R. J. Davis. 1999. The References JIP group of mitogen-activated protein kinase scaffold proteins. Mol. Cell. Biol. 1. Ihle, J. N., and I. M. Kerr. 1995. Jaks and Stats in signaling by the cytokine 19:7245. receptor superfamily. Trends Genet. 12:69. 30. Schaeffer, H. J., A. D. Catling, S. T. Eblen, L. S. Collier, A. Krauss, and 2. Kishimoto, T., T. Taga, and S. Akira. 1994. Cytokine signal transduction. Cell M. J. Weber. 1998. MP1: a MEK binding partner that enhances enzymatic acti- 76:253. vation of the MAP kinase cascade. Science 28:1668. 2994 RACK-1 AND IFN SIGNALING

31. Domanski, P., O. W. Nadeau, E. Fish, M. Kellum, L. C. Platanias, P. Pitha, and for binding of interferon-␣/␤ and for signal transduction. J. Biol. Chem. O. R. Colamonici. 1997. A region of the ␤ subunit of the interferon a receptor 270:3327. different from the box 1 interacts with Jak1 and is sufficient to activate the Jak- 36. Stahl, N., T. G. Boulton, T. Farruggella, N. Y. Ip, S. Davis, B. A. Witthuhn, Stat pathway and induce an antiviral state. J. Biol. Chem. 272:26388. F. W. Quelle, O. Silvennoinen, G. Barbieri, S. Pellegrini, et al. 1994. Association 32. Usacheva, A., S. Kotenko, M. M. Witte, and O. R. Colamonici. 2002. Two dis- and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL6␤ receptor compo- tinct domains within the N-terminal region of interact with cyto- nents. Science 263:92. kine receptors. J. Immunol. 169:1302. 37. Zhu, M.-H., J. A. Berry, S. M. Russell, and W. J. Leonard. 1998. Delineation of 33. Colamonici, O. R., H. Uyttendaele, P. Domanski, H. Yan, and J. J. Krolewski. the regions of the interleukin-2 (IL-2) receptor ␤ chain important for association 1994. p135tyk2, an interferon ␣ (IFN␣)-activated tyrosine kinase, associates with of Jak1 and Jak3. J. Biol. Chem. 273:10719. the IFN␣ receptor. J. Biol. Chem. 269:3518. 38. Yokota, S.-I., H. Saito, T. Kubota, N. Yokosawa, K.-I. Amano, and N. Fujii. 34. Colamonici, O., H. Yan, P. Domanski, R. Handa, D. Smalley, J. Mullersman, 2003. Measles virus suppresses interferon-␣ signaling pathway: suppression of M. Witte, K. Krishnan, and J. Krolewski. 1994. Direct binding to and tyrosine Jak1 phosphorylation and association of viral accessory proteins, C and V, with phosphorylation of the ␣ subunit of the type I interferon receptor by p135tyk2 interferon-␣ receptor complex. Virology 306:135. tyrosine kinase. Mol. Cell. Biol. 14:8133. 39. Kubota, T., N. Yokosawa, S.-I. Yokota, and N. Fujii. 2002. Association of 35. Velazquez, L., K. E. Mogensen, G. Barbieri, M. Fellous, G. Uze´, and Mumps virus V protein with RACK1 results in dissociation of STAT-1 from the S. Pellegrini. 1995. Distinct domains of the protein tyrosine kinase tyk2 required ␣ interferon receptor complex. J. Virol. 76:12676. Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021