© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 4190-4199 doi:10.1242/jcs.185421

RESEARCH ARTICLE Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response Håkan C. Steen1,*, Kevin P. Kotredes1,*, Shoko Nogusa2, Michele Y. Harris3, Siddharth Balachandran2 and Ana M. Gamero1,‡

ABSTRACT dimerization of the two STATs, that together with the DNA-binding Serine phosphorylation of STAT is an important post- IRF9 form the heterotrimeric IFN-stimulated factor 3 translational modification event that, in addition to tyrosine (ISGF3) complex (Fu et al., 1990, 1992; Improta et al., 1994). phosphorylation, is required for strong transcriptional activity. ISGF3 translocates to the nucleus and binds to the promoters of However, we recently showed that phosphorylation of STAT2 on multiple IFN-stimulated (ISGs) to activate their transcription S287 induced by type I (IFN-α and IFN-β), evoked the (Fu et al., 1990; Li et al., 1997a). The resulting gene products drive opposite effect. S287-STAT2 phosphorylation inhibited the biological the cellular responses to IFNs that include antiviral, antiproliferative effects of IFN-α. We now report the identification and characterization and, in certain instances, pro-apoptotic responses (Chawla-Sarkar of S734 on the C-terminal transactivation domain of STAT2 as a new et al., 2001; de Veer et al., 2001). phosphorylation site that can be induced by type I IFNs. IFN-α- The specific role of STAT2 in the ISGF3 complex is to provide its induced S734-STAT2 phosphorylation displayed different kinetics to transactivation domain (TAD) for full transcriptional activity (Li that of tyrosine phosphorylation. S734-STAT2 phosphorylation was et al., 1996). The STAT2 TAD is situated at the C-terminal end and ∼ – dependent on STAT2 tyrosine phosphorylation and JAK1 kinase spans 100 amino acid residues (amino acids 747 851) (Kraus activity. Mutation of S734-STAT2 to alanine (S734A) enhanced IFN- et al., 2003) and binds p300 and CBP (hereafter p300/CBP, also α-driven antiviral responses compared to those driven by wild-type known as EP300 and CREBBP, respectively) and GCN5 (also STAT2. Furthermore, DNA microarray analysis demonstrated that a known as KAT2A), both of which are involved in facilitating small subset of type I IFN stimulated genes (ISGs) was induced more transcription through their histone acetylation activities by IFNα in cells expressing S734A-STAT2 when compared to wild- (Bhattacharya et al., 1996; Paulson et al., 2002). Earlier studies type STAT2. Taken together, these studies identify phosphorylation have suggested that the most crucial region for transactivation of S734-STAT2 as a new regulatory mechanism that negatively resides between residues 800 and 832 because deletion of this controls the type I IFN-antiviral response by limiting the expression of region abolished the transcriptional activity of ISGF3 (Qureshi a select subset of antiviral ISGs. et al., 1996). Later studies have suggested T800 and Y833 as additional phosphorylation sites (Farrar et al., 2000; Hornbeck et al., KEY WORDS: STAT2, Serine phosphorylation, JAK1, , 2012; Shiromizu et al., 2013). Although mutation of Y833 to a Virus infection, Vesicular stomatitis phenylalanine residue impaired dimerization of STAT2–STAT4 induced by type I IFNs, evidence is lacking to support the biological INTRODUCTION significance of T800 and Y833 in their phosphorylated state Signal transducer and activator of transcription (STAT) 2 is a pivotal (Hornbeck et al, 2012; Shiromizu et al 2013). component of the type I interferon (IFN) signaling pathway (Leung Aside from tyrosine phosphorylation, serine phosphorylation et al., 1995; Steen and Gamero, 2013). Type I IFNs, which include modulates the transcriptional activity of the STATs. STATs are IFN-α,-β,-ε,-κ and -ω, activate the tyrosine phosphorylation of phosphorylated on serine residues located predominantly in the both STAT2 and STAT1 through engagement of the heterodimeric N-terminal region of the TAD, usually 20–30 amino acid residues type I IFN composed of IFNΑR1 and IFNΑR2 (Darnell, C-terminal of the conserved tyrosine (Decker and Kovarik, 2000). 1997; Pestka et al., 2004). IFN binding to its receptor triggers the STAT1, STAT3, STAT4, STAT5a and STAT5b all share a activation of protein tyrosine Janus kinases (JAK)1 and TYK2 conserved motif consisting of two prolines flanking either a single through reciprocal transphosphorylation in response to receptor serine residue or a serine residue preceded by a methionine residue, dimerization (Barbieri et al., 1994; Muller et al., 1993; Velazquez usually referred to as the ‘P(M)SP’ motif, that is targeted for et al., 1995; Yan et al., 1996). JAK1 and TYK2 phosphorylate phosphorylation. STAT2 and STAT6 are the exception as they lack STAT1 and STAT2 on a conserved tyrosine residue, resulting in the PMSP motif. STAT6, however, is phosphorylated at a serine residue in the TAD, but its function is to inhibit gene activation (Shirakawa et al., 2011). Most recently, we made the discovery that 1Dept. of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA. 2Immune Cell S287-STAT2, which is mapped to the coiled-coil domain, is Development and Host Defense Program, Fox Chase Cancer Center, Philadelphia, phosphorylated in response to IFN-α stimulation (Steen et al., PA 19111, USA. 3Dept. of Anatomy and Cell Biology, Lewis Katz School of 2013). In contrast to other STATs, but similarly to STAT6, Medicine, Temple University, Philadelphia, PA 19140, USA. *These authors contributed equally to this work phosphorylation of S734-STAT2 did not augment, but rather inhibited, gene transcription. ‡ Author for correspondence ([email protected]) In this study, we report that S734 is a new STAT2 A.M.G., 0000-0001-7843-1415 phosphorylation site targeted by type I IFN stimulation. This molecular event relies on JAK kinase activity and STAT2 tyrosine

Received 23 December 2015; Accepted 19 September 2016 phosphorylation. Phosphorylation of S734-STAT2 plays a specific Journal of Cell Science

4190 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4190-4199 doi:10.1242/jcs.185421 role in type I IFN signaling by contributing to the inactivation of acids. We established the specificity of the pS734-STAT2 antibody the antiviral, but not the anti-proliferative, response to IFN-α and in two ways. First, we showed the absence of a pS734-STAT2 signal IFN-β. Our findings indicate that phosphorylation of STAT2 at in immunoblots of protein lysates from parental U6A cells and U6A specific serine residues dictate the specificity and kinetics of an cells stably expressing mutant STAT2 in which S734 was changed IFN-α and IFN-β response. to alanine (S734A) (Fig. 2A; Fig. S1A). Of note, a second band was often detected migrating immediately below the pS734-STAT2- RESULTS specifc band. Given that this band was also detected in STAT2-null Identification of a new IFN-α-induced S734-STAT2 U6A cells and in S734A-STAT2-expressing U6A cells, we interpret phosphorylation by mass spectrometry this lower protein band to be non-specific. Second, to confirm that the To gain further insight into the regulation of type I IFN signaling observed protein band recognized by our pS734-STAT2 antibody by post-translational modifications of STAT2, we reconstituted was indeed a phosphorylated amino acid, immunoprecipitated STAT2-null U6A cells with human STAT2. We treated these cells wild-type (WT-)STAT2 was treated with or without lambda protein with or without IFN-α and immunoprecipitated STAT2 from these phosphatase (LPP). LPP treatment eliminated the protein band cells, which was then subjected to mass spectrometry. From this corresponding to pS734-STAT2 (Fig. 2B). With these two sequential analysis, we identified phosphorylation of S734 as a new IFN-α- approaches, we convincingly demonstrate the specificity of the induced post-translational modification of STAT2 (Fig. 1). S734 anti-pS734-STAT2 antibody. maps to the TAD of STAT2 (Fig. 1B) and appears to be evolutionarily restricted to primates (Fig. 1C). To confirm our S734-STAT2 phosphorylation by IFN-α is observed in multiple mass spectrometry data and show that STAT2 was indeed cell lines phosphorylated on S734 after stimulation with type I IFNs, we We consistently detected constitutive phosphorylation of S734- generated a polyclonal rabbit antibody against an immunopeptide STAT2 in resting U6A cells reconstituted with WT-STAT2. The containing phosphorylated (p)S734 and its surrounding amino level of phosphorylated S734-STAT2 was increased with IFN-α

Fig. 1. Mass spectrometry identifies S734 as a type I IFN-inducible phosphorylatable residue in human STAT2 that is conserved in primates. (A) Tandem mass spectrometry (MS/ MS) spectrum of STAT2 after treatment with IFN-α. +P (lower panel) indicates S734 as being phosphorylated and the underlined peptide mass (lower panel) indicates the presence of the peptide in the spectrum (upper panel). P, phosphoryl group (79.97 Da). Not all peaks are annotated in the mass spectrum. Matches were made with a mass tolerance of ±2 Da. (B) S734 is mapped to the transactivation domain (TAD) of STAT2. (C) Cross-species conservation of this phosphorylation site is seen in primate STAT2. Journal of Cell Science

4191 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4190-4199 doi:10.1242/jcs.185421

Fig. 2. STAT2 is phosphorylated at S734 in response to IFN-α treatment. (A) U6A cells stably expressing empty vector, WT-STAT2 or S734A-STAT2 were treated with human IFN-α (1000 U/ml) for the indicated times. Phosphorylation of S734-STAT2 was detected by western blot analysis using an affinity purified polyclonal antibody raised against this phosphorylated epitope. IFN-α-induced tyrosine phosphorylation of STAT2 (pY-STAT2) and STAT1 (pY-STAT1) together with total levels of STAT2 and STAT1 were also assessed by western blot analysis. (B) Immunoreactivity of the pS734-STAT2 antibody is confirmed by a lambda protein phosphatase (LPP) assay. (C–F) Immortalized hTERT cells (C) and human colorectal cancer cells F6-8 (D), T29 (E) and (F) Stat2−/− MEFs reconstituted with human STAT2 were stimulated with either human IFN-α (1000 U/ml) or murine IFN-β (1000 U/ml), accordingly, and examined as in A. GAPDH was used as an internal loading control. after 1 h of treatment that then peaked at 4 h (Fig. 2A). We next endogenous STAT2. No basal levels of phosphorylated S734- evaluated the basal phosphorylation and the kinetics of IFN-α- STAT2 were observed in immortalized human mammary epithelial induced phosphorylation of S734-STAT2 in cell lines that express hTERT-HME1 cells. In response to IFN-α treatment, induction of Journal of Cell Science

4192 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4190-4199 doi:10.1242/jcs.185421 serine phosphorylation was first detected at 8 h of Moreover, immunoprecipitation of STAT1 from U6A WT-STAT2 exposure that peaked around 12 h and decreased thereafter cells revealed that, in the absence of IFN-α treatment, STAT2 (Fig. 2C). As expected, given that STAT2 is an ISG, total STAT2 associated with STAT1 was serine phosphorylated at a basal level levels also increased over time in hTERT-HME1 cells. The (Fig. 3D). Treatment with IFN-α increased pS734-STAT2, but did observed decrease in pS734-STAT2 detected at 24 h did not not alter pS734-STAT2 binding to STAT1, indicating that pS734- correlate with elevated STAT2 levels, indicating at least a partial STAT2 is neither enriched in nor excluded from IFN-α-induced independence between STAT2 levels and S734-STAT2 STAT2–STAT1 dimers. Moreover, analysis of cytoplasmic and phosphorylation in hTERT-HME1 cells. Similarly, IFN-α nuclear extracts from IFN-α-treated 2fTGH cells (the parent of U6A treatment also induced S734-STAT2 phosphorylation in two cells) showed a time-dependent accumulation of nuclear pS734- human colon carcinoma cell lines (Fig. 2D,E). Additionally, we STAT2. After 1 h of IFN-α stimulation, the amount of cytoplasmic show that human STAT2 was phosphorylated on S734 when pS734-STAT2 was decreased, which coincides with an initial reconstituted in Stat2−/− mouse embryonic fibroblasts (MEFs) accumulation of nuclear pS734-STAT2. Between 4 h and 8 h of following treatment with murine IFN-β (Fig. 2F; Fig. S1B). It is IFN-α treatment, pS734-STAT2 peaks, and a higher level is seen in important to remark that in all the cell lines we studied, with the the nucleus with some detected in the cytoplasm, although at reduced exception of T29 cells, IFN-α-stimulated phosphorylation of S734- levels (Fig. 4A). We also detected pS734-STAT2 accumulation in the STAT2 peaked later when compared against Y690-STAT2 nucleus of IFN-α-treated hTERT cells (Fig. 4B). As expected, phosphorylation. Furthermore, we found no differences in the phosphorylation of S734-STAT2 in hTERT cells required a much kinetics of tyrosine phosphorylation of STAT2 and STAT1 between longer incubation time with IFN-α, as we previously determined in U6A cells expressing wild-type and mutant S734A-STAT2 at up to Fig. 2C. Our data, therefore, show that JAK1-dependent tyrosine 8 h of IFN-α treatment (Fig. 2A). However, when examined at 24 h, phosphorylation of STAT2 is a prerequisite for enhancing STAT2 we found STAT2 tyrosine phosphorylation to be prolonged in serine phosphorylation with optimal S734-STAT2 phosphorylation S734A-STAT2 U6A cells and barely detectable in WT-STAT2 U6A peaking when STAT2 is tyrosine phosphorylated. This finding cells (Fig. 7D), indicating that S734-STAT2 plays a role in correlated with a steady increase in the level of phosphorylated S734- regulating STAT2 activation. STAT2 in the nucleus. Further, U6A cells expressing the RKAA- STAT2 mutant showed intact IFN-α-induced pS734-STAT2 IFN-α can induce phosphorylation of STAT2 at S734 only (Fig. 3A). Thus, these results reveal that phosphorylation of S734- in the presence of JAK kinases and if its Y690 is STAT2 by type I IFNs must be occurring in the cytoplasm before phosphorylated STAT2 translocates to the nucleus. To define the signaling requirements for S734-STAT2 phosphorylation, we first examined the role of STAT2 tyrosine S734-STAT2 phosphorylation does not affect the IFN-α- phosphorylation as a potential pre-requisite event, as previously induced antiproliferative activity, but diminishes the IFN-α- demonstrated with STAT1 serine phosphorylation (Sadzak et al., induced antiviral state 2008). We chose two well-characterized STAT2 mutants for this To elucidate the biological relevance of phosphorylated S734-STAT2 analysis: Y690F-STAT2 and R409A/K415A-STAT2 (RKAA). The in type I IFN signaling, we compared the anti-proliferative responses Y690F-STAT2 mutant cannot be phosphorylated by type I IFNs and to IFN-α in U6A cells expressing WT-STAT2 or S734A-STAT2. is thus unable to translocate to the nucleus (Li et al., 1997b). The First, no significant differences in growth rates were observed RKAA-STAT2 mutant is retained in the cytosol due to a non- between U6A cells expressing WT-STAT2 and those expressing functioning nuclear localization signal, but preserves its capacity S734A-STAT2 (Fig. 5A). Second, the anti-proliferative effects of to become phosphorylated on tyrosine (Melen et al., 2001). IFN-α were also unaffected by S734A-STAT2. Similar results were Accordingly, we reconstituted STAT2-null U6A cells with WT-, obtained with the phosphomimetic mutant STAT2-S734D (Fig. 5B). RKAA- or Y690F-STAT2. The STAT2-reconstituted U6A cells were Next, we asked whether phosphorylation on S734 affected IFN- treated with or without IFN-α and assayed for pS734-STAT2 by α-mediated antiviral responses. To this end, we left untreated or pre- immunoblot analysis. We detected a basal level of pS734-STAT2 treated U6A cells expressing WT-STAT2 or S734A-STAT2 with with all three versions of STAT2 (Fig. 3A). In response to IFN-α 1000 U/ml of IFN-α, before infection with a prototypic IFN- treatment, RKAA-STAT2 showed an increase in S734 sensitive virus vesicular stomatitis virus (VSV) that also encodes for phosphorylation (Fig. 3A). In contrast, Y690F-STAT2 did not GFP expression (VSV-GFP) (Fig. 6A). Quantifying progeny virus undergo S734-STAT2 phosphorylation, supporting the notion that yield from these cells demonstrated that, although there was no tyrosine phosphorylation of STAT2 is necessary for the inducible detectable difference at 24 h between cells expressing WT-STAT2, phosphorylation of S734-STAT2 by type I IFNs. To test the potential and phospho-null or phosphomimetic STAT2 mutants (data not contribution of JAK kinases in the phosphorylation of S734-STAT2, shown), virion production at 36 h from S734A-STAT2-containing U6A cells expressing WT-STAT2 were pre-treated with pan-JAK cells was significantly reduced compared to cells expressing inhibitor followed by treatment with IFN-α. Western blot analysis WT-STAT2 (∼3×105 pfu ml−1 versus ∼4×106 pfu ml−1)or demonstrated that JAK activity was necessary for IFN-α-dependent phosphomimetic S734D-STAT2 mutant (Fig. 6A). Note that induction of pS734-STAT2 (Fig. 3B). To further validate these expression of S734A-STAT2 or WT-STAT2 in the absence of results, we evaluated pS734-STAT2 in JAK1-deficient U4A cells. IFN-α treatment rendered U6A cells similarly susceptible to VSV Lack of JAK1 not only impaired pS734-STAT2 by IFN-α, but also infection. It is also important to mention that S734A-STAT2 led to basal STAT2 serine phosphorylation (Fig. 3C). Collectively, these increased STAT2 tyrosine phosphorylation compared to WT- results identify tyrosine phosphorylation on Y690 as a prerequisite STAT2 in U6A cells pre-treated with IFN-α at 3 h but not at 36 h for enhancing basal S734 phosphorylation of STAT2 in response to post-VSV infection (Fig. S2). No differences were found in the level IFN-α treatment. Furthermore, IFN-α-induced S734-STAT2 of tyrosine-phosphorylated STAT1 under these conditions. phosphorylation appears to take place in the cytosol as evidenced Expression of S734D-STAT2 had no significant effect on VSV by the behavior of the RKAA-STAT2 mutant. yield at either 24 or 36 h post infection, compared to WT-STAT2- Journal of Cell Science

4193 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4190-4199 doi:10.1242/jcs.185421

Fig. 3. Phosphorylation of S734-STAT2 is dependent on STAT2 tyrosine phosphorylation and catalytically active JAK kinases. (A) U6A cells stably expressing WT-, R409A/K415A (RKAA)-, or Y690F-STAT2 were left untreated or treated for 1 and 4 h with IFN-α (1000 U/ml). Tyrosine and serine phosphorylation of STAT2, and GAPDH were assessed by western blot analysis. Data were quantified and shown as the ratio of pS734-STAT2 to STAT2 and pY690-STAT2 to STAT2. The arrow indicates a non-specific protein band (NS). (B) U6A cells expressing WT-STAT2 were pre-treated or not with pan-JAK inhibitor overnight (o/n) and (C) U4A cells and U6 cells expressing WT-STAT2 stimulated with IFN-α (1000 U/ml) for the specified times were evaluated for pS734- STAT2, pY-STAT2, pY-STAT1, STAT1, STAT2 and GAPDH as indicated. (D) STAT1 immune complexes obtained from U6A cells expressing WT-STAT2 that were left untreated or treated with IFN-α (1000 U/ml) were evaluated for interactions with pS734-STAT2 and total STAT2 by western blot analysis. expressing cells. We speculate that the effect of the phosphomimetic expression of VSV-GFP 36 h later by flow cytometry to mutant is masked by phosphorylation of S287-STAT2 because, determine the degree of viral infection. We found that compared as reported by us previously (Steen et al. 2013), it negatively to IFN-β-pre-treated Stat2−/− MEFs expressing human WT-STAT2, regulates type I IFN and because pS287-STAT2 led to a much IFN-β-pre-treated Stat2−/− MEFs expressing human S734A-STAT2 stronger phenotype than pS734-STAT2. To confirm our findings showed enhanced viral protection at 36 h post infection (Fig. 6B). using a different cell line, we infected Stat2−/− MEFs expressing Taken together, these results indicate that phosphorylation of either human WT-STAT2 or S734A-STAT2 that had been pre- STAT2 on S734 serves to negatively regulate the type I IFN antiviral treated with or without IFN-β with VSV-GFP. We quantified response. Journal of Cell Science

4194 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4190-4199 doi:10.1242/jcs.185421

Fig. 4. Subcellular localization of phosphorylated S734-STAT2. Nuclear and cytoplasmic fractions of human (A) 2fTGH and (B) hTERT cells treated with IFN- α (1000 U/ml) for the given times were evaluated for pS734-STAT2, pY-STAT2, pY-STAT1, STAT1 and STAT2 by western blot analysis as indicated. Presence of GAPDH in the cytoplasmic extract and presence of Lamin B1 in the nuclear extract confirmed purity of fractions. The arrow indicates the upper band corresponding to pS734-STAT2. Actin was used as an internal loading control. Fig. 5. Phosphorylation of S734-STAT2 does not affect the S734-STAT2 phosphorylation enhances the expression of a antiproliferative effects of IFN-α. (A) Growth rate of U6A cells stably subset of IFN-stimulated antiviral genes expressing WT-, S734A- or S734D-STAT2 was measured at 48 h and 72 h by We next conducted whole-genome DNA microarray analysis to an MTS assay. The inset shows a western blot analysis of STAT2 expression in identify potential differences in the transcriptional responses to IFN- U6A cells expressing empty vector, WT-, S734A or S734D-STAT2. (B) The α between WT-STAT2 and S734A-STAT2 that might explain how same panel of cells was treated with 100 U/ml or 1000 U/ml of IFN-α for 72 h phosphorylation of STAT2 on S734 regulated the type I IFN and cell viability determined by MTS assay. Results are presented as the percentage of viable cells in the presence of IFN-α compared against untreated antiviral state. We used total RNA extracted from U6A cells cells. Data shown are from three or four independent experiments and expressing WT-STAT2 or S734A-STAT2 that had been stimulated presented as mean±s.e.m. with or without IFN-α for 4 h. This genomic approach identified 10 ISGs that were induced more by IFN-α in U6A cells expressing 60 h, however, ISG15 protein levels were reduced in WT-STAT2 S734A-STAT2 than they were in cells expressing WT STAT2 cells, but remained elevated in S734A-STAT2 cells. In contrast, (Fig. 7A; full microarray data is available in the expression of OAS1 was found to be slightly elevated only at 24 h in Omnibus under accession number GSE57017). We then selected S734A-STAT2 cells when compared to WT-STAT2 cells. This representative genes from this list to validate by quantitative real- observation also correlated with increased STAT2 tyrosine time PCR (qPCR). At 6 h, we consistently detected an enhanced phosphorylation in cells expressing S734A-STAT2 at 24 h, induction of OAS1 and IFIT2 by IFN-α in U6A cells containing indicating that S734 regulates STAT2 activity. Thus, these results S734A-STAT2 compared to that in cells expressing WT-STAT2 support the idea that phosphorylation of S734-STAT2 contributes to (Fig. 7B). Similarly, we found that Stat2−/− MEFs reconstituted the transcriptional regulation of a small subset of ISGs with antiviral with human S734A-STAT2 and stimulated with murine IFN-β activity. showed enhanced transcription of Oas1b and Isg15 when compared to IFN-β-treated MEFs expressing human WT-STAT2 (Fig. 7C). DISCUSSION Although differences in Oas1b mRNA levels were not evident until Regulation of type I IFN signaling through STAT2 has remained 24 h after IFN-β stimulation, this could be attributed to the understudied chiefly because the diversity of STAT TADs and the activation of human STAT2 in mouse cells. To confirm these sequence divergence of the STAT2 TAD among different species results at the protein level, U6A cells reconstituted with either WT- have made it challenging to predict amino acids in STAT2 that STAT2 or S734A-STAT2 were stimulated with 1000 U/ml of IFN-α might be important for proper transcriptional activity (Park et al., for 24 and 60 h and analyzed by western blot analysis for ISG15 and 1999; Paulson et al., 1999). For example, serine phosphorylation OAS1 expression (Fig. 7D). We found ISG15 protein levels to be within the P(M)SP motifs found in several STAT transactivation similar in WT-STAT2 and S734A-STAT2 U6A cells at 24 h. At domains has been shown to be required for STAT-dependent gene Journal of Cell Science

4195 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4190-4199 doi:10.1242/jcs.185421

Fig. 6. S734A-STAT2 enhances IFN-α-induced protection against VSV infection. (A) The U6A cell panel was left untreated or pre-treated with IFN-α (1000 U/ml) for 16 h and then infected with GFP-tagged vesicular stomatitis virus (VSV) for 36 h. Viral titers after 36 h post-viral infection (h.p.i.) were determined by plaque assay. Viral titers are shown on a log scale. (B) Western blot analysis of STAT2 expression in Stat2−/− MEFs reconstituted with either human WT- or S734A-STAT2. (C) Stat2−/− MEFs expressing human WT- or S734A-STAT2 were pre-treated or not with murine IFN-β (1000 U/ml) for 16 h and VSV infection was quantified by flow cytometry analysis of GFP-positive cells. Data are shown as the mean±s.e.m. from three or four independent experiments. **P<0.01 (Student’s t-test). NS, not statistically significant. regulation. In contrast, STAT2 lacks this conserved motif, and this Our study provides new evidence that serine phosphorylation of opens the door to investigate the biological role of serine- STAT2 plays a pivotal role in selectively regulating the biological phosphorylated STAT2. actions of type I IFNs. We have identified a serine phosphorylation

Fig. 7. S734-STAT2 regulates the expression of a subset of ISGs. (A) Microarray analysis identified a small subset of ISGs for which mRNA levels were found to be enhanced in S734A-STAT2 U6A cells when compared against WT-STAT2 U6A cells after 4 h of IFN-α (1000 U/ml) treatment. Genes marked in A with an asterisk were subsequently validated by qPCR using (B) U6A or (C) Stat2−/− MEFs reconstituted with either human WT- or S734-STAT2 treated with or without IFN-α or IFN-β (1000 U/ml) for 6, 18 or 24 h. The mRNA levels were normalized to actin expression and calculated by using ΔΔCt method. Data are presented as the mean±s.e.m. fold change from untreated cells from three or four independent experiments. *P<0.05, **P<0.01 (Student’s t-test). (D) WT-STAT2 and S734A- STAT2 U6A cells were stimulated or not with IFN-α (1000 U/ml) for 24 or 60 h and levels of ISG15 and OAS1 as well as pY-STAT2, pY-STAT1, STAT1, STAT2 and

GAPDH as indicated, were assessed by western blot analysis. Journal of Cell Science

4196 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4190-4199 doi:10.1242/jcs.185421 site (S734) on STAT2 that resides in the TAD, but that is away from the crucial region previously mapped for transactivation. This new site appears to specifically regulate the antiviral activity of type I IFNs, without disturbing the anti-proliferative activity. We consistently show that S734-STAT2 is phosphorylated in response to IFN-α stimulation in several cell lines, with kinetics that are distinctly slower that those observed for STAT2 tyrosine phosphorylation. We found JAK1 activity and STAT2 tyrosine phosphorylation to be necessary for pS734-STAT2 induction. Furthermore, we present evidence that IFN- α-induced pS734-STAT2 translocates to the nucleus. Although the serine kinase responsible for phosphorylating S734 is yet unknown, with the use of prediction tools, we have identified two candidate serine kinases that we are currently evaluating for their role in pS734- STAT2 phosphorylation. The function of this newly discovered post-translational modification appears to differ from the S287 site we recently identified on STAT2. In response to IFN-α stimulation, disabling phosphorylation of S287- STAT2 has two consequences. First, it augments the growth inhibitory effects of type I IFN and, second, it protects cells from viral infection resulting in prolonged cell survival due to reduced virus replication (Steen et al., 2013), two features not entirely shared with S734-STAT2. Although additional studies are underway to determine the Fig. 8. Proposed model of S734-STAT2 phosphorylation in regulating the combinatory effect of STAT2 dually phosphorylated on serine antiviral activity of type I IFNs. Upon binding of IFN-α and IFN-β to its residues 287 and 734 in type I IFN signaling, we propose that S734- receptor, JAK1 and TYK2 become activated and tyrosine phosphorylate STAT2 phosphorylation has a specific contributory role in negatively STAT1 (pY701) and STAT2 (pY690). Activated STAT1 and STAT2, together with IRF9, as the ISGF3 complex, trigger the induction of antiviral ISGs. regulating the antiviral effects of type I IFN. Subsequently, tyrosine-phosphorylated STAT2 (pY690) is phosphorylated at How might phosphorylation of S734-STAT2 negatively regulate S734 (pS734) in a JAK-dependent manner, and this in turn, restricts the type I IFN signaling? Early experiments using U6A cells induction of ISGs with antiviral activity. reconstituted with C-terminally truncated versions of STAT2 showed that deletions up to amino acid 831 did not affect type I interaction with other proteins. In such a scenario, the IFN signaling and ISG induction (Qureshi et al., 1996). However, phosphorylated serine residue would increase the negative charge removing residues to amino acid 812 led to a partial reduction in on the acidic side, speculatively disrupting relevant STAT2–co- STAT2 function, and truncating STAT2 to residue 800 completely factor interactions at certain ISG loci and resulting in a dampened disrupted gene induction. The interaction between the STAT2-TAD transcriptional response. Because this particular post-translational and p300/CBP has been recently mapped by NMR and confined to modification seems to only affect the level of induction of a select amino acids 788–816 of STAT2, with some stabilizing, but non- subset of ISGs, we hypothesize that the proteins interacting with crucial interactions between residues 817 and 833 (Wojciak et al., STAT2 at the loci of these ISGs are what is providing selectivity that 2009). Owing to the deleterious effects of C-terminal truncations is not seen with other ISGs. after removing the first 50 residues, the importance of the Based on these observations together with our recent report on N-terminal part of the TAD (residues ∼700–800) has not been S287 phosphorylation of STAT2, we propose a preliminary model carefully studied. Compared to other STATs, STAT2 has a very (Fig. 8) in which dynamic S734 phosphorylation regulates the large TAD that includes the region C-terminal of Y690. Adding to antiviral activities of type I IFNs. Overall, this discovery highlights that, STAT2 is different from all other STATs in that it does not striking differences in the role of serine phosphorylation of stably interact with DNA (Bluyssen and Levy, 1997). In type I IFN STAT2 compared to other STATs, including STAT1, wherein signaling, this task is performed by STAT1 and IRF9, which bind phosphorylation on serine enhances, rather than antagonizes, the DNA directly while STAT2 contributes its transactivation function transcriptional response to type I IFNs. to the ISGF3 complex. Therefore, we can postulate that the first part MATERIALS AND METHODS of the STAT2-TAD (from residues ∼700–760) is required to bring – Cell culture the remaining 90 residues (761 851) into a proper conformation for STAT2-deficient human U6A fibrosarcoma cells (obtained from Dr Ana binding to co-activators such as p300/CBP, GCN5 and, most Costa-Pereira, Imperial College London, London, UK), STAT2-null mouse recently, RVB1 and RVB2 proteins (Gnatovskiy et al., 2013). embryonic fibroblasts (provided by Dr Chris Schindler, Columbia The S734-STAT2 location, ‘PELSLDLEP’, consists of several University, New York, NY), human colorectal carcinoma cells F6-8 and leucine residues (hydrophobic) and acidic residues flanked by two T29 (obtained from Dr Bert Vogelstein, Johns Hopkins University, proline residues. Interestingly, S734 is found within the mapped Baltimore, MD), and 293FT cells were cultured in Dulbecco’s modified nuclear export signal of STAT2 (Frahm et al., 2006). As a serine Eagle’s medium (DMEM; Cellgro Mediatech, Inc., Manassas, VA) phosphorylation motif, this was unknown, but remarkably very supplemented with 10% fetal calf serum (Gemini Bio-Products), 100 units/ml penicillin (Cellgro), 100 μgml−1 streptomycin (Cellgro), and similar to one present just a few residues upstream in a stretch that 1× GlutaMAX (Invitrogen, Waltham, MA). Human fibrosarcoma 2fTGH starts at amino acid 716 (PELESLELELG). It is worth mentioning cells and epithelial mammary cells hTERT-HME1 (kindly provided by Dr that WT-STAT2 and S734A-STAT2 showed identical kinetics in George Stark, Cleveland Clinic Foundation, Cleveland, OH) and 293FT their subcellular localization before and after IFN-β treatment (data cells purchased from Life Technologies (Grand Island, NY) were grown in not shown). The nature of the STAT2 TAD, which contains both an complete DMEM (Cellgro Mediatech). hTERT-HME1 cells required acidic side and an opposite hydrophobic side, might facilitate its DMEM further supplemented with mammary epithelium growth medium Journal of Cell Science

4197 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4190-4199 doi:10.1242/jcs.185421 containing bovine pituitary extract, hydrocortisone, insulin, epithelial SDS-PAGE. A protein band corresponding to the size of STAT2 (113 kDa) growth factor, and gentamicin and amphotericin-B (Clonetics, Basel, was visualized with Coomassie Blue stain and excised for further analysis. Switzerland). All cells were grown at 37°C in a humidified atmosphere Extracted proteins were treated with Proteinase K prior to mass spectrometry containing 5% CO2, authenticated and checked for contamination. analysis performed by ITSI Biosciences (Johnstown, PA) using an LTQ XL mass spectrometer (Thermo Scientific). Resulting mass spectra was searched Reagents and antibodies against a custom STAT2 human database from UniProt using Proteome Recombinant human IFN-α-2a (specific activity 2×107 units ml−1)was Discoverer (version 1.3, Thermo Scientific) and the SEAQUEST search purchased from PeproTech, Inc. (Rock Hill, NJ). Recombinant murine IFN- engine. Proteins were identified when two or more unique peptides had X- β was a kind gift from Biogen, Inc. (Cambridge, MA). JAK inhibitor was correlation scores greater than 1.5, 2.0 and 2.5 for respective peptide charge obtained from EMD Millipore (Bedford, MA). Rabbit anti-STAT2 (C-20, states of +1, +2, and +3, together with a delta correlation value of ≥0.1. cat. no. sc-476, 1:1000), rabbit anti-STAT1 (E-23, cat. no. sc-346, 1:1000) Carbamidomethyl was used as a static modification. Phosphorylated serine, and rabbit anti-Lamin B1 (C-5, cat. no. sc-365962, 1:1000) were purchased threonine, and tyrosine along with acetylated residues were variable from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-phospho- modifications. Peptide tolerance was set to ±5000 ppm and mass tolerance Y689-STAT2 (cat. no. 07-224, 1:1000) was obtained from Millipore, mouse was ±2 Da. anti-pY701-STAT1 (cat. no. 612233, 1:1000) was purchased from BD Biosciences, mouse anti-GAPDH (cat. no. 60004-1, 1:5000) and mouse MTS cell proliferation assay anti-actin (cat. no. 60008-1, 1:5000) antibodies were purchased from U6A cells seeded in flat-bottomed 96-well plates at a density of 500 cells per Proteintech Group, Inc. (Chicago, IL). Affinity purified rabbit polyclonal well were treated with or without IFN-α for 72 h at 37°C. Cell growth was anti-STAT2-pS734 was raised against the phospho-specific peptide determined by using CellTiter 96® AQueous One solution reagent VPEPEL(pS)LDLEPL; amino acids 728–740 in collaboration with (Promega, Madison, WI) according to the manufacturer’s protocol. Rockland Immunochemicals, Inc. (Gilbertsville, PA). Absorbance was measured at 490 nm using a VICTOR™X5 Multilabel Plate Reader (PerkinElmer Life Sciences, Waltham, MA). Data are shown as Plasmids, site-directed mutagenesis and transduction a percentage of the value of control (untreated cells). C-terminally Flag-tagged human STAT2 cDNA vector was obtained from Dr Curt Horvath (Northwestern University, Evanston, IL). Site-directed Virus infection mutagenesis was performed using the QuikChange Lightning (Agilent Cells seeded in 12-well plates were left untreated or treated with increasing Technologies, Santa Clara, CA) to construct R409A/K415A-(RKAA), doses of IFN-α for 16 h prior to infection with vesicular stomatitis virus Y690F- and S734A-STAT2 mutants. Mutant and wild-type STAT2 (VSV) with a GFP-expressing gene (Fernandez et al., 2002). Plaque- constructs were subcloned into the lentiviral expression vectors purified VSV (Indiana strain) was added to cells at a multiplicity of infection pCDH-CMV-MCS-EF1-Green and pCDH-CMV-MCS-EF1-RFP (System of 0.01 for U6A cells and 0.002 for Stat2−/− MEFs under serum-free Biosciences, Mountain View, CA). Lentiviruses were produced in 293FT medium conditions for 1 h at 37°C. Cells were washed twice with PBS cells using ViraPower Lentiviral Expression System (Life Technologies, followed by re-addition of complete DMEM. Progeny virion titers were Carlsbad, CA). U6A cells and Stat2−/− MEFs were transduced with quantified in culture supernatants by standard plaque assay on baby hamster lentivirus in the presence of 1 μgml−1 Polybrene (Sigma, St. Louis, MO) kidney (BHK-21) cells or by flow cytometry. and subjected to antibiotic selection by adding 2 μgml−1 puromycin to the growth medium at 72 h post infection. Quantitative real-time PCR Total RNA was extracted from cells using RNA Bee solution (Amsbio LLC, Western blotting, nuclear extract preparation and Lake Forest, CA). Contaminating DNA was removed (DNA-free Kit, Ambion) immunoprecipitation and cDNA was synthesized from RNA using the High Capacity cDNA Reverse Cells were lysed in Triton X-100 lysis buffer (50 mM Tris-HCl pH 7.5, Transcription Kit (Applied Biosystems, Carlsbad, CA). cDNA was combined 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100) supplemented with 1× with gene-specific FAM dye-labeled TaqMan MGB probes (Life Technologies) protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), 1 mM and 2× TaqMan Master Mix. Real-time analysis was performed using a 7300 sodium orthovanadate, 1 mM PMSF and 1× phosphatase inhibitor mixture 2 real-time PCR system (Applied Biosystems). Actin cDNA was used to and 3 (Sigma), for 20 min on ice. After centrifugation for 15 min at 4°C and normalize cDNA loading. Each sample was analyzed in triplicates and 15,000 g, supernatants were collected and protein concentration was normalized against actin. The ΔΔCt method was used to determine differences in determined with the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). gene transcription between WT-STAT2 and S734A-STAT2. Nuclear extracts were prepared as described previously (Steen et al., 2013). Immunoprecipitation assays were performed by first pre-clearing protein lysates Microarray gene analysis by incubation with protein-G–Sepharose (GE Healthcare, Little Chalfont, UK). STAT2-expressing U6A cells were seeded in triplicate and treated with or Pre-cleared lysates were then incubated with the indicated antibody overnight at without 1000 U ml−1 IFN-α for 4 h. Total RNA was isolated using RNA Bee 4°C and immunocomplexes collected by further incubation with protein-G– solution and further purified by RNeasy kit (Qiagen, Valencia, CA) according Sepharose for 2 h. Dephosphorylation of STAT2 was performed by adding to the manufacturer’s instructions. The quality of total RNA was assessed 400 U of lambda protein phosphatase (LPP; New England Biolabs, Ipswich, using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). MA) followed by incubation at 37°C for 30 min with constant agitation. RNA samples were labeled and hybridized to the Affymetrix Human Gene Lysates and immunoprecipitates were resolved on NuPAGE 4–12% Bis-Tris 2.0 ST Array (Affymetrix, Santa Clara, CA). For each condition, three gels (Life Technologies) and transferred to PVDF membranes (Millipore, biological replicate samples were used for microarray experiments. Scanned Temecula, CA). Membranes were blocked with Blocker Casein TBS (Thermo microarray images were analyzed using the Affymetrix Gene Expression Scientific, Rockford, IL) and incubated with the corresponding primary and Console with RMA (Robust Multi-array Average) normalization algorithm. horseradish peroxidase (HRP)-conjugated secondary antibodies in Tris- Further statistical analyses were performed using BRB-ArrayTools (Simon buffered saline with 0.2% Tween 20 (TBS-T) plus 3% bovine serum et al., 2007). Gene classification into ontology categories (GO) was albumin. Membranes were developed by chemiluminescence using performed using BLAST2GO® version 2.6.4 (Biobam Bioinformatics, SuperSignal West Pico or Femto (Thermo Scientific). Images were captured Valencia, Spain). The complete data files are available at NCBI, GEO with the Alpha-Innotech HD2 imaging system (ProteinSimple, San Jose, CA). accession number GSE57017.

Mass spectrometry Statistical analysis STAT2-reconstituted U6A cells were left untreated or treated with Prism software (GraphPad, San Diego, CA) was used for statistical analysis. 1000 U ml−1 IFN-α for 30 min and proteins were extracted with Triton Student’s t-test was applied to discern significant statistical differences

X-100 lysis buffer. STAT2 was immunoprecipitated and separated by between samples. P≤0.05 was considered significant. Journal of Cell Science

4198 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4190-4199 doi:10.1242/jcs.185421

Acknowledgements experimentally determined post-translational modifications in man and mouse. We thank Mr Michael Slifker for the analysis of the microarray data. Analyses were Nucleic Acids Res. 40, D261-D270. performed using the BRB-ArrayTools developed by Dr Richard Simon and BRB- Improta, T., Schindler, C., Horvath, C. M., Kerr, I. M., Stark, G. R. and Darnell, ArrayTools Development Team. J. E., Jr. (1994). ISGF-3 formation requires phosphorylated Stat91 protein, but Stat113 protein is phosphorylated independently of Stat91 Competing interests protein. Proc. Natl. Acad. Sci. USA 91, 4776-4780. Kraus, T. A., Lau, J. F., Parisien, J.-P. and Horvath, C. M. (2003). A hybrid IRF9- The authors declare no competing or financial interests. STAT2 protein recapitulates interferon-stimulated gene expression and antiviral response. J. Biol. Chem. 278, 13033-13038. Author contributions Leung, S., Qureshi, S. A., Kerr, I. M., Darnell, J. E., Jr and Stark, G. R. (1995). A.M.G. prepared the manuscript and designed experiments; H.C.S. and K.P.K. Role of STAT2 in the alpha interferon signaling pathway. Mol. Cell. Biol. 15, conducted and designed experiments, and helped write the manuscript; S.N. and 1312-1317. M.Y.H. conducted some of the experiments, and S.B. helped with manuscript Li, X., Leung, S., Qureshi, S., Darnell, J. E., Jr and Stark, G. R. (1996). Formation preparation and experimental design. of STAT1-STAT2 heterodimers and their role in the activation of IRF-1 gene transcription by interferon-α. J. Biol. Chem. 271, 5790-5794. Funding Li, X., Leung, S., Ker, I. M. and Stark, G. R. (1997a). Functional subdomains of This project was supported by grants to A.M.G. from the National Cancer Institute STAT2 required for preassociation with the alpha interferon receptor and for (RO1CA140499) and the Pennsylvania Department of Health. Deposited in PMC for signaling. Mol. Cell. Biol. 17, 2048-2056. release after 12 months. Li, X., Leung, S., Kerr, I. M. and Stark, G. R. (1997b). Functional subdomains of STAT2 required for preassociation with the alpha interferon receptor and for Data availability signaling. Mol. Cell. Biol. 17, 2048-2056. Melen, K., Kinnunen, L. and Julkunen, I. (2001). Arginine/lysine-rich structural The microarray data reported in this study are available from the Gene Expression element is involved in interferon-induced nuclear import of STATs. J. Biol. Chem. Omnibus database under accession number GSE57017 (https://www.ncbi.nlm.nih. 276, 16447-16455. gov/geo/query/acc.cgi?acc=GSE57017). Müller, M., Briscoe, J., Laxton, C., Guschin, D., Ziemiecki, A., Silvennoinen, O., Harpur, A. G., Barbieri, G., Withuhn, B. A., Schindler, C. et al. (1993). The Supplementary information protein tyrosine kinase JAK1 complements defects in the interferon-α/β and -γ Supplementary information available online at signal transduction. Nature 366, 129-135. http://jcs.biologists.org/lookup/doi/10.1242/jcs.185421.supplemental Park, C., Lecomte, M.-J. and Schindler, C. (1999). Murine Stat2 is uncharacteristically divergent. Nucleic Acids Res. 27, 4191-4199. References Paulson, M., Pisharody, S., Pan, L., Gaudagno, S., Mui, A. L. and Levy, D. E. Barbieri, G., Velazquez, L., Scrobogna, M., Fellous, M. and Pellegrini, S. (1994). (1999). Stat protein transactivation domains recruit p300/CBP through widely Activation of the protein tyrosine kinase tyk2 by interferon alpha/beta. divergent sequences. J. Biol. Chem. 274, 25343-25349. Eur. J. Biochem. 223, 427-435. Paulson, M., Press, C., Smith, E., Tanese, N. and Levy, D. E. (2002). IFN- Bhattacharya, S., Eckner, R., Grossman, S., Oldread, E., Arany, Z., D’Andrea, A. Stimulated transcription through a TBP-free acetyltransferase complex escapes and Livingston, D. M. (1996). Cooperation of Stat2 and p300/CBP in signalling viral shutoff. Nat. 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