View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Immunity Article Suppression of Cytokine Signaling by SOCS3: Characterization of the Mode of Inhibition and the Basis of Its Specificity Jeffrey J. Babon,1,2,5,* Nadia J. Kershaw,1,3,5 James M. Murphy,1,2,5 Leila N. Varghese,1,2 Artem Laktyushin,1 Samuel N. Young,1 Isabelle S. Lucet,4 Raymond S. Norton,1,2,6 and Nicos A. Nicola1,2,* 1Walter and Eliza Hall Institute of Medical Research, 1G Royal Pde, Parkville, 3052, VIC, Australia 2The University of Melbourne, Royal Parade, Parkville, 3050, VIC, Australia 3Ludwig Institute for Cancer Research, Royal Pde, Parkville, 3050, VIC, Australia 4Monash University, Wellington Rd, Clayton, 3800, VIC, Australia 5These authors contributed equally to this work 6Present address: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Australia *Correspondence: [email protected] (J.J.B.), [email protected] (N.A.N.) DOI 10.1016/j.immuni.2011.12.015 SUMMARY Genetic deletion of each individual JAK leads to various immunological and hematopoietic defects; however, aberrant Janus kinases (JAKs) are key effectors in controlling activation of JAKs can be likewise pathological. Three myelopro- immune responses and maintaining hematopoiesis. liferative disorders (polycythemia vera, essential thrombocythe- SOCS3 (suppressor of cytokine signaling-3) is a mia, and primary myelofibrosis) are caused by a single point major regulator of JAK signaling and here we investi- mutation in JAK2 (JAK2V617F)(James et al., 2005; Levine et al., gate the molecular basis of its mechanism of action. 2005) that renders the kinase constitutively active and results We found that SOCS3 bound and directly inhibited in cytokine-independent activation of JAK-based signaling path- ways. An even more severe phenotype results from activation of the catalytic domains of JAK1, JAK2, and TYK2 JAK by oncogenic fusion, for example TEL-JAK2, which has but not JAK3 via an evolutionarily conserved motif been studied because of its role in childhood T and B cell acute unique to JAKs. Mutation of this motif led to the lymphoblastic leukemia (Lacronique et al., 2000). formation of an active kinase that could not be In order to prevent aberrant proliferation, JAK activity is regu- inhibited by SOCS3. Surprisingly, we found that lated in a number of ways. The primary negative regulators of the SOCS3 simultaneously bound JAK and the cytokine JAKs are a family of proteins known as the suppressors of cyto- receptor to which it is attached, revealing how spec- kine signaling (SOCS) (Endo et al., 1997; Hilton et al., 1998; Naka ificity is generated in SOCS action and explaining et al., 1997; Starr et al., 1997) whose expression is induced by why SOCS3 inhibits only a subset of cytokines. JAK-STAT activation and they then inhibit the signaling cascade, Importantly, SOCS3 inhibited JAKs via a noncompet- creating a negative feedback loop. itive mechanism, making it a template for the devel- All eight SOCS proteins (SOCS1-7 and CIS) contain a central SH2 domain and a C-terminal SOCS box domain (Hilton et al., opment of specific and effective inhibitors to treat 1998), which interacts with elongins B and C and Cullin5 to cata- JAK-based immune and proliferative diseases. lyze the ubiquitination of bound signaling proteins (Babon et al., 2009; Kamizono et al., 2001; Zhang et al., 1999). Elegant studies INTRODUCTION performed by Yoshimura and colleagues (Sasaki et al., 1999; Yasukawa et al., 1999) showed that the two most potent Both hematopoiesis and the immune response are regulated suppressors of signaling, SOCS1 and SOCS3, contain a short by the action of cytokines through activation of the Janus motif, upstream of their SH2 domain, known as the KIR (kinase kinase-signal transducer and activator of transcription-sup- inhibitory region), which allows them to suppress signaling by pressor of cytokine signaling (JAK-STAT-SOCS) signal trans- direct inhibition of JAK catalytic activity. This is their dominant duction pathway (O’Shea and Murray, 2008). mode of action in vivo (Boyle et al., 2007; Zhang et al., 2001). There are four mammalian JAKs (JAK1-3 and TYK2), each Initial characterization of the KIR noted its amino acid sequence consisting of four domains (Figure S1 available online; Wilks similarity to the activation loop of JAKs (Sasaki et al., 1999; and Harpur, 1994). The N-terminal FERM domain binds constitu- Yasukawa et al., 1999). Like most tyrosine kinases, JAKs contain tively to the appropriate membrane-bound receptor whereas the an activation loop that blocks the catalytic cleft. Autophosphor- C-terminal kinase (catalytic) domain phosphorylates substrate ylation of this loop causes its translocation away from the cata- proteins. Between these are a noncanonical SH2 domain and lytic site and allows substrate access, thus activating the kinase. a pseudokinase domain, the most distinctive feature of the Consequently, it was proposed that the SH2 domain of SOCS1 JAK family. This domain has recently been shown to be catalyt- and SOCS3 binds the activation loop tyrosine phosphate and ically active (Ungureanu et al., 2011) and it regulates the activity the KIR acts as a pseudosubstrate to block the active site (Sasaki of the JH1 domain (Saharinen et al., 2000). et al., 1999; Yasukawa et al., 1999). Immunity 36, 239–250, February 24, 2012 ª2012 Elsevier Inc. 239 Immunity Mechanism of Inhibition of Janus Kinase by SOCS3 Despite the ability of SOCS proteins to bind to and inhibit to be a good substrate for JAK2JH1 phosphorylation. To verify JAKs, deletion of individual SOCS genes in mice has revealed that phosphorylation of SOCS3 was not by itself the cause of an exquisite specificity for particular cytokine-receptor combina- decreased gp130cyt phosphorylation, the entire reaction was tions rather than specific JAKs. For example, SOCS1 inhibits spotted onto nitrocellulose membranes, allowing total phos- interferon g signaling without affecting IL-6 signaling whereas phorylation of all components to be quantified. SOCS3 had the converse is true for SOCS3 (Alexander et al., 1999; Croker a clearly titratable inhibitory effect on JAK-catalyzed phosphor- et al., 2003), yet both cytokine receptor systems utilize the ylation with an IC50 of 1 mM(Figure 1C). same JAKs (JAK1 and JAK2) (Murray, 2007). Moreover, the A limiting feature of these assays was that the concentration of binding affinities of the SOCS3 SH2 domain for phosphorylated SOCS3 required to inhibit JAK2JH1 was similar to the concentra- JAK peptides is several logs lower than that for certain cytokine tion of substrate. To ensure that it was not a SOCS3-substrate receptor phosphopeptides, and this binding is important in intact interaction that was responsible for inhibiting the phosphoryla- cells (Nicholson et al., 2000). tion reaction, we adopted a more robust enzyme-inhibition assay In this study we dissect both the mechanism and specificity of format where [Substrate] > > [Inhibitor] > > [Enzyme]. These JAK inhibition by SOCS3 by using biochemical, structural, and assays used high concentrations of a peptide substrate, resi- kinetic methods and resolve these apparent discrepancies. dues 693–708 of STAT5b (Saharinen et al., 2003; Zhao et al., We show that SOCS3 directly inhibits JAK1, JAK2, and TYK2 2010). SOCS3 inhibited phosphorylation of this peptide but not JAK3 because of the presence of a conserved three- substrate with the same IC50 of 1 mM(Figure 1D). These results residue motif on the former three JAK family members. By indicate that SOCS3 functions by blocking the ability of JAK2 to utilizing two distinct binding surfaces, SOCS3 is able to bind to phosphorylate protein substrates and is therefore a direct inhib- JAK and the cytokine receptor to which it is attached simulta- itor of its catalytic activity. neously, explaining why SOCS3 is specific for cytokines that signal through particular receptors. Intriguingly, inhibition occurs A SOCS1-SOCS3 Chimera Is a More Potent Inhibitor via a mechanism in which SOCS3 does not compete with either than SOCS3 ATP or substrate. This makes SOCS3 a noncompetitive tyrosine Replacing the KIR of SOCS3 with the corresponding region from kinase inhibitor and a new template for the future development SOCS1 resulted in a chimeric construct (termed SOCS1-3) that of a class of small-molecule JAK inhibitors with distinct advan- inhibited JAK2 kinase activity with higher affinity than did wild- tages over the current ATP analogs used to treat JAK-based type SOCS3 (IC50 values of 0.15 ± .02 and 1.2 ± 0.1 mM, respec- diseases. tively; see Figure 1D), despite there being a difference of only four residues. A truncated construct of SOCS3, SOCS322-185, RESULTS that lacks the SOCS box and hence elonginB and C binding, was equally effective at inhibiting JAK2. These results show SOCS3 Inhibits Substrate Phosphorylation by the Kinase that the kinase inhibitory activity of SOCS3 relies solely upon Domain of JAK2 its KIR and extended SH2 domain and not its SOCS box domain. To examine SOCS3 inhibition of JAK kinase activity, we devel- In addition, the KIR from SOCS1, but not SOCS4 or SOCS5, can oped an in vitro kinase assay consisting of three purified, act as a functional replacement, making it likely that SOCS1 acts recombinant components: enzyme (JAK2 catalytic domain, in a similar manner to SOCS3. JAK2JH1), substrate (gp130 cytoplasmic domain, gp130cyt), and inhibitor (SOCS3). Various chimeras of SOCS3 were also Both the KIR and SH2 Domain of SOCS3 Are Required for produced that had the key residues in the KIR, L22-S29, Kinase Inhibition but Phosphotyrosine Binding Is Not replaced by the corresponding residues of SOCS1, SOCS4, Previous work had highlighted the importance of F25A within the or SOCS5 (these are designated SOCS1-3, SOCS4-3, and KIR and R71 within the SH2 domain for SOCS3 function (Nichol- SOCS5-3; see Figure S1A).