SOCS3 binds specific receptor–JAK complexes to control cytokine signaling by direct kinase inhibition

Journal name:
Nature Structural & Molecular Biology
Volume:
20,
Pages:
469–476
Year published:
DOI:
doi:10.1038/nsmb.2519
Received
Accepted
Published online

Abstract

The inhibitory protein SOCS3 plays a key part in the immune and hematopoietic systems by regulating signaling induced by specific cytokines. SOCS3 functions by inhibiting the catalytic activity of Janus kinases (JAKs) that initiate signaling within the cell. We determined the crystal structure of a ternary complex between mouse SOCS3, JAK2 (kinase domain) and a fragment of the interleukin-6 receptor β-chain. The structure shows that SOCS3 binds JAK2 and receptor simultaneously, using two opposing surfaces. While the phosphotyrosine-binding groove on the SOCS3 SH2 domain is occupied by receptor, JAK2 binds in a phosphoindependent manner to a noncanonical surface. The kinase-inhibitory region of SOCS3 occludes the substrate-binding groove on JAK2, and biochemical studies show that it blocks substrate association. These studies reveal that SOCS3 targets specific JAK–cytokine receptor pairs and explains the mechanism and specificity of SOCS action.

At a glance

Figures

  1. The structure of a JAK–SOCS–gp130 complex and the two interfaces.
    Figure 1: The structure of a JAKSOCSgp130 complex and the two interfaces.

    (a) Schematic of SOCS3 inhibiting JAK2 while bound to gp130 (shared receptor for IL-6, LIF, etc.). JAK2 consists of four distinct domains, FERM, SH2, JH2 and JH1. The JH1 domain is the C-terminal catalytic domain and interacts with SOCS3. The boxed region indicates the ternary complex structure solved in this work. (b) Ribbon diagram of the JAK2 (beige)–SOCS3 (green)–gp130 (black) complex. The gp130 peptide is located in the canonical phosphotyrosine-binding groove on the SH2 domain of SOCS3, while the opposing face of the SH2 domain contacts JAK2. (c) Diagram of SOCS3 binding to gp130 through the canonical phosphotyrosine-binding groove on the SH2 domain with the BC loop (Ser73–Phe79) of SOCS3 contacting both JAK2 and gp130 through opposing faces. The coordination of the phosphate moiety from gp130 pTyr757 is identical to that seen in the absence of JAK2. (d) Diagram of the short KIR motif of SOCS3 sitting in a groove bordered by the activation loop and GQM motif (yellow) of JAK2. (e) The molecular envelope of the JAK2SOCS3gp130 complex in solution calculated from SAXS data by performing ten independent DAMMIF ab initio bead reconstructions (gray spheres) superimposed with the complex crystal structure (in b). Top, same orientation as in b; bottom, top view. Data collection statistics and validation are shown in Supplementary Table 1.

  2. The SOCS3-JAK2 interaction.
    Figure 2: The SOCS3-JAK2 interaction.

    (a) The hydrophobic SOCS3-JAK2 interface. Important residues are labeled and a selection of van der Waals contacts shown as dotted lines. Color scheme as in Figure 1. (b) Diagram showing residues from the GQM motif and αG28 helix of JAK2 binding a concave hydrophobic surface on SOCS3 formed by the KIR, BC loop and ESS. JAK2 is shown in ribbon representation and SOCS3 as an electrostatic surface (±250 mV). The GQM motif and Phe1076 from αG of JAK2 are highlighted. (c) Comparison of the SOCS3 structure in isolation (PDB: 2HMH26, white) and in complex with JAK2 (this work, green). The ESS helix can be seen to undergo a translation of half a helical turn upon binding JAK2 while the KIR (which is unstructured in the absence of JAK2) adopts an extended structure. The orientation of SOCS3 is the same as in b. (d) Sequence conservation of the JAK2-binding interaction surface between SOCS1 and SOCS3. Conserved residues are shown in red.

  3. The JAK2-binding site is composed of the KIR, ESS and SH2 domain and is conserved between SOCS3 and SOCS1.
    Figure 3: The JAK2-binding site is composed of the KIR, ESS and SH2 domain and is conserved between SOCS3 and SOCS1.

    (a) Cut-away view of the JAK2-binding site on SOCS3. SOCS3 is shown as a ribbon diagram and JAK2 as an electrostatic surface (±250 mV). The SOCS3 KIR (Leu22–Ser29), which is unstructured in isolation, folds back underneath the BC loop and sits in a groove formed by the JAK2 activation loop and GQM motif (labeled). SOCS3 Phe25 sits in a deep hydrophobic pocket formed by residues from both SOCS3 and JAK2. The interface is mostly hydrophobic. (b) IC50 plots showing inhibition of JAK2 by SOCS3 point mutants. These assays highlight SOCS3 Phe25, Phe79 and Phe80 as being required for inhibition. The curves shown are an average of duplicate experiments. WT, wild type. (c) Inhibition of JAK2 by isolated KIR peptides from SOCS1. IC50, 0.1 mM. Error bars, s.d. (n = 3). (d) SOCS3-JAK2 interface highlights the conservation of residues in the JAK2-binding site. Yellow, conserved residues between SOCS3 and SOCS1. (e) Close-up of the interface in d. Color scheme as in d.

  4. The KIR is required for JAK binding.
    Figure 4: The KIR is required for JAK binding.

    (a) Co-precipitation experiments show that SOCS3 binds JAKJH1, provided that the kinase inhibitory region is intact. Lanes 2–6 show gradual loss of binding as the N-terminal residues of the SOCS3 KIR are removed, as well as when Phe25 is mutated to alanine (lane 7). The SOCS1-3 chimera is shown as a positive control. M, marker (kDa). (b) Co-precipitation experiments showing that SOCS3 binds activated (pTyr1007 or pTyr1007 and 1008) and dephosphorylated (Tyr1007 and 1008) JAK2JH1 with similar affinity. Dephosphorylated JAK2JH1 was coexpressed with the phosphatase PTP1B and used in co-precipitation experiments with SOCS3 as described in a. Western blot of this experiment probed with a pTyr1007/8-specific antibody shows that JAK2 co-expressed with PTB1B was >95% dephosphorylated. (c) Co-precipitation experiments showing that SOCS3 does not bind JAK2JH1 if the JAK2 GQM motif is mutated (G1071D). SOCS3F25A is shown as a negative control.

  5. SOCS3 inhibits JAK2 by blocking substrate binding.
    Figure 5: SOCS3 inhibits JAK2 by blocking substrate binding.

    (a) Model of a substrate peptide (white) bound to JAK2, based on the IRK–substrate–ATP structure (PDB: 1IR3), shows that the KIR of SOCS3 would block substrate binding. Leu22 is located where the P+1 residue would reside (both are shown in stick representation). (b) Schematic of the predicted overlap with substrate binding for the constructs and substrates used in these experiments. (c) Kinase-inhibition assays performed with constructs of SOCS3 truncated at the N-terminal end of the KIR, showing that constructs lacking one to three residues only partially inhibit JAK2 activity. Kinase-inhibition experiments using the standard STAT5b peptide (left) or a C-terminally truncated (Y+1) (right) as a substrate. Inhibition of STAT5bY+1 by SOCS3ΔN22–23 is only 50% complete under saturating conditions.

  6. Residues upstream of the SOCS3 KIR act as a pseudosubstrate.
    Figure 6: Residues upstream of the SOCS3 KIR act as a pseudosubstrate.

    (a) Radioactive kinase assays showing that constructs of SOCS3 with a tyrosine one to three residues upstream of the KIR are good substrates for JAK2. SDS-PAGE followed by autoradiography for reactions of 1 min (top) or 2 min (bottom) are shown. Experiments performed in the absence of SOCS3 are shown as a control. (b) As in a but with SOCS3 F25A mutants as controls to show that tyrosines upstream of the KIR are only good substrates for JAK2 when forced into proximity of the active site by the remainder of SOCS3. (c) Steady-state inhibition of JAK1 by full-length SOCS3. IC50 for full-length SOCS3 is identical to that of SOCS3 lacking the first 19 or 21 residues. (d) Steady-state inhibition of JAK2 by full-length SOCS3 gives noncompetitive inhibition as analyzed by Michaelis-Menten (top) and Dixon (bottom) plots. v, velocity. The averages of duplicate experiments are shown in each case.

  7. Comparison of three kinase inhibitors: SOCS3, PAK1IS and Grb14.
    Figure 7: Comparison of three kinase inhibitors: SOCS3, PAK1IS and Grb14.

    (a) SOCS3 binds a surface on the JAK2 kinase domain (shaded green) similar to that used by Grb14 on the insulin receptor kinase (magenta) and by the autoregulatory inhibitory-switch (IS) region of PAK1 (blue). (b) SOCS3 and Grb14 are tethered to their target kinase by a different surface. The SH2 domain of Grb14 binds the activation loop of IRK by using the canonical phosphotyrosine-binding groove. In contrast, SOCS3 binds JAK2 by using the opposite face of its SH2 domain, which leaves the canonical phosphotyrosine-binding groove available for binding receptor (black). (c) SOCS3, like Grb14, inhibits its target kinase by blocking substrate binding. The BPS region of Grb14 and the KIR of SOCS3 occupy the substrate-binding groove of their target kinases. Leu376 of Grb14 acts as the pseudosubstrate residue (asterisk).

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

NCBI Reference Sequence

Protein Data Bank

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Author information

Affiliations

  1. Department of Structural Biology, Walter and Eliza Hall Institute, Parkville, Australia.

    • Nadia J Kershaw,
    • Nicholas P D Liau,
    • Artem Laktyushin,
    • Eden L Whitlock &
    • Jeffrey J Babon
  2. Department of Cancer and Haematology, Walter and Eliza Hall Institute, Parkville, Australia.

    • James M Murphy,
    • Nicholas P D Liau,
    • Leila N Varghese,
    • Artem Laktyushin,
    • Eden L Whitlock,
    • Nicos A Nicola &
    • Jeffrey J Babon
  3. Department of Medical Biology, University of Melbourne, Parkville, Australia.

    • Nadia J Kershaw,
    • James M Murphy,
    • Nicholas P D Liau,
    • Leila N Varghese,
    • Nicos A Nicola &
    • Jeffrey J Babon
  4. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.

    • Isabelle S Lucet

Contributions

N.J.K. carried out crystallographic data collection, structure determination and refinement. J.J.B. and N.P.D.L. performed inhibition and co-precipitation assays. J.M.M. and L.N.V. carried out SAXS data collection and analysis. J.M.M., A.L., I.S.L. and E.L.W. performed protein expression and purification experiments. All authors commented on the manuscript . J.J.B., N.J.K. and N.A.N. designed experiments, analyzed data, supervised the project and wrote the paper.

Competing financial interests

N.A.N. is a founder and member of the scientific advisory board of MuriGen Pty Ltd.

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    Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Note

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