Structural basis for c-KIT inhibition by the suppressor of cytokine signaling 6 (SOCS6) ubiquitin ligase

Abstract

The c-KIT receptor tyrosine kinase mediates the cellular response to stem cell factor (SCF). Whereas c-KIT activity is important for the proliferation of hematopoietic cells, melanocytes and germ cells, uncontrolled c-KIT activity contributes to the growth of diverse human tumors. Suppressor of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its interaction with c-KIT pY568. The 1.45-Å crystal structure of SOCS6 SH2 domain bound to the c-KIT substrate peptide (c-KIT residues 564-574) revealed a highly complementary and specific interface giving rise to a high affinity interaction (K(d) = 0.3 μm). Interestingly, the SH2 binding pocket extends to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6 as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases.

FIGURE 1.

Regulation of SOCS6 turnover by its SH2 and SOCS box domains. A, HEK293T cells were transfected with a FLAG-tagged SOCS6 expression plasmid coding for WT or SOCS6 variants with point mutations in the SH2 domain (R409E) or the SOCS box (C504F) motif. In some cases SOCS6 was co-transfected with plasmids coding for elongin C and elongin B as indicated. B, CHX chase experiments were performed to analyze the effects of elongins B and C in SOCS6 stability. Expression levels of SOCS6 were analyzed by Western blot.

FIGURE 2.

SOCS6 interacts with c-KIT to regulate its ubiquitination and turnover. A, Flag-tagged SOCS6 and the R409E variants were expressed in HEK293T cells and immunoprecipitated using an anti-Flag antibody. The association with c-KIT, elongin C, elongin B, and cullin5 was analyzed by Western blot. Endogenous c-KIT was also immunoprecipitated and its tyrosine phosphorylation analyzed by Western blot. The expression levels of c-KIT and SOCS6 variants were analyzed in whole cell lysates (WCL) by Western blot. B, in vitro demonstration that SOCS6 is part of an ubiquitin ligase complex. HEK293T cells were transfected with a control, Flag-SOCS6 or the R409E variant expressing vector together with elongin B/C. Middle panel, an ubiquitination reaction was carried out in vitro in the presence of immunoprecipitated SOCS6 and exogenously added HA-ubiquitin, ATP, E1, and E2 as indicated. Protein samples were run on SDS-PAGE and high molecular weight HA-containing protein complexes were visualized by Western blot. Transfection efficiency was monitored by analysis of whole cell lysates (WCL). At the end of the ubiquitination reaction c-KIT was immunoprecipitated, and its ubiquitination status analyzed by a Western blot against HA-ubiquitin. The results are shown in the upper panel. C, amount of ubiquitinated c-KIT was analyzed in cells co-transfected with expression vectors for SOCS6 and HA-ubiquitin. D, CHX chase experiments to assess c-KIT turnover in cells overexpressing Flag-tagged SOCS6.

FIGURE 3.

Overall structure and affinity of the SOCS6/c-KIT peptide complex. A, ITC measurement of the c-KIT pY568 peptide binding to the SOCS6 SH2 domain. SOCS6 binds with K_d = 0.3 μm (K_b = 32.3 ± 4.2 × 10⁵ m⁻¹, ΔH^obs = −2.29 ± 0.03 kcal/mol, TΔS = 6.45 kcal/mol, ΔG = −8.74 kcal/mol, n = 1.02). A blank titration is colored blue and offset for clarity. B, ribbon diagram of the structure of the SOCS6/c-KIT complex. The ESS helix and SH2 domain are colored blue and orange, respectively. Part of the C-terminal linker to the SOCS box is colored red. Selected secondary structure elements are labeled. The c-KIT peptide is shown in stick representation and colored green. C, same view showing a surface representation of SOCS6 colored by electrostatic surface potential between −10 and +10 kcal/electron units.

FIGURE 4.

Specific interactions of the c-KIT phosphopeptide. A, c-KIT peptide (green) binds SOCS6 (orange) in an extended conformation. Electron density (2Fc-2Fo map) for the peptide is shown as a blue mesh contoured at 2σ. B, hydrogen bond interactions with c-KIT pY568 and Asn-567 (pY-1) are shown by a line of blue spheres. Asn-566 is not shown for clarity. C, hydrophobic and hydrogen bond interactions are shown with c-KIT Val-569, Tyr-570, and Ile-571 (pY+1 to +3). Two bound water molecules are shown as red spheres. D, view is changed by 180° to show the interactions of the C-terminal peptide residues Asp-572, Pro-573, and Thr-574 (pY+4 to +6).

FIGURE 5.

Schematic overview of the hydrogen bonding in the SOCS6/c-KIT complex. SOCS6 and c-KIT side chains are shown as sticks colored in gray and black, respectively. Residues in c-KIT are labeled in blue. Residue labels for SOCS6 are boxed and shaded blue for the EF and BG loop residues, orange for helices, and yellow for other residues in the SH2 β-structure. Water is shown as a sphere labeled W. Hydrogen bond interactions are shown by a dashed red line. Key hydrophobic interactions with the pY+1 to +3 positions are shown by a solid red line.

FIGURE 6.

Comparison with the SOCS3 and SHP2 SH2 domains. A, overlay of the crystal structures of the SOCS3 (blue, PDB 2HMH) and SOCS6 (orange) SH2 domains with the c-KIT peptide bound to SOCS6 shown in green. The contrasting structures of the EF and BG loops are labeled. B, overlay of the SHP2 N-terminal SH2 domain (blue, PDB 1AYA) and the SOCS6/c-KIT complex (orange/green) showing the similar loop arrangement. C, similar backbone conformations are observed for ligands of SHP2 (yellow, PDGFR pYTAVQP, PDB 1AYA) and SOCS6 (green, c-KIT pYVYIDP) between the pY and pY+5 positions. D, binding of GST-SOCS6-SH2 or GST to a SPOT peptide array representing phosphotyrosine sites in c-KIT and PDGFR.

FIGURE 7.

Domain organization of the SOCS C terminus. A, overlay of SOCS2 (PDB 2C9W), SOCS3 (PDB 2HMH), SOCS4 (PDB 2IZV), and SOCS6 (PDB 2VIF) showing the conserved packing of the SH2 C-terminal βG strand. Residues from a conserved LXXP(L/V) motif are shown in stick representation and colored as indicated. The structures C-terminal to this motif are not shown for clarity. Residue numbers are given for SOCS6 positions. B, overlay of the SOCS4 (green) and SOCS6 (orange) SH2 domain structures. The N-terminal ESS helix and C-terminal SOCS box (H1 helix) are additionally colored blue and red, respectively, in SOCS4. The ESS helix extends an additional turn before the SH2 in SOCS6. A conserved arginine mediating interactions between the N- and C-terminal regions is shown and labeled in SOCS6. C, structure-based sequence alignment of the SOCS SH2 C-terminal region. Secondary structure elements are labeled for SOCS6 and indicated across the alignment by orange (SH2) or red (SOCS box) box shading. A PEST insertion is present in CISH and SOCS3. The conserved LXXP(L/V) motif shown in A is shown in bold. The linker between βG and H1 is variable in length and longest in SOCS6. The αB helix is also unusually large in SOCS6.