The SOCS2 ubiquitin ligase complex regulates growth hormone receptor levels

Abstract

Growth Hormone is essential for the regulation of growth and the homeostatic control of intermediary metabolism. GH actions are mediated by the Growth Hormone Receptor; a member of the cytokine receptor super family that signals chiefly through the JAK2/STAT5 pathway. Target tissue responsiveness to GH is under regulatory control to avoid excessive and off-target effects upon GHR activation. The suppressor of cytokine signalling 2 (SOCS) is a key regulator of GHR sensitivity. This is clearly shown in mice where the SOCS2 gene has been inactivated, which show 30-40% increase in body length, a phenotype that is dependent on endogenous GH secretion. SOCS2 is a GH-stimulated, STAT5b-regulated gene that acts in a negative feedback loop to downregulate GHR signalling. Since the biochemical basis for these actions is poorly understood, we studied the molecular function of SOCS2. We demonstrated that SOCS2 is part of a multimeric complex with intrinsic ubiquitin ligase activity. Mutational analysis shows that the interaction with Elongin B/C controls SOCS2 protein turnover and affects its molecular activity. Increased GHR levels were observed in livers from SOCS2⁻/⁻ mice and in the absence of SOCS2 in in vitro experiments. We showed that SOCS2 regulates cellular GHR levels through direct ubiquitination and in a proteasomally dependent manner. We also confirmed the importance of the SOCS-box for the proper function of SOCS2. Finally, we identified two phosphotyrosine residues in the GHR to be responsible for the interaction with SOCS2, but only Y487 to account for the effects of SOCS2. The demonstration that SOCS2 is an ubiquitin ligase for the GHR unveils the molecular basis for its physiological actions.

Figure 1. SOCS2 forms a stable complex with Elongin B and C, Cullin 5 and Rbx2 mediated by the SOCS-box.

(A) Schematic of the various SOCS-constructs that were used. (B) Hek293T cells were transfected with Elongin B, Elongin C and Myc-Cullin5 and either an empty vector (lane 1), wildtype (lane 2 and 8) or a mutant form of FLAG-SOCS2 (lanes 3–7) as denoted in the picture. FLAG-SOCS2 and FLAG-SOCS2 mutants were immunoprecipitated with FLAG-beads (lanes 1–7), Mock-ip was immunoprecipitated with IgG. Western blots of immunoprecipitates and the lysate was performed as described in the Materials and Methods. (C) and (D) Western blot of whole cell lysate from HEK293T cells transfected with FLAG-SOCS2 and both, either or no Elongins as detailed in the figure. (E) HEK293T cells transfected with either FLAG-SOCS2 and both Elongins or FLAG-SOCS2 alone were treated with cycloheximide (100 µg/ml) for the times indicated in the figure before lysis. C denotes the control which was transfected with Empty vector instead of SOCS2.

Figure 2. SOCS2 exhibits E3 activity in the presence of E1, E2, Ubiquitin and ATP.

(A) The FLAG-tagged SOCS2-Elongin B/C complex was immunoprecipitated from HEK293T cells transfected with FLAG-SOCS2 and Elongin B/C or Empty vector and Elongin B/C. In vitro ubiquitination assay was performed as described in Materials and Methods except for the following: for lane 1 the assay was performed with immunoprecipitate from cells transfected with empty vector instead of SOCS2, lane 3 lacks the E1 enzyme, lane 4 lacks the E2 enzyme, lane 5 lacks Ub, lane 6 lacks ATP. Western blots of the samples were performed as described. (B) Immunoprecipitate from cells transfected with mutant variants of SOCS2 was used for the In vitro ubiquitination assay. Assay was performed as described in the Materials and Methods. Cells were transfected with Elongin B/C and either Empty vector (lane 1), FLAG-SOCS2 (lane 2) or a mutant form of FLAG-SOCS2 (lanes 3 and 4) as specified in the picture. Western blots of the samples were performed as described.

Figure 3. SOCS2 regulates GHR levels.

(A) HEK293T cells were transfected with Elongin B/C and Myc-GHR and either an Empty vector, FLAG-SOCS2 or a SOCS2 mutant version. C denotes the control which was tranfected with FLAG-SOCS2 and Elongin B/C but not GHR. Cells were starved for 4 hours in Serum-free media prior to treatment. Next, cells were treated with GH for the times specified in the figure and subsequently lysed as described in the Materials and Methods. Lysates were visualized by western blot. GHR was detected with an anti-Myc antibody. The immature form of the GHR (i) is visible after a short exposure of the film (top panel) whilst the mature form (m) is visible after a longer exposure (2nd panel from the top). (B) Co-Immunoprecipitation of GHR. HEK293T cells were transfected with Elongin B/C, FLAG-SOCS2 or a mutant version of SOCS2 together with Myc-GHR (lanes 1,2 and 7–10) or only FLAG-SOCS2 and Elongins (lanes 5,6) or Myc-GHR alone (lanes 3,4). Cells were starved and treated with GH for 0 (−) or 10 (+) minutes prior to lysis. Lysates were immunoprecipitated as described in the materials and methods and results visualized with western blot. GHR was detected with an anti-Myc antibody. (C) HEK293T cells transfected with either Myc-GHR, FLAG-SOCS2 and Elongin B/C or GHR and Elongin B/C alone were treated with cycloheximide (100 µg/ml) and GH (2 µg/ml) for the times indicated in the figure before lysis. Cell lysates were visualized by western blot. C denotes the control which was transfected with Empty vector instead of GHR. GHR was detected with an anti-GHR antibody.

Figure 4. GHR levels increase in the absence of SOCS2.

(A) HEK293T cells were transfected with GHR and either SOCS2 or Control siRNA. Cells were starved for 4 hours and then treated with GH for the times indicated in the picture. Cells were subsequently lysed as described in the Materials and Methods and lysates were visualized by western blot. GHR was detected with an anti-GHR antibody. (B) GHR-levels in SOCS2 KO mice. Liver lysates from SOCS2 KO mice and control mice were visualized by western blot and analyzed for GHR levels with an anti-GHR antibody.

Figure 5. SOCS2 targets the GHR for proteasomal degradation.

(A) Cells were transfected with GHR and Elongin B/C alone or in concert with FLAG-SOCS2. C denotes the control which was transfected with FLAG-SOCS2 and Elongin B/C. Cells were starved for 2 hours then treated with the indicated proteasomal inhibitors for 2 hours (20 µM MG132 or 5 nM Bortezomib (Bortez.)) followed by GH treatment as indicated. Cells were subsequently lysed as described in the Materials and Methods and lysates were visualized by western blot. GHR was detected with an anti-Myc antibody. (B) SOCS2 ubiquitinates the GHR in vitro. Cells were transfected with Elongin B/C and either Empty vector (Lane 1), FLAG-SOCS2 (Lane 5), GHR (Lane 4) or both (Lane 2 and 3). Cells were starved, treated with 5 nM Bortezomib for 2 hours and GH for 10 min followed by lysis. After lysis FLAG-tagged SOCS2 and proteins binding to it were immunoprecipitated with FLAG beads. In vitro Ub reaction was performed with E1/E2 (All except lane 3), ATP-system, Ub-aldehyde, HA-Ub and normal Ub in 100 µl. After the reaction proteins were eluted from the beads with a FLAG-peptide (100 µg/ml) and immunoprecipitated a scond time with α-Myc Ab. After IP, samples were boiled and visualized by western blot. GHR was detected with an anti-GHR antibody. Top three panels: samples post reaction. Bottom panel: samples after second IP.

Figure 6. SOCS2 downregulation of GHR-levels is mediated by Y487.

(A) HEK293T cells were transfected with Elongin B/C, Myc-GHR or a mutant version of the receptor alone or in concert with FLAG-SOCS2 as specified in the figure. C denotes the control which was transfected with Empty Vector. Cells were starved for 4 hours, treated with GH for the times indicated in the figure and lysed. Lysates were visualized by western blot as detailed in the Materials and Methods. GHR was detected with an anti-Myc antibody.