Figure 1.
SOCS5 can specifically block JAK1 and JAK2 autophosphorylation and the SOCS5 N-terminus is critical for inhibition of JAK.
(A) 293T cells were transfected with cDNA encoding Flag-tagged mouse JAK1 (+) in the presence or absence of cDNAs encoding Flag-tagged SOCS1-SOCS7. 293T cells were transfected with cDNA encoding (B) Flag-tagged JAK2, (C) JAK3 or TYK2 in the presence of either SOCS1 or SOCS5. (D & E) 293T cells were transfected with cDNA encoding Flag-tagged mouse JAK1 (+) in the presence or absence of cDNAs encoding Flag-tagged SOCS5 or various SOCS5 mutants with either N-terminal truncations (Δ369, Δ349, Δ313, Δ171, Δ110) or with His360 (H360A), the SH2 domain (mSH2) or SOCS box (mSB) mutated. (A–E) Cells were lysed and anti-Flag immunoprecipitates analyzed by Western blot with phospho-specific (JAK1: A, D & E; JAK2: B) or anti-phosphotyrosine antibodies (αPY) (JAK1, JAK3 & TYK2; C) (upper panels). The blots were stripped and reprobed with rat anti-Flag antibody (lower panels). (F) 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence or absence of cDNA encoding Flag-tagged JAK1, JAK2, JAK3 or TYK2. Cells were lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (top panel). The blot was stripped and reprobed with anti-Flag antibodies (middle panel). Cell lysates were blotted with anti-SOCS5 antibodies (bottom panel). Panels A, B, D and E are 10% acrylamide gels. Panels C and F are 4–12% gradient gels.
Figure 2.
SOCS5 inhibits JAK1 kinase activity.
(A) 293T cells were transfected with cDNA encoding Flag-tagged mouse JAK1 (+) in the presence or absence of cDNAs encoding Flag-tagged SOCS1 or SOCS5. Anti-Flag immunoprecipitates were incubated in the presence of 32P-γ-ATP at 37°C. Incorporation of 32P was visualised by autoradiography (top panel). Immunoprecipitates were analyzed by Western blot with anti-Flag antibodies (lower panel). (B) cDNAs encoding Flag-tagged SOCS1, SOCS3, SOCS5 or JAK1 were independently transfected into 293T cells. Proteins were immunoprecipitated using anti-Flag antibody, and eluted from the resin by competition with Flag peptide. Proteins were then mixed and an in vitro kinase assay performed. JAK1 autophosphorylation and phosphorylation of the GST-Jak2 activation peptide (substrate; GST-J) (top panel) were assessed by Western blotting with phospho-specific antibodies. A sample of the reaction mix was analyzed by Coomassie staining to show substrate input (lower panel).
Figure 3.
An N-terminal fragment corresponding to residues 175–244 of SOCS5 can directly bind JAK1.
(A) SPR analysis of SOCS5175–244 fragment binding to the JAK JH1 domain. Serially diluted JAK JH1 domains (62.5 nM–2 μM) were flowed over immobilised SOCS5175–244 protein. Upper panels represent sensorgrams showing the kinetics of binding. Lower panels show steady-state analysis. (B) 293T cells were transfected with the Stat6 reporter and increasing amounts of cDNA expressing Flag-tagged SOCS5 (3.13–100 ng) or SOCS5 lacking the conserved N-terminal fragment (9.5–300 ng; Δ175–244) and stimulated overnight with 10 ng/mL rhIL-4. Cells were lysed and induced luciferase activity measured and normalised according to Renilla activity. Data are expressed as arbitrary units and represent the mean of triplicates ± SD. Cell lysates were analyzed by Western blotting for Flag-tagged proteins (SOCS5 upper; Δ175–244 lower panel); images were generated from the same gel and exposure. (C) Recombinant SOCS5 JIR or SOCS3 was incubated with 20 nM JAK1 and GST-JAK2 activation peptide (substrate; GST-J) for 15 min in the presence of 2.5 mM Mg/32P-γ-ATP at 37°C. Incorporation of 32P was visualised by autoradiography (top panel) and protein input by SDS-PAGE and Coomassie staining (lower panel).
Figure 4.
SOCS5-SH2 domain binding analysis and identification of Shc-1 pY317 as a high affinity-potential binding target.
SPR analysis of phosphopeptide binding to the SOCS5-SH2 domain. A constant amount of recombinant SOCS5 was mixed with serially diluted phosphopeptides (0.4–10 µM) and flowed over immobilised Shc-1 pY317 peptide. The response units are expressed as a percentage of maximal binding in the absence of competitor and are plotted against the concentration of competitor peptide. Steady-state analysis at saturation of binding was used to derive the KD values for the respective phosphopeptides. Binding analysis of (A) JAK, Shc-1, or wild-type and (B) mutated EGF-R phosphopeptides. Phosphopeptide sequences and the respective KD values are shown in the right-hand side table. Yellow boxes highlight residues replaced by an alanine residue. (C) Structural model of the SOCS5-SH2-Shc-1 peptide complex. A homology model for the SOCS5-SH2 domain was built using the SOCS4 crystal structure as a template (PDB code 2IZV). The Shc-1 pY317 peptide was modelled from the SOCS3-gp130 crystal structure (PDB code 2HMH). Side chains were optimized using ICM-PRO (Molsoft). The backbone of the flexible EF and BG loops was fixed in the apo-SOCS4 conformation, but is likely to adjust on peptide binding to maximize interactions. Predicted hydrogen bonds are shown as dashed lines. (D) SOCS5 interacts with full-length Shc-1 protein. 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence (+) or absence of cDNA encoding Flag-tagged Shc-1 or alternatively, with cDNA encoding Flag-tagged SOCS5 alone. Cells were treated with 10 μM MG132 for 3.5 h prior to treatment with sodium pervanadate solution for 30 min. Cells were then lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (αSOCS5). The blots were stripped and reprobed with a phospho-specific antibody for Shc-1-Y317 (middle panel). Cell lysates were analyzed by Western blot with anti-SOCS5 (lower panel).
Figure 5.
Distinct domains within SOCS5 mediate interaction with JAK and Shc.
Schematic showing SOCS5 domain organization and the regions implicated in JAK interaction and inhibition (upper section) in comparison to those involved in inhibition of EGF-R signaling and binding to Shc-1 (lower section). *indicates regions of SOCS5 previously reported to be involved in interaction and degradation of the EGF-R (N-terminus and SOCS box, respectively) [7], [8]. JIR: JAK Interaction Region.