(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.

(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).

(A) SPR analysis of SOCS5^175–244 fragment binding to the JAK JH1 domain. Serially diluted JAK JH1 domains (62.5 nM–2 μM) were flowed over immobilised SOCS5^175–244 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).

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 K _D 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 K _D 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).

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) , . JIR: JAK Interaction Region.