Reconstruction of an active SOCS3-based E3 ubiquitin ligase complex in vitro: identification of the active components and JAK2 and gp130 as substrates

Abstract

SOCS3 (suppressor of cytokine signaling 3) inhibits the intracellular signaling cascade initiated by exposure of cells to cytokines. SOCS3 regulates signaling via two distinct mechanisms: directly inhibiting the catalytic activity of Janus kinases (JAKs) that initiate the intracellular signaling cascade and catalysing the ubiquitination of signaling components by recruiting components of an E3 ubiquitin ligase complex. Here we investigate the latter mode-of-action biochemically by reconstructing a SOCS3-based E3 ubiquitin ligase complex in vitro using fully purified, recombinant components and examining its ability to promote the ubiquitination of molecules involved in the cytokine signaling cascade. We show that SOCS3 is an active substrate recruitment module for a Cullin5-based E3 ligase and have defined the core protein components required for ubiquitination. SOCS3-induced polyubiquitination was rapid and could proceed through a number of different ubiquitin lysines. SOCS3 catalyzed the ubiquitination of both the IL-6 receptor common chain (gp130) and JAK2.

Figure 1. A SOCS3/Cullin5 based E3 ligase catalyses JAK2_JH1 ubiquitination

(A) SDS-PAGE analysis of in vitro ubiquitination system components. Cullin5-Rbx2 and SOCS3-elonginBC are produced separately as ternary complexes (lanes 1,2) and then mixed and further purified to achieve the final E3 ubiquitin ligase. Cullin5 is produced as two domains (NTD and CTD) and these associate non-covalently to form the functional scaffold. Recombinant E2 (UbcH5a) and purified bovine ubiquitin are shown in lanes 4,5 respectively. # marks indicate impurities (B) GST-JAK2 (JH1 domain) is a substrate for polyubiquitination but GST alone is not. SOCS3 based E3 ligase (2.5uM) was incubated with ubiquitin (50uM), human E1 (100nM), purified recombinant E2 (UbcH5a, 2.5uM) and either GST-JAK2 (JH1 domain, 5uM, lanes 1–4) or GST alone (lanes 5–8) in the presence of 2.5mM Mg/ATP at 37°C for 0, 5, 30, 60 minutes. Note that the cullin C-terminal domain was polyubiquitinated in each case (asterisks). (C) GST-JAK polyubiquitination was dependent upon the presence of E1, E2, ATP, ubiquitin and the KIR/SH2 domain of SOCS3. Each reaction component was absent as indicated above the gel. -SH2 indicates that the SH2 domain of SOCS3 was deleted resulting in a SOCS box only:elonginBC ternary complex. (D) Mutating the JAK binding site (F25A) in SOCS3 abrogates its ability to promote JAK ubiquitination The reactions were incubated for 0, 10, 20 and 60 minutes (left to right in each case). All results are visualised by Coomassie staining following SDS-PAGE. (E). Schematic diagram illustrating the components of the Cullin5 in vitro ubiquitination system. A model of the E3 ubiquitin ligase is shown on the right, adapted from PDB 4JGH.

Figure 2. Cullin is modified by ubiquitin on K724

(A) Ubiquitination reactions were performed in the absence of JAK2 to highlight Cullin5 ubiquitination.. The reactions were incubated for 0, 5, 10, 30, 60 and 120 minutes (left to right, upper panel). (B) Tryptic digest MS/MS analyses was used to identify ubiquitinated lysines on Cullin5 from the monoubiquitinated Cullin5 band shown in the left panel. (C) The use of methylated ubiquitin shows that Cullin5 is ubiquitinated on multiple lysines (asterisks). The reactions were incubated for 0, 5, 10, 30 minutes (left to right in each case). (D) Cullin K724R is self-ubiquitinated less efficiently than wild-type Cullin5 and is observable only after an extended incubation (60 minutes compared to 10 minutes, asterisks). The reactions were incubated for 0, 2, 5, 10, 30 and 60 minutes (left to right in each case). This mutant E3 ligase (right) was able to ubiquitinate JAK substrate as efficiently as wild-type. (E) Mutation of lysines 724, 727 and 728 to arginine abrogates Cullin auto-ubiquitination but has no effect on substrate ubiquitination. (F) Cullin ubiquitination does not impair E3 ligase function in vitro. The Cullin5 E3 ligase was auto-ubiquitinated prior to the addition of substrate (left) or incubated with buffer as a control (right). After 30 minutes, substrate (JAK2) and excess ubiquitin (50 μM final concentration) were added and substrate ubiquitination examined after the indicated times. Pre-incubation of the E3 ligase in this fashion resulted in at least 50% of the Cullin5 CTD becoming ubiquitinated (compare the intensity of the two asterisked bands, the lower band is unmodified Cul5(CTD) whilst the upper band is mono-ubiquitinated Cul5(CTD)))before the addition of substrate however no subsequent decrease in the rate of substrate ubiquitination was observed (compare the JAK-Ub_n bands in lanes 2–5 with lanes 7–10). All results are visualised by Coomassie staining following SDS-PAGE. The ubiquitinated reaction products indicated using larger font are those referred to in the main text.

Figure 3. Rbx1 and Rbx2 are active components of a SOCS3/Cullin5 based E3 ligase which catalyses poly-ubiquitination of multiple lysines in the presence of UbcH5a, UbcH5b or UbcH5c

(A) Ubiquitination reactions were performed as described in Figure 1 using either methylated (left) or wild-type (right) ubiquitin. The use of methylated ubiquitin indicates there are at least six individual lysines on GST-JAK2 (JH1) that are ubiquitinated in this system (asterisks). The reactions were incubated for 0, 10, 30, 60 and 120 minutes (left to right in each case). (B) Polyubiquitination of JAK was observed when ubiquitin with only a single lysine (K11, K48 or K63) was used. (C) A panel of different E2 enzymes were tested for the ability to promote JAK2 ubiquitination. Each E2 enzyme was added to a standard ubiquitination assay (see Figure 1) at a concentration of 2.5 μM and the reaction performed for 30 minutes at 37°C. Only UbcH5a, UbcH5b and UbcH5c were able to promote JAK ubiquitination. The results were visualised by Coomassie staining following SDS-PAGE. (D) Rbx1 and Rbx2 were both active RING finger components of a Cullin5/SOCS3 based E3 ligase. Experiments were performed under identical conditions to those in (A). (E) In the presence of Rbx2, mono-ubiquitinated Cullin5 accumulates to a greater extent than seen in the presence of Rbx1 (indicated by the black label to the right of the gel). Experimental conditions were the same as (D) only JAK was omitted. All results are visualised by Coomassie staining following SDS-PAGE. The ubiquitinated reaction products indicated using larger font at the right of each gel are those referred to in the main text.

Figure 4. Cullin5/Rbx2/SOCS3 based E3 ligase catalyses efficient gp130 polyubiquitination in a phosphorylation-dependent manner

(A) SOCS3 catalyses the ubiquitination of the gp130 cytoplasmic domain (gp130_cyt) when it is phosphorylated (left panel) but not in the absence of phosphorylation (center panel). Phosphorylation was achieved by including 500 nM JAK2_JH1 in the reaction. Note that whereas ubiquitination of JAK proceeds through a series of mostly mono- di- and tri-ubiquitinated intermediates (right panel), ubiquitination of gp130 is qualitatively different in that the intermediates are all highly poly-ubiquitinated. (B) Pre-phosphorylation of gp130 shows that its SOCS3/Cullin5-catalysed ubiquitination is highly efficient and proceeds through poly-ubiquitinated intermediates. Results are visualized by Coomassie staining (upper) and autoradiography (lower). (C) Schematic showing the gp130 cytoplasmic domain and the two truncation mutants tested here. (D) Lysines both N- and C-terminal to the SOCS3 binding site on gp130 are ubiquitinated. The ubiquitinated reaction products referred to in the main text are indicated using larger font in each panel.