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. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: Growth Factors. 2014 Jan 20;32(1):1–10. doi: 10.3109/08977194.2013.877005

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

Figure 4

(A) SOCS3 catalyses the ubiquitination of the gp130 cytoplasmic domain (gp130cyt) when it is phosphorylated (left panel) but not in the absence of phosphorylation (center panel). Phosphorylation was achieved by including 500 nM JAK2JH1 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.