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

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.
Mesh Terms:
Animals, Cell Line, Cloning, Molecular, Cullin Proteins, Cytokine Receptor gp130, Humans, Iron-Binding Proteins, Janus Kinase 2, Mice, Phosphorylation, Protein Binding, Signal Transduction, Spodoptera, Suppressor of Cytokine Signaling Proteins, Transcription Factors, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Ubiquitination, Ubiquitins
Growth Factors
Date: Feb. 01, 2014
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