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.
Division of Structural Biology, Walter and Eliza Hall Institute of Medical Research , Parkville, Victoria , Australia .
Nadia J
NJ
Kershaw
Artem
A
Laktyushin
Nicos A
NA
Nicola
Jeffrey J
JJ
Babon
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D044842:Cullin Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D050822:Cytokine Receptor gp130 / Q000235:genetics* / Q000378:metabolism; D000075065:Elongin; D006801:Humans; D033862:Iron-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D053614:Janus Kinase 2 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D000075687:NEDD8 Protein; D010766:Phosphorylation; D011485:Protein Binding; D015398:Signal Transduction / Q000235:genetics; D018411:Spodoptera / Q000166:cytology; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D044763:Ubiquitin-Conjugating Enzymes / Q000096:biosynthesis / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000235:genetics*; D054875:Ubiquitination / Q000235:genetics; D014452:Ubiquitins / Q000737:chemistry
D044842:Cullin Proteins; D000075065:Elongin; C494200:Il6st protein, mouse; D033862:Iron-Binding Proteins; D000075687:NEDD8 Protein; C513172:Nedd8 protein, mouse; C491411:Socs3 protein, mouse; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014157:Transcription Factors; D014452:Ubiquitins; C531145:cullin5 protein, mouse; D050822:Cytokine Receptor gp130; C490789:Ube2d1 protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2