title: The SOCS box encodes a hierarchy of affinities for Cullin5: implications for ubiquitin ligase formation and cytokine signalling suppression.
abstract: The SOCS (suppressors of cytokine signalling) family of proteins inhibits the cytokine-induced signalling cascade in part by promoting the ubiquitination of signalling intermediates that are then targeted for proteasomal degradation. This activity relies upon an interaction between the SOCS box domain, the adapter complex elonginBC and a member of the Cullin family, the scaffold protein of an E3 ubiquitin ligase. In this study, we dissected this interaction in vitro using purified components.We found that all eight SOCS proteins bound Cullin5 but required prior recruitment of elonginBC. Neither SOCS nor elonginBC bound Cullin5 when in isolation. Interestingly, the affinity of each SOCS-elonginBC complex for Cullin5 varied by 2 orders of magnitude across the SOCS family. Unexpectedly, the most potent suppressors of signalling, SOCS-1 and SOCS-3, bound most weakly to the E3 ligase scaffold, with affinities 100- and 10-fold lower, respectively, than the rest of the family. The remaining six SOCS proteins all bound Cullin5 with high affinity (K(d) of ~10 nM) due to a slower off-rate and hence a longer halflife of the complex. This difference in affinity may reflect a difference in mode of action as only SOCS-1 and SOCS-3 have been shown to suppress signalling using both SOCS box-dependent and SOCS box-independent mechanisms. This is not the case with the other six SOCS proteins, and our data imply the existence of two distinct subclasses of SOCS proteins with a high affinity for Cullin5, the E3 ligase scaffold, possibly reflecting complete dependence upon ubiquitination for suppression of cytokine signalling.
authors.affiliation: Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, Australia. babon@wehi.edu.au
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Babon
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Sabo
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Zhang
authors.forename: Nicos A
authors.initials: NA
authors.lastname: Nicola
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Norton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002151:Calorimetry; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D000071222:Suppressor of Cytokine Signaling 1 Protein; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: D044842:Cullin Proteins; D016207:Cytokines; C491590:Socs1 protein, mouse; C491411:Socs3 protein, mouse; D000071222:Suppressor of Cytokine Signaling 1 Protein; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; C531145:cullin5 protein, mouse; D044767:Ubiquitin-Protein Ligases