Identification of substrates for the Epac1-inducible E3 ubiquitin ligase component SOCS3

It is now accepted that there is a link between obesity and several diseases such as cardiovascular disease (CVD), diabetes, rheumatoid arthritis (RA), and atherosclerosis with the common initiating factor in pathogenesis being a state of low grade, chronic inflammation. This state, characterised by elevated levels of pro-inflammatory cytokines such as interleukin (IL) 6, leads to sustained activation of inflammatory signalling pathways such as the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and subsequently pathogenesis. Suppressor of cytokine signalling (SOCS) 3 is inducible by several stimuli including IL6 and 3'-5'-cyclic adenosine monophosphate (cAMP), and via these routes has been demonstrated to terminate IL6 signalling thus quenching JAK/STAT signalling and an inflammatory response. While SOCS3 was primarily characterised as a competitive inhibitor of intracellular signalling, it also functions as specificity factor for an elongin-cullin-SOCS (ECS)-type E3 ubiquitin ligase. In this role, it has been demonstrated to direct ubiquitin-mediated proteasomal degradation of several substrates and lysosomal routing. However, the full spectrum of SOCS3-dependently ubiquitinated substrates is unknown. Given that JAK/STAT signalling is critical in the development of chronic inflammatory disorders, delineating the role of SOCS3 as an E3 ligase might be therapeutically beneficial. However, given the broad range of SOCS3 stimuli, the availability of certain SOCS3 substrates might be conditional on the route of SOCS3 induction. Using a global proteomics approach, this study aimed to identify SOCS3-dependently ubiquitinated substrates in response to cAMP and thus elaborate on the already well-established role of cAMP in inflammation. Differentially stable isotope labelling of amino acids in cell culture (SILAC)-labelled, tandem affinity purified ubiquitinomes of wild type (WT) murine embryonic fibroblasts (MEFs) and SOCS3-/- MEFs, each expressing epitope-tagged forms of ubiquitin, were compared using mass spectrometry (MS) following cAMP-mediated SOCS3 induction. Using this approach, proteins modified by SOCS3 with the epitope-tagged form of ubiquitin should be enriched in WT MEFs but not SOCS3-/- MEFs. MaxQuant analysis of raw mass spectromeric data identified several candidate SOCS3 substrates. Of these, SOCS3 was found to interact with PTRF/cavin-1, a regulator of caveolae formation and stability. Other substrates were tested but with limited success. Co-immunoprecipitation studies showed that SOCS3 could precipitate cavin-1 however the interaction was reduced following the inhibition of protein tyrosine phosphatases (PTPs) using sodium orthovanadate and hydrogen peroxide. This was surprising since all known SOCS3 substrates are tyrosine-phosphorylated prior to interacting with SOCS3 via its Src-homology (SH) 2 domain. Consistent with this finding, SOCS3 did not interact with known cavin-1 tyrosine-phosphorylated peptides spotted on a peptide array. However, a full-length cavin-1 peptide array spotted with non-tyrosine-phosphorylated peptides showed specific interactions at multiple sites. It is proposed that this interaction might influence the localisation and stability of either protein. While SOCS3 was demonstrated to impact cavin-1 ubiquitination, the mechanism by which it does so or the functional consequence is still not clear. Immunoprecipitation of cavin-1 following the introduction of SOCS3 was accompanied by a shift in the polyubiquitin signal from a high molecular weight, seen with cavin-1 alone, to a low molecular weight. Furthermore, an enhanced K48-polyubiquitin signal was detectable in this low molecular weight fraction, which was focused around the molecular weight of cavin-1. It is not known if this ubiquitin signal is SOCS3-dependent. In conclusion, the project has identified and validated a novel substrate of SOCS3. However, the mechanism by which SOCS3 regulates cavin-1 ubiquitination or the biological function of the interaction is currently unknown