In vitro ubiquitination of cytokine signaling components.

: The eight SOCS (Suppressor of Cytokine Signaling) proteins encoded in the human genome all contain a C-terminal domain, the SOCS box, that allows them to function as E3 ubiquitin ligases and thereby catalyze the ubiquitination of components of the JAK/STAT signaling pathway. This activity is key to their function as cytokine signaling inhibitors as, once ubiquitinated, signaling molecules are degraded by the proteasome which allows the cell to return to its basal (unstimulated) state. SOCS based E3s are a subset of the CRL (Cullin-Ring-Ligase) family of ubiquitin ligases with the SOCS protein acting as the substrate recruitment module and interacting specifically with Cullin5, the E3 scaffold. Included here are protocols for the expression and purification of SOCS-based E3 complexes and their use in in vitro ubiquitination assays to characterize potential substrates. We have currently verified two components of the JAK/STAT pathway as substrates for ubiquitination using this method.