探討RACK1鷹架式結構蛋白對FBW2活性的調控

Abstract

FBW2 contains an F-box motif and is a substrate recognition subunit of SCF E3 ligase complex. The WD40 domain in FBW2 binds the substrate for ubiquitination and proteasomeal degradation. Although the SCF E3 ligase activity may be regulated by post-translational modification such as neddylation of its scaffold protein Cullin, it is possible that regulation of FBW2 activity may also affect the SCFFBW2 E3 ligase activity. In the present study, we used tandem affinity purification combined with LC/MS/MS to identify RACK1 (receptor for activated C-kinase 1) as an FBW2-associated protein that regulates FBW2-mediated ubiquitination. RACK1 is a scaffold protein, which contains seven WD40 repeats, and interacts with many proteins in multiple signal transduction pathways. In this study, we found RACK1 interacts with FBW2 in vivo and in vitro. By domain mapping analysis and in vitro competition experiments, we found that RACK1 interacts with the WD40 domain of FBW2 to block the interaction between FBW2 and its substrate GCM1. Accordingly, an in vivo ubiquitination assay showed that ubiquitination of exogenous GCM1 is enhanced in 293T cells when RACK1 is knocked down. Moreover, the protein level of endogenous GCM1 is decreased in RACK1-knockdown placental BeWo cells. In line with these observations, overexpression of RACK1 increases GCM1-mediated transcriptional activation. Overall, our results indicate that RACK1 may repress FBW2 activity by protecting its substrate from degradation and suggest a new role of RACK1 in regulation of E3 ligase activity.