Phosphorylation-dependent regulation of SCFFbx4 dimerization and activity involves a novel component, 14-3-3ε

Fbx4 is an F-box constituent of Skp-Cullin-F-box (SCF) ubiquitin ligases that directs ubiquitylation of cyclin D1. Ubiquitylation of cyclin D1 requires phosphorylation of both cyclin D1 and Fbx4 by GSK3 beta. GSK3 beta-mediated phosphorylation of Fbx4 Ser12 during the G1/S transition regulates Fbx4 dimerization, which in turn governs Fbx4-driven E3 ligase activity. In esophageal carcinomas that overexpress cyclin D1, Fbx4 is subject to inactivating mutations that specifically disrupt dimerization, highlighting the biological significance of this regulatory mechanism. In an effort to elucidate the mechanisms that regulate dimerization, we sought to identify proteins that differentially bind to wild-type Fbx4 versus a cancer-derived dimerization-deficient mutant. We provide evidence that phosphorylation of Ser12 generates a docking site for 14-3-3 epsilon. 14-3-3 epsilon binds to endogenous Fbx4 and this association is impaired by mutations that target either Ser8 or Ser12 in Fbx4, suggesting that this N-terminal motif in Fbx4 directs its interaction with 14-3-3 epsilon. Knockdown of 14-3-3 epsilon inhibited Fbx4 dimerization, reduced SCFFbx4 E3 ligase activity and stabilized cyclin D1. Collectively, the current results suggest a model wherein 14-3-3 epsilon binds to Ser12-phosphorylated Fbx4 to mediate dimerization and function. Oncogene (2011) 30, 1995-2002; doi:10.1038/onc.2010.584; published online 17 January 2011