Phosphorylation-dependent regulation of SCF(Fbx4) dimerization and activity involves a novel component, 14-3-3ɛ.
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 GSK3β. GSK3β-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 14-3-3ɛ. 14-3-3ɛ 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 14-3-3ɛ. Knockdown of 14-3-3ɛ inhibited Fbx4 dimerization, reduced SCF(Fbx4) E3 ligase activity and stabilized cyclin D1. Collectively, the current results suggest a model wherein 14-3-3ɛ binds to Ser12-phosphorylated Fbx4 to mediate dimerization and function.
The Leonard and Madlyn Abramson Family Cancer Research Institute and Cancer Center, University of Pennsylvania, Philadelphia, PA 19104-6160, USA.
O
O
Barbash
E K
EK
Lee
J A
JA
Diehl
D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D019938:Cyclin D1 / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004789:Enzyme Activation; D044783:F-Box Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016193:G1 Phase; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D051381:Rats; D016196:S Phase; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism; D012694:Serine; D054875:Ubiquitination
D048948:14-3-3 Proteins; D044783:F-Box Proteins; C508146:FBXO4 protein, human; D019938:Cyclin D1; D012694:Serine; D044843:SKP Cullin F-Box Protein Ligases
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