Nucleolar Targeting of the Fbw7 Ubiquitin Ligase by a Pseudosubstrate and Glycogen Synthase Kinase 3

Abstract

E3 ubiquitin ligases catalyze protein degradation by the ubiquitin-proteasome system, and their activity is tightly controlled. One level of regulation involves subcellular localization, and the Fbw7 tumor suppressor exemplifies this type of control. Fbw7 is the substrate-binding component of an SCF ubiquitin ligase that degrades critical oncoproteins. Alternative splicing produces three Fbw7 protein isoforms that occupy distinct compartments: Fbw7α is nucleoplasmic, Fbw7β is cytoplasmic, and Fbw7γ is nucleolar. We found that cancer-associated Fbw7 mutations that disrupt substrate binding prevent Fbw7γ nucleolar localization, implicating a substrate-like interaction in nucleolar targeting. We identified EBNA1-binding protein 2 (Ebp2) as the critical nucleolar factor that directly mediates Fbw7 nucleolar targeting. Ebp2 binds to Fbw7 like a substrate, and this is mediated by an Ebp2 degron that is phosphorylated by glycogen synthase kinase 3. However, despite these canonical substrate-like interactions, Fbw7 binding is largely uncoupled from Ebp2 turnover in vivo. Ebp2 thus acts like a pseudosubstrate that directly recruits Fbw7 to nucleoli.

ACKNOWLEDGMENTS

We thank Ning Zheng and his lab for assistance with the Fbw7 purification and provision of Cul1/Rbx1, UbcH5, and UBE1. We also thank Kathy Shire for preparing the anti-Ebp2 antibody.

This work was supported by NIH grants CA084069 and P01 HL 084205 (B.E.C.), 2T32 GM007270 (E.A.L.), and CIHR grant number12477 (L.F.).