Functional characterization of the three isoforms of Fbw7 (F-box and WD repeat domain containing 7) in ubiquitin dependent proteolysis.

Abstract

Fbw7 is the F-box protein of SCFFbw7 E3 ubiquitin ligase, which specifically associates with the substrates to be ubiquitinated. Substrates of Fbw7 play important roles in cell cycle regulation, proliferation, signal transduction and metabolism, which are related to tumor formation, suggesting that Fbw7 functions as a tumor suppressor. Fbw7 has three splicing variants α, β and γ, and the biological function of each isoform is not well understood. Our lab is interested in how the Fbw7 isoforms regulate cyclin E proteolysis and the cell cycle. By using mammalian and insect cell culture systems, I demonstrate that the three isoforms can form homo- and heterodimers in vivo and in vitro. The dimerization domain is located immediately upstream of the F-box motif, and it is highly conserved in all Fbw7 homologues and other related F-box proteins, indicating the dimerization may be common feature of a subset of F-box proteins. Abolishment of dimerization inhibits cyclin E proteolysis and leads to a prolonged half-life of cyclin E, although it does not affect Fbw7 binding to cyclin E or to the Cul-Rbx1-Skp1 E3 catalytic module. Cyclin E accumulation can be commonly found in many primary tumors and cancer cell lines. These results suggest a novel mechanism of how F-box proteins recognize their substrates. Fbw7 isoforms show different protein stabilities, where the α isoform is stable, but the β and γ isoforms are not. The stability of the β and γ isoforms is largely controlled by their N- terminal unique region. In order to better understand the mechanism regulating their stability, we performed a yeast two hybrid screen and identified SLP1 (stomatin like protein 1) as an Fbw7γ isoform specific interacting protein. SLP1 binds to the unique region of γ isoform, and stabilizes γ. We find that Cdk2 promotes the degradation of both SLP1 and the γ isoform, and this function of Cdk2 is dependent on its kinase activity. SLP1 also physically interacts with Cdk2 through its membrane association domain. These results support a model in which Fbw7γ and SLP1 are coordinately targeted for ubiquitin mediated degradation by Cdk2.