Abstract
Fbw7 is a tumor suppressor often deleted or mutated in human cancers. It serves as the substrate-recruiting subunit of a SCF ubiquitin ligase that targets numerous critical proteins for degradation, including oncoproteins and master transcription factors. Cyclin E was the first identified substrate of the SCFFbw7 ubiquitin ligase. In human cancers bearing FBXW7-gene mutations, deregulation of cyclin E turnover leads to its aberrant expression in mitosis. We investigated Fbw7 regulation in Xenopus eggs, which, although arrested in a mitotic-like phase, naturally express high levels of cyclin E. Here, we report that Fbw7α, the only Fbw7 isoform detected in eggs, is phosphorylated by PKC (protein kinase C) at a key residue (S18) in a manner coincident with Fbw7α inactivation. We show that this PKC-dependent phosphorylation and inactivation of Fbw7α also occurs in mitosis during human somatic cell cycles, and importantly is critical for Fbw7α stabilization itself upon nuclear envelope breakdown. Finally, we provide evidence that S18 phosphorylation, which lies within the intrinsically disordered N-terminal region specific to the α-isoform reduces the capacity of Fbw7α to dimerize and to bind cyclin E. Together, these findings implicate PKC in an evolutionarily-conserved pathway that aims to protect Fbw7α from degradation by keeping it transiently in a resting, inactive state.
Highlights
Cells rely on the ubiquitin-proteasome system to mediate the regulated degradation of protein and maintain cellular homeostasis
We identify one residue of Fbw7α (S18) that is phosphorylated by PKC in a manner coincident with its inactivation towards cyclin E in eggs arrested in metaphase II
As we wished to investigate the mechanism by which cyclin E can accumulate in a mitotic-like phase, our first question was to determine whether the Fbw7 isoforms are expressed in eggs (S1A Fig)
Summary
Cells rely on the ubiquitin-proteasome system to mediate the regulated degradation of protein and maintain cellular homeostasis. In this process, one key family of ubiquitin ligases are the SCF (Skp1/Cul-1/F-box) complexes, in which F-box-bearing proteins act as substrate-recruiting factors [1]. As a consequence of its critical role, alteration of Fbw functions leads to defects in cellular proliferation, differentiation, apoptosis and metabolism, and to the deregulation of numerous pathways with oncogenic potential [2, 29, 30]. Fbw is a haploinsufficient tumor suppressor [31], and deletions, promoter hypermethylation or mutations of the gene are found in many human cancers. Its role as a tumor suppressor was further demonstrated by genetic ablation of Fbw in mice (reviewed in [29, 30, 32])
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