Abstract

Identification of novel proteins that can potentially contribute to carcinogenesis is a requisite venture. Herein, we report the first biochemical characterization of the novel F-box and WD40 containing protein, FBXW4. We have identified interacting protein partners and demonstrated that FBXW4 is part of a ubiquitin ligase complex. Furthermore, the Fbxw4 locus is a common site of proviral insertion in a variety of retroviral insertional mutagenesis murine cancer models and Fbxw4 mRNA is highly expressed in the involuting murine mammary gland. To begin to characterize the biochemical function of Fbxw4, we used proteomic analysis to demonstrate that Fbxw4 interacts with Skp1 (SKP1), Cullin1 (CUL1), Ring-box1 (RBX1) and all components of the COP9 signalosome. All of these interactions are dependent on an intact F-box domain of Fbxw4. Furthermore, Fbxw4 is capable of interacting with ubiquitinated proteins within cells in an F-box dependent manner. Finally, we demonstrate that FBXW4 is mutated, lost and under-expressed in a variety of human cancer cell lines and clinical patient samples. Importantly, expression of FBXW4 correlates with survival of patients with non-small cell lung cancer. Taken together, we suggest that FBXW4 may be a novel tumor suppressor that regulates important cellular processes.

Highlights

  • Ubiquitin ligase complexes catalyze the conjugation of ubiquitin onto substrate proteins [1]

  • The F-box domain is responsible for directly interacting with Skp1, whereas the other domains contained within the protein are responsible for interacting with and bringing substrate proteins into proximity of the ubiquitin ligase [5]

  • We demonstrate that Fbxw4 is part of a ubiquitin ligase complex containing Skp1, Cullin1, Rbx1 and the COP9 signalosome

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Summary

Introduction

Ubiquitin ligase complexes catalyze the conjugation of ubiquitin onto substrate proteins [1]. Ubiquitin ligase complexes ( called E3 ubiquitin ligases) interact with an E1 (ubiquitin activating enzyme), and an E2 (ubiquitin conjugating enzyme) [1]. The E1 and E2 enzymes are generally shared between many E3 ligases and the E3 component is the specificity factor that interacts directly with substrate proteins. One type of E3 ubiquitin ligase complex, the SCF ubiquitin ligase complex, contains Skp, Cullin, ring-box and any one of more than seventy F-box containing proteins encoded in the genomes of higher eukaryotes [3,4]. The F-box domain is responsible for directly interacting with Skp, whereas the other domains contained within the protein are responsible for interacting with and bringing substrate proteins into proximity of the ubiquitin ligase [5]. Mutations that lead to a loss of function of FBXW7 or BTRCP lead to the stabilization of their cognate substrate proteins, Notch, MYC and CyclinE or Beta Catenin, respectively, all of which are well known oncogenes [6,9,11,12, 13,14,15]

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