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Abstract
Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6, enhances the level of cyclin D-Cdk6 complexes, and its overexpression causes Cdk6-dependent transformation of immortalised fibroblasts. Here, we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which, unexpectedly, it was unable to do despite high levels of Cdk6. Instead, reduction of Fbxo7 expression increased proliferation, decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27, and increased levels of S phase cyclins and Cdk2 activity. Also, Fbxo7 protein levels correlated inversely with those of CD43, suggesting direct regulation of its expression and, therefore, of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle, indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; however, decreased expression of Cdk6 also enhanced levels of CD43, indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7(LacZ) mice showed significantly increased pro-B cell and pro-erythroblast populations, consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells.
Highlights
During the cell cycle, ubiquitin-mediated proteolysis provides a swift and precise means to regulate the abundance of cell cycle regulatory proteins, including cyclins and cyclin-dependent kinase (Cdk) inhibitors
Analysis of cell cycle regulators showed that cells had decreased levels of p27, and increased levels of S phase cyclins and Cdk2 activity
Alterations to Cdk6 protein levels did not affect the cell cycle, indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; decreased expression of Cdk6 enhanced levels of CD43, indicating that expression of CD43 is independent of cell cycle regulation
Summary
Ubiquitin-mediated proteolysis provides a swift and precise means to regulate the abundance of cell cycle regulatory proteins, including cyclins and cyclin-dependent kinase (Cdk) inhibitors. SCF-type (Skp1– Cullin–F-box) E3 ubiquitin ligases are multi-subunit complexes that consisting of Skp, Cullin, Rbx and an F-box protein (FBP) (Ang and Wade, 2005; Deshaies, 1999; Jackson and Eldridge, 2002; Nakayama and Nakayama, 2005; Vodermaier, 2004) It is the FBP, which has a crucial role in recruiting the target substrate, usually directed by post-translational modification of the substrate (Cenciarelli et al, 1999; Hermand, 2006; Ho et al, 2008; Jin et al, 2004; Winston et al, 1999). Three FBPs – Fbxo, Fbxw, Fbxo31 – promote the ubiquitin-mediated degradation of cyclin D1 (Lin et al, 2006; Santra et al, 2009; Okabe et al, 2006)
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