Knockdown of Fbxo7 reveals its regulatory role in proliferation and differentiation of haematopoietic precursor cells

Abstract

Research Article 2175 IntroductionDuring 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. This mechanism for regulating the turnover of proteins is mediated through ubiquitin ligases, which transfer ubiquitin to target proteins, enabling their timely destruction (Hershko, 1997;Hershko and Ciechanover, 1998;Nakayama and Nakayama, 2006;Reed, 2006;Reed, 2003). E3 ubiquitin-ligases promote the specific attachment of poly-ubiquitin chains, which then triggers proteolysis by the 26S proteasome. SCF-type (Skp1-Cullin-F-box) E3 ubiquitin ligases are multi-subunit complexes that consisting of Skp1, Cullin, Rbx1 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 specifically 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). In cell cycle regulation, several FBPs that regulate G1-S phase regulators have been intensively studied. These include the prototypical S-phase kinaseassociated protein 2 (Skp2, also known as Fbxl1) and the F-box and WD repeat domain containing 7 (Fbxw7) that, respectively, regulate the levels of the Cdk inhibitors cyclin E and cyclindependent kinase inhibitor 1B (CDKN1B; also known as and hereafter referred to as p27). In addition, three FBPs -Fbxo4, Another FBP that interacts with G1-S regulatory proteins is Fbox protein 7 (Fbxo7). In contrast to the destabilising effects of other FBPs, Fbxo7 acts as a specific assembly factor for cyclin DCdk6 complexes. Fbxo7 interacts directly with both Cdk6 and p27, and cooperatively increases cyclin D3 interactions with Cdk6 in vitro (Laman et al., 2005). In vivo, Fbxo7 overexpression in immortalised murine fibroblasts leads to their Cdk6-dependent transformation. These cells had increased levels of cyclin D-Cdk6 complexes and E2F activity, and formed tumours in athymic nude mice (Laman et al., 2005). Many mitogen and cytokine signalling pathways converge on cyclin D-Cdk activity, which -when overstimulated -promotes oncogenesis. Because of its Cdk6-dependent transforming activity, we propose that Fbxo7 functions as an oncogene.In U2OS and NIH3T3 cells, Fbxo7 has shown selectivity for Cdk6 over Cdk2 and Cdk4. Although the biochemical properties of Cdk4 and Cdk6 are similar, more-recent studies have indicated that differences can be discerned in their selective binding to cofactors (Sugimoto et al., 1999;Laman et al., 2005), preference for phosphorylation sites in pRb in vitro (Takaki et al., 2005), sensitivities to INK4 family members (Tourigny et al., 2002;Jones et al., 2007) and in vivo partnering with D-type cyclins (Ely et al., 2005). Also, in studies of knockout mice, tissue-specific defects are seen: Cdk4-knockout mice have impair...

Defective erythropoiesis in a mouse model of reduced Fbxo7 expression due to decreased p27 expression

“…Fbxo7 expression was reduced using a previously validated shRNA construct [9]; cells were differentiated by DMSO treatment; and haemoglobin concentration was measured over time (Figure 3A). Control cells accumulated haemoglobin over 5 days, whereas cells expressing Fbxo7 shRNA (Fbxo7-sh) did not, demonstrating a requirement for Fbxo7 in MEL differentiation.…”

“…For IPs, 1 µg of anti-CDK2 or IgG, with 20 µl of Protein A/G agarose (Santa Cruz Biotechnology), or 20 µl of EZview anti-HA beads (Sigma) was used as previously described [9]. Beads were washed with kinase buffer (25 m m HEPES, pH 7.5; 5 m m MgCl 2 ; 2.5 m m MnCl 2 ; 0.5 m m DTT) prior to adding 0.75 µg of recombinant 6 × His-Rb (amino acids 792–928; ProSpec, Ness-Ziona, Israel), 37.5 µ m ATP, and 1 μCi of [γ- 33 P]ATP for 30 min at 30 °C.…”

“…This 69-member family is engaged in a range of activities, many of which are critically important for normal cellular functions [8]. We previously reported alterations in EB numbers in the bone marrow (BM) of mice with a disrupted Fbxo7 (F-box protein only 7) gene ( Fbxo7 LacZ/LacZ ) [9]. They showed increased numbers of early-stage EBs and decreased late-stage EBs [9].…”

The F-box Protein FBXO25 Promotes the Proteasome-dependent Degradation of ELK-1 Protein

“…It has been shown that the substrate degradation depends on its phosphorylation state (25), cell cycle, and intracellular localization (26). In addition, the functions of some FBPs are cell type-dependent (27)(28)(29). In an attempt to bypass these constraints and identify new SCF1(FBXO25) substrates, the ubiquitination in chip approach has been used.…”

Roles of F-box proteins in cancer

“…Fbxo7 −/− mice show increased pro-B cell and pro-erythroblast populations 135 , but no information has been obtained regarding the role of FBXO7 in tumorigenesis. Biochemically, FBXO7 catalyses the ubiquitylation of hepatoma upregulated protein (HURP; also known as DAP5) 136 ; HURP is a putative oncogene, as its overexpression has been observed in human hepatocellular carcinoma, colon cancer, breast cancer and transitional cell carcinoma 136 .…”