Opposing effects on the cell cycle of T lymphocytes by Fbxo7 via Cdk6 and p27.

G1 phase cell cycle proteins, such as cyclin-dependent kinase 6 (Cdk6) and its activating partners, the D-type cyclins, are important regulators of T-cell development and function. An F-box protein, called F-box only protein 7 (Fbxo7), acts as a cell cycle regulator by enhancing cyclin D-Cdk6 complex formation and stabilising levels of p27, a cyclin-dependent kinase inhibitor. We generated a murine model of reduced Fbxo7 expression to test its physiological role in multiple tissues and found that these mice displayed a pronounced thymic hypoplasia. Further analysis revealed that Fbxo7 differentially affected proliferation and apoptosis of thymocytes at various stages of differentiation in the thymus and also mature T-cell function and proliferation in the periphery. Paradoxically, Fbxo7-deficient immature thymocytes failed to undergo expansion in the thymus due to a lack of Cdk6 activity, while mature T cells showed enhanced proliferative capacity upon T-cell receptor engagement due to reduced p27 levels. Our studies reveal differential cell cycle regulation by Fbxo7 at different stages in T-cell development.

Gsk3ß and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease.

Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson's disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within specific functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deficiencies in SCFFbxo7/PARK15 ubiquitin ligase activity are mechanistically linked to early-onset PD. To date, relatively few substrates of the ligase have been identified. These include HURP (hepatoma up-regulated protein), whose ubiquitination results in proteasome-mediated degradation, and c-IAP1 (inhibitor of apoptosis protein 1), TNF receptor-associated factor 2 (TRAF2), and NRAGE, which are not destabilized as a result of ubiquitination. None of these substrates have been linked directly to PD, nor has it been determined whether they would directly engage neuronal cell death pathways. To discover ubiquitinated substrates of SCFFbxo7 implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. A total of 338 new targets were identified and from these we validated glycogen synthase kinase 3ß (Gsk3ß), which can phosphorylate α-synuclein, and translocase of outer mitochondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7 substrates both in vitro and in vivo Ubiquitin chain restriction analyses revealed that Fbxo7 modified Gsk3ß using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3ß activity, rather than its levels or localization. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our findings demonstrate that SCFFbxo7 has an impact directly on two proteins implicated in pathological processes leading to PD.

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

During the final stages of erythropoiesis, lineage-restricted progenitors mature over three to five cell divisions, culminating with withdrawal from the cell cycle and the loss of most organelles, including mitochondria and nuclei. Recent genome-wide association studies in human populations have associated several SNPs near or within FBXO7 with erythrocyte phenotypes. Fbxo7 encodes a multi-functional F-box protein known to bind p27 and participate in selective mitophagy. One SNP causes an amino acid substitution (Met115Ile) and is associated with smaller erythrocytes. We find that the less common IIe115 allele of Fbxo7 binds less efficiently to p27, and cells expressing this allele proliferate faster than cells expressing Met115. We show that an erythroleukaemic cell line with reduced Fbxo7 expression fails to stabilize p27 levels, exit the cell cycle, and produce haemoglobin. In addition, mice deficient in Fbxo7 expression are anaemic due to a reduction in erythrocyte numbers, and this is associated with lower p27 levels, increased numbers of late-stage erythroblasts with greater than 2N DNA content, and delayed mitophagy during terminal differentiation. Collectively, these data support an important physiological, cell cycle regulatory role for Fbxo7 during erythropoiesis.