Abstract
Proper activation of checkpoint during mitotic stress is an important mechanism to prevent genomic instability. Chfr (Check point protein with FHA (Forkhead-associated domain) and RING domains) is a ubiquitin-protein isopeptide ligase (E3) that is important for the control of an early mitotic checkpoint, which delays entry into metaphase in response to mitotic stress. Because several lines of evidence indicate that Chfr is a potential tumor suppressor, it is critically important for us to identify Chfr substrates and understand how Chfr may regulate these substrates, control mitotic transitions, and thus, act as a tumor suppressor in vivo. Here, we report the discovery of a new Chfr-associated protein Kif22, a chromokinesin that binds to both DNA and microtubules. We demonstrated that Kif22 is a novel substrate of Chfr. We showed that Chfr-mediated Kif22 down-regulation is critical for the maintenance of chromosome stability. Collectively, our results reveal a new substrate of Chfr that plays a role in the maintenance of genome integrity.
Highlights
Chfr delays the cell cycle progression at mitosis by inactivating cyclin B1-bound Cdc2 and exporting them from nucleus [6]
We reported the identification of another Chfr substrate as chromokinesin protein Kif22 and revealed that Kif22 overexpression contributes to chromosomal instability observed in Chfr-deficient cells
Kif22 overexpression results in chromosomal instability, analogous to cells with Chfr knockdown. These results suggest that at least one mechanism for Chfr functions in the maintenance of chromosomal stability and that tumor suppression could be through its regulation of Kif22 protein levels because both Chfr down-regulation and Kif22 overexpression result in chromosomal instability, which is a hallmark of tumorigenesis
Summary
Plasmids—Full-length Chfr and Kif were cloned into an S-protein/FLAG/SBP (streptavidin-binding protein) tripletagged destination vector using the Gateway cloning system (Invitrogen). Full-length Chfr and Kif were cloned to Myc-tagged mammalian expression destination vector and GST-tagged and MBP-tagged bacterial expression vector. Human mammary epithelial cells (HMEC) were maintained in MEGM complete medium supplemented with bovine pituitary extract. 293T-Chfr, 293T-Kif, MCF7-control shRNA, MCF7-Chfr shRNA, T47D-control shRNA, and T47D-Chfr shRNA stable cell lines were maintained in complete RPMI medium supplemented with 2 g/ml puromycin. HeLa-Myc Chfr stable cell lines were maintained in VOLUME 284 NUMBER 19 MAY 8, 2009. Chfr stable cells were lysed with NETN buffer The bound proteins were washed three times with NETN and eluted twice with 2 mg/ml biotin (Sigma) for 30 min at 4 °C. The eluates were incubated with S-protein-agarose (Novagen) for 1 h at 4 °C and washed three times with NETN
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