PMID:11807090

Citation	Kang, D, Chen, J, Wong, J and Fang, G (2002) The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition. J. Cell Biol. 156:249-59
Abstract	The checkpoint protein Chfr delays entry into mitosis, in the presence of mitotic stress (Scolnick, D.M., and T.D. Halazonetis. 2000. Nature. 406:430-435). We show here that Chfr is a ubiquitin ligase, both in vitro and in vivo. When transfected into HEK293T cells, Myc-Chfr promotes the formation of high molecular weight ubiquitin conjugates. The ring finger domain in Chfr is required for the ligase activity; this domain auto-ubiquitinates, and mutations of conserved residues in this domain abolish the ligase activity. Using Xenopus cell-free extracts, we demonstrated that Chfr delays the entry into mitosis by negatively regulating the activation of the Cdc2 kinase at the G2-M transition. Specifically, the Chfr pathway prolongs the phosphorylated state of tyrosine 15 in Cdc2. The Chfr-mediated cell cycle delay requires ubiquitin-dependent protein degradation, because inactivating mutations in Chfr, interference with poly-ubiquitination, and inhibition of proteasomes all abolish this delay in mitotic entry. The direct target of the Chfr pathway is Polo-like kinase 1 (Plk1). Ubiquitination of Plk1 by Chfr delays the activation of the Cdc25C phosphatase and the inactivation of the Wee1 kinase, leading to a delay in Cdc2 activation. Thus, the Chfr pathway represents a novel checkpoint pathway that regulates the entry into mitosis by ubiquitin-dependent proteolysis.
Links	PubMed PMC2199220 Online version:10.1083/jcb.200108016
Keywords	Animals; Blotting, Western; CDC2 Protein Kinase/antagonists & inhibitors; CDC2 Protein Kinase/metabolism; Cell Cycle Proteins/chemistry; Cell Cycle Proteins/metabolism; Cell Line; Cell-Free System; Enzyme Activation; G2 Phase; Humans; Ligases/chemistry; Ligases/metabolism; Maturation-Promoting Factor/metabolism; Mitosis; Neoplasm Proteins; Phosphorylation; Phosphotyrosine/metabolism; Protein Kinases/metabolism; Protein Processing, Post-Translational; Protein Structure, Tertiary; Protein-Serine-Threonine Kinases/metabolism; Proto-Oncogene Proteins; Ubiquitin/metabolism; Xenopus; Xenopus Proteins
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
XENLA:CHFR		GO:0051437: positive regulation of ubiquitin-protein ligase activity involved in regulation of mitotic cell cycle transition	ECO:0000314:		P		Figures 5 and 6	complete CACAO 10552
XENLA:CHFR		GO:0061630: ubiquitin protein ligase activity	ECO:0000314:		F		Figure 6	complete CACAO 10553

References

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