The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition.

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.
Mesh Terms:
Animals, Blotting, Western, CDC2 Protein Kinase, Cell Cycle Proteins, Cell Line, Cell-Free System, Enzyme Activation, G2 Phase, Humans, Ligases, Maturation-Promoting Factor, Mitosis, Neoplasm Proteins, Phosphorylation, Phosphotyrosine, Protein Kinases, Protein Processing, Post-Translational, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Ubiquitin, Xenopus, Xenopus Proteins
J. Cell Biol.
Date: Jan. 21, 2002
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