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

Kang D, Chen J, Wong J, Fang G - J. Cell Biol. (2002)

Bottom Line: Nature. 406:430-435).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA.

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.

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Chfr delays the activation of the Cdc2 kinase at the G2-M transition. (A and B) Xenopus interphase extracts were incubated with a buffer, Chfr, ChfrI306A, or ChfrW332A. Δ90 cyclin B was then added and the kinetics of the activation of the Cdc2 kinase was analyzed by measuring the phosphorylation of histone H1 with radioactive γ-ATP. The Cdc2 kinase activity was quantitated and plotted against time (B). The unit for kinase activity is arbitrary. In five separate experiments, the wild-type Chfr protein always delayed the activation of Cdc2 kinase. The levels of the Cdc2 activity at 60 min, from extracts treated with ChfrI306A and ChfrW332A, were slightly variable; in some experiments, these levels were close to, instead of higher than, that of the buffer control. (C) Xenopus mitotic extracts were incubated with a buffer, Chfr, or ChfrI306A, and the level of the Cdc2 kinase activity was analyzed using histone H1 as a substrate.
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fig5: Chfr delays the activation of the Cdc2 kinase at the G2-M transition. (A and B) Xenopus interphase extracts were incubated with a buffer, Chfr, ChfrI306A, or ChfrW332A. Δ90 cyclin B was then added and the kinetics of the activation of the Cdc2 kinase was analyzed by measuring the phosphorylation of histone H1 with radioactive γ-ATP. The Cdc2 kinase activity was quantitated and plotted against time (B). The unit for kinase activity is arbitrary. In five separate experiments, the wild-type Chfr protein always delayed the activation of Cdc2 kinase. The levels of the Cdc2 activity at 60 min, from extracts treated with ChfrI306A and ChfrW332A, were slightly variable; in some experiments, these levels were close to, instead of higher than, that of the buffer control. (C) Xenopus mitotic extracts were incubated with a buffer, Chfr, or ChfrI306A, and the level of the Cdc2 kinase activity was analyzed using histone H1 as a substrate.

Mentions: Entry into mitosis can be monitored in Xenopus extracts (Murray and Kirschner, 1989; Solomon et al., 1990). Addition of Δ90 cyclin B, a mutant cyclin B with the first 90 amino acids truncated, to interphase extracts efficiently activated the Cdc2 kinase activity and drove extracts into mitosis (Fig. 5 A, top). However, the addition of wild-type Chfr protein reduced the rate of Cdc2 activation and delayed entry into mitosis (Fig. 5, A and B). This inhibition of Cdc2 kinase activity required an active ligase, because the two inactive mutants, ChfrI306A and ChfrW332A, both failed to delay mitotic entry. Thus, Cdc2 is a key target for the Chfr pathway to control the entry into mitosis.


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

Kang D, Chen J, Wong J, Fang G - J. Cell Biol. (2002)

Chfr delays the activation of the Cdc2 kinase at the G2-M transition. (A and B) Xenopus interphase extracts were incubated with a buffer, Chfr, ChfrI306A, or ChfrW332A. Δ90 cyclin B was then added and the kinetics of the activation of the Cdc2 kinase was analyzed by measuring the phosphorylation of histone H1 with radioactive γ-ATP. The Cdc2 kinase activity was quantitated and plotted against time (B). The unit for kinase activity is arbitrary. In five separate experiments, the wild-type Chfr protein always delayed the activation of Cdc2 kinase. The levels of the Cdc2 activity at 60 min, from extracts treated with ChfrI306A and ChfrW332A, were slightly variable; in some experiments, these levels were close to, instead of higher than, that of the buffer control. (C) Xenopus mitotic extracts were incubated with a buffer, Chfr, or ChfrI306A, and the level of the Cdc2 kinase activity was analyzed using histone H1 as a substrate.
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fig5: Chfr delays the activation of the Cdc2 kinase at the G2-M transition. (A and B) Xenopus interphase extracts were incubated with a buffer, Chfr, ChfrI306A, or ChfrW332A. Δ90 cyclin B was then added and the kinetics of the activation of the Cdc2 kinase was analyzed by measuring the phosphorylation of histone H1 with radioactive γ-ATP. The Cdc2 kinase activity was quantitated and plotted against time (B). The unit for kinase activity is arbitrary. In five separate experiments, the wild-type Chfr protein always delayed the activation of Cdc2 kinase. The levels of the Cdc2 activity at 60 min, from extracts treated with ChfrI306A and ChfrW332A, were slightly variable; in some experiments, these levels were close to, instead of higher than, that of the buffer control. (C) Xenopus mitotic extracts were incubated with a buffer, Chfr, or ChfrI306A, and the level of the Cdc2 kinase activity was analyzed using histone H1 as a substrate.
Mentions: Entry into mitosis can be monitored in Xenopus extracts (Murray and Kirschner, 1989; Solomon et al., 1990). Addition of Δ90 cyclin B, a mutant cyclin B with the first 90 amino acids truncated, to interphase extracts efficiently activated the Cdc2 kinase activity and drove extracts into mitosis (Fig. 5 A, top). However, the addition of wild-type Chfr protein reduced the rate of Cdc2 activation and delayed entry into mitosis (Fig. 5, A and B). This inhibition of Cdc2 kinase activity required an active ligase, because the two inactive mutants, ChfrI306A and ChfrW332A, both failed to delay mitotic entry. Thus, Cdc2 is a key target for the Chfr pathway to control the entry into mitosis.

Bottom Line: Nature. 406:430-435).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA.

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.

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