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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 prolongs the phosphorylated state of Tyr15 in Cdc2 at the G2-M transition. (A) Xenopus interphase extracts were incubated with a buffer or Chfr. Δ90 cyclin B was then added and the activation of the Cdc2 kinase was analyzed by measuring the phosphorylation of histone H1. The kinetics of entry into mitosis was also measured by the level and the phosphorylation state of Cdc2, Cdc25C, Wee1, and Chk1 in Western blot analysis. Arrowheads point to nonspecific, cross-reacting bands. (B) Xenopus cycling extracts were incubated with a buffer or Chfr and the kinetics of entry into mitosis was measured by the Cdc2 kinase activity and by the phosphorylation state of Cdc25C and Wee1. Arrowheads point to nonspecific, cross-reacting bands.
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fig7: Chfr prolongs the phosphorylated state of Tyr15 in Cdc2 at the G2-M transition. (A) Xenopus interphase extracts were incubated with a buffer or Chfr. Δ90 cyclin B was then added and the activation of the Cdc2 kinase was analyzed by measuring the phosphorylation of histone H1. The kinetics of entry into mitosis was also measured by the level and the phosphorylation state of Cdc2, Cdc25C, Wee1, and Chk1 in Western blot analysis. Arrowheads point to nonspecific, cross-reacting bands. (B) Xenopus cycling extracts were incubated with a buffer or Chfr and the kinetics of entry into mitosis was measured by the Cdc2 kinase activity and by the phosphorylation state of Cdc25C and Wee1. Arrowheads point to nonspecific, cross-reacting bands.

Mentions: We investigated how the Cdc2 kinase activity is regulated by Chfr. When recombinant Chfr was incubated with interphase extracts and Δ90 cyclin B, the Chfr pathway delayed the removal of inhibitory phosphorylation on Cdc2, as analyzed by an antibody specific to the tyrosine 15–phosphorylated Cdc2 (Fig. 7 A). 25 min after addition of Δ90 cyclin B, Cdc2 from both the control extracts and the Chfr-treated extracts had significant levels of phosphorylated tyrosine. Interestingly, by 35 min, the level of phosphorylated tyrosine was greatly reduced in control extracts, correlating well with the activation of the Cdc2 kinase (Fig. 7 A). On the other hand, the level of phosphorylated tyrosine in Chfr-treated extracts remained very high, consistent with the observation that the Cdc2 kinase was not activated until 45 min after the addition of Δ90 cyclin B (Fig. 7 A). We conclude that Chfr delays mitotic entry by prolonging the phosphorylated state of Cdc2.


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 prolongs the phosphorylated state of Tyr15 in Cdc2 at the G2-M transition. (A) Xenopus interphase extracts were incubated with a buffer or Chfr. Δ90 cyclin B was then added and the activation of the Cdc2 kinase was analyzed by measuring the phosphorylation of histone H1. The kinetics of entry into mitosis was also measured by the level and the phosphorylation state of Cdc2, Cdc25C, Wee1, and Chk1 in Western blot analysis. Arrowheads point to nonspecific, cross-reacting bands. (B) Xenopus cycling extracts were incubated with a buffer or Chfr and the kinetics of entry into mitosis was measured by the Cdc2 kinase activity and by the phosphorylation state of Cdc25C and Wee1. Arrowheads point to nonspecific, cross-reacting bands.
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Related In: Results  -  Collection

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fig7: Chfr prolongs the phosphorylated state of Tyr15 in Cdc2 at the G2-M transition. (A) Xenopus interphase extracts were incubated with a buffer or Chfr. Δ90 cyclin B was then added and the activation of the Cdc2 kinase was analyzed by measuring the phosphorylation of histone H1. The kinetics of entry into mitosis was also measured by the level and the phosphorylation state of Cdc2, Cdc25C, Wee1, and Chk1 in Western blot analysis. Arrowheads point to nonspecific, cross-reacting bands. (B) Xenopus cycling extracts were incubated with a buffer or Chfr and the kinetics of entry into mitosis was measured by the Cdc2 kinase activity and by the phosphorylation state of Cdc25C and Wee1. Arrowheads point to nonspecific, cross-reacting bands.
Mentions: We investigated how the Cdc2 kinase activity is regulated by Chfr. When recombinant Chfr was incubated with interphase extracts and Δ90 cyclin B, the Chfr pathway delayed the removal of inhibitory phosphorylation on Cdc2, as analyzed by an antibody specific to the tyrosine 15–phosphorylated Cdc2 (Fig. 7 A). 25 min after addition of Δ90 cyclin B, Cdc2 from both the control extracts and the Chfr-treated extracts had significant levels of phosphorylated tyrosine. Interestingly, by 35 min, the level of phosphorylated tyrosine was greatly reduced in control extracts, correlating well with the activation of the Cdc2 kinase (Fig. 7 A). On the other hand, the level of phosphorylated tyrosine in Chfr-treated extracts remained very high, consistent with the observation that the Cdc2 kinase was not activated until 45 min after the addition of Δ90 cyclin B (Fig. 7 A). We conclude that Chfr delays mitotic entry by prolonging the phosphorylated state of Cdc2.

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.

Show MeSH