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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-mediated inhibition of Cdc2 requires ubiquitin-dependent protein degradation. (A) Xenopus interphase extracts were incubated with a buffer or Chfr in the presence or absence of methyl-ubiquitin and LLnL. Δ90 cyclin B was then added and the kinetics of the activation of the Cdc2 kinase was analyzed using histone H1 as a substrate. The exact kinetics of activation of Cdc2 in buffer control here are different from that in Fig. 5 due to variations between different extracts. (B) Methyl-ubiquitin and LLnL effectively inhibit ubiquitin-dependent proteolysis. Xenopus mitotic extracts were incubated with a buffer, methyl-ubiquitin, or LLnL. Radioactive cyclin B was then added and the kinetics of its degradation was analyzed by SDS-PAGE.
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fig6: Chfr-mediated inhibition of Cdc2 requires ubiquitin-dependent protein degradation. (A) Xenopus interphase extracts were incubated with a buffer or Chfr in the presence or absence of methyl-ubiquitin and LLnL. Δ90 cyclin B was then added and the kinetics of the activation of the Cdc2 kinase was analyzed using histone H1 as a substrate. The exact kinetics of activation of Cdc2 in buffer control here are different from that in Fig. 5 due to variations between different extracts. (B) Methyl-ubiquitin and LLnL effectively inhibit ubiquitin-dependent proteolysis. Xenopus mitotic extracts were incubated with a buffer, methyl-ubiquitin, or LLnL. Radioactive cyclin B was then added and the kinetics of its degradation was analyzed by SDS-PAGE.

Mentions: Chfr is a ubiquitin ligase and addition of an active ligase to extracts could have a nonspecific effect on the entry into mitosis if there is a specific requirement for ubiquitin-dependent proteolysis in the G2 to M transition. For example, active Chfr ligase may nonspecifically interfere with physiological ubiquitin-dependent proteolysis by depleting the ubiquitin pool or by competing for proteasome degradation in extracts. To establish whether there is a requirement for ubiquitin-dependent proteolysis at the G2 to M transition, we examined the effect of methylated ubiquitin (methyl-ubiquitin) and N-acetyl-Leu-Leu-norleucinal peptide (LLnL) on entry into mitosis. Methyl-ubiquitin interferes with the formation of poly-ubiquitin conjugates required for degradation and LLnL is an inhibitor of proteasomes. Neither methyl-ubiquitin nor LLnL delayed the mitotic entry, as measured by the kinetics of activation of the Cdc2 kinase (Fig. 6 A, left). The amount of methyl-ubiquitin and LLnL used in these experiments, when added to mitotic extracts, reduced the rate of cyclin degradation (Fig. 6 B), suggesting that both methyl-ubiquitin and LLnL interfere with ubiquitin-dependent proteolysis pathways in Xenopus extracts. We conclude that there is no requirement for ubiquitin-dependent proteolysis at the G2 to M transition under normal physiological conditions.


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-mediated inhibition of Cdc2 requires ubiquitin-dependent protein degradation. (A) Xenopus interphase extracts were incubated with a buffer or Chfr in the presence or absence of methyl-ubiquitin and LLnL. Δ90 cyclin B was then added and the kinetics of the activation of the Cdc2 kinase was analyzed using histone H1 as a substrate. The exact kinetics of activation of Cdc2 in buffer control here are different from that in Fig. 5 due to variations between different extracts. (B) Methyl-ubiquitin and LLnL effectively inhibit ubiquitin-dependent proteolysis. Xenopus mitotic extracts were incubated with a buffer, methyl-ubiquitin, or LLnL. Radioactive cyclin B was then added and the kinetics of its degradation was analyzed by SDS-PAGE.
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fig6: Chfr-mediated inhibition of Cdc2 requires ubiquitin-dependent protein degradation. (A) Xenopus interphase extracts were incubated with a buffer or Chfr in the presence or absence of methyl-ubiquitin and LLnL. Δ90 cyclin B was then added and the kinetics of the activation of the Cdc2 kinase was analyzed using histone H1 as a substrate. The exact kinetics of activation of Cdc2 in buffer control here are different from that in Fig. 5 due to variations between different extracts. (B) Methyl-ubiquitin and LLnL effectively inhibit ubiquitin-dependent proteolysis. Xenopus mitotic extracts were incubated with a buffer, methyl-ubiquitin, or LLnL. Radioactive cyclin B was then added and the kinetics of its degradation was analyzed by SDS-PAGE.
Mentions: Chfr is a ubiquitin ligase and addition of an active ligase to extracts could have a nonspecific effect on the entry into mitosis if there is a specific requirement for ubiquitin-dependent proteolysis in the G2 to M transition. For example, active Chfr ligase may nonspecifically interfere with physiological ubiquitin-dependent proteolysis by depleting the ubiquitin pool or by competing for proteasome degradation in extracts. To establish whether there is a requirement for ubiquitin-dependent proteolysis at the G2 to M transition, we examined the effect of methylated ubiquitin (methyl-ubiquitin) and N-acetyl-Leu-Leu-norleucinal peptide (LLnL) on entry into mitosis. Methyl-ubiquitin interferes with the formation of poly-ubiquitin conjugates required for degradation and LLnL is an inhibitor of proteasomes. Neither methyl-ubiquitin nor LLnL delayed the mitotic entry, as measured by the kinetics of activation of the Cdc2 kinase (Fig. 6 A, left). The amount of methyl-ubiquitin and LLnL used in these experiments, when added to mitotic extracts, reduced the rate of cyclin degradation (Fig. 6 B), suggesting that both methyl-ubiquitin and LLnL interfere with ubiquitin-dependent proteolysis pathways in Xenopus extracts. We conclude that there is no requirement for ubiquitin-dependent proteolysis at the G2 to M transition under normal physiological conditions.

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
Related in: MedlinePlus