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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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Plk1 is the substrate of Chfr ligase. (A) In vitro–translated Cdc25, Wee1, and Plk1 were incubated with Xenopus interphase extracts and ubiquitin, either in the presence or absence of recombinant Chfr, and the ubiquitination of these proteins was analyzed by SDS-PAGE. (B) In vitro–translated Cdc25, Wee1, and Plk1 were incubated with recombinant E1, E2, ubiquitin, and ATP, either in the presence or absence of recombinant Chfr, and the ubiquitination of these proteins was analyzed by SDS-PAGE. (C) Xenopus interphase extracts were incubated with a buffer or Chfr. 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. Duplicated samples were analyzed in Western blot with an antibody against Xenopus Plx1. Arrowheads point to nonspecific, cross-reacting bands. (D) Xenopus interphase extracts were incubated with GST–Ub plus a buffer or Chfr. Samples were collected at various time points to directly blot against an anti-Plx1 antibody. In addition, GST–Ub conjugates were purified with glutathione beads and then assayed by Western blot analysis using the anti-Plx1 antibody. Arrowheads point to nonspecific, cross-reacting bands. (E) Xenopus interphase extracts were incubated with recombinant ubiquitin (at 2 mg/ml) plus a buffer or Chfr for 5 min. Δ90 cyclin B was then added and the kinetics of entry into mitosis was measured by the Cdc2 kinase activity and by the phosphorylation state of Cdc2, Cdc25C, and Wee1. The degree of ubiquitination of Plx1 was assayed by Western blotting with an anti-Plx1 antibody. Arrowheads point to nonspecific, cross-reacting bands.
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fig8: Plk1 is the substrate of Chfr ligase. (A) In vitro–translated Cdc25, Wee1, and Plk1 were incubated with Xenopus interphase extracts and ubiquitin, either in the presence or absence of recombinant Chfr, and the ubiquitination of these proteins was analyzed by SDS-PAGE. (B) In vitro–translated Cdc25, Wee1, and Plk1 were incubated with recombinant E1, E2, ubiquitin, and ATP, either in the presence or absence of recombinant Chfr, and the ubiquitination of these proteins was analyzed by SDS-PAGE. (C) Xenopus interphase extracts were incubated with a buffer or Chfr. 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. Duplicated samples were analyzed in Western blot with an antibody against Xenopus Plx1. Arrowheads point to nonspecific, cross-reacting bands. (D) Xenopus interphase extracts were incubated with GST–Ub plus a buffer or Chfr. Samples were collected at various time points to directly blot against an anti-Plx1 antibody. In addition, GST–Ub conjugates were purified with glutathione beads and then assayed by Western blot analysis using the anti-Plx1 antibody. Arrowheads point to nonspecific, cross-reacting bands. (E) Xenopus interphase extracts were incubated with recombinant ubiquitin (at 2 mg/ml) plus a buffer or Chfr for 5 min. Δ90 cyclin B was then added and the kinetics of entry into mitosis was measured by the Cdc2 kinase activity and by the phosphorylation state of Cdc2, Cdc25C, and Wee1. The degree of ubiquitination of Plx1 was assayed by Western blotting with an anti-Plx1 antibody. Arrowheads point to nonspecific, cross-reacting bands.

Mentions: To identify the substrate of the Chfr ligases, we analyzed the ability of Chfr to ubiquitinate regulators of Cdc2 in Xenopus extracts. The addition of recombinant Chfr to interphase extracts did not change the stability of in vitro translated Cdc25C and Wee1 (Fig. 8 A), consistent with the Western blot analysis of the endogenous Cdc25C and Wee1 (Fig. 7). Thus, neither Cdc25C nor Wee1 is the substrate of Chfr. We next tested the stability of Plk1, a positive regulator for Cdc25C and a negative regulator for Wee1 at the G2 to M transition (King et al., 1994). When incubated with interphase extracts, in vitro–translated Plk1 was efficiently ubiquitinated in a Chfr-dependent manner (Fig. 8 A). Indeed, Plk1 is a substrate of Chfr. In an in vitro reconstituted reaction, Plk1, but not Cdc25C and Wee1, was quantitatively ubiquitinated in the presence of purified recombinant E1, Ubc4, and Chfr (Fig. 8 B).


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)

Plk1 is the substrate of Chfr ligase. (A) In vitro–translated Cdc25, Wee1, and Plk1 were incubated with Xenopus interphase extracts and ubiquitin, either in the presence or absence of recombinant Chfr, and the ubiquitination of these proteins was analyzed by SDS-PAGE. (B) In vitro–translated Cdc25, Wee1, and Plk1 were incubated with recombinant E1, E2, ubiquitin, and ATP, either in the presence or absence of recombinant Chfr, and the ubiquitination of these proteins was analyzed by SDS-PAGE. (C) Xenopus interphase extracts were incubated with a buffer or Chfr. 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. Duplicated samples were analyzed in Western blot with an antibody against Xenopus Plx1. Arrowheads point to nonspecific, cross-reacting bands. (D) Xenopus interphase extracts were incubated with GST–Ub plus a buffer or Chfr. Samples were collected at various time points to directly blot against an anti-Plx1 antibody. In addition, GST–Ub conjugates were purified with glutathione beads and then assayed by Western blot analysis using the anti-Plx1 antibody. Arrowheads point to nonspecific, cross-reacting bands. (E) Xenopus interphase extracts were incubated with recombinant ubiquitin (at 2 mg/ml) plus a buffer or Chfr for 5 min. Δ90 cyclin B was then added and the kinetics of entry into mitosis was measured by the Cdc2 kinase activity and by the phosphorylation state of Cdc2, Cdc25C, and Wee1. The degree of ubiquitination of Plx1 was assayed by Western blotting with an anti-Plx1 antibody. Arrowheads point to nonspecific, cross-reacting bands.
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fig8: Plk1 is the substrate of Chfr ligase. (A) In vitro–translated Cdc25, Wee1, and Plk1 were incubated with Xenopus interphase extracts and ubiquitin, either in the presence or absence of recombinant Chfr, and the ubiquitination of these proteins was analyzed by SDS-PAGE. (B) In vitro–translated Cdc25, Wee1, and Plk1 were incubated with recombinant E1, E2, ubiquitin, and ATP, either in the presence or absence of recombinant Chfr, and the ubiquitination of these proteins was analyzed by SDS-PAGE. (C) Xenopus interphase extracts were incubated with a buffer or Chfr. 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. Duplicated samples were analyzed in Western blot with an antibody against Xenopus Plx1. Arrowheads point to nonspecific, cross-reacting bands. (D) Xenopus interphase extracts were incubated with GST–Ub plus a buffer or Chfr. Samples were collected at various time points to directly blot against an anti-Plx1 antibody. In addition, GST–Ub conjugates were purified with glutathione beads and then assayed by Western blot analysis using the anti-Plx1 antibody. Arrowheads point to nonspecific, cross-reacting bands. (E) Xenopus interphase extracts were incubated with recombinant ubiquitin (at 2 mg/ml) plus a buffer or Chfr for 5 min. Δ90 cyclin B was then added and the kinetics of entry into mitosis was measured by the Cdc2 kinase activity and by the phosphorylation state of Cdc2, Cdc25C, and Wee1. The degree of ubiquitination of Plx1 was assayed by Western blotting with an anti-Plx1 antibody. Arrowheads point to nonspecific, cross-reacting bands.
Mentions: To identify the substrate of the Chfr ligases, we analyzed the ability of Chfr to ubiquitinate regulators of Cdc2 in Xenopus extracts. The addition of recombinant Chfr to interphase extracts did not change the stability of in vitro translated Cdc25C and Wee1 (Fig. 8 A), consistent with the Western blot analysis of the endogenous Cdc25C and Wee1 (Fig. 7). Thus, neither Cdc25C nor Wee1 is the substrate of Chfr. We next tested the stability of Plk1, a positive regulator for Cdc25C and a negative regulator for Wee1 at the G2 to M transition (King et al., 1994). When incubated with interphase extracts, in vitro–translated Plk1 was efficiently ubiquitinated in a Chfr-dependent manner (Fig. 8 A). Indeed, Plk1 is a substrate of Chfr. In an in vitro reconstituted reaction, Plk1, but not Cdc25C and Wee1, was quantitatively ubiquitinated in the presence of purified recombinant E1, Ubc4, and Chfr (Fig. 8 B).

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