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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 is a ubiquitin ligase. (A) Myc–Chfr (lanes 2) and control vector (lanes 1) were transfected into HEK293T cells and immunoprecipitated by an anti-Myc antibody. The immunoprecipitates were analyzed by Western blotting with an anti-Myc antibody (panel I) or with an anti-ubiquitin antibody (panel II). In addition, immunoprecipitates were incubated with radioactive ubiquitin in the presence of recombinant E1 and Ubc4 and assayed for ubiquitin ligase activity (panel III). The molecular weight markers for panels I–III are labeled on the left side of panel I. (B) Purified recombinant Chfr protein assayed by 12% SDS-PAGE. (C) Recombinant Chfr, at indicated final concentrations, was incubated with radioactive ubiquitin in the presence of E1 and Ubc4. The kinetics of the formation of the Chfr–Ub conjugates was assayed by 12% reducing SDS-PAGE. The arrows point to the wells of the stacking gel and the arrowheads indicate the junction between stacking and separation gels.
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fig1: Chfr is a ubiquitin ligase. (A) Myc–Chfr (lanes 2) and control vector (lanes 1) were transfected into HEK293T cells and immunoprecipitated by an anti-Myc antibody. The immunoprecipitates were analyzed by Western blotting with an anti-Myc antibody (panel I) or with an anti-ubiquitin antibody (panel II). In addition, immunoprecipitates were incubated with radioactive ubiquitin in the presence of recombinant E1 and Ubc4 and assayed for ubiquitin ligase activity (panel III). The molecular weight markers for panels I–III are labeled on the left side of panel I. (B) Purified recombinant Chfr protein assayed by 12% SDS-PAGE. (C) Recombinant Chfr, at indicated final concentrations, was incubated with radioactive ubiquitin in the presence of E1 and Ubc4. The kinetics of the formation of the Chfr–Ub conjugates was assayed by 12% reducing SDS-PAGE. The arrows point to the wells of the stacking gel and the arrowheads indicate the junction between stacking and separation gels.

Mentions: To test whether Chfr is a ubiquitin ligase, we transfected a Myc-tagged Chfr gene into HEK293T cells (Fig. 1 A, panel I). Myc–Chfr was immunopurified by an anti-Myc antibody and assayed by Western blotting with an anti-ubiquitin antibody. The Chfr immunoprecipitate, but not the immunoprecipitate from control transfected cells, contained a ubiquitinated protein(s) (Fig. 1 A, panel II). It remains to be determined whether the ubiquitinated protein is Chfr itself or another protein associated with Chfr. Although the majority of the Myc–Chfr protein did not comigrate with the ubiquitinated species (Fig. 1 A, compare panels I and II), we cannot exclude the possibility that only a minor portion of Myc–Chfr was ubiquitinated, and this minor species escaped detection in panel I.


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 is a ubiquitin ligase. (A) Myc–Chfr (lanes 2) and control vector (lanes 1) were transfected into HEK293T cells and immunoprecipitated by an anti-Myc antibody. The immunoprecipitates were analyzed by Western blotting with an anti-Myc antibody (panel I) or with an anti-ubiquitin antibody (panel II). In addition, immunoprecipitates were incubated with radioactive ubiquitin in the presence of recombinant E1 and Ubc4 and assayed for ubiquitin ligase activity (panel III). The molecular weight markers for panels I–III are labeled on the left side of panel I. (B) Purified recombinant Chfr protein assayed by 12% SDS-PAGE. (C) Recombinant Chfr, at indicated final concentrations, was incubated with radioactive ubiquitin in the presence of E1 and Ubc4. The kinetics of the formation of the Chfr–Ub conjugates was assayed by 12% reducing SDS-PAGE. The arrows point to the wells of the stacking gel and the arrowheads indicate the junction between stacking and separation gels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199220&req=5

fig1: Chfr is a ubiquitin ligase. (A) Myc–Chfr (lanes 2) and control vector (lanes 1) were transfected into HEK293T cells and immunoprecipitated by an anti-Myc antibody. The immunoprecipitates were analyzed by Western blotting with an anti-Myc antibody (panel I) or with an anti-ubiquitin antibody (panel II). In addition, immunoprecipitates were incubated with radioactive ubiquitin in the presence of recombinant E1 and Ubc4 and assayed for ubiquitin ligase activity (panel III). The molecular weight markers for panels I–III are labeled on the left side of panel I. (B) Purified recombinant Chfr protein assayed by 12% SDS-PAGE. (C) Recombinant Chfr, at indicated final concentrations, was incubated with radioactive ubiquitin in the presence of E1 and Ubc4. The kinetics of the formation of the Chfr–Ub conjugates was assayed by 12% reducing SDS-PAGE. The arrows point to the wells of the stacking gel and the arrowheads indicate the junction between stacking and separation gels.
Mentions: To test whether Chfr is a ubiquitin ligase, we transfected a Myc-tagged Chfr gene into HEK293T cells (Fig. 1 A, panel I). Myc–Chfr was immunopurified by an anti-Myc antibody and assayed by Western blotting with an anti-ubiquitin antibody. The Chfr immunoprecipitate, but not the immunoprecipitate from control transfected cells, contained a ubiquitinated protein(s) (Fig. 1 A, panel II). It remains to be determined whether the ubiquitinated protein is Chfr itself or another protein associated with Chfr. Although the majority of the Myc–Chfr protein did not comigrate with the ubiquitinated species (Fig. 1 A, compare panels I and II), we cannot exclude the possibility that only a minor portion of Myc–Chfr was ubiquitinated, and this minor species escaped detection in panel I.

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