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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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The ring finger domain is required for the Chfr ligase activity. (A) Alignment of the ring finger domains from APC11, Chfr, and c-Cbl. (B) Purified recombinant Chfr, ChfrI306A, and ChfrW332A, at concentrations indicated, were incubated with radioactive ubiquitin in the presence of E1 and Ubc4, and the kinetics of ubiquitin conjugate formation was followed by reducing SDS-PAGE. At the Chfr concentrations used here, all the ubiquitin conjugates stayed in the wells of the stacking gel and therefore only that portion of the gel is shown. (C–E) Myc–Chfr (lanes 2), Myc–ChfrI306A (lanes 3), Myc–ChfrW332A (lanes 4), 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 (C) or with an anti-ubiquitin antibody (D). In addition, immunoprecipitates were incubated with radioactive ubiquitin in the presence of recombinant E1 and Ubc4 and assayed for ubiquitin ligase activity (E). Arrow in E points to the wells of the stacking gel.
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fig3: The ring finger domain is required for the Chfr ligase activity. (A) Alignment of the ring finger domains from APC11, Chfr, and c-Cbl. (B) Purified recombinant Chfr, ChfrI306A, and ChfrW332A, at concentrations indicated, were incubated with radioactive ubiquitin in the presence of E1 and Ubc4, and the kinetics of ubiquitin conjugate formation was followed by reducing SDS-PAGE. At the Chfr concentrations used here, all the ubiquitin conjugates stayed in the wells of the stacking gel and therefore only that portion of the gel is shown. (C–E) Myc–Chfr (lanes 2), Myc–ChfrI306A (lanes 3), Myc–ChfrW332A (lanes 4), 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 (C) or with an anti-ubiquitin antibody (D). In addition, immunoprecipitates were incubated with radioactive ubiquitin in the presence of recombinant E1 and Ubc4 and assayed for ubiquitin ligase activity (E). Arrow in E points to the wells of the stacking gel.

Mentions: To confirm the importance of the RF domain in the ligase activity, we constructed two point mutations in the RF domain (Fig. 3 A). The mutated residues, Ile at amino acid 306 and Trp at amino acid 332, are well conserved within a subgroup of RF domains that act as ubiquitin ligases (Fig. 3 A). ChfrI306A and ChfrW332A were expressed in Sf9 cells and purified to homogeneity. When analyzed for auto-ubiquitination activity, both mutants at a low protein concentration (40 μg/ml) failed to auto-ubiquitinate, whereas the wild-type Chfr protein efficiently auto-ubiquitinated (Fig. 3 B). However, the mutant proteins did self-ubiquitinate at a higher protein concentration (200 μg/ml), albeit at a still lower efficiency than the wild-type protein.


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)

The ring finger domain is required for the Chfr ligase activity. (A) Alignment of the ring finger domains from APC11, Chfr, and c-Cbl. (B) Purified recombinant Chfr, ChfrI306A, and ChfrW332A, at concentrations indicated, were incubated with radioactive ubiquitin in the presence of E1 and Ubc4, and the kinetics of ubiquitin conjugate formation was followed by reducing SDS-PAGE. At the Chfr concentrations used here, all the ubiquitin conjugates stayed in the wells of the stacking gel and therefore only that portion of the gel is shown. (C–E) Myc–Chfr (lanes 2), Myc–ChfrI306A (lanes 3), Myc–ChfrW332A (lanes 4), 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 (C) or with an anti-ubiquitin antibody (D). In addition, immunoprecipitates were incubated with radioactive ubiquitin in the presence of recombinant E1 and Ubc4 and assayed for ubiquitin ligase activity (E). Arrow in E points to the wells of the stacking gel.
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fig3: The ring finger domain is required for the Chfr ligase activity. (A) Alignment of the ring finger domains from APC11, Chfr, and c-Cbl. (B) Purified recombinant Chfr, ChfrI306A, and ChfrW332A, at concentrations indicated, were incubated with radioactive ubiquitin in the presence of E1 and Ubc4, and the kinetics of ubiquitin conjugate formation was followed by reducing SDS-PAGE. At the Chfr concentrations used here, all the ubiquitin conjugates stayed in the wells of the stacking gel and therefore only that portion of the gel is shown. (C–E) Myc–Chfr (lanes 2), Myc–ChfrI306A (lanes 3), Myc–ChfrW332A (lanes 4), 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 (C) or with an anti-ubiquitin antibody (D). In addition, immunoprecipitates were incubated with radioactive ubiquitin in the presence of recombinant E1 and Ubc4 and assayed for ubiquitin ligase activity (E). Arrow in E points to the wells of the stacking gel.
Mentions: To confirm the importance of the RF domain in the ligase activity, we constructed two point mutations in the RF domain (Fig. 3 A). The mutated residues, Ile at amino acid 306 and Trp at amino acid 332, are well conserved within a subgroup of RF domains that act as ubiquitin ligases (Fig. 3 A). ChfrI306A and ChfrW332A were expressed in Sf9 cells and purified to homogeneity. When analyzed for auto-ubiquitination activity, both mutants at a low protein concentration (40 μg/ml) failed to auto-ubiquitinate, whereas the wild-type Chfr protein efficiently auto-ubiquitinated (Fig. 3 B). However, the mutant proteins did self-ubiquitinate at a higher protein concentration (200 μg/ml), albeit at a still lower efficiency than the wild-type protein.

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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