Kang D et al. (2002), The checkpoint protein Chfr is a ligase that ub...

XB-ART-7759

J Cell Biol 2002 Jan 21;1562:249-59. doi: 10.1083/jcb.200108016.

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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 .

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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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Species referenced: Xenopus
Genes referenced: cbl ccnb1.2 cdc25c cdk1 chek1 chfr myc plk1 pold1 rasgrf1 wee1

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	Figure 1. Chfr is a ubiquitin ligase. (A) MycâChfr (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 IâIII 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 ChfrâUb 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.
	Figure 2. The ring finger domain is sufficient to mediate the auto-ubiquitination reaction. (A) A schematic diagram for the domain structure of the Chfr protein and the deletion constructs used in this paper. (B) Chfr deletion mutants were expressed in E. coli and their expression levels were assayed by 12% SDS-PAGE. Lane 2, lysates of E.coli without Chfr mutant proteins. (C) Equal amounts of E. coli lysates expressing various recombinant proteins were incubated with radioactive ubiquitin in the presence of recombinant E1 and Ubc4 and the formation of ubiquitin conjugates was analyzed by reducing SDS-PAGE. (D) Purified ChfrF1 (400 Î¼g/ml final concentration), ChfrF2 (400 Î¼g/ml), and GSTâChfrF3 (200 Î¼g/ml) were incubated with radioactive ubiquitin in the presence of recombinant E1 and Ubc4 and the kinetics of ubiquitin conjugate formation was analyzed.
	Figure 3. 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. (CâE) MycâChfr (lanes 2), MycâChfrI306A (lanes 3), MycâChfrW332A (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.
	Figure 4. Chfr functions with Ubc4 and Ubc5, but not with UbcH7 and UbcH10. (A) A thioester assay for the activity of recombinant ubiquitin-conjugating enzymes. Recombinant E2s were incubated with E1 and then analyzed by nonreducing SDS-PAGE for E1 and E2s conjugated to ubiquitin through a thioester bond. Lane 1, E1 alone. (B) Purified recombinant Chfr (lanes 2â7) was incubated with radioactive ubiquitin in the presence of E1 and various E2s. The formation of the ubiquitinâChfr conjugates was analyzed by reducing SDS-PAGE (top) and the formation of ubiquitinâE2s conjugates linked through a thioester bond was assayed by nonreducing SDS-PAGE (bottom). Under the reaction conditions, all the ChfrâUb conjugates entered the separating gel. Arrow indicates the junction between the stacking and separating gels. Arrowhead points to an E1âUb conjugate that is also present in control lanes (1 and 2). Lane 1, E1 alone. Lane 2, E1 and Chfr.
	Figure 5. Chfr delays the activation of the Cdc2 kinase at the G2-M transition. (A and B) Xenopus interphase extracts were incubated with a buffer, Chfr, ChfrI306A, or ChfrW332A. Î90 cyclin B was then added and the kinetics of the activation of the Cdc2 kinase was analyzed by measuring the phosphorylation of histone H1 with radioactive Î³-ATP. The Cdc2 kinase activity was quantitated and plotted against time (B). The unit for kinase activity is arbitrary. In five separate experiments, the wild-type Chfr protein always delayed the activation of Cdc2 kinase. The levels of the Cdc2 activity at 60 min, from extracts treated with ChfrI306A and ChfrW332A, were slightly variable; in some experiments, these levels were close to, instead of higher than, that of the buffer control. (C) Xenopus mitotic extracts were incubated with a buffer, Chfr, or ChfrI306A, and the level of the Cdc2 kinase activity was analyzed using histone H1 as a substrate.
	Figure 6. 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. Î90 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.
	Figure 7. 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. Î90 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.
	Figure 8. Plk1 is the substrate of Chfr ligase. (A) In vitroâtranslated 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 vitroâtranslated 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. Î90 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 GSTâUb plus a buffer or Chfr. Samples were collected at various time points to directly blot against an anti-Plx1 antibody. In addition, GSTâUb 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. Î90 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.

References [+] :

Buschmann, SUMO-1 modification of Mdm2 prevents its self-ubiquitination and increases Mdm2 ability to ubiquitinate p53. 2000, Pubmed