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DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7.

Yamada A, Duffy B, Perry JA, Kornbluth S - J. Cell Biol. (2004)

Bottom Line: Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction.Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase.These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

ABSTRACT
G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.

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Excess xHsl7 restores inhibition of oocyte maturation induced by Wee1 injection and xHsl7 does not affect Wee1 kinase activity. (A) 50 stage VI oocytes were injected with 40 ng of mRNAs encoding β-globin (open square), FLAG-xHsl7 (open circle), HA-Wee1 (closed square), and 40 ng each of FLAG-xHsl7 and HA-Wee1 mRNAs together (closed circle). After a 12-h incubation, they were treated with progesterone and scored for the percentage of GVBD. (B) Ultra S Xenopus egg extract (purified cytosol) was either mock depleted, depleted of xHsl7, or supplemented with recombinant xHsl7. ATP-regenerating system was then added along with sodium vanadate to inhibit dephosphorylation of Cdc2. Recombinant human cyclin B1 was then added and the reaction was allowed to proceed for 10 min. xWee1 activity was determined by assaying the phosphorylation level of Cdc2 by immunoblotting with anti-phospo Cdc2.
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fig5: Excess xHsl7 restores inhibition of oocyte maturation induced by Wee1 injection and xHsl7 does not affect Wee1 kinase activity. (A) 50 stage VI oocytes were injected with 40 ng of mRNAs encoding β-globin (open square), FLAG-xHsl7 (open circle), HA-Wee1 (closed square), and 40 ng each of FLAG-xHsl7 and HA-Wee1 mRNAs together (closed circle). After a 12-h incubation, they were treated with progesterone and scored for the percentage of GVBD. (B) Ultra S Xenopus egg extract (purified cytosol) was either mock depleted, depleted of xHsl7, or supplemented with recombinant xHsl7. ATP-regenerating system was then added along with sodium vanadate to inhibit dephosphorylation of Cdc2. Recombinant human cyclin B1 was then added and the reaction was allowed to proceed for 10 min. xWee1 activity was determined by assaying the phosphorylation level of Cdc2 by immunoblotting with anti-phospo Cdc2.

Mentions: Given the ability of xHsl7 to bind Wee1, we wished to determine whether the effect of xHsl7 overexpression on mitotic entry was Wee1 mediated. Initially, we found that immunodepletion of Wee1 or addition of excess xHsl7 produced similar accelerations in mitotic entry (unpublished data). These data precluded an experiment in egg extracts to determine whether xHsl7 could accelerate mitotic entry in the absence of Wee1, as the kinetics of cyclin B accumulation in the cycling extract make it difficult to observe cell cycle accelerations greater than those seen with depletion of Wee1 alone. Consequently, to determine if xHsl7 effects were Wee1 mediated, we took advantage of the fact that G2-arrested stage VI Xenopus oocytes, which contain endogenous xHsl7 at levels similar to that found in egg extracts, lack endogenous Wee1 (Nakajo et al., 2000). Oocytes were injected with mRNA for the overexpression of FLAG-tagged xHsl7 and then treated with progesterone, which stimulates entry into M phase, as monitored by breakdown of the nuclear envelope, or germinal vesicle breakdown (GVBD). As shown in Fig. 5 A, when compared with injection of control β-globin mRNA, xHsl7 mRNA injection produced no acceleration in progesterone-induced GVBD. However, when we introduced mRNA encoding Xenopus Wee1 into oocytes, we observed an inhibition of GVBD, likely to due tyrosine phosphorylation of stockpiled Cdc2–cyclin B complexes. This Wee1-induced delay in GVBD could be almost entirely reversed by production of excess xHsl7 (Fig. 5 A), demonstrating that xHsl7 can regulate M phase progression in the oocyte milieu, but only in the presence of Wee1.


DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7.

Yamada A, Duffy B, Perry JA, Kornbluth S - J. Cell Biol. (2004)

Excess xHsl7 restores inhibition of oocyte maturation induced by Wee1 injection and xHsl7 does not affect Wee1 kinase activity. (A) 50 stage VI oocytes were injected with 40 ng of mRNAs encoding β-globin (open square), FLAG-xHsl7 (open circle), HA-Wee1 (closed square), and 40 ng each of FLAG-xHsl7 and HA-Wee1 mRNAs together (closed circle). After a 12-h incubation, they were treated with progesterone and scored for the percentage of GVBD. (B) Ultra S Xenopus egg extract (purified cytosol) was either mock depleted, depleted of xHsl7, or supplemented with recombinant xHsl7. ATP-regenerating system was then added along with sodium vanadate to inhibit dephosphorylation of Cdc2. Recombinant human cyclin B1 was then added and the reaction was allowed to proceed for 10 min. xWee1 activity was determined by assaying the phosphorylation level of Cdc2 by immunoblotting with anti-phospo Cdc2.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172454&req=5

fig5: Excess xHsl7 restores inhibition of oocyte maturation induced by Wee1 injection and xHsl7 does not affect Wee1 kinase activity. (A) 50 stage VI oocytes were injected with 40 ng of mRNAs encoding β-globin (open square), FLAG-xHsl7 (open circle), HA-Wee1 (closed square), and 40 ng each of FLAG-xHsl7 and HA-Wee1 mRNAs together (closed circle). After a 12-h incubation, they were treated with progesterone and scored for the percentage of GVBD. (B) Ultra S Xenopus egg extract (purified cytosol) was either mock depleted, depleted of xHsl7, or supplemented with recombinant xHsl7. ATP-regenerating system was then added along with sodium vanadate to inhibit dephosphorylation of Cdc2. Recombinant human cyclin B1 was then added and the reaction was allowed to proceed for 10 min. xWee1 activity was determined by assaying the phosphorylation level of Cdc2 by immunoblotting with anti-phospo Cdc2.
Mentions: Given the ability of xHsl7 to bind Wee1, we wished to determine whether the effect of xHsl7 overexpression on mitotic entry was Wee1 mediated. Initially, we found that immunodepletion of Wee1 or addition of excess xHsl7 produced similar accelerations in mitotic entry (unpublished data). These data precluded an experiment in egg extracts to determine whether xHsl7 could accelerate mitotic entry in the absence of Wee1, as the kinetics of cyclin B accumulation in the cycling extract make it difficult to observe cell cycle accelerations greater than those seen with depletion of Wee1 alone. Consequently, to determine if xHsl7 effects were Wee1 mediated, we took advantage of the fact that G2-arrested stage VI Xenopus oocytes, which contain endogenous xHsl7 at levels similar to that found in egg extracts, lack endogenous Wee1 (Nakajo et al., 2000). Oocytes were injected with mRNA for the overexpression of FLAG-tagged xHsl7 and then treated with progesterone, which stimulates entry into M phase, as monitored by breakdown of the nuclear envelope, or germinal vesicle breakdown (GVBD). As shown in Fig. 5 A, when compared with injection of control β-globin mRNA, xHsl7 mRNA injection produced no acceleration in progesterone-induced GVBD. However, when we introduced mRNA encoding Xenopus Wee1 into oocytes, we observed an inhibition of GVBD, likely to due tyrosine phosphorylation of stockpiled Cdc2–cyclin B complexes. This Wee1-induced delay in GVBD could be almost entirely reversed by production of excess xHsl7 (Fig. 5 A), demonstrating that xHsl7 can regulate M phase progression in the oocyte milieu, but only in the presence of Wee1.

Bottom Line: Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction.Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase.These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

ABSTRACT
G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.

Show MeSH
Related in: MedlinePlus