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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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Xenopus Hsl7 interacts with Wee1 in Xenopus egg extracts. (A) HeLa cell lysate (10 μl), Xenopus egg extracts (2 μl), or GST-xHsl7 were resolved by SDS-PAGE and immunoblotted with anti-JBP1. (B) Immunoprecipitates formed using affinity-purified anti-Wee1 or control IgG were immunoblotted with anti-JBP1. (C) GST or GST-xHsl7 coupled to glutathione Sepharose was incubated in Xenopus egg extract and immunoblotted with anti-xWee1.
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fig3: Xenopus Hsl7 interacts with Wee1 in Xenopus egg extracts. (A) HeLa cell lysate (10 μl), Xenopus egg extracts (2 μl), or GST-xHsl7 were resolved by SDS-PAGE and immunoblotted with anti-JBP1. (B) Immunoprecipitates formed using affinity-purified anti-Wee1 or control IgG were immunoblotted with anti-JBP1. (C) GST or GST-xHsl7 coupled to glutathione Sepharose was incubated in Xenopus egg extract and immunoblotted with anti-xWee1.

Mentions: Using a commercially available antibody directed against the human Hsl7 homologue, JBP1, we were readily able to detect endogenous xHsl7 (Fig. 3 A). Moreover, this antibody could detect xHsl7 co-immunoprecipitated with endogenous Wee1 (Fig. 3 B). This interaction was further confirmed by retrieval of Wee1 from egg extracts by recombinant GST–xHsl7 linked to glutatione sepharose (Fig. 3 C). These data suggested that xHsl7 could positively regulate mitotic entry, perhaps through an interaction with Wee1. Given these data, we wished to assess the consequences of xHsl7 removal or inactivation on entry into mitosis. Therefore, we used Hsl7 antibodies to immunodeplete xHsl7 from cycling extracts. As shown in Fig. 4 A, extracts depleted with control IgG underwent two rounds of mitosis, as measured by peaks of histone H1-directed kinase activity, whereas extracts depleted of xHsl7 were entirely blocked in their ability to enter mitosis. To examine the role of xHsl7 using another approach, we added anti-Hsl7 antibodies directly to cycling extracts with the goal of interfering with xHsl7 function. Consistent with a role for xHsl7 in promoting mitotic entry, we found that addition of purified anti-Hsl7 antibodies, but not control IgG, to cycling egg extracts was able to significantly delay entry into mitosis as measured by histone H1-directed kinase activity assays and microsocopic observation of chromsome condensation/nuclear envelope breakdown (Fig. 4, B and C). Moreover, readdition of recombinant xHsl7 restored the normal timing of mitotic entry, demonstrating that the effects of the anti-Hsl7 antibody were specific (Fig. 4, B and C). These data suggest that xHsl7 is required for efficient entry into M phase in Xenopus.


DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7.

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

Xenopus Hsl7 interacts with Wee1 in Xenopus egg extracts. (A) HeLa cell lysate (10 μl), Xenopus egg extracts (2 μl), or GST-xHsl7 were resolved by SDS-PAGE and immunoblotted with anti-JBP1. (B) Immunoprecipitates formed using affinity-purified anti-Wee1 or control IgG were immunoblotted with anti-JBP1. (C) GST or GST-xHsl7 coupled to glutathione Sepharose was incubated in Xenopus egg extract and immunoblotted with anti-xWee1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172454&req=5

fig3: Xenopus Hsl7 interacts with Wee1 in Xenopus egg extracts. (A) HeLa cell lysate (10 μl), Xenopus egg extracts (2 μl), or GST-xHsl7 were resolved by SDS-PAGE and immunoblotted with anti-JBP1. (B) Immunoprecipitates formed using affinity-purified anti-Wee1 or control IgG were immunoblotted with anti-JBP1. (C) GST or GST-xHsl7 coupled to glutathione Sepharose was incubated in Xenopus egg extract and immunoblotted with anti-xWee1.
Mentions: Using a commercially available antibody directed against the human Hsl7 homologue, JBP1, we were readily able to detect endogenous xHsl7 (Fig. 3 A). Moreover, this antibody could detect xHsl7 co-immunoprecipitated with endogenous Wee1 (Fig. 3 B). This interaction was further confirmed by retrieval of Wee1 from egg extracts by recombinant GST–xHsl7 linked to glutatione sepharose (Fig. 3 C). These data suggested that xHsl7 could positively regulate mitotic entry, perhaps through an interaction with Wee1. Given these data, we wished to assess the consequences of xHsl7 removal or inactivation on entry into mitosis. Therefore, we used Hsl7 antibodies to immunodeplete xHsl7 from cycling extracts. As shown in Fig. 4 A, extracts depleted with control IgG underwent two rounds of mitosis, as measured by peaks of histone H1-directed kinase activity, whereas extracts depleted of xHsl7 were entirely blocked in their ability to enter mitosis. To examine the role of xHsl7 using another approach, we added anti-Hsl7 antibodies directly to cycling extracts with the goal of interfering with xHsl7 function. Consistent with a role for xHsl7 in promoting mitotic entry, we found that addition of purified anti-Hsl7 antibodies, but not control IgG, to cycling egg extracts was able to significantly delay entry into mitosis as measured by histone H1-directed kinase activity assays and microsocopic observation of chromsome condensation/nuclear envelope breakdown (Fig. 4, B and C). Moreover, readdition of recombinant xHsl7 restored the normal timing of mitotic entry, demonstrating that the effects of the anti-Hsl7 antibody were specific (Fig. 4, B and C). These data suggest that xHsl7 is required for efficient entry into M phase in Xenopus.

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