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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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Hsl7 promotes intranuclear Wee1 degradation. (A) Xenopus stage VI oocytes were injected with 40 ng of β-globin or FLAG-xHsl7 mRNAs and incubated in the presence of 50 μM roscovitine. 12 h later, they were injected again with 35S-labeled Wee1 protein. After the second injection, they were treated with progesterone (1 μM) and roscovitine in modified Barth's + Ca buffer. At the indicated times after treatment, five oocytes were manually dissected into nuclear (left) and cytoplasmic (right) fractions, and analyzed by SDS-PAGE and autoradiography. The graph represents a quantitation of the data above showing the fraction of Wee1 remaining in nuclei or cytosol. Square, β-globin; circle, FLAG-xHsl7 mRNA injection. The bars to the left of the panel denote the mobility shift of Wee1. (B) 40 ng mRNAs of FLAG-xHsl7 (circle) and FLAG-xHsl7 metyltransferase mutant (triangle) were injected into oocytes. The Wee1 degradation assay was performed as described in panel A. The graph shows the fraction of Wee1 remaining in nuclei. (C) Oocytes were injected as in panel A, but after the second injection, they were treated with leptomycin B (200 nM) for 2 h. Progesterone (1 μM) was then added to induce oocyte maturation. At the indicated times after progesterone treatment, five oocytes were manually fractionated and analyzed by SDS-PAGE followed by autoradiography. The graph shows the fraction of Wee1 remaining in nuclei. Square, β-globin; circle, FLAG-xHsl7 mRNA injection. (D) The experiment in Fig. 5 A was repeated as described except that MG132 was injected into the oocytes along with the indicated mRNAs. Open square, β-globin; open circle, FLAG-xHsl7; closed square, HA-Wee1 mRNA injection; closed circle, FLAG-xHsl7 and HA-Wee1 mRNAs coinjection. (E) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2 and 3) were incubated in cycling extracts in the presence (lanes 1 and 3) or absence (lane 2) of sperm chromatin. After 60 min, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting. (F) Interphase (lanes 1 and 3) or mitotic (lane 2) egg extracts were incubated in the presence of sperm chromatin for 60 min, and immunoprecipitated with anti-Wee1 antibody (lanes 1 and 2) or control antibody (lane 3) bound to protein A–Sepharose beads. Immunoprecipitates were then blotted with anti-Hsl7 antibodies.
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fig6: Hsl7 promotes intranuclear Wee1 degradation. (A) Xenopus stage VI oocytes were injected with 40 ng of β-globin or FLAG-xHsl7 mRNAs and incubated in the presence of 50 μM roscovitine. 12 h later, they were injected again with 35S-labeled Wee1 protein. After the second injection, they were treated with progesterone (1 μM) and roscovitine in modified Barth's + Ca buffer. At the indicated times after treatment, five oocytes were manually dissected into nuclear (left) and cytoplasmic (right) fractions, and analyzed by SDS-PAGE and autoradiography. The graph represents a quantitation of the data above showing the fraction of Wee1 remaining in nuclei or cytosol. Square, β-globin; circle, FLAG-xHsl7 mRNA injection. The bars to the left of the panel denote the mobility shift of Wee1. (B) 40 ng mRNAs of FLAG-xHsl7 (circle) and FLAG-xHsl7 metyltransferase mutant (triangle) were injected into oocytes. The Wee1 degradation assay was performed as described in panel A. The graph shows the fraction of Wee1 remaining in nuclei. (C) Oocytes were injected as in panel A, but after the second injection, they were treated with leptomycin B (200 nM) for 2 h. Progesterone (1 μM) was then added to induce oocyte maturation. At the indicated times after progesterone treatment, five oocytes were manually fractionated and analyzed by SDS-PAGE followed by autoradiography. The graph shows the fraction of Wee1 remaining in nuclei. Square, β-globin; circle, FLAG-xHsl7 mRNA injection. (D) The experiment in Fig. 5 A was repeated as described except that MG132 was injected into the oocytes along with the indicated mRNAs. Open square, β-globin; open circle, FLAG-xHsl7; closed square, HA-Wee1 mRNA injection; closed circle, FLAG-xHsl7 and HA-Wee1 mRNAs coinjection. (E) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2 and 3) were incubated in cycling extracts in the presence (lanes 1 and 3) or absence (lane 2) of sperm chromatin. After 60 min, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting. (F) Interphase (lanes 1 and 3) or mitotic (lane 2) egg extracts were incubated in the presence of sperm chromatin for 60 min, and immunoprecipitated with anti-Wee1 antibody (lanes 1 and 2) or control antibody (lane 3) bound to protein A–Sepharose beads. Immunoprecipitates were then blotted with anti-Hsl7 antibodies.

Mentions: It has been reported that the DNA replication checkpoint stabilizes Wee1 within nuclei, where it would otherwise be degraded at the time of mitotic entry (Michael and Newport, 1998). In examining xHsl7-mediated Wee1 degradation in oocytes injected with both excess xHsl7 mRNA and radiolabeled in vitro translated Wee1, we noted that xHsl7 overproduction promoted specific loss of the nuclear Wee1, as detected by SDS-PAGE and autoradiography of manually dissected oocyte nuclei (Fig. 6 A; nuclear envelope breakdown was prevented by addition of the Cdc2 inhibitor roscovitine). Although Wee1 was also lost from the cytoplasm during these experiments, this loss was not stimulated by xHsl7 overproduction. Moreover, since Wee1 is rapidly transported into oocyte nuclei, we suspect that much of the loss of cytoplasmic Wee1 reflects nuclear import (unpublished data). As in S. cerevisiae, xHsl7-stimulated loss of Wee1 was not abrogated by disruption of the well-conserved residues responsible for methyltransferase activity (Fig. 6 B), nor was it inhibited by leptomycin B treatment to stop Crm1-mediated nuclear export (Fig. 6 C; note the increase in nuclear Wee1 in control injections due to inhibition of nuclear export. Xenopus Hsl7 injection counteracts even the added burden of imported Wee1 that cannot be exported). These data are consistent with data reported previously suggesting intranuclear Wee1 degradation, particularly as nuclear export of Swe1 depends on the Crm1 homologue, Expo1 (Lew, D.J., personal communication).


DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7.

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

Hsl7 promotes intranuclear Wee1 degradation. (A) Xenopus stage VI oocytes were injected with 40 ng of β-globin or FLAG-xHsl7 mRNAs and incubated in the presence of 50 μM roscovitine. 12 h later, they were injected again with 35S-labeled Wee1 protein. After the second injection, they were treated with progesterone (1 μM) and roscovitine in modified Barth's + Ca buffer. At the indicated times after treatment, five oocytes were manually dissected into nuclear (left) and cytoplasmic (right) fractions, and analyzed by SDS-PAGE and autoradiography. The graph represents a quantitation of the data above showing the fraction of Wee1 remaining in nuclei or cytosol. Square, β-globin; circle, FLAG-xHsl7 mRNA injection. The bars to the left of the panel denote the mobility shift of Wee1. (B) 40 ng mRNAs of FLAG-xHsl7 (circle) and FLAG-xHsl7 metyltransferase mutant (triangle) were injected into oocytes. The Wee1 degradation assay was performed as described in panel A. The graph shows the fraction of Wee1 remaining in nuclei. (C) Oocytes were injected as in panel A, but after the second injection, they were treated with leptomycin B (200 nM) for 2 h. Progesterone (1 μM) was then added to induce oocyte maturation. At the indicated times after progesterone treatment, five oocytes were manually fractionated and analyzed by SDS-PAGE followed by autoradiography. The graph shows the fraction of Wee1 remaining in nuclei. Square, β-globin; circle, FLAG-xHsl7 mRNA injection. (D) The experiment in Fig. 5 A was repeated as described except that MG132 was injected into the oocytes along with the indicated mRNAs. Open square, β-globin; open circle, FLAG-xHsl7; closed square, HA-Wee1 mRNA injection; closed circle, FLAG-xHsl7 and HA-Wee1 mRNAs coinjection. (E) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2 and 3) were incubated in cycling extracts in the presence (lanes 1 and 3) or absence (lane 2) of sperm chromatin. After 60 min, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting. (F) Interphase (lanes 1 and 3) or mitotic (lane 2) egg extracts were incubated in the presence of sperm chromatin for 60 min, and immunoprecipitated with anti-Wee1 antibody (lanes 1 and 2) or control antibody (lane 3) bound to protein A–Sepharose beads. Immunoprecipitates were then blotted with anti-Hsl7 antibodies.
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fig6: Hsl7 promotes intranuclear Wee1 degradation. (A) Xenopus stage VI oocytes were injected with 40 ng of β-globin or FLAG-xHsl7 mRNAs and incubated in the presence of 50 μM roscovitine. 12 h later, they were injected again with 35S-labeled Wee1 protein. After the second injection, they were treated with progesterone (1 μM) and roscovitine in modified Barth's + Ca buffer. At the indicated times after treatment, five oocytes were manually dissected into nuclear (left) and cytoplasmic (right) fractions, and analyzed by SDS-PAGE and autoradiography. The graph represents a quantitation of the data above showing the fraction of Wee1 remaining in nuclei or cytosol. Square, β-globin; circle, FLAG-xHsl7 mRNA injection. The bars to the left of the panel denote the mobility shift of Wee1. (B) 40 ng mRNAs of FLAG-xHsl7 (circle) and FLAG-xHsl7 metyltransferase mutant (triangle) were injected into oocytes. The Wee1 degradation assay was performed as described in panel A. The graph shows the fraction of Wee1 remaining in nuclei. (C) Oocytes were injected as in panel A, but after the second injection, they were treated with leptomycin B (200 nM) for 2 h. Progesterone (1 μM) was then added to induce oocyte maturation. At the indicated times after progesterone treatment, five oocytes were manually fractionated and analyzed by SDS-PAGE followed by autoradiography. The graph shows the fraction of Wee1 remaining in nuclei. Square, β-globin; circle, FLAG-xHsl7 mRNA injection. (D) The experiment in Fig. 5 A was repeated as described except that MG132 was injected into the oocytes along with the indicated mRNAs. Open square, β-globin; open circle, FLAG-xHsl7; closed square, HA-Wee1 mRNA injection; closed circle, FLAG-xHsl7 and HA-Wee1 mRNAs coinjection. (E) Buffer (lane 1) or HA-Wee1 mRNA (lanes 2 and 3) were incubated in cycling extracts in the presence (lanes 1 and 3) or absence (lane 2) of sperm chromatin. After 60 min, HA-Wee1 protein was isolated with anti-HA beads and bound proteins were analyzed by anti-Hsl7 immunoblotting. (F) Interphase (lanes 1 and 3) or mitotic (lane 2) egg extracts were incubated in the presence of sperm chromatin for 60 min, and immunoprecipitated with anti-Wee1 antibody (lanes 1 and 2) or control antibody (lane 3) bound to protein A–Sepharose beads. Immunoprecipitates were then blotted with anti-Hsl7 antibodies.
Mentions: It has been reported that the DNA replication checkpoint stabilizes Wee1 within nuclei, where it would otherwise be degraded at the time of mitotic entry (Michael and Newport, 1998). In examining xHsl7-mediated Wee1 degradation in oocytes injected with both excess xHsl7 mRNA and radiolabeled in vitro translated Wee1, we noted that xHsl7 overproduction promoted specific loss of the nuclear Wee1, as detected by SDS-PAGE and autoradiography of manually dissected oocyte nuclei (Fig. 6 A; nuclear envelope breakdown was prevented by addition of the Cdc2 inhibitor roscovitine). Although Wee1 was also lost from the cytoplasm during these experiments, this loss was not stimulated by xHsl7 overproduction. Moreover, since Wee1 is rapidly transported into oocyte nuclei, we suspect that much of the loss of cytoplasmic Wee1 reflects nuclear import (unpublished data). As in S. cerevisiae, xHsl7-stimulated loss of Wee1 was not abrogated by disruption of the well-conserved residues responsible for methyltransferase activity (Fig. 6 B), nor was it inhibited by leptomycin B treatment to stop Crm1-mediated nuclear export (Fig. 6 C; note the increase in nuclear Wee1 in control injections due to inhibition of nuclear export. Xenopus Hsl7 injection counteracts even the added burden of imported Wee1 that cannot be exported). These data are consistent with data reported previously suggesting intranuclear Wee1 degradation, particularly as nuclear export of Swe1 depends on the Crm1 homologue, Expo1 (Lew, D.J., personal communication).

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