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CDK phosphorylation of SLD-2 is required for replication initiation and germline development in C. elegans.

Gaggioli V, Zeiser E, Rivers D, Bradshaw CR, Ahringer J, Zegerman P - J. Cell Biol. (2014)

Bottom Line: We demonstrate that SLD-2 is required for replication initiation and the nuclear retention of a critical component of the replicative helicase CDC-45 in embryos.By mutation of the CDK sites in sld-2, we show that CDK phosphorylation of SLD-2 is essential in C. elegans.These results determine an essential function of CDK in metazoa and identify a developmental role for regulated SLD-2 phosphorylation.

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Affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, 2 Department of Genetics, and 3 Department of Zoology, University of Cambridge, Cambridge CB2 1QN, England, UK.

ABSTRACT
Cyclin-dependent kinase (CDK) plays a vital role in proliferation control across eukaryotes. Despite this, how CDK mediates cell cycle and developmental transitions in metazoa is poorly understood. In this paper, we identify orthologues of Sld2, a CDK target that is important for DNA replication in yeast, and characterize SLD-2 in the nematode worm Caenorhabditis elegans. We demonstrate that SLD-2 is required for replication initiation and the nuclear retention of a critical component of the replicative helicase CDC-45 in embryos. SLD-2 is a CDK target in vivo, and phosphorylation regulates the interaction with another replication factor, MUS-101. By mutation of the CDK sites in sld-2, we show that CDK phosphorylation of SLD-2 is essential in C. elegans. Finally, using a phosphomimicking sld-2 mutant, we demonstrate that timely CDK phosphorylation of SLD-2 is an important control mechanism to allow normal proliferation in the germline. These results determine an essential function of CDK in metazoa and identify a developmental role for regulated SLD-2 phosphorylation.

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C. elegans SLD-2 is an essential CDK target. (A) Selective growth medium of yeast two-hybrid analysis between MUS-101 (679–1,182; bait) and SLD-2 mutants (prey). (B) GST pull-down with GST–MUS-101 (679–1,182) and T7/6HIS SLD-2. The GST–MUS-101 lane (−) is with glutathione–Sepharose beads alone. (C) The length of time from P0 nuclear envelope breakdown until division of the P1 blastomere in N2 and transgenic worms either uninjected or after sld-2 RNAi injection. These data are the means of five separate experiments. For N2 and sld-2 wild type, n = 7; for 8A, n = 11; and for 8D, n = 14. (D) Percentage of embryonic lethality at 25°C of uninjected (n = 12–15) and sld-2 RNAi-injected adult worms (n = 40) from 2–24 h. These data are the means of three experiments. Error bars are SEMs. WT, wild type.
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fig5: C. elegans SLD-2 is an essential CDK target. (A) Selective growth medium of yeast two-hybrid analysis between MUS-101 (679–1,182; bait) and SLD-2 mutants (prey). (B) GST pull-down with GST–MUS-101 (679–1,182) and T7/6HIS SLD-2. The GST–MUS-101 lane (−) is with glutathione–Sepharose beads alone. (C) The length of time from P0 nuclear envelope breakdown until division of the P1 blastomere in N2 and transgenic worms either uninjected or after sld-2 RNAi injection. These data are the means of five separate experiments. For N2 and sld-2 wild type, n = 7; for 8A, n = 11; and for 8D, n = 14. (D) Percentage of embryonic lethality at 25°C of uninjected (n = 12–15) and sld-2 RNAi-injected adult worms (n = 40) from 2–24 h. These data are the means of three experiments. Error bars are SEMs. WT, wild type.

Mentions: Having demonstrated that SLD-2 is targeted by CDK and that this phosphorylation event mediates the interaction with MUS-101, we next set out to address whether this CDK phosphorylation of SLD-2 is important in vivo. Although the SLD-2 8A mutant does not interact with MUS-101 either in vitro (Fig. 4 F) or by yeast two-hybrid analysis (Fig. 5 A), the SLD-2 8D mutant interacts with MUS-101 both in a yeast two-hybrid analysis (Fig. 5 A) and in a pull-down in the absence of CDK activity (Fig. 5 B). From this, we conclude that the 8D mutant is an effective phosphomimic for the interaction between SLD-2 and MUS-101. As a result, if CDK-dependent regulation of SLD-2 is essential in C. elegans, mutation of the CDK sites in sld-2 to alanine should not be compatible with viability, but mutation of these CDK sites to aspartic acid, which acts as a phosphomimetic (Fig. 5, A and B), should be viable. To test this, we analyzed the activity of transgenes expressing sld-2 wild type or the CDK mutant 8A and 8D versions of sld-2 (described in Fig. 4 C). These sld-2 transgenes are codon altered to be refractory to RNAi of endogenous sld-2, and we confirmed that these transgenes are similarly expressed and that their levels are indeed unaffected by RNAi of endogenous sld-2 (Fig. S3).


CDK phosphorylation of SLD-2 is required for replication initiation and germline development in C. elegans.

Gaggioli V, Zeiser E, Rivers D, Bradshaw CR, Ahringer J, Zegerman P - J. Cell Biol. (2014)

C. elegans SLD-2 is an essential CDK target. (A) Selective growth medium of yeast two-hybrid analysis between MUS-101 (679–1,182; bait) and SLD-2 mutants (prey). (B) GST pull-down with GST–MUS-101 (679–1,182) and T7/6HIS SLD-2. The GST–MUS-101 lane (−) is with glutathione–Sepharose beads alone. (C) The length of time from P0 nuclear envelope breakdown until division of the P1 blastomere in N2 and transgenic worms either uninjected or after sld-2 RNAi injection. These data are the means of five separate experiments. For N2 and sld-2 wild type, n = 7; for 8A, n = 11; and for 8D, n = 14. (D) Percentage of embryonic lethality at 25°C of uninjected (n = 12–15) and sld-2 RNAi-injected adult worms (n = 40) from 2–24 h. These data are the means of three experiments. Error bars are SEMs. WT, wild type.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC3926958&req=5

fig5: C. elegans SLD-2 is an essential CDK target. (A) Selective growth medium of yeast two-hybrid analysis between MUS-101 (679–1,182; bait) and SLD-2 mutants (prey). (B) GST pull-down with GST–MUS-101 (679–1,182) and T7/6HIS SLD-2. The GST–MUS-101 lane (−) is with glutathione–Sepharose beads alone. (C) The length of time from P0 nuclear envelope breakdown until division of the P1 blastomere in N2 and transgenic worms either uninjected or after sld-2 RNAi injection. These data are the means of five separate experiments. For N2 and sld-2 wild type, n = 7; for 8A, n = 11; and for 8D, n = 14. (D) Percentage of embryonic lethality at 25°C of uninjected (n = 12–15) and sld-2 RNAi-injected adult worms (n = 40) from 2–24 h. These data are the means of three experiments. Error bars are SEMs. WT, wild type.
Mentions: Having demonstrated that SLD-2 is targeted by CDK and that this phosphorylation event mediates the interaction with MUS-101, we next set out to address whether this CDK phosphorylation of SLD-2 is important in vivo. Although the SLD-2 8A mutant does not interact with MUS-101 either in vitro (Fig. 4 F) or by yeast two-hybrid analysis (Fig. 5 A), the SLD-2 8D mutant interacts with MUS-101 both in a yeast two-hybrid analysis (Fig. 5 A) and in a pull-down in the absence of CDK activity (Fig. 5 B). From this, we conclude that the 8D mutant is an effective phosphomimic for the interaction between SLD-2 and MUS-101. As a result, if CDK-dependent regulation of SLD-2 is essential in C. elegans, mutation of the CDK sites in sld-2 to alanine should not be compatible with viability, but mutation of these CDK sites to aspartic acid, which acts as a phosphomimetic (Fig. 5, A and B), should be viable. To test this, we analyzed the activity of transgenes expressing sld-2 wild type or the CDK mutant 8A and 8D versions of sld-2 (described in Fig. 4 C). These sld-2 transgenes are codon altered to be refractory to RNAi of endogenous sld-2, and we confirmed that these transgenes are similarly expressed and that their levels are indeed unaffected by RNAi of endogenous sld-2 (Fig. S3).

Bottom Line: We demonstrate that SLD-2 is required for replication initiation and the nuclear retention of a critical component of the replicative helicase CDC-45 in embryos.By mutation of the CDK sites in sld-2, we show that CDK phosphorylation of SLD-2 is essential in C. elegans.These results determine an essential function of CDK in metazoa and identify a developmental role for regulated SLD-2 phosphorylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, 2 Department of Genetics, and 3 Department of Zoology, University of Cambridge, Cambridge CB2 1QN, England, UK.

ABSTRACT
Cyclin-dependent kinase (CDK) plays a vital role in proliferation control across eukaryotes. Despite this, how CDK mediates cell cycle and developmental transitions in metazoa is poorly understood. In this paper, we identify orthologues of Sld2, a CDK target that is important for DNA replication in yeast, and characterize SLD-2 in the nematode worm Caenorhabditis elegans. We demonstrate that SLD-2 is required for replication initiation and the nuclear retention of a critical component of the replicative helicase CDC-45 in embryos. SLD-2 is a CDK target in vivo, and phosphorylation regulates the interaction with another replication factor, MUS-101. By mutation of the CDK sites in sld-2, we show that CDK phosphorylation of SLD-2 is essential in C. elegans. Finally, using a phosphomimicking sld-2 mutant, we demonstrate that timely CDK phosphorylation of SLD-2 is an important control mechanism to allow normal proliferation in the germline. These results determine an essential function of CDK in metazoa and identify a developmental role for regulated SLD-2 phosphorylation.

Show MeSH
Related in: MedlinePlus