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CDK-dependent nuclear localization of B-cyclin Clb1 promotes FEAR activation during meiosis I in budding yeast.

Tibbles KL, Sarkar S, Novak B, Arumugam P - PLoS ONE (2013)

Bottom Line: While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity.Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway.We discuss the significance of our results in relation to regulation of exit from meiosis I.

View Article: PubMed Central - PubMed

Affiliation: University of Warwick, Coventry, United Kingdom.

ABSTRACT
Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1's phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.

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Fusion of NES/NLS sequences to Clb1 alters its nuclear localization without affecting its kinase activity.A) Schematic showing the variants of Clb1 constructed to alter its nuclear localization. Tandem copies of Nuclear Localization sequences (NLS) or Nuclear Export Sequences (NES) followed by a single copy of HA epitope was added the C-terminus of Clb1. B) Diploid cells bearing either wild type Clb1 or NES/NLS –tagged Clb1 and containing CDC20 under the CLB2 promoter were arrested in metaphase I by transferring them to SPM for 7 h. Clb1 localization was determined by immunofluorescence. Note that Clb1 is diffuse in the NES-tagged strain but nuclear in wild type/NLS-tagged strains. C) Cells bearing either wild type Clb1 or NES/NLS –tagged Clb1 and containing CDC20 under the repressible MET3 promoter were arrested in metaphase by Cdc20 depletion and Clb1localization was determined by immunofluorescence Notice that Clb1 is nuclear in the NLS-tagged strain but delocalized in wild type and NES-tagged strains (images of cells after 2 hours in methionine are shown). D) CLB1-ha PDS1-myc18 cdc28-as, CLB1-NES-ha PDS1-myc18 cdc28-as and CLB1-NLS-ha PDS1-myc18 cdc28-as cells were induced to enter meiosis by transferring them to SPM. Samples were taken hourly to determine Clb1 localisation. E) Tagged Clb1 was immunoprecipitated from mitotic cultures of PCLB2CDC20 cdc28-as, CLB1-ha PCLB2CDC20 cdc28-as, CLB1-NES-ha PCLB2CDC20 cdc28-as, and CLB1-NLS-ha PCLB2CDC20 cdc28-as cells (Figure S4). Purified Clb1 was incubated with 35 µM histone and 50 µM ATP (0.25 µCi/µL of γ-P32-ATP) in kinase buffer for 40 minutes, with samples taken at 0, 10, 20 and 40 minutes in the presence or absence of the inhibitor 1-NM-PP1. Samples were analysed by SDS-PAGE and gels were dried, exposed to phosphorimager screen and the signals were quantified using IMAGE Quant.
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pone-0079001-g005: Fusion of NES/NLS sequences to Clb1 alters its nuclear localization without affecting its kinase activity.A) Schematic showing the variants of Clb1 constructed to alter its nuclear localization. Tandem copies of Nuclear Localization sequences (NLS) or Nuclear Export Sequences (NES) followed by a single copy of HA epitope was added the C-terminus of Clb1. B) Diploid cells bearing either wild type Clb1 or NES/NLS –tagged Clb1 and containing CDC20 under the CLB2 promoter were arrested in metaphase I by transferring them to SPM for 7 h. Clb1 localization was determined by immunofluorescence. Note that Clb1 is diffuse in the NES-tagged strain but nuclear in wild type/NLS-tagged strains. C) Cells bearing either wild type Clb1 or NES/NLS –tagged Clb1 and containing CDC20 under the repressible MET3 promoter were arrested in metaphase by Cdc20 depletion and Clb1localization was determined by immunofluorescence Notice that Clb1 is nuclear in the NLS-tagged strain but delocalized in wild type and NES-tagged strains (images of cells after 2 hours in methionine are shown). D) CLB1-ha PDS1-myc18 cdc28-as, CLB1-NES-ha PDS1-myc18 cdc28-as and CLB1-NLS-ha PDS1-myc18 cdc28-as cells were induced to enter meiosis by transferring them to SPM. Samples were taken hourly to determine Clb1 localisation. E) Tagged Clb1 was immunoprecipitated from mitotic cultures of PCLB2CDC20 cdc28-as, CLB1-ha PCLB2CDC20 cdc28-as, CLB1-NES-ha PCLB2CDC20 cdc28-as, and CLB1-NLS-ha PCLB2CDC20 cdc28-as cells (Figure S4). Purified Clb1 was incubated with 35 µM histone and 50 µM ATP (0.25 µCi/µL of γ-P32-ATP) in kinase buffer for 40 minutes, with samples taken at 0, 10, 20 and 40 minutes in the presence or absence of the inhibitor 1-NM-PP1. Samples were analysed by SDS-PAGE and gels were dried, exposed to phosphorimager screen and the signals were quantified using IMAGE Quant.

Mentions: To determine the functional significance of Clb1 nuclear localisation, the protein was tagged with ectopic Nuclear Localisation Sequences (NLS) or Nuclear Export Sequences (NES), and 6 copies of HA epitope. In each case, two sequences were added in tandem to overcome any endogenous localisation signals (Figure 5A). The tandem NLS tag leads to nuclear localisation of Clb1 during mitosis (Figure 5C). In contrast the tandem NES severely reduced the nuclear localisation of Clb1 during meiosis I (Figure 5B, D). Crucially, the fusion of NLS/NES had little or no effect on Clb1-CDK activity (Figure 5E, Figure S4B). The clb1Δ strains sporulate poorly, producing only 15% tetrads in comparison to 70% for wild-type cells (Figure S4C). Expression of NES/NLS-tagged Clb1 does not affect sporulation efficiency in otherwise wild-type cells (Figure S4C), suggesting that the localization mutants are not inactivated, and that nuclear localisation of Clb1 is not essential for meiotic nuclear divisions. Spore viability was also largely unaffected by the mutations: CLB1-ha and CLB1-NES-ha strains have viabilities of 97%, whereas the CLB1-NLS-ha strain has a spore viability of 85%.


CDK-dependent nuclear localization of B-cyclin Clb1 promotes FEAR activation during meiosis I in budding yeast.

Tibbles KL, Sarkar S, Novak B, Arumugam P - PLoS ONE (2013)

Fusion of NES/NLS sequences to Clb1 alters its nuclear localization without affecting its kinase activity.A) Schematic showing the variants of Clb1 constructed to alter its nuclear localization. Tandem copies of Nuclear Localization sequences (NLS) or Nuclear Export Sequences (NES) followed by a single copy of HA epitope was added the C-terminus of Clb1. B) Diploid cells bearing either wild type Clb1 or NES/NLS –tagged Clb1 and containing CDC20 under the CLB2 promoter were arrested in metaphase I by transferring them to SPM for 7 h. Clb1 localization was determined by immunofluorescence. Note that Clb1 is diffuse in the NES-tagged strain but nuclear in wild type/NLS-tagged strains. C) Cells bearing either wild type Clb1 or NES/NLS –tagged Clb1 and containing CDC20 under the repressible MET3 promoter were arrested in metaphase by Cdc20 depletion and Clb1localization was determined by immunofluorescence Notice that Clb1 is nuclear in the NLS-tagged strain but delocalized in wild type and NES-tagged strains (images of cells after 2 hours in methionine are shown). D) CLB1-ha PDS1-myc18 cdc28-as, CLB1-NES-ha PDS1-myc18 cdc28-as and CLB1-NLS-ha PDS1-myc18 cdc28-as cells were induced to enter meiosis by transferring them to SPM. Samples were taken hourly to determine Clb1 localisation. E) Tagged Clb1 was immunoprecipitated from mitotic cultures of PCLB2CDC20 cdc28-as, CLB1-ha PCLB2CDC20 cdc28-as, CLB1-NES-ha PCLB2CDC20 cdc28-as, and CLB1-NLS-ha PCLB2CDC20 cdc28-as cells (Figure S4). Purified Clb1 was incubated with 35 µM histone and 50 µM ATP (0.25 µCi/µL of γ-P32-ATP) in kinase buffer for 40 minutes, with samples taken at 0, 10, 20 and 40 minutes in the presence or absence of the inhibitor 1-NM-PP1. Samples were analysed by SDS-PAGE and gels were dried, exposed to phosphorimager screen and the signals were quantified using IMAGE Quant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815228&req=5

pone-0079001-g005: Fusion of NES/NLS sequences to Clb1 alters its nuclear localization without affecting its kinase activity.A) Schematic showing the variants of Clb1 constructed to alter its nuclear localization. Tandem copies of Nuclear Localization sequences (NLS) or Nuclear Export Sequences (NES) followed by a single copy of HA epitope was added the C-terminus of Clb1. B) Diploid cells bearing either wild type Clb1 or NES/NLS –tagged Clb1 and containing CDC20 under the CLB2 promoter were arrested in metaphase I by transferring them to SPM for 7 h. Clb1 localization was determined by immunofluorescence. Note that Clb1 is diffuse in the NES-tagged strain but nuclear in wild type/NLS-tagged strains. C) Cells bearing either wild type Clb1 or NES/NLS –tagged Clb1 and containing CDC20 under the repressible MET3 promoter were arrested in metaphase by Cdc20 depletion and Clb1localization was determined by immunofluorescence Notice that Clb1 is nuclear in the NLS-tagged strain but delocalized in wild type and NES-tagged strains (images of cells after 2 hours in methionine are shown). D) CLB1-ha PDS1-myc18 cdc28-as, CLB1-NES-ha PDS1-myc18 cdc28-as and CLB1-NLS-ha PDS1-myc18 cdc28-as cells were induced to enter meiosis by transferring them to SPM. Samples were taken hourly to determine Clb1 localisation. E) Tagged Clb1 was immunoprecipitated from mitotic cultures of PCLB2CDC20 cdc28-as, CLB1-ha PCLB2CDC20 cdc28-as, CLB1-NES-ha PCLB2CDC20 cdc28-as, and CLB1-NLS-ha PCLB2CDC20 cdc28-as cells (Figure S4). Purified Clb1 was incubated with 35 µM histone and 50 µM ATP (0.25 µCi/µL of γ-P32-ATP) in kinase buffer for 40 minutes, with samples taken at 0, 10, 20 and 40 minutes in the presence or absence of the inhibitor 1-NM-PP1. Samples were analysed by SDS-PAGE and gels were dried, exposed to phosphorimager screen and the signals were quantified using IMAGE Quant.
Mentions: To determine the functional significance of Clb1 nuclear localisation, the protein was tagged with ectopic Nuclear Localisation Sequences (NLS) or Nuclear Export Sequences (NES), and 6 copies of HA epitope. In each case, two sequences were added in tandem to overcome any endogenous localisation signals (Figure 5A). The tandem NLS tag leads to nuclear localisation of Clb1 during mitosis (Figure 5C). In contrast the tandem NES severely reduced the nuclear localisation of Clb1 during meiosis I (Figure 5B, D). Crucially, the fusion of NLS/NES had little or no effect on Clb1-CDK activity (Figure 5E, Figure S4B). The clb1Δ strains sporulate poorly, producing only 15% tetrads in comparison to 70% for wild-type cells (Figure S4C). Expression of NES/NLS-tagged Clb1 does not affect sporulation efficiency in otherwise wild-type cells (Figure S4C), suggesting that the localization mutants are not inactivated, and that nuclear localisation of Clb1 is not essential for meiotic nuclear divisions. Spore viability was also largely unaffected by the mutations: CLB1-ha and CLB1-NES-ha strains have viabilities of 97%, whereas the CLB1-NLS-ha strain has a spore viability of 85%.

Bottom Line: While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity.Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway.We discuss the significance of our results in relation to regulation of exit from meiosis I.

View Article: PubMed Central - PubMed

Affiliation: University of Warwick, Coventry, United Kingdom.

ABSTRACT
Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1's phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.

Show MeSH
Related in: MedlinePlus