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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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Clb1 is phosphorylated and localizes to the nucleus during metaphase I.Cultures of CLB1-myc9, PCLB2CDC55 CLB1-myc9, PCLB2CDC20 CLB1-myc9 and PCLB2CDC20 PCLB2CDC55 CLB1-myc9 strains were induced to enter meiosis by transferring them to SPM. A) Modification of Clb1 was assayed by subjecting whole cell extracts from the above cultures to SDS-PAGE followed by Western analysis using an anti-myc antibody. Tubulin served as a loading control. B) Cells were fixed and examined for Clb1-Myc localisation by in situ immunofluorescence. Proportion of cells with nuclear Clb1 (green), cytoplasmic Clb1 (red) and no Clb1 signals (blue) are indicated. A representative image of a cell belonging to each of the three categories is shown below the graphs. C) Clb1 was immunoprecipitated from a culture of PCLB2CDC20 CLB1-myc9 cdc28-as cells following 8 hours into SPM and then were either mock treated or incubated with λ-phosphatase alone or with λ-phosphatase + phosphatase inhibitors at 30°C for 30 minutes. Samples were analysed by SDS-PAGE followed by Western blotting. Asterisk indicates a Clb1 cleavage fragment.
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pone-0079001-g001: Clb1 is phosphorylated and localizes to the nucleus during metaphase I.Cultures of CLB1-myc9, PCLB2CDC55 CLB1-myc9, PCLB2CDC20 CLB1-myc9 and PCLB2CDC20 PCLB2CDC55 CLB1-myc9 strains were induced to enter meiosis by transferring them to SPM. A) Modification of Clb1 was assayed by subjecting whole cell extracts from the above cultures to SDS-PAGE followed by Western analysis using an anti-myc antibody. Tubulin served as a loading control. B) Cells were fixed and examined for Clb1-Myc localisation by in situ immunofluorescence. Proportion of cells with nuclear Clb1 (green), cytoplasmic Clb1 (red) and no Clb1 signals (blue) are indicated. A representative image of a cell belonging to each of the three categories is shown below the graphs. C) Clb1 was immunoprecipitated from a culture of PCLB2CDC20 CLB1-myc9 cdc28-as cells following 8 hours into SPM and then were either mock treated or incubated with λ-phosphatase alone or with λ-phosphatase + phosphatase inhibitors at 30°C for 30 minutes. Samples were analysed by SDS-PAGE followed by Western blotting. Asterisk indicates a Clb1 cleavage fragment.

Mentions: Clb1 has been observed to be post-translationally modified and localized to the nucleus during meiosis I [22], [23]. We first tested whether progression through anaphase I was required for post-translational modification and nuclear localisation of Clb1 during meiosis. We constructed Clb1-tagged strains containing the CDC20 (responsible for activating the anaphase promoting complex) either under the endogenous promoter or the mitosis-specific promoter PCLB2[25]. We induced cells to enter meiosis by transferring them to sporulation medium (SPM). While wild type cells underwent two rounds of nuclear division and formed 60% tetrads after 12 h in SPM, the PCLB2CDC20 cells remained mononucleate during the entire experiment (Figure S1). After 6 h into SPM, Clb1 was modified and localized to the nucleus in both wild type and PCLB2CDC20 cells (Figure 1A and 1B). This indicates that progression through anaphase I is not required for Clb1 modification and nuclear localization. However, in contrast to wild type cells, Clb1 modification and nuclear localization was sustained in PCLB2CDC20 cells (Figure 1A and 1B). This suggests that passage through anaphase I reverses the modification and nuclear localization of Clb1. One possibility is that activation of FEAR during anaphase I might abolish nuclear localization and modification of Clb1. Consistent with this possibility, nuclear localization of Clb1 during meiosis is retained in FEAR mutants [22]. If FEAR was required for delocalization of Clb1, then its premature activation should prevent Clb1 nuclear localization. Since PCLB2CDC55 cells release Cdc14 prematurely from the nucleolus but undergo other cell cycle events like securin degradation, cohesin cleavage and Clb3 expression (specific to meiosis II) normally, we monitored Clb1 nuclear localization and modification in PCLB2CDC55 cells [24]. Clb1 nuclear localisation and modification was maintained in both PCLB2CDC55 and PCLB2CDC55 PCLB2CDC20 strains (Figure 1A and 1B) suggesting that Clb1 nuclear localization and modification is not sensitive to premature FEAR activation [24].


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)

Clb1 is phosphorylated and localizes to the nucleus during metaphase I.Cultures of CLB1-myc9, PCLB2CDC55 CLB1-myc9, PCLB2CDC20 CLB1-myc9 and PCLB2CDC20 PCLB2CDC55 CLB1-myc9 strains were induced to enter meiosis by transferring them to SPM. A) Modification of Clb1 was assayed by subjecting whole cell extracts from the above cultures to SDS-PAGE followed by Western analysis using an anti-myc antibody. Tubulin served as a loading control. B) Cells were fixed and examined for Clb1-Myc localisation by in situ immunofluorescence. Proportion of cells with nuclear Clb1 (green), cytoplasmic Clb1 (red) and no Clb1 signals (blue) are indicated. A representative image of a cell belonging to each of the three categories is shown below the graphs. C) Clb1 was immunoprecipitated from a culture of PCLB2CDC20 CLB1-myc9 cdc28-as cells following 8 hours into SPM and then were either mock treated or incubated with λ-phosphatase alone or with λ-phosphatase + phosphatase inhibitors at 30°C for 30 minutes. Samples were analysed by SDS-PAGE followed by Western blotting. Asterisk indicates a Clb1 cleavage fragment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815228&req=5

pone-0079001-g001: Clb1 is phosphorylated and localizes to the nucleus during metaphase I.Cultures of CLB1-myc9, PCLB2CDC55 CLB1-myc9, PCLB2CDC20 CLB1-myc9 and PCLB2CDC20 PCLB2CDC55 CLB1-myc9 strains were induced to enter meiosis by transferring them to SPM. A) Modification of Clb1 was assayed by subjecting whole cell extracts from the above cultures to SDS-PAGE followed by Western analysis using an anti-myc antibody. Tubulin served as a loading control. B) Cells were fixed and examined for Clb1-Myc localisation by in situ immunofluorescence. Proportion of cells with nuclear Clb1 (green), cytoplasmic Clb1 (red) and no Clb1 signals (blue) are indicated. A representative image of a cell belonging to each of the three categories is shown below the graphs. C) Clb1 was immunoprecipitated from a culture of PCLB2CDC20 CLB1-myc9 cdc28-as cells following 8 hours into SPM and then were either mock treated or incubated with λ-phosphatase alone or with λ-phosphatase + phosphatase inhibitors at 30°C for 30 minutes. Samples were analysed by SDS-PAGE followed by Western blotting. Asterisk indicates a Clb1 cleavage fragment.
Mentions: Clb1 has been observed to be post-translationally modified and localized to the nucleus during meiosis I [22], [23]. We first tested whether progression through anaphase I was required for post-translational modification and nuclear localisation of Clb1 during meiosis. We constructed Clb1-tagged strains containing the CDC20 (responsible for activating the anaphase promoting complex) either under the endogenous promoter or the mitosis-specific promoter PCLB2[25]. We induced cells to enter meiosis by transferring them to sporulation medium (SPM). While wild type cells underwent two rounds of nuclear division and formed 60% tetrads after 12 h in SPM, the PCLB2CDC20 cells remained mononucleate during the entire experiment (Figure S1). After 6 h into SPM, Clb1 was modified and localized to the nucleus in both wild type and PCLB2CDC20 cells (Figure 1A and 1B). This indicates that progression through anaphase I is not required for Clb1 modification and nuclear localization. However, in contrast to wild type cells, Clb1 modification and nuclear localization was sustained in PCLB2CDC20 cells (Figure 1A and 1B). This suggests that passage through anaphase I reverses the modification and nuclear localization of Clb1. One possibility is that activation of FEAR during anaphase I might abolish nuclear localization and modification of Clb1. Consistent with this possibility, nuclear localization of Clb1 during meiosis is retained in FEAR mutants [22]. If FEAR was required for delocalization of Clb1, then its premature activation should prevent Clb1 nuclear localization. Since PCLB2CDC55 cells release Cdc14 prematurely from the nucleolus but undergo other cell cycle events like securin degradation, cohesin cleavage and Clb3 expression (specific to meiosis II) normally, we monitored Clb1 nuclear localization and modification in PCLB2CDC55 cells [24]. Clb1 nuclear localisation and modification was maintained in both PCLB2CDC55 and PCLB2CDC55 PCLB2CDC20 strains (Figure 1A and 1B) suggesting that Clb1 nuclear localization and modification is not sensitive to premature FEAR activation [24].

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