Limits...
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.

Show MeSH
Cdc5 is required for phosphorylation, but not nuclear localisation, of Clb1 during meiosis I.PCLB2CDC20 CLB1-myc9 and PCLB2CDC5 PCLB2CDC20 CLB1-myc9 cells were induced to enter meiosis by transferring them to SPM. A) Samples were taken hourly throughout the time course for in situ immunofluorescence to determine Clb1 localisation (green-nuclear, red-cytoplasmic, blue-no signal). B) Samples were taken hourly from 4 hours for preparing whole cell extracts. Whole cell extracts were analysed by Western blotting using anti-myc (Clb1), anti-Cdc5 and anti-tubulin antibodies. Asterisk represents a cross-reactive band seen in anti-Cdc5 blots.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815228&req=5

pone-0079001-g004: Cdc5 is required for phosphorylation, but not nuclear localisation, of Clb1 during meiosis I.PCLB2CDC20 CLB1-myc9 and PCLB2CDC5 PCLB2CDC20 CLB1-myc9 cells were induced to enter meiosis by transferring them to SPM. A) Samples were taken hourly throughout the time course for in situ immunofluorescence to determine Clb1 localisation (green-nuclear, red-cytoplasmic, blue-no signal). B) Samples were taken hourly from 4 hours for preparing whole cell extracts. Whole cell extracts were analysed by Western blotting using anti-myc (Clb1), anti-Cdc5 and anti-tubulin antibodies. Asterisk represents a cross-reactive band seen in anti-Cdc5 blots.

Mentions: CDK is not meiosis-specific; to explain the meiosis-specific nature of the phosphorylation, another regulatory step must be involved. Cdc5 is also active during meiosis and has a number of meiosis-specific functions [30], [31]. Cdc5 expression also coincides with Clb1 phosphorylation (Figure S3). The requirement for Cdc5 activity was examined by depleting Cdc5 in meiosis by replacing the endogenous promoter with that of CLB2. PCLB2CDC5 cells undergoing meiosis expressed Clb1, but it was not phosphorylated (Figure 4A), indicating that Cdc5 is required for phosphorylation of Clb1. However, Clb1 was still concentrated in the nucleus in PCLB2CDC5 cells (Figure 4B), indicating that Cdc5 activity is not required for nuclear localisation of Clb1. This also shows that Clb1 nuclear localisation is independent of its phosphorylation. Since Cdc5 is required for exit from meiotic prophase [31], our results also indicate that exit from prophase is not required for nuclear localisation of Clb1.


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)

Cdc5 is required for phosphorylation, but not nuclear localisation, of Clb1 during meiosis I.PCLB2CDC20 CLB1-myc9 and PCLB2CDC5 PCLB2CDC20 CLB1-myc9 cells were induced to enter meiosis by transferring them to SPM. A) Samples were taken hourly throughout the time course for in situ immunofluorescence to determine Clb1 localisation (green-nuclear, red-cytoplasmic, blue-no signal). B) Samples were taken hourly from 4 hours for preparing whole cell extracts. Whole cell extracts were analysed by Western blotting using anti-myc (Clb1), anti-Cdc5 and anti-tubulin antibodies. Asterisk represents a cross-reactive band seen in anti-Cdc5 blots.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079001-g004: Cdc5 is required for phosphorylation, but not nuclear localisation, of Clb1 during meiosis I.PCLB2CDC20 CLB1-myc9 and PCLB2CDC5 PCLB2CDC20 CLB1-myc9 cells were induced to enter meiosis by transferring them to SPM. A) Samples were taken hourly throughout the time course for in situ immunofluorescence to determine Clb1 localisation (green-nuclear, red-cytoplasmic, blue-no signal). B) Samples were taken hourly from 4 hours for preparing whole cell extracts. Whole cell extracts were analysed by Western blotting using anti-myc (Clb1), anti-Cdc5 and anti-tubulin antibodies. Asterisk represents a cross-reactive band seen in anti-Cdc5 blots.
Mentions: CDK is not meiosis-specific; to explain the meiosis-specific nature of the phosphorylation, another regulatory step must be involved. Cdc5 is also active during meiosis and has a number of meiosis-specific functions [30], [31]. Cdc5 expression also coincides with Clb1 phosphorylation (Figure S3). The requirement for Cdc5 activity was examined by depleting Cdc5 in meiosis by replacing the endogenous promoter with that of CLB2. PCLB2CDC5 cells undergoing meiosis expressed Clb1, but it was not phosphorylated (Figure 4A), indicating that Cdc5 is required for phosphorylation of Clb1. However, Clb1 was still concentrated in the nucleus in PCLB2CDC5 cells (Figure 4B), indicating that Cdc5 activity is not required for nuclear localisation of Clb1. This also shows that Clb1 nuclear localisation is independent of its phosphorylation. Since Cdc5 is required for exit from meiotic prophase [31], our results also indicate that exit from prophase is not required for nuclear localisation of Clb1.

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