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Two Distinct Cdc2 Pools Regulate Cell Cycle Progression and the DNA Damage Response in the Fission Yeast S.pombe.

Caspari T, Hilditch V - PLoS ONE (2015)

Bottom Line: The fifth phospho-form has a low affinity for cyclin B and is neither Y15 nor T14 modified.We also show that the G146D mutation in Cdc2.1w, which renders Cdc2 insensitive to Wee1 inhibition, is aberrantly modified in a Wee1-dependent manner.In conclusion, our work adds support to the idea that two distinct Cdc2 pools regulate cell cycle progression and the response to DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Genome Biology Group, School of Medical Sciences, Bangor University, Bangor, LL57 2UW, Wales, United Kingdom.

ABSTRACT
The activity of Cdc2 (CDK1) kinase, which coordinates cell cycle progression and DNA break repair, is blocked upon its phosphorylation at tyrosine 15 (Y15) by Wee1 kinase in the presence of DNA damage. How Cdc2 can support DNA repair whilst being inactivated by the DNA damage checkpoint remains to be explained. Human CDK1 is phosphorylated by Myt1 kinase at threonine 14 (T14) close to its ATP binding site before being modified at threonine 161 (T167Sp) in its T-loop by the CDK-activating kinase (CAK). While modification of T161 promotes association with the cyclin partner, phosphorylation of T14 inhibits the CDK1-cyclin complex. This inhibition is further enforced by the modification of Y15 by Wee1 in the presence of DNA lesions. In S.pombe, the dominant inhibition of Cdc2 is provided by the phosphorylation of Y15 and only a small amount of Cdc2 is modified at T14 when cells are in S phase. Unlike human cells, both inhibitory modifications are executed by Wee1. Using the novel IEFPT technology, which combines isoelectric focusing (IEF) with Phos-tag SDS electrophoresis (PT), we report here that S.pombe Cdc2 kinase exists in seven forms. While five forms are phosphorylated, two species are not. Four phospho-forms associate with cyclin B (Cdc13) of which only two are modified at Y15 by Wee1. Interestingly, only one Y15-modified species carries also the T14 modification. The fifth phospho-form has a low affinity for cyclin B and is neither Y15 nor T14 modified. The two unphosphorylated forms may contribute directly to the DNA damage response as only they associate with the DNA damage checkpoint kinase Chk1. Interestingly, cyclin B is also present in the unphosphorylated pool. We also show that the G146D mutation in Cdc2.1w, which renders Cdc2 insensitive to Wee1 inhibition, is aberrantly modified in a Wee1-dependent manner. In conclusion, our work adds support to the idea that two distinct Cdc2 pools regulate cell cycle progression and the response to DNA damage.

No MeSH data available.


Related in: MedlinePlus

Wee1 kinase contributes to the aberrant modification of Cdc2.1w.(A) IEFPT analysis: IEF strips were placed on top of a 10% Phos-tag SDS page. The IEF strip separates Cdc2 by its isoelectric values and the Phos-tag SDS page by its degree of phosphorylation. (B) IEFPT analysis of total extracts prepared from the indicated strains. The mobility of the Cdc2 bands H, M and U is indicated. The top panel shows an example of the normal IEF pattern of wild type cells. The two wild type and cdc2.1w experiments were conducted independently. The arrows indicate the acidic shift of forms A and B in the cdc2.1w mutant, its reversal in the cdc2.1w Δwee1 strain, and the additional, highly phosphorylated species in the H range in the cdc2.1w Δwee1 strain. (C-E) Phos-tag analysis of total protein extracts prepared from the indicated strains.
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pone.0130748.g002: Wee1 kinase contributes to the aberrant modification of Cdc2.1w.(A) IEFPT analysis: IEF strips were placed on top of a 10% Phos-tag SDS page. The IEF strip separates Cdc2 by its isoelectric values and the Phos-tag SDS page by its degree of phosphorylation. (B) IEFPT analysis of total extracts prepared from the indicated strains. The mobility of the Cdc2 bands H, M and U is indicated. The top panel shows an example of the normal IEF pattern of wild type cells. The two wild type and cdc2.1w experiments were conducted independently. The arrows indicate the acidic shift of forms A and B in the cdc2.1w mutant, its reversal in the cdc2.1w Δwee1 strain, and the additional, highly phosphorylated species in the H range in the cdc2.1w Δwee1 strain. (C-E) Phos-tag analysis of total protein extracts prepared from the indicated strains.

Mentions: To correlate the IEF forms A-F with the experiments conducted by Potashkin and Beach, we combined IEF with Phos-tag SDS electrophoresis (IEFPT) as this should confirm the phosphorylation status of these forms (Fig 2A). As expected, the four negative forms C to F migrated more slowly at the height of the H band, whereas the two least negative species (A+B) migrated faster at the level of the unmodified U band (Fig 2B). While these observations are consistent with the previous report [27], we observed one additional spot at the medium phosphorylated M level. This led us to conclude that spots A and B represent the two unphosphorylated Cdc2 species and forms C to F the four phosphorylated forms described by Potashkin and Beach. Since the A and B species have distinct isoelectric values, but are not phosphorylated, other post-translational modifications must contribute to their charge difference. For example, S.cerevisiae Cdc28Cdc2 is acetylated at lysine-40 (K33 in human and S.pombe Cdc2) which removes one positive charge and is crucial for its function [35]. We probed affinity-purified Cdc2 samples with an antibody that detects lysine residues acetylated at their ε-amino group, but failed to detect a positive signal (not shown). This negative result could however be due to the limited specificity of this antibody.


Two Distinct Cdc2 Pools Regulate Cell Cycle Progression and the DNA Damage Response in the Fission Yeast S.pombe.

Caspari T, Hilditch V - PLoS ONE (2015)

Wee1 kinase contributes to the aberrant modification of Cdc2.1w.(A) IEFPT analysis: IEF strips were placed on top of a 10% Phos-tag SDS page. The IEF strip separates Cdc2 by its isoelectric values and the Phos-tag SDS page by its degree of phosphorylation. (B) IEFPT analysis of total extracts prepared from the indicated strains. The mobility of the Cdc2 bands H, M and U is indicated. The top panel shows an example of the normal IEF pattern of wild type cells. The two wild type and cdc2.1w experiments were conducted independently. The arrows indicate the acidic shift of forms A and B in the cdc2.1w mutant, its reversal in the cdc2.1w Δwee1 strain, and the additional, highly phosphorylated species in the H range in the cdc2.1w Δwee1 strain. (C-E) Phos-tag analysis of total protein extracts prepared from the indicated strains.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488491&req=5

pone.0130748.g002: Wee1 kinase contributes to the aberrant modification of Cdc2.1w.(A) IEFPT analysis: IEF strips were placed on top of a 10% Phos-tag SDS page. The IEF strip separates Cdc2 by its isoelectric values and the Phos-tag SDS page by its degree of phosphorylation. (B) IEFPT analysis of total extracts prepared from the indicated strains. The mobility of the Cdc2 bands H, M and U is indicated. The top panel shows an example of the normal IEF pattern of wild type cells. The two wild type and cdc2.1w experiments were conducted independently. The arrows indicate the acidic shift of forms A and B in the cdc2.1w mutant, its reversal in the cdc2.1w Δwee1 strain, and the additional, highly phosphorylated species in the H range in the cdc2.1w Δwee1 strain. (C-E) Phos-tag analysis of total protein extracts prepared from the indicated strains.
Mentions: To correlate the IEF forms A-F with the experiments conducted by Potashkin and Beach, we combined IEF with Phos-tag SDS electrophoresis (IEFPT) as this should confirm the phosphorylation status of these forms (Fig 2A). As expected, the four negative forms C to F migrated more slowly at the height of the H band, whereas the two least negative species (A+B) migrated faster at the level of the unmodified U band (Fig 2B). While these observations are consistent with the previous report [27], we observed one additional spot at the medium phosphorylated M level. This led us to conclude that spots A and B represent the two unphosphorylated Cdc2 species and forms C to F the four phosphorylated forms described by Potashkin and Beach. Since the A and B species have distinct isoelectric values, but are not phosphorylated, other post-translational modifications must contribute to their charge difference. For example, S.cerevisiae Cdc28Cdc2 is acetylated at lysine-40 (K33 in human and S.pombe Cdc2) which removes one positive charge and is crucial for its function [35]. We probed affinity-purified Cdc2 samples with an antibody that detects lysine residues acetylated at their ε-amino group, but failed to detect a positive signal (not shown). This negative result could however be due to the limited specificity of this antibody.

Bottom Line: The fifth phospho-form has a low affinity for cyclin B and is neither Y15 nor T14 modified.We also show that the G146D mutation in Cdc2.1w, which renders Cdc2 insensitive to Wee1 inhibition, is aberrantly modified in a Wee1-dependent manner.In conclusion, our work adds support to the idea that two distinct Cdc2 pools regulate cell cycle progression and the response to DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Genome Biology Group, School of Medical Sciences, Bangor University, Bangor, LL57 2UW, Wales, United Kingdom.

ABSTRACT
The activity of Cdc2 (CDK1) kinase, which coordinates cell cycle progression and DNA break repair, is blocked upon its phosphorylation at tyrosine 15 (Y15) by Wee1 kinase in the presence of DNA damage. How Cdc2 can support DNA repair whilst being inactivated by the DNA damage checkpoint remains to be explained. Human CDK1 is phosphorylated by Myt1 kinase at threonine 14 (T14) close to its ATP binding site before being modified at threonine 161 (T167Sp) in its T-loop by the CDK-activating kinase (CAK). While modification of T161 promotes association with the cyclin partner, phosphorylation of T14 inhibits the CDK1-cyclin complex. This inhibition is further enforced by the modification of Y15 by Wee1 in the presence of DNA lesions. In S.pombe, the dominant inhibition of Cdc2 is provided by the phosphorylation of Y15 and only a small amount of Cdc2 is modified at T14 when cells are in S phase. Unlike human cells, both inhibitory modifications are executed by Wee1. Using the novel IEFPT technology, which combines isoelectric focusing (IEF) with Phos-tag SDS electrophoresis (PT), we report here that S.pombe Cdc2 kinase exists in seven forms. While five forms are phosphorylated, two species are not. Four phospho-forms associate with cyclin B (Cdc13) of which only two are modified at Y15 by Wee1. Interestingly, only one Y15-modified species carries also the T14 modification. The fifth phospho-form has a low affinity for cyclin B and is neither Y15 nor T14 modified. The two unphosphorylated forms may contribute directly to the DNA damage response as only they associate with the DNA damage checkpoint kinase Chk1. Interestingly, cyclin B is also present in the unphosphorylated pool. We also show that the G146D mutation in Cdc2.1w, which renders Cdc2 insensitive to Wee1 inhibition, is aberrantly modified in a Wee1-dependent manner. In conclusion, our work adds support to the idea that two distinct Cdc2 pools regulate cell cycle progression and the response to DNA damage.

No MeSH data available.


Related in: MedlinePlus