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Phosphorylation of dis2 protein phosphatase at the C-terminal cdc2 consensus and its potential role in cell cycle
H Yamano1, K Ishii, M Yanagida
1Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Abstract:
We show that the fission yeast dis2 protein phosphatase, which is highly similar to mammalian type 1 phosphatase, is a phosphoprotein containing phosphoserine (phospho-S) and threonine (phospho-T). It has several phosphorylation sites, two of which locate in the C-terminus. Phospho-T was abolished in the alanine substitution mutant at the C-terminal T316, which is conserved as a residue in the cdc2 consensus, TPPR, in a number of type 1-like phosphatases. In G2-arrested cdc2-L7 cells, the degree of T316 phosphorylation was reduced, whereas it was enhanced in metaphase-arrested nuc2-663 mutant cells. Phospho-T was produced in dis2 by fission yeast cdc2 kinase, but not in the substitution mutant A316, indicating that the T316 residue was the site for cdc2 kinase in vitro. Phosphatase activity of wild type dis2 was reduced by incubation with cdc2 kinase, but that of mutant dis2-A316 was not. Phosphorylation of T316 hence has a potential significance in cell cycle control in conjunction with cdc2 kinase activation and inactivation. Overexpression phenotypes of wild type dis2+, sds21+ and mutant dis2-A316, sds21-TPPR genes were consistent with negative regulation of dis2 by phosphorylation. This type of regulation would explain why cells harboring the dis2-11 mutation enter mitosis but fail to exit from it.
Insights
Fission yeast dis2 protein phosphatase is phosphorylated by cdc2 kinase at threonine 316, regulating its activity and cell cycle progression. This phosphorylation is crucial for proper cell division, preventing mitotic exit failure.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The fission yeast dis2 protein phosphatase shares similarities with mammalian type 1 phosphatase.
- Protein phosphorylation plays a critical role in regulating cellular processes, including cell cycle control.
Purpose of the Study:
- To investigate the phosphorylation status and regulatory mechanisms of the fission yeast dis2 protein phosphatase.
- To determine the role of specific phosphorylation sites in dis2 phosphatase activity and cell cycle function.
Main Methods:
- Analysis of dis2 protein phosphorylation using phosphoserine and phosphothreonine detection.
- Site-directed mutagenesis to create alanine substitution mutants at potential phosphorylation sites (e.g., T316A).
- In vitro kinase assays using fission yeast cdc2 kinase and dis2 phosphatase.
- Assessment of phosphatase activity in wild-type and mutant dis2 proteins.
- Analysis of overexpression phenotypes for wild-type and mutant genes (dis2+, sds21+, dis2-A316, sds21-TPPR).
Main Results:
- Fission yeast dis2 is a phosphoprotein containing phosphoserine and phosphothreonine, with phosphorylation sites in the C-terminus.
- Threonine 316 (T316) in the C-terminus is a phosphorylation site targeted by cdc2 kinase.
- Phosphorylation of T316 reduces dis2 phosphatase activity, suggesting negative regulation.
- Mutant dis2 lacking T316 phosphorylation (dis2-A316) exhibits altered phosphatase activity and overexpression phenotypes.
- Cell cycle progression is affected, with implications for mitotic exit control.
Conclusions:
- Phosphorylation of dis2 protein phosphatase by cdc2 kinase at T316 is a key regulatory mechanism.
- This phosphorylation negatively regulates dis2 activity, impacting cell cycle control.
- Dysregulation of dis2 phosphorylation may lead to cell division defects, such as failure to exit mitosis.