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Isoform-specific phosphorylation of fission yeast type 2C protein phosphatase
T Kobayashi1, M Sadaie, M Ohnishi
1Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575, Japan.
Abstract:
Protein phosphatase 2C (PP2C) is one of the four major protein serine/threonine phosphatases of eukaryotes and is implicated in the regulation of various cellular functions. With the goal of elucidating the mechanism responsible for regulating PP2C functions, we investigated the significance of phosphorylation of fission yeast Ptc1, Ptc2, and Ptc3, the yeast orthologs of mammalian PP2C. Both Ptc2 and Ptc3 but not Ptc1 were phosphorylated stoichiometrically by casein kinase II on serine residues at their carboxy-terminal regions. Mutational analysis of Ptc2 and Ptc3 revealed that serine residues of the conserved sequence (Ser-X-Ser-X-X-Glu/Asp) of these proteins were the phosphorylation sites. Interestingly, the activities of Ptc2 and Ptc3 were decreased 25 +/- 7.5% and increased 55 +/- 3.7%, respectively, by phosphorylation. In addition, the same site(s) of Ptc2 was phosphorylated when the protein was expressed in fission yeast cells. These results suggest that phosphorylation of PP2C plays important physiological roles in fission yeast cells.
Insights
Phosphorylation of fission yeast protein phosphatase 2C (PP2C) enzymes Ptc2 and Ptc3 by casein kinase II regulates their activity. This study reveals key phosphorylation sites and their impact on enzyme function, suggesting significant physiological roles.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Protein phosphatase 2C (PP2C) is a crucial eukaryotic enzyme family involved in diverse cellular processes.
- Understanding the regulation of PP2C activity is vital for comprehending cellular signaling pathways.
Purpose of the Study:
- To investigate the role of phosphorylation in regulating the function of fission yeast PP2C orthologs: Ptc1, Ptc2, and Ptc3.
- To identify specific phosphorylation sites and their impact on enzyme activity.
Main Methods:
- Utilized casein kinase II to phosphorylate Ptc1, Ptc2, and Ptc3 in vitro.
- Performed mutational analysis to pinpoint serine residues as phosphorylation sites.
- Assessed changes in Ptc2 and Ptc3 enzyme activity following phosphorylation.
Main Results:
- Ptc2 and Ptc3, but not Ptc1, were stoichiometrically phosphorylated by casein kinase II at their carboxy-terminal regions.
- Identified conserved serine residues (Ser-X-Ser-X-X-Glu/Asp) as key phosphorylation sites.
- Phosphorylation decreased Ptc2 activity by 25% and increased Ptc3 activity by 55%.
Conclusions:
- Phosphorylation significantly modulates the enzymatic activity of fission yeast PP2C proteins.
- These findings highlight the physiological importance of PP2C phosphorylation in cellular regulation.
- The identified phosphorylation sites and their effects provide insights into PP2C-mediated signaling networks.