1994 Fiscal Year Final Research Report Summary
Regulation of gene expression by protein kinase in yeast
| Project/Area Number |
04404001
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
遺伝学
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| Research Institution | University of Tokyo |
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Principal Investigator |
TOH-E Akio University of Tokyo, Graduate School of Science, Professor, 大学院・理学系研究科, 教授 (90029249)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUI Yasushi Universy of Tokyo, Graduate School of Science, Assistant Professor, 大学院・理学系研究科, 助手 (50229407)
KIKUCHI Yoshiko University of Tokyo, Graduate School of Science, Associate Professor, 大学院・理学系研究科, 助教授 (00138124)
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| Project Period (FY) |
1992 – 1994
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| Keywords | protein kinase / PHU system / Pho85 / PHO80 / Saccharomyces cerevisiae |
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| Research Abstract |
PHO85 was first identified as a negative regulator of the PHO system in budding yeast. It encodes a protein kinase that is highly homologous to cyclindependent kinases (CDKs). Since a pho85DELTA mutation causes not only a constitutive expression of the yeast acid phosphatase but defects in normal growth rate and in the utilization of carbon sources, we assume that the Pho85 kinase mediates a signal from the nutrient availability to START progression that is regulated by the Cdc28 kinase. To test this idea, we carried out genetic and biochemical analyzes of the Pho85 kinase.
PHO80 is another negative regulator of the PHO system, functioning together with PHO85 to repress PHO5. By an in vitro kinase assay using kappa-casein as substrate, we found that hyperproduced Pho80 stimulates the Pho85 kinase activity and that an immunoprecipitate containing a tagged Pho80 possessed a kinase activity that was dependent of functional PHO85. These results indicate that the Pho80 protein interacts with the Pho85 kinase to regulate its activity.
To reveal the functional difference between Pho85 and Cdc28, we analyzed the functional domain (s) of the Pho85 kinase. We constructed various point mutants containing mutations in the conserved regions of CDKs. Both a Y18,22F mutation which occurred in the region corresponding to the negative phosphorylation site of cdc2, and a E53A mutation that resides in the PSTAIRE sequence are nonfunctional in vivo as well as in vitro and are dominant negative against the wild type Pho85. On the other hand, YTH (Cdc28 type), FTS,or FAA mutations in F165S166S167, the region corresponding to an active phosphorylation site of cdc2, are functional in vivo. These results indicate that Y18,22 and the PSTAIRE sequence are important for PHO85 function whereas S166 is not, and imply that the function of each domain could be different among CDKs in spite of its conservation.
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[Publications] Fujino,M.,Nishizawa,M.,Yoor,S.-J.,Ozuchi,T.,and Toh.e,A: "Characterization of Pho85 Kinase of Sacharoryces cerevisiae" Phosphate in microorganisms,cellirlar and Molecular Biology Fd.A.Torriani Gorin,F,Yagil,S,Silver. 70-75 (1994)
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[Publications] Kikuchi,Y.,Oka,Y.,Kobayashi,M.,Uesono,Y.,Toh.e,A.,Kikuchi,A: "A new yeast gene CHTR2,required for growth at high temperature,is needed for recovery from making pheromone-induced GI auert" Mol.Gen.Genet.245. 107-116 (1994)
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[Publications] Fujino, M., Nishizawa, M., Yoon, S.-J., Oguchi, T., and Toh-e, A.: "Characterization of Pho85 kinase of Saccharomyces cerevisiae." Eds.A.Torriani-Gorini, E.Yagil, and S.Silver ASM,Washington, USA.70-75 (1994)
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[Publications] Kikuchi, Y., Oka, Y., Kobayashi, M., Uesono, Y., Toh-e, A., and Kikauchi, A.: "A new yeast gene HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest." Mol.Gen.Genet.245. 107-116 (1994)
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