1996 Fiscal Year Final Research Report Summary
Regulation Mechanism of Cyclin Dependent Kinase Activity in Mammalian Cells
| Project/Area Number |
06454597
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | TOKYO UNIVERSITY OF PHARMACY AND LIFE SCIENCE |
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Principal Investigator |
YASUDA Hideyo Tokyo University of Pharmacy and Life Science, School of Life Science, Professor, 生命科学部, 教授 (40111554)
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| Project Period (FY) |
1994 – 1996
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| Keywords | cdc2 kinase / cdk2 / cyclin / weel kinase / 14-3-3 / cell cycle |
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| Research Abstract |
To identify proteins which bind to mouse weel kinase, the yeast " two-hybrid"system was used with a mouse cDNA library. Using the carboxyl half of weel kinase, the 14-3-3 zeta protein was isolated. Recombinat 14-3-3 zeta was demonstrated to bind to weel kinase in vitro. When both weel kinase and 14-3-3 zeta were transfected into COS-1 cells, they formed a complex in a cell. The weel kinase phosphorylated by cdc2 kinase also bound to 14-3-3 zeta ptotein. 14-3-3 zeta protein did not affect the activity of weel kinase. Further, cyclin B/cdc2 kinase, weel kinase and 14-3-3 zeta ptotein form a complex. The sequence of weel kinase necessary for the binding was tested by two hybrid system expressing several lengthes of peptides of weel kinase. The entire kinase domain and a sequence in the carboxyl terminus was thought to be necessary for the binding. The function of 14-3-3 zeta protein remained to be elucidated in relationto the regulation of G2 to M phase transition through weel kinase.
The weel kinase phosphorylates only Y-15 residue of cdc2 kinase and T-14 phosphorylation is observed in a intact cell. The other kinase which phosphorylates T-14 of cdc2 kinase must exist. We tried to isolate the T-14 kinase by use of RT-PCR method using DNA sequencest of weel kinase as PCR primers. We isolated mouse mytl kinase which phosphorylate both T-14 and Y-15 in vitro. Cdk2 was also phophorylated by this mytl kinase less than was done by the weel kinase. The weel kinase located in nucleus and mytl kinase located in cytoplasm.
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[Publications] Matsukawa, T., Kitagawa, M., Katayama, K., Nagai, Y., Hayashi, T., Yasuda, H.and Ohba, Y.: "The existence of Thyroid hormone responsive element, TRE,was confirmed in the first intron of rat alpha1-acid glycoprotein gene.." J.Biochem.119. 934-939 (1996)
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[Publications] Nagai, Y., Kaneda, S., Nomura, K., Yasudam, H., Seno, T.and Yamao, F.: "Ubiquitin-activating enzyme, El, is phosphorylated in mammalian cells by protein kinase Cdc2." J.Cell Sci.108. 2145-2152 (1995)
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[Publications] Ohkubo, Y., Kishimoto, T., Nakata, T., Yasuda, H., and Endo, T.: "SV40 large T antigen reinduces the cell cycle in terminally differentiated myotubes through inducing cdk2, cdc2, and their partner cyclins." Expt.Cell Res.214. 270-278 (1994)
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「研究成果報告書概要(欧文)」より
