Recognition of CDK inhibitor p21 by the Ubiquitin-proteasome pathway
| Project/Area Number |
13672430
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory medicine
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| Research Institution | Showa University |
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Principal Investigator |
FUKUCHI Kunihiko Showa University, School of Medicine, Department of Clinical Pathology, Associate professor, 医学部, 助教授 (70181287)
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| Co-Investigator(Kenkyū-buntansha) |
GOMI Kunihide Showa University, School of Medicine, Department of Clinical Pathology, Professor, 医学部, 教授 (60053980)
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| Project Period (FY) |
2001 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2004: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | p21 / ubiquitin / cyclin / DNA damage / apoptosis / caspase / CDK / His tag / Cyclin kinase inhibitor / リン酸化 / cyclin kinase inhibitor |
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| Research Abstract |
Cyclin kinase inhibitor p21 is a multifunctional protein which binds and inhibits cyclin kinases and PCNA, and also inhibits the process of apoptosis. Because the expression of p21 determines the fate of cells after DNA damage, the analysis of the regulatory mechanism for its expression and of its binding to cellular protein should be critical issue to be clarified. In this study, we analyzed three themes.
1. The regulatory region for the p21-degradation by the ubiquitin-proteasome was determined by analysis of its ubiquitination and stability of various deletion mutants. As a result, it became clear that C-terminal 148-157 as region is essential for the efficient ubiquitination.
2. The association of p21 and cyclinA after DNA damage was analyzed using various p21 mutants. CyclinA binding to p21 after DNA damage required neither cyclin binding motif at N-terminal nor cyclin binding motif at C-teminal. CDK2 binding region at 49-71 as were appeared to be essential region for CyclinA-p21 binding.
3. Effects of p21 on the regulation of apoptosis were analyzed. To define the apoptosis-regulatory function of p21, we constructed cells that stably express C-terminal deletion mutants of p21, 1-157, 1-147 or 1-128, and evaluated the apoptotic response of these cells. The AnnexinV positive cell fraction after y-irradiation did not increase in cells expressing 1-157. Consistently, an increase of caspase3 activity or the active form of caspase3 was not observed in cells expressing 1-157, but was prominent in cells expressing 1-128 and 1-147. Further, the activity of caspase9 was suppressed in y-irradiated cells expressing 1-157. Our data indicate that the 1-157 region of p21 inhibits apoptosis caused by y-irradiation by reducing the activity of caspase9 and caspase3, and the 148-157 region is critical for its inhibiting activity.
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Report
(5 results)
Research Products
(11 results)
