| Project/Area Number |
09558086
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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 | 展開研究 |
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| Research Field |
Functional biochemistry
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| Research Institution | HIROSHIMA UNIVERSITY |
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Principal Investigator |
TAKEDA Masao Hiroshima University, Faculty of Medicine, Professor, 医学部, 教授 (40030853)
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| Co-Investigator(Kenkyū-buntansha) |
NISHITO Yasumasa Hiroshima University, Faculty of Medicine, Research Associate, 医学部, 助手 (40284187)
TANABE Osamu Hiroshima University, Faculty of Medicine, Assistant Professor, 医学部, 講師 (70221398)
USUI Hirofumi Hiroshima University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (40127618)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Protein phosphorylation / Protein phosphatase 2A / Serine / threonine protein phosphatase / Regulatory subunits / 74kDa subunit B" / B" binding proteins / Inhibitor peptides / Human erythrocytes / トレオニンホフファターゼ / 74kDaB^<11>サブユニット / B^<11>結合タンパク質 |
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| Research Abstract |
1. Identification of splicing variants of the B" subunit of protein phosphatase 2A
Two cDNAs for splicing variants of the B" regulatory subunit of protein phosphatase 2A (PP2A) were identified from human cerebral cortex. These variants lacked a 32- or 106-residue sequence of B" near the N-terminus. GST-fusion proteins of the variants were expressed in F.coli using pGEX vector. Preparation of various deletion mutants of B" in GST fusion is also in progress. Regions of B", essential for binding to the AC dimer of PP2A and inhibiting its catalytic activity, will be determined using the GST fusion proteins. Based on the primary structure of the regions, inhibitory peptides for PP2A will be designed.
2. Activation of B"AC trimer by phosphorylation of B" by A-kinase
B" in B"AC trimer of PP2A was phosphorylated by A-kinase at Ser-60, 75 and 573 with concomitant increase in its phosphatase activity toward Hi and H2B histones. Therefore, the regions around the phosphorylation sites may be involved in suppression of CA activity by B". Based on the primary structure of the regions, inhibitory peptides for PP2A will be designed.
2. Identification of B" binding proteins by yeast two-hybrid screening
To identify proteins which regulate activity or localization of B"AC, B" binding proteins were sought by yeast two-hybrid screening. Human cerebral cortex cDNA library which can express proteins fused to GAL4 activation domain was prepared. The library was introduced into a yeast strain, which expresses human B" fused to GAL4 DNA binding domain, and was screened for HIS3 expression. Out of over 1.2 million clones, 78 positive were isolated. Determination of the regions essential for interaction between B" and the binding partners is in progress. Based on the primary structure of the regions, inhibitory peptides for B"AC will be designed.
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