| Project/Area Number |
04454156
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Kobe University |
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Principal Investigator |
KATAOKA Tohru Kobe Univ.Sch.Med., Dept.Physiology II, Professor, 医学部, 教授 (40144472)
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| Co-Investigator(Kenkyū-buntansha) |
KATAOKA Yuriko Kobe Univ.Sch.Med., Dept.Physiology II, Instructor, 医学部, 助手 (50233739)
SUZUKI Noboru Kobe Univ.Sch.Med., Dept.Physiology II, Instructor, 医学部, 助手 (00202135)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1993: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1992: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Keywords | Adenylyl Cyclase / Cyclase-Associated Protein / Budding Yeast / Ribosomal Protein / Cyclic AMP / Ras Protein / リボゾーム蛋白質 / CAP / rasがん遺伝子 / ras蛋白質 / cAMP / GTP結合蛋白質 |
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| Research Abstract |
In budding yeast adenylyl cyclase is a downstream effector of Ras proteins. We found adenylyl cyclase forms a complex with two cyclase-associated proteins ; 70-kDa CAP and 50-kDa p50, and analyzed their structures and functions.
1. CAP is a bifunctional protein ; its N-terminal region is required for association with adenylyl cyclase, while its C-terminal region is homologous to mammalian actin-binding protein ASP-56 and presumably involved in cytoskeletal regulation. Gene-disruption and mutational studies showed that CAP is not essential for interaction of cyclase with Ras proteins. We assigned a CAP-binding site of cyclase to its C-terminal domain and created mutants which lost the ability to bind CAP.Yeast cells whose chromosomal cyclase genes were replaced by the CAP-nonbinding mutants posessed cyclase activity fully responsive to Ras in vitro. However, in the activated RAS2^<Vall9> background they did not exhibit abnormal phenotypes and exaggerated cAMP response to glucouse, which
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were characteristic of RAS2^<Vall9> cells carrying wild-type cyclase. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the cAMP response. These results suggested that the association with CAP, although not involved in response to wild-type Ras, is required for the exaggerated response of cyclase to activated Ras. Thus, we found a way to suppress the exaggerated action of activated Ras without affecting that of wild-type Ras. Mechanisms underlying this are currently under investigation.
2. p50 binds to leucine-rich repeats domain of adenylyl cyclase, a Ras-binding site. p50 was purified, and N-terminal amino acid sequence determaned. The result indicated p50 was identical with a ribosomal large subunit protein, L3. The function of L3 is presently unknown except for involvement in tricodermin-resistance. We are preparing antibody against L3 for analysis of its function. We speculate that the association of L3 with cyclase may represent a link between Ras pathway and protein synthesis machinery. Less
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