| Project/Area Number |
11470234
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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 |
Metabolomics
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| Research Institution | Yamaguchi University |
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Principal Investigator |
OKA Yoshitomo Yamaguchi University, School of Medicine, Professor, 医学部, 教授 (70175256)
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| Co-Investigator(Kenkyū-buntansha) |
MASUJIMA Tsutomu Hiroshima University Hospital, Professor, 医学部, 教授 (10136054)
KATAGIRI Hideki Tokyo University Hospital, Clinical Fellow, 医学部・附属病院, 医員(臨床)
AOKI Minoru Yamaguchi University, School of Medicine, Research Associate, 医学部, 助手 (70304475)
TANAKA Keiji Yamaguchi University Hospital, Clinical Fellow, 医学部・附属病院, 医員(臨床)
INOUE Hiroshi Yamaguchi University Hospital, Research Associate, 医学部・附属病院, 助手 (20294639)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2000: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1999: ¥11,000,000 (Direct Cost: ¥11,000,000)
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| Keywords | Insulin / glucose transport / GLUT4 / PI3 kinase / Akt1 / PDK1 / アデノウイルスベクター / Akt / PH domain |
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| Research Abstract |
To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulin-stimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK 1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulin-stimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.
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