The cloning and characterization of the 50 kDa protein which is associated with PDE3B and phosphorylated by insulin
| Project/Area Number |
17590939
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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 |
Metabolomics
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| Research Institution | Ehime University |
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Principal Investigator |
MAKINO Hideichi Ehime University, Graduate School of Medicine, 大学院・医学系研究科, 教授 (50009578)
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| Co-Investigator(Kenkyū-buntansha) |
OSAWA Haruhiko Ehime University, Graduate School of Medicine, 大学院・医学系研究科, 助教授 (90294800)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | PDE3B / adipocytes / insulin / phosphorylation / insulin resistance / interaction |
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| Research Abstract |
We found the 50kDa protein (p50) which is associated with phosphodiesterase 3B(PDE3B) and phosphorylated by insulin in PI 3-kinase dependent manner on serine and threonine residues. To identify p50, we analyzed 80 clones which had been positive in the first screening of the yeast two-hybrid screening. The yeast two-hybrid screening had been performed to get PDE3B-associated protein, and 14-3-3 beta had been identified as PDE3B-associated protein by the screening.
Of 80 clones, 11 clones were positive in the second screening of the yeast two-hybrid system. GST pull-down assay was performed to check the in vitro interaction between the positive clones and PDE3B using GST fusion protein of each positive clone as a bait and recombinant PDE3B synthesized by reticulocyte lysate system as a prey. The in vitro interaction between recombinant PBE3B and each of 11 clones was confirmed. GST fusion protein of 7 clones interacted with endogenous PDE3B of rat adipocytes. We made polyclonal antibodies to each 11 clones for the further analysis.
On the other hand, we investigated the kinase of p50. The chromatographic analysis of cytosol fraction from insulin-stimulated rat adipocytes revealed that the same fraction phosphorylated both p50 and PDE3B and that the fraction included phosphorylated Akt.
In this study, we got 7 clones as candidates of p50. Some kinds of proteins other than p50 and 14-3-3 beta are suggested to interact with PDE3B. The chromatographic analysis suggested that the kinase of p50 was Akt, which was identified as a PDE3B kinase..
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Report
(3 results)
Research Products
(3 results)
