| Project/Area Number |
12670103
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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 |
General medical chemistry
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| Research Institution | Kagawa University |
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Principal Investigator |
KAMESHITA isamu Kagawa University, Faculty of Agriculture Professor, 農学部, 教授 (60127941)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIDA atsuhiko Asahikawa Medical College, Assistant Professor, 医学部, 助手 (90212886)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Protein phosphorylation / Protein phosphatase / Protein kinase / Calmodulin-dependent protein kinase / enzyme regulation / cDNA cloning / ポリカチオン / 細胞内局在 / ホスホペプチド / 基質特異性 |
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| Research Abstract |
CaM-dependent protein kinase phosphatase (CaMKPase) is an enzyme that dephosphorylates and regulates CaM-dependent protein kinases (CaMK). In this study, regulatory mechanisms of CaMKPase have been investigated.
(1) Substrate specificity of CaMKPase
In order to elucidate the mechanism of substrate recognition by CaMKPase, synthetic phosphopeptides were used for kinetic analysis as model substrates. The data suggest that substrate specificity of CaMKPase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site.
(2) Identification and characterization of CaMKP-N
Novel CaMKPase related protein, CaMKP-N, was cloned and characterized. CaMKP-N consisted of 757 amino acid residues with molecular weight of 84,176. CaMKP-N specifically dephosphorylated CaMKs and is stimulated by polycations. This enzyme is expressed abundantly in brain tissue and localized in nucleus. These results indicates that CaMKP-N dephophorylates CaMKIV and nuclear CaMKII, whereas CaMKPase dephosphorylates CaMKI and cytosolic CaMKII.
(3) Stimulation of CaMKPase by polycations
One of the prominent features of CaMKPase is stimulation of phosphatase activity by polycations such as poly(Lys). Various binding experiments suggested that the formation of a tightly associated ternary complex consisting of CaMKPase, poly(Lys), and phosphorylated CaMK is essential for stimulation. Poly(Lys) failed to stimulate a CaMKPase mutant in which a Glu cluster corresponding to residue 101-109 in the N-terminal domain was deleted, and the mutant could not interact with the polycations. Thus, the Glu cluster appeared to be the biding site for polycations and to play a pivotal role in the polycation stimulation of CaMKPase activity.
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