Regulation of ion-transporters by A-kinase regulated by via anchoring protein
| Project/Area Number |
11671851
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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 |
Functional basic dentistry
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| Research Institution | Meikai University |
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Principal Investigator |
KURIHARA Kinji Meikai University School of Dentisrty, Physiology, Instructor, 歯学部, 助手 (10170086)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Na^+, K^+-ATPase / Na^+-K^+-2Cl^- cotransporter / ion-transport / A-kinase / anchoring protein / phosphorylation / parotid gland / acinar cell / Na^+K^+-ATPase / Na^+, K^+-2Cl^-cortransporter |
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| Research Abstract |
Existence of A-kinase anchoring proteins (AKAPs) in parotid glands (PGs)
1. Membrane proteins including Na^+, K^+-ATPase and Na^+-K^+-2Cl^- cotransporter in isolated basolateral membrane vesicles (BLMVs) were phosphorylated by membrane-bound protein kinase A (PKA) in the presence of cAMP, suggesting the involvement of AKAPs in these phosphorylations.
2. mRNAs and proteins of AKAP-150 were detected in PGs significantly by RT-PCR and Western blot analysis, whereas AKAP-95 and AKAP-220 were less.
3. AKAP-150 of BLMVs was co-immunoprecipitated with the RII subunit by anti-RII antibody. These results indicate that AKAP-150 in BLMVs is functional and associated with the endogenous RII subunit.
Phosphorylation of Na^+, K^+-ATPase via AKAP/PKA complex
1. Inhibition of the RII binding to AKAP by HT-31 peptide resulted in a decrease in membrane-bound PKA-dependent phosphorylation of Na^+, K^+-ATPase.
2. Phosphorylation of Na^+, K^+-ATPase by the AKAP/PKA was associated with a decrease in the Na^+, K^+
… More
-ATPase activity
Mechanism of phosphorylation of Na^+-K^+-2Cl^- cotransporter N-terminus via cAMP/PKA pathway
1. Bumetanide-sensitive Na^+-K^+-2Cl^- cotransport activity in rat PG acini was upregulated with isoproterenol. The increase in its activity was accompanied by phosphorylation of the Na^+-K^+-2Cl^- cotransporter protein and by an increase in the number of high affinity bumetanide building sites.
2. The phosphorylation site of Na^+-K^+-2Cl^- cotransporter was on its N-terminus by isoproterenol-stimulation of acinar cells.
3. N-terminus phosphorylation never occurred by in vitro incubation of the Na^+-K^+-2Cl^- cotransporter in BLM preparation with exogenous PKA or AKAP/PKA, and neither its activity nor its bumetanide binding were changed.
4. When acinar cells were mildly permeabilized with digitonin, N-terminus of the Na^+-K^+-2Cl^- cotransporter was clearly phosphorylated upon the addition of cAMP. These results indicated that Na^+-K^+-2Cl^- cotransporter was regulated by the phosphorylation on its N-terminus and that PKA did not directly phosphorylate the cotransporter N-terminus but via protein phosphorylation cascade triggered by PKA. Less
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Report
(4 results)
Research Products
(22 results)
