2005 Fiscal Year Final Research Report Summary
Regulation of ion-transporters by A-kinase regulated via anchoring protein
| Project/Area Number |
14571775
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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 Dentistry, Instructor, 歯学部, 助手 (10170086)
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| Co-Investigator(Kenkyū-buntansha) |
NAKANISHI Nobuo Meikai University, School of Dentistry, Lecturer, 歯学部, 講師 (20118574)
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| Project Period (FY) |
2002 – 2005
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| Keywords | anchoring protein / AKAP / A-kinase / phosphorylation / Na / K-ATPase / Na / K / 2Cl cotransporter / NKCC1 / salivary gland |
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| Research Abstract |
Phosphorylation of Na^+, K^+-ATPase via AKAP/PKA complex
1.We have examined the expression of A-kinase anchoring protein (AKAP)/PKA complex in the three major salivary glands, i.e., the parotid gland (PG), submandibular gland (SMG), and sublingual gland (SLG), of the rat. AKAP-150 mRNA and RII regulatory subunit of PKA were clearly detected in the PG, but they were hardly detectable in either the SMG or SLG.
2.Incubation with [γ-^<32>P]ATP revealed that Na^+,K^+-ATPase in the PG membranes was quickly phosphorylated upon the addition of cAMP, whereas the ATPases in the membranes from SMG and SLG were not, suggesting phosphorylation of Na^+,K^+-ATPase by AKAP/PKA are characteristics specific to PG among the three major salivary glands.
Phosphorylation of Na^+/K^+/2Cl^- cotransporter (NKCC1) N-terminus via cAMP/PKA pathway
1.We have previously shown that Na^+,K^+,2Cl^--cotransport activity in parotid acinar cells is dramatically upregulated by N-terminus phosphorylation. However we showed here that N-terminus was not phosphorylated by PKA directly in vitro system.
2.The phosphorylation of N-terminus was mimicked by the addition of cAMP to permeabilized acini without stimulation of β-adrenergic receptors. This result indicates that the N-terminus was not phosphorylated by PKA directly, however it was clearly phosphorylated by PKA cascade triggered by PKA activation.
3.The cAMP dependent phosphorylation of the N-terminus was blocked competitively by the addition of partial N-terminus peptides including Thr-208 into permeabilized acini. This result indicated that functions of NKCC1 were regulated by the phosphorylation of Thr-208 on the N-terminus.
4.We carried out cloning of NKCC1 gene and prepared E.Coli transfected its gene. We are going to prepare the plasmids modified to Ala from Thr at various phosphorylation sites, i.e. Thr-203, -208 and -221 in N-terminus and at PKA consensus site.
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