1997 Fiscal Year Final Research Report Summary
ROLE OF THE NOVEL PHOSPHOLYLATION IN TRANSPORT PUMPS
| Project/Area Number |
08044047
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Field |
Functional biochemistry
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
TANIGUCHI Kazuya HOKKAIDO UNIV., GRADUATE SCHOOL OF SCIENCE,PROFESSOR, 大学院・理学研究科, 教授 (40028204)
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| Co-Investigator(Kenkyū-buntansha) |
POST Robert L UNIVERSITY OF PENNSYLVANIA,SCHOOL OF MEDICINE,PROFESSOR, 医学部, 教授
MARDH Sven LINKOPING UNIVERSITY,FACULTY OF HEALTH SCIENCES,PROFESSOR, 生命科学部, 教授
FROEHLICH Jeffrey P NIA,LAB.OF CARDIOVASCULAR SCIENCE,CHIEF, 疫年学研究センター・部長, 教授
SCHONER Wilhelm JUSTUS-LIEBIG UNIVERSITY,INSTITUT FUR BIOCHEMIE UND ENDOKRINOLOQIE,PROFESSOR, 生化学・内分泌学, 教授
KAYA Shunji HOKKAIDO UNIV., GRADUATE OF SCHOOL OF SCIENCE ASSISTANT PROFESSOR, 大学院・理学研究科, 助教授 (90186023)
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| Project Period (FY) |
1996 – 1997
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| Keywords | phosphorylation / dephosphorylation / protein kinase / protein phosphatases / H-pump / H,K-ATPase |
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| Research Abstract |
H/K-ATPase preparations (G_1) of pig stomach showed Ca dependent Ser-27 (k_<1/2>=0.6muM) phosphorylation and independent Tyr-10 and -7 phosphorylation of the catalytic subunit of H/K-ATPase preferentially. Addition of a detergent, 3-[(3-Cholamidepropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) or a specific PKC inhibitor abolished the Ca dependent phosphorylation. The addition of CHAPS permitted to phosphorylate synthetic copolymer substrates and N-terminal domains of alpha-chain of H/K-ATPase fused with maltose binding protein. Western blotting analysis and invitro kinase assay using fusion proteins and others showed that the Ser-kinase recognized with antibodies against PKCalpha and PKCgamma and the Tyr-kinase with that against N-terminal part of SRC but not against C-terminal part of SRC are responsible for these phosphorylation. The Tyr-kinase was neither recognized with antibody against Yes nor Lyn. Column chromatographic separation of CHAPS solubilized kinases and the reactvities of sample with antibodies indicated that the apparent molecular weight of the Ser kinase was 80 kDa and that of Tyrkinase was 60 kDa. Breakdown of phosphoserine of the fusion protein by G1 was biphasically inhibited by okadaic acid with little effect of Mg and Ca suggesting the presence of protein phosphatase-1 and 2A but neither -2B nor -2C.The presence of phosphotyrosine phosphatase, SHP-2 but not SHP-1 wasimmunologically detected. These data and others strongly suggested that conventional PKCs, a novel SRC-related Tyr-kinase and phosphatases present in near N-terminal membrane domain of H/K-ATPase are participating specific phosphorylation and dephosphorylation of Ser-27 and Tyr-10 and -7.
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