| Project/Area Number |
17590212
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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 pharmacology
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| Research Institution | Gunma University |
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Principal Investigator |
NAKAMURA Akio Gunma University, Molecular and Cellular Pharmacology, Associate Professor, 大学院医学系研究科, 講師 (30282388)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIKAWA Ryoki Gunma University, Molecular and Cellular Pharmacology, Associate Professor, 大学院医学系研究科, 講師 (20212863)
KOHAMA Kazuhiro Gunma University, Molecular and Cellular Pharmacology, Professor, 大学院医学系研究科, 教授 (30101116)
KISHI Hiroko Yamaguchi University, Molecular and Physiology and Medical Bioregulation, Associate Professor, 大学院医学系研究科, 講師 (40359899)
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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 |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Smooth Muscle / Myosin / Actin / Contraction / Myosin Light chain kinase / Phosphorylation / kinase / recombinant / 平滑筋 / 収縮制御 / キナーゼ / プロテオーム / アクトミオシン |
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| Research Abstract |
Smooth muscle myosin light chain kinase (SmMLCK), family of calmodulin-dependent protein kinases, catalyzes the phosphorylation of regulatory light chain (MLc 20) of myosin, and plays an important role in activating actomyosin-linked contractility in smooth muscle cell. SmMLCK also exhibits actin-binding and myosin-binding activities in addition to this kinase activity. These are called non-kinase activity. The former inhibits myosin ATPase activity, and latter activates. In order to clarify these molecule mechanisms, we tried making the recombinant SmMLCK in E.coli. We succeeded in the high-level expression (5-10 mg/L culture medium) of full-length recombinant SmMLCK by new bacterial expression system using the cold shock promoter. Recombinant SmMLCK was a soluble form and it phosphorylated MLc20. It also had non-kinase activity such as actin-binding and -bundling activities. Recombinant SmMLCK, with the properties known for SmMLCK, was quantifiably expressed and is the first step in analyzing the structure and function of MLCK. It is also reported that SmMLCK is regulated by several kinases, but it is not clear how upstream signaling regulates SmMLCK. We found that the purified SmMLCK from the bovine stomach was already partially phosphorylated by endogenous kinase. Usually the recombinant SmMLCK is not phosphorylated in bacteria. Therefore we examined which kind of kinase phosphorylated MLCK by using the recombinant SmMLCK and various Ser/Thr kinases. Recombinant SmMLCK was phosphorylated by MAPK, PAK, PKA, PKC and ROCK1 phosphorylated in the absence of Ca/calmodulin. Except for the PKA, other kinase could not incorporate into recombinant SmMLCK in the presence of Ca/calmodulin. We determined these phosphorylation sites by the 4000 Q Trap LC/MS/MS. The phosphorylation of MLCK by those upstream kinases may be physiologically important in regulating of actomyosin-linked contractility in smooth muscle cell.
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