2004 Fiscal Year Final Research Report Summary
Significance of the binding between syntaxin and CaMKII using the knock-in mice strategy
| Project/Area Number |
15300123
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Niigata University |
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Principal Investigator |
IGARASHI Mchihiro Niigata University, Graduate School of Medical and Dental Science, Professor, 大学院・医歯学総合研究科, 教授 (50193173)
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| Co-Investigator(Kenkyū-buntansha) |
WATANABE Michitoshi Niigata University, Graduate School of Medical and Dental Science, Assistant, 大学院・医歯学総合研究科, 助手 (40303127)
ABE Haruki Niigata University, Graduate School of Medical and Dental Science, Professor, 大学院・医歯学総合研究科, 教授 (40018875)
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| Project Period (FY) |
2003 – 2004
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| Keywords | Syntaxin / Calmodulin / Myosin-V / Exocytosis / SNARE mechanism / Calcium / Knock-in mice / Structural analysis |
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| Research Abstract |
1)Knock-in mice : We have tried to produce the knock-in mice, which have the point mutation preventing from syntaxin to bind CaMKII. In the brain, three are two distinct isoforms known as syntaxin-1A and 1B ; thus, we have tried the two targeting vectors corresponding to these two isoforms. We have already succeeded in producing these vectors. By screening of the ES cells, we have confirmed the presence of the ES cells inbtoduced by the syntaxin-1A targeting vector.
2)We found another Ca2+-dependent syntaxin-binding protein, myosin-V, a molecular motor along the actin filament. Our group revealed that this interaction requires submicromolar Ca2+. This binding did not change the myosin-V motility itself, however, myosin-V ATPase activity was kept low. Using amperometry, we succeeded in showing the reduction of exocytosis when the myosin-V binding site derived from syntaxin was microinjected into chromaffin cells. Thus, we concluded that this interaction is involved in the efficiency of exocytosis.
3)We have characterized the interaction between syntaxin isoforms, which are localized in non-neuronal cells, and CaMKII. CaMKII was bound to syntaxin-2 to some extent, and it was not bound to syntaxin-3 or syntaxin-4. Syntaxin-2 interacted with CaMKII in a Ca2+-dependent manner, as well as syntaxin-1A. We also three important residues for the CaMKII binding. We considered that syntaxin-2 physiologically interacts with non-neuronal isoforms of CaMKII in non-neuronal cells, and that this interaction may contribute to the roles of syntaxin 2-bearing cells.
4)We investigated the phosphorylation sites of SCG10,a growth cone-specific microtubule-depolymerizing protein. Using the mutants, we succeeded in producing the SCG10 with
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