| Co-Investigator(Kenkyū-buntansha) |
ISHIDA Atsuhiko Asahikawa Medical College, Biochemistry, Instructor, 医学部, 助手 (90212886)
KATOH Tsuyoshi Asahikawa Medical College, Biochemistry, Assistant Professor, 医学部, 助教授 (60194833)
KAMESHITA Isamu Asahikawa Medical College, Biochemistry, Assistant Professor, 医学部, 助教授 (60127941)
KITANI Takako Asahikawa Medical College, Biochemistry, Research Associate, 医学部, 教務職員 (70101417)
TAKEUCHI Masayuki Asahikawa Medical College, Biochemistry, Instructor, 医学部, 助手 (40226999)
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| Research Abstract |
1. Regulation of CaM-kinase II
Ca^<2+>/calmodulin-dependent protein kinase (CaM-kinase) II is activated through autophosphorylation of Thr-286. We purified a new protein phosphatase (named CaM-kinase phosphatase), which dephosphorylates the phosphorylated Thr-286 restoring the activated CaM-kinase II to its original state, from rat brain and studied the properties of CaM-kinase phosphatase.
The binding affinity for calmodulin of CaM-kinase II was studied using surface plasmon resonance. The dissociation rate constant of CaM-kinase II for calmodulin was decreased upon autophosphorylation of Thr-286 by approximately 10, 000-fold, suggesting that CaM-kinase II, once activated, can keep calmodulin even in the presence of many calmodulin-binding proteins. Thus, CaM-kinase II, once activated, may keep the highest activity in the cell until cytosolic Ca^<2+> is completely pumped out of the cell.
2. Regulation of CaM-kinases IV and I
CaM-kinases IV and I which have been activated upon phosphorylat
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ion of Thr-196 and Thr-177, respectively, by an upstream CaM-kinase kinase were deactivated by incubation with CaM-kinase phosphatase. CaM-kinase phosphatase specifically dephosphorylated phosphorylated Thr-286, Thr-196, and Thr-177 of CaM-kinases II, IV, and I, respectively. Thus, the activities of the three multifunctional CaM-kinases, such as CaM-kinases I, II, and IV, are all regulated by phosphorylation and dephosphorylation catalyzed by a specific protein kinase and phosphatase.
3. Identification of CaM-kinase phosphatase N
A protein (named CaM-kinase phosphatase N) showing a sequence homology to CaM-kinase phosphatase was found by a database search. CaM-kinase phosphatase N has a substrate specificity and other catalytic properties very similar to those of CaM-kinase phosphatase. However, CaM-kinase phosphatase is distributed in the cytosol but CaM-kinase phosphatase N is distributed in the cell nuclei.
4. Possible model for CaM-kinase cascade
On the basis of the results that tissue and subcellular distributions of CaM-kinases, CaM-kinase kinases, and CaM-kinase phosphatases were examined, a possible model for CaM-kinase cascade was proposed. Less
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