1995 Fiscal Year Final Research Report Summary
Regulation of Multifunctional Calmodulin-dependent Protein Kinase IV by Phosphorylation
| Project/Area Number |
06680570
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Structural biochemistry
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| Research Institution | Asahikawa Medical College |
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Principal Investigator |
KAMESHITA Isamu Asahikawa Medical College, Associate Professor, 医学部, 助教授 (60127941)
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| Co-Investigator(Kenkyū-buntansha) |
FUJISAWA Hitoshi Asahikawa Medical College, Professor, 医学部, 教授 (10027039)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Protein Kinase / Calmodulin / Protein Phosphorylation / Signal Transduction / Central Nervous System |
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| Research Abstract |
(1) Regulation of Calmodulin-dependent Protein Kinase IV (CaMKIV) by Phosphorylation : In the previous studies, CaMKIV was reported to be activated by autophosphorylation. In this study, however, the enzyme was found to be activated by the other calmodulin-dependent protein kinase (CaMKIV kinase) included in purified preparation of CaMKIV.Phosphrylation sites determined in CaMKIV were multiple Ser residues at N-terminal Ser/Thr rich-region, Ser^<196> at middle part of the enzyme, and Ser^<437> at C-terminal region. Recent results suggested that the phosphorylation site responsible for enzyme activation was Ser^<196>. Physiological significance of the other phosphorylation sites are now under investigation.
(2) Characterization of CaMBP64 from Rat Cerebral Cortex : A novel calmodulin-binding protein (CaMBP64) was isolated in the process of purification of CaMKIV from rat cerebral cortex. CaMBP64 was a brain-specific dimeric protein with subunit weight of 64 kDa. This protein served as a good substrate for calmodulin-dependent protein kinase II and cAMP-dependent protein kinase. Amino acid sequence of major phosphorylation site was determined to be SQPSFQWRQPSLDVDVGD.In order to determibe whole amino acid sequence of this protein, molecular cloning was carried out. From the homology search of 535 amino acid sequence, this protein was identified as rat isoform of 63 kDa calmodulin-dependent cyclic nucleotide phsophodiesterase.
(3) New Method for Detection of Sequence Specific Protein Kinases after SDS-Polyacrylamide Gel Electrophoresis : A new method for detection of protein kinase activities toward synthetic oligopeptides in gel was developed. When in-gel renaturation assay was carried out using polyacrylamide gel containing synthetic oligepeptide conjugated to poly-L-lysine, sequence specific protein kinases could be detected. This method will be useful for the analysis of cross-talks between protein kinase involved in calmodulin-dependent protein kinase cascade.
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