Regulation of Multifunctional Calmodulin-dependent Protein Kinase IV by Phosphorylation

(1)通过磷酸化调节钙调素依赖性蛋白激酶IV（CaMKIV）：在先前的研究中，CaMKIV被报道通过自磷酸化激活。在本研究中，发现该酶可被纯化的CaMKIV制剂中的另一种钙调蛋白依赖性蛋白激酶（CaMKIV激酶）激活。在CaMKIV中测定的磷酸化位点为N端Ser/Thr富集区的多个Ser残基，<196>酶中部的Ser^，和C端的Ser^<437>。最近的结果表明，负责酶激活的磷酸化位点是Ser^<196>。其他磷酸化位点的生理意义正在研究中。(2)从大鼠大脑皮层中鉴定CaMBP 64：在从大鼠大脑皮层中纯化CaMKIV的过程中分离到一种新的钙调素结合蛋白（CaMBP 64）。CaMBP 64是脑特异性二聚体蛋白，亚基重量为64 kDa。该蛋白质作为钙调素依赖性蛋白激酶II和cAMP依赖性蛋白激酶的良好底物。经测定其主要磷酸化位点的氨基酸序列为SQPSFQWRQPSLDVDVGD。从535个氨基酸序列的同源性搜索，该蛋白被确定为大鼠的63 kDa钙调素依赖性环核苷酸磷酸二酯酶的异构体。(3)SDS-聚丙烯酰胺凝胶电泳检测序列特异性蛋白激酶的新方法：建立了一种检测凝胶中蛋白激酶活性的新方法。当使用含有与多聚赖氨酸缀合的合成寡肽的聚丙烯酰胺凝胶进行凝胶内复性分析时，可以检测序列特异性蛋白激酶。该方法可用于钙调素依赖性蛋白激酶级联反应中蛋白激酶间的交叉作用分析。

(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.