Regulation of the Function of CNS by Ca^<2+>/calmodulin-dependent Multifunctional Protein Kinases

钙调素依赖性蛋白激酶II(CaM-Kinase II，CaM-Kinase II)和钙调蛋白依赖性蛋白激酶IV(CaM-Kinase IV，CaM-Kinase IV)在脑内含量特别丰富，与cAMP依赖的蛋白激酶和蛋白激酶C一样，是第二信使反应型多功能蛋白激酶，在中枢神经系统(CNS)的信号转导中发挥着重要作用。本研究的重点是CaM-Kinase II和CaM-Kinase IV活性的调节机制。CaM-Kinase II的自身抑制结构域在没有钙离子/钙调素的情况下能够保持无酶的活性，被发现具有很强的稳定性。因此，在细胞中，当不需要其活性时，CaM-Kinase II似乎处于不活跃和稳定的状态。另一方面，CaM-Kinase II活性所需的钙调素使该酶失活。在CaM-Kinase IV中也观察到了这种相反的两种作用，尽管与CaM-Kinase II相比，CaM-Kinase IV的失活是可逆的。对来自大脑和肝脏的CaM-Kinase II克隆的详细序列分析表明，通过选择性剪接从CaM-Kinase II的α、β、γ和Delta基因中产生许多CaM-Kinase II亚型的可能机制。我们研究的最突出的结果是在大脑中发现了CaM-Kinase IV激酶，它通过磷酸化激活了CaM-Kinase IV 30-40倍。从大鼠大脑皮质中分离纯化出明显均一的CaM-KK IV，并对其分子和催化性质进行了研究。CaM-Kinase IV是一种钙离子/钙调素依赖的蛋白激酶，能特异性地磷酸化CaM-Kinase IV。从人T细胞系Jurkat细胞的cDNA文库中克隆了人CaM-Kinase IV基因，并测定了其核苷酸序列和氨基酸序列。

Ca^<2+>/calmodulin-dependent protein kinase II (CaM-kinase II) and IV (CaM-kinase IV), which exist particularly abundantly in the brain, are second messenger-responsive multifunctional protein kinases, just like cAMP-dependent protein kinase and protein kinase C,and therefore these protein kinases are thought to play important roles in signal transduction in the central nervous system (CNS). The present study focuses our attention on regulatory mechanisms of the activities of CaM-kinase II and CaM-kinase IV.The autoinhibitory domain of CaM-kinase II,which is known to keep the enzymeless active in the absence of Ca^<2+>/calmodulin, was found to strongly stabilize the enzyme. Thus, CaM-kinase II appears to be in an inactive and stable state in a cell when its activity is not necessary. Ca^<2+>/calmodulin required for the activity of CaM-kinase II,on the other hand, inactivated the enzyme. Such two opposite effects of calmodulin were also observed with CaM-kinase IV,although the inactivation of CaM-kinase IV by Ca^<2+>/calmodulin was reversible in contrast to the case of CaM-kinase II.Detailed sequence analysis of CaM-kinase II cDNA clones from the brain and liver suggested a possible mechanism for producing many CaM-kinase II isoforms from the alpha, beta, gamma, and delta genes of CaM-kinase II by alternative splicing.The most prominent result of our study is the discovery of CaM-kinase IV kinase in the brain which activates CaM-kinase IV 30- to 40-fold by phosphorylation. CaM-kinase IV kinase was purified from rat cerebral cortex to apparent homogeneity upon SDS-polyacrylamide gel electrophoresis, and its molecular and catalytic properties were examined. CaM-kinase IV kinase was a Ca^<2+>/calmodulin-dependent protein kinase which phosphorylated CaM-kinase IV specifically. Human CaM-kinase IV cDNA was cloned from a cDNA library of a Jurkat cell, a cell line of human T-cell, and its nucleotide sequence and the deduced amino acid sequence were determined.