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

Ca^<2+>/钙调素依赖性蛋白激酶II （CaM-kinase II）和IV （CaM-kinase IV）是第二信使反应的多功能蛋白激酶，与camp依赖性蛋白激酶和蛋白激酶C一样，在大脑中大量存在，因此这些蛋白激酶被认为在中枢神经系统（CNS）的信号转导中起重要作用。本研究主要关注CaM-kinase II和CaM-kinase iv活性的调控机制，发现CaM-kinase II的自抑制结构域在缺乏Ca^<2+>/钙调蛋白的情况下保持无酶活性，并对CaM-kinase II具有很强的稳定性。因此，cam -激酶II在细胞中似乎处于不活跃和稳定的状态，当它的活性是不必要的。另一方面，CaM-kinase II活性所需的Ca^<2+>/calmodulin使酶失活。钙调素的这两种相反作用在cam -激酶IV中也被观察到，尽管与cam -激酶II相比，Ca^<2+>/钙调素对cam -激酶IV的失活是可逆的。对来自大脑和肝脏的CaM-kinase II cDNA克隆的详细序列分析表明，CaM-kinase II的α、β、γ和δ基因通过选择性剪接产生许多CaM-kinase II亚型的可能机制。我们的研究最突出的结果是在大脑中发现了cam -激酶IV激酶，该激酶通过磷酸化激活cam -激酶IV 30- 40倍。从大鼠大脑皮层中纯化CaM-kinase IV激酶，经sds -聚丙烯酰胺凝胶电泳检测其均质性，并对其分子和催化性能进行了表征。CaM-kinase IV激酶是一种Ca^<2+>/钙调素依赖性蛋白激酶，可特异性磷酸化CaM-kinase IV。从人t细胞Jurkat细胞系cDNA文库中克隆人CaM-kinase IV cDNA，测定其核苷酸序列和推导出的氨基酸序列。

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.