Enzymatic study of CaM kinase phosphatase for elucidation of its biological function

CaMKP是一种Ser/Thr蛋白磷酸酶，可使多功能CaMKI、II和IV去磷酸化并调节。CaMKP属于PPM家族，与PP 2Calpha在催化结构域的同源性为28%。大鼠CaMKP具有独特的N端150个氨基酸的聚谷氨酸簇结构，为了研究CaMKP的催化和调节特性，对重组CaMKP进行了突变分析。分析表明，N端序列是催化活性酶形成所必需的，聚谷氨酸簇是聚阳离子激活CaMKP的关键，PPM家族中保守的氨基酸残基可能对CaMKP的催化活性起关键作用。我们使用二维远蛋白质印迹技术鉴定了甘油醛-3-磷酸脱氢酶（GAPDH）和果糖二磷酸醛缩酶作为大鼠脑可溶性部分中CaMKP的主要结合伴侣，结合肽质量指纹分析。我们分析了这些相互作用的亲和力。在GST下拉测定中与GAPDH相关的野生型CaMKP-谷胱甘肽S-转移酶（GST）。缺失分析表明CaMKP催化结构域的N端负责与GAPDH的结合，CaMKP-N几乎只存在于脑中，在羧基端区域具有核定位信号。检测CaMKP-N在脑中的分布。Western blot分析表明，大部分的CaMKP-N在大脑中存在的形式，其中羧基端段含有核定位信号被删除。大鼠脑组织的免疫组织化学研究表明，CaMKP-N主要存在于细胞质中，但在整个中枢神经系统的细胞核中有一点，虽然在一些大的神经元中主要发生在细胞核中。

CaMKP is a Ser/Thr protein phosphatase that dephosphorylates and regulates multifunctional CaMKI,II, and IV. CaMKP belongs to PPM family with homology to PP2Calpha being 28% in the catalytic domain. Rat CaMKP has a unique N-terminal sequence of about 150 amino acids containing poly(Glu) cluster.To investigate catalytic and regulatory properties of CaMKP, mutational analysis of recombinant CaMKP was carried out. The analysis suggested that N-terminal sequences are essential for formation of the catalytically active enzyme, that poly(Glu) cluster is responsible for activation of CaMKP by polycations, and that amino acid residues conserved in PPM family may play crucial roles in the catalytic activity of CaMKP.To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions. Wild type CaMKP-glutathione S-transferase (GST) associated with GAPDH in a GST pull-down assay. Deletion analysis suggested that the N-terminal side of the catalytic domain of CaMKP is responsible for the binding to GAPDH.CaMKP-N, occurring almost exclusively in the brain, has nuclear localization signals in the carboxyl-terminal region. The distribution of CaMKP-N in the brain was examined. Western blot analysis indicated that the majority of CaMKP-N in the brain exists in a form in which the carboxyl-terminal segment containing nuclear localization signals is deleted. Immunohistochemical studies of the rat brain indicated that CaMKP-N is present mostly in the cytoplasm but a little in the nucleus throughout the central nervous system, although occurring mostly in the nucleus in some large neurons.