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

CaMKP是一种丝氨酸/苏氨酸蛋白磷酸酶，能去磷酸化和调节多功能的CaMKI、II和IV。CaMKP属于PPM家族，在催化区与PP2Calpha的同源性为28%。为了研究CaMKP的催化和调控特性，对重组CaMKP进行了突变分析。分析表明，N-末端序列是催化活性酶形成的关键，聚(Glu)簇负责阳离子激活CaMKP，PPM家族中保守的氨基酸残基可能在CaMKP的催化活性中起关键作用。为了阐明CaMKP的生理意义，我们利用二维Far-Western印迹技术结合多肽质量指纹分析，在大鼠脑内的一个可溶部分中确定了CaMKP的主要结合伙伴是甘油醛-3-磷酸脱氢酶(GAPDH)和果糖二磷酸缩醛缩醛酶。我们分析了这些互动的亲和力。野生型谷胱甘肽S转移酶在谷胱甘肽转移酶下拉分析中与谷胱甘肽脱氢酶相关。缺失分析表明，CaMKP催化结构域的N端负责与GAPDH的结合。CaMKP-N几乎只在大脑中出现，在羧基末端有核定位信号。观察CaMKP-N在脑内的分布。Western印迹分析表明，脑内CaMKP-N大部分以含有核定位信号的羧基末端缺失的形式存在。对大鼠脑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也有少量存在，尽管在一些大型神经元中主要存在于细胞核中。

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.