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

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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.