Signal Transduction Mediated by Multifunctional Ca^<2+>/calmodulin-dependent Protein Kinases

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1. Regulation of CaM-kinase IICa^<2+>/calmodulin-dependent protein kinase (CaM-kinase) II is activated through autophosphorylation of Thr-286. We purified a new protein phosphatase (named CaM-kinase phosphatase), which dephosphorylates the phosphorylated Thr-286 restoring the activated CaM-kinase II to its original state, from rat brain and studied the properties of CaM-kinase phosphatase.The binding affinity for calmodulin of CaM-kinase II was studied using surface plasmon resonance. The dissociation rate constant of CaM-kinase II for calmodulin was decreased upon autophosphorylation of Thr-286 by approximately 10, 000-fold, suggesting that CaM-kinase II, once activated, can keep calmodulin even in the presence of many calmodulin-binding proteins. Thus, CaM-kinase II, once activated, may keep the highest activity in the cell until cytosolic Ca^<2+> is completely pumped out of the cell.2. Regulation of CaM-kinases IV and ICaM-kinases IV and I which have been activated upon phosphorylat … More ion of Thr-196 and Thr-177, respectively, by an upstream CaM-kinase kinase were deactivated by incubation with CaM-kinase phosphatase. CaM-kinase phosphatase specifically dephosphorylated phosphorylated Thr-286, Thr-196, and Thr-177 of CaM-kinases II, IV, and I, respectively. Thus, the activities of the three multifunctional CaM-kinases, such as CaM-kinases I, II, and IV, are all regulated by phosphorylation and dephosphorylation catalyzed by a specific protein kinase and phosphatase.3. Identification of CaM-kinase phosphatase NA protein (named CaM-kinase phosphatase N) showing a sequence homology to CaM-kinase phosphatase was found by a database search. CaM-kinase phosphatase N has a substrate specificity and other catalytic properties very similar to those of CaM-kinase phosphatase. However, CaM-kinase phosphatase is distributed in the cytosol but CaM-kinase phosphatase N is distributed in the cell nuclei.4. Possible model for CaM-kinase cascadeOn the basis of the results that tissue and subcellular distributions of CaM-kinases, CaM-kinase kinases, and CaM-kinase phosphatases were examined, a possible model for CaM-kinase cascade was proposed. Less