Post-translational excision of the carboxyl-terminal segment of CaM kinase phosphatase N and its cytosolic occurrence in the brain.

Ca2+/Calmodulin-dependent protein kinase (CaM kinase) phosphatase, occurring in the cytoplasm of all tissues, dephosphorylates and thereby deactivates multifunctional CaM kinases, such as CaM kinases I, II and IV. In contrast, CaM kinase phosphatase N has been reported to occur almost exclusively in the brain and to be localized in the nucleus in the transfected COS-7 cells, as examined immunocytochemically with antibodies against the carboxyl-terminal segment of the enzyme, indicating its involvement in the deactivation of CaM kinase IV. Here, we show that the majority of the naturally occurring CaM kinase phosphatase N in the brain exists not in the intact form of the enzyme (83.4 kDa) but in a form (61.1 kDa) in which the carboxyl-terminal segment containing nuclear localization signals is deleted, and that it 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. Strong immunostaining of the enzyme was also observed at postsynaptic density. These findings suggest that CaM kinase phosphatase N is involved in the regulation of not only CaM kinase IV but also CaM kinases II and I.

Growth phase-dependent changes in the subcellular localization of pre-B-cell colony-enhancing factor.

A cDNA encoding the homolog of the human pre-B-cell colony-enhancing factor (PBEF), a cytokine-like secreted protein, was isolated from a rat cDNA library. This protein existed in both the cytoplasm and nucleus of the cells, and the amount was higher in the cytoplasm than in the nucleus of proliferating PC-12 and Swiss 3T3 cells but higher in the nucleus than in the cytoplasm of the PC-12 cells treated with nerve growth factor and the 3T3 cells grown to a confluent state. Thus, the so-called PBEF is not a cytokine-like secreted protein but an intracellular protein associated with the cell cycle.

Subcellular distributions of rat CaM kinase phosphatase N and other members of the CaM kinase regulatory system.

Ca2+/Calmodulin-dependent protein kinase (CaM kinase) regulatory system is composed of multifunctional CaM kinases such as CaM kinases IV and I, upstream CaM kinases such as CaM kinase kinases alpha and beta, which activate multifunctional CaM kinases, and CaM kinase phosphatases such as CaM kinase phosphatase and CaM kinase phosphatase N, which deactivate the activated multifunctional CaM kinases. To understand the combinations of CaM kinases I and IV, CaM kinase kinases alpha and beta, and CaM kinase phosphatases, the locations of the enzymes in the cell were examined by immunocytochemical studies of cultured cells. The results indicate that CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase occur in the cytoplasm and that CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N occur inside the cellular nucleus, suggesting that there are at least two different sets of CaM kinase regulatory systems, one consisting of CaM kinase I, CaM kinase kinase beta, and CaM kinase phosphatase in the cytoplasm and the other consisting of CaM kinase IV, CaM kinase kinase alpha (and CaM kinase kinase beta in some cell types and tissues), and CaM kinase phosphatase N in the nucleus.

Stimulation of Ca(2+)/calmodulin-dependent protein kinase phosphatase by polycations.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). One of the prominent features of CaMKPase is stimulation of phosphatase activity by polycations such as poly-L-lysine (poly(Lys)). Using various polycations, basicity and molecular weight of the polymer proved to be important for the stimulation. Surface plasmon resonance (SPR) analysis showed that CaMKIV(T196D), which mimics CaMKPase substrate, and CaMKPase could form tight complexes with poly(Lys). Pull-down binding experiments suggested that the formation of a tightly associated ternary complex consisting of CaMKPase, poly(Lys), and phosphorylated CaMKIV is essential for stimulation. Dilution experiments also supported this contention. Poly(Lys) failed to stimulate a CaMKPase mutant in which a Glu cluster corresponding to residues 101-109 in the N-terminal domain was deleted, and the mutant could not interact with poly(Lys) in the presence of Mn(2+). Thus, the Glu cluster appeared to be the binding site for polycations and to play a pivotal role in the polycation stimulation of CaMKPase activity.

Phosphorylation of calmodulin by Ca2+/calmodulin-dependent protein kinase IV.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly-L-Lys [poly(Lys)]-dependent manner. Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase alpha, and cAMP-dependent protein kinase did not phosphorylate CaM. Polycations such as poly(Lys) were required for the phosphorylation. The optimum concentration of poly(Lys) for the phosphorylation of 1 microM CaM was about 10 microg/ml, but poly(Lys) strongly inhibited CaM-kinase IV activity toward syntide-2 at this concentration, suggesting that the phosphorylation of CaM is not due to simple activation of the catalytic activity. Poly-L-Arg could partially substitute for poly(Lys), but protamine, spermine, and poly-L-Glu/Lys/Tyr (6/3/1) could not. When phosphorylation was carried out in the presence of poly(Lys) having various molecular weights, poly(Lys) with a higher molecular weight resulted in a higher degree of phosphorylation. Binding experiments using fluorescence polarization suggested that poly(Lys) mediates interaction between the CaM-kinase IV/CaM complex and another CaM. The 32P-labeled CaM was digested with BrCN and Achromobacter protease I, and the resulting peptides were purified by reversed-phase HPLC. Automated Edman sequence analysis of the peptides, together with phosphoamino acid analysis, indicated that the major phosphorylation site was Thr44. Activation of CaM-kinase II by the phosphorylated CaM was significantly lower than that by the nonphosphorylated CaM. Thus, CaM-kinase IV activated by binding Ca2+/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM.