Takako Kitani

Growth phase‐dependent changes in the subcellular localization of pre‐B‐cell colony‐enhancing factor1

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.

Calmodulin-dependent Protein Kinase II Potentiates Transcriptional Activation through Activating Transcription Factor 1 but Not cAMP Response Element-binding Protein

Activating transcription factor 1 (ATF1) and the cAMP response element-binding protein (CREB) are members of the CREB/ATF family implicated in cAMP- and calcium-induced transcriptional activation. Although ATF1 and CREB share extensive homology, the function of ATF1 is poorly understood. Its phosphorylation state and activation by Ca2+- and calmodulin-dependent protein kinase (CaMK) II were therefore examined. Phosphopeptide mapping analysis and Western blotting studies demonstrated that in vitro, CaMK II phosphorylates only Ser63 (corresponding to Ser133 of CREB), which is essential for the activation, and not Ser72 (corresponding to Ser142 of CREB), which is a negative regulation site. Both ATF1 and CREB bound CBP in a phosphorylation-dependent manner. As expected from these in vitro studies, transient transfection studies revealed that ATF1 is activated by CaMK II. Our findings suggest that CaMK II mediates transactivation of cAMP responsive genes via ATF1.

Critical amino acid residues of AIP, a highly specific inhibitory peptide of calmodulin-dependent protein kinase II

The importance of the individual amino acid residues of AIP (KKALRRQEAVDAL), a highly specific inhibitor of calmodulin‐dependent protein kinase II (CaMKII), was studied. Replacement of Arg6, Gln7, or Ala9 by other amino acid residues produced a marked increase in the IC50 value. Leu4 and Val10 were also sensitive to replacement, but some hydrophobic amino acids could substitute for these residues. Although replacement of Ala3, Glu8, Ala12, and Leu13 by other residues produced no significant increase in the IC50, the substitution of Lys for Ala3 decreased the IC50. An AIP analog (KK LRRQEA DAY), in which Ala3 and Val10 were replaced with Lys and Phe, respectively, showed an IC50 value as low as 4 nM, suggesting that it is a useful tool for studying the physiological roles of CaMKII.

Localization of the mRNAs for two isoforms of Ca2+/calmodulin-dependent protein kinase kinases in the adult rat brain.

Ca2+/calmodulin-dependent protein kinase (CaM kinase) I and IV are thought to be activated by CaM kinase kinases (CaMKK). We examined the distribution of mRNAs for two isoforms (alpha and beta) of CaMKKs in the brain by in situ hybridization histochemistry. In the adult rat brain, CaMKK alpha mRNAs are widely distributed throughout the brain, whereas CaMKK beta mRNAs are restricted to some neuronal populations, particularly the cerebellar granule cells.

Distribution of Ca2+/calmodulin-dependent protein kinase α in the rat central nervous system: an immunohistochemical study

Abstract Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated by Cam-kinase IV kinase. We provided a rabbit antiserum against 20 amino acid residues at the carboxyl-terminal end of CaM-kinase IV kinase, and examined regional and intracellular distribution of CaM-kinase IV kinase immunohistochemically in the central nervous system of the rat by light and electron microscopy. The immunoreactivity was found in cellular nuclei of virtually all neurons. However, the immunoreactivity was weak in the nuclei of the granule cells in the cerebellar cortex, although the nuclei of the granule cells were reported to contain high CaM-kinase IV activity. Thus, it was suggested that other types of CaM-kinase IV kinase might exist in the cerebellum, and the present CaM-kinase IV kinase was named as CaM-kinase kinase α.

Purification and characterization of antizyme inhibitor of ornithine decarboxylase from rat liver

A protein inhibiting a protein inhibitor (antizyme) to ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (ODC), antizyme inhibitor, was purified from the liver cytosol of thioacetamide-treated rats by procedures including antizyme affinity chromatography. Overall purification was roughly estimated to be about 17,000,000-fold and recovery was about 2.4%. The purified preparation showed one major protein band and a faint band corresponding in mobility to molecular weights of 51,000 and 53,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Judging from the ornithine decarboxylase activity of the final preparation, the faint band may be ornithine decarboxylase. The apparent molecular weight of antizyme inhibitor estimated by gel filtration on Sephacryl S-200 was approx. 62,000, indicating that antizyme inhibitor may be composed of a single polypeptide chain. In order to examine the question of whether antizyme inhibitor is a protein derived from ornithine decarboxylase, an inactive ornithine decarboxylase, in an immunotitration study and analysis of the binding to antizyme were investigated. The results indicate that antizyme inhibitor may be a protein distinct from ornithine decarboxylase.

Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase kinase β in the rat central nervous system

We examined regional and intracellular distribution of Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaM-KK beta), which activated Ca(2+)/calmodulin-dependent protein kinase I and IV (CaM-K I and IV) immunohistochemically in the central nervous system of the rat by light and electron microscopy. Although most neurons in the brain and spinal cord exhibited the immunoreactivity, no labeled neurons were observed in the globus pallidus or entopeduncular nucleus, and only a small number of neurons showed weak immunoreactivity in the substantia nigra pars reticulata. In general, the immunoreactivity was observed both in the cytoplasm and cellular nucleus, although the immunoreactivity was not found in the cellular nucleus in some large neurons such as in the mesencephalic trigeminal nucleus, lateral vestibular nucleus or gigant cellular reticular formation. As to motoneurons of the cranial nerve nuclei and the anterior horn of the spinal cord, they revealed the immunoreactivity both in the cytoplasm and nucleus. The reaction product appeared as fine granules in the cytoplasm and nucleus under light microscopy. Electron microscopic observations confirmed that the reaction product was localized mainly on the Golgi apparatus or on the nuclear chromatin. Immunolabeling for antibody against CaM-KK beta was discussed with the distribution of CaM-K I, IV and another CaM-KK, CaM-KK alpha, in the central nervous system.

Stimulation of Ca2+/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.

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 α and β, 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 α and β, 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 β, and CaM kinase phosphatase occur in the cytoplasm and that CaM kinase IV, CaM kinase kinase α (and CaM kinase kinase β 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 β, and CaM kinase phosphatase in the cytoplasm and the other consisting of CaM kinase IV, CaM kinase kinase α (and CaM kinase kinase β in some cell types and tissues), and CaM kinase phosphatase N in the nucleus.

Immunohistochemical study of the distribution of Ca2+/calmodulin-dependent protein kinase phosphatase in the rat central nervous system

Abstract Distribution of Ca2+/calmodulin-dependent protein kinase phosphatase (CaM-K Pase) which dephosphorylate multifunctional Ca2+/calmodulin-dependent protein kinases (CaM-kinases) in the rat brain and spinal cord were examined immunohistochemically by using an antibody against this enzyme. CaM-K Pase was localized only in the cytoplasm as has been investigated in PC 12 cells, and was never observed in the nucleus. Immunostainability varied from cell group to cell group. Mitral cells in the olfactory bulb, pyramidal neurons in the fifth layer of the cerebral cortex, hippocampal and striatal interneurons, dorsal and ventral pallidal, entopeduncular, and the reticular part of the substantia nigra neurons were intensely immunolabeled. Motoneurons in all the cranial nerve nuclei and the anterior horn of the spinal cord also revealed intense immunolabeling. On the contrary, pyramidal neurons in the Ammon’s horn of the hippocampal formation, granule cells in the olfactory bulb, dentate gyrus and cerebellar cortex, Purkinje cells, neurons in the medial habenular nucleus and the inferior olivary nucleus have not shown immunoreactivity. Axons in the white matter or nerve root of the cranial nerve nuclei were immunolabeled. Glial cells in the white matter also showed immunostaining. Because the substrate of CaM-K Pase is multifunctional CaM-kinase II, I and IV, localization of each CaM-kinase was compared with that of CaM-K Pase. The distribution of CaM-K Pase and these CaM-kinases was found to overlap in various regions in the brain and spinal cord. It was concluded, therefore, that CaM-K Pase could regulate the activity of these CaM-kinases by dephosphorylation, when they existed together in neurons.