Hideyuki Yamamoto

Purification and Characterization of a Ca2+‐ and Calmodulin‐Dependent Protein Kinase from Rat Brain

Abstract: A Ca2+‐ and calmodulin‐dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800‐fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S‐300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited Km values of 109 and 30 μM for ATP and chicken gizzard myosin light chain, respectively, and Ka values of 12 nM and 1.9 μM for brain calmodulin and Ca2+, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine‐rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a Ca2+‐ and calmodulin‐dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunit of cyclic AMP‐dependent protein kinase. Our findings indicate that there is a multifunctional Ca2+‐ and calmodulin‐dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins.

Ca2+– and Calmodulin‐Dependent Phosphorylation of Microtubule‐Associated Protein 2 and t Factor, and Inhibition of Microtubule Assembly

Abstract: Microtubule‐associated proteins (MAPs) were phosphorylated by a Ca2+– and calmodulin‐dependent protein kinase from rat brain cytosol. The maximal amount of phosphate incorporated into MAPs was 25 nmol of phosphate/mg protein. A Ka value of the enzyme for calmodulin was 57.0 nM, with MAPs as substrates. Among MAPs, MAP2 and t factor were phosphorylated in a Ca2 +‐and calmodulin‐dependent manner. The phosphorylation of MAPs led to an inhibition of microtubule assembly in accordance with its degree. This reaction was dependent on addition of the enzyme, Ca2+, and calmodulin, and had a greater effect on the initial rate of microtubule assembly rather than on the final extent. The critical tubulin concentration for microtubule assembly was unchanged by the MAPs phosphorylation. Therefore assembly and disassembly of brain microtubule are regulated by the Ca2+‐ and calmodulin‐dependent protein kinase that requires only a nanomolar concentration of calmodulin for activation.

Ca2+, Calmodulin‐Dependent Regulation of Microtubule Formation via Phosphorylation of Microtubule‐Associated Protein 2, τ Factor, and Tubulin, and Comparison with the Cyclic AMP‐Dependent Phosphorylation

Abstract: Isolated microtubule‐associated protein 2 (MAP2), τ factor, and tubulin were phosphorylated by a purified Ca2+, calmodulin‐dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and τ factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, τ factor, and tubulin were also phosphorylated by the catalytic subunit of type‐II cyclic AMP‐dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The Km values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, τ factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca2+, calmodulin system via phosphorylation of MAP2, τ factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.

Dephosphorylation of microtubule-associated protein 2, τ factor, and tubulin by calcineurin

Abstract: Calcineurin dephosphorylated microtubule‐associated protein 2 (MAP2) and τ factor phosphorylated by cyclic AMP‐dependent and Ca2+, calmodulin‐dependent protein kinases from the brain. Tubulin, only phosphorylated by the Ca2+, calmodulin‐dependent protein kinase, served as substrate for calcineurin. The concentrations of calmodulin required to give half‐maximal activation of calcineurin were 21 and 16 nM with MAP2 and τ factor as substrates, respectively. The Km and Vmaxvalues were in ranges of 1–3 μM and 0.4–1.7 μmol/mg/ min, respectively, for MAP2 and τ factor. The Km value for tubulin was in a similar range, but the Vmax value was lower. The peptide map analysis revealed that calcineurin dephosphorylated MAP2 and τ factor universally, but not in a site‐specific manner. The autophosphorylated Ca2+, calmodulin‐dependent protein kinase was not dephosphorylated by calcineurin. These results suggest that calcineurin plays an important role in the functions of microtubules via dephosphorylation.

Staurosporine: An Effective Inhibitor for Ca2+/Calmodulin‐Dependent Protein Kinase II

Abstract: We investigated the effect of staurosporine on Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) purified from rat brain. (a) Staurosporine (10–100 nM) inhibited the activity of CaM kinase II. The half‐maximal and maximal inhibitory concentrations were 20 and 100 nM, respectively. (b) The inhibition with staurosporine was of the noncompetitive type with respect to ATP, calmodulin, and phosphate acceptor (β‐casein). (c) Staurosporine suppressed the autophosphorylation of α‐ and β‐subunits of CaM kinase II at concentrations similar to those at which the enzyme activity was inhibited. (d) Staurosporine also attenuated the Ca2+ calmodulin‐independent activity of the autophosphorylated CaM kinase II. These results suggest that staurosporine inhibits CaM kinase II by interacting with the catalytic domain, distinct from the ATP‐binding site or substrate‐binding site, of the enzyme and that staurosporine is an effective inhibitor for CaM kinase II in the cell system.

A human homolog of Drosophila warts tumor suppressor, h-warts, localized to mitotic apparatus and specifically phosphorylated during mitosis

We identified a human homolog of Drosophila warts tumor suppressor gene, termed h‐warts, which was mapped at chromosome 6q24‐25.1. The h‐warts protein has a serine/threonine kinase domain and is localized to centrosomes in interphase cells. However, it becomes localized to the mitotic apparatus, including spindle pole bodies, mitotic spindle, and midbody, in a highly dynamic manner during mitosis. Furthermore, h‐warts is specifically phosphorylated in cells at mitotic phase, most likely by Cdc2 kinase. These findings suggest that h‐warts functions as a component of the mitotic apparatus and is involved in proper progression of mitosis.

Interaction of NE-dlg/SAP102, a Neuronal and Endocrine Tissue-specific Membrane-associated Guanylate Kinase Protein, with Calmodulin and PSD-95/SAP90 A POSSIBLE REGULATORY ROLE IN MOLECULAR CLUSTERING AT SYNAPTIC SITES

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends ofN-methyl-d-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic α-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a K d value of 44 nm. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.

Involvement of protein kinase C in superoxide anion-induced activation of nuclear factor-κB in human endothelial cells

Objective: Nuclear factor-kappa B (NF-κB) plays an important role in the regulation of redox-sensitive genes which are related to the pathogenesis of various vascular diseases. Although oxygen free-radicals are known to activate NF-κB, the signaling pathway of oxygen free radical-induced NF-κB activation remains largely unclear. Thus, this study was performed to examine the possible involvement of protein kinase C (PKC) in the oxygen free radical-induced NF-κB activation in human umbilical vein endothelial cells (HUVECs). Methods: Superoxide anion was generated by xanthine and xanthine oxidase. An electrophoretic mobility shift assay (EMSA) was performed using a κB-motif oligonucleotide and nuclear extracts from HUVECs. Immunoblot analysis using an antibody against IκBα, phosphorylated by IκBα kinase, or myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylated by protein kinase C was carried out. An NF-κB luciferase reporter gene assay was also performed. Results: The treatment of the cells with superoxide anion for 60 min increased the NF-κB/DNA binding activity. Immunoblot analysis showed that superoxide anion induced phosphorylation of IκBα within 10 min. Furthermore, phosphorylation of MARCKS occurred more rapidly than phosphorylation of IκBα. Pretreatment of the cells with calphostin C (100–400 nmol/l) and chelerythrine chloride (5–10 μmol/l), inhibitors of PKC, abolished the superoxide anion-induced NF-κB activation. Down-regulation of endogenous PKC by long-term exposure to phorbol 12-myristate 13-acetate decreased the superoxide anion-induced NF-κB activation to a basal level. Superoxide anion induced the luciferase reporter gene and this induction was completely inhibited by calphostin C (200 nmol/l) and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron). Conclusion: These results suggest that PKC is involved in the activation of NF-κB by superoxide anion in human endothelial cells.

Dephosphorylation of r Factor by Protein Phosphatase 2A in Synaptosomal Cytosol Fractions, and Inhibition by Aluminum

When the synaptosomal cytosol fraction from rat brain was chromatographed on a DEAE‐cellulose column and assayed for protein phosphatases for τ factor and histone H1, two peaks of activities, termed peak 1 (major) and peak 2 (minor), were separated. Each peak was in a single form on Sephacryl S‐300 column chromatography. Both peaks 1 and 2 dephosphorylated τ factor phosphorylated by Ca2+/calmodulin‐dependent protein kinase II and the catalytic subunit of cyclic AMP‐dependent protein kinase. The Km values were in the range of 0.42–0.84 μM for τ factor. There were no differences in kinetic properties of dephosphorylation between the substrates phosphorylated by the two kinases. The phosphatase activities did not depend on Ca2+, Mn2+, and Mg2+. Immunoprecipitation and immunoblotting analysis using polyclonal antibodies to the catalytic subunit of brain protein phosphatase 2A revealed that both protein phosphatases are the holoenzymic forms of protein phosphatase 2A. Aluminum chloride inhibited the activities of both peaks 1 and 2 with IC50 values of 40–60 μM. These results suggest that dephosphorylation of r factor in presynaptic nerve terminals is controlled mainly by protein phosphatase 2A and that the neurotoxic effect of aluminum seems to be related mostly to inhibition of dephosphorylation of τ factor

Identification of the Isoforms of Ca2+/Calmodulin‐Dependent Protein Kinase II in Rat Astrocytes and Their Subcellular Localization

Abstract: Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT‐PCR followed by the “nested” PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was δ2. Immunostaining suggested that the δ2 isoform was localized predominantly at the Golgi apparatus. The localization of the δ2 isoform at the Golgi apparatus was also observed in NG108‐15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108‐15 cells. In contrast to the αB and δ3 isoforms that entered the nucleus, as reported, the γA isoform was excluded from the nucleus in the transfected NG108‐15 cells. These results suggest that the 15‐amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the γA isoform.