Eishichi Miyamoto

Role of MAP kinase in neurons

Extracellular stimuli such as neurotransmitters, neurotrophins, and growth factors in the brain regulate critical cellular events, including synaptic transmission, neuronal plasticity, morphological differentiation and survival. Although many such stimuli trigger Ser/Thr-kinase and tyrosine-kinase cascades, the extracellular signal-regulated kinases, ERK1 and ERK2, prototypic members of the mitogen-activated protein (MAP) kinase family, are most attractive candidates among protein kinases that mediate morphological differentiation and promote survival in neurons. ERK1 and ERK2 are abundant in the central nervous system (CNS) and are activated during various physiological and pathological events such as brain ischemia and epilepsy. In cultured hippocampal neurons, simulation of glutamate receptors can activate ERK signaling, for which elevation of intracellular Ca2+ is required. In addition, brain-derived neurotrophic factor and growth factors also induce the ERK signaling and here, receptor-coupled tyrosine kinase activation has an association. We describe herein intracellular cascades of ERK signaling through neurotransmitters and neurotrophic factors. Putative functional implications of ERK and other MAP-kinase family members in the central nervous system are give attention.

Activation of Akt/Protein Kinase B Contributes to Induction of Ischemic Tolerance in the CA1 Subfield of Gerbil Hippocampus

Apoptosis plays an important role in delayed neuronal cell death after cerebral ischemia. Activation of Akt/protein kinase B has been recently reported to prevent apoptosis in several cell types. In this article the authors examine whether induction of ischemic tolerance resulting from a sublethal ischemic insult requires Akt activation. Sublethal ischemia gradually and persistently stimulated phosphorylation of Akt-Ser-473 in the hippocampal CA1 region after reperfusion. After lethal ischemia, phosphorylation of Akt-Ser-473 showed no obvious decrease in preconditioned gerbils but a marked decrease in nonconditioned gerbils. Changes in Akt-Ser-473 phosphorylation were correlated with changes in Akt activities, as measured by an in vitro kinase assay. Intracerebral ventricular infusion of wortmannin before preconditioning blocked both the increase in Akt-Ser-473 phosphorylation in a dose-dependent manner and the neuroprotective action of preconditioning. These results suggest that Akt activation is induced by a sublethal ischemic insult in gerbil hippocampus and contributes to neuroprotective ischemic tolerance in CA1 pyramidal neurons.

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.

Immunohistochemical Localization of Ca2+/Calmodulin-Dependent Protein Kinase II in Rat Brain and Various Tissues

Abstract: Polyclonal antibodies against Ca2+/calmodulin‐de‐pendent protein kinase II (CaM kinase II) of rat brain were prepared by immunizing rabbits and then purified by antigen‐affinity column. The antibodies which recognized both sub‐units of the enzyme with MrS 49K and 60K were used for the study on the distribution of CaM kinase II in formalin‐fixed, paraffin‐embedded tissues. In the brain, a light‐microscopic study demonstrated strong immunoreactivity in neu‐ronal somata and dendrites and weak immunoreactivity in nuclei. The densely stained regions included cerebral cortex, hippocampal formation, striatum, substantia nigra, and cer‐ebellar cortex. In substantia nigra, neurites were stained, but not neuronal somata. Electron microscopy revealed that the immunoreactive product was highly concentrated at the postsynaptic densities. In addition to neurons, weak immunoreactivity was also demonstrated in glial cells, such as as‐trocytes and ependymal cells of ventricles and epithelial cells of choroid plexus. In other tissues, strong immunoreactivity was observed in the islet of pancreas and moderate immunoreactivity in skeletal muscle and kidney tubules. Immunoreactivity was demonstrated in all of the tissues tested. The results suggest that CaM kinase II is widely distributed in the tissues.

Activation of mitogen-activated protein kinase in cultured rat hippocampal neurons by stimulation of glutamate receptors.

Abstract: Mitogen‐activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non‐NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down‐regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca2+ ionophore, remained intact. Lavendustin A, a tyrosine kinase inhibitor, was without effect. In experiments with 32P‐labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf‐1, the c‐raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC‐dependent and the Ca2+‐dependent pathways and that the activation of Raf‐1 is not involved.

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.

The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay.

Calcineurin is the calcium (divalent cations)-dependent calmodulin-stimulated phosphoprotein phosphatase which is capable of dephosphorylating various substrate proteins. The subcellular and regional distribution of calcineurin in the rat brain has been studied by light and electron microscopic immunohistochemistry using antiserum against calcineurin. Immunoreactivity was observed in many neurons but was not detected in glial cells, such as astrocytes, oligodendrocytes and ependymal cells by the PAP method. Light microscopy demonstrates strong immunoreactivity in neuronal somata and neurites. By electron microscopy, calcineurin immunoreactivity was found to be present in dendrites including postsynaptic densities, somata, spines, axons and terminals. Calcineurin immunoreactivity was present in neurons throughout the brain, but a marked regional variation in strength of the immunoreactivity was observed. The caudatoputamen, hippocampal formation, and substantia nigra were strongly stained. Cerebral and cerebellar neocortex showed moderate immunoreactivity. In substantia nigra and globus pallidus, only neurites were stained, but neuronal somata not. The staining of the substantia nigra was thought to be due to that of the nerve terminals originating from the caudatoputamen, in view of the findings by cerebral hemitransection and electron microscopic immunohistochemistry. We developed an enzyme-immunoassay (EIA) for calcineurin. The sensitivity of the EIA was 1 ng (13 fmol) of calcineurin. We determined the level of calcineurin in various regions of the rat brain. The caudate nucleus, putamen and hippocampal formation showed a high concentration of calcineurin. The results are consistent with those obtained by immunohistochemistry.

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.