A group of glycosphingolipids found in an invertebrate: their structures and biological significance.

A novel group of glycosphingolipids was identified in the nervous tissue and skin of the mollusc, Aplysia kurodai, which lacks gangliosides. More than 30 glycolipids were detected on HPTLC plates and the structures of 9 major glycolipids were determined. They were pentaosylglycosphingolipids and their common core structure was GalNAcα1→3Galß1→4Glcß1→1ceramide, except for one glycolipid in which Galß of the core structure was replaced by Galα. 3-O-MeGalß or 4-O-MeGlcNAcα or 3,4-O-carboxyethylideneGalß was at their non-reducing ends. Galα or Fucα binds to Gal of the core structure at 2C as a side chain sugar. One to three 2-aminoethylphosphonic acids and/or phosphoethanolamine link to the glycolipids. Immunohistochemically, glycolipids having carboxyethylideneGal at their non-reducing ends were localized exclusively in nerve bundles. Glycolipids activated cAMP-dependent protein kinase in the rat brain and may directly activate cAMP-dependent protein kinase in a manner similar, but not identical, to that of cAMP. The biological functions of glycolipids may share neurobiological functions proposed for gangliosides in vertebrates.

Molecular mechanism of neuronal plasticity: induction and maintenance of long-term potentiation in the hippocampus.

Recent studies have demonstrated that activation of enzymes can be observed in living cells in response to stimulation with neurotransmitters, hormones, growth factors, and so forth. Thus, the activation of enzymes was shown to be closely related to the dynamic states of various cell functions. The development of new experimental methodologies has enabled researchers to study the molecular basis of neuronal plasticity in living cells. In 1973, Bliss and his associates identified the phenomena of long-term potentiation (LTP). Since it was thought to be a model for neuronal plasticity such as learning and memory, its molecular mechanism has been extensively investigated. The mechanism was found to involve a signal transduction cascade that includes release of glutamate, activation of the NMDA glutamate receptors, Ca(2+) entry, and activations of Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) II and IV and mitogen-activated protein kinase (MAPK). Consequently, AMPA glutamate receptors were activated by phosphorylation by CaM kinase II, resulting in an increase of Ca(2+) entry into postsynaptic neurons. Furthermore, activation of CaM kinase IV and MAPK increased phosphorylation of CREB (cyclic AMP response element binding protein) and expression of c-Fos by stimulation of gene expression. These results suggest that LTP induction and maintenance would be models of short- and long-term memory, respectively.

Differential activation of the luteinizing hormone beta-subunit promoter by activin and gonadotropin-releasing hormone: a role for the mitogen-activated protein kinase signaling pathway in LbetaT2 gonadotrophs.

LH consists of alpha- and beta-subunits, and synthesis of the beta-subunit has been reported to be the rate-limiting step in LH production. In this study, we found that activin A increased both the LHbeta mRNA level and LH content in cells of the gonadotroph cell line, LbetaT2. We next examined the effects of activin A and GnRH on LHbeta promoter activity by reporter gene assay and compared the signal transduction pathways. Activin A and GnRH activated the LHbeta promoter, and the response to a combination of activin A and GnRH was higher than that to activin A or GnRH alone. The effects of activin A and GnRH were specifically inhibited by inhibin-like peptide and antide, a GnRH antagonist, respectively. The activation of the LHbeta promoter by GnRH was inhibited by PD098059 and U0126, MAP kinase kinase (MEK) inhibitors. In contrast, these protein kinase inhibitors did not inhibit the activin A-induced activation. GnRH, but not activin A, activated MAP kinase in LbetaT2 cells. Overexpression of constitutively active MEK1 or MEK kinase activated both MAP kinase and the LHbeta promoter. Furthermore, GnRH, but not activin A, strongly induced SRE-mediated transcription, a known target of the MAP kinase pathway. These results suggest that GnRH activates the LHbeta promoter via the MAP kinase pathway and that activin A-induced activation of the LHbeta promoter is independent of the MAP kinase pathway.

Chronic carbamazepine treatment increases myristoylated alanine-rich C kinase substrate phosphorylation in the rat cerebral cortex via down-regulation of calcineurin A alpha.

Carbamazepine (CBZ) is generally used as a mood-stabilizing drug for the treatment of bipolar disorders. However, little is known about the molecular mechanisms of CBZ actions in the brain, which account for this therapeutic profile. In the present study, we examined the effects of chronic CBZ treatment on the protein kinase C (PKC) pathway. Male Wistar rats received injections of CBZ once daily for 3-5 weeks. The protein levels of PKC isozymes, calcineurin Aalpha subunit (CaN-Aalpha) and myristoylated alanine-rich C kinase substrate (MARCKS), and phosphorylation of MARCKS in the rat cerebral cortex were determined by immunoblot analysis. The content of CaN-Aalpha mRNA was determined by Northern blot analysis. Nomicr; significant changes were observed in PKC alpha, beta, gamma, delta and epsilon in the cytosol and membrane fractions after 5 weeks of CBZ treatment. There were no significant changes in the actin-binding PKCepsilon. Interestingly, phosphorylation of MARCKS was increased more than twofold, while no significant changes were observed in MARCKS protein level in the cytosol fraction. Furthermore, CaN-Aalpha was significantly decreased at both the protein and mRNA levels. The level of MARCKS phosphorylation is reportedly regulated by the balance between PKC-mediated phosphorylation and CaN-mediated dephosphorylation. Our results indicate that chronic CBZ treatment increases MARCKS phosphorylation via decreasing the content of CaN-Aalpha. Phosphorylation of MARCKS has been reported to play an important role in the release of neurotransmitters, such as noradrenaline and serotonin. Therefore, the increase in phosphorylation of MARCKS observed only after chronic CBZ treatment may be related to the mood-stabilizing effects of CBZ.

New aspects of neurotransmitter release and exocytosis: involvement of Ca2+/calmodulin-dependent phosphorylation of synapsin I in insulin exocytosis.

The exocytosis of insulin from pancreatic beta-cells is closely related to intracellular elevation of Ca(2+). The effects of Ca(2+) may be mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Four subunits of CaMKII, termed alpha, beta, gamma, and delta, are encoded by distinct genes, and various isoforms of these subunits exist as different splicing variants. In the brain, phosphorylation of synapsin I by the alpha isoform induces neurotransmitter release. In order to clarify whether phosphorylation of synapsin I by CaMKII was involved in insulin exocytosis, we cloned the isoforms of CaMKII and synapsin I from mouse insulinoma MIN6 cells. We found that beta'e and delta2 are the major isoforms of CaMKII and that synapsin Ib is a major isoform of synapsin I in MIN6 cells. It was interesting that delta2 and synapsin Ib were co-localized with insulin secretory granules in the cells. Treatment of MIN6 cells with glucose and tolbutamide rapidly activated CaMKII. Immunoblot analysis with two antibodies against synapsin I phosphorylated by CaMKII demonstrated the increase in phosphorylation of synapsin I by the secretagogues. Furthermore, the secretagogue-induced phosphorylation of synapsin I and insulin secretion were potentiated by transient overexpression of the beta'e or delta2 isoform. These results suggest that activation of CaMKII and the concomitant phosphorylation of synapsin I induce insulin exocytosis from pancreatic beta-cells.

Characterization of alphaT3-1 cells stably transfected with luteininzing hormone beta-subunit complementary deoxyribonucleic acid.

Luteinizing hormone (LH) consists of alpha- and beta-subunits, and synthesis and secretion of LH are regulated by gonadotropin-releasing hormone (GnRH). In order to examine the molecular mechanisms by which GnRH regulates LH secretion, we transfected alphaT3-1 cells with rat LHbeta-subunit cDNA under the control of a constitutive promoter and established a stable cell line of LH2 cells which secreted LH in response to GnRH. Pulsatile and continuous GnRH pretreatments increased gene expression of the alpha-subunit and synthesis of LH, and enhanced the LH secretion by brief treatments with GnRH and 56 mM KCl. The LH secretions were partially blocked by elimination of extracellular Ca2+. GnRH-induced LH secretion was completely inhibited by calphostin C (a protein kinase C inhibitor) and 1 microM wortmannin. In contrast to the GnRH induction, high K+-induced LH secretion was inhibited by KN93, a Ca2+/calmodulin-dependent protein kinase II inhibitor, as well as by 1 microM wortmannin. We also confirmed that activation of cAMP-pathway induced LH secretion, but activation of mitogen-activated protein (MAP) kinase pathway was not involved in LH secretion. These results suggest that GnRH directly regulates LH secretion as well as LHbeta-subunit synthesis, and that LH2 cells are a useful model for the study of LH secretion induced by several secretagogues.

The X-linked inhibitor of apoptosis protein inhibits taxol-induced apoptosis in LNCaP cells.

To clarify the roles of the X-linked inhibitor of apoptosis protein (XIAP), we investigated the effects of XIAP overexpression on taxol-induced cell growth arrest and apoptosis in the prostate cancer cell line (LNCaP). After the transfection of XIAP cDNA into LNCaP cells, we established clonal cell lines that overexpressed XIAP and examined the taxol effects on growth and apoptosis by 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and flow cytometric analysis. The effects of XIAP overexpression on caspase-3 were examined by immunoblot analysis and activity assay. The interaction between XIAP and caspase-3 in LNCaP cells was examined by cotransfection with myc-XIAP and caspase-3-HA cDNAs followed by immunoprecipitation and immunoblot analysis. Large amounts of XIAP were expressed in the established cell lines. Although the growth rates were reduced in a dose- and time-dependent manner by taxol, these effects were significantly decreased in XIAP stably overexpressing cell lines. In addition, we found that taxol treatment induced the cleavage of pro-caspase-3, followed by apoptosis, and that the overexpression of XIAP inhibited apoptosis by attenuating the cleavage of pro-caspase-3 and caspase-3 activity. Interestingly, XIAP bound to pro-caspase-3 in LNCaP cells transiently cotransfected with myc-XIAP and caspase-3-HA cDNAs, and this interaction was inhibited by taxol treatment. These results suggest that the overexpression of XIAP inhibits taxol-induced apoptosis through the decrease of caspase-3 activity and inhibition of the processing of pro-caspase-3.

Acute changes in the axonal cytoskeleton after mild stretching of the rat brachial plexus.

We have developed an animal model to investigate acute changes in the axonal cytoskeleton caused by a mild stretching of the peripheral nerve in the upper limbs of rats. Rat forelimbs were continuously stretched at 2 N for 1 h. Thereafter, a part of the brachial plexus and median nerve were harvested and processed for electron microscopic analysis. The total number of microtubules in the brachial plexus decreased to 55% of that of the control animals (p<0.05) without change in the number of neurofilaments. No significant changes in microtubules or neurofilaments were observed in the median nerve. By Western blotting analysis, the amount of tau protein in the stretch group significantly decreased in the brachial plexus but not in the median nerve. However, no significant changes in the amount of tubulin protein were observed in either the brachial plexus or median nerve. These results suggest that the microtubules were depolymerized by stretching of the brachial plexus and that the depolymerization may have been mediated by the decrease in the tau protein.

Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites.

The paired helical filaments (PHF) found in Alzheimer's disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain.

Activation of the rat dopamine D2 receptor promoter by mitogen-activated protein kinase and Ca2+/calmodulin-dependent protein kinase II pathways.

To investigate regulation of D2 receptor (D2R) gene expression by protein kinases, we evaluated effects of constitutively active MAPK kinase kinase (MEKK), Ca2+/calmodulin-dependent protein kinase (CaMK) II, CaMKIV and cyclic AMP-dependent protein kinase (PKA) on D2R promoter activity using luciferase reporter gene assays. A 1.5-kbp fragment containing the rat D2R promoter was cloned upstream of the reporter and transfected into D2R-expressing NB2A cells or nonexpressing NG108-15 and C6 glioma cells. MEKK and CaMKII, but not CaMKIV and PKA, increased promoter activity 4.5- and 1.5-fold, respectively, in NB2A cells. Inhibitory effects of a MEK inhibitor and lack of effect by dominant negative (DN)-JNK1 or DN-p38MAPK revealed that ERK but not JNK and p38MAPK is involved in MEKK-induced promoter activation. Deletion and mutation of the promoter revealed that the MEKK-responsive region was Sp1 site B between nucleotides -56 and -47. Overexpression of Sp1 suppressed promoter activity without affecting MEKK-induced activation. Interestingly, overexpression of Zif268 increased promoter activity through the region between nucleotides -56 and -36. Increased activity by Zif268 was additive with CaMKII-induced activation but not with activation induced by MEKK. Co-transfection with CaMKII stimulated nuclear translocation of Zif268. These results suggest that ERK and CaMKII positively regulate the D2R promoter and that Zif268 is a potential transcription factor for the CaMKII-dependent pathway.

Ca2+/calmodulin-dependent protein kinase II-dependent long-term potentiation in the rat suprachiasmatic nucleus and its inhibition by melatonin.

We recently reported that Ca(2+)/calmodulin-dependent protein (CaM) kinase II is involved in light-induced phase delays and Per gene induction in the suprachiasmatic nucleus (SCN). To clarify the activation mechanisms of CaM kinase II by glutamate receptor stimulation in the SCN, we documented CaM kinase II activation following induction of long-term potentiation (LTP) in the rat SCN. High-frequency stimulation (100 Hz, 1 sec) applied to the optic nerve resulted in LTP of a postsynaptic field potential in the rat SCN. Unlike LTP in the hippocampal CA1 region, LTP onset in the SCN was slow and partly dependent on N-methyl-D-aspartate receptor activation. LTP induction in the SCN was completely inhibited by treatment with a nitric oxide synthase inhibitor or with a specific CaM kinase II inhibitor. Immunoblotting analysis using phosphospecific antibodies against autophosphorylated CaM kinase II revealed that LTP induction was accompanied by an increase in autophosphorylation. After high-frequency stimulation, we could visualize activation of CaM kinase II in vasoactive intestinal polypeptide-positive neurons in the SCN by immunohistochemistry. Treatment with cyclosporin A, a calcineurin inhibitor, potentiated LTP induction in the rat SCN. Interestingly, treatment with melatonin totally prevented LTP induction, without changes in basal synaptic transmission. Analyses of phosphorylation of CaM kinase II, mitogen-activated protein kinase, and cAMP-responsive element binding protein revealed that stimulatory and inhibitory effects on CaM kinase II autophosphorylation underlie the effects of cyclosporin A and melatonin, respectively. These results suggest that CaM kinase II plays critical roles in LTP induction in the SCN and that melatonin has inhibitory effects on synaptic plasticity through CaM kinase II.

Analysis on the promoter region of exon IV brain-derived neurotrophic factor in NG108-15 cells.

We have reported that the nuclear isoforms of Ca2+/calmodulin-dependent protein kinase II (CaM KII) are involved in the expression of the exon IV-containing brain-derived neurotrophic factor (BDNF) mRNA. We document here the cis-elements and transcription factors responsive to CaM KII in the activation of the promoter upstream of the exon IV (exon IV-BDNF promoter). Effects of constitutively active mutants of CaM KIV, MAPK kinase kinase (MEKK) and protein kinase A (PKA) on the exon IV-BDNF promoter activity were also evaluated by transfection and luciferase assay. The exon IV-BDNF promoter activity was increased by transfection with CaM KII, MEKK and PKA, but not by CaM KIV. Deletion and mutational analysis of the promoter revealed that the region between nucleotides -56 and -27 was responsive to CaM KII, which contained a CCAAT-box in the region between nucleotides -56 and -43. Expression of C/EBPbeta increased the promoter activity and potentiated the effects of CaM KII. The region between nucleotides -79 and -56 was responsive to MEKK, in which both a GA-rich sequence and a GC-box were included. Expression of Sp1 increased the promoter activity, which was further enhanced by transfection with MEKK. The region between nucleotides -43 and -27 was responsive to both PKA and CaM KII, but the transcription factors involved in the region remained unclear. These results suggest that the promoter of the exon IV-BDNF is at least regulated by CaM KII, MEKK and PKA, and that C/EBP/beta and Sp1 are potential transcription factors activated by CaM KII and MEKK, respectively.

Regulation of gonadotropin alpha subunit gene expression by dopamine D(2) receptor agonist in clonal mouse gonadotroph alphaT3-1 cells.

Pituitary prolactin biosynthesis is negatively regulated by hypothalamic dopamine through D(2) receptors in pituitary lactotrophs, but little is known about the direct effect of dopamine on gonadotrophs. In this study, the clonal gonadotroph-derived cell line, alphaT3-1, was used to examine whether gene expression of the pituitary gonadotropin alpha subunit, stimulated with GnRH or pituitary adenylate cyclase-activating polypeptide (PACAP), was controlled by dopamine D(2) receptor. Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated the presence of dopamine D(2) receptors in alphaT3-1 cells. Both GnRH and PACAP increased alpha subunit gene expression. GnRH-induced alpha subunit gene expression was not affected by quinpirol, a specific dopamine D(2) receptor agonist. In contrast, PACAP-induced gene expression was significantly lower in the presence of quinpirol. The roles of extracellular signal-regulated kinase (ERK) and cAMP in the expression of the alpha subunit gene were examined. GnRH activated ERK, but PACAP did not, and the activation was not inhibited by quinpirol. GnRH-induced alpha subunit gene expression was completely inhibited by an ERK inhibitor, PD098059. Cyclic AMP accumulation in alphaT3-1 cells was increased by treatment with PACAP, and quinpirol inhibited this effect. GnRH did not affect cAMP production in these cells. These results suggest that in alphaT3-1 cells, dopamine D(2) receptors negatively regulate pituitary alpha subunit gene expression in association with the cAMP-dependent pathway, but not with the ERK pathway.

Phosphorylation of neuronal nitric oxide synthase at Ser847 by CaM-KII in the hippocampus of rat brain after transient forebrain ischemia.

The authors previously demonstrated that Ca2+/calmodulin (CaM)-dependent protein kinase IIalpha (CaM-KIIalpha) can phosphorylate neuronal nitric oxide synthase (nNOS) at Ser847 and attenuate NOS activity in neuronal cells. In the present study, they established that forebrain ischemia causes an increase in the phosphorylation of nNOS at Ser847 in the hippocampus. This nNOS phosphorylation appeared to be catalyzed by CaM-KII: (1) it correlated with the autophosphorylation of CaM-KIIalpha; (2) it was blocked by the CaM-KII inhibitor, KN-93; and (3) nNOS and CaM-KIIalpha were found to coexist in the hippocampus. Examination of the spatial relation between nNOS and CaM-KIIalpha in the brain revealed coexistence in the hippocampus but not in the cortex during reperfusion, with a concomitant increase in autophosphorylation of CaM-KIIalpha. The phosphorylation of nNOS at Ser847 probably takes place in nonpyramidal hippocampal neurons, which increased after 30 minutes of reperfusion in the hippocampus, whereas no significant increase was detected in the cortex. An intraventricular injection of KN-93 significantly decreased the phosphorylation of nNOS in the hippocampus. These results point to CaM-KII as a protein kinase, which by its colocalization may attenuate the activity of nNOS through its Ser847 phosphorylation, and may thus contribute to promotion of tolerance to postischemic damage in hippocampal neurons.

Synapsin I is phosphorylated at Ser603 by p21-activated kinases (PAKs) in vitro and in PC12 cells stimulated with bradykinin.

The function of synapsin I is regulated by phosphorylation of the molecule at multiple sites; among them, the Ser(603) residue (site 3) is considered to be a pivotal site targeted by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Although phosphorylation of the Ser(603) residue responds to several kinds of stimuli, it is unlikely that many or all of the stimuli activate the CaMKII-involved pathway. Among the several stimulants tested in PC12 cells, bradykinin evoked the phosphorylation of Ser(603) without inducing the autophosphorylation of CaMKII, which was determined using phosphorylation site-specific antibodies against phospho-Ser(603)-synapsin I (pS603-Syn I-Ab) and phospho-Thr(286/287)-CaMKII. The bradykinin-evoked phosphorylation of Ser(603) was not suppressed by the CaMKII inhibitor KN62, whereas high KCl-evoked phosphorylation was accompanied by CaMKII autophosphorylation and inhibited by KN62. Thus, we attempted to identify Ser(603) kinase(s) besides CaMKII. We consequently detected four and three fractions with Ca(2+)/calmodulin-independent Ser(603) kinase activity on the DEAE column chromatography of bovine brain homogenate and PC12 cell lysate, respectively, two of which were purified and identified by amino acid sequence of proteolytic fragments as p21-activated kinase (PAK) 1 and PAK3. The immunoprecipitants from bovine brain homogenate with anti-PAK1 and PAK3 antibodies incorporated (32)P into synapsin I in a Cdc42/GTPgammaS-dependent manner, and its phosphorylation site was confirmed as Ser(603) using pS603-Syn I-Ab. Additionally, recombinant GST-PAK2 could phosphorylate the Ser(603) residue in the presence of Cdc42/GTPgammaS. Finally, we confirmed by immunocytochemical analysis that the transfection of constitutively active rat alphaPAK (PAK1) in PC12 cells evokes the phosphorylation of Ser(603) even in the resting mutant cells and enhances it in the bradykinin-stimulated cells, whereas that of dominant-negative alphaPAK quenches the phosphorylation. These results raise the possibility that Ser(603) on synapsin I is alternatively phosphorylated by PAKs, not only by CaMKII, in neuronal cells in response to some stimulants.

Decreased akt activity is associated with activation of forkhead transcription factor after transient forebrain ischemia in gerbil hippocampus.

The authors recently reported that sodium orthovanadate rescues cells from delayed neuronal death in gerbil hippocampus after transient forebrain ischemia through phosphatidylinositol 3-kinase-protein kinase B (Akt) pathway (Kawano et al., 2001). In the current study, they demonstrated that the activation of FKHR, a Forkhead transcription factor and a substrate for Akt, preceded delayed neuronal death in CA1 regions after transient forebrain ischemia. Adult Mongolian gerbils were subjected to 5-minute forebrain ischemia. Immunoblotting analysis with anti-phospho-FKHR antibody showed that phosphorylation of FKHR at serine-256 in the CA1 region decreased immediately after and 0.5 and 1 hour after reperfusion. The dephosphorylation of FKHR was correlated with the decreased Akt activity. Intracerebroventricular injection of orthovanadate 30 minutes before ischemia inhibited dephosphorylation of FKHR after reperfusion, and blocked delayed neuronal death in the CA1 region. Gel mobility shift analysis using nuclear extracts from the CA1 region prepared immediately after reperfusion revealed increases in DNA binding activity for the FKHR-responsive element on the Fas ligand promoter. The orthovanadate injection administered before ischemia inhibited its binding activity. Two days after reperfusion, expression of Fas ligand increased in the CA1 region and the orthovanadate injection inhibited this increased expression. These results suggest that the inactivation of Akt results in the activation of FKHR and, in turn, relates to the expression of Fas ligand in the CA1 region after transient forebrain ischemia.

Activation of Ca2+/calmodulin-dependent protein kinase I in cultured rat hippocampal neurons.

We have focused on activation mechanisms of calcium/calmodulin-dependent protein kinase (CaM) kinase I in the hippocampal neurons and compared them with that of CaM kinase IV. Increased activation of CaM kinase I occurred by stimulation with glutamate and depolarization in cultured rat hippocampal neurons. Similar to CaM kinases II and IV, CaM kinase I was essentially activated by stimulation with the NMDA receptor. Although both CaM kinases I and IV seem to be activated by CaM kinase kinase, the activation of CaM kinase I was persistent during stimulation with glutamate in contrast to a transient activation of CaM kinase IV. In addition, CaM kinase I was activated in a lower concentration of glutamate than that of CaM kinase IV. Depolarization-induced activation of CaM kinase I was also evident in the cultured neurons and was largely blocked by nifedipine. In the experiment with 32P-labeled cells, phosphorylation of CaM kinase I was stimulated by glutamate treatment and depolarization. The glutamate- and depolarization-induced phosphorylation was inhibited by the NMDA receptor antagonist and nifedipine, respectively. These results suggest that, although CaM kinases I and IV are activated by the NMDA receptor and depolarization stimulation, these kinase activities are differently regulated in the hippocampal neurons.

MCM3-binding GANP DNA-primase is associated with a novel phosphatase component G5PR.

BACKGROUND: GANP, carrying DNA-primase and MCM3-binding domains, is up-regulated in germinal centre B cells. To understand the regulatory function of GANP upon MCM complex, we searched for GANP-associated molecules by yeast two-hybrid screening. RESULTS: Using the 1 kb fragment (G5) of the ganp cDNA, we identified a clone named G5PR that is structurally homologous to known regulatory subunits of protein phosphatases (PPases) and determined the association of G5PR with GANP in vivo in the DNA transfectant. G5PR is associated with protein phosphatase 5 (PP5) through its tetratricopeptide-repeat (TPR) domain. Pull-down assays demonstrated that G5PR is also associated with protein phosphatase 2A (PP2A), the complex of A subunit (PR65) and the catalytic (C) subunit (PP2Ac), similar to the B" subunit. The G5PR-associated complex had phosphatase activity on casein, histone H1 and MCM3 in vitro, but the addition of G5PR did not stimulate or inhibit the phosphatase activities of PP5 and PP2A. The cellular localization of G5PR in transfected cells varies during cell cycling, appearing in the nucleus during prophase, in the peri-chromatin during mitotic phase, and in the cytoplasm after cell division. CONCLUSION: G5PR is capable of recruiting two kinds of PPases, PP5 and PP2A, into the GANP/MCM3 complex, which might regulate its phosphorylation state during cell cycle progression.

Activation of nuclear Ca(2+)/calmodulin-dependent protein kinase II and brain-derived neurotrophic factor gene expression by stimulation of dopamine D2 receptor in transfected NG108-15 cells.

We identified the isoforms of Ca(2+) /calmodulin-dependent protein kinase II (CaM kinase II) subunits in rat striatum. All four subunits of CaM kinase II alpha, beta, gamma and delta were detected including the isoforms of alphaB, gammaA, gammaA', gammaA.B, delta3 and delta7 with nuclear localization signal. We established NG108-15 cells with the stably expressed dopamine D2L receptor (D2LR, long form), which is an alternative splicing variant. The cells were termed NGD2L. Immunostaining demonstrated that D2LR was localized in plasma membranes. Calcium imaging with fluo-3 AM revealed that quinpirole, a D2R agonist, increased the intracellular Ca(2+), which was blocked by treatment with sulpiride and pertussis toxin in NGD2L cells, but not in mock cells. Furthermore, stimulation of D2LR with quinpirole in NGD2L cells activated the nuclear isoform of CaM kinase II. Stimulation of D2LR increased the expression of exon III- and IV-BDNF mRNA. Overexpression of CaM kinase II delta3 increased exon IV- but not exon III-BDNF mRNA. These results suggest that D2R is involved in the activation of the nuclear isoform of CaM kinase II and thereby in stimulation of gene expression through Ca(2+) signaling.