T Yoshihara, Y Kadota, Yoshiyuki Yoshimura, Y Tatano, N Takeuchi, H Okitsu, Atsushi Umemoto, Takashi Yamauchi and Kouji Itou : Proteomic alteration in gastic adenocarcinomas from Japanese patients, Molecular Cancer, Vol.5, No.75, 75, 2006.
(要約)
Gastric adenocarcinomas comprise one of the common types of cancers in Asian countries including Japan. Comprehensive protein profiling of paired surgical specimens of primary gastric adenocarcinomas and nontumor mucosae derived from Japanese patients was carried out by means of two-dimensional gel electrophoresis (2D-EP) and liquid chromatography-electrospray ionic tandem mass spectrometry (LC-ESI-MS) to establish gastric cancer-specific proteins as putative clinical biomarkers and molecular targets for chemotherapy. Relatively common alterations in protein expression were revealed in the tumor tissues. Increases in manganese dismutase and nonhistone chromosomal protein HMG-1 (HMG-1) were observed, while decreases in carbonic anhydrases I and II, glutatione-S-transferase and foveolin precursor (gastrokine-1) (FOV), an 18-kDa stomach-specific protein with putative tumor suppressor activity, were detected. RT-PCR analysis also revealed significant down-regulation of FOV mRNA expression in tumor tissues. A possible pathological role for down-regulation of FOV in gastric carcinogenesis was demonstrated. Evaluation of the specific decreases in gene and protein expression of FOV in patients may be utilized as clinical biomarkers for effective diagnosis and assessment of gastric cancer.
(キーワード)
Adenocarcinoma / Aged / Asian Continental Ancestry Group / Chromatography, Liquid / Electrophoresis, Gel, Two-Dimensional / Female / Gene Expression Regulation, Neoplastic / Humans / Japan / Male / Middle Aged / Proteome / Proteomics / RNA, Messenger / Reverse Transcriptase Polymerase Chain Reaction / Stomach Neoplasms / Tandem Mass Spectrometry
Takenori Yamamoto, Akiko Yamada, Masahiro Watanabe, Yuya Yoshimura, Naoshi Yamazaki, Yoshiyuki Yoshimura, Takashi Yamauchi, Masatoshi Kataoka, Toshihiko Nagata, Hiroshi Terada and Yasuo Shinohara : VDAC1, having a shorter N-terminus than VDAC2 but showing the same migration in an SDS-polyacrylamide gel, is the predominant form expressed in mitochondria of various tissues., Journal of Proteome Research, Vol.5, No.12, 3336-3344, 2006.
(要約)
The voltage-dependent anion channel (VDAC) is a pore-forming protein expressed in the outer membrane of eukaryotic mitochondria. Three isoforms of it, i.e., VDAC1, VDAC2, and VDAC3, are known to be expressed in mammals; however, the question as to which is the main isoform in mitochondria is still unanswered. To address this question, we first prepared standard VDACs by using a bacterial expression system and raised various antibodies against them by using synthetic peptides as immunogens. Of the three bacterially expressed VDAC isoforms, VDAC3 showed faster migration in SDS-polyacrylamide gels than VDAC1 and VDAC2, although VDAC2 is longer than VDAC1 and VDAC3, due to a 12-amino acid extension of its N-terminal region. Even with careful structural characterization of the expressed VDACs by LC-MS/MS analysis, serious structural modifications of VDACs causing changes in their migration in SDS-polyacrylamide gels were not detected. Next, immunoreactivities of the raised antibodies toward these bacterially expressed VDAC isoforms were evaluated. Trials to prepare specific antibodies against the three individual VDAC isoforms were not successful except in the case of VDAC1. However, using a synthetic peptide corresponding to the highly conserved region among the three VDACs, we were successful in preparing an antibody showing essentially equal immunoreactivities toward all three VDACs. When mitochondrial outer membrane proteins of various rat tissues were subjected to 2-dimensional electrophoresis followed by immunoblotting with this antibody, six immunoreactive protein spots were detected. These spots were characterized by LC-MS/MS analysis, and the signal intensities among the spots were compared. As a result, the signal intensity of the spot representing VDAC1 was the highest, and thus, VDAC1 was concluded to be the most abundantly expressed of the three VDAC isoforms in mammalian mitochondria.
(キーワード)
Amino Acid Sequence / Animals / Base Sequence / Chromatography, Liquid / DNA Primers / Electrophoresis, Gel, Two-Dimensional / Electrophoresis, Polyacrylamide Gel / Immunoblotting / Mass Spectrometry / Mitochondrial Proteins / Molecular Sequence Data / Protein Isoforms / Rats / Sequence Analysis, DNA / Voltage-Dependent Anion Channel 1
O-B. Tian, T. Suzuki, Takashi Yamauchi, H. Sakagami and Yoshiyuki Yoshimura : Interaction of LDL receptor-related protein 4 (LRP4) with postsynaptic scaffold proteins via its C-terminal PDZ domain-binding motif, and its regulation by Ca2+/calmodulin-dependent protein kinase II., The European Journal of Neuroscience, Vol.23, No.11, 2864-2876, 2006.
(要約)
We cloned here a full-length cDNA of Dem26[Tian et al. (1999)Mol. Brain Res., 72, 147-157], a member of the low-density lipoprotein (LDL) receptor gene family from the rat brain. We originally named the corresponding protein synaptic LDL receptor-related protein (synLRP) [Tian et al. (2002) Soc. Neurosci. Abstr., 28, 405] and have renamed it LRP4 to accord it systematic nomenclature (GenBank(TM) accession no. AB073317). LRP4 protein interacted with postsynaptic scaffold proteins such as postsynaptic density (PSD)-95 via its C-terminal tail sequence, and associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor subunit. The mRNA of LRP4 was localized to dendrites, as well as somas, of neuronal cells, and the full-length protein of 250 kDa was highly concentrated in the brain and localized to various subcellular compartments in the brain, including synaptic fractions. Immunocytochemical study using cultured cortical neurons suggested surface localization in the neuronal cells both in somas and dendrites. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylated the C-terminal cytoplasmic region of LRP4 at Ser1887 and Ser1900, and the phosphorylation at the latter site suppressed the interaction of the protein with PSD-95 and synapse-associated protein 97 (SAP97). These findings suggest a postsynaptic role for LRP4, a putative endocytic multiligand receptor, and a mechanism in which CaMKII regulates PDZ-dependent protein-protein interactions and receptor dynamics.
Mariko Tsukane and Takashi Yamauchi : Increase in apoptosis with neural differentiation and shortening of the lifespan of P19 cells overexpressing tau, Neurochemistry International, Vol.48, No.4, 243-254, 2006.
(要約)
Apoptosis or programmed cell death is considered to be involved in neurodegenerative disorders including Alzheimer's disease (AD). AD is characterized by intracellular aggregates of hyperphosphorylated tau, a microtubule-associated protein. To investigate the effect of the overexpression of tau in P19 cells, we engineered P19 wild-type cells (P19wt) stably expressing human tau441 (P19tau). When P19tau cells were induced to undergo neural differentiation by treatment with retinoic acid (RA), a remarkable increase in apoptosis was observed. However, in the undifferentiated state, there was no notable difference of phenotype between P19wt and P19tau cells. Additionally, we found that tau dissociated from microtubules, and co-localized with the RA receptor (RAR) at nucleoli. Further, the lifespan of the differentiated P19tau cells was shorter than that of P19wt cells, and the re-treatment of differentiated P19wt cells with RA resulted in a reduction of lifespan. These observations suggested that tau affects RA signaling in apoptosis and lifespan during the neural differentiation induced by RA treatment.
Takumi Sakurada, Kazuko Mima, Akira Kurisaki, Hiromu Sugino and Takashi Yamauchi : Neuronal cell type-specific promoter of the α CaM kinase II gene is activated by Zic2, a Zic family zinc finger protein, Neuroscience Research, Vol.53, No.3, 323-330, 2005.
(要約)
To understand the neuronal cell type-specific expression of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), we investigated binding proteins that specifically activated the promoter of the alpha isoform of CaM kinase II (alpha CaM kinase II). Proteins that bind the promoter sequence were found in rat brain nuclear extract by electrophoretic mobility shift assay. Then, we screened for binding proteins in a mouse brain cDNA library using the yeast one-hybrid system. Zic2, a Zic family zinc finger transcription factor, was identified as one of the binding proteins. To investigate the effect of Zic2 on the promoter activity, Zic2 cDNA was expressed with a luciferase reporter gene containing a neuronal cell type-specific promoter of alpha CaM kinase II in neuronal and non-neuronal cells. The promoter activity of alpha CaM kinase II was enhanced 1.3-5-fold in cultured neuronal cells by Zic2. The activation was varied among neuronal cell types. Zic2 also increased the promoter activity in non-neuronal cells, although the relative luciferase activites in non-neuronal cells were lower than those in neuronal cell lines. These results indicated that Zic2 was one of the proteins binding to, and regulating the activity of, the promoter of alpha CaM kinase II.
(キーワード)
Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) / Gene expression / Neuronal cell / Promoter-binding protein / Yeast one-hybrid system / Zic2 / Zinc finger protein
Yuka Takaoka, Tomiyoshi Setsu, Kazuyo Misaki, Takashi Yamauchi and Toshio Terashima : Expression of reelin in the dorsal cochlear nucleus of the mouse, Brain Research. Developmental Brain Research, Vol.159, No.2, 127-134, 2005.
(要約)
The cytoarchitecture of dorsal cochlear nucleus (DCN), characterized by a distinct laminar structure similar to the cerebellar cortex of the normal mouse, is known to be disrupted in the Reelin-deficient mouse, reeler. Here, we have reexamined both the cytoarchitecture and myeloarchitecture of this nucleus and described expression pattern of Reelin protein during perinatal periods. Reelin-immunopositive granule cells were firstly recognized in the external granular layer of the DCN at embryological day 16 (E16). Next, we examined the cytoarchitecture of the DCN of the normal and reeler mice with Ca2+/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha) immunostaining. CaMKIIalpha-immunoreactive cartwheel cells were laminarly distributed in the layer II of the normal DCN, but scattered throughout the reeler DCN. Injection of retrograde tracer, Fluoro-Gold (FG) into the inferior colliculus of the reeler mouse resulted in that retrogradely labeled neurons in the DCN were radially scattered instead of being confined to a single layer as seen in the normal mouse. To examine whether CaMKIIalpha-immunopositive cartwheel cells are neurons projecting to the inferior colliculus or not, double labeling with CaMKIIalpha immunohistochemistry and retrograde labeling with an injection of FG into the inferior colliculus were made, which revealed that CaMKIIalpha-immunoreactive cartwheel cells do not send axons to the inferior colliculus. The present findings imply that Reelin may have some roles in the formation of laminar structures of the DCN.
Chihiro Yoshizaki, Mariko Tsukane and Takashi Yamauchi : Overexpression of tau leads to the stimulation of neurite outgrowth, the activation of caspase 3 activity, and accumulation and phosphorylation of tau in neuroblastoma cells on cAMP treatment, Neuroscience Research, Vol.49, No.4, 363-371, 2004.
(要約)
To explore changes to the tau molecule in Alzheimer's disease, we studied the effect of tau expression in stably transfected neuroblastoma x glioma hybrid NG108-15 cells (tau cells). Tau cells had a similar shape to, but more neurites than, wild type NG108-15 cells (wild type cells). When treated with cAMP, tau cells began to form neurites within 2h. After that, these neurites became longer and thicker than those of wild type cells. An accumulation and increased phosphorylation of tau were observed after 8 h and caspase 3 activity was increased after 4 h in tau cells, but not in wild type cells, upon treatment with cAMP. Caspase 3 activity was activated after the initiation of morphological change, and before the accumulation of tau in tau cells. Under these conditions, apoptotic cell death was not observed and tau was colocalized with tubulin. However, the accumulated tau molecules did not associate with tubulin and were dislocated around and in the nuclei of tau cells. These observations have implications for the cellular causes of Alzheimer's disease where the accumulation and mislocation of tau occur concomitant with neuronal degeneration.
Hiroko Sugiura, Ken Iwata, Masato Matsuoka, Hiroshi Hayashi, Takako Takemiya, Shin Yasuda, Masumi Ichikawa, Takashi Yamauchi, Patrick Mehlen, Tatsuya Haga and Kanato Yamagata : Inhibitory Role of Endophilin 3 in Receptor-mediated Endocytosis, The Journal of Biological Chemistry, Vol.279, No.22, 23343-23348, 2004.
(要約)
Endophilin 1 (Endo1) participates in synaptic vesicle biogenesis through interactions of its Src homology 3 domain with the polyphosphoinositide phosphatase Synaptojanin and the GTPase Dynamin. Endo1 has also been reported to affect endocytosis by converting membrane curvature via its lysophosphatidic acid acyltransferase activity. Here we report that a closely related isoform of Endo1, Endo3, inhibits clathrin-mediated endocytosis. Mutational analyses showed that the variable region of Endo3 is important in regulating transferrin endocytosis. In the brain, Endo3 is co-localized with dopamine D2 receptor in olfactory nerve terminals and inhibits its clathrin-mediated endocytosis in COS-7 cells. Furthermore, overexpression of Endo3 in an olfactory epithelium-derived cell line suppressed dopamine D2 receptor-mediated endocytosis and therefore accelerated its dopamine-induced differentiation. These results indicate that Endo3 may act as a negative regulator of clathrin-mediated endocytosis in brain neurons.
(キーワード)
Adaptor Proteins, Signal Transducing / Animals / Carrier Proteins / Clathrin / Endocytosis / Nerve Tissue Proteins / Protein Isoforms / Rats / Signal Transduction
Yoshiyuki Yoshimura, Yoshio Yamauchi, Takashi Shinkawa, Masato Taoka, Hitomi Donai, Hitomi Donai, Toshiaki Isobe and Takashi Yamauchi : Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography-tandem mass spectrometry, Journal of Neurochemistry, Vol.88, No.3, 759-768, 2004.
(要約)
Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale two-dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S-carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC-MS/MS system in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G-protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell-cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery.
(キーワード)
excitatory synapse / liquid chromatography / protein composition / 質量分析法 (mass spectrometry) / postsynaptic density / プロテオミクス (proteomics)
Yoshiyuki Yoshimura, Tatsuya Ichinose and Takashi Yamauchi : Phosphorylation of tau protein to sites found in Alzheimer's disease brain is catalyzed by Ca2+/calmodulin-dependent protein kinase II as demonstrated tandem mass spectrometry, Neuroscience Letters, Vol.353, No.3, 185-188, 2003.
(要約)
Neuronal Ca2+/calmodulin-dependent protein kinase II (CaMKII) is one of the most abundant protein kinases in the brain, and phosphorylates a broad range of substrate proteins. The phosphorylation of microtubule tau by CaMKII was investigated using tandem mass spectrometry (MS/MS). Recombinant human tau was phosphorylated at Thr212, Ser214, Ser262, and Ser356 by CaMKII. The phosphorylation of these sites is found in paired helical filament (PHF)-tau. In addition to these sites, Ser131 and Thr135 were phosphorylated by CaMKII. Phosphorylation at Ser131, Thr135, Thr212 and Ser214 by CaMKII has not been reported previously. Thr212 and Ser214 are in the consensus phosphorylation sequence of CaMKII (RXXS/T), and non-fetal-type phosphorylation sites of tau. Non-fetal-type phosphorylation may produce PHF-tau. These results suggested that CaMKII is involved in the phosphorylation of tau in Alzheimer's disease brain.
(キーワード)
Ca2+/calmodulin-dependent protein kinase II (CaMKII) / Tau / リン酸化 (phosphorylation) / Alzheimers disease / Paired helical filaments
Hitomi Donai, Hiroko Sugiura, Daisuke Ara, Yoshiyuki Yoshimura, Kanato Yamagata and Takashi Yamauchi : Interaction of Arc with CaM kinase II and stimulation of neurite extension by Arc in neuroblastoma cells expressing CaM kinase II, Neuroscience Research, Vol.47, No.4, 399-408, 2003.
(要約)
We investigated the relationship between Arc (activity-regulated cytoskeleton-associated protein) and Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). Arc and CaM kinase II were concentrated in the postsynaptic density. These proteins were accumulated after electroconvulsive treatment. Arc increased about 2.5-fold within 30 min and was maintained at this level for 8h after the stimulation. CaM kinase II also increased within 30 min and remained at this level for at least 24h. The interaction of Arc with CaM kinase II was demonstrated using GST-Arc fusion protein, and confirmed in neuroblastoma cells by immunoprecipitation. We examined the function of Arc by introducing Arc cDNA into neuroblastoma cells expressing CaM kinase II. The cells expressing both Arc and CaM kinase II had longer neurites than those expressing CaM kinase II alone. Arc itself did not promote neurite outgrowth. The growth of neurites by Arc was completely blocked by treatment with KN62, an inhibitor of CaM kinases. These results indicated that Arc potentiated the action of CaM kinase II for neurite extension.
(キーワード)
Ca2+/calmodulin-dependent protein kinase II (CaMKII) / Postsynaptic density / Arc / Neurite extension / Neuroblastoma
Mutsuki Amano, Takako Kaneko, Akio Maeda, Masanori Nakayama, Masaaki Ito, Takashi Yamauchi, Hideyuki Goto, Yuko Fukata, Noriko Oshiro, Azusa Shinohara, Akihiro Iwamatsu and Kozo Kaibuchi : Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase, Journal of Neurochemistry, Vol.87, No.3, 780-790, 2003.
(要約)
Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.
(キーワード)
protein phosphorylation / 情報伝達機構 (signal transduction)
Nozomi Kawakami, Katsuhiro Miyoshi, Shuhei Horio, Yoshiyuki Yoshimura, Takashi Yamauchi and Hiroyuki Fukui : Direct phosphorylation of histamine H1 receptor by various protein kinases in vitro., Methods and Findings in Experimental and Clinical Pharmacology, Vol.25, No.9, 685-693, 2003.
(要約)
Phosphorylation of G protein-coupled receptors (GPCRs) by various kinases is suggested to be an important step in initiating receptor desensitization. Some reports have indirectly demonstrated the involvement of protein kinase C (PKC)-mediated receptor phosphorylation in the desensitization of the histamine H1 receptor (H1R). In this study, human c-myc-epitope-tagged H1R (hm mcH1R) was expressed in Sf9 cells, and an in vitro approach was taken to obtain direct evidence that H1R could be phosphorylated by various kinases. When hm mcH1R, which had been immunoprecipitated with anti-c-myc antibody from Sf9 cell membranes, was incubated with PKC, cAMP-dependent protein kinase (PKA), calcium/calmodulin-dependent protein kinase II (CaMKII) or cGMP-dependent protein kinase (PKG), the immunoprecipitated receptor was phosphorylated by these kinases. Membrane-bound hm mcH1R, whose conformation is closer to its physiological state than that of the immunoprecipitated receptor, was also phosphorylated by PKC, PKA, CaMKII and PKG. Phosphorylation of immunoprecipitated and membrane-bound hm mcH1R was inhibited by kinase inhibitors. These data are the first demonstration of the phosphorylation of H1R by four protein kinases, i.e., PKC, PKA, CaMKII and PKG, and provide fundamental information to help us further understand the relationship between H1R phosphorylation and desensitization of this receptor.
(キーワード)
Animals / Baculoviridae / Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Calcium-Calmodulin-Dependent Protein Kinases / Catalytic Domain / Cattle / Cell Line / Cyclic AMP-Dependent Protein Kinases / Cyclic GMP-Dependent Protein Kinases / Humans / リン酸化 (phosphorylation) / Protein Binding / Pyrilamine / Receptors, Histamine H1 / Spodoptera
Akihiro Ohyama, Kohei Hosaka, Yoshiaki komiya, Kimio Akagawa, Emiko Yamauchi, Hisaaki Taniguchi, Nobuyuki Sasagawa, Konosuke Kumakura, Sumiko Mochida, Takashi Yamauchi and Michihiro Igarashi : Regulation of exocytosis through Ca2+/ATP-dependent binding of autophosphorylated Ca2+/calmodulin-activated protein kinase II to syntaxin 1A., The Journal of Neuroscience, Vol.22, No.9, 3342-3351, 2002.
(要約)
Syntaxin 1A/HPC-1 is a key component of the exocytotic molecular machinery, namely, the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor mechanism. Although >10 syntaxin-binding proteins have been identified, they cannot completely explain the regulation of exocytosis. Thus, novel proteins may interact with syntaxin. Because exocytosis requires both Ca2+ and ATP, we searched for Ca2+/ATP-dependent syntaxin-binding proteins from the rat brain and discovered Ca2+/calmodulin-activated protein kinase II (CaMKII)-alpha. At Ca2+ concentrations of >10(-6) m, only autophosphorylated CaMKII bound to syntaxin. Bound CaMKII was released from syntaxin by EGTA or by phosphatase, indicating that the binding is reversible. CaMKII bound to the linker domain of syntaxin, unlike any other known syntaxin-binding proteins. CaMKII-syntaxin complexes were also detected in synaptosomes by immunoprecipitation, and when reconstituted in vitro, they recruited larger amounts of synaptotagmin and SNAP-25 than syntaxin alone. The microinjected CaMKII-binding domain of syntaxin specifically affected exocytosis in chromaffin cells and in neurons. These results indicate that the Ca2+/ATP-dependent binding of CaMKII to syntaxin is an important process in the regulation of exocytosis.
Yoshiyuki Yoshimura, Takashi Shinkawa, Masato Taoka, Kana Kobayashi, Toshiaki Isobe and Takashi Yamauchi : Identification of protein substrates of Ca2+/calmodulin-dependent protein kinase in the postsynaptic density by protein sequencing and mass spectrometry, Biochemical and Biophysical Research Communications, Vol.290, No.3, 948-954, 2002.
(要約)
Previously we detected more than 28 PSD proteins to be phosphorylated by CaM kinase II, and identified 14 protein substrates (Yoshimura, Y., Aoi, T., Yamauchi, T., Mol. Brain Res. 81, 118-128, 2000). In the present study, the remaining substrates were analyzed by protein sequencing and mass spectrometry. We found 6 proteins not previously known to be substrates of CaM kinase II, namely PSD95-associated protein, SAP97, TOAD-64, TNF receptor-associated protein, insulin-receptor tyrosine kinase 58/53 kDa substrate, and homer 1b.
Yoshimi Sogawa, Yoshiyuki Yoshimura and Takashi Yamauchi : Investigation of the Ca2+-independent form of Ca2+/calmodulin-dependent protein kinase II in neurite outgrowth, Brain Research. Brain Research Protocols, Vol.8, No.3, 159-169, 2001.
(要約)
Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) plays important roles in the control of nerve functions in response to intracellular Ca(2+) (for reviews [Annu. Rev. Physiol. 57 (1995) 417-445; Trends Neurosci. 17 (1994) 406-412]). Brief Ca(2+) signals activate CaM kinase II, and stimulate an autophosphorylation of Thr-286 which allows the kinase to maintain its activated state even after the Ca(2+) concentration has returned to basal levels [J. Biol. Chem. 264 (1989) 16759-16763; Neuron 3 (1989) 59-70; J. Biochem. 109 (1991) 137-143]. Autophosphorylation of CaM kinase II occurs in situ, but it occurs relatively quickly, within just a few minutes [Endocrinology 134 (1994) 2245-2250; J. Biol. Chem. 268 (1993) 7863-7867; J. Biol. Chem. 265 (1990) 18055-18058]. In the present study, we investigated the involvement of the autophosphorylated/Ca(2+)-independent form of CaM kinase II in neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the alpha isoform of CaM kinase II (Nb2a/alpha cells) were stimulated by plating, they formed neurites. The autophosphorylation of Thr-286 and appearance of Ca(2+)-independent activity preceded the neurite formation. The effect of mutating of the kinase autophosphorylation site replacing Thr-286 with Ala (alpha T286A kinase) or Asp (alpha T286D kinase) was examined. alpha T286A kinase was not converted to a Ca(2+)-independent form, and alpha T286D kinase had Ca(2+)-independent activity significantly as an autophosphorylated kinase. Cells expressing alpha T286D kinase had much longer neurites than Nb2a/alpha cells, whereas cells with alpha T286A kinase did not form neurites. These results indicated that the Ca(2+)-independent form of CaM kinase II autophosphorylated at Thr-286 is involved in neurite outgrowth.
(キーワード)
Animals / Calcium / Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Calcium-Calmodulin-Dependent Protein Kinases / Cell Line / DNA, Complementary / Fluorescent Dyes / Green Fluorescent Proteins / Immunoblotting / Indicators and Reagents / Isoenzymes / Luminescent Proteins / Mice / Microscopy, Fluorescence / Mutation / Neurites / Neuroblastoma / リン酸化 (phosphorylation) / Transfection
Hitomi Donai, Hisayo Morinaga and Takashi Yamauchi : Genomic organization and neuronal cell type specific promoter activity of β isoform of Ca2+/calmodulin dependent protein kinase II of rat brain, Brain Research. Molecular Brain Research, Vol.94, No.1-2, 35-47, 2001.
(要約)
The gene encoding the beta isoform of rat Ca(2+)/calmodulin-dependent protein kinase II was cloned, and its exon-intron organization was analyzed. The gene consisted of 21 exons spanning more than 80 kilobase pairs and the coding sequence was made up of 20 exons. Each discrete functional unit, such as the ATP-binding site, the autophosphorylation site responsible for Ca(2+)-independent activity, the calmodulin binding site, and the link structure, was encoded by a single exon. All splice junction sequences flanking the introns conformed to the consensus splice junction sequence and the GT-AG splice rule. The site of transcription initiation was -78 bases from the initiation codon as determined by 5' RACE analysis. The promoter activity of the gene was analyzed using neuroblastomas, as well as non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5'-upstream region -66 to -35 bp from the transcription initiation site. Silence elements were found further upstream at -222 to -123 bp and -576 to -323 bp. A protein bound to the -66 to -35 region was found in the nuclear extract of rat brain, including the cerebellum, forebrain, and brainstem, by gel mobility shift assay.
(キーワード)
Ca2+/calmodulin-dependent protein kinase II / Gene structure / Promoter activity / Neuronal cell
Masato Urushihara and Takashi Yamauchi : Role of β isoform-specific insertions of Ca2+/calmodulin-dependent protein kinase II, European Journal of Biochemistry, Vol.268, No.17, 4802-4808, 2001.
(要約)
Alpha and beta isoforms of Ca2+/calmodulin-dependent protein kinase II (alpha and beta CaM kinase II, respectively) are highly conserved except for beta-specific insertions 1 and 2, located at amino acids 316-340 and 354-392, respectively. To investigate the role of these beta-specific insertions, we prepared the deletion mutants betaDelta1, betaDelta2 and betaDelta1/2, which lacked insertions 1, 2 and both, respectively. These mutant DNAs were expressed in neuroblastoma cells and compared with the wild-type enzyme. Green fluorescent protein tagged CaM kinase II was used to further explore the distribution of the kinase in living cells. Most (80%) of wild-type beta and mutant betaDelta1 were located in the particulate fraction, and distributed in the cell body and neurites, forming punctate or spot-like structures in the neurites. Mutants betaDelta2 and betaDelta1/2 were distributed in almost equal amounts in the soluble and particulate fractions. They were concentrated in the base of neurites and only partlially distributed throughout neurites, indicating that their transport to neurites was impaired. Beta(1-410), a deletion mutant of the association domain with a monomeric form, was located primarily in the soluble fraction. These results indicate that insertion 2, the association domain, and the oligomeric form of beta CaM kinase II play an important role in the cellular distribution of beta CaM kinase II.
(キーワード)
Animals / Calcium / Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Calcium-Calmodulin-Dependent Protein Kinases / Calmodulin / Green Fluorescent Proteins / Isoenzymes / Luminescent Proteins / Mice / Mutation / Tumor Cells, Cultured
Kazuko Mima, Shogo Deguchi and Takashi Yamauchi : Characterization of 5' flanking region of α isoform of rat Ca2+/calmodulin-dependent protein kinase II gene and neuronal cell type specific promoter activity, Neuroscience Letters, Vol.307, No.2, 117-121, 2001.
(要約)
The 5' flanking region of the alpha isoform of the rat Ca2+/calmodulin-dependent protein kinase II (alpha CaM kinase II) gene was isolated in 2.3 kbp of genomic sequence. Functional analysis of alpha CaM kinase II promoter deletion mutants fused to a reporter gene in neuroblastoma, including N18TG2, NG108-15, and CAD cells revealed strong transcriptional activity localized 100-145 bp, and a potent silencer 199-275 bp upstream of the transcription start site. The promoter is inactive in non-neuronal cells including BALB/c 3T3, Chinese hamster ovary, HT1080, and C6 glioma cells. These results indicated that the alpha CaM kinase II gene is transcribed from a tissue-specific promoter which is under intense negative control.
(キーワード)
Ca2+/calmodulin-dependent protein kinase II / Promotor activity / Neuronal cell / Gene expression / 5 flanking region
Hitomi Donai, Takahiro Murakami, Takahito Amano, Yoshimi Sogawa and Takashi Yamauchi : Induction and alternative splicing of δ isoform of Ca2+/calmodulin-dependent protein kinase II during neural differentiation of P19 embryonal carcinoma cells and during brain development, Brain Research. Molecular Brain Research, Vol.85, No.1-2, 189-199, 2000.
Hitomi Donai, Miki Nakamura, Yoshimi Sogawa, James T. K. Wang, Masato Urushihara and Takashi Yamauchi : Involvement of Ca2+/calmodulin-dependent protein kinase II in neurite outgrowth induced by cAMP treatment and serum deprivation in a central nervous system cell line, CAD derived from rat brain, Neuroscience Letters, Vol.293, No.2, 111-114, 2000.
(要約)
A central nervous system (CNS) cell line, CAD, is known to differentiate in the absence of serum. This cell line was found to differentiate by the treatment of cAMP. Expression of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) was induced to about 2-fold or more on day 1, and was continued at a high level for 5 days after the exposure to differentiating conditions. Neurite extension was stimulated from day 1 and continued for 5 days, suggesting that CaM kinase II activity is correlated with neurite outgrowth. Of the four distinct isoforms (alpha, beta, gamma, and delta) of the kinase, the delta isoform was the major isoform in CAD cells. The splicing pattern of this isoform in the differentiated cells differed from that in undifferentiated cells, suggesting that expression of CaM kinase II is regulated during neural differentiation.
(キーワード)
Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) / CNS cell line (CAD) / Neural differentiation / Neurite outgrowth / Alternative splicing / Isoform
Yoshimi Sagawa, Yoshiyuki Yoshimura, Akira Otaka and Takashi Yamauchi : Ca2+-independent activity of Ca2+/calmodulin-dependent protein kinase II involved in stimulation of neurite outgrowth in neuroblastoma cells, Brain Research, Vol.881, No.2, 165-175, 2000.
(要約)
We investigated the involvement of Ca(2+)-independent activity of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) in stimulation of neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the alpha isoform of CaM kinase II (Nb2a/alpha cells) were stimulated by plating, they changed shape from round to flattened, and began to form neurites within 15 min. Numbers of cells bearing neurites increased from 15 min to about 2 h. Neurite length increased markedly from 30 min to 2 h after stimulation. Ca(2+)-independent activity of CaM kinase II increased immediately after stimulation, peaked at about 30 min, and then gradually decreased. Autophosphorylation of Thr-286 followed the same time course as the increase in Ca(2+)-independent activity. The autophosphorylation and appearance of Ca(2+)-independent activity preceded the formation of neurites. The effect of mutation of the autophosphorylation site in the kinase whose Thr-286 was replaced with Ala (alphaT286A kinase) or Asp (alphaT286D kinase) was examined. alphaT286A kinase was not converted to a Ca(2+)-independent form, and alphaT286D kinase had Ca(2+)-independent activity significantly as an autophosphorylated kinase. Cells expressing alphaT286A kinase did not form neurites, and were indistinguishable from control Nb2a cells. Cells expressing alphaT286D kinase had much longer neurites than Nb2a/alpha cells expressing the wild type kinase, although the initiation of neurite outgrowth was very late. These results indicated that Ca(2+)-independent activity of the kinase autophosphorylated at Thr-286 involves for neurite outgrowth.
(キーワード)
Ca2+/calmodulin-dependent protein kinase II / Autophosphorylation / Neurite outgrowth / Ca2+-independent activity / Neuroblastoma
Yoshiyuki Yoshimura, Chiaki Aoi and Takashi Yamauchi : Investigation of protein substrates of Ca2+/calmodulin-dependent protein kinase II translocated to the postsynaptic density, Brain Research. Molecular Brain Research, Vol.81, No.1-2, 118-128, 2000.
(要約)
To elucidate the physiological significance of the translocation of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II), we investigated substrates of CaM kinase II in the postsynaptic density (PSD). PSD proteins were phosphorylated by CaM kinase II of its PSD complex, and separated by two-dimensional gel electrophoresis. More than 28 proteins were phosphorylated under experimental conditions. Proteins corresponding to CaM kinase II substrates were excised from the gels, eluted electrophoretically, and then sequenced. Several substrates were identified, including PSD95, SAP90, alpha-internexin, neurofilament L chain, cAMP phosphodiesterase, and alpha- and beta-tubulin. Some substrates were also identified by immunoblotting, including N-methyl-D-aspartic acid (NMDA) receptor 2B subunit, 1-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor 1 (GluR1), neurofilament H chain and dynamin. PSD95, SAP90, dynamin, and alpha-internexin were demonstrated for the first time to be substrates of CaM kinase II. NMDA receptor 2B subunit and GluR1 existed as major substrates in the PSD. Moreover, translocation of CaM kinase II was inhibited by phosphorylation of PSD proteins. These results suggest that CaM kinase II plays important roles in the regulation of synaptic functions through phosphorylation of PSD proteins.
(キーワード)
Ca2+/calmodulin-dependent protein kinase II / Postsynaptic density / リン酸化 (phosphorylation) / Translocation / Two-dimensional gel electrophoresis
Takashi Yamauchi : Neuronal Ca2+/Calmodulin-Dependent Protein Kinase II-Discovery, Progress in a Quarter of a Century, and Perspective: Implication for Learning and Memory, Biological & Pharmaceutical Bulletin, Vol.28, No.8, 1342-1354, Aug. 2005.
(要約)
Much has been learned about the activity-dependent synaptic modifications that are thought to underlie memory storage, but the mechanism by which these modifications are stored remains unclear. A good candidate for the storage mechanism is Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). CaM kinase II is one of the most prominent protein kinases, present in essentially every tissue but most concentrated in brain. Although it has been about a quarter of a century since the finding, CaM kinase II has been of the major interest in the region of brain science. It plays a multifunctional role in many intracellular events, and the expression of the enzyme is carefully regulated in brain regions and during brain development. Neuronal CaM kinase II regulates important neuronal functions, including neurotransmitter synthesis, neurotransmitter release, modulation of ion channel activity, cellular transport, cell morphology and neurite extension, synaptic plasticity, learning and memory, and gene expression. Studies concerning this kinase have provided insight into the molecular basis of nerve functions, especially learning and memory, and indicate one direction for studies in the field of neuroscience. This review presents the molecular structure, properties and functions of CaM kinase II, as a major component of neurons, based mainly developed on findings made in our laboratory.
(キーワード)
Ca2+/calmodulin-dependent protein kinase II / memory / protein phosphorylation / Ca2+ signaling; plasticity / postsynaptic density
Takashi Yamauchi : Molecular constituents and phosphorylation-dependent regulation of the post-synaptic density, Mass Spectrometry Reviews, Vol.21, No.4, 266-286, Jul. 2002.
(キーワード)
Ca2+/calmodulin-dependent protein kinase II (CaM KII) / post-synaptic density / phosphorylation / synaptic plasticity / long-term potentiation / glutamate receptor / proteomics analysis
国際会議:
1.
Shunsuke Izaki, Masashi Takano, Yoshito Kadota, Takashi Yamauchi, Atsushi Umemoto, Toshinori Oka and Kouji Itou : Glycoproteomic alteration in gastric and colorectal adenocarcinomas detected on lectin blotting., IUBMB, Kyoto, Jun. 2006.