Localization of diacylglycerol kinase γ and -ɛ in neurons
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S196 Abstracts / Neuroscience Research 58S (2007) S1–S244 P3-c0 2 Characterization of SCS as a potent activator for the CK1-mediated phosphorylation of tau protein and its associated proteins from rat brain Kanzo Suzuki, Fumitaka Kawakami, Hisashi Sasaki, Kenzo Ohtsuki Laboratory of Genetical Biochemistry and Signal Biology, Graduate School of Medical Sciences, Kitasato University, Japan Purpose: Recently, we reported that casein kinase 1 (CK1) phophorylates two functional basic proteins [myelin basic protein (MBP) and tau protein (TP)] in the presence of two sulfated lipids [sulfatide and cholesterol-3- sulfate (CH-3S), SCS] in vitro. The present in vitro study has been carried out to characterize the SCS-dependent phosphorylation of TP and its associated proteins by CK1 in the TP fraction from rat brain. Results and Discussion: It was found that (i), in the presence of SCS, CK1 phosphorylated TP and its associated proteins (p82 and p55) in the par- tially purified TP fraction; and (ii) p82 and p55 was identified as eIF-4B and syndapin 1, respectively. These results suggest that the accumulated high level of SCS preferentially induces the CK1-mediated phosphoryla- tion of TP, eIF-4B and syndapin 1, which are involved in the mechanisms of various neuronal diseases, including Alzheimer’s disease. P3-c0 4 Activation of the c-fos promoter is modulated by PKA in PC12 cells Takashi Sugiyama, Hirobumi Suzuki Advanced Core Technology Department, Olympus Corporation, Japan The c-fos protooncogene is thought to contribute to the regulation of nor- mal cellular proliferation and differentiation. It has been reported that application of NGF, which leads to cellular differentiation, induced a tran- sient c-fos gene expression in PC12 cells. NGF activates the c-fos promoter through two well characterized regulatory elements, the serum response element (SRE) and the cAMP response element (CRE). Many kinases are involved in the transcription through these elements, a contribution of PKA, however, has not been investigated in detail. Using a luminescence imaging system and a human c-fos promoter-driven luciferase expression vector, we analyzed physiological roles of PKA in NGF-induced c-fos pro- moter activation in a single cell level. Inhibitors for PKA enhanced the increase of the c-fos promoter activity without any effects to MEK/MAPK cascade. Mutational analysis of the CRE and/or the NFB consensus sequence in the regulatory region of the c-fos gene revealed that c-fos promoter activity might be modulated by both CREB and NFB. Finally, we propose a molecular mechanism under the modulation of the c-fos promoter by PKA. P3-c0 5 Tyrosin kinase is involved in the mGluR1-mediated inward current in the cerebellar molecular layer interneurons Hideo Kubota 1 , Moritoshi Hirono 2 , Kunihiko Obata 2 1 Supply Unit, Faculty of Medicine, Tokyo Medical and Dental University Hospital, Tokyo, Japan; 2 Neuronal Circuit Mechanisms Research Group, BSI, RIKEN, Wako, Japan Activation of mGluR1 induces an excitatory slow inward current in the cerebellar molecular layer interneurons (MLIs), such as basket and stellate cells. We addressed the underlying mechanism with whole-cell patch-clamp recordings. Pressure application of the group I mGluR ago- nist DHPG produced a slow inward current in MLIs, which was blocked by an mGluR1 antagonist, CPCCOEt. Bath-applied protein tyrosin kinase inhibitors, genistein and AG490, considerably reduced the DHPG-induced inward current, although infusion of a PLC inhibitor U73122 into MLIs only partially inhibited the current amplitude. Furthermore, perfusion of a selective Src kinase inhibitor PP2 significantly suppressed the inward current. These results suggest that mGluR1 activation in MLIs induces the inward current through a Src-dependent pathway, which is different from the mGluR1-TRPC1 signaling cascade in cerebellar Purkinje cells. Research funds: KAKENHI (16700344) P3-c0 6 Distinct cellular expression of PKC and PKC in par- ticular interneurons of the mouse telencephalon Eriko Miura, Yoshiyuki Kubota, Masahiko Watanabe Department of Anatomy, Hokkaido University Graduate School of Medicine, Sapporo, Japan Protein kinase C (PKC) is classified as a family of closely related ser- ine/threonine protein kinases that regulate various biological events within the cell. PKC and PKC, members of classical PKC, are Ca 2+ - dependent and activated by diacylglycerol (DAG) in the process of a series of phosphorylation events. In this study, we studied cellular expression of PKC and PKC in the mouse striatum, cortex, and hippocampus. PKC and PKC were more or less coexpressed in principal neurons, including medium spiny neurons in the striatum, cortical neurons in the layer II/III, and pyramidal cells and granule cells in the hippocampus. However, PKC was expressed predominantly in cholinergic interneurons and parvalbu- min (PV)-positive interneurons in the striatum, layer V non-pyramidal neurons and PV-positive interneurons in the cortex, and nNOS-positive interneurons in the hippocampus. In contrast, pyramidal cells in the layer V selectively expressed PKC. Therefore, particular interneurons pref- erentially express PKC, whereas most principal neurons express both PKC and PKC. P3-c0 8 Localization of diacylglycerol kinase and - in neurons Yasukazu Hozumi, Kaoru Goto Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata, Japan Diacylglycerol kinase (DGK) is involved in intracellular signal transduc- tion as a regulator of a second messenger, diacylglycerol. To address the functional implications of DGK isozymes in the brain, we have raised specific antibodies against DGK and -. By immunoblot analy- sis, we found that immunoreactivities against DGK and - were detected in the light membrane/microsome enriched fraction of rat brains, sug- gesting that these isozymes localize to the internal membrane system. Immunohistochemical examination on the cerebellum revealed that immunoreactivity for DGK was detected as punctate or reticular struc- tures in the cytoplasm and dendrites, but not in the axon, of Purkinje cells. The immunoreactivity was also detected diffusely in the cytoplasm of cerebellar granule cells. On the other hand, DGK-immunoreactivity was observed as granular structures in the perinuclear region, dendrites and axon of Purkinje cells, but not in cerebellar granule cells. These data sug- gest that DGK and - play different roles in distinct internal membrane system in neurons. Research funds: KAKENHI (18700337) P3-c0 9 Lipid components in the detergent-resistant mem- brane microdomain from rat synaptic plasma membrane Shohei Maekawa 1 , Daisuke Matsuura 1 , Katsutoshi Taguchi 1 , Hitoshi Yagisawa 2 1 Department of Biology, Kobe-University, Kobe, Japan; 2 Graduate School of Life Science, University of Hyogo, Hyogo, Japan Membrane microdomains are recovered as a detergent-resistant low-density membrane fraction (DRM) after Triton extraction and centrifugation. Since specific recovery of phosphatidylinositol 4,5- bisphosphate (PIP2) in DRM is known in cultured cells, the DRM is considered as a signal-processing platform. Little information is however available on the PIP2 content in the DRM prepared from brain, for the RI-labeling of live animals are not practical. In this study, we assayed the amounts of PIP2 in SPM and SPM-derived DRM using the PH domain of phospholipase C-delta1 fused to GST. Extracted lipids were separated on HPTLC and heat-transferred to PVDF membrane. After blocking, anti- GST and ECL system was used to measure the bound GST. About 10% of PIP2 in SPM was recovered in DRM and no specific localization was detected. In contrast, nearly half amounts of sphingomyelin and choles- terol, and about 30% of phosphatidylcholine or phosphatidylethanolamine were recovered in DRM. Research funds: KAKENHI (16370062)
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196 Abstracts / Neuroscience
3-c0� 2 Characterization of SCS as a potent activator for theK1-mediated phosphorylation of tau protein and its associatedroteins from rat brainanzo Suzuki, Fumitaka Kawakami, Hisashi Sasaki, Kenzo Ohtsuki
Laboratory of Genetical Biochemistry and Signal Biology, Graduatechool of Medical Sciences, Kitasato University, Japan
urpose: Recently, we reported that casein kinase 1 (CK1) phophorylateswo functional basic proteins [myelin basic protein (MBP) and tau proteinTP)] in the presence of two sulfated lipids [sulfatide and cholesterol-3-ulfate (CH-3S), SCS] in vitro. The present in vitro study has been carriedut to characterize the SCS-dependent phosphorylation of TP and itsssociated proteins by CK1 in the TP fraction from rat brain.esults and Discussion: It was found that (i), in the presence of SCS, CK1hosphorylated TP and its associated proteins (p82 and p55) in the par-ially purified TP fraction; and (ii) p82 and p55 was identified as eIF-4Bnd syndapin 1, respectively. These results suggest that the accumulatedigh level of SCS preferentially induces the CK1-mediated phosphoryla-ion of TP, eIF-4B and syndapin 1, which are involved in the mechanismsf various neuronal diseases, including Alzheimer’s disease.
3-c0� 4 Activation of the c-fos promoter is modulated by PKAn PC12 cellsakashi Sugiyama, Hirobumi Suzuki
Advanced Core Technology Department, Olympus Corporation,apan
he c-fos protooncogene is thought to contribute to the regulation of nor-al cellular proliferation and differentiation. It has been reported that
pplication of NGF, which leads to cellular differentiation, induced a tran-ient c-fos gene expression in PC12 cells. NGF activates the c-fos promoterhrough two well characterized regulatory elements, the serum responselement (SRE) and the cAMP response element (CRE). Many kinases arenvolved in the transcription through these elements, a contribution ofKA, however, has not been investigated in detail. Using a luminescence
maging system and a human c-fos promoter-driven luciferase expressionector, we analyzed physiological roles of PKA in NGF-induced c-fos pro-oter activation in a single cell level. Inhibitors for PKA enhanced the
ncrease of the c-fos promoter activity without any effects to MEK/MAPKascade. Mutational analysis of the CRE and/or the NFB consensusequence in the regulatory region of the c-fos gene revealed that c-fosromoter activity might be modulated by both CREB and NFB. Finally,e propose a molecular mechanism under the modulation of the c-fosromoter by PKA.
3-c0� 5 Tyrosin kinase is involved in the mGluR1-mediatednward current in the cerebellar molecular layer interneuronsideo Kubota1, Moritoshi Hirono2, Kunihiko Obata2
Supply Unit, Faculty of Medicine, Tokyo Medical and Dentalniversity Hospital, Tokyo, Japan; 2 Neuronal Circuit Mechanismsesearch Group, BSI, RIKEN, Wako, Japan
ctivation of mGluR1 induces an excitatory slow inward current inhe cerebellar molecular layer interneurons (MLIs), such as basket andtellate cells. We addressed the underlying mechanism with whole-cellatch-clamp recordings. Pressure application of the group I mGluR ago-ist DHPG produced a slow inward current in MLIs, which was blockedy an mGluR1 antagonist, CPCCOEt. Bath-applied protein tyrosin kinase
nhibitors, genistein and AG490, considerably reduced the DHPG-inducednward current, although infusion of a PLC inhibitor U73122 into MLIsnly partially inhibited the current amplitude. Furthermore, perfusion of
selective Src kinase inhibitor PP2 significantly suppressed the inwardurrent. These results suggest that mGluR1 activation in MLIs induceshe inward current through a Src-dependent pathway, which is differentrom the mGluR1-TRPC1 signaling cascade in cerebellar Purkinje cells.
esearch funds: KAKENHI (16700344)
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3-c0� 6 Distinct cellular expression of PKC� and PKC� in par-icular interneurons of the mouse telencephalonriko Miura, Yoshiyuki Kubota, Masahiko Watanabe
Department of Anatomy, Hokkaido University Graduate School ofedicine, Sapporo, Japan
rotein kinase C (PKC) is classified as a family of closely related ser-ne/threonine protein kinases that regulate various biological events
ithin the cell. PKC� and PKC�, members of classical PKC, are Ca2+-ependent and activated by diacylglycerol (DAG) in the process of a seriesf phosphorylation events. In this study, we studied cellular expression ofKC� and PKC� in the mouse striatum, cortex, and hippocampus. PKC�nd PKC� were more or less coexpressed in principal neurons, includingedium spiny neurons in the striatum, cortical neurons in the layer II/III,
nd pyramidal cells and granule cells in the hippocampus. However, PKC�as expressed predominantly in cholinergic interneurons and parvalbu-in (PV)-positive interneurons in the striatum, layer V non-pyramidal
eurons and PV-positive interneurons in the cortex, and nNOS-positiventerneurons in the hippocampus. In contrast, pyramidal cells in the layer
selectively expressed PKC�. Therefore, particular interneurons pref-rentially express PKC�, whereas most principal neurons express bothKC� and PKC�.
3-c0� 8 Localization of diacylglycerol kinase � and -� in neuronsasukazu Hozumi, Kaoru Goto
Department of Anatomy and Cell Biology, Yamagata Universitychool of Medicine, Yamagata, Japan
iacylglycerol kinase (DGK) is involved in intracellular signal transduc-ion as a regulator of a second messenger, diacylglycerol. To addresshe functional implications of DGK isozymes in the brain, we haveaised specific antibodies against DGK� and -�. By immunoblot analy-is, we found that immunoreactivities against DGK� and -� were detectedn the light membrane/microsome enriched fraction of rat brains, sug-esting that these isozymes localize to the internal membrane system.mmunohistochemical examination on the cerebellum revealed thatmmunoreactivity for DGK� was detected as punctate or reticular struc-ures in the cytoplasm and dendrites, but not in the axon, of Purkinje cells.he immunoreactivity was also detected diffusely in the cytoplasm oferebellar granule cells. On the other hand, DGK�-immunoreactivity wasbserved as granular structures in the perinuclear region, dendrites andxon of Purkinje cells, but not in cerebellar granule cells. These data sug-est that DGK� and -� play different roles in distinct internal membraneystem in neurons.
esearch funds: KAKENHI (18700337)
3-c0� 9 Lipid components in the detergent-resistant mem-rane microdomain from rat synaptic plasma membranehohei Maekawa1, Daisuke Matsuura1, Katsutoshi Taguchi1,itoshi Yagisawa2
Department of Biology, Kobe-University, Kobe, Japan; 2 Graduatechool of Life Science, University of Hyogo, Hyogo, Japan
embrane microdomains are recovered as a detergent-resistantow-density membrane fraction (DRM) after Triton extraction andentrifugation. Since specific recovery of phosphatidylinositol 4,5-isphosphate (PIP2) in DRM is known in cultured cells, the DRM isonsidered as a signal-processing platform. Little information is howevervailable on the PIP2 content in the DRM prepared from brain, for theI-labeling of live animals are not practical. In this study, we assayed themounts of PIP2 in SPM and SPM-derived DRM using the PH domain ofhospholipase C-delta1 fused to GST. Extracted lipids were separatedn HPTLC and heat-transferred to PVDF membrane. After blocking, anti-ST and ECL system was used to measure the bound GST. About 10%
f PIP2 in SPM was recovered in DRM and no specific localization wasetected. In contrast, nearly half amounts of sphingomyelin and choles-erol, and about 30% of phosphatidylcholine or phosphatidylethanolamineere recovered in DRM.esearch funds: KAKENHI (16370062)
