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Lipid components in the detergent-resistant membrane microdomain from rat synaptic plasma membrane
S196
Abstracts / Neuroscience Research 58S (2007) S1–S244
2 Characterization of SCS as a potent activator for the P3-c0 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-3sulfate (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 partially 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 phosphorylation of TP, eIF-4B and syndapin 1, which are involved in the mechanisms of various neuronal diseases, including Alzheimer’s disease.
4 Activation of the c-fos promoter is modulated by PKA P3-c0 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 normal cellular proliferation and differentiation. It has been reported that application of NGF, which leads to cellular differentiation, induced a transient 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 promoter 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.
6 Distinct cellular expression of PKC␣ and PKC␥ in parP3-c0 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 serine/threonine protein kinases that regulate various biological events within the cell. PKC␣ and PKC␥, members of classical PKC, are Ca2+ 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 parvalbumin (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 preferentially express PKC␣, whereas most principal neurons express both PKC␣ and PKC␥.
8 Localization of diacylglycerol kinase ␥ and - in neurons P3-c0 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 transduction 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 analysis, we found that immunoreactivities against DGK␥ and - were detected in the light membrane/microsome enriched fraction of rat brains, suggesting 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 structures 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 suggest that DGK␥ and - play different roles in distinct internal membrane system in neurons. Research funds: KAKENHI (18700337)
5 Tyrosin kinase is involved in the mGluR1-mediated P3-c0 inward current in the cerebellar molecular layer interneurons
9 Lipid components in the detergent-resistant memP3-c0
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
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
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 agonist 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.
Membrane microdomains are recovered as a detergent-resistant low-density membrane fraction (DRM) after Triton extraction and centrifugation. Since specific recovery of phosphatidylinositol 4,5bisphosphate (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, antiGST 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 cholesterol, and about 30% of phosphatidylcholine or phosphatidylethanolamine were recovered in DRM.
Research funds: KAKENHI (16700344)
brane microdomain from rat synaptic plasma membrane
Research funds: KAKENHI (16370062)
Abstracts / Neuroscience Research 58S (2007) S1–S244
2 Characterization of SCS as a potent activator for the P3-c0 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-3sulfate (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 partially 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 phosphorylation of TP, eIF-4B and syndapin 1, which are involved in the mechanisms of various neuronal diseases, including Alzheimer’s disease.
4 Activation of the c-fos promoter is modulated by PKA P3-c0 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 normal cellular proliferation and differentiation. It has been reported that application of NGF, which leads to cellular differentiation, induced a transient 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 promoter 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.
6 Distinct cellular expression of PKC␣ and PKC␥ in parP3-c0 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 serine/threonine protein kinases that regulate various biological events within the cell. PKC␣ and PKC␥, members of classical PKC, are Ca2+ 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 parvalbumin (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 preferentially express PKC␣, whereas most principal neurons express both PKC␣ and PKC␥.
8 Localization of diacylglycerol kinase ␥ and - in neurons P3-c0 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 transduction 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 analysis, we found that immunoreactivities against DGK␥ and - were detected in the light membrane/microsome enriched fraction of rat brains, suggesting 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 structures 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 suggest that DGK␥ and - play different roles in distinct internal membrane system in neurons. Research funds: KAKENHI (18700337)
5 Tyrosin kinase is involved in the mGluR1-mediated P3-c0 inward current in the cerebellar molecular layer interneurons
9 Lipid components in the detergent-resistant memP3-c0
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
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
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 agonist 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.
Membrane microdomains are recovered as a detergent-resistant low-density membrane fraction (DRM) after Triton extraction and centrifugation. Since specific recovery of phosphatidylinositol 4,5bisphosphate (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, antiGST 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 cholesterol, and about 30% of phosphatidylcholine or phosphatidylethanolamine were recovered in DRM.
Research funds: KAKENHI (16700344)
brane microdomain from rat synaptic plasma membrane
Research funds: KAKENHI (16370062)