Functional and structural diversity of variable domain of Ca^<2+>/calmodulin-dependent protein kinase II

Ca^2/钙调蛋白依赖性蛋白激酶II可变域的功能和结构多样性

• 批准号: 11680758
• 负责人: YAMAMOTO Hideyuki
• 金额: $ 2.24万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680758/
• 关键词: Astrocyte; Gene expression; Insulin secretion; NG108-15 cells; Ca^<2+>; calmodulin-dependent protein kinase II; Synapsin I; Brain-derived neurotrophic factor; MIN6 cells; カルモデュリン; インスリン分泌細胞; 可変領域; ゴルジ体; 小胞体

Ca^<2+>/calmodulin-dependent protein kinase II (CaM kinase II) is widely distributed and may be involved in a variety of Ca^<2+>-mediated cellular processes. Four subunits, termed α, β, γ, δ, are encoded by distinct genes in eukaryotes. All subunits have high homology in the N-terminal catalytic domain and in the regulatory domain comprising calmodulin-binding and autoinhibitory sites. Various isoforms of these subunits exist as different splicing variants. The isoforms differ mainly at the end of the regulatory domain (variable domain). In this study, we identified isoforms in brain and various cultured cells with immunochemical and molecular biological techniques. In addition, their physiological roles in these cells were examined. 1. We prepared a specific antibody to δ subunit and found that δ3 was abundant in the nucleus in cerebellar granule cells. We transiently overexpressed the nuclear isoforms, αB and δ3, in NG108-15 cells and found that these isoforms were involved in the expression of brain-derived neurotrophic factor. 2. We identified all isoforms in cultured astrocytes and NG108-15 cells. Among the isoforms identified, the most abundant isoform was δ2. Immunostaining suggested that δ2 was localized predominantly at the Golgi apparatus in both cells. 3. When we examined the isoforms in mouse insulinoma MIN6 cells, the abundant isoforms were β'e and δ2 at both mRNA and protein levels. We found that δ2 and synapsin I colocalized with insulin secretory granules. We confirmed that overexpression of β'e and δ2 enhanced the phosphorylation of synapsin I and insulin secretion. These results suggest that each isoform is localized at the specific site in the cells and plays a critical role in the coupling of Ca^<2+> signals to specific cellular responces.

Ca^2+/钙调蛋白依赖性蛋白激酶II(CaM激酶II)分布广泛并且可能参与多种Ca^2+介导的细胞过程，称为α、β、γ、 δ，由真核生物中的不同基因编码，在 N 端催化结构域和包含钙调蛋白结合和自身抑制位点的调节结构域中具有高度同源性。这些亚基以不同的剪接变体形式存在。在本研究中，我们通过免疫化学和分子生物学技术鉴定了大脑和各种培养细胞中的同工型。 1.我们制备了针对 δ 亚基的特异性抗体，发现 δ3 在小脑颗粒细胞的细胞核中含量丰富。 αB 和 δ3，在 NG108-15 细胞中，发现这些异构体参与脑源性神经营养因子 2 的表达。我们鉴定了培养的星形胶质细胞和 NG108-15 细胞中的所有异构体。免疫染色表明 δ2 主要位于两种细胞的高尔基体 3 中。在小鼠胰岛素瘤 MIN6 细胞中，丰富的亚型是 β'e 和 δ2，我们发现 δ2 和突触蛋白 I 与胰岛素分泌颗粒共定位，我们证实 β'e 和 δ2 的过度表达增强了 β'e 和 δ2 的磷酸化。这些结果表明，每种亚型都位于细胞的特定位点，并在突触蛋白 I 和胰岛素分泌的偶联中发挥着关键作用。 Ca ^ 2+ 向特定细胞反应发出信号。

项目成果

M.Nakai, K.Hojo, K.Yagi, N.Saito, T.Taniguchi, A.Terashima, T.Kawamata, T.Hashimoto, K.Maeda, M.Gschwendt, H.Yamamoto, E.Miyamoto and C.Tanaka: "Amyloid β protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling p

M.Nakai、K.Hojo、K.Yagi、N.Saito、T.Taniguchi、A.Terashima、T.Kawamata、T.Hashimoto、K.Maeda、M.Gschwendt、H.Yamamoto、E.Miyamoto 和 C. Tanaka：“β 淀粉样蛋白 (25-35) 通过酪氨酸激酶激活的蛋白激酶 C 信号传导使 MARCKS 磷酸化

Nakai,M.: "Amyloid β protein (25-35) phosphorylates MARKS throtgh tyrosine kinase-activated protein kinase C signaling pathway in microglia."J. Naurochem.. 72. 1179-1186 (1999)

Nakai, M.：“β 淀粉样蛋白 (25-35) 通过小胶质细胞中酪氨酸激酶激活的蛋白激酶 C 信号通路磷酸化 MARKS。J. Naurochem.. 72. 1179-1186 (1999)

J.Liu, K.Fukunaga, H.Yamamoto, K.Nishi and E.Miyamoto: "Differential roles of Ca^<2+>/calmodulin-dependent protein kinase II and mitogen-activated protein kinase activation in hippocampal long-term potentiation."J.Neurosci.. 19. 8292-8299 (1999)

J.Liu、K.Fukunaga、H.Yamamoto、K.Nishi 和 E.Miyamoto：“Ca^2/钙调蛋白依赖性蛋白激酶 II 和丝裂原激活蛋白激酶激活在海马长时程增强中的不同作用。

Y.Nishiyama, T.Hirota, T.Morisaki, T.Hara, T.Marumoto, S.Iida, K.Makino, H.Yamamoto, T.Hiraoka, N.Kitamura and H.Saya: "A human homolog of Drosophila warts tumor suppressor, h-warts, localized to mitotic apparatus and specifically phosphorylated during mi

Y.Nishiyama、T.Hirota、T.Morisaki、T.Hara、T.Marumoto、S.Iida、K.Makino、H.Yamamoto、T.Hiraoka、N.Kitamura 和 H.Saya：“果蝇的人类同源物

Liu,J.: "Differential roles of Ca^<2+>/calmodulin-dependent protein kinase II and mitogen-activated protein kinase activation in hippocampal long-term potentiation."J. Neurosci.. 19. 8292-8299 (1999)

Liu，J.：“Ca ^2 /钙调蛋白依赖性蛋白激酶II和丝裂原激活蛋白激酶激活在海马长时程增强中的不同作用。”J。