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）被广泛分布，可能与各种Ca^<2+> - 介导的细胞过程有关。四个称为α，β，γ，δ的亚基由真核生物中的不同基因编码。所有亚基在N末端催化结构域以及完成钙调蛋白结合和自身抑制性位点的调节结构域中均具有较高的同源性。这些亚基的各种同工型作为不同的剪接变体存在。同工型主要在调节域（可变域）的末端不同。在这项研究中，我们鉴定了具有免疫化学和分子生物学技术的大脑和各种培养细胞中的同工型。此外，检查了它们在这些细胞中的身体作用。 1。我们制备了δ亚基的特异性抗体，发现小脑颗粒细胞的细胞核中δ3丰富。我们在NG108-15细胞中瞬时过表达了核同工型αB和δ3，发现这些同工型与脑衍生的神经营养因子的表达有关。 2。我们确定了培养的星形胶质细胞和NG108-15细胞中的所有同工型。在确定的同工型中，最丰富的同工型为Δ2。免疫染色表明，δ2主要在两个细胞的高尔基体中定位。 3。当我们检查小鼠胰岛素瘤MIN6细胞中的同工型时，在mRNA和蛋白质水平上，富集的同工型为β'e和Δ2。我们发现δ2和突触I与胰岛素分泌颗粒共定位。我们证实，β'e和δ2的过表达增强了突触素I和胰岛素分泌的磷酸化。这些结果表明，每个同工型都位于细胞中的特定位点，并且在CA^<2+>信号与特定细胞反应的偶联中起着至关重要的作用。

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

N.Inagaki, M.Nishizawa, N.Arimura, H.Yamamoto, Y.Takeuchi, E.Miyamoto, K.Kaibuchi and M.Inagaki: "Activation of Ca^<2+>/calmodulin-dependent protein kinase II within post-synaptic dendritic spines of cultured hippocampal neurons."J.Biol.Chem.. 275. 27165-

N.Inagaki、M.Nishizawa、N.Arimura、H.Yamamoto、Y.Takeuchi、E.Miyamoto、K.Kaibuchi 和 M.Inagaki：“Ca^<2>/钙调蛋白依赖性蛋白激酶 II 的激活