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.

钙调素依赖的蛋白激酶II(CaM Kinase II)分布广泛，可能参与钙离子介导的多种细胞过程。真核生物中有四个亚基，称为α，β，γ，δ，由不同的基因编码。所有亚基在N-末端催化结构域和包括钙调蛋白结合和自身抑制位点的调节域中具有高度的同源性。这些亚基的各种异构体以不同的剪接变体存在。异构体主要在调节结构域(可变结构域)的末端不同。在这项研究中，我们用免疫化学和分子生物学技术鉴定了脑和各种培养细胞中的异构体。此外，还检测了它们在这些细胞中的生理作用。1.制备了抗δ亚基的抗体，发现δ-3在小脑颗粒细胞胞核内大量表达。我们在NG108-15细胞中瞬时过表达核异构体αB和δ3，发现这些异构体参与脑源性神经营养因子的表达。2.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。在已鉴定的亚型中，最丰富的亚型是δ-2。免疫组织化学染色显示，δ-2主要定位于两种细胞的高尔基体。3.研究了小鼠胰岛素瘤MIN6细胞中β‘e和δ2亚型的表达，发现δ’e和β‘2亚型与胰岛素分泌颗粒共存，证实了δ’e和突触素2的过表达促进了突触素I的磷酸化和胰岛素的分泌。这些结果表明，每种亚型都定位于细胞中特定的位置，在细胞内钙离子和2+-GT信号的偶联中起重要作用。

项目成果

Y. Takeuchi, H. Yamamoto, T. Miyakawa and E. Miyamoto: "Increase of brain-derived neurotrophic factor gene expression in NG108-15 cells by the nuclear isoforms of Ca^<2+>/calmodulin-dependent protein kinase II"J. Neurochem.. 74. 1913-1922 (2000)

Y. Takeuchi、H. Yamamoto、T. Miyakawa 和 E. Miyamoto：“Ca^2 >/钙调蛋白依赖性蛋白激酶 II 的核亚型增加 NG108-15 细胞中脑源性神经营养因子基因表达”J

S. Ohmori, N. Sakai, Y. Shirai, H. Yamamoto, E. Miyamoto, N. Shimizu and N. Saito: "Importance of protein kinase C targeting for the phosphorylation of its substrate, myristoylated alanine-rich C-kinase substrate"J. Biol. Chem.. 275. 26449-26457 (2000)

S. Ohmori、N. Sakai、Y. Shirai、H. Yamamoto、E. Miyamoto、N. Shimizu 和 N. Saito：“蛋白激酶 C 靶向对其底物（富含肉豆蔻酰化丙氨酸的 C 激酶底物）磷酸化的重要性

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)

Y. Takeuchi: "Nuclear localization of the δ subunit of Ca^<2+>/calmodulin-dependent protein kinase II in rat cerebellar granule cells"J. Neurochem.. 72. 815-825 (1999)

Y. Takeuchi：“大鼠小脑颗粒细胞中 Ca^2+/钙调蛋白依赖性蛋白激酶 II 的 δ 亚基的核定位”J. Neurochem.. 72. 815-825 (1999)