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+/钙调素依赖性蛋白激酶II（CaM kinase II）广泛分布，可能参与多种Ca^2+介导的细胞过程。在真核生物中，α、β、γ、δ四个亚基由不同的基因编码。所有亚基在N-末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。这些亚基的各种同种型作为不同的剪接变体存在。亚型的差异主要在调节结构域（可变结构域）的末端。在这项研究中，我们确定了异构体在脑和各种培养细胞的免疫化学和分子生物学技术。此外，还研究了它们在这些细胞中的生理作用。1.我们制备了δ亚基的特异性抗体，发现δ3在小脑颗粒细胞的核中含量丰富。我们在NG 108 -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NG 108 -15细胞中的所有亚型。在鉴定的同种型中，最丰富的同种型是δ2。免疫组化结果表明，δ2主要定位于两种细胞的高尔基体。3.当我们检测小鼠胰岛素瘤MIN 6细胞中的同种型时，在mRNA和蛋白质水平上丰富的同种型是β 2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的过表达增强了突触蛋白I的磷酸化和胰岛素分泌。这些结果表明，每个异构体都定位于细胞中的特定位点，并在Ca^2+信号与特定细胞反应的偶联中起关键作用。

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)