Establishment and Analysis of mice disrupted with DNAM-1 gene

DNAM-1基因破坏小鼠的建立及分析

• 批准号: 10833002
• 负责人: SHIBUYA Akira
• 金额: $ 1.86万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10833002/
• 关键词: DNAM-1; Adhesion Molecules; Lymphocyte; 細胞障害性リンパ球

DNAM-1 is a signal transducing adhesion molecule involved in the cytolytic function mediated by CTL and NK cells. Cross-linking DNAM-1 with anti-DNAM-1 mAb induces cytolysis mediated by CTL and NK cells and also results in tyrosine phosphorylation of the DNAM-1 molecule. We studied regulation of DNAM-1-mediated signaling and adhesion. We report here that specific inhibitors of PKC activity prevented DNAM-1-mediated cytolytic activation of NK cells. Adhesion of DNAM-1 to its ligand does not require divalent cations, such as magnesium or calcium, and is regulated by PKC, as demonstrated by augmentation of DNAM-1 adhesion by PMA and inhibition by specific PKC inhibitors. Mutation of the putative PKC binding site in the cytoplasmic domain of DNAM-1 (SerィイD1329ィエD1 to PheィイD1329ィエD1) prevents ligand binding and PMA-induced serine phosphorylation of the DNAM-1 receptor. These results indicate that PKC phosphorylates SerィイD1329ィエD1 of DNAM-1 and plays a critical role for both DNAM-1 adhesion and signaling.Whereas ligation of DNAM-1 adhesion molecule triggers cytotoxicity mediated by normal NK and T cells, this function was defective in NK cell clones from leukocyte adhesion deficiency syndrome. However, genetic reconstitution of cell surface expression of LFA-1 restored the ability of DNAM-1 to initiate anti-DNAM-1 mAb induced cytotoxicity, indicating a functional relationship between DNAM-1 and LFA-1. Further studies demonstrated that LFA-1 physically associates with DNAM-1 in NK cells and anti-CD3 mAb stimulated T cells, for which serine phosphorylation of DNAM-1 plays a critical role. In addition, cross-linking of LFA-1 induces tyrosine phosphorylation of DNAM-1, for which the fyn protein tyrosine kinase is responsible. These results indicate that DNAM-1 is involved in the LFA-1-mediated intracellular signals.

DNAM-1 是一种信号转导粘附分子，参与 CTL 和 NK 细胞介导的溶细胞功能。 DNAM-1 与抗 DNAM-1 mAb 交联可诱导 CTL 和 NK 细胞介导的细胞溶解作用，还导致 DNAM-1 分子的酪氨酸磷酸化。我们研究了 DNAM-1 介导的信号传导和粘附的调节。我们在此报告，PKC 活性的特异性抑制剂可阻止 DNAM-1 介导的 NK 细胞的溶细胞激活。 DNAM-1 与其配体的粘附不需要二价阳离子（例如镁或钙），并且受 PKC 调节，PMA 增强 DNAM-1 粘附和特定 PKC 抑制剂抑制的作用证明了这一点。 DNAM-1 胞质结构域中推定的 PKC 结合位点（SeriiD1329ィエD1 变为 PheiiD1329ィエD1）的突变可防止配体结合和 PMA 诱导的 DNAM-1 受体丝氨酸磷酸化。这些结果表明，PKC 磷酸化 DNAM-1 的 SerィイD1329ィエD1，并对 DNAM-1 粘附和信号传导发挥关键作用。虽然 DNAM-1 粘附分子的连接会触发正常 NK 和 T 细胞介导的细胞毒性，但这种功能在白细胞粘附缺陷综合征的 NK 细胞克隆中存在缺陷。然而，LFA-1 细胞表面表达的基因重建恢复了 DNAM-1 启动抗 DNAM-1 mAb 诱导的细胞毒性的能力，表明 DNAM-1 和 LFA-1 之间存在功能关系。进一步的研究表明，LFA-1 与 NK 细胞和抗 CD3 mAb 刺激的 T 细胞中的 DNAM-1 物理结合，其中 DNAM-1 的丝氨酸磷酸化起着关键作用。此外，LFA-1 的交联会诱导 DNAM-1 的酪氨酸磷酸化，这是由 fyn 蛋白酪氨酸激酶负责的。这些结果表明 DNAM-1 参与 LFA-1 介导的细胞内信号。

项目成果

Shibuya, A, et al: "Protein kinase C is involved in the regulation of both signaling and adresion mediated DNAM-1"J. Immunology. 161. 1671-1676 (1998)

Shibuya, A, 等人：“蛋白激酶 C 参与信号传导和邻接介导的 DNAM-1 的调节”J.

Shibuya K., Lanier LL., Phillips JH., Ochs D., Shimizu K., Nakayama E., Nakauchi H., Shibuya A.: "Physical and functional association of LFA-1 with DNAM-1 adhesion molecule."Immunity. 11. 615-623 (1999)

Shibuya K.、Lanier LL.、Phillips JH.、Ochs D.、Shimizu K.、Nakayama E.、Nakauchi H.、Shibuya A.：“LFA-1 与 DNAM-1 粘附分子的物理和功能关联。”

Shibuya A., Lanier LL., Phillips JH.: "Protein kinase C is involved in the regulation of both signaling and adhesion mediated by DNAM-1 receptor."J. Immunol.. 161. 1671-1676 (1998)

Shibuya A.、Lanier LL.、Phillips JH.：“蛋白激酶 C 参与 DNAM-1 受体介导的信号传导和粘附的调节。”J.

Shibuya K.et al.: "Physical and functional association of LFA-1 with DNAM-1 adhesion molecule"Immunity. 11. 615-623 (1999)

Shibuya K.等人：“LFA-1 与 DNAM-1 粘附分子的物理和功能关联”免疫。

Shibuya A.et al.: "Protein-kinase C in involved in the regulation of both signaling and adhesion mediated DNAM-1"J. Immunology. 161. 1671-1676 (1998)

Shibuya A.et al.：“蛋白激酶 C 参与信号传导和粘附介导的 DNAM-1 的调节”J.