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是参与CTL和NK细胞介导的细胞溶解功能的信号转导粘附分子。DNAM-1与抗DNAM-1 mAb的交联诱导CTL和NK细胞介导的细胞溶解，并且还导致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。我们在这里报告，PKC活性的特异性抑制剂阻止DNAM-1介导的NK细胞的细胞溶解活化。DNAM-1与其配体的粘附不需要二价阳离子，如镁或钙，并且受PKC调节，如PMA增强DNAM-1粘附和特异性PKC抑制剂抑制所证明的。DNAM-1胞质结构域中推定的PKC结合位点突变（丝氨酸蛋白酶D1329丝氨酸蛋白酶D1突变为苯丙氨酸蛋白酶D1329丝氨酸蛋白酶D1）可阻止配体结合和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的丝氨酸蛋白酶D1329丝氨酸蛋白酶D1，并在DNAM-1粘附和信号传导中起关键作用，而连接DNAM-1粘附分子触发正常NK和T细胞介导的细胞毒性，这一功能在白细胞粘附缺陷综合征的NK细胞克隆中是缺陷的。然而，LFA-1的细胞表面表达的遗传重建恢复了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之间的功能关系。进一步的研究表明，在NK细胞和抗CD 3 mAb刺激的T细胞中，LFA-1与DNAM-1物理缔合，其中DNAM-1的丝氨酸磷酸化起关键作用。此外，LFA-1的交联诱导DNAM-1的酪氨酸磷酸化，fyn蛋白酪氨酸激酶对此负责。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.