Establishment and Analysis of mice disrupted with DNAM-1 gene

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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.