Identification of Fyn as the binding partner for the WASP N-terminal domain in T cells.

Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling. In transgenic (Tg) mice, over-expression of the WASP N-terminal region (exons 1-5) including the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain and anti-WASP-EVH1 single-chain variable fragment (scFv) intracellular expressed antibodies (intrabodies) impairs IL-2 production in activated T cells. However, it largely remains unknown that how this domain transduces TCR signaling. Here, we demonstrate for the first time that the WASP N-terminal domain specifically associates with the Fyn SH3 domain; the interaction was uncovered by screening a λgt11 cDNA expression library obtained from the mouse T-cell line KKF. The interaction between Fyn and WASP was inhibited by over-expression of the WASP N-terminal domain and anti-WASP-EVH1 scFv intrabodies in gene-transfected NIH3T3 cells and T cells derived from these Tg mice. WASP-interacting protein binding to the EVH1 domain of WASP was also inhibited in these Tg mice T cells. Furthermore, tyrosine phosphorylation of WASP and nuclear translocation of nuclear factor of activated T cells following TCR stimulation was severely inhibited by over-expression of the WASP N-terminal domain. These observations strongly suggest that the WASP N-terminal domain plays a pivotal role in the TCR signaling cascade by binding to Fyn.