The kinase, SH3, and SH2 domains of Lck play critical roles in T-cell activation after ZAP-70 membrane localization

Antigenic stimulation of the T-cell antigen receptor initiates signal transduction through the immunoreceptor tyrosine-based activation motifs (ITAMs). When its two tyrosines are phosphorylated, ITAM forms a binding site for ZAP-70, one of the cytoplasmic protein tyrosine kinases essential for T- cell activation. The signaling process that follows ZAP-70 binding to ITAM has been analyzed by the construction of fusion proteins that localize ZAP- 70 to the plasma membrane. We found that membrane-localized forms of ZAP-70 induce late signaling events such as activation of nuclear factor of activated T cells without any stimulation. This activity was observed only when Lck was expressed and functional. In addition, each mutation that affects the function of Lck in the kinase, Src homology 2 (SH2), and SH3 domains greatly impaired the signaling ability of the chimeric protein. Therefore, Lck functions in multiple manners in T-cell activation for the steps following ZAP-70 binding to ITAM.