In addition to serving as a scaffold via auto-phosphorylation, ZAP-70 also phosphorylates a restricted set of substrates following TCR stimulation - including LAT and SLP-76. These substrates have been recognized to play pivotal role in TCR signaling by releasing second messengers. When phosphorylated, LAT and SLP-76 act as adaptor proteins which serve as nucleation points for the construction of a higher order signalosome: GADS, PLC-gamma1 and GRB2 bind to the LAT on the phosphorylated tyrosine residues (steps 8 and 13). SLP-76 and SOS are then moved to the signalosome by interacting with the SH3 domains of GRB2 and GADS via their proline rich sequences (step 9). Three SLP-76 acidic domain N-term tyrosine residues are phosphorylated by ZAP-70, once SLP-76 binds to GADS (step 10). These phospho-tyrosine residues act as binding sites to the SH2 domains of PLC-gamma1, Vav and Itk (steps 11 and 12).
PLC-gamma1 is activated by dual phosphorylation on the tyrosine residues at positions 771, 783 and 1254 by Itk and ZAP-70 (step 14). Phosphorylated PLC-gamma1 subsequently detaches from LAT and SLP-76 and translocates to the plasma membrane by binding to phosphatidylinositol-4,5-bisphosphate (PIP2) via its PH domain (step 15). PLC-gamma1 goes on to hydrolyse PIP2 to second messengers DAG and IP3. These second messengers are involved in PKC and NF-kB activation and calcium mobilization (step 16).