ZAP70/Syk kinase expression is required for CD2-dependent LAT phosphorylation.
Wild-type Jurkat cells and the ZAP70/Syk-deficient clone P116 (107 cells/test) were left unstimulated or stimulated with either T112+T113 or OKT3 for 5 minutes. PNLs were immunoprecipitated with rabbit anti-LAT antibody or incubated with protein A–agarose alone (data not shown), resolved by SDS-PAGE, and immunoblotted with the antiphosphotyrosine 4G10 mAb. Analysis of LAT tyrosine phosphorylation is shown (upper panel). Immunoprecipitation of comparable amounts of LAT protein was confirmed by stripping the membrane and reblotting with anti-LAT–specific antibody (lower panel). Note that the anti-LAT antibody recognizes the tyrosine phosphorylated form of the protein less efficiently. Results are representative of 4 independent experiments.
