Figure 2.  TCR-induced phosphorylation of WASp Y291 is required for induction of NFAT activity, actin polymerization, and immunological synapse formation. (A) Lysates prepared from resting or TCR-stimulated Jurkat T cells at the indicated times after stimulation were immunoprecipitated with anti-WASp antibody or control IgG subjected to SDS-PAGE and sequential immunoblotting analysis with anti-pTyr and anti-WASp antibodies. (B) Lysates prepared from wild-type (WT) or WAS−/−ΔGBD thymocytes at the indicated times after stimulation were immunoprecipitated with anti-WASp antibody or control IgG and the complexes were then subjected to SDS-PAGE and sequential immunoblotting analysis with anti-pTyr and anti-WASp antibodies. (C) Jurkat cells were transfected with pEGFP vector containing either the wild-type (WT) WASp cDNA or WASp cDNAs carrying each of the indicated Y→F substitutions. Lysates prepared from the TCR-stimulated cells were immunoprecipitated with anti-GFP antibody and the immunoprecipitated proteins were then subjected to SDS-PAGE and sequential immunoblotting analysis with anti-pTyr and anti-GFP antibodies. (D) Thymocytes from WAS−/− mice were cotransfected with pEGFP vectors containing WASp or the indicated WASp tyrosine phenylalanine (Y→F) mutant cDNAs and an NFAT luciferase reporter vector. At 4 h after transfection, cells were stimulated with anti-CD3 and anti-CD28 antibodies. After 8 h of incubation, cells were lysed and assayed by luminometry for luciferase expression. Values represent the means (± SEM) of three assays and the results are representative of four independent experiments. (E) Thymocytes from WAS−/− mice were transfected with pEGFP-WASp, WASpY291F, WASpY212F, or WASpY88F and the cells were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies for 30 min on ice followed by cross-linking with anti–hamster Ig secondary antibody. Cells were fixed with 5% paraformaldehyde and F-actin content was quantified by flow cytometric analysis of FITC phalloidin–stained cells. The results are representative of three independent experiments. (F) Lymphocytes from WAS−/−/OT-II mice were either untreated (a) or transfected with pEGFP-WASp (b), pEGFP-WASpY102F (c), or pEGFP-WASpY291F (d) and the cells were then incubated with OVA329–339–pulsed LB27.4 cells, fixed, and stained for actin and PKC-θ, and then visualized by immunofluorescent microscopy. The images on the far right of each panel represent merges of the other three images within the panel. Data shown are representative of four independent experiments.