Figure 3.  Fyn binds and phosphorylates WASp after TCR engagement. (A) Lymphocytes from Itk−/−, Fyn−/−, and wild-type mice as well as JCam-1 (Lck-deficient) and Lck-reconstituted JCam-1 (JCam + Lck) cells were stimulated with anti-CD3/anti-CD28 antibodies and cell lysates were then prepared and immunoprecipitated with anti-WASp antibody. Complexes were subjected to SDS-PAGE and sequential immunoblotting analysis with anti-pTyr and anti-WASp antibodies. (B) Lysates were prepared from anti-CD3/anti-CD28–stimulated Jurkat cell transfectants expressing cDNAs for wild-type Fyn and FynK296M alone or in combination. After immunoprecipitation with anti-WASp antibody, complexes were subjected to immunoblotting analysis using anti-pTyr and then anti-WASp antibodies (top two panels). Fyn expression levels in the transfected cells lysates are shown in the bottom panel. (C) Lysates were prepared from unstimulated (U) or stimulated (S) Jurkat cells cotransfected with Fyn, WASp, and one of either the cdc42 wild-type (WT), the cdc42-V12, or N17 mutant cDNAs. Lysate proteins were immunoprecipitated with anti-WASp antibody and subjected to SDS-PAGE and sequential immunoblotting with anti-pTyr and anti-WASp antibodies (top two panels). Expression of Fyn and cdc42 in the lysates is shown in the bottom two panels. (D) Jurkat cells were stimulated for the indicated time periods with anti-CD3 and anti-CD28 antibodies, lysed, and the lysate proteins were immunoprecipitated with anti-WASp antibody. Complexes, lysate proteins, and IgG were subjected to SDS-PAGE and sequential immunoblotting analysis with anti-Fyn and anti-WASp antibodies. (E) Purified His-tagged Fyn fusion protein was incubated with GST-WASp, GST-WASpΔPro, or GST fusion proteins bound to glutathione sepharose beads. The complexes were washed and subjected to SDS-PAGE and sequential immunoblotting with anti-Fyn and anti-GST antibodies. (F) Jurkat cells were stimulated, lysed, and the lysate proteins were subjected to anti-Fyn antibody immunoprecipitation. Immunoprecipitates were then incubated in kinase buffer containing 10 μg [γ-32P] ATP and either GST, GST-WASp, GST-WASpΔPro, or GST-WASp Y291F fusion proteins bound to glutathione sepharose beads. Complexes were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and phosphorylation was analyzed by autoradiography. The position of phosphorylated GST-WASp is indicated. This result is representative of four independent assays.