Identification of the phosphorylation site of WASP by Btk. (A) A schematic representation of the structure of WASP and its functional domains (pleckstrin homology domain [PH], the GTPase binding domain [GBD], and proline rich regions [Poly-Pro]) and the distribution of the tyrosine residues. The positions and amino acid numbers of tyrosine residues are indicated (arrows). (B) The flanking amino acid sequences of each tyrosine residue in WASP and of tyrosine 223 (the autophosphorylation site) of Btk are listed. (C) Coexpression of Btk and mutant WASP constructs. Cotransfection of pEF-BOS/wild-type Btk with pcDNA3/wild-type WASP (lane 1), pcDNA3/Y51F (lane 2), pcDNA3/Y83F (lane 3), pcDNA3/Y88F (lane 4), pcDNA3/Y102F (lane 5), pcDNA3/Y107F (lane 6), pcDNA3/Y212F (lane 7), or pcDNA3/Y291F mutant WASP (lane 8) was performed with lipofectamine. Cells were harvested after 48 hours and lysed with Triton X-100 lysis buffer. After preclearance, lysates were immunoprecipitated with the anti-WASP antibody 503 and immunoblotted with the antiphosphotyrosine (APT) antibody 4G10 (top). The same membrane was reprobed with the anti-WASP antibody 503 (middle). To detect the expression of Btk proteins, the total cell lysates (TCL) were loaded and immunoblotted with the anti-Btk antibody 43-3B (bottom). (D) Association of wild-type or Y291F mutant WASP with Btk. Transfection of pEF-BOS/wild-type Btk as a control (lane 1) or cotransfection of pEF-BOS/wild-type Btk with T7 epitope-tagged WASP (lane 2) or T7 epitope-tagged Y291F (lane 3) into 293T cells was performed to transiently express the proteins. Cells were harvested after 48 hours and lysed with digitonin lysis buffer. After preclearance, lysates were immunoprecipitated with the anti-T7 tag antibody and immunoblotted with the anti-Btk antibody 43-3B (top), followed by reprobing with the anti-WASP antibody 503 (middle). To detect the expression of Btk proteins, the total cell lysates (TCL) were also loaded and immunoblotted with the anti-Btk antibody 43-3B (bottom).