The cell surface molecule 2B4 recruits the

XLP gene product SAP after phosphorylation of the tyrosine residues in its cytoplasmic tail. (A) Comparison of the interactions between 2B4/SAP and SLAM/SAP in yeast cells. To detect binding between the cytoplasmic tail of 2B4 and SAP, a yeast 2-hybrid system was adapted to measure interactions with phospho-proteins. To this end, mutations of c-fyn were cotransfected with SAP into the yeast cell. Binding between 2 proteins in yeast cell extracts was detected by a β-galactosidase assay, as described in “Materials and methods.” For each construct, at least 3 independent colonies were tested in the β-galactosidase assay. Open bars: cells transfected with empty pBRIDGE vector and pGAD424 encoding the cytoplasmic tail of 2B4 or SLAM; solid bars: cells transfected with pBRIDGE-SAP and pGAD424 encoding the cytoplasmic tail of 2B4 or SLAM; hatched bars: cells transfected with pBRIDGE-SAP and Fyn_{420, 531 Y-F} and with pGAD424 encoding the cytoplasmic tail of 2B4 or SLAM; dotted bars: cells transfected with pBRIDGE-SAP and Fyn _{420, 531 Y-F, 176 R-Q} and with pGAD424 encoding the cytoplasmic tail of 2B4 or SLAM. Control, empty pGAD424 with pBRIDGE encoding the indicated DNA sequences. (B) SAP binds to phosphorylated 2B4 in NK cell line. Interactions between SAP and 2B4 in the YT cell line were studied by immunoprecipitation of 2B4 followed by Western blot analysis with anti-SAP. YT cells (50 × 10⁶ cells/mL) were biotinylated and then incubated in the presence or absence of 1 mM pervanadate. Cells were lysed in detergent, and 2B4 was immunoprecipitated with the 2B4 specific monoclonal antibody C1.7 (α-2B4). After SDS-PAGE, proteins were transferred to a PVDF membrane and were identified with streptavidin, antiphosphotyrosine (α-PY), or antihuman SAP 10C4.2 (α-SAP). CONTROL, immunoprecipitation with an irrelevant monoclonal antibody. (C) SAP binds to nonphosphorylated and phosphorylated SLAM in a T-cell line. Interactions between SAP and SLAM were studied in the T-cell transfectant cell line EL-4/SLAM4 by immunoprecipitation of SLAM, followed by Western blot analysis with anti-SAP. EL-4/SLAM4 cells (20 × 10⁶ cells/mL) expressing human SLAM were treated with pervanadate, as described in panel B, and SLAM was immunoprecipitated by an antihuman SLAM monoclonal antibody (α-SLAM).2 After SDS-PAGE, proteins were transferred to a PVDF membrane and identified by Western blot analysis with a rabbit antihuman SLAM antibody, antiphosphotyrosine (α-PY), or antihuman SAP (α-SAP) 10C4.2 CONTROL, immunoprecipitation with an irrelevant monoclonal antibody.