PLD2 interacts via its PX domain with Syk in vitro, an interaction that is dependent on Syk tyrosine 317. (A) Following the incubation of recombinant hexahistidine (His6)–PLD2 with recombinant Syk, Syk was immunoprecipitated with anti-Syk antibody, and immunoblots were prepared for detection of His6-PLD2 and Syk as shown. (B) Structures of the individual fragments as well as the PX and PH domains of human PLD2 that were prepared as GST fusion proteins for studies shown in panels C-F. Numbers indicate the terminal amino acid of each nonoverlapping fragment (F) as described in “Materials and methods.” The initial and terminal amino acids of the PH and PX are also indicated (C-F). The GST fusion proteins were incubated with recombinant Syk (C-D), endogenous Syk (E), and the indicated mutants (tyrosine to phenylalanine) of myc-Syk (F). Lysates of RBL-2H3 cells and of cells made to overexpress the myc-Syk mutants were used as sources of endogenous Syk and mutated myc-Syk, respectively (see “Materials and methods”). Immunoblots of the protein precipitates were probed with antibodies against Syk, Myc, and GST as shown. (G) Cells were made to overexpress wild-type or the Y317F mutant of myc-Syk along with HA-PLD2. Cells were stimulated or not with antigen (Ag). HA-PLD2 was immunoprecipitated with anti-HA antibody, and HA-PLD2 and coimmunoprecipitated myc-Syk were detected by immunoblotting and use of antimyc and HA antibodies.
