DEP-1 Y1311 and Y1320 are major phosphorylation sites that associate with Src via its SH2 domain. (A) Diagram representing the potential SFK tyrosine phosphorylation sites in the intracellular domain of DEP-1 according to published consensus sequences or Scansite predictions (http://scansite.mit.edu/). FN indicates fibronectin-type III-like repeats; EC, extracellular; TM, transmembrane; and IC, intracellular. (B) HEK 293 cells were transfected with “nonmutated” (−) Myr-DEP-1 D/A or the various Y/F mutants. Their tyrosine phosphorylation was detected by immunoblotting (IB) total cell lysates (TCL) with the PY99 antibody (PY). Expression levels of transfected Myr-DEP-1 were detected with a Myc antibody. (C) HEK 293 cells were transfected with Myr-DEP-1 mutated at both Y1311 and Y1320, which is not detectably tyrosine phosphorylated. (D) DEP-1 associates with the Src SH2 domain via its phosphorylated Y1311 and Y1320 residues. Lysates of HEK 293 cells transfected with the indicated constructs were incubated with GST-Src-SH2, GST-Src-SH3, or with GST alone. Association of DEP-1 with Src domains was detected by immunoblotting with the MYC antibody. The equivalent amount of GST fusion proteins used in the pull-down assay is detected with a GST antibody. (Bottom panel) Lysates of transfected HEK 293 cells were immunoblotted with the MYC antibody to show the similar expression level of DEP-1 constructs. (E) In vitro association of recombinant Src with the purified intracellular domain of DEP-1 (DEP-1_IC) previously phosphorylated by Src in vitro. DEP-1_IC was immunoprecipitated with DEP-1 goat antibodies and associated Src was immunodetected using the 36D10 clone. (F) The association of DEP-1 C/S and DEP-1 C/S Y/F mutants to Src was investigated in HEK 293T cells cotransfected with constitutively active Src, to induce maximal tyrosine phosphorylation of DEP-1. The coprecipitation of Src with DEP-1 and the equal expression of Src Y527F were detected by immunoblotting with a Src antibody (GD11 clone). (G) Mutation in the Src SH2 domain abrogates its ability to associate with DEP-1. HEK 293T cells were cotransfected with DEP-1 C/S and activated Src Y527F or the Src Y527F/R175L SH2 domain mutant. Src was immunoprecipitated with the mouse GD11 mouse clone, and associated DEP-1 was detected with the goat antibody. Immunoblotting of total cell lysates with the DEP-1 goat antibody or the Src GD11 antibody reveal that equal amounts of Src and DEP-1 constructs were expressed. All results are representative of at least 3 independent experiments.