SRC binds two nonoverlapping regions of DLC1 and phosphorylates two tyrosines in DLC1. (A) Schematic representation of DLC1 domains, DLC1 mutants, and DLC1 fragments used in this study. All constructs were GFP-tagged. (B) SRC binds to DLC1. N-terminal and C-terminal fragments. Lysates from HEK 293T cells transfected with indicated DLC1 constructs were IP with SRC antibody followed by IB with GFP (top) and SRC (middle) antibodies. Expression of DLC1 constructs (bottom). (C–E) Experimental conditions were similar to B. (C) SRC binds to DLC1 amino acids 80–200, but not 1–110 and 400–500 N-terminal DLC1 fragments. (D and E) SRC binds to the Rho-GAP domain (amino acids 800–900) of DLC1, but not the C-terminal fragment (amino acids 850–1091). (F) Saracatinib reduces tyrosine phosphorylation of DLC1 (pTyrosine) without reducing total DLC1. Lysates from H1703 and H157 lines treated without or with saracatinib were IP with DLC1 antibodies followed by IB with pTyrosine (top) or DLC1 (bottom) antibodies. Whole cell extract (WCE) input is the same, and it is shown in Fig. 1. (G) Phosphorylation of DLC1 S129, Y451, and Y701 in cells. DLC1 phosphopeptides from extracts of HEK 293T cells transfected with SRC were detected by mass spectrometry. The phosphorylation signals were fully localized to the indicated serine and tyrosines in phosphopeptides. GI, GenInfo identifier number. (H) Recombinant SRC strongly phosphorylates DLC1-WT, from transfected HEK 293T cells, in vitro, while single mutants (DLC1-Y451F or DLC1-Y701F) were weakly phosphorylated, and phosphorylation of the combined DLC1-2F mutant (DLC1-Y451F,Y701F) was barely detectable. (I) Top: Two other SFKs, FYN and YES, phosphorylate DLC1, but less efficiently than SRC. Bottom: Expression of DLC1 constructs.