Ordered multisite Tyr phosphorylation is required for full Draper activation. (A) Engulfment of 10% PS beads by S2 cells expressing Draper-GFP, Draper-Ala11-GFP, or a GFP control was assessed after a 45-min incubation. The number of beads ingested per cell for three biological replicates is shown (mean ± SD). (B) Alignment of residues mutated to Phe in Draper-ΔITAM. When a BG505-labeled reporter protein comes into close proximity to a rhodamine-labeled liposome, the 505-nm fluorescence signal is quenched. If quenching occurs after addition of ATP, it is most likely due to the Src42a-dependent phosphorylation of Draper’s cytoplasmic Tyr residues and effector recruitment. The results shown in this panel reveal that direct binding of BG505-Shark-tSH2 to Draper’s tail is dependent on an intact ITAM on Draper. His₁₀-Draper cytosolic domain or His₁₀-DraperY934F/Y949F (ΔITAM) receptor tail was ligated to 0.5% rhodamine-PE liposomes, and ATP-dependent quenching was assessed. (C) Draper-ΔITAM displays a kinetic delay and reduced phagocytic proficiency relative to Draper-GFP, while Draper’s ITAM (ITAM only) is sufficient for partial engulfment activity. S2 cells transfected with Draper-GFP or the indicated mutants were incubated with 10% PS beads, allowed to settle, and imaged at the indicated time points. The number of beads fully ingested per cell for three independent biological replicates is shown (mean ± SD). (D) On-liposome phosphorylation reactions to assess Draper activation by its cognate Src family kinase Src42a. His₁₀-Draper cytoplasmic domain or 10×his-DraperY934F/Y949F (ΔITAM) receptor tail (both at 1 µM) were ligated to liposomes doped with DGS-Ni-NTA and incubated with 86 nM Src42a and 1 mM ATP for 30 min. Reactions were quenched with SDS-PAGE buffer containing DTT and 2-Me and boiled for 10 min at 95°C. Samples were immunoblotted with pTyr antibody or His₁₀ antibody. Quantification of phosphorylation of WT or ΔITAM by Src42a was determined as the ratio of total pTyr signal over total His₁₀ signal internally for each lane. Samples were visualized using Li-Cor infrared (IR) imaging. (E) 1 µM His₁₀-Draper cytoplasmic domain was phosphorylated using 1 nM on-membrane Src42a and samples quenched using 10 mM EDTA and 8 M urea. Reactions were digested for 2D mass spectrometry at 30-s and 60-s time points (see Materials and methods). 2D mass spectrometry peptide counts for each Tyr residue on Draper’s cytoplasmic tail are shown. pTyr and total peptide counts for each time point are shown (30 s in blue and 60 s in red). pTyr in the right column indicates the ratio of (pTyr peptides at 60 s/all peptides) relative to the (pTyr peptides at 30 s/all peptides). A ratio above 1.0 indicates preferential phosphorylation at the later 60-s time point. “Not covered” indicates no peptides were detected for a residue in 2D mass spectrometry. “No pTyr” indicates that no peptides with the +79.9663 D shift characteristic of phosphorylation were detected. Asterisks indicate Tyr residues that compose the Draper ITAM.