Tyr572, Tyr591, and Tyr919 residues of FLT3 were responsible for phosphorylation-dependent binding to Lnk. (A) Diagram of the structure and localization of indicated tyrosines of FLT3 protein. (B) Beads with indicated immobilized peptides were incubated with cell lysates from Lnk-transfected COS-1 cells. After washing, the bound proteins were processed for Western blotting using anti-V5 Ab. (C) Immobilized phosphopeptides and their corresponding nonphosphorylated peptides were used to pull-down Lnk proteins and then were processed as above. (D-E) Immobilized phosphorylated and nonphosphorylated peptides, as above, were used to pull-down recombinant GST-fusion Lnk-domains, which were then processed for Western blotting using an anti-GST Ab. (F-G) Expression and phosphorylation of GST-fusion proteins of various domains of FLT3 in TKX1 E coli cells and the GST-fusion proteins were subsequently used to pull-down of cellular lysates from 293T cells overexpressing Lnk. GST indicates the GST control protein without fusion. DH5α indicates nonphosphorylated GST-fusion proteins expressed in DH5α E coli cells. (H) Pull-down of cellular lysates from 293T cells overexpressing either Lnk or the RE mutant with GST-FLT3-Kin2 proteins expressed and phosphorylated from TKX1 E coli cells. Input indicates that equal amounts of Lnk and RE mutant were used. (I) Pull-down of cellular lysates from 293T cells overexpressing Lnk with either wild-type or Y919F-mutated GST-FLT3-Kin2 proteins expressed and phosphorylated from TKX1 E coli cells. After pull-down, beads containing protein complex were processed for Western blotting using the indicated Abs: anti-phosphotyrosine (P-Ty), anti-GST, and anti-V5. Asterisk indicates nonspecific products. (J) COS-1 cells were cotransfected with Lnk and the FLT3-WT, FLT3-Y572/Y919F, or FLT3-Y591F/Y919F expression vectors. Cells were serum starved and stimulated with FL for 5 minutes. Lysates were immunoprecipitated with anti-FLT3 Abs and analyzed by Western blotting.
