pY589 and pY599 are binding sites for Src family kinases and are involved in activation of Lyn. (A) The 32D cells were metabolically labeled with [³⁵S] methionine/[³⁵S] cysteine for 4 hours. Lysates were subjected to an affinity chromatography with the indicated immobilized peptides as described in “Affinity fishing of proteins with immobilized peptides.” Bound proteins were eluted, separated by SDS-PAGE, and detected by exposure on a PhosphorImager. The letters a, b, and c highlight proteins of the sizes 55, 52, and 45 kDa, respectively, showing phosphospecific interaction with pY589, whereas d, e, f, and g point to proteins of the sizes 70, 55, 52, and 45 kDa, respectively, that specifically interact with pY599. (B) Total 32D cell lysate was incubated with the following immobilized synthetic peptides: Y589 (lane A), pY589 (lanes B-D), Y599 (lane E), or pY599 (lanes F-H). To prove phosphoselectivity of found interactions, soluble phosphorylated or unphosphorylated peptides (0.5 mg/mL, Y589, lane C; pY589, lane D; Y599, lane G; pY599, lane H) were included in the reactions to compete for binding proteins. Bound proteins were eluted, separated on a 10% SDS-PAGE, immunoblotted, and probed for Lyn, Fgr, and Hck. Each lane was densitometrically evaluated and relative intensities (RIs) are depicted under each lane. Data of 1 of 3 performed experiments with consistent results are displayed. (C) The 32D cells were incubated in the presence or absence of FL, followed by lysis and pulldown (PD) with a GST fusion protein containing the SH2 domain of Src. Samples were separated by SDS-PAGE, electrotransferred to Immobilon P, and probed with an antibody against GST. In parallel, an aliquot of each cell lysate was immunoprecipitated with an antibody against Flt3, separated by SDS gel electrophoresis, electrotransferred to Immobilon P, and probed with an antibody against Flt3 to verify equal loading. (D) Lysates from 32D cells expressing either wild-type, Y589F, or Y599F mutant Flt3 were stimulated with FL for 10 minutes, lysed, and subjected to immunoprecipitation with an antibody against Flt3. After washing of immunoprecipitates, proteins were separated by SDS-PAGE, immunoblotted, and probed for Lyn, Fgr, or Hck, respectively. The filter was consecutively stripped and reprobed with an Flt3 antibody to ensure equal loading and a phosphotyrosine antibody to ensure equal ligand stimulation. (E) WT-Flt3, Y589F-Flt3, and Y599F-Flt3-32D cells were starved followed by stimulation for 7 minutes with FL (100 ng/mL) and subsequent lysis. Lyn was immunoprecipitated from the cell lysates and subjected to an in vitro kinase assay with enolase as exogenous substrate. Phosphorylated enolase was run out on a 10% gel, blotted, and visualized on a PhosphorImager as well as quantified densitometrically. Results are means of 3 independent experiments and depicted as fold Lyn activation upon ligand binding (▪) compared with unstimulated cells (□). Error bars indicate SEM of 3 different experiments. (F) Phosphorylation of Cbl is impaired in Y589F-Flt3 and Y599F-Flt3 32D cells. The 32D infectants were starved and stimulated with FL (100 ng/mL) for the indicated periods of time. After lysis, Cbl was immunoprecipitated, run on a gel, and probed against phosphotyrosine. To ensure equal protein loading, the membrane was stripped and reprobed for Cbl.