Lnk SH2 domain associates with the juxtamembrane domain of Kit receptor. (A) Schematic representation of GST-Lnk fusion proteins. *R364M mutation. (B) BaF3 cells expressing WT Kit receptor were unstimulated (−) or stimulated (+) with SCF (100 ng/mL) for 1 minute and incubated with GST alone or different GST-fused Lnk domains. Immunoblotting with anti-pTyr and anti-Kit antibodies allowed detection of bound activated Kit protein. TCL indicates total cell lysate. (C) 13-mer peptides containing either phosphorylated (pY) or nonphosphorylated (Y) tyrosine residues from Kit intracellular domain were synthesized on a peptide spot array. The resulting membrane was incubated with either GST-Lnk SH2 protein (0.5 μM) or GST alone as a control. Positive spots with GST are circled in black. Immunoblotting with anti-GST antibodies identified the interacting peptides. JM indicates juxtamembrane; KI, kinase insert; TK2, distal kinase domain; and C-tail, carboxyl-tail. Numbers indicate tyrosine position in Kit. (D) BMMC lysates were incubated with 2 nmol peptides containing nonphosphorylated (Y567Y569) and either one (pY567 or pY569) or both tyrosines phosphorylated (pY567pY569). Lnk binding was revealed with anti-Lnk antibodies. Lysate is used as a control. (E) Lysates from BaF3 cells expressing either WT or mutant Kit Y567F or Y569F receptors were prepared as in panel B and incubated with GST-Lnk SH2 fusion protein (top panel). Associated Kit was subsequently detected by immunoblotting with anti-pTyr antibodies. Immunoprecipitation (IP) with anti-Kit antibodies showed phosphorylation and expression of Kit proteins (bottom panels).