PI 3-kinase and SHP-1 coprecipitate with mutant Kit.

(A) The C57 mouse mast cell line expressed WT (WT) Kit. C2 and BR lines were derived from spontaneous MCTs; the C2 cell line contains a 48-bp tandem duplication in the exon 11 (the JM domain) of Kit; the BR cell line contains a point mutation (Leu575Pro) in exon 11 of Kit. The P815 line has a point mutation in the catalytic domain (Asp816Tyr) of Kit. TM indicates transmembrane domain; TK1, tyrosine kinase domain 1; KI, kinase insert; and TK2, tyrosine kinase domain 2. (B) The above-described mast cell lines were unstained, stained with a phycoerythrin-labeled isotype-matched control antibody, or the ACK45 anti-KIT monoclonal antibody (PharMingen). Cells were analyzed by flow cytometry on a Becton Dickinson FACScan. Mutation in either the JM domain or the catalytic domain of Kit did not alter cell surface expression of the protein. (C) Cells were cultured in serum-free medium for 2 hours before treatment with SCF (+) at 100 ng/mL and immunoprecipitation with anti-Kit antibody. Western blots were performed for phosphotyrosine, PI 3-kinase, SHP-1, and Kit according to procedures outlined in “Materials and methods.”