Fig. 3. Molecular identification of pp145 as ß-subunit of c-Met protein. (A) Immunopurification of pp145. Whole cell extracts were prepared from carcinoma 5637 cells cultured in serum-starved conditions for 24 hours. The extracts (–FCS, 300 µg/lane) were subjected to immunoprecipitation with anti-phosphotyrosine antibody and the immunoprecipitates were analyzed by silver staining. A control immunoprecipitate prepared from the normally grown cell extracts (+ FCS, 300 µg/lane) was also analyzed. The position of a 145 kDa protein (pp145) is indicated. (B) MS identification of four peptide fragments annotated to be part of c-Met. Mass values of four peptide fragments were obtained from the 145 kDa tyrosine-phosphorylated protein digested with trypsin (see Materials and Methods for detail) and that were annotated to be part of the human p145^met using the MASCOT database search algorithm. Also shown are amino acid numbers (start-end) and expected (expt) as well as calculated mass values (calc) for each peptide fragment. Note that a peptide fragment annotated to be the amino acid residues 988-1004 contained mass value equivalent to one phosphate. (C) Schematic structure of p145^met. The whole amino acid sequence (1390 amino acids) of the human p145^met is shown. The amino acid sequences matching those of the known partial sequences by mass spectrometry analysis (see panel B) are shown in red. Also shown are the N-terminal signal sequence (residues 1-24, underlined), a C-terminal end of potential proteolytic cleavage site (residues 303-307, arrowhead), transmembrane region (residues 933-955, dashed line), catalytic domain (residues 1078-1345, squared), and tyrosine phosphorylation sites (residues 1003, 1230, 1234, 1235, 1313, 1349 and 1365, underlined in bold).