Figure 1.

Tyrosine 1196 of SOS1 is the major target residue of ABL kinase. (a) SOS1 immuno-purified from SOS1-expressing 293T cells (1 mg lysates) with an anti-SOS1 antibody was in vitro phosphorylated with ABL kinase, resolved on SDS-Page and detected by Coomassie staining or by immunoblotting (IB) using the indicated antibody. SOS1 band was digested with trypsin. Phosphorylated peptides were enriched with titanium oxide beads and analyzed by tandem mass spectrometry. Left: the diagram shows the detected fragments of the phosphorylated pYSISDR peptide. Fragments are presented according to their mass/charge ratio (m/z) and their intensity (relative abundance). The intensity of the highest peak corresponds to a relative abundance of 100%. Right: The possible fragmentation of the peptide is shown. The peak (pY) in the diagram (left) with an m/z of 216,07 corresponds to the a₁ fragment on the right. Mw markers are indicated on the side of the immunoblots. (b) WT-SOS1 and Y1196F-SOS1 immunopurified from SOS1-overexpressing 293T cells (1 mg lysate) were incubated in the presence or absence (Control) of ABL kinase and ³²P-ATP and subjected to SDS-polyacrylamide gel electrophoresis. After PonceauS staining, the SOS1 band was excised and digested with trypsin. Oxidized peptides were separated by electrophoresis and thin layer chromatography.