Identification of Tyr438 as the major in vitro c-Src phosphorylation site in human gelsolin: A mass spectrometric approach

Gelsolin is an actin-binding protein (82 kDa) consisting of six repeated se gments (S1-S6), each approximately 120 residues long. It interacts with pho spholipids and we previously showed that phosphatidylinositol 4,5-bisphosph ate promotes phosphorylation of gelsolin by the tyrosine kinase c-Src. We u sed a combination of different methods, such as thin-layer chromatography a nd anti-phosphotyrosine-agarose immunoprecipitation of phosphopeptides comb ined with matrix assisted laser desorption ionization-mass spectrometry (MA LDI-MS) and post source decay (PSD) analysis, to identify the phosphorylati on sites in gelsolin. The major phosphorylation site (Tyr438) was located i n subdomain 4 (S4). Phosphorylation of gelsolin in the gelsolin-actin(2) co mplex was inhibited by 90%. Gelsolin phosphorylation by c-Src in the presen ce of lysophosphatidic acid also revealed Tyr438 as the most prominent sire . Additional minor sites were found using the anti-phosphotyrosine bead imm unoprecipitation method followed by MALDI-MS and PSD analysis. These sites, representing similar to 5% of the total phosphate incorporation, were iden tified as Tyr59, Tyr382, Tyr576, and Tyr624. Based on these results we gene rated antibodies which specifically recognize Tyr438 phosphorylated gelsoli n.