Gelsolin is an actin-binding protein (82 kDa) consisting
of six repeated segments (S1–S6), each approximately
120 residues long. It interacts with phospholipids and
we previously showed that phosphatidylinositol 4,5-bisphosphate
promotes phosphorylation of gelsolin by the tyrosine kinase
c-Src. We used a combination of different methods, such
as thin-layer chromatography and anti-phosphotyrosine-agarose
immunoprecipitation of phosphopeptides combined with matrix
assisted laser desorption ionization-mass spectrometry
(MALDI-MS) and post source decay (PSD) analysis, to identify
the phosphorylation sites in gelsolin. The major phosphorylation
site (Tyr438) was located in subdomain 4 (S4). Phosphorylation
of gelsolin in the gelsolin-actin2 complex was
inhibited by 90%. Gelsolin phosphorylation by c-Src in
the presence of lysophosphatidic acid also revealed Tyr438
as the most prominent site. Additional minor sites were
found using the anti-phosphotyrosine bead immunoprecipitation
method followed by MALDI-MS and PSD analysis. These sites,
representing ∼5% of the total phosphate incorporation,
were identified as Tyr59, Tyr382, Tyr576, and Tyr624. Based
on these results we generated antibodies which specifically
recognize Tyr438 phosphorylated gelsolin.