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. 1999 Jan;8(1):234–241. doi: 10.1110/ps.8.1.234

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

V De Corte 1, H Demol 1, M Goethals 1, J Van Damme 1, J Gettemans 1, J Vandekerckhove 1
PMCID: PMC2144107  PMID: 10210201

Abstract

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 approximately 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.

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Selected References

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