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Identification of Tyr438 as the major in vitro c‐Src phosphorylation site in human gelsolin: A mass spectrometric approach
Author(s) -
Corte Veerle De,
Demol Hans,
Goethals Mark,
Damme Jozef Van,
Gettemans Jan,
Vandekerckhove Joël
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.1.234
Subject(s) - gelsolin , phosphorylation , immunoprecipitation , tyrosine phosphorylation , chemistry , actin , kinase , biochemistry , dephosphorylation , tyrosine , microbiology and biotechnology , phosphatidylinositol , biology , phosphatase , gene
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‐actin 2 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.