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 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.
De Corte
V
V
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ghent, Belgium.
Demol
H
H
Goethals
M
M
Van Damme
J
J
Gettemans
J
J
Vandekerckhove
J
J
D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D019281:Dimerization; D018260:Gelsolin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008246:Lysophospholipids / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000378:metabolism*
D000199:Actins; D018260:Gelsolin; D008246:Lysophospholipids; D019269:Phosphatidylinositol 4,5-Diphosphate; D014443:Tyrosine; D016392:Proto-Oncogene Proteins pp60(c-src)