Hostname: page-component-78c5997874-m6dg7 Total loading time: 0 Render date: 2024-11-03T00:21:37.151Z Has data issue: false hasContentIssue false

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

Published online by Cambridge University Press:  01 January 1999

VEERLE DE CORTE
Affiliation:
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganckstraat 35, B-9000 Ghent, Belgium
HANS DEMOL
Affiliation:
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganckstraat 35, B-9000 Ghent, Belgium
MARK GOETHALS
Affiliation:
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganckstraat 35, B-9000 Ghent, Belgium
JOZEF VAN DAMME
Affiliation:
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganckstraat 35, B-9000 Ghent, Belgium
JAN GETTEMANS
Affiliation:
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganckstraat 35, B-9000 Ghent, Belgium
JOËL VANDEKERCKHOVE
Affiliation:
Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganckstraat 35, B-9000 Ghent, Belgium
Get access

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 ∼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.

Type
Research Article
Copyright
© 1999 The Protein Society

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)