Integrin-dependent translocation of LASP-1 to the cytoskeleton of activated platelets correlates with LASP-1 phosphorylation at tyrosine 171 by Src-kinase

Julia Traenka; Christof R Hauck; Urs Lewandrowski; Albert Sickmann; Stepan Gambaryan; Petra Thalheimer; Elke Butt

doi:10.1160/TH09-03-0143

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2009; 102(03): 520-528
DOI: 10.1160/TH09-03-0143

Platelets and Blood Cells

Schattauer GmbH

Integrin-dependent translocation of LASP-1 to the cytoskeleton of activated platelets correlates with LASP-1 phosphorylation at tyrosine 171 by Src-kinase

Julia Traenka

¹Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Wuerzburg, Germany

,

Christof R Hauck

²Institute of Cell Biology, University of Konstanz, Konstanz, Germany

,

Urs Lewandrowski

³Department of Bioanalytics, ISAS Institute for Analytical Sciences, Bunsen, Dortmund, Germany

,

Albert Sickmann

³Department of Bioanalytics, ISAS Institute for Analytical Sciences, Bunsen, Dortmund, Germany

,

Stepan Gambaryan

¹Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Wuerzburg, Germany

⁴Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Science, St. Petersburg, Russia

,

Petra Thalheimer

¹Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Wuerzburg, Germany

,

Elke Butt

¹Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Wuerzburg, Germany

› Author Affiliations

Abstract

Summary

During platelet adhesion, the complex cytoskeletal structure is rearranged resulting in the formation of F-actin-based filopodia and lamellipodia. Stimulatory platelet signalling pathways include binding of integrin α_IIbβ₃ to fibrinogen followed by activation of protein tyrosine kinases (PTK) and phosphorylation of downstream signalling proteins. In this study, we demonstrate that the scaffolding and F-actin binding protein LASP-1 undergoes tyrosine phosphorylation in thrombin-stimulated human platelets. By means of specific inhibitors we identified Src-kinase as the primary enzyme phosphorylating LASP-1 in intact cells. These data were confirmed in platelet model cells (A5-CHO cells), constitutively expressing integrin α_IIbβ₃. Fibrinogen-mediated cell stimulation resulted in a similar tyrosine phosphorylation of transiently transfected LASP-1. Site directed mutagenesis identified tyrosine 171 as the Src-kinase phosphorylation site. Immunofluorescence microscopic analysis of these cells revealed a relocation of LASP-1 to focal contacts and the leading edge of the membrane upon fibrinogen activation and tyrosine 171 phosphorylation. This translocation was also seen in adherent platelets. Concomitant with adhesion, LASP-1 translocated from the cytosol along the arms of the pseudopodia into the leading lamellae of the spreading platelets, indicating a crucial role of the protein in platelet cytoskeleton rearrangement.

Keywords

LASP-1 - Src-kinase - adhesion - tyrosine phosphorylation - platelets

Full Text

References

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