title: Integrin-dependent translocation of LASP-1 to the cytoskeleton of activated platelets correlates with LASP-1 phosphorylation at tyrosine 171 by Src-kinase.
abstract: 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 alpha(IIb)beta(3) 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 alpha(IIb)beta(3). 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.
authors.affiliation: Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, D-97080 Wuerzburg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Traenka
authors.forename: Christof R
authors.initials: CR
authors.lastname: Hauck
authors.forename: Urs
authors.initials: U
authors.lastname: Lewandrowski
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
authors.forename: Stepan
authors.initials: S
authors.lastname: Gambaryan
authors.forename: Petra
authors.initials: P
authors.lastname: Thalheimer
authors.forename: Elke
authors.initials: E
authors.lastname: Butt
mesh_terms: D000199:Actins / Q000737:chemistry; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D060588:LIM Domain Proteins; D010766:Phosphorylation; D010973:Platelet Adhesiveness; D021381:Protein Transport; D014443:Tyrosine / Q000737:chemistry*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D016023:Integrins; C096353:LASP1 protein, human; D060588:LIM Domain Proteins; D014443:Tyrosine; D019061:src-Family Kinases