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, Inhaltsverzeichnis

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

› Institutsangaben

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

Volltext

Referenzen

References
1 Felsenfeld DP, Schwartzberg PL, Venegas A. et al. Selective regulation of integrin--cytoskeleton interactions by the tyrosine kinase Src. Nature cell Biol 1999; 01: 200-206.
2 Shattil SJ. Integrins and Src: dynamic duo of adhesion signalling. Trends Cell Biol 2005; 15: 399-403.
3 Vielreicher M, Harms G, Butt E. et al. Dynamic interaction between Src and C-terminal Src kinase in integrin alphaIIbbeta3-mediated signalling to the cytoskeleton. J Biol Chem 2007; 282: 33623-33631.
4 Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases. Ann Rev Cell Devel Biol 1997; 13: 513-609.
5 Bodnar RJ, Xi X, Li Z. et al. Regulation of glycoprotein Ib-IX-von Willebrand factor interaction by cAMP-dependent protein kinase-mediated phosphorylation at Ser 166 of glycoprotein Ib(beta). J Biol Chem 2002; 277: 47080-47087.
6 Harbeck B, Huttelmaier S, Schluter K. et al. Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin. J Biol Chem 2000; 275: 30817-30825.
7 Butt E, Gambaryan S, Gottfert N. et al. Actin binding of human LIM and SH3 protein is regulated by cGMP- and cAMP-dependent protein kinase phosphorylation on serine 146. J Biol Chem 2003; 278: 15601-15607.
8 Chew CS, Chen X, Parente Jr JA. et al. Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo. J Cell Sci 2002; 115: 4787-4799.
9 Rachlin AS, Otey CA. Identification of palladin isoforms and characterisation of an isoform-specific interaction between Lasp-1 and palladin. J Cell Sci 2006; 119: 995-1004.
10 Keicher C, Gambaryan S, Schulze E. et al. Phosphorylation of mouse LASP-1 on threonine 156 by cAMP- and cGMP-dependent protein kinase. Biochem Biophys Res Commun 2004; 324: 308-316.
11 Haag M. Interakting Partners of Pro-Interleukin-16. Berlin: FU-Berlin; 2007
12 Li B, Zhuang L, Trueb B. Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1. J Biol Chem 2004; 279: 20401-20410.
13 Nakagawa H, Terasaki AG, Suzuki H. et al. Shortterm retention of actin filament binding proteins on lamellipodial actin bundles. FEBS Letters 2006; 580: 3223-3228.
14 Panaviene Z, Moncman CL. Linker region of nebulin family members plays an important role in targeting these molecules to cellular structures. Cell Tissue Res 2007; 327: 353-369.
15 Spence HJ, McGarry L, Chew CS. et al. AP-1 differentially expressed proteins Krp1 and fibronectin cooperatively enhance Rho-ROCK-independent mesenchymal invasion by altering the function, localisation, and activity of nondifferentially expressed proteins. Mol Cell Biol 2006; 26: 1480-1495.
16 Lin YH, Park ZY, Lin D. et al. Regulation of cell migration and survival by focal adhesion targeting of Lasp-1. J Cell Biol 2004; 165: 421-432.
17 Zahedi RP, Lewandrowski U, Wiesner J. et al. Phosphoproteome of resting human platelets. J Proteome Res 2008; 07: 526-534.
18 Walter U, Miller P, Wilson F. et al. Immunological distinction between guanosine 3’:5’-monophosphatedependent and adenosine 3’:5’-monophosphate-dependent protein kinases. J Biol Chem 1980; 255: 3757-3762.
19 O’Toole TE, Loftus JC, Du XP. et al. Affinity modulation of the alpha IIb beta 3 integrin (platelet GPIIbIIIa) is an intrinsic property of the receptor. Cell Regul 1990; 01: 883-893.
20 O’Toole TE, Katagiri Y, Faull RJ. et al. Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol 1994; 124: 1047-1059.
21 Hauck CR, Hsia DA, Ilic D. et al. v-Src SH3-enhanced interaction with focal adhesion kinase at beta 1 integrin-containing invadopodia promotes cell invasion. J Biol Chem 2002; 277: 12487-12490.
22 Shattil SJ, Newman PJ. Integrins: dynamic scaffolds for adhesion and signalling in platelets. Blood 2004; 104: 1606-1615.
23 Hanke JH, Gardner JP, Dow RL. et al. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem 1996; 271: 695-701.
24 Buchdunger E, Zimmermann J, Mett H. et al. Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative. Cancer Res 1996; 56: 100-104.
25 Blom N, Gammeltoft S, Brunak S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 1999; 294: 1351-1362.
26 Grunewald TG, Kammerer U, Schulze E. et al. Silencing of LASP-1 influences zyxin localisation, inhibits proliferation and reduces migration in breast cancer cells. Exp Cell Res 2006; 312: 974-982.
27 Yeatman TJ. A renaissance for SRC. Nature Rev 2004; 04: 470-480.
28 Schreiber V, Moog-Lutz C, Regnier CH. et al. Lasp-1, a novel type of actin-binding protein accumulating in cell membrane extensions. Mol Med 1998; 04: 675-687.
29 Chew CS, Parente Jr JA, Chen X. et al. The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signalling pathway with dynamic membrane restructuring activities in ion transporting epithelia. J Cell Sci 2000; 113: 2035-2045.
30 Chew CS, Chen X, Bollag RJ. et al. Targeted disruption of the Lasp1 gene is linked to increases in histamine-stimulated gastric HCl secretion. Am J Physiol Gastrointest Liver Physiol 2008; 295: G37-G44.