Introduction The Tat protein is a cationic 86-101 amino acid polypeptide that acts as the main transactivating factor of HIV-1 (Gatignol and Jeang, 2000). Direct and indirect evidence indicate that Tat is actively released by HIV-1-infected cells (Noonan and Albini, 2000). Extracellular Tat targets different types of uninfected cells, including endothelial cells (ECs) (Rusnati and Presta, 2002), causing a variety of biological effects possibly related to distinct AIDS-associated pathologies, including neuropathies (Dewhurst et al., 1996), immune suppression (Caputo et al., 1999; Noonan and Albini, 2000) and increased tumorigenesis in AIDS patients (Caputo et al., 1999). The mechanisms by which Tat exerts its pathological effects are manifold and mediated by various signalling receptors in target cells, including the vascular endothelial growth factor (VEGF) receptor 2 (KDR) (Albini et al., 1996), chemochine receptors (Albini et al., 1998), and integrins (Barillari et al., 1999a; Barillari et al., 1999b; Fiorelli et al., 1999; Mitola et al., 2000). Integrins are a family of heterodimeric receptors that, unlike growth factor receptors, lack intrinsic tyrosine kinase activity. Yet, an early event during integrin signalling is the tyrosine phosphorylation of the non-receptor tyrosine kinase focal adhesion kinase (FAK) (Kumar, 1998; Schlaepfer et al., 1999) that, in turn, leads to the activation of the GTPase RhoA and/or pp60 src in different cell types (Palazzo et al., 2004; Sharma- Walia et al., 2004; Zhai et al., 2003). In ECs, this signal transduction pathway can be activated upon  v  3 integrin engagement and leads to nuclear translocation of NF-B and cell survival (Scatena et al., 1998). Accordingly, integrins regulate EC proliferation in vitro (Eliceiri, 2001) and angiogenesis in vivo (Varner and Cheresh, 1996). In ECs, Tat interacts with  v  3 and  5  1 integrins (Barillari et al., 1999a; Barillari et al., 1999b; Fiorelli et al., 1999; Mitola et al., 2000). This interaction is required by Tat to induce neovascularization in vivo and chemotactic and mitogenic activity in cultured ECs (Barillari et al., 1999a; Barillari et al., 1999b; Mitola et al., 2000). Also, integrins mediate the adhesion and spreading of ECs to substratum- immobilized Tat (Barillari et al., 1999a; Barillari et al., 1999b; Fiorelli et al., 1999; Mitola et al., 2000). However, the molecular bases and biological relevance of this process remain poorly elucidated. Relevant to this point, Tat accumulates in the extracellular matrix (ECM) as an immobilized protein (Chang et al., 1997) by interacting with heparan sulphate proteoglycans (HSPGs) (Chang et al., 1997; Tyagi et al., 2001). These findings suggest that the concentration of Tat can increase in the microenvironment, 3949 Once in the extracellular environment, the transactivator protein HIV-1 Tat exerts several pleiotropic effects by interacting with different cellular receptors, including integrin  v  3 . Real-time surface plasmon resonance analysis reveals that Tat/ V  3 interaction occurs with rapid kinetics (association and dissociation rates equal to 1.1610 7 M –1 s –1 and 3.7810 –1 s –1 , respectively) and high affinity (dissociation constant = 32 nM). Through this interaction, substratum-immobilized Tat promotes adhesion and motogenic activity in endothelial cells. Also,  v  3 /Tat interaction triggers the activation of focal adhesion kinase, RhoA and pp60 src . Overexpression of the dominant negative form of focal adhesion kinase, but not of an inactive Leu 1034 Ser substitution mutant isoform, impairs the activation of focal adhesion kinase and RhoA, but not that of pp60 src , without affecting endothelial cell adhesion and spreading.  v  3 /Tat interaction triggers the activation of NF-B in endothelial cells in a focal adhesion kinase-, RhoA- and pp60 src -dependent manner, as shown in dominant negative focal adhesion kinase transfectants or using specific pharmacological inhibitors. Finally, the activation of focal adhesion kinase, RhoA, NF-B and pp60 src are required to mediate the motogenic activity of Tat in endothelial cells. Since Tat accumulates in an immobilized form in the extracellular matrix, these results provide new biochemical and biological insights about  v  3 /Tat interaction exploitable for the design of anti-Tat strategies. Key words: HIV-1 Tat,  v  3 integrin, endothelium, FAK, NF-B Summary  v  3 -integrin-dependent activation of focal adhesion kinase mediates NF-B activation and motogenic activity by HIV-1 Tat in endothelial cells Chiara Urbinati 1 , Antonella Bugatti 1 , Mauro Giacca 2 , David Schlaepfer 3 , Marco Presta 1 and Marco Rusnati 1, * 1 General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, viale Europe 11, 25123 Brescia, Italy 2 International Center for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy 3 The Scripps Research Institute, Department of Immunology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA *Author for correspondence (e-mail: rusnati@med.unibs.it) Accepted 3 June 2005 Journal of Cell Science 118, 3949-3958 Published by The Company of Biologists 2005 doi:10.1242/jcs.02518 Research Article Journal฀of฀Cell฀Science