P21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. Science. 283: 2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alphavbeta5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alphavbeta5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alphavbeta5. Functionally, PAK4 induced integrin alphavbeta5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alphavbeta5 and selectively promotes integrin alphavbeta5-mediated cell migration.