beta-Catenin, a component of Wnt signaling, plays a key role in colorectal carcinogenesis. The phosphorylation status of beta-catenin determines its fate and affects its cellular function, and serine 675 (S675) was previously identified as a common target of p21-activated kinase 1 (PAK1) and protein kinase A. In the present study, we explored the PAK1-specific phosphorylation site(s) in p-catenin. Active PAK1 T423E but not inactive PAK1 K299R interacted with and phosphorylated beta-catenin. Mutagenesis followed by a kinase assay revealed that PAK1 phosphorylated S663 in addition to S675, and an anti-phospho-beta-catenin(S663) antibody detected the phosphorylation of S663 downstream of PAK1 in various human colon cancer cells. Furthermore, the Wnt3a-stimulated S663 phosphorylation was inhibited by the PAK1-specific inhibitor, IPA-3, but not by H-89 or LY294002. The non-phosphorylatable mutant forms of beta-catenin, S663A, S675A and S663/675A, showed similar defects in their PAK1-induced TCF/LEF transactivation, whereas the phosphomimetic form of beta-catenin, S663D, demonstrated a transcriptional activity that was comparable to that of beta-catenin S675D and beta-catenin S663D/S675D. Taken together, these results provide evidence that PAK1 specifically phosphorylates beta-catenin at S663 and that this phosphorylation is essential for the PAK1-mediated transcriptional activation of beta-catenin. Crown Copyright (C) 2012 Published by Elsevier Inc. All rights reserved.