The Rho-associated coiled-coil kinase (ROCK) family of proteins, including ROCK1 and ROCK2, are key regulators of actin and intermediate filament morphology. The newly discovered ROCK substrate Tubulin polymerization promoting protein 1 (TPPP1) promotes microtubule polymerization and inhibits the activity of Histone deacetylase 6 (HDAC6). The effect of TPPP1 on HDAC6 activity is inhibited by ROCK signaling. Moreover, it was recently demonstrated that ROCK activity increases the cellular expression of the oncogene beta-catenin, which is a HDAC6 substrate. In this study, we investigated the interplay between ROCK-TPPP1-HDAC6 signaling and beta-catenin expression. We demonstrate that beta-catenin expression is increased with ROCK signaling activation and is reduced with increased TPPP1 expression in U2OS cells. Further investigation revealed that ROCK-mediated TPPP1 phosphorylation, which prevents its binding to HDAC6, negates TPPP1-mediated reduction in beta-catenin expression. We also show that increased HDAC6 activity resulting from ROCK signaling activation reduced beta-catenin acetylation at Lys-49, which was also accompanied by its decreased phosphorylation by Caesin kinase 1 (CK1) and Glycogen synthase kinase 3 beta (GSK3 beta), thus preventing its proteasomal degradation. Overall, our results suggest that ROCK regulates beta-catenin stability in cells via preventing TPPP1-mediated inhibition of HDAC6 activity, to reduce its acetylation and degradation via phosphorylation by CK1 and GSK3 beta. (c) 2013 Elsevier Inc. All rights reserved.