Protein kinase C alpha (PKC alpha) phosphorylates the Ser33/37/Thr41 residues of beta-catenin, which lacks a typical PKC alpha canonical sequence, but little is known about its underlying mechanism. Here we showed that Ser33/Ser37/Thr41 of beta-catenin fragments encompassing the armadillo repeats 1-5 (beta-catenin(1-781), beta-catenin(1-682), and beta-catenin(1-422)) are phosphorylated by PKC alpha whereas beta catenin(1-138) lacking these repeats is not phosphorylated. Binding-site analysis revealed that PKC alpha directly interacts with beta-catenin through the sites on the armadillo repeats 1-5. In addition, axin fragments (365-500), which interacts with beta-catenin through armadillo repeats 3-5, disrupted PKC alpha/beta-catenin association and inhibited beta-catenin phosphorylation by PKC alpha. In HEK293 cells, the levels of beta-catenin(1-781) and beta-catenin(1-422) were decreased whereas the amount of beta-catenin(1-138) was unchanged by pharmacological stimulation of PKC alpha. Our results suggest that the association of PKC alpha with the armadillo repeats of beta-catenin placed the Ser33137/Thr41 residues of beta-catenin in close proximity to PKC alpha, thereby facilitating PKC alpha-mediated beta-catenin phosphorylation. (C) 2014 Elsevier Inc. All rights reserved.