Functional correlates of mutations in β-catenin exon 3 phosphorylation sites

beta-Catenin-mediated signaling can be constitutively activated by truncation or mutation of serine and threonine residues in exon 3. Mutations in this region are observed in many human tumors. Examination of the locations of these mutations reveals interesting patterns; specifically, Ser(45) and Thr(41) appear more frequently in malignant tumors, and Ser(37) and Ser(33) are more common in benign entities. To test whether these patterns represent functional differences in beta-catenin signaling mechanisms, we generated mutations of each of these residues. Stable transformation of Madin-Darby canine kidney cells showed a transformed phenotype with each of the four mutations, as assessed by growth in soft agar and collagen. Functional assays including proliferation assays, cell shedding assays, and wounding assays demonstrated two groups. Ser(45) and Thr(41) represent a more transformed phenotype, whereas Ser(37) and Ser(33) behaved similarly to the vector in these assays. Assessment of downstream genes demonstrated increased activation of the beta-catenin target gene cyclin D1 by Ser(45). Finally, we examined the kinase activity of IkappaB kinase-alpha and found that this kinase, unlike glycogen synthase kinase-3beta, appears to preferentially phosphorylate Ser(45) and Thr(41), independent of priming by casein kinase-1. We conclude that these sites may represent an alternative (non-wnt) signaling pathway, which may be inappropriately activated in tumors with mutations of these residues.