Functional correlates of mutation of the Asp32 and Gly34 residues of beta-catenin.

Beta-catenin is a multifunctional protein involved in both cadherin-mediated adhesion and the wnt signaling cascade. Mutations in exon 3 of beta-catenin have been identified in many cancers. In addition to disruption of key serine and threonine residues, mutations are frequently reported in other residues in exon 3 that are not kinase substrates. The most frequently mutated nonserine/threonine residues are D32 and G34. Since D32 and G34 are part of the ubiquitination destruction motif, DSGPhiXS, we hypothesize that this motif may contribute to disruption of beta-catenin homeostasis and lead to cellular transformation. We demonstrate that the mutants D32A and G34A exhibit no change in phosphorylation by GSK3beta, but display reduced ubiquitination compared to wild-type and S33A mutant beta-catenin. To assess the functional implications of these mutations, we created stable MDCK cell lines expressing these constructs. We found that stable cell lines harboring D32A-mutated beta-catenin were highly transformed, while S33A and G34 demonstrated only weak transforming properties in our assays. Despite altered ubiquitination status and increased transformation, the D32A mutant cell line does not display transcriptional activation of standard target genes. Therefore, D32A mutation may mediate transformation by an alternative beta-catenin-mediated signaling pathway.

Functional correlates of mutations in beta-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, Ser45 and Thr41 appear more frequently in malignant tumors, and Ser37 and Ser33 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. Ser45 and Thr41 represent a more transformed phenotype, whereas Ser37 and Ser33 behaved similarly to the vector in these assays. Assessment of downstream genes demonstrated increased activation of the beta-catenin target gene cyclin D1 by Ser45. Finally, we examined the kinase activity of I kappa B kinase-alpha and found that this kinase, unlike glycogen synthase kinase-3 beta, appears to preferentially phosphorylate Ser45 and Thr41, 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.