Lysine residues Lys-19 and Lys-49 of β-catenin regulate its levels and function in T cell factor transcriptional activation and neoplastic transformation

Abstract

Wnt signaling regulates cell fate determination, proliferation, and survival, among other processes. Certain Wnt ligands stabilize the β-catenin protein, leading to the ability of β-catenin to activate T cell factor-regulated genes. In the absence of Wnts, β-catenin is phosphorylated at defined serine and threonine residues in its amino (N) terminus. The phosphorylated β-catenin is recognized by a β-transducin repeat-containing protein (βTrCP) and associated ubiquitin ligase components. The serine/ threonine residues and βTrCP-binding site in the N-terminal region of β-catenin constitute a key regulatory motif targeted by somatic mutations in human cancers, resulting in constitutive stabilization of the mutant β-catenin proteins. Structural studies have implicated β-catenin lysine 19 as the major target for βTrCP-dependent ubiquitination, but Lys-19 mutations in cancer have not been reported. We studied the consequences of single amino acid substitutions of the only 2 lysine residues in the N-terminal 130 amino acids of β-catenin. Mutation of Lys-19 minimally affected β-catenin levels and functional activity, and mutation of Lys-49 led to reduced β-catenin levels and function. In contrast, β-catenin proteins with substitutions at both Lys-19 and Lys-49 positions were present at elevated levels and had the ability to potently activate T cell factor-dependent transcription and promote neoplastic transformation. We further more demonstrate that the K19/K49 double mutant forms of β-catenin are stabilized as a result of reduced βTrCP-dependent ubiquitination. Our findings suggest that Lys-19 is a primary in vivo site of βTrCP-dependent ubiquitination and Lys-49 may be a secondary or cryptic site. Moreover, our results inform understanding of why single amino acid substitutions at lysine 19 or 49 have not been reported in human cancer. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.