Lysine residues Lys-19 and Lys-49 of beta-catenin regulate its levels and function in T cell factor transcriptional activation and neoplastic transformation.
Wnt signaling regulates cell fate determination, proliferation, and survival, among other processes. Certain Wnt ligands stabilize the beta-catenin protein, leading to the ability of beta-catenin to activate T cell factor-regulated genes. In the absence of Wnts, beta-catenin is phosphorylated at defined serine and threonine residues in its amino (N) terminus. The phosphorylated beta-catenin is recognized by a beta-transducin repeat-containing protein (betaTrCP) and associated ubiquitin ligase components. The serine/threonine residues and betaTrCP-binding site in the N-terminal region of beta-catenin constitute a key regulatory motif targeted by somatic mutations in human cancers, resulting in constitutive stabilization of the mutant beta-catenin proteins. Structural studies have implicated beta-catenin lysine 19 as the major target for betaTrCP-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 beta-catenin. Mutation of Lys-19 minimally affected beta-catenin levels and functional activity, and mutation of Lys-49 led to reduced beta-catenin levels and function. In contrast, beta-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 furthermore demonstrate that the K19/K49 double mutant forms of beta-catenin are stabilized as a result of reduced betaTrCP-dependent ubiquitination. Our findings suggest that Lys-19 is a primary in vivo site of betaTrCP-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.
Winer
Ira S
IS
Cell and Molecular Biology Graduate Program, Division of Molecular Medicine and Genetics, University of Michigan School of Medicine, Ann Arbor, Michigan 48109, USA.
Bommer
Guido T
GT
Gonik
Nathan
N
Fearon
Eric R
ER
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology; D051820:TCF Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D025801:Ubiquitin / Q000378:metabolism; D051153:Wnt Proteins / Q000378:metabolism; D051176:beta Catenin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; D025801:Ubiquitin; D051153:Wnt Proteins; D051176:beta Catenin; D008239:Lysine