Analysis of beta-catenin aggregation and localization using GFP fusion proteins: nuclear import of alpha-catenin by the beta-catenin/Tcf complex.
beta-Catenin plays essential roles in cell adhesion, by associating with cadherins, and as a signaling molecule, by interacting with the Tcf/LEF-1 family of transcription factors. In order to study the protein-protein interactions of beta-catenin in living cells, we fused it to green fluorescent protein (GFP). GFP-beta-catenin was incorporated into cell junctions but also accumulated in the nucleus, where it formed rod-like structures. The carboxyl-terminal armadillo repeats of GFP-beta-catenin were sufficient for nuclear localization, but formation of rods required the armadillo repeats and sequences in both the amino- and the carboxyl-terminal domains. Rod formation was prevented by coexpression of N-cadherin, APC, and Tcf-4, which bind to the armadillo repeats of beta-catenin, but not by coexpression of alpha-catenin, although alpha-catenin expression did prevent accumulation of beta-catenin in the nucleus. Interestingly, when alpha-catenin, beta-catenin, and Tcf-4 were coexpressed they colocalized in the nucleus, and this correlated with a decrease in beta-catenin/Tcf-dependent transcriptional activity. These results indicate that binding of beta-catenin to Tcf-4 overrides the function of alpha-catenin to sequester beta-catenin in the cytoplasm and suggest that alpha-catenin can regulate beta-catenin signaling in the nucleus.
Giannini
A L
AL
MRC Laboratory for Molecular Cell Biology, University College London, London, WC1E 6BT.
Vivanco
M M
MM
Kypta
R M
RM
D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; D015534:Trans-Activators; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors / Q000378:metabolism; D051178:alpha Catenin; D051176:beta Catenin
D003598:Cytoskeletal Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D051820:TCF Transcription Factors; D015534:Trans-Activators; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors; D051178:alpha Catenin; D051176:beta Catenin; D049452:Green Fluorescent Proteins