Abstract
beta-Catenin plays a central role in the establishment and regulation of adherens junctions because it interacts with E-cadherin and, through alpha-catenin, with the actin cytoskeleton. beta-Catenin is composed of three domains: a central armadillo repeat domain and two N- and C-terminal tails. The C-tail interacts with the armadillo domain and limits its ability to bind E-cadherin and other cofactors. The two beta-catenin tails are mutually inter-regulated because the C-tail is also necessary for binding of the N-tail to the armadillo domain. Moreover, the N-tail restricts the interaction of the C-tail with the central domain. Depletion of either of the two tails has consequences for the binding of factors at the other end: deletion of the C-tail increases alpha-catenin binding, whereas deletion of the N-tail blocks E-cadherin interaction to the armadillo repeats. As an effect of the interconnection of the tails, the association of alpha-catenin and E-cadherin to beta-catenin is interdependent. Thus, binding of alpha-catenin to the N-tail, through conformational changes that affect the C-tail, facilitates the association of E-cadherin. These results indicate that different cofactors of beta-catenin bind coordinately to this protein and indicate how the two terminal ends of beta-catenin exquisitely modulate intermolecular binding within junctional complexes.
Highlights
-Catenin plays a central role in the establishment and through the interaction with members of the T cell transcription regulation of adherens junctions because it interacts with factor family [3]
E-cadherin and, through ␣-catenin, with the actin cy- teins, several other transcriptional factors have been reported to toskeleton. -Catenin is composed of three domains: a interact with -catenin, presumably modulating its positive accentral armadillo repeat domain and two N- and C- tivity on the transcription of several target genes [4]
Expression of Recombinant Proteins—The preparation of all the plasmids codifying the different -catenin forms as glutathione S-transferase (GST) fusion proteins has been described previously [7, 11] except for the following cases: GST-arm(120 – 683) and GST--cat-(120 –750); DNA fragments corresponding to amino acids 120 – 683 or 120 –750 of murine -catenin were amplified by PCR using oligonucleotides corresponding to the nucleotide sequences 358 –372 and 2035–2047 or 358 –372 and 2260 –2242 and containing BamHI and XhoI sites at their ends
Summary
Reagents—The following -catenin monoclonal antibodies were used in this study: -catenin C terminus (Transduction Laboratories, Lexington, KY); the epitope recognized by this antibody has been mapped to residues 696 –750 using different deletion mutants of this protein (not shown); -catenin C-end (Calbiochem), epitope recognized, 769 –781 (according the manufacturer); -catenin armadillo core, epitope recognized, 422– 685 (according the manufacturer) and -catenin N terminus, raised against the first 100 amino acids (both from Alexis Biochemicals, San Diego, CA). Expression of Recombinant Proteins—The preparation of all the plasmids codifying the different -catenin forms (deletion and point mutants) as glutathione S-transferase (GST) fusion proteins has been described previously [7, 11] except for the following cases: GST-arm(120 – 683) and GST--cat-(120 –750); DNA fragments corresponding to amino acids 120 – 683 or 120 –750 of murine -catenin were amplified by PCR using oligonucleotides corresponding to the nucleotide sequences 358 –372 and 2035–2047 or 358 –372 and 2260 –2242 and containing BamHI and XhoI sites at their ends. Binding assays were performed as described [7, 11], and protein complexes bound to the Sepharose were analyzed by Western blot using specific mAbs against -catenin (see above), ␣-catenin, E-cadherin (cytosolic domain), TBP (the three from Transduction Laboratories) or GST (Amersham Biosciences) as control. The results were reproduced when a higher concentration of protease was used, all the forms presented an accelerated rate of degradation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
