Wnt signaling and phosphorylation status of β-catenin: importance of the correct antibody tools

To the editor:

Two recent papers in Blood provide the first indications that deregulated Wnt signaling plays a role in the development of acute lymphoblastic leukemias (ALLs).^1,2  Activating mutations in the critical Wnt mediator β-catenin lead to the development of thymic lymphomas, the murine counterpart of T-ALL.¹  The Wnt signaling pathway plays a key role in the development of various cell types and is subject to strict molecular regulation. The localization and consequently the function of β-catenin is regulated by Ser/Thr and possibly Tyr phosphorylation. Nuclear β-catenin serves as a coactivator for Tcf-mediated transcription of target genes that play important roles in leukemic progression.

Given the importance of the phosphorylation status of β-catenin, we have previously generated an antibody specific for β-catenin dephosphorylated at residues Ser37 and Thr41. Wnt signals specifically increase the levels of dephosphorylated β-catenin as detected with this antibody.^3,4  The antibody, named antiactive β-catenin (ABC), is produced by hybridoma clone 8E7. Another antibody claimed to recognize the dephosphorylated form is termed 8E4 (www.upstate.com).

Using 8E4 instead of 8E7, various investigators have studied activation of the Wnt pathway. Gottardi and Gumbiner⁵  report that dephosphorylated β-catenin readily interacts with both cytoplasmic cadherins and nuclear Tcf. Derksen et al⁶  and Diks et al⁷  report very similar patterns of bands in Western blots using 8E4 and a pan–β-catenin antibody, whereas Jamieson et al⁸  using 8E7 antibody show that Wnt signaling is activated in blast crises of chronic myelogenous leukemia (CML).

These conclusions critically depend on the specificity of the antibodies. We therefore mapped the antigenic epitope recognized by the 8E4 and 8E7 antibodies using a set of N-terminal deletion clones of β-catenin covering the regulatory region that is the target of the Wnt pathway kinases (Ser33 to Ser45; Figure 1A). Using these constructs, 8E4 was found to recognize all deleted forms (Figure 1B), implying that it does not recognize the regulatory region of β-catenin. These results are at odds with the claims on the company's website (http://www.upstate.com) that this antibody was raised against amino acids 27-37 of human β-catenin. As published previously, the 8E7 epitope mapped directly C-terminal to amino acid 35.³ 

It is unfortunate that these antibodies with such similar names, commercialized by the same company, have strikingly different specificities. Our observations imply that the 8E4 antibody does not possess the advertised specificity for the dephosphorylated regulatory region of β-catenin and should not be used to study activity of the Wnt pathway, for instance, on cytospin preparations of cells suspected for hematologic malignancies.