Non-phosphorylated b-catenin in Wnt signaling

Abstract

The Wnt signaling cascade plays an important role in development and carcinogenesis. Under non-signaling conditions, a large cytoplasmic complex, consisting of the kinase GSK-3?, the gene product of the adenomatous poliposis coli gene (APC), axin and ?-catenin, results in the phosphorylation of ß-catenin. Phosphorylated ?-catenin is ubiquitinated and subsequently degraded by the proteasome. Upon Wnt binding to its receptors Frz and LRP, the complex dissociates, ?-catenin is no longer degraded, and accumulates. It translocates to the nucleus, where it forms a bipartite transcription factor with a member of the TCF family and results in target gene transcription. In this thesis the generation of a monoclonal antibody (?ABC) is described, which is specific for the N-terminally non-phosphorylated ?-catenin. This tool proved to be very useful in answering some remaining questions about the regulation of ?-catenin. First we established, that Wnt signaling results in the accumulation of non-phosphorylated ?-catenin. Another widely accepted idea, that GSK-3? phosphorylates ?-catenin at the N-terminus was proven as well as the notion that phosphorylation precedes ubiquitination. Secondly, we showed that mere accumulation of ?-catenin is not sufficient for signaling, rather that the phosphorylation status of ?-catenin is not only important for its degradation but also for the signaling capacity. In chapter 4 we used ?ABC to investigate the role of Wnt signaling and ?-catenin in the pathogenesis of breast carcinoma. We stained breast tumor samples with ?ABC and found to our surprise that 70% of the samples tested, were positive for non-phosphorylated ?-catenin in the nucleus. It was reported previously that in breast tumor cell lines, ?-catenin/Tcf mediated signaling was only found in 1 of 26 cell lines. Testing cell lysates of theses lines with ?ABC confirmed these data. In Chapter 5, we tried to unravel more of the mechanisms involved in ?-catenin phosphorylation by GSK-3?. First, the answer was given to the question whether ?-catenin is actively dephosphorylated or needs to be synthesized de novo. The latter was proven to be the case, which made us question the given half life of ?-catenin as stated in literature. Therefore we determined the half life of the signaling pool of ?-catenin in two ways. The half life was estimated to be 17 minutes. Next the serines and threonine, which are phosphorylated by GSK-3? were substituted with aspartic acid residues or mutations were introduced which were found in tumors. Aspartic acid can mimic phosphorylation, which was the case for S37 and T41, however, when S33 and S45 were substituted with the aspartic acid residues, ?-catenin was as active as all mutations found in tumors. These mutant constructs were subsequently used to determine the relative order of phosphorylation using ?ABC and ?S29NP. We could show that the phosphorylation is likely to start at the 3’ serine, moving upwards. In chapter 6 we describe the presence of two additional serines which phosphorylation status is regulated by Wnt signaling. Introducing mutations found in tumors in these serines 23 and 29, however, did neither increase the signaling activity of the protein nor enhance the transformational potential. In conclusion this thesis describes the development of an antibody specific for the signaling active form of ?-catenin, with which some of the still unanswered questions concerning ?-catenin regulation are answered.