Studies on histidine phosphorylation in Wnt/beta-catenin signaling
Biochemistry
Final Report Abstract
Wnt/β-Catenin signaling is a key pathway involved in embryonic development and cancer. It is activated by secreted proteins named Wnts that stimulate target cells by binding to receptors on the cell surface. This starts a cascade of molecular events that in the end leads to the entry of the protein β-catenin into the nucleus, which activates different target genes by stimulating their transcription. Depending on the cellular context, Wnt β-catenin signaling induces proliferation, differentiation, and migration of cells, in particular stem cells. Mutations in Wnt pathway components can lead to different forms of cancer, in particular colorectal cancer. The mechanism that control β-catenin are therefore important for understanding metazoan biology, but also disease. A central mechanism that regulates β-catenin is its degradation by a so-called destruction complex. Here, several proteins based on the scaffold axin cooperate to phosphorylate bcatenin, which is then degraded by proteasomes within the cell. Wnts counteract β-catenin degradation by activating Dishevelled proteins, abbreviated Dvl1, Dvl32, and Dvl3 in vertebrates. Dvls interact with axin in so called puncta that are round and dynamic assemblies of Dvl proteins in the cytoplasm, which are mediated by head-to-tail polymerization of the DIX domain of Dvl at its N-terminus. Puncta formation is essential for Dvl activity and inhibition of the axin based destruction complex. In this projected we started out to analyze a possible biochemical role of histidine phosphorylation of Dvl2 for which we had indications from previous work because antibodies that detect phosphohistidines stained Dvl2 puncta. However, through detailed work it turned out that we were not able not identify a specific histidine in Dvl2 that was phosphorylated, and also couldn’t discover any other component associated with Dvl2 that would carry the phosphohistidine signal. Thus, a role of histidine phosphorylation in Dvl2 puncta as initially envisioned is not supported by our data. Through experiments we found that mutations of specific amino acids in a special part of Dvl, the DEP domain dramatically altered puncta shape and led to loss of Wnt activity of Dvl2, suggesting that in line with the literature the DEP domain is involved in control of puncta formation. However, full deletion of the DEP domain of Dvl2 reduced puncta formation but did not completely abolish it suggesting that other parts of the Dvl2 protein play a role as well. Indeed, by further studies we could identify three domains of Dvl2, namely CD1, LCR, and CD2 that promote puncta formation and collaborate with the DIX domain in this process. Thus, deletion of CD1, LCR, and CD2 impaired puncta formation of DEP-less constructs whereas fusion of these parts to the DIX domain triggered puncta formation. Taken together, we have discovered new domains in Dvl2 involved in puncta formation, which contributes to understanding its three dimensional assembly in the cell, and provides a new basis for understanding Dvl function in the Wnt pathway.
