Degradation of cytosolic β-catenin by the APC/Axin1 destruction complex represents the key regulated step of the Wnt pathway. It is incompletely understood how the Axin1 complex exerts its Wnt-regulated function. Here, we examine the mechanism of Wnt signaling under endogenous levels of the Axin1 complex. Our results demonstrate that β-catenin is not only phosphorylated inside the Axin1 complex, but also ubiquinated and degraded via the proteasome, all within an intact Axin1 complex. In disagreement with current views, we find neither a disassembly of the complex nor an inhibition of phosphorylation of Axin1-bound β-catenin upon Wnt signaling. Similar observations are made in primary intestinal epithelium and in colorectal cancer cell lines carrying activating Wnt pathway mutations. Wnt signaling suppresses β-catenin ubiquitination normally occurring within the complex, leading to complex saturation by accumulated phospho-β-catenin. Subsequently, newly synthesized β-catenin can accumulate in a free cytosolic form and engage nuclear TCF transcription factors.

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Wnt signalling maintains the undifferentiated state of intestinal crypt/progenitor cells through the TCF4/b-catenin-activating transcriptional complex. In colorectal cancer, activating mutations in Wnt pathway components lead to inappropriate activation of the TCF4/b-catenin transcriptional programme and tumourigenesis. The mechanisms by which TCF4/b-catenin activate key target genes are not well understood. Using a proteomics approach, we identified Tnik, a member of the germinal centre kinase family as a Tcf4 interactor in the proliferative crypts of mouse small intestine. Tnik is recruited to promoters of Wnt target genes in mouse crypts and in Ls174T colorectal cancer cells in a b-catenin-dependent manner. Depletion of TNIK and expression of TNIK kinase mutants abrogated TCF-LEF transcription, highlighting the essential function of the kinase activity in Wnt target gene activation. In vitro binding and kinase assays show that TNIK directly binds both TCF4 and b-catenin and phosphorylates TCF4. siRNA depletion of TNIK followed by expression array analysis showed that TNIK is an essential, specific activator of Wnt transcriptional programme. This kinase may present an attractive candidate for drug targeting in colorectal cancer.

Mutational activation of the phosphatidylinositol 3-kinase (PI3K) pathway occurs in a wide variety of tumors, whereas activating Wnt pathway mutants are predominantly found in colon cancer. Because GSK3 is a key component of both pathways, it is widely assumed that active PI3K signaling feeds positively into the Wnt pathway by protein kinase B (PKB)-mediatefd inhibition of GSK3. In addition, PKB has been proposed to modulate the canonical Wnt signaling through direct stabilization and nuclear localization of ␤-catenin. Here, we show that compartmentalization by Axin of GSK3 prohibits cross-talk between the PI3K and Wnt pathways and that Wnt-mediated transcriptional activity is not modulated by activation of the PI3K/PKB pathway.Developmental signaling cascades typically transduce signals from the cell surface onto regulatory sequences of nuclear target genes. In the simplest model, signals transduced through different pathways are integrated at the level of the regulatory elements of individual genes. Such regulatory elements may be viewed as assemblies of cis-acting response elements that are tailored to create the unique expression pattern for each gene. However, numerous studies propose that signaling pathways may interact at any stage between the plasma membrane and the nucleus. One mechanism by which such cross-talk may occur involves the sharing of a common component between two different pathways. It is often tacitly assumed that such shared components are equally accessible to all pertinent pathways.Glycogen synthase kinase 3-␣ and -␤, collectively termed GSK3, are constitutively active serine/threonine kinases (1). GSK3 features in two signaling pathways that are of particular importance in cancer. GSK3 is a downstream component of the phosphoinositide 3-OH kinase (PI3K) 2 pathway (2, 3). Growth signals, activated Ras proteins, or loss of the phosphatase and tensin homolog (PTEN) all activate PI3K, which in turn phosphorylates and activates protein kinase B (PKB) (3). Active PKB phosphorylates GSK3␣ on Ser-21 (4) and GSK3␤ on Ser-9 (5), in both cases leading to inhibition of the constitutive kinase activity. GSK3 is also a component of the Wnt cascade (6). GSK3 is bound by Axin (Axis inhibition protein) (7) and phosphorylates ␤-catenin, thus targeting it for ubiquitination and degradation by the proteasome. Wnt signaling is assumed to block GSK3-mediated ␤-catenin phosphorylation, leading to the accumulation and nuclear translocation of ␤-catenin (6). It remains unclear how the Wnt cascade controls the activity of the dedicated Axin1-bound GSK3 pool. A recent genetic experiment has demonstrated that removal of the inhibitory serines from the two GSK3 proteins has no effect on Wnt signaling (8).Although an early study proposed that the two pathways do not cross-talk at the level of GSK3 (9), a multitude of papers have since appeared that are based on the premise that a single pool of GSK3 is targeted by both signals (see supplemental Table S1). Moreover, direct stabilization of ␤-catenin by the PI3K/PKB...

The liver parenchyma is composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists regarding the cellular origin of human liver parenchymal tissue generation during embryonic development, homeostasis or repair. Here we report the existence of a hepatobiliary hybrid progenitor (HHyP) population in human foetal liver using single-cell RNA sequencing. HHyPs are anatomically restricted to the ductal plate of foetal liver and maintain a transcriptional profile distinct from foetal hepatocytes, mature hepatocytes and mature BECs. In addition, molecular heterogeneity within the EpCAM + population of freshly isolated foetal and adult human liver identifies diverse gene expression signatures of hepatic and biliary lineage potential. Finally, we FACS isolate foetal HHyPs and confirm their hybrid progenitor phenotype in vivo. Our study suggests that hepatobiliary progenitor cells previously identified in mice also exist in humans, and can be distinguished from other parenchymal populations, including mature BECs, by distinct gene expression profiles.