The transcription program that is responsible for the pluripotency of human ESCs (hESCs) is believed to be comaintained by exogenous fibroblast growth factor-2 (FGF-2), which activates FGF receptors (FGFRs) and stimulates the mitogen-activated protein kinase (MAPK) pathway. However, the same pathway is stimulated by insulin receptors, insulin-like growth factor 1 receptors, and epidermal growth factor receptors. This mechanism is further complicated by intracrine FGF signals. Thus, the molecular mechanisms by which FGF-2 promotes the undifferentiated growth of hESCs are unclear. Here we show that, in undifferentiated hESCs, exogenous FGF-2 stimulated the expression of stem cell genes while suppressing cell death and apoptosis genes. Inhibition of autocrine FGF signaling caused upregulation of differentiation-related genes and downregulation of stem cell genes. Thus, exogenous FGF-2 reinforced the pluripotency maintenance program of intracrine FGF-2 signaling. Consistent with this hypothesis, expression of endogenous FGF-2 decreased during hESC differentiation and FGF-2 knockdown-induced hESC differentiation. In addition, FGF-2 signaling via FGFR2 activated MAPK kinase/extracellular signal-regulated kinase and AKT kinases, protected hESC from stress-induced cell death, and increased hESC adhesion and cloning efficiency. This stimulation of self-renewal, cell survival, and adhesion by exogenous and endogenous FGF-2 may synergize to maintain the undifferentiated growth of hESCs. Stem Cells 2009;27:1847–1857

Amer1/WTX binds to the tumor suppressor adenomatous polyposis coli and acts as an inhibitor of Wnt signaling by inducing ␤-catenin degradation. We show here that Amer1 directly interacts with the armadillo repeats of ␤-catenin via a domain consisting of repeated arginine-glutamic acid-alanine (REA) motifs, and that Amer1 assembles the ␤-catenin destruction complex at the plasma membrane by recruiting ␤-catenin, adenomatous polyposis coli, and Axin/Conductin. Deletion or specific mutations of the membrane binding domain of Amer1 abolish its membrane localization and abrogate negative control of Wnt signaling, which can be restored by artificial targeting of Amer1 to the plasma membrane. In line, a natural splice variant of Amer1 lacking the plasma membrane localization domain is deficient for Wnt inhibition. Knockdown of Amer1 leads to the activation of Wnt target genes, preferentially in dense compared with sparse cell cultures, suggesting that Amer1 function is regulated by cell contacts. Amer1 stabilizes Axin and counteracts Wnt-induced degradation of Axin, which requires membrane localization of Amer1. The data suggest that Amer1 exerts its negative regulatory role in Wnt signaling by acting as a scaffold protein for the ␤-catenin destruction complex and promoting stabilization of Axin at the plasma membrane.The canonical Wnt/␤-catenin signaling pathway is a key pathway in embryonic development and disease. Aberrant activation of Wnt signaling leads to the development of tumors (1, 2). The binding of Wnt ligands to the transmembrane receptors Frizzled (Fz) 2 and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) leads to the stabilization of cytoplasmic ␤-catenin, which enters the nucleus and activates target gene expression by interacting with DNA-binding proteins of the T-cell factor/lymphoid enhancer factor (TCF/lymphoid enhancer factor) family (3, 4). In the absence of Wnts, ␤-catenin is degraded in the proteasome after its ubiquitination by the E3 ligase ␤-transducin repeat-containing protein (␤-TrCP), which recognizes ␤-catenin phosphorylated at specific N-terminal residues. ␤-catenin phosphorylation is accomplished by the coordinated action of CK1 and glycogen synthase kinase 3 (GSK3) and takes place in a multiprotein complex assembled by the scaffold proteins adenomatous polyposis coli (APC) and Axin or its homologue Axin2/Conductin (4, 5). Because Axin binds to ␤-catenin, GSK, CK1, and APC, and APC has multiple binding sites for ␤-catenin, it is generally believed that one function of this ␤-catenin destruction complex is to bring ␤-catenin into close vicinity to the kinases, thereby allowing efficient phosphorylation (6).It is generally thought that ␤-catenin phosphorylation and degradation is a constitutive process, keeping the amount of ␤-catenin in the cytoplasm and nucleus low in the absence of Wnts. Wnt binding to the receptor complex leads to the recruitment of Axin to the plasma membrane and phosphorylation of LRP5/6 at cytoplasmic PPPSPXS motifs (7-10). Phosphorylated PPPSPXS m...

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3b (GSK3b) and casein kinase 1c (CK1c) is a key step in Wnt/b-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P 2 -binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P 2 . Amer1 translocates to the plasma membrane in a PtdIns(4,5)P 2 -dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P 2 . Amer1 binds CK1c, recruits Axin and GSK3b to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/b-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns (4,5)P 2 leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.

Shb (Src homology 2 protein B) is an adapter protein downstream of the vascular endothelial growth factor receptor receptor-2

Recent experiments have unravelled novel signal transduction pathways that involve the SRC homology 2 (SH2) domain adapter protein SHB. SHB is ubiquitously expressed and contains proline rich motifs, a phosphotyrosine binding (PTB) domain, tyrosine phosphorylation sites and an SH2 domain and serves a role in generating signaling complexes in response to tyrosine kinase activation. SHB mediates certain responses in platelet-derived growth factor (PDGF) receptor-, fibroblast growth factor (FGF) receptor-, neural growth factor (NGF) receptor TRKA-, T cell receptor-, interleukin-2 (IL-2) receptor- and focal adhesion kinase- (FAK) signaling. Upstream of SHB in some cells lies the SRC-like FYN-Related Kinase FRK/RAK (also named BSK/IYK or GTK). FRK/RAK and SHB exert similar effects when overexpressed in rat phaeochromocytoma (PC12) and beta-cells, where they both induce PC12 cell differentiation and beta-cell proliferation. Furthermore, beta-cell apoptosis is augmented by these proteins under conditions that cause beta-cell degeneration. The FRK/RAK-SHB responses involve FAK and insulin receptor substrates (IRS) -1 and -2. Besides regulating apoptosis, proliferation and differentiation, SHB is also a component of the T cell receptor (TCR) signaling response. In Jurkat T cells, SHB links several signaling components with the TCR and is thus required for IL-2 production. In endothelial cells, SHB both promotes apoptosis under conditions that are anti-angiogenic, but is also required for proper mitogenicity, spreading and tubular morphogenesis. In embryonic stem cells, dominant-negative SHB (R522K) prevents early cavitation of embryoid bodies and reduces differentiation to cells expressing albumin, amylase, insulin and glucagon, suggesting a role of SHB in development. In summary, SHB is a versatile signal transduction molecule that produces diverse biological responses in different cell types under various conditions. SHB operates downstream of GTK in cells that express this kinase.

The Wnt signaling pathway is required during embryonic development and for the maintenance of homeostasis in adult tissues. However, aberrant activation of the pathway is implicated in a number of human disorders, including cancer of the gastrointestinal tract, breast, liver, melanoma, and hematologic malignancies. In this study, we identified monensin, a polyether ionophore antibiotic, as a potent inhibitor of Wnt signaling. The inhibitory effect of monensin on the Wnt/b-catenin signaling cascade was observed in mammalian cells stimulated with Wnt ligands, glycogen synthase kinase-3 inhibitors, and in cells transfected with b-catenin expression constructs. Furthermore, monensin suppressed the Wnt-dependent tail fin regeneration in zebrafish and Wnt-or b-catenin-induced formation of secondary body axis in Xenopus embryos. In Wnt3a-activated HEK293 cells, monensin blocked the phoshorylation of Wnt coreceptor low-density lipoprotein receptor related protein 6 and promoted its degradation. In human colorectal carcinoma cells displaying deregulated Wnt signaling, monensin reduced the intracellular levels of b-catenin. The reduction attenuated the expression of Wnt signaling target genes such as cyclin D1 and SP5 and decreased the cell proliferation rate. In multiple intestinal neoplasia (Min) mice, daily administration of monensin suppressed progression of the intestinal tumors without any sign of toxicity on normal mucosa. Our data suggest monensin as a prospective anticancer drug for therapy of neoplasia with deregulated Wnt signaling. Mol Cancer Ther; 13(4); 812-22. Ó2014 AACR.

IRESite is an exhaustive, manually annotated non-redundant relational database focused on the IRES elements (Internal Ribosome Entry Site) and containing information not available in the primary public databases. IRES elements were originally found in eukaryotic viruses hijacking initiation of translation of their host. Later on, they were also discovered in 5′-untranslated regions of some eukaryotic mRNA molecules. Currently, IRESite presents up to 92 biologically relevant aspects of every experiment, e.g. the nature of an IRES element, its functionality/defectivity, origin, size, sequence, structure, its relative position with respect to surrounding protein coding regions, positive/negative controls used in the experiment, the reporter genes used to monitor IRES activity, the measured reporter protein yields/activities, and references to original publications as well as cross-references to other databases, and also comments from submitters and our curators. Furthermore, the site presents the known similarities to rRNA sequences as well as RNA–protein interactions. Special care is given to the annotation of promoter-like regions. The annotated data in IRESite are bound to mostly complete, full-length mRNA, and whenever possible, accompanied by original plasmid vector sequences. New data can be submitted through the publicly available web-based interface at and are curated by a team of lab-experienced biologists.

T-cell factor 4 (TCF4), together with β-catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.