Wnt/β-catenin signaling controls numerous steps in normal animal development and can also cause cancer if inappropriately activated. In the absence of Wnt, β-catenin is targeted continuously for proteasomal degradation by the Axin destruction complex, whose activity is blocked upon Wnt stimulation by Dishevelled, which recruits Axin to the plasma membrane and assembles it into a signalosome. This key event during Wnt signal transduction depends on dynamic headto-tail polymerization by the DIX domain of Dishevelled. Here, we use rescue assays in Drosophila tissues and functional assays in human cells to show that polymerization-blocking mutations in the DIX domain of Axin disable its effector function in down-regulating Armadillo/β-catenin and its response to Dishevelled during Wnt signaling. Intriguingly, NMR spectroscopy revealed that the purified DIX domains of the two proteins interact with each other directly through their polymerization interfaces, whereby the same residues mediate both homo-and heterotypic interactions. This result implies that Dishevelled has the potential to act as a "natural" dominantnegative, binding to the polymerization interface of Axin's DIX domain to interfere with its self-assembly, thereby blocking its effector function.T he Wnt effector β-catenin is a transcriptional coactivator that controls numerous cell fates in normal animal development and tissue homeostasis, and it can also mutate to a potent oncogene (1, 2). In the absence of a Wnt signal, β-catenin binds to the adenomatous polyposis coli (APC) tumor suppressor and is thus recruited to the Axin destruction complex, which promotes its phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β) to target it for proteasomal degradation. Phosphorylation of β-catenin depends critically on a scaffolding effect afforded by Axin, which binds simultaneously to GSK3β and its β-catenin substrate through a central domain (3, 4). Upon Wnt stimulation, Dishevelled (Dsh in flies, or Dvl in mammals) interacts with Axin to recruit it to the plasma membrane (PM) (5), where Dvl assembles a stable signalosome in which it stimulates the phosphorylation of multiple motifs in the cytoplasmic tail of the LRP6 coreceptor (6-8). One of these phosphorylated motifs (phospho-PPPSPXS/T) acts as a direct competitive inhibitor of GSK3β (9, 10), blocking its activity toward β-catenin, thus allowing unphosphorylated β-catenin to accumulate and operate a transcriptional switch in the nucleus-the key functional output of Wnt/β-catenin signaling in normal development and in disease (1, 2).Axin contains two structured and conserved domains at its termini that mediate additional functional interactions: through its N-terminal RGS domain, it binds directly to APC (11, 12), whereas its C terminus contains a DIX domain that mediates Axin homodimerization (13-15) and that is also required but not sufficient for Axin's interaction with . The DIX domain is found only in two other protein families-namely in Dsh/Dvl proteins (see Results) and in ...

The single protein caveolar coat complex comprises only cavins and caveolins, coats the caveolar bulb, and is probably responsible for creating caveolae.

SummaryWnt/-catenin signalling controls cell fates in development, tissue homeostasis and cancer. Wnt binding to Frizzled receptors triggers recruitment of Dishevelled to the plasma membrane and formation of a signalosome containing the LRP5/6 co-receptor, whose cytoplasmic tail (ctail) thus becomes phosphorylated at multiple PPP(S/T)Px(S/T) motifs. These then directly inhibit GSK3, which results in -catenin accumulation and signalling. Here, we revisit previous epistasis experiments, and show that Dishevelled signals through LRP5/6 in human cells and Drosophila embryos. To recapitulate this signalling event, and to define its functional elements, we fused the Dishevelled DIX domain to the LRP6 ctail, which forms cytoplasmic signalosomes with potent signalling activity mediated by its PPP(S/T)Px(S/T) motifs. Their phosphorylation and activity depends critically on DIX-mediated polymerization, and on multiple stability elements in the LRP6 ctail, including the T1479 epitope upstream of the membrane-proximal PPP(S/T)Px(S/T) motif. Thus, stable polymerization emerges as a key principle underlying the function of Dishevelled-dependent signalosomes.

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1−/− mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo.