Wnt regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector β-catenin (β-cat). The multiprotein destruction complex binds and phosphorylates β-cat and transfers it to the SCF-TrCP E3-ubiquitin ligase for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing β-cat. Recent work clarified underlying mechanisms, but important questions remain. We explore β-cat transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin\u27s RGS domain plays an important role. We find that elevating Dishevelled levels i

Abelson kinase acts as a robust, multifunctional scaffold in regulating embryonic morphogenesis

Abelson family kinases (Abl) are key regulators of cell behavior and the cytoskeleton during development and in leukemia. Abl's SH3, SH2, and tyrosine kinase domains are joined via a linker to an F-actin-binding domain (FABD). Research on Abl's roles in cell culture led to several hypotheses for its mechanism of action: 1) Abl phosphorylates other proteins, modulating their activity. 2) Abl directly regulates the cytoskeleton via its cytoskeletal interaction domains, and/or 3) Abl is a scaffold for a signaling complex. The importance of these roles during normal development remains untested. We tested these mechanistic hypotheses during Drosophila morphogenesis using a series of mutants to examine Abl's many cell biological roles. Strikingly, Abl lacking the FABD fully rescued morphogenesis, cell shape change, actin regulation, and viability, while kinase dead Abl, though reduced in function, retained substantial rescuing ability in some but not all Abl functions. We also tested the function of four conserved motifs in the linker region, revealing a key role for a conserved PXXP motif known to bind Crk and Abi. We propose Abl acts as a robust multi-domain scaffold with different protein motifs and activities contributing differentially to diverse cellular behaviors

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Frs2α and Shp2 signal independently of Gab to mediate FGF signaling in lens development

Fibroblast growth factor (FGF) signaling requires a plethora of adaptor proteins to elicit downstream responses, but the functional significances of these docking proteins remain controversial. In this study, we used lens development as a model to investigate Frs2α and its structurally related scaffolding proteins, Gab1 and Gab2, in FGF signaling. We show that genetic ablation of Frs2α alone has a modest effect, but additional deletion of tyrosine phosphatase Shp2 causes a complete arrest of lens vesicle development. Biochemical evidence suggests that this Frs2α-Shp2 synergy reflects their epistatic relationship in the FGF signaling cascade, as opposed to compensatory or parallel functions of these two proteins. Genetic interaction experiments further demonstrate that direct binding of Shp2 to Frs2α is necessary for activation of ERK signaling, whereas constitutive activation of either Shp2 or Kras signaling can compensate for the absence of Frs2α in lens development. By contrast, knockout of Gab1 and Gab2 failed to disrupt FGF signaling in vitro and lens development in vivo. These results establish the Frs2α-Shp2 complex as the key mediator of FGF signaling in lens development

Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo

<div><p>Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in <i>Drosophila</i> embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.</p></div

The destruction complex contains thousands of APC2 or Axin molecules after over-expression in SW480 cells, and 10-100s of Axin molecules in vivo in embryos.

<p>(A) Representative images of live samples used for fluorescence comparisons to calculate GFP molecule numbers. Each panel is scaled to the same size and brightness. Ndc80:GFP assembles into a structure containing ~306 GFP molecules while Mif2:GFP assembles into a structure containing ~58 GFP molecules. (B) Pattern of Axin:GFP accumulation and localization in a live embryo. Comparison to our fixed samples allowed identification of regions receiving Wg signal (dimmer puncta) or not receiving Wg signal (brighter puncta). (C-E) Estimated number of GFP molecules per punctum. Each dot = an individual punctum analyzed. Means and standard deviation are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007339#pgen.1007339.s016" target="_blank">S8 Table</a>. (C) GFP Molecule counts from SW480 colorectal cancer cells expressing Axin:GFP alone, Axin:GFP plus RFP:APC2, or GFP:APC2 in addition to Axin:RFP. (D-E) GFP molecule counts <i>in vivo</i> from stage 9 embryos expressing RFP and Axin:GFP under the control of MatGAL4 (Mat RFP&Axin). (E) Quantification of puncta GFP molecule counts from D, after being separated into those in presumptive regions receiving or not receiving Wg signals (as in B). Statistical analysis via an unpaired t-test.</p

Dsh accumulates at similar levels to Axin and APC2, and co-localizes with Axin puncta in Wg-ON but not Wg-OFF cells.

<p>(A) Immunoblot with anti-Dsh antibody, wildtype embryos and three different lines expressing Dsh:GFP driven by its endogenous promotor. Tubulin was the loading control. (B) Left. Immunoblot with anti-GFP antibody, embryos expressing GFP:APC2 driven by its endogenous promotor, Zyg Axin:GFP, or expressing Dsh:GFP driven by its endogenous promotor. Right. Quantification of levels of Dsh:GFP normalized to those of Zyg Axin:GFP. (C-G) Stage 9 embryos, anterior to the left. (C) Dsh localization in a wildtype embryo. There is subtle enrichment of Dsh at the membrane in cells receiving Wg signal (double arrows). (D) In embryos expressing Axin:GFP at 9x endogenous levels (Mat Axin), Dsh co-localizes with membrane associated Axin puncta in Wg-ON cells (white arrows) but not with cytoplasmic Axin puncta in Wg-OFF cells (blue arrowheads). (E) A similar pattern of Dsh recruitment is seen when Axin:GFP is expressed at 4x endogenous levels (MatAxin&RFP). Insets show higher magnification views of region in box. (F,G) Stage 9 wildtype embryo (F) versus embryo overexpressing Dsh (G), using MatGAL4 to drive UAS-Dsh:Myc (Mat Dsh). The elevation of Dsh levels is apparent, but there is little or no effect on Arm regulation. (H) Immunoblot with anti-Dsh antibody. Wildtype embryos versus embryos overexpressing Dsh (Mat Dsh). Tubulin is a loading control. (I) Immunoblot with anti-Axin antibodies and quantification. Axin levels remain unchanged after Dsh overexpression. A one-way t-test was used to assess the significance of difference in Axin levels.</p

Elevating Axin produces dose-sensitive inhibition of Wg signaling, while increasing APC2 levels does not.

<p>(A) Embryonic viability of indicated genotypes. (B,C) Assessing the effect of elevating Axin or APC2 levels on Wg-regulated cell fates. (B) Range of cuticle phenotypes of embryos/larvae of each genotype—since not all genotypes are lethal, phenotypes include those of hatched larvae. (C) Representative images of cuticle phenotypes used in B. Anterior to the top. 1: Wildtype. 2: 1–2 merged denticle belts (brackets). 3: 3–4 merged denticle belts. 4: Most denticle belts merged, mouth parts still present. 5: <i>wg</i> null phenotype–denticle lawn and no head (arrow). (D) Quantification of number of rows of En-expressing cells per segment. Embryos analyzed: WT-21, AxinRNAi-5, APC2^g10- 5, wg^IG22–14, Zyg Axin- 9, Mat RFP&Axin- 11, Mat Axin- 18, Mat APC2- 12. * = p<0.05 using a one-way ANOVA test. (E-J) Representative images, En expression, as quantified in D. Anterior to the left.</p

Endogenous APC2 and Axin proteins accumulate at similar levels.

<p>(A) mRNA levels (RNAseq) of <i>APC1</i> (light blue), <i>APC2</i> (blue), and <i>Axin</i> (yellow) during <i>Drosophila</i> embryogenesis. Levels are Fragments Per Kilobase of transcript per Million mapped reads (FPKM). (B-D) Immunoblots, 4-8hr old <i>Drosophila</i> embryos. Tubulin is loading control. n = # of blots quantified (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007339#pgen.1007339.s009" target="_blank">S1 Table</a>). (B) Anti-Axin antibody. Endogenous Axin levels versus those in Axin RNAi or Zyg Axin:GFP embryos. Endogenous Axin runs as doublet ~75kDa (red arrowheads) while Axin:GFP runs at ~105kDa (yellow arrowhead). * = background band. (C) Anti-APC2 antibody. Endogenous APC2 levels versus those of a GFP:APC2 transgene expressed under its endogenous promoter in an <i>APC2</i> null (<i>APC2</i>^<i>g10</i>) background. (D) Anti-GFP antibody. Relative levels of GFP:APC2 expressed under its endogenous promoter versus Zyg Axin:GFP.</p

Elevating APC2 levels increases the ability of endogenous Wg signaling to turn down the destruction complex, thus increasing Arm levels in cells receiving Wg.

<p>(A-D) Fixed Stage 9 embryos. Anterior to the left. (A-C) Representative images, wildtype (A) or Mat GFP:APC2 embryos with higher (B) or lower (C) levels of GFP:APC2 expression. Elevating APC2 levels increases levels of Arm specifically in cells receiving Wg signal. (D) Close-up, embryo expressing elevated levels of GFP:APC2. The boundary of cells with elevated levels of Arm is quite sharp, and does not expand much farther than the cells adjacent to those expressing Wg. (E) Elevating APC2 levels increases Arm accumulation in Wg stripes but does not affect Arm levels in interstripes. Box and whisker plot (as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007339#pgen.1007339.g004" target="_blank">Fig 4I and 4K</a>), comparing Arm accumulation levels in Wg-expressing stripes versus Arm in the interstripes in wildtype or Mat GFP:APC2 embryos imaged on the same slide. (F) Difference in Arm accumulation between the Wg stripes and interstripes within individual embryos. Each point = a single embryo. (G) Plots of Arm accumulation pattern over 2 segments. Dot plots = raw data from 3 separate embryos. Line graphs underneath = averages of these data. Elevating APC2 levels exaggerates and sharpens the Arm stripes. (H) Immunoblotting with anti-Axin antibodies reveals that embryos overexpressing APC2 have no change in Axin levels. Statistical analysis: a paired t-test was used to assess the significance between intragroup values in E, and an unpaired t-test was used to determine the significance between intergroup values in E and F. A one-way t-test was used to assess the significance of difference in Axin levels in H. ns, not significant i.e. p≥ 0.05. ** = p<0.01. *** = p<0.001. **** = p<0.0001.</p