Comparative Genetic Screens in Human Cells Reveal New Regulatory Mechanisms in WNT Signaling

The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling β-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the β-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems

The ADP-Ribose Polymerase Tankyrase Regulates Adult Intestinal Stem Cell Proliferation During Homeostasis in Drosophila

Wnt/β-catenin signaling controls intestinal stem cell (ISC) proliferation, and is aberrantly activated in colorectal cancer. Inhibitors of the ADP-ribose polymerase Tankyrase (Tnks) have become lead therapeutic candidates for Wnt-driven cancers, following the recent discovery that Tnks targets Axin, a negative regulator of Wnt signaling, for proteolysis. Initial reports indicated that Tnks is important for Wnt pathway activation in cultured human cell lines. However, the requirement for Tnks in physiological settings has been less clear, as subsequent studies in mice, fish and flies suggested that Tnks was either entirely dispensable for Wnt-dependent processes in vivo, or alternatively, had tissue-specific roles. Here, using null alleles, we demonstrate that the regulation of Axin by the highly conserved Drosophila Tnks homolog is essential for the control of ISC proliferation. Furthermore, in the adult intestine, where activity of the Wingless pathway is graded and peaks at each compartmental boundary, Tnks is dispensable for signaling in regions where pathway activity is high, but essential where pathway activity is relatively low. Finally, as observed previously for Wingless pathway components, Tnks activity in absorptive enterocytes controls the proliferation of neighboring ISCs non-autonomously by regulating JAK/STAT signaling. These findings reveal the requirement for Tnks in the control of ISC proliferation and suggest an essential role in the amplification of Wnt signaling, with relevance for development, homeostasis and cancer

Wingless stimulation results in increased membrane-associated ADP-ribosylated Axin

<p>(A, left panel) Subcellular fractionation of lysates from S2R+ cells treated with either control medium (CTR) or Wingless conditioned medium (Wg CM) for 1 hour. The total cell lysates (T), cytoplasmic (C), and membrane (M) fractions were analyzed by SDS-PAGE. Immunoblotting with phosphorylated LRP6 antibody shows activation of the Wnt pathway following treatment with Wg CM. Immunoblotting with Axin antibody shows no significant shift from the cytoplasmic fraction to membrane fraction. Tubulin, a cytoplasmic marker, and Nervana, a membrane marker, were used to assay the efficiency of fractionation. Asterisk indicates a non-specific band with p-LRP6 antibody. (A, right panel) Following cell fractionation, GST-WWE pulldown was performed with cytoplasmic (C) and membrane (M) fractions. Immunoblotting with Axin antibody reveals that the majority of ADP-ribosylated Axin is in the membrane fraction, with little ADP-ribosylated Axin detected in the cytoplasm. Phospho-LRP6 is pulled down by GST-WWE in the membrane fraction in response to Wingless. (B) Model for dual roles of membrane-associated Axin. In the absence of Wnt stimulation, a pool of Axin is localized in membrane-proximal puncta, which might represent the sites of the destruction complex. Membrane-associated Axin is targeted for degradation by Tnks in the unstimulated state, which contributes to maintaining Axin at the limiting concentrations important for regulation of Wnt signaling. Wnt stimulation induces a rapid increase in the level of Axin, and in particular the membrane-associated ADP-ribosylated Axin pool. ADP-ribosylation enhances the interaction of Axin with phospho-LRP6/Arrow, and thus promotes the activation of signaling. R: ADP-ribosylation.</p

Dual Roles for Membrane Association of Drosophila Axin in Wnt Signaling

<div><p>Deregulation of the Wnt signal transduction pathway underlies numerous congenital disorders and cancers. Axin, a concentration-limiting scaffold protein, facilitates assembly of a “destruction complex” that prevents signaling in the unstimulated state and a plasma membrane-associated “signalosome” that activates signaling following Wnt stimulation. In the classical model, Axin is cytoplasmic under basal conditions, but relocates to the cell membrane after Wnt exposure; however, due to the very low levels of endogenous Axin, this model is based largely on examination of Axin at supraphysiological levels. Here, we analyze the subcellular distribution of endogenous Drosophila Axin <i>in vivo</i> and find that a pool of Axin localizes to cell membrane proximal puncta even in the absence of Wnt stimulation. Axin localization in these puncta is dependent on the destruction complex component Adenomatous polyposis coli (Apc). In the unstimulated state, the membrane association of Axin increases its Tankyrase-dependent ADP-ribosylation and consequent proteasomal degradation to control its basal levels. Furthermore, Wnt stimulation does not result in a bulk redistribution of Axin from cytoplasmic to membrane pools, but causes an initial increase of Axin in both of these pools, with concomitant changes in two post-translational modifications, followed by Axin proteolysis hours later. Finally, the ADP-ribosylated Axin that increases rapidly following Wnt stimulation is membrane associated. We conclude that even in the unstimulated state, a pool of Axin forms membrane-proximal puncta that are dependent on Apc, and that membrane association regulates both Axin levels and Axin’s role in the rapid activation of signaling that follows Wnt exposure.</p></div

Membrane-association promotes Axin degradation through Tankyrase-dependent ADP-ribosylation

<p>(A) Lysates from third instar larvae expressing indicated transgenes with the <i>C765-Gal4</i> driver were analyzed by immunoblotting. Myr-Axin-V5 is present at much lower levels than Axin-V5 or Myr^G-A-Axin-V5. Kinesin was used as a loading control. (B) Quantification of the relative levels of indicated proteins. Results were obtained from four independent experiments with representative blot shown in (A). Values indicate mean ± SD. *** <i>p</i><0.001, ns: not significant (t-test). (C) S2R+ cells were transfected with the indicated plasmids. Lysates were analyzed by SDS-PAGE. Immunoblotting with V5 antibody revealed that Myr-AxinΔTBD-V5 is present at higher levels than Myr-Axin-V5. Tubulin was used as a loading control. (D) Lysates from third instar larvae of indicated genotypes were analyzed by immunoblotting. Transgene was expressed with the <i>C765-Gal4</i> driver. Eliminating Tnks restores the protein levels of Myr-Axin-V5. Kinesin was used as a loading control. (E) Quantification of the relative protein levels of Axin-V5 or Myr-Axin-V5 from lysates of third instar larvae of indicated genotype. Results were obtained from three independent experiments with a representative blot shown in (D). Values indicate mean ± SD. * p = 0.0163, ns: not significant (t-test). (F) Use of GST-WWE pull-down assay for detection of ADP-ribosylated Axin. Lysates from third instar larvae expressing <i>Axin-V5</i> with the <i>C765-Gal4</i> driver were incubated with GST-WWE or GST-WWE^R163A beads. Axin-V5 is pulled down by GST-WWE, but not GST-WWE^R163A, suggesting this assay specifically detects ADP-ribosylated Axin. (G) GST-WWE pulldown from lysates of third instar larvae expressing indicated transgene with the <i>C765-Gal4</i> driver. ADP-ribosylation of Axin is abolished in <i>Tnks</i>^<i>19</i> null mutants. Total indicates total larval lysates and pd indicates samples pulled down with GST-WWE beads. (H) GST-WWE pulldown from lysates of third instar larvae of indicated genotypes. Myr-Axin-V5 is highly ADP-ribosylated by comparison with Axin-V5 or Myr^G-A-Axin-V5.</p

Axin is localized at cell membrane-proximal puncta independently of Wingless pathway activation

<p>(A-I) Confocal images of third instar larval wing imaginal discs stained with antibodies indicated at bottom right; genotypes at left margin. (A-C) Wing disc stained with β-gal (A, magenta), Axin (B, green), and Arm (C, blue) antibodies. <i>Axin</i>^<i>18</i> null mutant clones (marked by the absence of β-gal, -/- in A) demonstrate the specificity of the Axin antibody. Armadillo marks the adherens junctions, which are present at the boundary between the apical and basolateral membrane, and also accumulates in the cytoplasm in <i>Axin</i> mutant clones. At this apical level, Axin staining is diffuse in the cytoplasm of all cells. (D-I) Axin staining at basolateral levels in the wing disc. <i>Axin</i>^<i>18</i> null mutant clones are marked by the absence of β-gal (D) or by dashed line (G). At this level, Axin antibody reveals specific staining that partially overlaps the basolateral membrane marker Fas III (I). Higher magnification views of the boxed area in (H) reveals endogenous Axin is present in puncta at or near the plasma membrane (G’-I’). Images were taken at the periphery of the wing discs. (J-L) Wild-type pupal wing (~28 hrs after pupa formation) double labeled with α-Dlg (J) and α-Axin (K). Endogenous Axin is also present in puncta proximal to the cell membrane in pupal wing (L). (M) Subcellular fractionation of lysates from S2R+ cells. The total lysates, cytoplasmic, and membrane fractions were analyzed by SDS-PAGE. Immunoblotting with Axin antibody revealed that Axin is present in both the cytoplasmic and membrane fractions. The efficiency of the fractionation was assayed by the presence of Arrow and Tubulin, membrane and cytoplasmic markers, respectively. (N) Quantification of the distribution of endogenous Axin in S2R+ cells. Results were obtained from four independent experiments, with a representative blot shown in (M). Values indicate mean ± SD. (O) Subcellular fractionation of lysates from 0–2.5 hour wild-type embryos. (P) Quantification of the distribution of endogenous Axin in 0–2.5 hour wild-type embryos. Results were obtained from four independent experiments, with a representative blot shown in (O). Values indicate mean ± SD. Scale bar: 5μm.</p

An <i>in vivo</i> system for analysis of membrane-associated Axin

<p>(A-B) Confocal images of adult midguts expressing <i>Axin-V5</i> or <i>Myr-Axin-V5</i> using the <i>Myo1A-Gal4</i> driver. Midguts were stained with anti-Axin (magenta) and DAPI (blue). (C) Subcellular fractionation of lysates from S2R+ cells transfected with indicated plasmids. The total cell lysate (T), cytoplasmic (C) and membrane (M) fractions were analyzed by immunoblotting with V5 antibody. Myr-Axin-V5 is present mainly in the membrane fraction, whereas Axin-V5 is present in both the cytoplasmic and membrane fractions. The efficiency of the fractionation was assayed by the presence of Arrow and Tubulin, membrane and cytoplasmic markers, respectively. (D) Total Axin levels in wing imaginal discs expressing indicated transgenes with the <i>C765-Gal4</i> driver. <i>C765-Gal4</i> flies were used as control. (E) Quantification of the relative total Axin protein levels in wing discs with indicated genotypes. Results were obtained from four independent experiments with a representative blot shown in (D). Values indicate mean ± SD. (F-K) Expressing <i>Axin-V5</i> or <i>Myr-Axin-V5</i> with the <i>C765-Gal4</i> driver in larval wing discs does not disrupt expression of the Wingless target gene <i>senseless</i> (F-H), or the morphology of adult wings (I-K). Yellow arrows in (F-H) indicate the dorsoventral boundary of the larval wing disc. Boxed areas in (I-K) are shown in (I’-K’). 15–20 flies of each genotype were examined. Scale bar: 20μm.</p

Apc is required for the localization of Axin to puncta juxtaposed with cell membrane.

<p>Images of third instar larval wing imaginal discs stained with indicated antibodies; genotypes at left margin. (A-C) Apc2 antibody revealed specific staining of endogenous Apc2 (green), which is absent in <i>Apc2</i>^{<i>19</i>.<i>3</i>} null mutant clones (marked by the absence of β-gal (magenta), -/- in A). (D-F) Double staining with Fas III (magenta) and Apc2 antibodies indicated that Apc2 partially overlaps Fas III and is enriched at cell cortex. (G-I) Double staining with Apc2 and Axin (magenta) antibodies reveals that Apc2 is present at some membrane-proximal Axin puncta (white arrow), whereas distinct Apc2 or Axin puncta are also observed (yellow and red arrowheads respectively). (J-N) Wing disc triple labeled with β-gal (J, magenta), Axin (K, green) and Fas III (M, red) antibodies. Merge of Axin and β-gal is shown in (L), and merge of Axin and Fas III is in (N). <i>Apc2</i>^{<i>19</i>.<i>3</i>} homozygous null mutant clones are marked by the absence of β-gal (-/- in J). Endogenous Axin staining indicates reduced Axin puncta at the basolateral membrane in <i>Apc2</i>^{<i>19</i>.<i>3</i>} null mutant clones (K, L, N). Fas III localization is not disrupted in <i>Apc2</i>^{<i>19</i>.<i>3</i>} mutant clones (M). (O-S) Wing discs bearing <i>Apc1</i>^<i>Q8</i> null mutant clones (marked by the absence of β-gal staining, -/- in O) were stained with β-gal (O), Axin (P) and Fas III (R) antibodies. Merge of Axin and β-gal is shown in (Q), and merge of Axin and Fas III is in (S). Axin puncta are reduced at the basolateral membrane in <i>Apc1</i>^<i>Q8</i> mutant clones (P, Q, S). Fas III localization is not disrupted in <i>Apc1</i>^<i>Q8</i> mutant clones (R). Scale bar: 5μm.</p

Axin membrane association does not require Tnks-dependent ADP-ribosylation

<p>(A) Subcellular fractionation of lysates from 0–2.5 hour old wild-type embryos and <i>Tnks</i>^<i>503</i> null mutant embryos. The total lysates (T), cytoplasmic (C), and membrane (M) fractions were analyzed by SDS-PAGE. Immunoblotting with Axin antibody revealed that Axin is present in both the membrane and cytoplasmic fractions in both wild-type embryos and <i>Tnks</i>^<i>503</i> null mutants. The efficiency of the fractionation was assayed by the presence of Arrow and Tubulin, membrane and cytoplasmic markers, respectively. (B) Subcellular fractionation of lysates from S2R+ cells transfected with the indicated plasmids. The total lysates (T), cytoplasmic (C), and membrane (M) fractions were analyzed by SDS-PAGE. Immunoblot with V5 antibody revealed that Axin-V5 and AxinΔTBD-V5 are localized in both the membrane and cytoplasmic compartments. The efficiency of the fractionation was assayed by the presence of Arrow and Tubulin, membrane and cytoplasmic markers respectively.</p

Axin phosphorylation in both Wnt-off and Wnt-on states requires the tumor suppressor APC

<div><p>The aberrant activation of Wnt signal transduction initiates the development of 90% of colorectal cancers, the majority of which arise from inactivation of the tumor suppressor Adenomatous polyposis coli (APC). In the classical model for Wnt signaling, the primary role of APC is to act, together with the concentration-limiting scaffold protein Axin, in a “destruction complex” that directs the phosphorylation and consequent proteasomal degradation of the transcriptional activator β-catenin, thereby preventing signaling in the Wnt-off state. Following Wnt stimulation, Axin is recruited to a multiprotein “signalosome” required for pathway activation. Whereas it is well-documented that APC is essential in the destruction complex, APC’s role in this complex remains elusive. Here, we demonstrate in Drosophila that Axin exists in two distinct phosphorylation states in Wnt-off and Wnt-on conditions, respectively, that underlie its roles in the destruction complex and signalosome. These two Axin phosphorylation states are catalyzed by glycogen synthase kinase 3 (GSK3), and unexpectedly, completely dependent on APC in both unstimulated and Wnt-stimulated conditions. In a major revision of the classical model, we show that APC is essential not only in the destruction complex, but also for the rapid transition in Axin that occurs after Wnt stimulation and Axin’s subsequent association with the Wnt co-receptor LRP6/Arrow, one of the earliest steps in pathway activation. We propose that this novel requirement for APC in Axin regulation through phosphorylation both prevents signaling in the Wnt-off state and promotes signaling immediately following Wnt stimulation.</p></div