Compensatory flux changes within an endocytic trafficking network maintain thermal robustness of Notch signaling.

Developmental signaling is remarkably robust to environmental variation, including temperature. For example, in ectothermic animals such as Drosophila, Notch signaling is maintained within functional limits across a wide temperature range. We combine experimental and computational approaches to show that temperature compensation of Notch signaling is achieved by an unexpected variety of endocytic-dependent routes to Notch activation which, when superimposed on ligand-induced activation, act as a robustness module. Thermal compensation arises through an altered balance of fluxes within competing trafficking routes, coupled with temperature-dependent ubiquitination of Notch. This flexible ensemble of trafficking routes supports Notch signaling at low temperature but can be switched to restrain Notch signaling at high temperature and thus compensates for the inherent temperature sensitivity of ligand-induced activation. The outcome is to extend the physiological range over which normal development can occur. Similar mechanisms may provide thermal robustness for other developmental signals.

SRC-induced disassembly of adherens junctions requires localized phosphorylation and degradation of the rac activator tiam1.

The Rac activator Tiam1 is required for adherens junction (AJ) maintenance, and its depletion results in AJ disassembly. Conversely, the oncoprotein Src potently induces AJ disassembly and epithelial-mesenchymal transition (EMT). Here, we show that Tiam1 is phosphorylated on Y384 by Src. This occurs predominantly at AJs, is required for Src-induced AJ disassembly and cell migration, and creates a docking site on Tiam1 for Grb2. We find that Tiam1 is associated with ERK. Following recruitment of the Grb2-Sos1 complex, ERK becomes activated and triggers the localized degradation of Tiam1 at AJs, likely involving calpain proteases. Furthermore, we demonstrate that, in human tumors, Y384 phosphorylation positively correlates with Src activity, and total Tiam1 levels are inversely correlated. Thus, our data implicate Tiam1 phosphorylation and consequent degradation in Src-mediated EMT and resultant cell motility and establish a paradigm for regulating local concentrations of Rho-GEFs.

RasGAP mediates neuronal survival in Drosophila through direct regulation of Rab5-dependent endocytosis.

The GTPase Ras can either promote or inhibit cell survival. Inactivating mutations in Drosophila RasGAP (encoded by vap), a Ras GTPase-activating protein, lead to age-related brain degeneration. Genetic interactions implicate the epidermal growth factor receptor (EGFR)-Ras pathway in promoting neurodegeneration but the mechanism is not known. Here, we show that the Src homology 2 (SH2) domains of RasGAP are essential for its neuroprotective function. By using affinity purification and mass spectrometry, we identify a complex containing RasGAP together with Sprint, which is a Ras effector and putative activator of the endocytic GTPase Rab5. Formation of the RasGAP-Sprint complex requires the SH2 domains of RasGAP and tyrosine phosphorylation of Sprint. RasGAP and Sprint colocalize with Rab5-positive early endosomes but not with Rab7-positive late endosomes. We demonstrate a key role for this interaction in neurodegeneration: mutation of Sprint (or Rab5) suppresses neuronal cell death caused by the loss of RasGAP. These results indicate that the long-term survival of adult neurons in Drosophila is crucially dependent on the activities of two GTPases, Ras and Rab5, regulated by the interplay of RasGAP and Sprint.

Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains.

Different membrane microdomain compositions provide unique environments that can regulate signaling receptor function. We identify microdomains on the endosome membrane of Drosophila endosomes, enriched in lipid-raft or clathrin/ESCRT-0, which are associated with Notch activation by distinct, ligand-independent mechanisms. Transfer of Notch between microdomains is regulated by Deltex and Suppressor of deltex ubiquitin ligases and is limited by a gate-keeper role for ESCRT complexes. Ubiquitination of Notch by Deltex recruits it to the clathrin/ESCRT-0 microdomain and enhances Notch activation by an ADAM10-independent/TRPML-dependent mechanism. This requirement for Deltex is bypassed by the downregulation of ESCRT-III. In contrast, while ESCRT-I depletion also activates Notch, it does so by an ADAM10-dependent/TRPML-independent mechanism and Notch is retained in the lipid raft-like microdomain. In the absence of such endosomal perturbation, different activating Notch mutations also localize to different microdomains and are activated by different mechanisms. Our findings demonstrate the interplay between Notch regulators, endosomal trafficking components, and Notch genetics, which defines membrane locations and activation mechanisms.

The Rac activator STEF (Tiam2) regulates cell migration by microtubule-mediated focal adhesion disassembly.

Focal adhesion (FA) disassembly required for optimal cell migration is mediated by microtubules (MTs); targeting of FAs by MTs coincides with their disassembly. Regrowth of MTs, induced by removal of the MT destabilizer nocodazole, activates the Rho-like GTPase Rac, concomitant with FA disassembly. Here, we show that the Rac guanine nucleotide exchange factor (GEF) Sif and Tiam1-like exchange factor (STEF) is responsible for Rac activation during MT regrowth. Importantly, STEF is required for multiple targeting of FAs by MTs. As a result, FAs in STEF-knockdown cells have a reduced disassembly rate and are consequently enlarged. This leads to reduced speed of migration. Together, these findings suggest a new role for STEF in FA disassembly and cell migration through MT-mediated mechanisms.

Targeting ligand-dependent wnt pathway dysregulation in gastrointestinal cancers through porcupine inhibition.

Gastrointestinal cancers are responsible for more cancer deaths than any other system of the body. This review summarises how Wnt pathway dysregulation contributes to the development of the most common gastrointestinal cancers, with a particular focus on the nature and frequency of upstream pathway aberrations. Tumors with upstream aberrations maintain a dependency on the presence of functional Wnt ligand, and are predicted to be tractable to inhibitors of Porcupine, an enzyme that plays a key role in Wnt secretion. We summarise available pre-clinical efficacy data from Porcupine inhibitors in vitro and in vivo, as well as potential toxicities and the data from early phase clinical trials. We appraise the rationale for biomarker-defined targeted approaches, as well as outlining future opportunities for combination with other therapeutics.

The Wnt Pathway Inhibitor RXC004 Blocks Tumor Growth and Reverses Immune Evasion in Wnt Ligand-dependent Cancer Models.

Wnt signaling is implicated in the etiology of gastrointestinal tract cancers. Targeting Wnt signaling is challenging due to on-target toxicity concerns and lack of druggable pathway components. We describe the discovery and characterization of RXC004, a potent and selective inhibitor of the membrane-bound o-acyl transferase Porcupine, essential for Wnt ligand secretion. Absorption, distribution, metabolism, and excretion and safety pharmacology studies were conducted with RXC004 in vitro, and pharmacokinetic exposure assessed in vivo. RXC004 effects on proliferation and tumor metabolism were explored in genetically defined colorectal and pancreatic cancer models in vitro and in vivo. RXC004 effects on immune evasion were assessed in B16F10 immune "cold" and CT26 immune "hot" murine syngeneic models, and in human cell cocultures. RXC004 showed a promising pharmacokinetic profile, inhibited Wnt ligand palmitoylation, secretion, and pathway activation, and demonstrated potent antiproliferative effects in Wnt ligand-dependent (RNF43-mutant or RSPO3-fusion) colorectal and pancreatic cell lines. Reduced tumor growth and increased cancer cell differentiation were observed in SNU-1411 (RSPO3-fusion), AsPC1 and HPAF-II (both RNF43-mutant) xenograft models, with a therapeutic window versus Wnt homeostatic functions. Additional effects of RXC004 on tumor cell metabolism were confirmed in vitro and in vivo by glucose uptake and 18fluorodeoxyglucose-PET, respectively. RXC004 stimulated host tumor immunity; reducing resident myeloid-derived suppressor cells within B16F10 tumors and synergizing with anti-programmed cell death protein-1 (PD-1) to increase CD8+/regulatory T cell ratios within CT26 tumors. Moreover, RXC004 reversed the immunosuppressive effects of HPAF-II cells cocultured with human peripheral blood mononuclear cells, confirming the multiple anticancer mechanisms of this compound, which has progressed into phase II clinical trials. Significance: Wnt pathway dysregulation drives many gastrointestinal cancers; however, there are no approved therapies that target the pathway. RXC004 has demonstrated the potential to block both tumor growth and tumor immune evasion in a genetically defined, clinically actionable subpopulation of Wnt ligand-dependent gastrointestinal cancers. The clinical utility of RXC004, and other Porcupine inhibitors, in such Wnt ligand-dependent cancers is currently being assessed in patient trials.

A modified tandem affinity purification technique identifies that 14-3-3 proteins interact with Tiam1, an interaction which controls Tiam1 stability.

The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 has important functions in multiple cellular processes including proliferation, apoptosis and adherens junction maintenance. Here we describe a modified tandem affinity purification (TAP) technique that we have applied to specifically enrich Tiam1-containing protein complexes from mammalian cells. Using this technique in conjunction with LC-MS/MS mass spectrometry, we have identified additional Tiam1-interacting proteins not seen with the standard technique, and have identified multiple 14-3-3 family members as Tiam1 interactors. We confirm the Tiam1/14-3-3 protein interaction by GST-pulldown and coimmunoprecipitation experiments, show that it is phosphorylation-dependent, and that they colocalize in cells. The interaction is largely dependent on the N-terminal region of Tiam1; within this region, there are four putative phospho-serine-containing 14-3-3 binding motifs, and we confirm that two of them (Ser172 and Ser231) are phosphorylated in cells using mass spectrometry. Moreover, we show that phosphorylation at three of these motifs (containing Ser60, Ser172 and Ser231) is required for the binding of 14-3-3 proteins to this region of Tiam1. We show that phosphorylation of these sites does not affect Tiam1 activity; significantly however, we demonstrate that phosphorylation of the Ser60-containing motif is required for the degradation of Tiam1. Thus, we have established and proven methodology that allows the identification of additional protein-protein interactions in mammalian cells, resulting in the discovery of a novel mechanism of regulating Tiam1 stability.

The Drosophila ZO-1 protein Polychaetoid suppresses Deltex-regulated Notch activity to modulate germline stem cell niche formation.

The developmental signalling protein Notch can be proteolytically activated following ligand-interaction at the cell surface, or can be activated independently of its ligands, following Deltex (Dx)-induced Notch endocytosis and trafficking to the lysosomal membrane. The means by which different pools of Notch are directed towards these alternative outcomes remains poorly understood. We found that the Drosophila ZO-1 protein Polychaetoid (Pyd) suppresses specifically the Dx-induced form of Notch activation both in vivo and in cell culture assays. In vivo we confirmed the physiological relevance and direction of the Pyd/Dx interaction by showing that the expanded ovary stem cell niche phenotypes of pyd mutants require the presence of functional Dx and other components that are specific to the Dx-induced Notch activation mechanism. In S2 cells we found that Pyd can form a complex with Dx and Notch at the cell surface and reduce Dx-induced Notch endocytosis. Similar to other known activities of ZO-1 family proteins, the action of Pyd on Dx-induced endocytosis and signalling was found to be cell density dependent. Thus, together, our results suggest an alternative means by which external cues can tune Notch signalling through Pyd regulation of Dx-induced Notch trafficking.

p120 Ras GTPase-activating protein associates with fibroblast growth factor receptors in Drosophila.

Btl (breathless) and Htl (heartless), the two FGFRs (fibroblast growth factor receptors) in Drosophila melanogaster, control cell migration and differentiation in the developing embryo. These receptors signal through the conserved Ras/mitogen-activated protein kinase pathway, but how they regulate Ras activity is not known. The present study shows that there is a direct interaction between p120 RasGAP (Ras GTPase-activating protein), a negative regulator of Ras, and activated FGFRs in Drosophila. The interaction is dependent on the SH2 (Src homology 2) domains of RasGAP, which have been shown to interact with a phosphotyrosine residue within the consensus sequence (phospho)YXXPXD. A potential binding site that matches this consensus is found in both Btl and Htl, located between the transmembrane and kinase domains of each receptor. A peptide corresponding to this region was capable of binding RasGAP only when the tyrosine residue was phosphorylated. This tyrosine residue appears to be conserved in human FGFR-1 and mediates the association with the adapter protein CrkII, but no association between dCrk (Drosophila homologue of CrkII) and the activated FGFRs was detected. RasGAP was a substrate of the activated FGFR kinase domain, and mutation of the tyrosine residue within the potential binding site on the receptor prevented tyrosine phosphorylation of RasGAP. RasGAP attenuated FGFR signalling in vivo and this ability was dependent on both its SH2 domains and its GAP activity. On the basis of these results, we propose that RasGAP is directly recruited into activated FGFRs in Drosophila and plays a role in regulating the strength of signalling through Ras and the mitogen-activated protein kinase pathway.

Tiam1-Rac signaling counteracts Eg5 during bipolar spindle assembly to facilitate chromosome congression.

Centrosome separation, critical for bipolar spindle formation and subsequent chromosome segregation during mitosis, occurs via distinct prophase and prometaphase pathways. Kinesin-5 (Eg5), a microtubule (MT) motor, pushes centrosomes apart during bipolar spindle assembly; its suppression results in monopolar spindles and mitotic arrest. Forces that antagonize Eg5 in prophase are unknown. Here we identify a new force generating mechanism mediated by the guanine nucleotide exchange factor (GEF) Tiam1, dependent on its ability to activate the GTPase Rac. We reveal that Tiam1 and Rac localize to centrosomes during prophase and prometaphase, and Tiam1, acting through Rac, ordinarily retards centrosome separation. Importantly, both Tiam1-depleted cells in culture and Rac1-deficient epithelial cells in vivo escape the mitotic arrest induced by Eg5 suppression. Moreover, Tiam1-depleted cells transit more slowly through prometaphase and display increased chromosome congression errors. Significantly, Eg5 suppression in Tiam1-depleted cells rectifies not only their increased centrosome separation but also their chromosome congression errors and mitotic delay. These findings identify Tiam1-Rac signaling as the first antagonist of centrosome separation during prophase, demonstrate its requirement in balancing Eg5-induced forces during bipolar spindle assembly in vitro and in vivo, and show that proper centrosome separation in prophase facilitates subsequent chromosome congression.