Wnt ligands signal through b-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through b-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. We demonstrate here that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). We identify Ror2 Ser 864 as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, we provide evidence that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways.[Keywords: Ror1/2; LRP5/6; Wnt3a; Wnt5a; receptor activation; noncanonical Wnt signaling] Supplemental material is available at http://www.genesdev.org.

The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 (ref. 1) and its Drosophila orthologue, Draper 2 . The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells 3 , developmentally pruned axons 4,5 and dendrites 6 , and axons undergoing Wallerian degeneration 7 . Here we show that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70, binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. We show that Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. We also show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. We propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells 8,9 . Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade.Developing tissues produce excessive numbers of cells and selectively destroy a subpopulation through programmed cell death to regulate growth. Rapid clearance of cell corpses is essential for maintaining tissue homeostasis and preventing the release of potentially cytotoxic or antigenic molecules from dying cells, and defects in cell corpse clearance are closely associated with autoimmune and inflammatory diseases 10-13 . In C. elegans the CED-1 receptor is expressed in engulfing cells, where it acts to recognize cell corpses and drive their phagocytosis 1 . CED-1 promotes engulfment through an intracellular NPXY motif, a binding site for proteins containing a phosphotyrosine-binding (PTB) domain, and a YXXL motif, a potential interaction site for proteins containing SH2 domains 1 . The PTB domain adaptor protein CED-6 can bind the NPXY motif of , is required for cell corpse Glia are the primary phagocytic cell type in the developing and mature brain. Glia rapidly engulf neuronal cell corpses produced during development, as well as neuronal debris generated during axon pruning 19,20 or during Wallerian degeneration in the adult brain 21 . In Drosophila, glial phagocytosis of these engulfment targets requires Draper, the fly orthologue of CED-1 (refs 2 , 4-6 ). Draper, like CED-1, contains 15 extracellular atypical epidermal growth factor (EGF) repeats, a single transmembrane domain, and NPXY an...

SUMMARY Intratumor mutational heterogeneity has been documented in primary non-small-cell lung cancer. Here, we elucidate mechanisms of tumor evolution and heterogeneity in metastatic thoracic tumors (lung adenocarcinoma and thymic carcinoma) using whole-exome and transcriptome sequencing, SNP array for copy-number alterations (CNAs), and mass-spectrometry-based quantitative proteomics of metastases obtained by rapid autopsy. APOBEC mutagenesis, promoted by increased expression of APOBEC3 region transcripts and associated with a high-risk APOBEC3 germline variant, correlated with mutational tumor heterogeneity. TP53 mutation status was associated with APOBEC hypermutator status. Interferon pathways were enriched in tumors with high APOBEC mutagenesis and IFN-γ-induced expression of APOBEC3B in lung adenocarcinoma cells, suggesting that the immune microenvironment may promote mutational heterogeneity. CNAs occurring late in tumor evolution correlated with downstream transcriptomic and proteomic heterogeneity, although global proteomic heterogeneity was significantly greater than transcriptomic and CNA heterogeneity. These results illustrate key mechanisms underlying multi-dimensional heterogeneity in metastatic thoracic tumors.

Somatic mutations in the epidermal growth factor receptor (EGFR) kinase domain drive lung adenocarcinoma. We have previously identified MIG6, an inhibitor of ERBB signaling and a potential tumor suppressor, as a target for phosphorylation by mutant EGFRs. Here we demonstrate that Mig6 is a tumor suppressor for the initiation and progression of mutant EGFR-driven lung adenocarcinoma in mouse models. Mutant EGFR-induced lung tumor formation was accelerated in Mig6-deficient mice, even with Mig6 haploinsufficiency. We demonstrate that constitutive phosphorylation of MIG6 at Y394/395 in EGFR-mutant human lung adenocarcinoma cell lines is associated with an increased interaction of MIG6 with mutant EGFR, which may stabilize EGFR protein. MIG6 also fails to promote mutant EGFR degradation. We propose a model whereby increased tyrosine phosphorylation of MIG6 decreases its capacity to inhibit mutant EGFR. Nonetheless, the residual inhibition is sufficient for Mig6 to delay mutant EGFR-driven tumor initiation and progression in mouse models.