Novel Developmental Regulation of Bmp and nodal signaling by Tril

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Our long term goal is to understand how Bmp and Tgf-ß/nodal signaling is regulated in development and disease. Imbalances in Bmp and Tgf-ß/nodal signaling lead to birth defects and cancers, and yet knowledge of how the equilibrium between these pathways is maintained is incomplete. We identified Tril as a protein that coordinately regulates Bmp and nodal signaling and propose that it does so through a novel mechanism involving Toll-like receptors (Tlrs). Tlrs play evolutionarily conserved roles in mediating innate immune responses. The proposed studies will provide the first demonstration that Tlr signaling is also required for early patterning in vertebrates, and will set the stage for future studies that ask whether Tril or downstream signaling components play a protective and/or causal role in congenital anomalies and disease. Our published studies show that Tril triggers degradation of Smad7 to enhance Bmp signaling during gastrulation in Xenopus, and our new data show that Tril simultaneously inhibits nodal signaling. Our data support the hypothesis that Tril serves as a co-receptor for Tlrs to initiate a signaling cascade in which Irak kinases activate Pellino2. Pellino2 then stimulates Rnf12- mediated degradation of Smad7 to enhance Bmp signaling, and Nedd4L-mediated degradation of nodal signaling components to inhibit nodal signaling. We will test this hypothesis as follows (1) Determine how Tril regulates Nedd4l to inhibit nodal signaling. We will compare the kinetics of nodal pathway inactivation, analyze steady state levels of nodal receptors and ask whether Nedd4l is sufficient to rescue nodal signaling in control and Tril deficient Xenopus embryos and mouse embryonic fibroblasts (MEFs). We will test whether Tril prevents proteosomal degradation and/or stimulates post-translational modification of Nedd4l. (2) Determine whether and how Tril regulates Rnf12 to mediate degradation of Smad7. We will test whether the ability of Tril to trigger degradation of Smad7 is impaired in embryos or in MEFs in which expression of Rnf12 is knocked down, and conversely, whether Rnf12-mediated degradation of Smad7 is impaired in Tril morphants and in tril mutant MEFs. We will test whether steady state levels, subcellular localization and/or post-translational modification of Rnf1 is altered in Tril morphant embryos and tril mutant MEFs. (3) Determine whether Tril activates a Tlr-signaling cascade to regulate Bmp and nodal signaling. We will down-regulate the function of Tlr3, Tlr4, Iraks and Pellino2 in embryos and in MEFs and ask whether Bmp signaling is impaired and nodal signaling is enhanced, and we will upregulate the function of each protein and ask whether this rescues Bmp and nodal signaling in Tril morphant embryos and tril mutant MEFs. We will test whether Tril is required to regulate Pellino2 phosphorylation, subcellular localization and/or binding to Smad7 and Nedd4l, and will use rescue assays to determine if phosphorylation of Pellino2 and/or its ubiquitin ligase activity are required for function during embryogenesis.