Ubquitination of Costal 2 by the Ubr3 E3 ligase is required for proper Hedgehog signaling. Tongchao Li¹, Nikos Giagtzoglou², Junkai Fan⁷, Jianhang Jia⁷, Sinya Yamamoto¹, Wu-Lin Charng¹, Manish Jaiwal², Hector Sandoval², Vafa Bayat^1,5, Bo Xiong¹, Ke Zhang³, Gabriela David¹, Andy Groves^1,2,4, Hugo Bellen^1,2,3,4,6. 1) Program in Developmental Biology; 2) Department of Molecular and Human Genetics; 3) Program in Structural and Computational Biology & Molecular Biophysics; 4) Department of Neuroscience; 5) Medical Scientist Training Program; 6) Howard Hughes Medical Institute, Neurological Research Institute at Baylor College of Medicine, Houston, Texas; 7) Markey Cancer Center, University of Kentucky, Lexington, KY.

   Hedgehog (Hh) signaling affects cell proliferation, cell differentiation and wound healing. Loss of Hh signaling leads to developmental disorders including holoprosencephaly, craniofacial defects, polydactyly and skeletal malformations whereas aberrant activation of Hh signaling causes polydactyly, multiple cancers including basal cell carcinoma (BCC), medulloblastoma, and rhabdomyosarcoma. Here we report the isolation of the Drosophila homolog of UBR3, a member of the UBR superfamily of E3 ubiquitin ligases, from a forward genetic screen aimed at identifying genes that regulate organogenesis of sensory organs. ubr3 homozygous mutants are first instar larval lethal. Mosaic clones show that loss of ubr3 causes a loss of Hh signaling at the boundary of anterior/posterior compartments of the developing eye imaginal and wing discs. Ubr3 functions as a novel regulator of Hh by promoting the activation of the pathway. Loss of ubr3 results in the upregulation of Costal2 (Cos2), a Kinesin-related motor protein that negatively regulates Hh signaling. Cos2 is necessary and sufficient to suppress Hh signaling, indicating the importance of controlling correct protein levels of Cos2. We show that Ubr3 binds to the amino terminal motor domain of Cos2 with its UBR domain and that it polyubiquitinates Cos2, promoting its degradation. In summary, we identified a novel E3 ligase that acts as a positive regulator of Hh signaling revealing a critical regulatory mechanism to control the protein levels of Cos2.