Hedgehog (Hh) proteins function as morphogens and play critical roles in pattern formation and cell growth control. Dysregulation and dysfunction of the Hh signaling pathway induces diverse types of cancers. The seven-pass transmembrane protein Smoothened (Smo) is required in both insects and mammals for transduction of the Hh signal. Our strategy is to use Drosophila as a simple and genetically tractable model system to explore the mechanisms of Hh signal transduction by the receptor complex. The long-term goal of our research is to elucidate how Hh signals are sensed and transmitted to control downstream biological events that ultimately govern cell growth and patterning. Although many components in the Hh pathway have been identified, how the Hh signal is transduced through Patched (Ptc) to Smo is still unclear. We have discovered that Smo activation requires phosphorylation by multiple kinases, including protein kinase A (PKA), casein kinase 1 (CK1), casein kinase 2 (CK2), and G protein-coupled receptor kinase 2 (Gprk2), and that phosphorylation leads to increased Smo cell surface levels and signaling activity. We show that Smo transduces Hh signals by directly recruiting a downstream Fused-Costal2 (Fu-Cos2) complex, and that Fus- Cos2 protein complex promotes hyperphosphorylation of Smo, uncovering a feedback mechanism essential for optimal Hh pathway activation. Furthermore, we have identified protein phosphatase PP4 as a negative regulator of Smo. Results from our studies have suggested that the cell surface accumulation and intracellular trafficking of Smo play critical roles in regulating Hh-induced Smo activation. However, many questions persist regarding the mechanism controlling Smo localization and trafficking. To begin address these questions, we have recently discovered that the deubiquitinase USP8 promotes Smo signaling activity by preventing Smo ubiquitination and regulating Smo subcellular localization. Combined with the preliminary studies in the project, our findings provide new tools and hypotheses for investigating the mechanisms of Smo signaling. In this project, our central hypothesis is that Smo activation downstream of Hh is tightly controlled by its subcellular localization, in which a novel kinase and small molecules promotes Hh signaling by enhancing the cell surface accumulation of Smo whereas ubiquitin inactivates Smo by promoting Smo endocytosis and degradation. We will use a combination of genetic and biochemical approaches in three Specific Aims: 1) To determine how a newly identified kinase regulates Smo phosphorylation and basolateral membrane localization; 2) to investigate the molecular mechanisms by which small molecules activate Smo in response to Hh stimulation; 3) to determine how Hh regulates the ubiquitin-mediated endosomal sorting of Smo via a novel E3 ligase of Smo.

Public Health Relevance

Hedgehog (Hh) signaling is known for its role in directing processes such as cell growth, proliferation, and differentiation during embryogenesis. Abnormal activation of Smo, an atypical GPCR in the receptor complex, results cancers such as basal cell carcinoma (BCC) and medulloblastoma. Investigation of the Smo signaling mechanisms, the goal of this project, will provide both insights into fundamental developmental problems and new strategies to design preventive and curative tools and treatments for cancers caused by deregulated Hh signaling.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Development - 1 Study Section (DEV1)
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Melillo, Amanda A
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University of Kentucky
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Jiang, Kai; Liu, Yajuan; Zhang, Jie et al. (2018) An intracellular activation of Smoothened that is independent of Hedgehog stimulation in Drosophila. J Cell Sci 131:
Wu, Yadi; Wang, Yu; Lin, Yiwei et al. (2017) Dub3 inhibition suppresses breast cancer invasion and metastasis by promoting Snail1 degradation. Nat Commun 8:14228
Liu, Yi; Zhang, Cuiping; Li, Zhenyu et al. (2017) Latexin Inactivation Enhances Survival and Long-Term Engraftment of Hematopoietic Stem Cells and Expands the Entire Hematopoietic System in Mice. Stem Cell Reports 8:991-1004
Zhang, Jie; Liu, Yajuan; Jiang, Kai et al. (2017) SUMO regulates the activity of Smoothened and Costal-2 in Drosophila Hedgehog signaling. Sci Rep 7:42749
Jiang, Kai; Liu, Yajuan; Fan, Junkai et al. (2016) PI(4)P Promotes Phosphorylation and Conformational Change of Smoothened through Interaction with Its C-terminal Tail. PLoS Biol 14:e1002375
Li, Jing; Song, Jun; Zaytseva, Yekaterina Y et al. (2016) An obligatory role for neurotensin in high-fat-diet-induced obesity. Nature 533:411-5
Li, Tongchao; Fan, Junkai; Blanco-Sánchez, Bernardo et al. (2016) Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 through Poly-ubiquitination. PLoS Genet 12:e1006054
Jiang, Kai; Jia, Jianhang (2015) Smoothened regulation in response to Hedgehog stimulation. Front Biol (Beijing) 10:475-486
Shi, Jiandang; Liu, Yajuan; Xu, Xuehe et al. (2015) Deubiquitinase USP47/UBP64E Regulates ?-Catenin Ubiquitination and Degradation and Plays a Positive Role in Wnt Signaling. Mol Cell Biol 35:3301-11
Jiang, Kai; Jia, Jianhang (2015) Analysis of Smoothened Phosphorylation and Activation in Cultured Cells and Wing Discs of Drosophila. Methods Mol Biol 1322:45-60

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