The Regulation of Smoothened in Hedgehog Signaling

Hedgehog (Hh) proteins function as morphogenes 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 Smo negative regulators, such as phosphatase (PP4) and ubiquitin. We recently discovered USP8 as a deubiquitinase that prevents Smo ubiquitination, changes Smo subcellular localization, and promotes Smo signaling activity. The cell surface accumulation and intracellular trafficking of Smo have been critical issues regarding Smo activation by Hh. Our findings provide new tools and hypotheses to investigate the mechanisms of Smo activation at the cell membrane. In this project, our central hypothesis is that small molecules activate Smo by inducing a conformational switch and cell surface accumulation of Smo, whereas ubiquitin inactivates Smo by promoting Smo endocytosis and degradation. To test this, 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.