Studies of Smoothened in Hedgehog Signaling Pathway
The Hedgehog (Hh) family of morphogens controls cell growth and patterning in both vertebrates and invertebrates. Malfunction of Hh signaling has been implicated in numerous human disorders. As the Hh signal transducer, the seven-transmembrane protein Smoothened (Smo) is highly regulated. It is still a mystery how Smo transduces graded Hh signal to downstream components. Although Smo shares some structural similarity with G protein coupled receptors (GPCR), there is little evidence that G proteins are involved in Hh signal transduction in physiological settings. A kinesin like protein Costal2 (Cos2) and a serine/threonine kinase Fused (Fu) form complexes with the transcription factor Cubitus-interrupts (Ci), which is essential for Hh signal transduction. However, how Smo transduces Hh signal to this complex is still not clear. In this study, we found that Smo interacts with Cos2-Fu complex through its C-terminal tail, which is essential for the Hh pathway activation. In response to Hh, Smo is phosphorylated and accumulated on the cell surface. However, the kinases responsible for Hh induced Smo phosphorylation are still unknown. It is also not clear whether phosphorylation regulates Smo activity or not. In this study, I found that protein kinase A (PKA) and casein kinase I (CKI) regulate Smo cell surface accumulation and activity in response to Hh. PKA and CKI phosphorylate Smo directly at multiple sites which form three clusters in Smo C-terminal tail. In cooperation with Jianhang, we found that phosphorylation deficient forms of Smo failed to accumulate on the cell surface and were unable to transduce Hh signal. By contrast, phosphorylation mimicking forms of Smo have increased cell surface accumulation and constitutive activity. In addition, we also found the levels of Smo cell surface accumulation and activity correlate with its phosphorylation levels, suggesting that the graded Smo activity may be regulated by differential phosphorylation of its C-terminal tail. Furthermore, I have identified multiple Arginine clusters in Smo the C-terminal tail that negatively regulate Smo activity by preventing Smo cell surface accumulation and keeping Smo C-terminal tail in a closed inactive conformation maintained by intramolecular electrostatic interactions. I have also found that the number of arginine clusters is reversely correlated with Smo cell-surface expression and activity. I also provided evidence that phosphorylation antagonizes the negative effects of the Arginines by neutralizing the positive charges they carry, which lets Smo C-terminal tail adopts an open and active conformation and promotes Smo cell surface accumulation. Based on these data, we proposed that multiple arginine clusters provide a way to finetune Smo activity in response to different Hh levels by differentially phosphorylating Smo C-terminal tail. This study also showed that Gprk2, a G protein coupled receptor kinase (GRK), plays a positive role in regulating Hh signalling. I provided evidence that Gprk2 interacts with Smo Ctail. Furthermore, I identified a new CKI phosphorylation cluster that appears to be critical for Smo endocytosis and activation.