Studies Of Smoothened In Hedgehog Signaling Pathway

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Title: Studies Of Smoothened In Hedgehog Signaling Pathway
Author: Tong, Chao
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .5 /306
Date: 2006-12-20

Citation

Studies Of Smoothened In Hedgehog Signaling Pathway. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /306 .

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