The hedgehog (hh) family members control many aspects of development in both vertebrates and invertebrates. Hh signaling is associated with important biological phenomena such as patterning, cell growth, and morphogenesis. Abnormal activation of this pathway has been observed in several types of human 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. 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. We showed that Smo transduces the Hh signals by directly recruiting a Costal2-Fused (Cos2-Fu) complex. We also showed that Smo activation requires phosphorylation by protein kinase A (PKA) and casein kinase I (CKI), and that phosphorylation leads to increased Smo cell-surface levels and signaling activity. We recently uncovered a feedback mechanism by which Fu promotes Smo hyperphosphorylation and cell-surface accumulation by antagonizing Cos2. These findings provide new tools and hypotheses to investigate the mechanisms of Hh signaling at the cell membrane. In this project, our central hypothesis is that Smo cell-surface accumulation and signaling activity are regulated by multiple kinases, while phosphatase(s) act as inhibitory components to attenuate Smo activation. To test our hypothesis, we will use a combination of genetic and biochemical approaches in three Specific Aims: 1) To further determine if differential Smo phosphorylation transduces gradient Hh activity;2) to investigate the molecular mechanisms by which G protein-coupled receptor kinase 2 (GRK2) is involved in Hh signaling;and 3) to determine the role of phosphatase in Smo dephosphorylation and inactivation.

Public Health Relevance

Abnormal Smoothened (Smo) activation results cancers such as basal cell carcinoma (BCC) and medulloblastoma. Investigation of the Smo signaling mechanisms will provide both insights into fundamental developmental problems and new avenues for developing diagnostic tools and therapeutical treatments of cancers. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Project Narrative

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079684-05
Application #
8286239
Study Section
Development - 1 Study Section (DEV1)
Program Officer
Hoodbhoy, Tanya
Project Start
2008-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$283,813
Indirect Cost
$92,693
Name
University of Kentucky
Department
Biochemistry
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Jiang, Kai; Liu, Yajuan; Fan, Junkai et al. (2014) Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila. Proc Natl Acad Sci U S A 111:E4842-50
Schwend, Tyler; Jin, Zhigang; Jiang, Kai et al. (2013) Stabilization of speckle-type POZ protein (Spop) by Daz interacting protein 1 (Dzip1) is essential for Gli turnover and the proper output of Hedgehog signaling. J Biol Chem 288:32809-20
Xia, Ruohan; Jia, Hongge; Fan, Junkai et al. (2012) USP8 promotes smoothened signaling by preventing its ubiquitination and changing its subcellular localization. PLoS Biol 10:e1001238
Fan, Junkai; Liu, Yajuan; Jia, Jianhang (2012) Hh-induced Smoothened conformational switch is mediated by differential phosphorylation at its C-terminal tail in a dose- and position-dependent manner. Dev Biol 366:172-84
Chen, Yongbin; Sasai, Noriaki; Ma, Guoqiang et al. (2011) Sonic Hedgehog dependent phosphorylation by CK1? and GRK2 is required for ciliary accumulation and activation of smoothened. PLoS Biol 9:e1001083
Jin, Zhigang; Mei, Wenyan; Strack, Stefan et al. (2011) The antagonistic action of B56-containing protein phosphatase 2As and casein kinase 2 controls the phosphorylation and Gli turnover function of Daz interacting protein 1. J Biol Chem 286:36171-9
Shi, Qing; Li, Shuang; Jia, Jianhang et al. (2011) The Hedgehog-induced Smoothened conformational switch assembles a signaling complex that activates Fused by promoting its dimerization and phosphorylation. Development 138:4219-31
Jia, Hongge; Liu, Yajuan; Xia, Ruohan et al. (2010) Casein kinase 2 promotes Hedgehog signaling by regulating both smoothened and Cubitus interruptus. J Biol Chem 285:37218-26
Jia, Hongge; Liu, Yajuan; Yan, Wei et al. (2009) PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation. Development 136:307-16
Zhang, Wen; Yang, Jun; Liu, Yajuan et al. (2009) PR55 alpha, a regulatory subunit of PP2A, specifically regulates PP2A-mediated beta-catenin dephosphorylation. J Biol Chem 284:22649-56