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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM079684-06A1
Application #
8628290
Study Section
Development - 1 Study Section (DEV1)
Program Officer
Gaillard, Shawn R
Project Start
2008-07-01
Project End
2018-06-30
Budget Start
2014-09-19
Budget End
2015-06-30
Support Year
6
Fiscal Year
2014
Total Cost
$307,125
Indirect Cost
$102,375
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