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Review
. 2013 Feb;23(2):186-200.
doi: 10.1038/cr.2013.10. Epub 2013 Jan 22.

Decoding the phosphorylation code in Hedgehog signal transduction

Yongbin Chen  1 , Jin Jiang
Affiliations
Review

Decoding the phosphorylation code in Hedgehog signal transduction

Yongbin Chen et al. Cell Res. 2013 Feb.

Abstract

Hedgehog (Hh) signaling plays pivotal roles in embryonic development and adult tissue homeostasis, and its deregulation leads to numerous human disorders including cancer. Binding of Hh to Patched (Ptc), a twelve-transmembrane protein, alleviates its inhibition of Smoothened (Smo), a seven-transmembrane protein related to G-protein-coupled receptors (GPCRs), leading to Smo phosphorylation and activation. Smo acts through intracellular signaling complexes to convert the latent transcription factor Cubitus interruptus (Ci)/Gli from a truncated repressor to a full-length activator, leading to derepression/activation of Hh target genes. Increasing evidence suggests that phosphorylation participates in almost every step in the signal relay from Smo to Ci/Gli, and that differential phosphorylation of several key pathway components may be crucial for translating the Hh morphogen gradient into graded pathway activities. In this review, we focus on the multifaceted roles that phosphorylation plays in Hh signal transduction, and discuss the conservation and difference between Drosophila and mammalian Hh signaling mechanisms.

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Figures

Figure 1
Figure 1
Hh signal transduction in Drosophila and mammalian systems. Hh protein is lipid-modified. Drosophila and mammalian HSPGs, Dally, Dally-like (Dlp), GPC3, GPC4 and GPC6 modulate Hh signaling. In the absence of Hh, Ptc inhibits Smo, allowing CiF/GliF to be phosphorylated by multiple kinases and targeted for Slimb/β-TRCP-mediated proteolysis to generate CiR/GliR. In Drosophila, the kinesin-like protein Cos2 acts as a molecular scaffold to bridge Ci to its kinases. In the presence of Hh, Ptc inhibition of Smo is released, which triggers Smo phosphorylation by PKA, CK1 and Gprk2/GRK2, leading to its cell surface accumulation and activation. Smo recruits Cos2-Fu complex to the cell surface, and dissociates Cos2-Ci-kinase complexes to inhibit Ci phosphorylation and processing. In the presence of high levels of Hh, Fu converts CiF into CiA by antagonizing Sufu inhibition. CiA is unstable and degraded by the HIB-mediated Ub/proteasome pathway. Fu-Cos2 also regulates Smo phosphorylation in a feedback loop mechanism. In mammals, Hh induces Smo phosphorylation by CK1 and GRK2, leading to its ciliary accumulation (not shown here) and activation. Sufu is a major whereas Kif7 a minor inhibitor of Gli proteins. The HIB homolog SPOP is responsible for degrading Gli proteins in the absence of Sufu. CiF/GliF: full length Ci/Gli; CiA/GliA: activator form of Ci/Gli; CiR/GliR: repressor form of Ci/Gli. Adapted from reference.
Figure 2
Figure 2
Ci/Gli phosphorylation and proteolysis. (A) In the absence of Hh, CiF/GliFis sequentially phosphorylated by PKA, GSK3 and CK1, and targeted to Ub/proteasome-mediated proteolysis through Slimb/β-TRCP to generate a truncated repressor CiR/GliR. (B) Diagrams of Ci, mouse Gli2 and human Gli3 showing the PKA/GSK3/CK1 phosphorylation clusters in Ci/Gli and the Slimb/β-TRCP binding consensus site. Putative Slimb/β-TRCP binding sites in Ci/Gli are underlined. Grey and blue boxes denote Zn-finger DNA binding domain and transactivation domain, respectively. ub: ubiquitin.
Figure 3
Figure 3
Phosphorylation of Smo regulates its subcellular localization and conformation in both Drosophila and mammalian systems. (A) Diagrams showing the phosphorylation sites in Drosophila (top) and mammalian Smo (bottom). The Arg motifs adjacent to phosphorylation clusters in Drosophila Smo are indicated in red. The red box in Drosophila Smo C-tail denotes the SAID domain. (B) Diagrams of closed inactive (left) and open active (right) conformations of Drosophila Smo. The closed conformation is maintained by intramolecular electrostatic interactions between multiple Arg motifs in the SAID domain and multiple acidic clusters in the C-terminal region of Smo. Hh-induced phosphorylation by PKA and CK1 neutralizes the positive charges and disrupts the intramolecular electrostatic interactions, leading to the opening and dimerization of Smo C-tail. Binding of GRK2 to Smo C-tail stabilizes its dimerization. (C) In Drosophila, Smo phosphorylation by PKA and CK1 leads to its cell surface accumulation and active conformation whereas in mammals, Smo phosphorylation by CK1 and GRK2 leads to its ciliary accumulation and active conformation. SAID: Smo autoinhibitory domain. Adapted from the references,,.
Figure 4
Figure 4
A model for Smo phosphorylation and conformational switch leading to Hh pathway activation in Drosophila. Hh morphogen gradient is translated into Smo phosphorylation and activity gradients, which are in turn translated into Fu/Cos2 phosphorylation and activity gradients. Smo phosphorylation induces an open conformation of Smo C-tail, which facilitates the recruitment of Cos2/Fu. In addition, dimerization of Smo C-tail leads to clustering of the bound Cos2/Fu, resulting in Fu phosphorylation and activation. Activated Fu inhibits CiR production by dissociating Cos2-Ci-kinase complexes and stimulates CiA formation likely by dissociating Sufu from Ci. Adapted from the reference.
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