Gli Phosphorylation Code in Hedgehog Signal Transduction

Abstract

Hedgehog (Hh) family of secreted proteins governs many key processes in embryonic development and adult tissue homeostasis in species ranging from insects to human. Deregulation of Hh signaling has been implicated in a wide range of human diseases including birth defect and cancer. Hh signaling pathway culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli from a repressor form (Ci^R/Gli^R) into an activator form (Ci^A/Gli^A). Both the production of Ci^R/Gli^R in the absence of Hh and the formation of Ci^A/Gli^A in response to Hh are regulated by phosphorylation. Whereas previous studies demonstrated that sequential phosphorylation by protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1) at multiple Ser/Thr clusters in the C-terminal region of Ci/Gli targets it for proteolytic processing to generate Ci^R/Gli^R, recent studies revealed that phosphorylation of Ci/Gli by the Fused (Fu)/Unc-51 like kinase (Ulk) family kinases Fu/Ulk3/Stk36 and other kinases contributes to Ci/Gli activation. Fu/Ulk3/Stk36-mediated phosphorylation of Ci/Gli is stimulated by Hh, leading to altered interaction between Ci/Gli and the Hh pathway repressor Sufu. Here we review our current understanding of how various Ci/Gli phosphorylation events are regulated and how they influence Hh signal transduction.

Keywords: CK1; Ci; Fu; GSK3; Gli; Hedgehog; PKA; Ulk3.

FIGURE 1

The hedgehog signaling transduction in Drosophila and vertebrates. (A) In Drosophila, the 12-span transmembrane protein Ptc represses the GPCR-family protein Smo in the absence of Hh ligand, leading to the degradation of Smo by the ubiquitylation pathway. Full-length Ci (Ci^F) forms a complex with the kinesin-like protein Cos2 and the Ser/Thr kinase Fu, which recruits PKA, GSK3, and CK1 to phosphorylate Ci. Phosphorylation of Ci^F targets it for Slimb-mediated ubiquitination, followed by proteasome-mediated proteolysis to generate Ci^R. Binding of Hh to Ptc releases its inhibition of Smo, allowing Smo to be phosphorylated by PKA, CK1 and GRK2. Activated Smo not only blocks Ci^F phosphorylation by PKA/GSK3/CK1 and thus the production of Ci^R but also converts Ci^F into the activator form (Ci^A) through activating the Fu kinase, which directly phosphorylates Ci^F to release its inhibition by Sufu. (B) Vertebrate Hh signal transduction depends on primary cilia. In the absence of Hh, both Ptch1 and Gpr161 (a GPCR coupled to G⍺s) are localized in primary cilia where Ptch1 prevents Smo ciliary localization and Gpr161 activates PKA. Full-length Gli (Gli^F; mainly Gli3 and Gli2) is phosphorylated by PKA, GSK3, and CK1, which targets it for β-TRCP-mediated ubiquitination, followed by proteasome-mediated proteolysis to generate Gli^R. Binding of Hh to Ptch1 inhibits its activity and promotes its ciliary exit, allowing Smo to be phosphorylated by CK1 and GRK2 and accumulated in primary cilia. Activated Smo inhibits Ci phosphorylation by PKA and the production of Gli^R in part by promoting Gpr161 ciliary exit. In addition, activated Smo converts Gli^F into Gli^A at least in part through Ulk3 and STK36, which phosphorylate Gli^F to attenuate the inhibition by Sufu.

FIGURE 2

Ci/Gli phosphorylation code. (A) Ci/Gli is phosphorylated on different sites by distinct sets of kinases depending on the Hh signaling states. In the absence of Hh, phosphorylation of Ci^F/Gli^F by PKA/CK1/GSK3 negatively regulates Hh signaling by converting Ci^F/Gli^F into Ci^R/Gli^R whereas in the presence of Hh, phosphorylation of Ci^F/Gli^F by Fu/Ulk3/Stk36 plays a positive role by promoting the formation of Ci^A/Gli^A. (B–C) Schematic representation of Ci and Gli showing the PKA/CK1/GSK3 phosphorylation sites (B) and Fu/Ulk3/Stk36 phosphorylation sites (C). Individual kinase sites are color coded. The underlines indicate the Slimb/β-TRCP binding sites in Ci/Gli. The green box represents the Zn-finger DNA-binding domain whereas the yellow box indicates the transactivation domain.