A Broadly Conserved G-Protein-Coupled Receptor Kinase Phosphorylation Mechanism Controls Drosophila Smoothened Activity

Abstract

Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a C-terminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized.

Dose-dependent transduction of Hedgehog relies on phosphorylation-based feedback between the G-protein-coupled receptor Smoothened and the kinase Fused

“…The extent of phosphorylation of the PKA/ CKI clusters depends on HH levels and controls in a graded manner SMO clustering, the degree of conformational change in its cytotail, its accumulation at the cell surface and its signaling activity Su et al, 2011;Zhao et al, 2007). Moreover, CKIγ/Gilgamesh (Gish), G-protein-coupled receptor kinase 2 (GPRK2/GRK2), Casein kinase II and atypical protein kinase C increase the ability of SMO to transduce high levels of HH by phosphorylating residues present in the membrane-proximal and central regions of the SMO cyto-tail (Jia et al, 2010;Jiang et al, 2014;Li et al, 2016;Maier et al, 2014). In vertebrates, SMO phosphorylation has been reported to depend on GRK2 and CKIγ (Chen et al, 2004).…”

Smoothened Regulation: A Tale of Two Signals

“…The essential GRK2 phosphorylation sites are localized to two clusters in a well conserved membrane proximal region of the tail. Phosphorylation at these clusters is required for high-level Smo signaling, and in vertebrates, serves to recruit β-arrestin to initiate its internalization through clathrin-coated pits [43-45]. Although phosphorylation by the Drosophila GRK2 homolog does trigger the eventual internalization of Smo, it does not appear to be through recruitment of the Drosophila arrestin Kurtz [42].…”

“…V231M, L412 and F460L affect TM domains 3, 5 and 6, which are essential pivot regions for activation of stereotypical GPCRs, and likely activate Smo by mimicking its ligand-induced active conformation [94]. The carboxyl-terminal tail mutation R562Q is adjacent to the first conserved GRK2 phosphorylation cluster, and could potentially trigger ligand-independent signaling through altering conformation of this important regulatory domain [43]. …”

Novel Insights into the Role of the Smoothened Cysteine Rich Domain in Hedgehog Signalling

“…Phosphorylation neutralizes the charge on the basic residues and leads to a conformational switch which brings the cytoplasmic loop of the adjacent Smo receptor in close proximity and facilitates downstream signalling [68]. In the presence of Hh, Drosophila Smo is phosphorylated by PKA, CK1, CK2 and the G protein coupled receptor kinase (GRK2) [69][70][71][72]. The vertebrate protein kinases include CK1 and GRK2 [73].…”

“…Smoothened in the plasma membrane is phosphorylated by the protein kinases PKA, CK1 and GPRK2 [69][70][71][72]. Phosphorylation triggers a conformational switch in the cytoplasmic domains of the Smo receptor triggering downstream Hh signalling [68].…”