Smoothened and ciliary GPCRs regulate ciliary protein kinase A activity involved in Hedgehog signal transduction

by Thi D. Nguyen, Mia J. Konjikusic, Lorenzo M. Del Castillo, Roshanak Irannejad, Jeremy F. Reiter Hedgehog (HH) signaling in vertebrates is dependent on the primary cilium, an organelle that scaffolds signal transduction. HH signals induce ciliary enrichment of Smoothened (SMO) and ciliary departure of the G protein-coupled receptor (GPCR) GPR161 to trigger GLI activation of the HH transcriptional program. Recently, SMO has been shown to inhibit protein kinase A (PKA). To test the hypothesis that SMO inhibits PKA at cilia to activate the HH signal transduction pathway, we developed a ciliary PKA reporter. Ciliary PKA activity was graded during zebrafish development. Activation of the HH signal transduction pathway by either Sonic hedgehog (SHH) or SMO agonist (SAG) inhibited ciliary PKA activity. Blocking SMO phosphorylation by GRK2/3 prevented ciliary SMO from inhibiting ciliary PKA activity. The SMO C-terminal PKA pseudosubstrate site was critical for SMO-mediated inhibition of cili