The Basal Ciliary but not Cytosol PKA Specifically Regulates HH Pathway Downstream of Smoothened

Effectors of the vertebrate Hedgehog (HH) signaling pathway are organized through primary cilia (PC) that grow and retract in lockstep with the cell cycle in response to extracellular signals. Protein kinase A (PKA), a kinase with ubiquitous distribution in most cells, functions as a specific negative regulator of the HH pathway. Its functional specificity in the HH pathway has been suggested to be controlled by cAMP in the PC. However, the regulation of PKA and its functions in PC is still poorly elucidated, partially due to the lack of observation of PKA localization in PC and inconsistency in the fluctuation of ciliary cAMP with HH pathway activity. Herein, we have developed an effective biosensor for monitoring real-time PKA activity in the PC of both cultured cells and zebrafish embryos, using a ciliary specific localized FRET-based A-kinase activity probe (Nphp3N-AKAR2-CR). Although the PKA catalytic subunit (PKA-C) was not observed in PC, ciliary PKA basal activity in cells was detected by Nphp3N-AKAR2-CR. Notably, its activity was modestly increased both by inhibition and activation of the HH pathway at the level of Smoothened (Smo). In addition, we have found that only ciliary-targeted PKA and not cytosolic PKA, can specifically modulate the HH pathway, even when the integrity of the PC is disrupted. Our results suggest that a basal level of PKA activity in the PC maintains the HH pathway in the OFF state and that PKA mediated inhibition is countered by changes in GLI concentration in the PC.