AI Insight
This study demonstrates that Hedgehog signaling in vertebrates is regulated by protein kinase A (PKA) activity within primary cilia. The researchers developed a ciliary PKA reporter and found that Smoothened (SMO), when phosphorylated by GRK2/3, inhibits ciliary PKA activity through a pseudosubstrate mechanism, while the GPCR GPR161 promotes ciliary PKA activity by recruiting the PKA catalytic subunit through an AKAP domain. The balance between SMO-mediated PKA inhibition and GPR161-mediated PKA activation determines whether Hedgehog signaling is activated or suppressed.
Why it matters
Understanding the molecular mechanisms of Hedgehog signaling has important implications for developmental biology and cancer research, as aberrant Hedgehog pathway activity is linked to birth defects and various cancers. This work identifies specific molecular targets that could potentially be exploited for therapeutic interventions in Hedgehog-related diseases.
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 ciliary PKA activity. A ciliary GPCR, SSTR3, activated ciliary PKA and induced HH transcriptional responses in NIH-3T3 cells via a different mechanism: activation of Gαi/o. A different ciliary GPCR, GPR161, possesses an A-Kinase Anchoring Protein (AKAP), which we found was critical for the ciliary localization of the catalytic subunit of PKA (PKA-C) to promote ciliary PKA activity. We propose that HH signal transduction is inhibited by GPR161-mediated ciliary enrichment of PKA-C, and activated by GRK2/3-phosphorylated SMO inhibition of ciliary PKA activity.