Protein Kinase A Catalytic subunit:Smoothened interface ‐ Role of a parallel holoenzyme in negative feedback in Hedgehog Signaling

Abstract

During signal transduction in the Hedgehog pathway, the G protein‐coupled receptor (GPCR) Smoothened (SMO) signals intracellularly via a novel mechanism involving sequestration of the Protein Kinase A (PKA) catalytic (C)‐subunit at the membrane. Activation of Hedgehog signaling leads to negative feedback regulation mediated through inhibition of PKA‐C by SMO at the ciliary membrane. While it is known that PKA‐C interacts with SMO at its C‐terminal proximal cytoplasmic tail domain (pCT), the dynamics of PKA‐C:SMO interactions is poorly understood. Here, we report a map of the PKA‐C:SMO interface by Amide Hydrogen Deuterium Exchange Mass spectrometry (HDXMS) over 1‐10 min timescales. SMO binding on PKA‐C resulted in global decreases in deuterium exchange (Dex). Peptides spanning the kinase activation loop (188‐198), P+1 loop (199‐211) and pseudo‐substrate recognition site between β5 and αD (129‐145) showed significant protection (>0.5 Da) when bound to SMO, identifying these regions as the SMO binding interface on PKA‐C. Interestingly, the footprint of SMO binding on PKA‐C was identical to that of the RIa regulatory subunit in PKA‐I holoenzymes. Furthermore, increased Dex (0.4 Da) was observed at the region spanning αC and β4 (83‐100), indicating that SMO binding destabilizes the active kinase conformation to allosterically inhibit PKA‐C. Correspondingly on SMO, PKA‐C binding elicited only low magnitudes of Dex protection (0.4 Da) in regions flanking the pseudo substrate site (573‐590, 622‐631). Increased Dex (0.4 Da) was observed at the sterol binding pocket (96‐112) in the extracellular Cysteine rich domain, indicating that PKA‐C binding may allosterically impact SMO activation by modulating its ability to bind sterols. A significant increase in Dex (0.9 Da) was observed at SMO helix 8 (537‐546) which is positioned parallel to the membrane, indicating that C binding promoted a disordered helix 8 conformation. We speculate that the pCT exists in a membrane‐bound or folded conformation in free SMO, but PKA‐C interaction switches the binding mode of pCT leading to a significantly Dex‐protected state. Thus, PKA‐C:SMO interactions reveal an unexpected intersection between cyclic nucleotide signaling and the Hedgehog pathway, defining a novel mechanism of PKA‐C regulation beyond the canonical PKA signalosome.