Short talk:
Direct Interaction of Smoothened and PKA Opens Up a New Window into Hedgehog Signaling

Jan Bröckel1, Daniela Bertinetti1, Jessica Bruystens2, Benjamin Myers3, Susan Taylor2, Friedrich Herberg1

1Universität Kassel, Institut für Biologie, Abteilung Biochemie, Kassel, Germany,
2University of California, San Diego, United States,
3University of Utah, Salt Lake City,

Hedgehog signaling is important for the development of a complex organism and tissue homeostasis in adults. Dysregulation leads to severe developmental defects or cancer. The extracellular morphogen hedgehog (Hh) activates Hh gene transcription via a network of spatiotemporally regulated components. A central, yet poorly understood step of the Hh cascade is the inhibition of cAMP-dependent protein kinase (PKA) by the G-protein coupled receptor (GPCR) Smoothened (SMO). Active SMO inhibits the phosphorylation of the transcriptional regulator GLI by the catalytic subunit of PKA (PKA-C). Canonically, PKA is inhibited at low cAMP levels and GPCRs regulate intracellular cAMP levels by the regulation of adenylyl cyclases. Although SMO couples inhibitory G-proteins, those are not responsible for Hh mediated PKA-C inhibition by SMO. The recently demonstrated membrane recruitment of PKA-C by the SMO C-tail suggests a direct interaction. We employed Surface Plasmon Resonance (SPR) to characterize wildtype and mutant PKA-C binding to the recombinant SMO C-tail. An activity assay for PKA was then used to verify the inhibition of PKA-C by the SMO C-tail in vitro. It could be shown that PKA-C binds to a sequence in the SMO C-tail which resembles the consensus recognition sequence of PKA-C found in other PKA pseudosubstrate inhibitors. The SMO C-tail also inhibits phosphorylation of peptide substrates by PKA. These results suggest a direct inhibition of PKA-C by the SMO C-tail which acts as a pseudosubstrate and physically separates PKA-C from its substrate GLI. Our results point to a novel mechanism of PKA regulation by GPCRs, allowing SMO to transduce Hh signals. This provides a new perspective on the spatiotemporal regulation of PKA.

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