Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits

Abstract

The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein–coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.

A PKA Inhibitor Motif within Smoothened Controls Hedgehog Signal Transduction

“…Alexa 647-conjugated M1 FLAG antibody and M1 FLAG affinity resin were prepared inhouse. Control or ShhN conditioned medium was prepared from stably transfected HEK293 cells as previously described 24 . Dual-luciferase assay was obtained from Promega.…”

“…Proteins were separated by SDS-PAGE on a 4-20% Stain-Free TGX gel (BioRad), and total protein in lysate or eluate was visualized via Stain Free imaging. To detect PKA-Ca-YFP, inputs and eluates were transferred to PVDF membranes and processed for immunoblotting with anti-GFP antibodies as previously described 24 . In-gel Pro-Q Diamond assay to detect phosphoprotein was performed according to the manufacturer's instructions, as previously described 24 .…”

“…We next assessed the impact of the SMO PKI motif on PKA-C activity in cells using a model PKA-C substrate, the cyclic AMP response element binding protein (CREB) transcription factor 45 , and a CREB transcriptional reporter assay. This experimental paradigm entails overexpression of PKA-C at levels exceeding those of endogenous PKA-R, thereby minimizing potentially confounding contributions from heterotrimeric G protein-and cAMP-containing cascades 24 . Under these conditions, wild type SMO674 blocked PKA-C-mediated CREB reporter activation while SMO674 harboring the WRR mutation failed to do so (Fig.…”

“…Several lines of evidence support this proposal. First, SMO mutations known to reduce PKA-C interactions in vivo, including deletion of a predicted amphipathic helix spanning residues 570-581 or alanine substitution of GRK2/3 phosphorylation sites 24 , map to non-PKI regions of the pCT (summarized in Fig. 4a).…”

“…4a). Second, SMO pCT interactions with PKA-C are enhanced by the distal SMO C-tail (dCT, residues 658-793) 24 , demonstrating that this region harbors additional PKA-C binding determinants. Accordingly, in HEK293 cells, PKA-C interactions with SMO containing its fulllength C-tail are only partially blocked by the WRR mutation (Fig.…”