Smoothened Transduces Hedgehog Signals via Activity-Dependent Sequestration of PKA Catalytic Subunits
- Organism: Homo sapiens
- Instrument: Orbitrap Exploris 480
PRM, Phosphorylation, Smoothened
Lab head: Ruth Huttenhain
Submitter: Ruth Huttenhain
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.
To identify GRK2/3 phosphorylation sites in SMO, the Flag-tagged receptor was purified from HEK293 cells stimulated with SAG21k, KAADcyc, or Cmpd101. The purified protein extracts from different conditions were denatured, reduced and alkylated. For digestion, proteins were incubated with (1) trypsin at 37 °C overnight, (2) chymotrypsin at 25°C overnight or (3) consecutively with chymotrypsin at 25°C overnight followed by trypsin both at 37 °C for 4 hours. The resulting peptide sampels were initially analyzed by DDA to generate a spectral library and subsequently all samples were analyzed in PRM mode for targeted quantification of phosphosites.
Created on 1/29/21, 4:14 PM