The Hedgehog (Hh) signaling is one of the essential signaling pathways during embryogenesis and in adults. Hh signal transduction relies on primary cilium, a specialized cell surface organelle viewed as the hub of cell signaling. Protein kinase A (PKA) has been recognized as a potent negative regulator of the Hh pathway, raising the question of how such a ubiquitous kinase specifically regulates one signaling pathway. We reviewed recent genetic, molecular and biochemical studies that have advanced our mechanistic understanding of PKA's role in Hh signaling in vertebrates, focusing on the compartmentalized PKA at the centrosome and in the primary cilium. We outlined the recently developed genetic and optical tools that can be harvested to study PKA activities during the course of Hh signal transduction.

Mutations in the hedgehog (Hh) signaling are implicated in birth defects and cancers, including medulloblastoma (MB), one of the most malignant pediatric brain tumors. Current Hh inhibitors face the challenge of drug resistance and tumor relapse, urging new insights in the Hh pathway regulation. Our previous study revealed how PDE4D controls global levels of cAMP in the cytoplasm to positively regulate Hh signaling; in the present study, we found that a specific isoform PDE4D3 is tethered to the centrosome by Myomegalin (Mmg), a centrosome/Golgi-associated protein. Mmg loss dislocates PDE4D3 from the centrosome, leading to local PKA overactivation and inhibition of the Hh signaling, leaving other PKA-related pathways unaffected. Mmg loss suppresses the proliferation of granule neuron precursors and blocks the growth of MB in mouse model. Our findings specify a new regulatory mechanism of the Hh pathway and highlight an exciting therapeutic avenue for Hh-related cancers with reduced side effects.

Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.

The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. Here we show that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, we demonstrate that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.