PKAc-directed interaction and phosphorylation of Ptc is required for Hh signaling inhibition in Drosophila

Wenqing Wu,Yajie Gao,Shuo Yuan,Yi Lu,Jialin Fan,Dahua Chen,Hailong Wu,Yun Zhao

doi:10.1038/s41421-019-0112-z

Abstract

Ptc is a gatekeeper to avoid abnormal Hh signaling activation, but the key regulators involved in Ptc-mediated inhibition remain largely unknown. Here, we identify PKAc as a key regulator required for Ptc inhibitory function. In the absence of Hh, PKAc physically interacts with Ptc and phosphorylates Ptc at Ser-1150 and -1183 residues. The presence of Hh unleashes PKAc from Ptc and activates Hh signaling. By combining both in vitro and in vivo functional assays, we demonstrate that such Ptc–PKAc interaction and Ptc phosphorylation are both important for Ptc inhibitory function. Interestingly, we further demonstrate that PKAc is subjected to palmitoylation, contributing to its kinase activity on plasma membrane. Based on those novel findings, we establish a working model on Ptc inhibitory function: In the absence of Hh, PKAc interacts with and phosphorylates Ptc to ensure its inhibitory function; and Hh presence releases PKAc from Ptc, resulting in Hh signaling activation.

Highlights

As one of the most evolutionarily conserved morphogen pathways, the Hedgehog (Hh) signaling pathway plays vital roles in embryo development, tissue regeneration, and homeostasis[1,2]
Transmembrane receptor Ptc interacts with PKAc Since protein kinase A (PKA) is deeply involved in Hh signaling transduction and shows the potential capacity of localizing at plasma membrane, we sought to test whether PKA functions as a novel regulator involved in Ptc-mediated Hh signaling inhibition
In contrast to the SmoPKAc interaction which is enhanced in the presence of Hh (Fig. 1c), the Ptc–PKAc interaction was significantly diminished in the presence of Hh (S2 cells were simultaneously transfected with Hh plasmid and treated with Hh condition medium to maximize Hh stimulation) (Fig. 1d)

Summary

Introduction

As one of the most evolutionarily conserved morphogen pathways, the Hedgehog (Hh) signaling pathway plays vital roles in embryo development, tissue regeneration, and homeostasis[1,2]. One of the key features which distinguishes Hh signaling with other signaling pathways is the existence of an important inhibitory receptor, a 12transmembrane protein Patched (Ptc). In the absence of ligands, Ptc inhibits the activation of a downstream seven-transmembrane signal transducer, Smoothened (Smo), and silences the Hh signaling[5]. Hh and releases Smo from inhibition, resulting in the activation of Hh signaling as evidenced by accumulation of Ci full-length and induction of downstream target genes of Hh signaling including dpp, ptc, and en[5]. The inhibitory function of Ptc is critical in regulating the switch-off and -on of Hh signaling and is inevitably subjected to precise regulation at multiple levels to guarantee the appropriate Hh signaling activity in Hhresponsive cells

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Conclusion