Hedgehog Pathway Activity Downstream of Smoothened is regulated specifically by Basal Ciliary PKA

Hongyu Zhang, Shujing Chen, Zhuoya Huang, Jin Ben, Guangxin Chen, Changxin Wu, Haibo Xie, Philip Ingham, Zhonghua Zhao
2024 · doi:10.1101/2024.12.30.630707
preprint OA: closed
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This study developed a biosensor to show that basal PKA activity within primary cilia, not cytosolic PKA, regulates the Hedgehog pathway, independent of Smoothened activation.

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Abstract

Effectors of the vertebrate Hedgehog (HH) signaling pathway are organized through primary cilia (PC) that grow and retract in lockstep with the cell cycle in response to extracellular signals. Protein kinase A (PKA), a kinase with ubiquitous distribution in most cells, functions as a specific negative regulator of the HH pathway. Its functional specificity in the HH pathway has been suggested to be controlled by cAMP in the PC. However, the regulation of PKA and its functions in PC remain unclear, in part due to the lack of observation of PKA localization in PC during HH resting state as well as conflicting reports of the dynamic changes of cAMP in cilia and HH pathway activity. To address this issue, we have developed a ciliary-localized FRET-based A-kinase activity probe (Nphp3N-AKAR2-CR) as an improved biosensor for monitoring real-time PKA activity in the PC of both cultured cells and living zebrafish embryos. Although the PKA catalytic subunit (PKA-C) was not observed in PC, basal PKA activity in cells could be detected with this probe. In addition, we have found that only ciliary-targeted PKA and not cytosolic PKA, can modulate the HH pathway, even when the integrity of the PC is disrupted. Notably, ciliary PKA activity was barely changed either by inhibition or activation of the HH pathway at the level of Smoothened (SMO), the obligate HH signal transducer. Moreover, we found that even low concentration of the adenylyl cyclase agonist forskolin (FSK) can efficiently inhibit the HH pathway in the presence of the constitutively active variant SMOA1, suggesting that the activation of the HH pathway by SMO may not be due solely to direct regulation of PKA activity in the PC.

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Funding

funders
[{'doi': '10.13039/501100001809', 'name': 'National Natural Science Foundation of China', 'awards': ['31970738']}, {'doi': '10.13039/501100001809', 'name': 'National Natural Science Foundation of China', 'awards': ['U21A20389']}, {'doi': '10.13039/501100001809', 'name': 'National Natural Science Foundation of China', 'awards': ['81960286']}, {'doi': None, 'name': 'Central Guidance on Local Science and Technology Development Fund of Shanxi Province', 'awards': ['YDZJSX2024D009']}]

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