Endothelial PKA activity regulates angiogenesis by limiting autophagy through phosphorylation of ATG16L1
- VIB, Belgium
- INSERM UMR-970, France
- Max Delbrück Center for Molecular Medicine, Germany
- Luxembourg Institute of Health, Luxembourg
Abstract
The cAMP-dependent protein kinase A (PKA) regulates various cellular functions in health and disease. In endothelial cells PKA activity promotes vessel maturation and limits tip cell formation. Here, we used a chemical genetic screen to identify endothelial-specific direct substrates of PKA in human umbilical vein endothelial cells (HUVEC) that may mediate these effects. Amongst several candidates, we identified ATG16L1, a regulator of autophagy, as novel target of PKA. Biochemical validation, mass spectrometry and peptide spot arrays revealed that PKA phosphorylates ATG16L1α at Ser268 and ATG16L1β at Ser269, driving phosphorylation-dependent degradation of ATG16L1 protein. Reducing PKA activity increased ATG16L1 protein levels and endothelial autophagy. Mouse in vivo genetics and pharmacological experiments demonstrated that autophagy inhibition partially rescues vascular hypersprouting caused by PKA deficiency. Together these results indicate that endothelial PKA activity mediates a critical switch from active sprouting to quiescence in part through phosphorylation of ATG16L1, which in turn reduces endothelial autophagy.
Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012975. All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for figures 3, 4 and 5.
Article and author information
Author details
Anne-Clemence Vion
Funding
Fonds vor Wetenschappelijk Onderzoek (G.0742.15N)
- Holger Gerhardt
Else-Kroener Stiftung (2014_A26)
- Pavel Nedvetsky
- Holger Gerhardt
European Research Council (311719 REshape)
- Holger Gerhardt
Deutsche Forschungsgemeinschaft (DFG KL1415/7-1)
- Enno Klussmann
Deutsche Forschungsgemeinschaft (394046635 - SFB 1365)
- Enno Klussmann
Bundes Ministerium für Bildung und Forschung (16GW0179K)
- Enno Klussmann
VIB (Tech Watch Q3 2015)
- Pavel Nedvetsky
- Holger Gerhardt
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experimental procedures were approved by the Institutional Animal Care and Research Advisory Committee of the University of Leuven (application P249/2014) and performed according to the European guidelines.
Copyright
© 2019, Zhao et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Endothelial PKA activity regulates angiogenesis by limiting autophagy through phosphorylation of ATG16L1
eLife 8:e46380.
https://doi.org/10.7554/eLife.46380
Further reading
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N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.
