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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- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
