1. Biochemistry and Chemical Biology
  2. Cell Biology
Download icon

Endothelial PKA activity regulates angiogenesis by limiting autophagy through phosphorylation of ATG16L1

  1. Xiaocheng Zhao
  2. Pavel Nedvetsky
  3. Fabio Stanchi
  4. Anne-Clemence Vion
  5. Oliver Popp
  6. Kerstin Zühlke
  7. Gunnar Dittmar
  8. Enno Klussmann
  9. Holger Gerhardt  Is a corresponding author
  1. VIB, Belgium
  2. INSERM UMR-970, France
  3. Max Delbrück Center for Molecular Medicine, Germany
  4. Luxembourg Institute of Health, Luxembourg
Research Article
  • Cited 8
  • Views 1,804
  • Annotations
Cite this article as: eLife 2019;8:e46380 doi: 10.7554/eLife.46380

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.

Article and author information

Author details

  1. Xiaocheng Zhao

    Vascular Patterning Laboratory, Center for Cancer Biology, VIB, Leuven, Belgium
    Competing interests
    No competing interests declared.

  2. Pavel Nedvetsky

    Vascular Patterning Laboratory, Center for Cancer Biology, VIB, Leuven, Belgium
    Competing interests
    No competing interests declared.

  3. Fabio Stanchi

    Vascular Patterning Laboratory, Center for Cancer Biology, VIB, Leuven, Belgium
    Competing interests
    No competing interests declared.

  4. Anne-Clemence Vion

    Paris Cardiovascular Research Center, INSERM UMR-970, Paris, France
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2788-2512

  5. Oliver Popp

    Proteomics, Max Delbrück Center for Molecular Medicine, Berlin, Germany
    Competing interests
    No competing interests declared.

  6. Kerstin Zühlke

    Anchored Signaling Lab, Max Delbrück Center for Molecular Medicine, Berlin, Germany
    Competing interests
    No competing interests declared.

  7. Gunnar Dittmar

    Department of Oncology, Luxembourg Institute of Health, Luxembourg, Luxembourg
    Competing interests
    No competing interests declared.

  8. Enno Klussmann

    Anchored Signaling Lab, Max Delbrück Center for Molecular Medicine, Berlin, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4004-5003

  9. Holger Gerhardt

    Integrative Vascular Biology Lab, Max Delbrück Center for Molecular Medicine, Berlin, Germany
    For correspondence
    holger.gerhardt@mdc-berlin.de
    Competing interests
    Holger Gerhardt, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3030-0384

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.

Reviewing Editor

  1. Gou Young Koh, Institute of Basic Science and Korea Advanced Institute of Science and Technology (KAIST), Korea (South), Republic of

Publication history

  1. Received: February 25, 2019
  2. Accepted: October 1, 2019
  3. Accepted Manuscript published: October 3, 2019 (version 1)
  4. Version of Record published: October 17, 2019 (version 2)
  5. Version of Record updated: April 27, 2020 (version 3)

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.

Metrics

  • 1,804
    Page views
  • 382
    Downloads
  • 8
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Categories and tags

Research organisms

Further reading

    1. Biochemistry and Chemical Biology

    Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning

    Phil Huss et al.
    Research Article Updated

    The interaction between a bacteriophage and its host is mediated by the phage's receptor binding protein (RBP). Despite its fundamental role in governing phage activity and host range, molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP (1660 variants) by developing a high-throughput, locus-specific, phage engineering method. This rich dataset allowed us to cross compare functional profiles across hosts to precisely identify regions of functional importance, many of which were previously unknown. Substitution patterns showed host-specific differences in position and physicochemical properties of mutations, revealing molecular adaptation to individual hosts. We discovered gain-of-function variants against resistant hosts and host-constricting variants that eliminated certain hosts. To demonstrate therapeutic utility, we engineered highly active T7 variants against a urinary tract pathogen. Our approach presents a generalized framework for characterizing sequence–function relationships in many phage–bacterial systems.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

    Mikel Garcia-Marcos
    Research Article Updated

    It has become evident that activation of heterotrimeric G-proteins by cytoplasmic proteins that are not G-protein-coupled receptors (GPCRs) plays a role in physiology and disease. Despite sharing the same biochemical guanine nucleotide exchange factor (GEF) activity as GPCRs in vitro, the mechanisms by which these cytoplasmic proteins trigger G-protein-dependent signaling in cells have not been elucidated. Heterotrimeric G-proteins can give rise to two active signaling species, Gα-GTP and dissociated Gβγ, with different downstream effectors, but how non-receptor GEFs affect the levels of these two species in cells is not known. Here, a systematic comparison of GPCRs and three unrelated non-receptor proteins with GEF activity in vitro (GIV/Girdin, AGS1/Dexras1, and Ric-8A) revealed high divergence in their contribution to generating Gα-GTP and free Gβγ in cells directly measured with live-cell biosensors. These findings demonstrate fundamental differences in how receptor and non-receptor G-protein activators promote signaling in cells despite sharing similar biochemical activities in vitro.