1. Biochemistry and Chemical Biology
  2. Cell Biology
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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. University Hospital Münster, Germany
  3. Max-Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Germany
  4. Paris Descartes University, France
  5. LIH Luxembourg Institute of Health, Luxembourg
  6. DZHK (German Center for Cardiovascular Research), Germany
  7. Berlin Institute of Health (BIH), Germany
Research Article
Cite this article as: eLife 2019;8:e46380 doi: 10.7554/eLife.46380
7 figures, 2 tables and 2 additional files

Figures

Figure 1 with 1 supplement
Identification of direct substrates of PKACα.

(A) Strategy for labeling, immunoprecipitation and identifying of PKACα substrates in HUVEC lysates. (B) Thio-phosphorylation of PKA substrates in HUVEC lysates expressing WT-PKACα or AS-PKACα for mass spectrometry analysis. Left panel (input) shows western blot analysis of lysates after alkylation before immunoprecipitation, middle panel (Eluate) shows western blot analysis of the eluted proteins from the immunoprecipitation beads, right panel (Eluate) shows the silver staining of the same samples as middle panel. (C) Validation of the six PKACα substrates identified in the chemical genetic approach screen by overexpressing of potential substrates and WT-PKACα (or AS-PKACα) in 293Tcells, and labeling the substrate in 293 T cell lysates. Western blots are representative of two independent experiments.

Figure 1—figure supplement 1
Screen for the best N6-substituted ATPγS analog as a phosphodonor of AS-PKACα.

(A) Kinase assay in HUVEC lysates using WT-PKACα and AS-PKACα. All the western blot membranes were exposed at the same time. (B) Structural formulas of 7 different N6-substituted ATPγS analogs.

Figure 2 with 1 supplement
PKACα phosphorylates ATG16L1α at S268 and ATG16L1β at S269.

(A) Peptide SPOT assay of ATG16L1 phosphorylation sites screening. (B) Amino acid sequence including potential PKACα phosphorylation sites in ATG16L1α and ATG16L1β according to the peptide SPOT assay result of Figure 2A. (C–D) Identification of PKACα phosphorylation site in ATG16L1α (C) and ATG16L1β (D). Analysis was performed as in Figure 1C. Western blots are representative of two independent experiments. (E–F) Flag tagged ATG16L1α and ATG16L1β were thio-phosphorylated by AS-PKACα and purified twice using M2 beads and thioP antibody coupled beads, followed by mass spectrometric analysis. LC-MS/MS spectra of the PKA- phosphorylated ATG16L1α tryptic peptide pSVSSFPVPQDNVDTHPGSGK and ATG16L1β tryptic peptide RLpSQPAGGLLDSITNIFGR. The results demonstrate that PKA phosphorylated ATG16L1α at S268 and phosphorylated ATG16L1β at S269.

Figure 2—figure supplement 1
Thiophophorylation is converted to normal phosphoryation by 1% TFA acid–promoted hydrolysis.

(A–B) ATG16L1α (A) and ATG16L1β (B) thiophosphorylated and immunoprecopitated as in Figure 1C, then treated with or without 1% TFA at 37°C for 4 hr.

PKACα mediated phosphorylation of ATG16L1 facilitates its degradation whereas PKA deficiency stabilizes ATG16L1.

(A–A'') HUVECs infected with Flag ATG16L1α (WT or S268A) were treated with 250 µM 6-bnz-cAMP and 20 µg/ml CHX at the time points indicated when they reached confluence (A). Quantifications of Flag ATG16L1α WT (A') and S268A (A'') expression. (B–B'') HUVECs infected with Flag ATG16L1β (WT or S269A) were treated with 250 µM 6-bnz-cAMP and 20 µg/ml CHX at the time points indicated when they reached confluence (B). Quantifications of Flag ATG16L1β WT (B') and S269A (B'') expression. (C–C'') HUVECs infected with GFP ATG16L1α (WT or S268D) were treated with 20 µg/ml CHX at the time points indicated when they reached confluence (C). Quantifications of GFP ATG16L1α WT (C') and S268D (C'') expression. (D–D'') HUVECs infected with GFP ATG16L1β (WT or S269D) were treated with 20 µg/ml CHX at the time points indicated when they reached confluence (D). Quantifications of GFP ATG16L1β WT (D') and S269D (D'') expression. (E–E') HUVECs infected with shRNA (scramble or shPKACα) virus were lysed in RIPA buffer and proteins were analyzed by western blot using indicated antibodies (E). Quantifications of indicated protein expression (E'). (F–F') HUVECs treated with DMSO (control) and 500 µM 6-bnz-cAMP were lysed in RIPA buffer and proteins were analyzed by western blot using indicated antibodies (F). Quantifications of indicated protein expression (F'). (G–G') Endothelial cells isolated from mice (wild type or dnPKAiEC) were lysed in RIPA buffer and ATG16L1 protein was analyzed by western blot (G). Quantifications of indicated ATG16L1 expression (G'). Data present the mean ± SD of 3 independent experiments. *P<0,05; **P<0,01; ***P<0,001; ****P<0,0001.

Figure 4 with 1 supplement
Autophagy inhibition partially rescues retinal vascular hypersprouting caused by PKA deficiency.

(A–F) Mice were injected with tamoxifen from P1 to P3, then retinas were collected at P6. Isolectin B4 and ESM1 staining of P6 retinas isolated from wtPKA with wtATG5 or ATG5ECKO mice and dnPKAiEC with wtATG5 or ATG5ECKO mice. Representative images are shown (A,D). Quantifications of radial expansion (B), sprouts per 100µm (C), vascular area (E) and ESM1 positive area (F) per field of retinal fronts. 8-10 retinas were measured for each group, *P<0,05; **P<0,01; ***P<0,001; ****P<0,0001.

Figure 4—source data 1

Values for quantification of radial expansion (Figure 4B), sprouts per 100 µm (Figure 4C), vascular area (Figure 4E) and ESM1 positive area (Figure 4F) per field of retinal fronts.

https://cdn.elifesciences.org/articles/46380/elife-46380-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Autophagy inhibition partially rescues retinal vascular hypersprouting caused by PKA deficiency.

(A–B) Mice were injected with tamoxifen from P1 to P3, and retinas were collected at P6. Isolectin B4 and ESM1 staining of P6 retinas isolated from wtPKA and dnPKAiEC mice treated with PBS or CQ. Representative images are shown (A,B). (C–D) Quantifications of vascular area (C) and ESM1 positive area (D) per field of retinal fronts. 30–35 fields (images) taken from 6 to 7 retinas were measured for each group, ****p<0,0001.

Figure 4—figure supplement 1—source data 1

Values for vascular area (Figure 4—figure supplement 1C) and ESM1 positive area (Figure 4—figure supplement 1D) per field of retinal fronts.

https://cdn.elifesciences.org/articles/46380/elife-46380-fig4-figsupp1-data1-v3.xlsx
Autophagy inhibition partially rescues retinal vascular hypersprouting caused by PKA deficiency through reducing endothelial cell number but not the ratio of proliferation endothelial cells and apoptosis of endothelial cells.

(A–D) Mice were injected with tamoxifen from P1 to P3, 50 µl 1 mg/ml EdU were I.P injected 2 hr before retinas collecting at P6. CD31 and ERG staining of P6 retinas isolated from wtPKA with wtATG5 or ATG5ECKO mice and dnPKAiEC with wtATG5 or ATG5ECKO mice followed by EdU Click-iT 647 dye labeling. Representative images are shown (A). Quantifications of endothelial cells (ERG positive cells) (B), proliferating endothelial cells (EdU and ERG positive cells) (C) and ratio of proliferating endothelial cells (EdU and ERG positive cells/ERG positive cells) (D). 6–9 retinas were measured for each group, *p<0,05; **p<0,01; ***p<0,001; ****p<0,0001. (E–F) Mice were injected with tamoxifen from P1 to P3, then retinas were collected at P6. CD31 and Cleaved caspase 3 staining of P6 retinas isolated from wtPKA with wtATG5 or ATG5ECKO mice and dnPKAiEC with wtATG5 or ATG5ECKO mice. Representative images are shown (E). Quantifications of endothelial apoptosis (F). 6–8 retinas were measured for each group.

Figure 5—source data 1

Values for quantification of endothelial cells (ERG positive cells) (Figure 5B), proliferating endothelial cells (EdU and ERG positive cells) (figure 5C), ratio of proliferating endothelial cells (EdU and ERG positive cells/ERG positive cells) (Figure 5D) and endothelial apoptosis (Figure 5F).

https://cdn.elifesciences.org/articles/46380/elife-46380-fig5-data1-v3.xlsx
Author response image 1
Spot array assay testing additional kinases predicted to phosphorylate the identified PKA sites on ATG16L1.
Author response image 2
HUVECs treated with PBS(control) and 10 µM isopropanol were lysed in RIPA buffer and proteins were analyzed by western blot using indicated antibodies.

Tables

Table 1
List of PKACa substrates.

Proteins are listed according to the log of fold changes of AS-PKACα to WT- PKACα. Two independent experiments have been done to prepare the PKACα substrates samples for mass spectrometric analysis.

UniprotProtein.namesGene.namesPeptidesLog ratio AS/WT
experiment1experiment2
Q6AI12Ankyrin repeat domain-containing protein 40ANKRD4091010
Q6P6C2RNA demethylase ALKBH5ALKBH561010
Q9NRY4Rho GTPase-activating protein 35ARHGAP3591010
E7EVC7Autophagy-related protein 16–1ATG16L181010
J3KPC8Serine/threonine-protein kinase SIK3SIK3;KIAA099951010
A1 × 283SH3 and PX domain-containing protein 2BSH3PXD2B41010
Q8IWZ8SURP and G-patch domain-containing protein 1SUGP151010
Q9UJX5Anaphase-promoting complex subunit 4ANAPC451010
O43719HIV Tat-specific factor 1HTATSF141010
O95644-5Nuclear factor of activated T-cells, cytoplasmic 1NFATC151010
G8JLI6Prolyl 3-hydroxylase 3LEPREL231010
F8W781Zinc finger CCCH domain-containing protein 13ZC3H1331010
Q9BZL4Protein phosphatase 1 regulatory subunit 12CPPP1R12C216,044402747,30701515
O14974Protein phosphatase 1 regulatory subunit 12APPP1R12A265,727960347,12654716
Q00537Cyclin-dependent kinase 17CDK17316,378673816,39216838
Q9Y4G8Rap guanine nucleotide exchange factor 2RAPGEF221106,17455504
Q9BYB0SH3 and multiple ankyrin repeat domains protein 3SHANK3325,265914215,6389181
J3KSW8Myosin phosphatase Rho-interacting proteinMPRIP184,613984775,61155414
P31323cAMP-dependent protein kinase type II-beta regulatory subunitPRKAR2B197,050771055,33509437
P13861cAMP-dependent protein kinase type II-alpha regulatory subunitPRKAR2A245,428419985,04010629
Q14980-2Nuclear mitotic apparatus protein 1NUMA1613,456259694,47466712
O15056Synaptojanin-2SYNJ2134,640226554,46069701
J3KNX9Unconventional myosin-XVIIIaMYO18A10104,43208178
Q86UU1-2Pleckstrin homology-like domain family B member 1PHLDB1195,41052434,10782285
P28715DNA repair protein complementing XP-G cellsERCC5;BIVM-ERCC583,35718264,09305592
P12270Nucleoprotein TPRTPR1043,33334724,04477536
Q15111Inactive phospholipase C-like protein 1;Phosphoinositide phospholipase CPLCL110103,32188704
Q9HD67Unconventional myosin-XMYO10394,139734153,21827463
Q14185Dedicator of cytokinesis protein 1DOCK1344,504134263,18515106
O75116Rho-associated protein kinase 2ROCK2293,277018643,08277835
Table 1—source data 1

The full list of proteins identified in both experiments is provided.

https://cdn.elifesciences.org/articles/46380/elife-46380-table1-data1-v3.docx
Key resources table
Reagent
type
(species) or
resource
DesignationSource
or reference
IdentifiersAdditional information
Strain, strain background (Mus musculus)Prkar1αtm2GsmPMID: 21533282
Strain, strain background (Mus musculus)Tg(Cdh5-cre/ERT2)1RhaMGI:3848980
Genetic reagent (Mus musculus)ATG5flox/floxPMID: 16625204
Cell line (human)HUVECPromoCell and freshly isolated cells
Cell line (human)HEK293TATCC
Transfected construct (Mus)pRRL.CMV.flag- PKACαThis paper
Transfected construct (Mus)pRRL.CMV.flag- PKACαM120GThis paper
Transfected construct (human)pECE-M2-PPP1R12AAddgene:31658
Transfected construct (human)EGFPC1-huNFATc1EE-WTAddgene: 24219
Transfected construct (human)pDESTmycDDX17, pRRL.CMV.flag-DDX17Addgene: 19876
This paper
Transfected construct (human)pMRX-IP/SECFP-hATG16L1
pRRL.CMV.flag- ATG16L1α
Addgene: 58994
This paper
Transfected construct (human)pRRL.CMV.flag- ATG16L1βThis paper
Transfected construct (human)pRRL.CMV.GFP- ATG5This paper
Transfected construct (human)pRRL.CMV.flag- ATG16L1α S268AThis paper
Transfected construct (human)pRRL.CMV.flag- ATG16L1β S269AThis paper
Transfected construct (human)pRRL.CMV.flag- ATG16L1β S287AThis paper
Transfected construct (human)pRRL.CMV.flag- ATG16L1β S269A and S287AThis paper
Transfected construct (human)pRRL.CMV.GFP- ATG16L1αThis paper
Transfected construct (human)pRRL.CMV.GFP- ATG16L1α S268DThis paper
Transfected construct (human)pRRL.CMV.GFP- ATG16L1βThis paper
Transfected construct (human)pRRL.CMV.GFP- ATG16L1β S269DThis paper
Transfected construct (human)pLKO.1-TRC cloning shRNA vectorAddgene: 10878
Transfected construct (human)pLKO.1-TRC shPKACαThis paper
AntibodyAnti-Thiophosphate ester antibody [51-8]Abcam: ab92570WB 1:5000
AntibodyAnti-Thiophosphate ester antibody [51-8]Abcam: ab133473IP
AntibodyGAPDH (14C10) Rabbit mAbcell signaling: #2118WB 1:1000
AntibodyAtg16L1 (D6D5) Rabbit mAbcell signaling: #8089WB 1:1000
AntibodyLC3B (D11) XP Rabbit mAbcell signaling: #3868WB 1:1000
AntibodyPKA C-α Antibodycell signaling: #4782WB 1:1000
AntibodySQSTM1/p62 (D5E2) Rabbit mAbcell signaling: #8025WB 1:1000
AntibodyPhospho-PKA Substrate (RRXS*/T*) (100G7E) Rabbit mAbcell signaling: #9624WB 1:1000
AntibodyGFP Tag Polyclonal AntibodyInvitrogen: A11122WB 1:1000
Antibodygoat anti-Actin(c-11)Santa Cruz Biotechnology: sc-1615WB 1:2000
AntibodyANTI-FLAG antibody produced in rabbitSigma: F7425WB 1:1000
AntibodyMonoclonal ANTI-FLAG M2 antibody produced in mouseSigma: F3165WB 1:1000
AntibodyAnti-rabbit IgG, HRP-linked Antibodycell signaling: #7074WB 1:2000
Antibodychicken anti-goat IgG-HRPSanta Cruz Biotechnology: sc-516086WB 1:5000
AntibodyPeroxidase AffiniPure Donkey Anti-Mouse IgG (H+L)Jackson Immuno Research: 715-035-151WB 1:2000
AntibodyIsolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor 488 ConjugateThermo Fisher: I21411IF 1:100
AntibodyMouse Endocan/ESM-1 AntibodyR and D: AF1999IF 1:100
AntibodyDonkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555Thermo Fisher: A21432IF 1:500
AntibodyRabbit anti-ERGAbcam: ab92513IF 1:500
Antibodyrabbit anti-cleaved caspase 3R and D: AF835IF 1:200
AntibodyRat anti-CD31BD Pharmingen: BD553370IF 1:200
IP
AntibodyDonkey anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488Thermo Fisher: A21208IF 1:500
AntibodyDonkey anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555Thermo Fisher: A31572IF 1:500
Commercial assay or kitQ5 Site-Directed Mutagenesis KitNEB: E0554S
Commercial assay or kitLenti-X p24 Rapid Titer KitClontech:632200
Commercial assay or kitPierce Silver Stain KitThermo Fisher:24600
Commercial assay or kitClick-iT EdU Alexa
Fluor 647 Imaging Kit
Thermo fishier:C10340
Chemical compound, drugX-tremeGENE HP DNA transfection reagentRoche
Chemical compound, drug6-cHe-ATPγSBiolog:C127
Chemical compound, drugSp-8-CPT-cAMPSBiolog:C012
Chemical compound, drug6-bnz-cAMPBiolog:C009
Software, algorithmimage Jimage J
Software, algorithmGraphPad Prism 7GraphPad Prism 7
OtherRecombinant Protein G AgaroseInvitrogen
Othersheep anti-Rat IgG-coupled DynabeadsInvitrogen

Additional files

Supplementary file 1

Array map of spot-synthesized 25-mer overlapping peptides covering the entire ATG16L1 protein.

https://cdn.elifesciences.org/articles/46380/elife-46380-supp1-v3.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/46380/elife-46380-transrepform-v3.docx

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