cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior.
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 ATG16L1a at Ser268 and ATG16L1b at Ser269, driving phosphorylationdependent 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 Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Introduction Angiogenesis is the process of new blood vessels formation from pre-existing vessels via sprouting and remodeling. Blood vessels are crucial for tissue growth and physiology in vertebrates since they are the pipelines for oxygen and nutrients supply and for immune cell distribution. Inadequate vessel formation and maintenance as well as abnormal vascular remodeling cause, accompany or aggravate many disease processes including myocardial infarction, stroke, cancer, and inflammatory disorders (Geudens and Gerhardt, 2011; Potente et al., 2011). Sprouting angiogenesis is a multistep process encompassing sprout initiation, elongation, anastomosis and final vascular network formation (Geudens and Gerhardt, 2011). Multiple molecular pathways have been identified to regulate sprouting angiogenesis; the most investigated are vascular endothelial growth factor (VEGF) and Notch/delta-like 4 (DLL4) signaling. Whereas VEGF initiates angiogenesis through activating VEGF receptor 2 (VEGFR2), thereby guiding angiogenic sprouting and tip cell formation as well as stalk cell proliferation (Ferrara et al., 2003; Gerhardt et al., 2003), Notch and its ligand delta-like 4 (DLL4) limit tip cell formation and angiogenic sprouting (Hellström et al., 2007; Leslie et al., 2007; Lobov et al., 2007; Suchting et al., 2007). VEGF and Notch/DLL4 pathways co-operate to form a fine-tuned feedback loop to balance tip and stalk cells during developmental angiogenesis and to maintain vascular stabilization (Hellström et al., 2007; Leslie et al., 2007; Lobov et al., 2007; Suchting et al., 2007). In addition, PI3K/Akt (Lee et al., 2014), MAP4K4 (Vitorino et al., 2015), ephrins and Eph receptors (Cheng et al., 2002), hedgehog signaling (Pola et al., 2001),YAP/TAZ (Kim et al., 2017; Neto et al., 2018),BMP signaling (Vion et al., 2018) and other pathways also regulate various aspects of angiogenesis. We identified endothelial cAMP-dependent protein kinase A (PKA) as a critical regulator of angiogenesis during vascular development in vivo. Inhibition of endothelial PKA results in hypersprouting and increased numbers of tip cells, indicating that PKA regulates the transition from sprouting to quiescent vessels (Nedvetsky et al., 2016). However, the PKA targets mediating these effects remained unknown. Here, we established a chemical genetics approach based on the mutation of a 'gatekeeper residue' of the ATP-binding pocket of a protein kinase to identify endothelial-specific substrates of PKA. We verified autophagy-related-protein-16-like 1 (ATG16L1) as a substrate of endothelial PKA and identified that phosphorylation of ATG16L1 facilitates its degradation. in vivo experiments showed that autophagy inhibition partially rescued the hypersprouting and increased tip cell numbers caused by PKA deficiency. Results Screen for novel substrates of endothelial PKA In order to identify direct substrates of endothelial PKA, we employed a chemical genetics approach (Allen et al., 2005). The ATP-binding pocket of kinases contains a conserved 'gatekeeper residue', which in naturally occurring wild type (WT) kinases is usually a methionine or phenylalanine. In engineered analogue specific (AS) kinases, this 'gatekeeper residue' is replaced with a smaller amino acid (glycine or alanine), enabling the AS-kinases to accept ATP analogues (or ATPγS analogues) that are modified at the N6 position with bulky groups as (thio-)phosphodonors. In contrast, WT-kinases poorly use these analogues. Once the substrates are thiophosphorylated by AS-kinases, they can be further alkylated and therefore recognized by a thiophosphate ester-specific antibody (Alaimo et al., 2001; Allen et al., 2005; Allen et al., 2007; Banko et al., 2011). To generate AS-PKACα, we mutated the methionine 120 to a glycine residue. Testing seven different variants of N6-substituted bulky ATPγS analogues, we identified 6-cHe-ATPγS as the best thiophosphodonor for AS-PKACα substrates in HUVEC lysates (Figure 1—figure supplement 1). To identify endothelial substrates of PKACα, HUVECs expressing WT-PKACα or AS-PKACα were lysed in kinase lysis buffer (KLB) and the thiophosphorylation reaction with 6-cHe-ATPγS was performed. After alkylation with p-Nitrobenzyl mesylate (PNBM), thiophosphorylated proteins were immunoprecipitated with the thioP antibody coupled to rProtein G Agarose beads (Figure 1A). For quality control, one thirtieth of the protein on agarose beads was eluted for western blot analysis, and the same amount of protein was used for gel silver staining (Figure 1B). The rest was subjected to mass spectrometry analysis. Two independent experiments were performed. Candidate endothelial PKA targets were identified as peptides that were at least 2-fold (log ratio(AS/WT)>1) enriched in the AS-PKACα samples compared to WT- PKACα samples in both experiments. Figure 1 with 1 supplement see all Download asset Open asset 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. The ninety-seven proteins identified in both experiments (Table 1—source data 1) included several known PKA substrates such as NFATC1 (Niswender et al., 2002), VASP (Anton et al., 2014; Butt et al., 1994; Profirovic et al., 2005), PRKAR2A and PRKAR2B (Manni et al., 2008) indicating that the chemical genetic screen worked well to identify PKA substrates. Thirty proteins with at least 8-fold enrichment (log ratio(AS/WT)>3) are listed according to their log ratio (AS/WT) value in Table 1, presenting the most likely direct substrates of PKA in this screen. 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/WTexperiment1experiment2Q6AI12Ankyrin repeat domain-containing protein 40ANKRD4091010Q6P6C2RNA demethylase ALKBH5ALKBH561010Q9NRY4Rho GTPase-activating protein 35ARHGAP3591010E7EVC7Autophagy-related protein 16–1ATG16L181010J3KPC8Serine/threonine-protein kinase SIK3SIK3;KIAA099951010A1 × 283SH3 and PX domain-containing protein 2BSH3PXD2B41010Q8IWZ8SURP and G-patch domain-containing protein 1SUGP151010Q9UJX5Anaphase-promoting complex subunit 4ANAPC451010O43719HIV Tat-specific factor 1HTATSF141010O95644-5Nuclear factor of activated T-cells, cytoplasmic 1NFATC151010G8JLI6Prolyl 3-hydroxylase 3LEPREL231010F8W781Zinc finger CCCH domain-containing protein 13ZC3H1331010Q9BZL4Protein phosphatase 1 regulatory subunit 12CPPP1R12C216,044402747,30701515O14974Protein phosphatase 1 regulatory subunit 12APPP1R12A265,727960347,12654716Q00537Cyclin-dependent kinase 17CDK17316,378673816,39216838Q9Y4G8Rap guanine nucleotide exchange factor 2RAPGEF221106,17455504Q9BYB0SH3 and multiple ankyrin repeat domains protein 3SHANK3325,265914215,6389181J3KSW8Myosin phosphatase Rho-interacting proteinMPRIP184,613984775,61155414P31323cAMP-dependent protein kinase type II-beta regulatory subunitPRKAR2B197,050771055,33509437P13861cAMP-dependent protein kinase type II-alpha regulatory subunitPRKAR2A245,428419985,04010629Q14980-2Nuclear mitotic apparatus protein 1NUMA1613,456259694,47466712O15056Synaptojanin-2SYNJ2134,640226554,46069701J3KNX9Unconventional myosin-XVIIIaMYO18A10104,43208178Q86UU1-2Pleckstrin homology-like domain family B member 1PHLDB1195,41052434,10782285P28715DNA repair protein complementing XP-G cellsERCC5;BIVM-ERCC583,35718264,09305592P12270Nucleoprotein TPRTPR1043,33334724,04477536Q15111Inactive phospholipase C-like protein 1;Phosphoinositide phospholipase CPLCL110103,32188704Q9HD67Unconventional myosin-XMYO10394,139734153,21827463Q14185Dedicator of cytokinesis protein 1DOCK1344,504134263,18515106O75116Rho-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 Download elife-46380-table1-data1-v3.docx To validate novel candidate PKACα substrates, we overexpressed WT-PKACα or AS-PKACα together with flag- or GFP- tagged candidate proteins in 293 T cells, and used the 6-cHe-ATPγS as thiophosphate donor to thiophosphorylate substrates in lysates as described above. Lysate immunoprecipitation was carried out with M2 anti-flag beads or anti-GFP antibody coupled agarose beads, and the immune complexes were probed by western blot using thiophosphate antibody. The known PKA substrate NFATC1 served as a positive control. Five selected new candidate proteins (PPP1R12C, ATG16L1α, DDX17, ANKRD40 and ATG5) out of ninety-seven proteins were tested; four of these five proteins were confirmed to be thiophosphorylated by AS-PKACα, indicating that they are indeed direct substrates of AS-PKACα (Figure 1C). Only ATG5 was not thiophosphorylated by AS-PKACα in the validation of the screen (Figure 1C). Bioinformatic analysis of ATG5 amino acids sequence also failed to identify a consensus PKA substrate motif (R-R/K-X-S/T;K/R-X1-2-S/T) (Kennelly and Krebs, 1991). Since ATG5 directly binds to ATG16L1 (Matsushita et al., 2007; Mizushima et al., 1999), it likely co-precipitated with ATG16L1 in our screen. ATG5 and ATG16L1 are conserved core components of the autophagy process, and PKA activity has been shown to negatively regulate autophagy in S. Cervisiae and mammalian cells through phosphorylation of ATG1/ULK1 (Mizushima, 2010). ATG16L1 however has not previously been identified as a PKA target, prompting us to further investigate this interaction and the potential regulatory role of PKA and autophagy in endothelial sprouting. Pkacα phosphorylates ATG16L1α at S268 and ATG16L1β at S269 To identify the PKACα phosphorylation sites in ATG16L1, we spot-synthesized 25-mer overlapping peptides that cover the entire ATG16L1 protein. The peptide array was subjected to an in vitro PKA phosphorylation assay. Three peptides of the ATG16L1α and 7 peptides of ATG16L1β were phosphorylated compared to the negative control (Figure 2A, Supplementary file 1). The common amino acid sequences included in the phosphorylated peptides predicted Ser268 in ATG16L1α and Ser269 as well as Ser287 in ATG16L1β as potential PKA phosphorylation sites (Figure 2B). Indeed serine to alanine mutation S268A in ATG16L1α and S269A but not S287A mutation in ATG16L1β resulted in a loss of AS-PKACα thiophosphorylation of these two ATG16L1 isoforms (Figure 2C and D). Moreover, LC-MS/MS analysis demonstrated phosphorylation of ATG16L1α at S268 and of ATG16L1β at S269 (Figure 2E and F). Of note, thiophosphorylation was converted to normal phosphorylation by 1% TFA acid-catalyzed hydrolysis during sample preparation for LC-MS/MS, thus identifying the target sites as phosphorylated, not thiophosphorylated (Figure 2—figure supplement 1). Together, these results demonstrate that S268 and S269 are the PKACα phosphorylation sites of ATG16L1α and ATG16L1β, respectively. Figure 2 with 1 supplement see all Download asset Open asset 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. Pkacα regulates ATG16L1 by phosphorylation-dependent degradation Phosphorylation is one of the most widespread types of post-translational modification, and is crucial for signal transduction (Hunter, 1995; Manning et al., 2002; Ubersax and Ferrell, 2007). Previous research demonstrated that phosphorylation can regulate protein degradation by controlling its stabilization (Bullen et al., 2016; Geng et al., 2009; Hwang et al., 2009). To determine whether phosphorylation of ATG16L1α by PKA regulates protein stability, we overexpressed ATG16L1αWT and the mutant ATG16L1αS268A in HUVECs. To activate PKA, HUVECs were treated with the PKA specific activator 6-Bnz-cAMP. Using cycloheximide (CHX) to prevent new protein synthesis allowed us to detect the degradation of ATG16L1αWT and ATG16L1αS268A over time. Western blot analysis showed that most of the ATG16L1αWT degraded after 12 hr, whereas ATG16L1αS268A remained largely stable (Figure 3A), suggesting that the phosphorylation of ATG16L1α at site Ser268 by PKA promotes degradation. For ATG16L1β, Ser269 phosphorylation exhibited a similar function (Figure 3B). In addition, the phosphomimetic site mutants ATG16L1αS268D and ATG16L1βS269D were less stable than wild type ATG16L1α and ATG16L1β (Figure 3C–3D), supporting the idea that phosphorylation of ATG16L1α on site S268 and ATG16L1β on site S269 by PKA promotes degradation. In accordance, depleting PKACα in HUVECs by shRNA led to accumulation of ATG16L1 (Figure 3E) whereas activating PKA by 6-Bnz-cAMP caused ATG16L1 reduction (Figure 3F). Moreover, in PKA inhibited endothelial cells isolated from dnPKAiEC mice (Nedvetsky et al., 2016), ATG16L1 protein was also increased compared to endothelial cells isolated from corresponding Cdh5-CreERT2 control mice (Figure 3G). dnPKAiEC is the short denomination for Prkar1aTg/+ mice carrying a single floxed dominant-negative Prkar1a allele, (the regulatory subunit Prkar1a of PKA is an endogenous inhibitor of PKA) crossed with Cdh5-CreERT2 mice expressing tamoxifen inducible Cre recombinase under control of endothelial specific Cdh5 promotor (Nedvetsky et al., 2016). Taken together, these results indicate that PKACα regulates ATG16L1 by phosphorylation-dependent degradation. Figure 3 Download asset Open asset 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 3—source data 1 Values for quantification of indicated protein expression in Figure 3A, B, C, D, E, F and G. https://cdn.elifesciences.org/articles/46380/elife-46380-fig3-data1-v3.xlsx Download elife-46380-fig3-data1-v3.xlsx Inhibition of autophagy partially normalizes the vascular phenotype caused by PKA-deficiency ATG16L1 is an important component of the ATG16L1-ATG5-ATG12 protein complex, required for LC3 lipidation and autophagosome formation. Both LC3 lipidation and autophagosome formation represent essential steps in autophagy (Kuma et al., 2002; Levine and Kroemer, 2008; Matsushita et al., 2007; Mizushima et al., 2003; Mizushima et al., 1999). Accumulation of ATG16L1 upon PKA knock down resulted in increased levels of the positive autophagy marker LC3II whilst reducing the negative autophagy marker p62 in HUVECs (Figure 3E) whereas depleting ATG16L1 by 6-Bnz-cAMP mediated PKA activation resulted in decreased levels of LC3II whilst increasing p62 in HUVECs (Figure 3F). Since ATG16L1 protein levels in endothelial cells isolated from dnPKA mice were also increased, we hypothesized that increased autophagy in endothelial cells may contribute to the vascular phenotype in these mice. If so, inhibiting autophagy could potentially normalize vascular hypersprouting in dnPKAiEC mice. To test this hypothesis, we crossed the ATG5 conditional knock out mice ATG5ECKO (ATG5flox/flox mice crossed with Cdh5-CreERT2 mice) with dnPKAiEC mice, performed retinal staining for isolectin B4 (IB4, membrane staining) and the tip cell specific marker ESM1, and quantified the IB4 and ESM1 positive areas. No significant difference were observed between Cdh5-CreERT2 control mice and ATG5ECKO mice on both the vascular plexus and tip cells. However, ATG5ECKO partially rescued both the hyperdense vascular plexus front and the increasing tip cells in dnPKAiEC mice. Retinal stainings demonstrated that ATG5 deletion in endothelial cells in vivo, which shuts down ATG5-dependent autophagy, partially normalizes the hypersprouting phenotype of dnPKAiEC mice (Figure 4A–4F). Autophagy inhibitor chloroquine (CQ) treatment confirmed that autophagy inhibition can partially rescue both the hyperdense vascular plexus front and the increasing tip cells in dnPKAiEC mice (Figure 4—figure supplement 1) . Further more, although the ratio of proliferating endothelial cells was not significant different in retinas of the four groups of mice (Cdh5-CreERT2 control; ATG5ECKO;dnPKAiEC; and dnPKAiEC ATG5ECKO), the total number of endothelial cells and proliferating endothelial cells was deceased in dnPKAiEC ATG5ECKO compared to dnPKAiEC mouse retinas (Figure 5A–5D). Also the low levels of apoptotic endothelial cells showed no significant differences (Figure 5E–5F), suggesting that neither the rate of proliferation nor the frequency of apoptosis are drivers of PKA and ATG5 dependent vascular density and sprouting phenotypes. ATG5 deletion in endothelial cells instead appears to normalize the hypersprouting phenotype of dnPKAiEC mice by reducing the number of endothelial tip cells cells as well as the total number of proliferating endothelial cells. Although speculative at this time, a shorter cell cycle or more rounds of cycling per cell in the case of increased autophagy would for example explain the increased number of cells with unchanged ratio of proliferating endothelial cells. Altogether our results suggest that PKA regulates the switch from sprouting to stabilization of nascent vascular plexuses by limiting endothelial autophagy levels via ATG16L1 degradation. Figure 4 with 1 supplement see all Download asset Open asset 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 Download elife-46380-fig4-data1-v3.xlsx Figure 5 Download asset Open asset 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 Download elife-46380-fig5-data1-v3.xlsx Discussion Our chemical genetic screen and biochemical analysis identified ATG16L1 as a novel target of PKA activity in endothelial cells. The combined results demonstrate that PKA activity inhibits autophagy in cultured human umbilical vein endothelial cells (HUVEC) via the phosphorylation of ATG16L1, which accelerates its degradation. In cultured bovine aortic endothelial cells, induction of autophagy by overexpression of ATG5 has been shown to promote in vitro vascular tubulogenesis, whereas ATG5 silencing suppressed this morphogenic behavior (Du et al., 2012). In mice, inhibition of autophagy by bafilomycin, or genetic beclin heterozygosity as well as ATG5 knockout impairs angiogenesis post myocardial infarction, whereas the angiogenic factor AGGF1 enhances therapeutic angiogenesis through JNK-mediated stimulation of endothelial autophagy (Lu et al., 2016). Angiogenesis during tissue regeneration in a burn wound model also relied on induction of endothelial autophagy, by driving AMPK/AKT/mTOR signaling (Liang et al., 2018), together suggesting that autophagy regulation may represent a critical determinant of the extent of vascular sprouting. The identification of ATG16L1 as a direct target of PKA therefore raises the hypothesis that the dramatic hypersprouting phenotype in dnPKAiEC mouse retinas deficient in endothelial PKA activity may result from exuberant endothelial activation of autophagy. Both genetic endothelial inactivation of ATG5 and chemical inhibition of autophagy partially normalized the hypersprouting phenotype in dnPKAiEC mice, suggesting that indeed the activation of autophagy in dnPKAiEC mice contributes to vascular hypersprouting. However, the failure to fully normalize vascular patterning by autophagy inhibition indicates that additional PKA targets and mechanisms may be involved. Our mass spectrometry analysis identified a list of presumptive endothelial PKA substrates, which will potentially also be involved in angiogenesis. For example, we identified RAPGEF2 as a candidate target, deficiency of which causes embryonic lethality at E11.5 due to yolk sac vascular defects (Satyanarayana et al., 2010), very similar to the yolk sac phenotype in dnPKAiEC embryos (Nedvetsky et al., 2016). Similarly the potential target Rock2 has a well known role in regulating endothelial functions in angiogenesis (Liu et al., 2018; Montalvo et al., 2013; Seto et al., 2016; Shimizu et al., 2013). Further studies will need to validate all the listed targets and establish which of these exert critical endothelial functions, and under what conditions. An alternative explanation for the partial rescue of the dnPKAiEC phenotype by inhibition of autophagy could lie in additional functions of ATG16L1 itself. Although ATG16L1 plays an essential role in autophagy, and is part of a larger protein complex ATG16L1-ATG5-ATG12 that is necessary for autophagy, ATG16L1 is also involved in the production of inflammatory cytokines IL-1β and IL-18 and exerts anti-inflammatory functions during intestinal inflammation (Cadwell et al., 2008; Diamanti et al., 2017; Saitoh et al., 2008; Sorbara et al., 2013). IL-1β promotes angiogenesis by activating VEGF production during tumor progression (Carmi et al., 2013; Voronov et al., 2003), while IL-18 suppresses angiogenesis in cancer (Cao et al., 1999; Xing et al., 2016; Yang et al., 2010). How ATG16L1 regulates angiogenesis through inflammatory cytokines and whether this regulation operates downstream of PKA activity in vivo requires further investigation. Intriguingly, our rescue experiments show that in wild type mice, inhibition of autophagy has no significant effect on developmental retinal angiogenesis. This could indicate that autophagy in developmental angiogenesis, unlike in pathological angiogenesis and post-ischemic tissue responses, is not very active. Although, to our knowledge, this is the first identification of ATG16L1 as a PKA target and the first indication that PKA regulates autophagy in endothelial cells, PKA has previously been identified as regulator of autophagy. For example, PKA reduces autophagy through phosphorylation of ATG13 in Saccharomyces cerevisiae (Hundsrucker et al., 2006), and through phosphorylation of LC3 in neurons (Cherra et al., 2010). In our research, ATG16L1 was identified as a novel direct PKA substrate in endothelial cells, but not ATG13 or LC3. Mechanistically, the phosphorylation of ATG16L1 by PKA accelerates its degradation, and consequently decreases autophagy levels in endothelial cells. The finding of different components of the autophagy pathway as targets of PKA identified in yeast and various vertebrate cell populations raises the intriguing possibility that although the principle regulatory logic of PKA in autophagy is conserved, different protein targets mediate this effect in different cells or organisms. In addition, or alternatively, this regulation carries multiple levels of redundancy, and the individual studies simply identify the most prevalent targets within the respective cell types. The fact that also ATG16L1 comes in two splice variants that are both targeted by PKA in endothelial cells lends some strength to this idea. Interestingly, ATG16L1 can itself be regulated by multiple phosphorylation events by distinct kinases, with opposing effects on protein stability and autophagy. ATG16L1 can be phosphorylat
Abstract The cAMP-dependent protein kinase A (PKA) regulates a plethora of cellular functions in health and disease. During angiogenesis, PKA activity in endothelial cells controls the transition from sprouting to vessel maturation and limits tip cell formation independently of Notch signaling. The molecular PKA targets mediating these effects remain unknown. We report a chemical genetics screen identifying endothelial-specific substrates of PKA in human umbilical vein endothelial cells (HUVEC). 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. The phosphorylations drive degradation of ATG16L1 protein. Knocking down PKA or inhibiting its activity increased ATG16L1 protein levels and endothelial autophagy. In vivo genetics and pharmacological experiments demonstrated that autophagy inhibition partially rescues vascular hypersprouting caused by PKA deficiency. We propose that endothelial PKA activity restricts active sprouting by reducing endothelial autophagy through phosphorylation of ATG16L1.
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