Vascular Patterning Laboratory, Center for Cancer Biology, VIB, Leuven, Belgium.
Vascular Patterning Laboratory, Center for Cancer Biology, Department of Oncology, VIB, Leuven, Belgium.
Elife. 2019 Oct 3;8:e46380. doi: 10.7554/eLife.46380.
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.
环腺苷酸（cAMP）依赖性蛋白激酶 A（PKA）在健康和疾病中调节各种细胞功能。在内皮细胞中，PKA 活性促进血管成熟并限制尖端细胞的形成。在这里，我们使用化学遗传学筛选鉴定了人脐静脉内皮细胞（HUVEC）中 PKA 的内皮特异性直接底物，这些底物可能介导这些作用。在几个候选物中，我们发现自噬的调节因子 ATG16L1 是 PKA 的新靶标。生化验证、质谱和肽点阵列显示，PKA 使 ATG16L1α 在 Ser268 和 ATG16L1β 在 Ser269 处磷酸化，从而驱动 ATG16L1 蛋白的磷酸化依赖性降解。降低 PKA 活性会增加 ATG16L1 蛋白水平和内皮自噬。小鼠体内遗传学和药理学实验表明，自噬抑制部分挽救了由 PKA 缺乏引起的血管过度发芽。这些结果表明，内皮 PKA 活性介导了从活跃发芽到静止的关键转换，部分是通过 ATG16L1 的磷酸化，进而减少内皮自噬。
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