Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, 07743 Jena, Germany.
MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital, London W12 0NN, U.K.
Biochem J. 2020 Sep 18;477(17):3453-3469. doi: 10.1042/BCJ20200555.
Activation of AMP-activated protein kinase (AMPK) in endothelial cells by vascular endothelial growth factor (VEGF) via the Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) represents a pro-angiogenic pathway, whose regulation and function is incompletely understood. This study investigates whether the VEGF/AMPK pathway is regulated by cAMP-mediated signalling. We show that cAMP elevation in endothelial cells by forskolin, an activator of the adenylate cyclase, and/or 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, triggers protein kinase A (PKA)-mediated phosphorylation of CaMKK2 (serine residues S495, S511) and AMPK (S487). Phosphorylation of CaMKK2 by PKA led to an inhibition of its activity as measured in CaMKK2 immunoprecipitates of forskolin/IBMX-treated cells. This inhibition was linked to phosphorylation of S495, since it was not seen in cells expressing a non-phosphorylatable CaMKK2 S495C mutant. Phosphorylation of S511 alone in these cells was not able to inhibit CaMKK2 activity. Moreover, phosphorylation of AMPK at S487 was not sufficient to inhibit VEGF-induced AMPK activation in cells, in which PKA-mediated CaMKK2 inhibition was prevented by expression of the CaMKK2 S495C mutant. cAMP elevation in endothelial cells reduced basal and VEGF-induced acetyl-CoA carboxylase (ACC) phosphorylation at S79 even if AMPK was not inhibited. Together, this study reveals a novel regulatory mechanism of VEGF-induced AMPK activation by cAMP/PKA, which may explain, in part, inhibitory effects of PKA on angiogenic sprouting and play a role in balancing pro- and anti-angiogenic mechanisms in order to ensure functional angiogenesis.
血管内皮生长因子（VEGF）通过钙/钙调蛋白依赖性蛋白激酶激酶 2（CaMKK2）激活内皮细胞中的 AMP 激活的蛋白激酶（AMPK）代表了一种促血管生成途径，其调节和功能尚不完全清楚。本研究探讨了 VEGF/AMPK 途径是否受 cAMP 介导的信号转导调节。我们表明，内皮细胞中的 cAMP 升高通过佛司可林（一种激活腺嘌呤核苷酸环化酶的物质）和/或 3-异丁基-1-甲基黄嘌呤（IBMX，一种磷酸二酯酶抑制剂）实现，这触发了蛋白激酶 A（PKA）介导的 CaMKK2（丝氨酸残基 S495、S511）和 AMPK（S487）的磷酸化。PKA 对 CaMKK2 的磷酸化导致其在佛司可林/IBMX 处理细胞的 CaMKK2 免疫沉淀物中活性受到抑制。这种抑制与 S495 的磷酸化有关，因为在表达非磷酸化 CaMKK2 S495C 突变体的细胞中观察不到这种抑制。在这些细胞中，S511 的单独磷酸化不足以抑制 CaMKK2 活性。此外，在 PKA 介导的 CaMKK2 抑制被 CaMKK2 S495C 突变体的表达所阻止的细胞中，AMPK 丝氨酸 487 的磷酸化不足以抑制 VEGF 诱导的 AMPK 激活。内皮细胞中的 cAMP 升高即使在没有抑制 AMPK 的情况下，也降低了基础和 VEGF 诱导的乙酰辅酶 A 羧化酶（ACC）在 S79 处的磷酸化。总之，本研究揭示了 cAMP/PKA 对 VEGF 诱导的 AMPK 激活的新的调节机制，这部分解释了 PKA 对血管生成发芽的抑制作用，并在平衡促血管生成和抗血管生成机制方面发挥作用，以确保功能性血管生成。
