Protein kinase A negatively regulates VEGF-induced AMPK activation by phosphorylating CaMKK2 at serine 495

Abstract

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 mutant (S495C). 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.

The 14-3-3 Proteins as Important Allosteric Regulators of Protein Kinases

“…14-3-3 protein recruitment by CaMKK1 partly suppresses its catalytic activity, whereas CaMKK2 activity remains unaltered, suggesting different regulation mechanisms for these isoforms [3,70,71,74]. Moreover, it has recently been shown that phosphorylation of CaMKK2 at Ser 495 is involved in regulating VEGF-induced AMPK activation, a pathway shown to regulate angiogenesis [75].…”

Adenosine monophosphate activated protein kinase contributes to skeletal muscle health through the control of mitochondrial function

“…Protein kinase C (PKC) results in phosphorylation at AMPKα1 Ser487, thereby inhibiting AMPK activity ( Heathcote et al, 2016 ). Vascular endothelial growth factor (VEGF) activates AMPK through CaMKK2 in endothelial cells, but protein kinase A (PKA) inhibits AMPK activation by phosphorylation at Ser495 ( Spengler et al, 2020 ). Further investigation will likely reveal more types of kinases and ensure better understanding of their important roles in AMPK activation and inhibition.…”

Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes

“…This signal transduction results in the regulation of many important physiological and pathological processes. 6 − 16 …”

“…Upon activation, CAMKK2 phosphorylates and activates its substrates including CAMK1, CAMK4, AMP-activated protein kinase, (AMPK) and in some cases AKT. This signal transduction results in the regulation of many important physiological and pathological processes. − …”