Related Experiment Video
Updated: Aug 6, 2026

Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader
Published on: May 24, 2024
Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving
Nadine Stahmann1, Angela Woods, David Carling
1Institute of Molecular Cell Biology, Friedrich Schiller University of Jena, Nonnenplan 2, 07743 Jena, Germany.
Abstract:
AMP-activated protein kinase (AMPK) is a sensor of cellular energy state in response to metabolic stress and other regulatory signals. AMPK is controlled by upstream kinases which have recently been identified as LKB1 or Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca2+-dependent mechanism involving the thrombin receptor protease-activated receptor 1 and Gq-protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKKbeta using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKKbeta was the responsible AMPK kinase. In contrast, downregulation of LKB1 did not affect thrombin-induced AMPK activation but abolished phosphorylation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside. Thrombin stimulation led to phosphorylation of acetyl coenzyme A carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), two downstream targets of AMPK. Inhibition or downregulation of CaMKKbeta or AMPK abolished phosphorylation of ACC in response to thrombin but had no effect on eNOS phosphorylation, indicating that thrombin-stimulated phosphorylation of eNOS is not mediated by AMPK. Our results underline the role of Ca2+ as a regulator of AMPK activation in response to a physiologic stimulation. We also demonstrate that endothelial cells possess two pathways to activate AMPK, one Ca2+/CaMKKbeta dependent and one AMP/LKB1 dependent.
Related Concept Videos
cAMP-dependent Protein Kinase Pathways
Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
Amplifying Signals via Enzymatic Cascade
Amplifying Signals via Second Messengers
MAPK Signaling Cascades
Extrinsic and Intrinsic Pathways of Hemostasis
The Extrinsic Pathway
The extrinsic pathway of coagulation is typically initiated by tissue damage that exposes blood to tissue factor (TF), a protein released by the damaged tissue cells outside the blood vessels—this interaction with TF triggers biochemical reactions involving specific clotting factors. The key player here is Factor VII, which forms a...
