4.5 Article

Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase β

Journal

MOLECULAR AND CELLULAR BIOLOGY
Volume 26, Issue 16, Pages 5933-5945

Publisher

AMER SOC MICROBIOLOGY
DOI: 10.1128/MCB.00383-06

Keywords

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Funding

  1. Medical Research Council [MC_U120027537] Funding Source: researchfish
  2. MRC [MC_U120027537] Funding Source: UKRI
  3. Medical Research Council [MC_U120027537] Funding Source: Medline

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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 (CaMKK beta). 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 G.-protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKK beta using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKK beta 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 CaMKK beta 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+/CaMKK beta dependent and one AMP/LKB1 dependent.

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