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 ␤ (CaMKK␤). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca 2؉ -dependent mechanism involving the thrombin receptor protease-activated receptor 1 and G q -protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKK␤ using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKK␤ 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␤ 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 Ca 2؉ 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 Ca 2؉ /CaMKK␤ dependent and one AMP/LKB1 dependent.AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase composed of a catalytic ␣ subunit and regulatory ␤ and ␥ subunits (6, 16). AMPK has been shown to function as a sensor of the energy state of the cell. It is activated by a rise in the AMP/ATP ratio following a fall of intracellular ATP and initiates a series of changes aimed at regulating energy balance at the cellular level. These processes include the inhibition of ATP-requiring anabolic pathways and the stimulation of ATP-generating catabolic pathways as well as changes in gene and protein expression (6, 16). Additionally, AMPK acts as a regulator of the whole-body energy metabolism by mediating the effects of hormones such as adiponectin, leptin, or ghrelin (28).AMPK activation requires phosphorylation of threonine 172 in the activation loop of the ␣ subunit (18). Two AMPKactivating kinases have been identified recently. LKB1, a tumor suppressor kinase, in complex with two accessory subunits, STRAD and MO25, has been shown to phosphorylate AMPK (19,23,38,46). Ca 2ϩ /calmodulin-dependent protein kinase kinase ␤ (CaMKK␤) has also been identified as an AMPK kinase (20,25,45). In addition to phosphorylation, AMPK is allosterically activated by binding of AMP, and this can also promote phosphorylation of threonine 172 (21). However, AMPK can be activated in an AMP-independent manner as shown with hyperosmotic stress or with the antidiabetic drug metformin (14). The finding that CaMKK␤ acts upstream of AMPK suggests that in addition ...

No abstract

No abstract