Thrombin Activates AMP-Activated Protein Kinase in Endothelial Cells via a Pathway Involving Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase β

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 ␤ (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 ...

Store-operated Ca2+ Entry (SOCE) Induced by Protease-activated Receptor-1 Mediates STIM1 Protein Phosphorylation to Inhibit SOCE in Endothelial Cells through AMP-activated Protein Kinase and p38β Mitogen-activated Protein Kinase

“…It is known that Ca 2ϩ entry signaling activates CaMKK␤ to induce AMPK␣ phosphorylation in endothelial cells (15,22). In this study, we observed that pretreatment with CaMKK␤ inhibitor STO-609 suppressed thrombin-induced AMPK␣1 phosphorylation indicating that CaMKK␤ is essential to activate AMPK␣1 in endothelial cells.…”

“…The upstream-activating enzyme AMPK kinase or the closely related tumor suppressor kinase LKB1 can phosphorylate the catalytic ␣-subunit of AMPK in an AMP-dependent manner (23,34). AMPK can also be activated in an AMP-independent manner, which involves CaMKK␤ (22,23). An increase in intracellular Ca 2ϩ is required for CaMKK␤ activation (22).…”

“…AMPK can also be activated in an AMP-independent manner, which involves CaMKK␤ (22,23). An increase in intracellular Ca 2ϩ is required for CaMKK␤ activation (22). The pharmacological AMPK activator, AICAR, has been shown to phosphorylate AMPK subunit ␣ and activate AMPK both in vitro and in vivo (23,34).…”

“…Microvessel Permeability-Studies from our laboratory (15) and others (22) showed that Ca 2ϩ entry signal activates AMPK in endothelial cells. Therefore, we tested whether AMPK signaling is involved in modulating SOCE in endothelial cells.…”

Role of AMP‐activated protein kinase in the metabolic syndrome and in heart disease

“…The CaMKKs are expressed at highest levels in neural tissue [19], and the distribution of this Ca 2+ -mediated pathway for AMPK activation in other cell types is still being investigated. It clearly occurs in neurones in response to activation of voltage-gated Ca 2+ channels [16], in T lymphocytes in response to stimulation of the antigen receptor [20], and in endothelial cells in response to activation of the thrombin receptor, which is coupled via Gq to release of inositol trisphosphate and hence of Ca 2+ from the endoplasmic reticulum [21].…”