Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta.
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.
Stahmann
Nadine
N
Institute of Molecular Cell Biology, Friedrich Schiller University of Jena, Nonnenplan 2, 07743 Jena, Germany.
Woods
Angela
A
Carling
David
D
Heller
Regine
R
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034622:RNA Interference; D044463:Receptor, PAR-1 / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism
D009097:Multienzyme Complexes; D044463:Receptor, PAR-1; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; C517698:CAMKK2 protein, human; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D010738:Type C Phospholipases; D013917:Thrombin; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000103:Acetyl-CoA Carboxylase