Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells.
We previously reported the phosphoinositide 3-kinase-dependent activation of the 5'-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Czeta (PKCzeta) (Thr410/403) and translocation of cytosolic PKCzeta into the membrane. Further, inhibition of PKCzeta abrogated ONOO- -induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCzeta mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCzeta is upstream of AMPK activation. In contrast, ONOO- activated PKCzeta in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCzeta-enhanced AMPK activation. In vitro, recombinant PKCzeta phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO- -induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCzeta significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCzeta can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1.
Xie
Zhonglin
Z
Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104, USA.
Dong
Yunzhou
Y
Zhang
Miao
M
Cui
Mei-Zhen
MZ
Cohen
Richard A
RA
Riek
Uwe
U
Neumann
Dietbert
D
Schlattner
Uwe
U
Zou
Ming-Hui
MH
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D000818:Animals; D002417:Cattle; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004734:Energy Metabolism; D004789:Enzyme Activation / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D051381:Rats; D000099221:Protein Kinase C zeta
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D009097:Multienzyme Complexes; D052250:Nitric Oxide Synthase Type III; D030421:Peroxynitrous Acid; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D000099221:Protein Kinase C zeta; C109790:STK11 protein, human