Activation of protein kinase Cζ 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 Thr(172) of AMPK and Ser(1179) 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 C zeta (PKC zeta) ( Thr(410/403)) and translocation of cytosolic PKC zeta into the membrane. Further, inhibition of PKC zeta abrogated ONOO--induced AMPK-Thr(172) phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKC zeta mutant enhanced the phosphorylation of AMPK-Thr(172), suggesting that PKC zeta is upstream of AMPK activation. In contrast, ONOO- activated PKC zeta in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr(172) nor AMPK activity. These data suggest that LKB1 is required for PKC zeta-enhanced AMPK activation. In vitro, recombinant PKC zeta phosphorylated LKB1 at Ser(428), resulting in phosphorylation of AMPK at Thr(172). Further, direct mutation of Ser(428) 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 PKC zeta significantly attenuated the phosphorylation of both LKB1-Ser(428) and AMPK-Thr(172) that were enhanced by ONOO-. Taken together, we conclude that PKC zeta can regulate AMPK activity by increasing the Ser(428) phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr(172) phosphorylation by LKB1.