Xie Zhonglin, Dong Yunzhou, Zhang Miao, Cui Mei-Zhen, Cohen Richard A, Riek Uwe, Neumann Dietbert, Schlattner Uwe, Zou Ming-Hui
Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104, USA.
J Biol Chem. 2006 Mar 10;281(10):6366-75. doi: 10.1074/jbc.M511178200. Epub 2006 Jan 9.
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.
我们之前报道过，在培养的内皮细胞中，过氧亚硝酸根（ONOO-）和缺氧复氧可通过磷酸肌醇3激酶依赖性途径激活5'-AMP激活蛋白激酶（AMPK）。在此我们展示了该途径激活的分子机制。将牛主动脉内皮细胞暴露于ONOO-后，AMPK的苏氨酸172（Thr172）和内皮型一氧化氮合酶（一种已知的AMPK下游酶）的丝氨酸1179（Ser1179）的磷酸化显著增加。此外，ONOO-激活AMPK伴随着蛋白激酶Cζ（PKCζ）（苏氨酸410/403）磷酸化增加以及胞质PKCζ转位至细胞膜。进一步研究发现，抑制PKCζ可消除ONOO-诱导的AMPK-Thr172磷酸化，内皮型一氧化氮合酶的情况也如此。此外，组成型活性PKCζ突变体的过表达增强了AMPK-Thr172的磷酸化，表明PKCζ位于AMPK激活的上游。相反，ONOO-可激活LKB1缺陷的HeLa-S3细胞中的PKCζ，但对AMPK-Thr172和AMPK活性均无影响。这些数据表明，LKB1是PKCζ增强AMPK激活所必需的。在体外，重组PKCζ使LKB1的丝氨酸428（Ser428）磷酸化，导致AMPK的苏氨酸172磷酸化。此外，将LKB1的Ser428直接突变为丙氨酸，与激酶失活的LKB1突变体一样，可消除ONOO-诱导的AMPK激活。在源自人、大鼠和小鼠的几种细胞类型中，抑制PKCζ可显著减弱ONOO-增强的LKB1-Ser428和AMPK-Thr172的磷酸化。综上所述，我们得出结论：PKCζ可通过增加LKB1的Ser428磷酸化来调节AMPK活性，导致LKB1与AMPK结合，随后LKB1使AMPK的Thr172磷酸化。
