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1 REGULATION OF LKB1-DEPENDENT ADENOSINE MONOPHOSPHATE-ACTIVATED PROTEIN KINASE BY PROTEIN KINASE C-z R.
  1. Z. Xie,
  2. B. Davis,
  3. M. Zhang,
  4. M-H Zou*
  1. *Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, OK

Abstract

We previously reported the PI-3 kinase-dependent activation of the 59-AMP-activated kinase (AMPK) by peroxynitrite (ONOO2) and hypoxia-reoxygenation (H/R) in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway involves atypical protein kinase C (PKC)-z, a known downstream enzyme of PI-3 kinase, which leads to AMPK activation via LKB1. Exposure of bovine aortic endothelial cells (BAEC) to ONOO2 significantly increased the phosphorylation of both Thr172 of AMPK and the Ser1179 of endothelial nitric oxide synthase (eNOS), a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO2 was accompanied by increased phosphorylation of PKC-z (Thr410/403) and translocation of cytosolic PKC-z into the membrane. Further, inhibition of PKC-z with either pharmacological (PKC-z pseuosubstrates, PKC-z-PS) or genetic inhibitors (PKC-z dominant negative mutants, PKC-z-DN) abrogated ONOO2 -induced AMPK-Thr172 phosphorylation as that of eNOS. Furthermore, overexpression of a constitutively active PKC-z mutant (PKC-z-CA) enhanced the phosphorylation of AMPK-Thr172, suggesting that PKC-z is upstream of AMPK activation. In contrast, ONOO2 activated PKC-z in LKB1-deficient Hela-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKC-z-enhanced AMPK activation. In vitro, recombinant PKC-z phosphorylated both LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. In several cell types originating from human, rat, and mouse, inhibition of PKC-z significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO2. Taken together, we conclude that PKC-z 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.

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