Phosphorylation of LKB1 at serine 428 by protein kinase C-zeta is required for metformin-enhanced activation of the AMP-activated protein kinase in endothelial cells.
BACKGROUND
Metformin, one of most commonly used antidiabetes drugs, is reported to exert its therapeutic effects by activating AMP-activated protein kinase (AMPK); however, the mechanism by which metformin activates AMPK is poorly defined. The objective of the present study was to determine how metformin activates AMPK in endothelial cells.


RESULTS
Exposure of human umbilical vein endothelial cells or bovine aortic endothelial cells to metformin significantly increased AMPK activity and the phosphorylation of both AMPK at Thr172 and LKB1 at Ser428, an AMPK kinase, which was paralleled by increased activation of protein kinase C (PKC)-zeta, as evidenced by increased activity, phosphorylation (Thr410/403), and nuclear translocation of PKC-zeta. Consistently, either pharmacological or genetic inhibition of PKC-zeta ablated metformin-enhanced phosphorylation of both AMPK-Thr172 and LKB1-Ser428, suggesting that PKC-zeta might act as an upstream kinase for LKB1. Furthermore, adenoviral overexpression of LKB1 kinase-dead mutants abolished but LKB1 wild-type overexpression enhanced the effects of metformin on AMPK in bovine aortic endothelial cells. In addition, metformin increased the phosphorylation and nuclear export of LKB1 into the cytosols as well as the association of AMPK with LKB1 in bovine aortic endothelial cells. Similarly, overexpression of LKB1 wild-type but not LKB1 S428A mutants (serine replaced by alanine) restored the effects of metformin on AMPK in LKB1-deficient HeLa-S3 cells, suggesting that Ser428 phosphorylation of LKB1 is required for metformin-enhanced AMPK activation. Moreover, LKB1 S428A, like kinase-dead LKB1 D194A, abolished metformin-enhanced LKB1 translocation as well as the association of LKB1 with AMPK in HeLa-S3 cells. Finally, inhibition of PKC-zeta abolished metformin-enhanced coimmunoprecipitation of LKB1 with both AMPKalpha1 and AMPKalpha2.


CONCLUSIONS
We conclude that PKC-zeta phosphorylates LKB1 at Ser428, resulting in LKB1 nuclear export and hence AMPK activation.
Xie
Zhonglin
Z
Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Dong
Yunzhou
Y
Scholz
Roland
R
Neumann
Dietbert
D
Zou
Ming-Hui
MH
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D021581:Active Transport, Cell Nucleus; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D003062:Codon / Q000187:drug effects; D003600:Cytosol / Q000201:enzymology; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D008687:Metformin / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D017354:Point Mutation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D000099221:Protein Kinase C zeta
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase; D003062:Codon; D007004:Hypoglycemic Agents; D008687:Metformin; D009097:Multienzyme Complexes; D010768:Phosphoserine; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D000099221:Protein Kinase C zeta; C109790:STK11 protein, human