Identification of the Serine 307 of LKB1 as a Novel Phosphorylation Site Essential for Its Nucleocytoplasmic Transport and Endothelial Cell Angiogenesis

Abstract

LKB1, a master kinase that controls at least 13 downstream protein kinases including the AMP-activated protein kinase (AMPK), resides mainly in the nucleus. A key step in LKB1 activation is its export from the nucleus to the cytoplasm. Here, we identified S307 of LKB1 as a putative novel phosphorylation site which is essential for its nucleocytoplasmic transport. In a cell-free system, recombinant PKC-ζ phosphorylates LKB1 at S307. AMPK-activating agents stimulate PKC-ζ activity and LKB1 phosphorylation at S307 in endothelial cells, hepatocytes, skeletal muscle cells, and vascular smooth muscle cells. Like the kinase-dead LKB1 D194A mutant (mutation of Asp194 to Ala), the constitutively nucleus-localized LKB1 SL26 mutant and the LKB1 S307A mutant (Ser307 to Ala) exhibit a decreased association with STRADα. Interestingly, the PKC-ζ consensus sequence surrounding LKB1 S307 is disrupted in the LKB1 SL26 mutant, thus providing a likely molecular explanation for this mutation causing LKB1 dysfunction. In addition, LKB1 nucleocytoplasmic transport and AMPK activation in response to peroxynitrite are markedly reduced by pharmacological inhibition of CRM1, which normally facilitates nuclear export of LKB1-STRAD complexes. In comparison to the LKB1 wild type, the S307A mutant complexes show reduced association with CRM1. Finally, adenoviral overexpression of wild-type LKB1 suppresses, while the LKB1 S307A mutant increases, tube formation and hydrogen peroxide-enhanced apoptosis in cultured endothelial cells. Taken together, our results suggest that, in multiple cell types the signaling pathways engaged by several physiological stimuli converge upon PKC-ζ-dependent LKB1 phosphorylation at S307, which directs the nucleocytoplasmic transport of LKB1 and consequent AMPK activation.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank Lily Q. Dong for providing the LKB1 SL26 construct. We also thank the reviewers for excellent suggestions.

This study was supported by funding from the following: NIH (HL079584, HL080499, HL074399, HL089920, and HL096032 to M.Z. and 1P20RR024215-01 to Z.X. and M.Z.), the American Heart Association (scientist development grant to Z.X.), the Juvenile Diabetes Research Foundation (M.Z.), the Oklahoma Center for the Advancement of Science and Technology (M.Z.), the American Diabetes Association (M.Z.), the Travis Endowed Chair of the University of Oklahoma Health Science Center (M.Z.), the “Innerschweizer Krebsliga” (D.N.), an ETH graduate student grant for R.S. (D.N. and Theo Wallimann), and the European Union FP6 program (EXGENESIS, LSHM-CT-2004-005272). M. H. Zou is a recipient of the National Established Investigator Award of the American Heart Association.