Identification of the serine 307 of LKB1 as a novel phosphorylation site essential for its nucleocytoplasmic transport and endothelial cell angiogenesis.
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-zeta phosphorylates LKB1 at S307. AMPK-activating agents stimulate PKC-zeta 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 STRAD alpha. Interestingly, the PKC-zeta 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-zeta-dependent LKB1 phosphorylation at S307, which directs the nucleocytoplasmic transport of LKB1 and consequent AMPK activation.
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Zhonglin
Z
Xie
Yunzhou
Y
Dong
Junhua
J
Zhang
Roland
R
Scholz
Dietbert
D
Neumann
Ming-Hui
MH
Zou
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002460:Cell Line; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000378:metabolism; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism; D008687:Metformin / Q000378:metabolism; D051379:Mice; D018919:Neovascularization, Physiologic / Q000502:physiology*; D016877:Oxidants / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D000097606:Exportin 1 Protein; D000099221:Protein Kinase C zeta
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D033942:Adaptor Proteins, Vesicular Transport; D006861:Hydrogen Peroxide; D007004:Hypoglycemic Agents; D028884:Karyopherins; D008687:Metformin; D016877:Oxidants; D030421:Peroxynitrous Acid; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D012694:Serine; D000097606:Exportin 1 Protein; D000099221:Protein Kinase C zeta; C476400:STRADA protein, human; C109790:STK11 protein, human