Identification of a nuclear export signal in the catalytic subunit of AMP-activated protein kinase.
The metabolic regulator AMP-activated protein kinase (AMPK) maintains cellular homeostasis through regulation of proteins involved in energy-producing and -consuming pathways. Although AMPK phosphorylation targets include cytoplasmic and nuclear proteins, the precise mechanisms that regulate AMPK localization, and thus its access to these substrates, are unclear. We identify highly conserved carboxy-terminal hydrophobic amino acids that function as a leptomycin B-sensitive, CRM1-dependent nuclear export sequence (NES) in the AMPK catalytic subunit (AMPKα). When this sequence is modified AMPKα shows increased nuclear localization via a Ran-dependent import pathway. Cytoplasmic localization can be restored by substituting well-defined snurportin-1 or protein kinase A inhibitor (PKIA) CRM1-binding NESs into AMPKα. We demonstrate a functional requirement in vivo for the AMPKα carboxy-terminal NES, as transgenic Drosophila expressing AMPKα lacking this NES fail to rescue lethality of AMPKα null mutant flies and show decreased activation loop phosphorylation under heat-shock stress. Sequestered to the nucleus, this truncated protein shows highly reduced phosphorylation at the key Thr172 activation residue, suggesting that AMPK activation predominantly occurs in the cytoplasm under unstressed conditions. Thus, modulation of CRM1-mediated export of AMPKα via its C-terminal NES provides an additional mechanism for cells to use in the regulation of AMPK activity and localization.
Kazgan
Nevzat
N
Neuroscience Center, University of North Carolina Chapel Hill School of Medicine, Chapel Hill, NC 27599-7250, USA.
Williams
Tyisha
T
Forsberg
Lawrence J
LJ
Brenman
Jay E
JE
D055372:AMP-Activated Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004331:Drosophila melanogaster / Q000378:metabolism; D004789:Enzyme Activation; D018869:Heat-Shock Response; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D049790:Nuclear Export Signals; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D013329:Structure-Activity Relationship; D020931:ran GTP-Binding Protein / Q000378:metabolism
D000596:Amino Acids; D007527:Isoenzymes; D050505:Mutant Proteins; D049790:Nuclear Export Signals; D021122:Protein Subunits; D055372:AMP-Activated Protein Kinases; D020931:ran GTP-Binding Protein