A novel CRM1-mediated nuclear export signal governs nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase following genotoxic stress.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.
Brown
Victor M
VM
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
Krynetski
Eugene Y
EY
Krynetskaia
Natalia F
NF
Grieger
Dara
D
Mukatira
Suraj T
ST
Murti
Kuruganti G
KG
Slaughter
Clive A
CA
Park
Hee-Won
HW
Evans
William E
WE
D021581:Active Transport, Cell Nucleus; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000911:Antibodies, Monoclonal; D017209:Apoptosis; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D002845:Chromatography; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D028884:Karyopherins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019913:Nuclear Localization Signals; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry; D015534:Trans-Activators / Q000737:chemistry; D014162:Transfection; D000097606:Exportin 1 Protein
D000409:Alanine; D000596:Amino Acids; D000911:Antibodies, Monoclonal; D004247:DNA; D000939:Epitopes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D008239:Lysine; D019913:Nuclear Localization Signals; D010455:Peptides; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D017415:Ribonucleoprotein, U5 Small Nuclear; D015534:Trans-Activators; D000097606:Exportin 1 Protein