ABSTRACT
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.
Subject(s)
Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear , Alanine/chemistry , Amino Acid Sequence , Amino Acids/chemistry , Antibodies, Monoclonal , Apoptosis , Cell Line, Tumor , Chromatography , Cytosol/metabolism , DNA/metabolism , Epitopes/chemistry , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Lysine/chemistry , Microscopy, Confocal , Microscopy, Fluorescence , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Nuclear Localization Signals , Peptides/chemistry , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Ribonucleoprotein, U5 Small Nuclear/chemistry , Trans-Activators/chemistry , Transfection , Exportin 1 ProteinABSTRACT
Translocations interrupting the mixed lineage leukemia gene (MLL) occur in 7-10% of acute lymphoblastic leukemia (ALL) and 5-6% of acute myeloid leukemia (AML) cases. One of these translocations, t(11;15)(q23;q14), occurs rarely in both ALL and AML. The gene on chromosome 15, AF15q14, was cloned recently in a patient with AML-M4. We have identified the same gene in a de novo T-ALL patient. However, both the MLL and AF15q14 breakpoints in these patients differed: in the previously reported AML-M4, both gene breaks were within exons, while in our ALL case the MLL break is intronic and the AF15q14 break is exonic. The MLL-AF15q14 fusion described previously shares no AF15q14 residues in common with the chimera reported here. The fusion proteins also differ with respect to MLL--the previously described fusion contains 55 extra amino acids as its MLL break is in exon 11, while the chimera we report breaks in intron 9. Contrary to the originally described normal AF15q14 (5925-bp cDNA encoding a 1833-aa protein), we identify a 7542-bp cDNA and a 2342-aa AF15q14 protein. AF15q14 appears identical to an mRNA previously found to be expressed in melanoma rendered nontumorigenic by microcell-mediated introduction of normal chromosome 6, suggesting the gene may function normally to suppress cell growth and/or enhance maturation.
