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. 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. In addition to its integral role in glycolysis, converting glyceraldehyde-3-phosphate (GAPDH) 1The abbreviations used are: GAPDHglyceraldehyde-3-phosphate dehydrogenaseCRM1exportin1 or chromosome region maintenanceALLacute lymphoblastic leukemiamAbmonoclonal antibodyMPmercaptopurineTGthioguanineFPLCfast protein liquid chromatographyDTTdithiothreitolHRPhorseradish peroxidaseGFPgreen fluorescent proteinEGFPenhanced GFPPIpropidium iodideNESnuclear export signalLMBleptomycin BFIVfeline immunodeficiency virus. into 1,3-bisphosphoglycerate, GAPDH has been shown to have diverse biological functions, including as a protein that signals apoptosis (1Vaudry D. Falluel-Morel A. Leuillet S. Vaudry H. Gonzalez B.J. Science. 2003; 300: 1532-1534Crossref PubMed Scopus (54) Google Scholar). GAPDH also participates in membrane, cytoplasmic, and nuclear functions for endocytosis, mRNA regulation, tRNA export, DNA replication, and DNA repair (2Sirover M.A. J. Cell. Biochem. 1997; 66: 133-140Crossref PubMed Scopus (223) Google Scholar, 3Sirover M.A. Biochim. Biophys. Acta. 1999; 1432: 159-184Crossref PubMed Scopus (709) Google Scholar). Some species, including humans and mouse, contain more than one functional GAPDH gene and a diversity of pseudogenes (4Ishitani R. Tajima H. Takata H. Tsuchiya K. Kuwae T. Yamada M. Takahashi H. Tatton N.A. Katsube N. Prog. Neuropsychopharmacol. Biol. Psychiatry. 2003; 27: 291-301Crossref PubMed Scopus (50) Google Scholar). In its monomeric form, GAPDH has a molecular mass of ∼37 kDa, however, within cells, it exists mainly as a tetramer comprising four identical 37-kDa subunits (3Sirover M.A. Biochim. Biophys. Acta. 1999; 1432: 159-184Crossref PubMed Scopus (709) Google Scholar, 5Berry M.D. Boulton A.A. J. Neurosci. Res. 2000; 60: 150-154Crossref PubMed Scopus (104) Google Scholar). GAPDH is located in multiple cellular compartments, including the plasma membrane, nucleus, and cytosol (6Schmitz H.D. Eur. J. Cell Biol. 2001; 80: 419-427Crossref PubMed Scopus (55) Google Scholar). glyceraldehyde-3-phosphate dehydrogenase exportin1 or chromosome region maintenance acute lymphoblastic leukemia monoclonal antibody mercaptopurine thioguanine fast protein liquid chromatography dithiothreitol horseradish peroxidase green fluorescent protein enhanced GFP propidium iodide nuclear export signal leptomycin B feline immunodeficiency virus. GAPDH plays an important role in stress response leading to apoptosis (5Berry M.D. Boulton A.A. J. Neurosci. Res. 2000; 60: 150-154Crossref PubMed Scopus (104) Google Scholar, 7Saunders P.A. Chen R.W. Chuang D.M. J. Neurochem. 1999; 72: 925-932Crossref PubMed Scopus (112) Google Scholar), with the cytoplasmic to nuclear translocation of GAPDH preceding the onset of apoptosis (8Ishitani R. Tanaka M. Sunaga K. Katsube N. Chuang D.M. Mol. Pharmacol. 1998; 53: 701-707Crossref PubMed Scopus (143) Google Scholar, 9Ishitani R. Sunaga K. Tanaka M. Aishita H. Chuang D.M. Mol. Pharmacol. 1997; 51: 542-550Crossref PubMed Scopus (71) Google Scholar). K+ depolarization (7Saunders P.A. Chen R.W. Chuang D.M. J. Neurochem. 1999; 72: 925-932Crossref PubMed Scopus (112) Google Scholar, 9Ishitani R. Sunaga K. Tanaka M. Aishita H. Chuang D.M. Mol. Pharmacol. 1997; 51: 542-550Crossref PubMed Scopus (71) Google Scholar), serum withdrawal (6Schmitz H.D. Eur. J. Cell Biol. 2001; 80: 419-427Crossref PubMed Scopus (55) Google Scholar), aging of cultures (10Ishitani R. Kimura M. Sunaga K. Katsube N. Tanaka M. Chuang D.M. J. Pharmacol. Exp. Ther. 1996; 278: 447-454PubMed Google Scholar), or treatment with anticancer agents such as mercaptopurine or cytosine arabinoside (8Ishitani R. Tanaka M. Sunaga K. Katsube N. Chuang D.M. Mol. Pharmacol. 1998; 53: 701-707Crossref PubMed Scopus (143) Google Scholar, 10Ishitani R. Kimura M. Sunaga K. Katsube N. Tanaka M. Chuang D.M. J. Pharmacol. Exp. Ther. 1996; 278: 447-454PubMed Google Scholar, 11Saunders P.A. Chalecka-Franaszek E. Chuang D.M. J. Neurochem. 1997; 69: 1820-1828Crossref PubMed Scopus (82) Google Scholar, 12Krynetski E.Y. Krynetskaia N.F. Gallo A.E. Murti K.G. Evans W.E. Mol. Pharmacol. 2001; 59: 367-374Crossref PubMed Scopus (54) Google Scholar) cause nuclear accumulation of GAPDH. An increase in nuclear GAPDH is required for its apoptotic effects, which appear to be upstream from events that mediate apoptotic degradation (13Carlile G.W. Chalmers-Redman R.M. Tatton N.A. Pong A. Borden K.E. Tatton W.G. Mol. Pharmacol. 2000; 57: 2-12PubMed Google Scholar), and the nuclear accumulation of GAPDH precedes chromatin condensation, nuclear fragmentation, and a decline in mitochondrial membrane protein (14Dastoor Z. Dreyer J.L. J. Cell Sci. 2001; 114: 1643-1653Crossref PubMed Google Scholar). This is consistent with the reported involvement of GAPDH in apoptosis of primary cultures of cerebellar neurons following nuclear translocation (9Ishitani R. Sunaga K. Tanaka M. Aishita H. Chuang D.M. Mol. Pharmacol. 1997; 51: 542-550Crossref PubMed Scopus (71) Google Scholar) and the induction of intranuclear translocation of GAPDH by treatment of cells with thiopurines (12Krynetski E.Y. Krynetskaia N.F. Gallo A.E. Murti K.G. Evans W.E. Mol. Pharmacol. 2001; 59: 367-374Crossref PubMed Scopus (54) Google Scholar). Moreover, significant correlation has been shown between basal intranuclear GAPDH in acute lymphoblastic leukemia (ALL) cell lines and sensitivity to thiopurine treatment (12Krynetski E.Y. Krynetskaia N.F. Gallo A.E. Murti K.G. Evans W.E. Mol. Pharmacol. 2001; 59: 367-374Crossref PubMed Scopus (54) Google Scholar). Recently, GAPDH was identified as a component of a nuclear protein complex that recognizes duplex DNA into which fraudulent nucleosides (e.g. thioguanosine, cytosine arabinoside, or 5-fluorouridine) have been incorporated (15Krynetski E.Y. Krynetskaia N.F. Bianchi M.E. Evans W.E. Cancer Res. 2003; 63: 100-106PubMed Google Scholar). In vitro treatment of the complex with monoclonal anti-GAPDH antibody (anti-GAPDH mAb) resulted in its dissociation (12Krynetski E.Y. Krynetskaia N.F. Gallo A.E. Murti K.G. Evans W.E. Mol. Pharmacol. 2001; 59: 367-374Crossref PubMed Scopus (54) Google Scholar). This observation led us to hypothesize that the corresponding epitope recognized by anti-GAPDH mAb is localized at or near the surface involved in protein-protein interactions and provided the basis for the present study to characterize the region(s) of the GAPDH polypeptide chain involved in protein-protein interactions. We identified the region of GAPDH, which constitutes the anti-GAPDH mAb binding site, and demonstrated involvement of this region in binding with other nuclear proteins. Finally, we demonstrated that this region interacts with components of the nuclear export system and defines intracellular localization of GAPDH. Elucidating the mechanism of nuclear targeting of GAPDH has identified a novel nuclear export signal and provided new insights into disease pathogenesis and drug-induced apoptosis. Cell Cultures and Nuclear Extract Precipitation—Colon adenocarcinoma cell lines SW620 and DLD1 were obtained from ATCC (Manassas, VA). Cell lines were grown in RPMI 1640 (BioWhittaker, Walkersville, MD) medium supplemented with 10% fetal bovine serum (Invitrogen, Palo Alto, CA) and 1.0% l-glutamine. Cytotoxic effects of thiopurines (Sigma, St. Louis MO) were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (16Pieters R. Huismans D.R. Leyva A. Veerman A.J. Cancer Lett. 1988; 41: 323-332Crossref PubMed Scopus (130) Google Scholar) after incubation of SW620 and DLD1 cells with mercaptopurine (MP, 0.001-180 μm) or thioguanine (TG, 0.001-100 μm) for 3-6 of or were determined by at and were by a were obtained by a to the cell of determined in were using an of of the addition of or In thiopurines were to the in a to a of or in the human leukemia cell was obtained from ATCC (Manassas, the human leukemia and cell lines were obtained from the of and Cell Cultures Cell and were determined in in a using were with an of Nuclear from human acute lymphoblastic leukemia cells or were to R.M. Res. PubMed Scopus Google Scholar). was determined by using protein of GAPDH from human St. GAPDH protein and nuclear protein from cells were by in at at of was by at were the and to using an and and by were with anti-GAPDH monoclonal antibody CA) N.F. E.Y. Evans W.E. Mol. Pharmacol. 2003; PubMed Scopus Google Scholar). glycolytic was by assay at the assay was in in the of and using human GAPDH or cellular or nuclear were for at of mAb the GAPDH polypeptide sequence were using CA) in the for using and of was to using monoclonal antibody CA) at a of and antibody CA) at a of of was with anti-GAPDH monoclonal anti-GAPDH and the complex with was by incubation with were using a at or nuclear from cells was with or in and in a for at was with of and with of were and by and of localized by following were from the and with from the were to by liquid mass using an mass CA) with a liquid chromatography system were by chromatography using a with by were to the basis of of the protein using the was by from from human leukemia a site, a and the the of the sequence of the GAPDH a binding and the the of the sequence of the GAPDH modified GAPDH was into to the (6Schmitz H.D. Eur. J. Cell Biol. 2001; 80: 419-427Crossref PubMed Scopus (55) Google Scholar, H.D. J. Cell Biol. PubMed Scopus Google Scholar). of the Palo Alto, CA) the region for was into (Invitrogen, CA) using in and Scholar). and of the were by mutagenesis of were by DNA at the for and at St. of the the and or and GAPDH GAPDH which the however, it of the putative NES. which the and the four amino of the putative NES. which the amino of the putative NES and the of the of GAPDH. In the amino including within the putative NES, were with four a of the was in which was to was using (Invitrogen, to the of the GFP in the cell lines was by was used to from cells, and the cells were grown in Palo Alto, CA) were were in a of medium supplemented with and at and the cells cells were and grown in a the medium supplemented with 10% fetal bovine serum and cell was as by and protein was determined using the was using a of the by of assay was to of cell and the was by of and of was at for the the were by at for and the was to a at of antibody was to the and the was at for of were to the by incubation at with was by at for with assay four and in of (Invitrogen, and at for of and of was to the and the was and the were to a membrane by membrane was with mAb antibody were using and membrane was and with antibody and as antibody were using a scanning with and GAPDH was using anti-GAPDH as primary antibody and as and the were with propidium iodide cells were with were with of in the or and in the or using a the green and were using the and the were and in nuclear and cytoplasmic were using a cells were used in the to the nuclear and cytoplasmic of nucleus or nuclear is by the to propidium of GAPDH and cells, GAPDH was localized mainly in the cytosol of adenocarcinoma cells and as by and following of thiopurine treatment or GAPDH in the nucleus, with from the the of GAPDH in SW620 cells after of treatment with GAPDH was localized in the of DLD1 cells the to GAPDH, as in with of DLD1 cells the with thiopurine resulted in of GAPDH, with accumulation in the nucleus and from the after of thiopurine treatment and with This was to that for GAPDH was used as a to the of truncation or mutation the localization of GAPDH. In with SW620 and DLD1 cells GFP was present in and nucleus not treatment of SW620 and DLD1 cells the cellular of not of the within the GAPDH was used to the amino acid sequences of that the epitope of GAPDH recognized by anti-GAPDH and corresponding to and the at and with the other resulted in with monoclonal antibody did not in indicating the of with not the sequences of and and of GAPDH in binding of anti-GAPDH with encompassing amino acids of GAPDH not NES in of cellular from cells with to resulted in binding of GAPDH, as by and and at least four were by of the mass of the identified it as GAPDH identified sequence within GAPDH is conserved and and amino acid and as in This GAPDH sequence is to Rev protein that has an nuclear export signal and of Nuclear chromatography a of and GAPDH from human used as that and GAPDH were as with a molecular mass of with molecular mass than were chromatography of nuclear from cells and in and of the that GAPDH was present in or molecular in the nuclear of of nuclear protein from a of nuclear protein for a molecular protein complex the of the more than and of GAPDH, including the and B and of in the nuclear and cellular from and cell lines that GAPDH in the nucleus of and cells was with cellular of the NES of scanning using sequence the of the region in of the putative NES between cellular DLD1 cells the and demonstrated cytoplasmic localization of the as by green GFP and is of nuclear by the the was localized in nucleus and of DLD1 cells and In DLD1 cells nuclear accumulation of the GAPDH and Similar of GAPDH was in cells the and and and DLD1 cells the nuclear accumulation of the GAPDH and the mainly nuclear localization of the GAPDH and Similar of GAPDH was in cells the not of the in DLD1 cells resulted in nuclear accumulation of GAPDH in the of thiopurine treatment and localization of was to that by DLD1 cells and and and and of Nuclear of or of GAPDH, which in the nucleus, did not nuclear localization of after thiopurine the treatment of cells the protein with thiopurine or resulted in nuclear with intracellular accumulation of GAPDH was in cells the following thiopurine Similar were for SW620 and DLD1 cell B of from the for with leptomycin B an of nuclear export, resulted in of GAPDH from the to the nucleus in DLD1 cells and B with and Similar were obtained SW620 cells were treated with not of GAPDH and using antibody that protein was present in the cell and in the antibody from DLD1 cells with the with antibody that CRM1 was present in the indicating that CRM1 was with the protein In the of obtained from DLD1 cells the using and that GFP and CRM1 were present in the cell was with CRM1 was not was for SW620 cells not of the cellular from DLD1 cells the with antibody and with that CRM1 was present in the cellular CRM1 was not with the protein Similar were obtained with SW620 cells intranuclear localization of GAPDH after treatment and its involvement in DNA repair have been demonstrated A. A.A. Sci. S. A. 1997; PubMed Scopus Google Scholar), but is the involved in nuclear localization of GAPDH. Nuclear accumulation of GAPDH after of treatment with the genotoxic agents or thiopurine (2Sirover M.A. J. Cell. Biochem. 1997; 66: 133-140Crossref PubMed Scopus (223) Google Scholar, 12Krynetski E.Y. Krynetskaia N.F. Gallo A.E. Murti K.G. Evans W.E. Mol. Pharmacol. 2001; 59: 367-374Crossref PubMed Scopus (54) Google Scholar, A. A.A. Sci. S. A. 1997; PubMed Scopus Google Scholar) or following other of stress (14Dastoor Z. Dreyer J.L. J. Cell Sci. 2001; 114: 1643-1653Crossref PubMed Google Scholar) and is by apoptotic cell (13Carlile G.W. Chalmers-Redman R.M. Tatton N.A. Pong A. Borden K.E. Tatton W.G. Mol. Pharmacol. 2000; 57: 2-12PubMed Google Scholar). of GAPDH is essential for induction of apoptosis of cerebellar cells (5Berry M.D. Boulton A.A. J. Neurosci. Res. 2000; 60: 150-154Crossref PubMed Scopus (104) Google Scholar), and the of nuclear GAPDH has been to the sensitivity of human leukemia cells to thiopurine treatment (12Krynetski E.Y. Krynetskaia N.F. Gallo A.E. Murti K.G. Evans W.E. Mol. Pharmacol. 2001; 59: 367-374Crossref PubMed Scopus (54) Google Scholar). treatment with or we of GAPDH or nuclear export from the to the nucleus in and cell lines In we the of and monomeric of GAPDH, with molecular in the nuclear of acute lymphoblastic cells B and GAPDH glycolytic with GAPDH consistent with functions of nuclear GAPDH. We demonstrated that GAPDH is a of a protein complex that recognizes modified DNA (15Krynetski E.Y. Krynetskaia N.F. Bianchi M.E. Evans W.E. Cancer Res. 2003; 63: 100-106PubMed Google Scholar), and this complex was by an anti-GAPDH monoclonal antibody (15Krynetski E.Y. Krynetskaia N.F. Bianchi M.E. Evans W.E. Cancer Res. 2003; 63: 100-106PubMed Google Scholar). protein-protein within GAPDH, other than involved in have not been GAPDH is to a of protein-protein (15Krynetski E.Y. Krynetskaia N.F. Bianchi M.E. Evans W.E. Cancer Res. 2003; 63: 100-106PubMed Google Scholar, J.L. M.A. PubMed Scopus Google Scholar). binding of GAPDH to and a role of GAPDH in with of (4Ishitani R. Tajima H. Takata H. Tsuchiya K. Kuwae T. Yamada M. Takahashi H. Tatton N.A. Katsube N. Prog. Neuropsychopharmacol. Biol. Psychiatry. 2003; 27: 291-301Crossref PubMed Scopus (50) Google Scholar, J.L. M.A. PubMed Scopus Google Scholar). This to led us to that its an important role in multiple biological functions of GAPDH within the In localization of GAPDH in intracellular plays an important role in its biological the binding within GAPDH that interacts with a monoclonal which the we used the of the GAPDH polypeptide This identified two and located in the C-terminal region of the protein. we with the sequence and demonstrated by that binding between GAPDH and this as a domain not we that this region of the GAPDH is involved in protein-protein interactions and be a region of a of GAPDH that this is the subunits within the GAPDH tetramer the role of the identified in the intranuclear localization of GAPDH, we and in which the amino acids this sequence were modified or and cell into this sequence by mutagenesis caused nuclear accumulation of GAPDH in the of genotoxic that this the of a nuclear export signal than involved in nuclear and In the which the C-terminal of the polypeptide chain that the putative NES, was localized in the nucleus the protein which the four amino acids of the putative NES, also intranuclear localization in the of treatment and protein which C-terminal of the GAPDH including the amino acids of this in the nucleus of SW620 and DLD1 cell lines in the of thiopurine treatment caused nuclear of the it the of the other or not mechanism and biological of nuclear of the protein to be Alanine scanning of the putative NES by amino acid residues by demonstrated the complex of this NES. of the sequence and with in the sequence did not intracellular of or of the sequences and with resulted in intranuclear accumulation of GAPDH, to of nuclear export This observation within the two of amino acids are important for with nuclear export and the sequence of amino acids located between two is not This NES sequence to nuclear localization to characterize the role of in the NES, we a mutation located in the of the within the NES. In with with this resulted in nuclear accumulation of GAPDH and (7Saunders P.A. Chen R.W. Chuang D.M. J. Neurochem. 1999; 72: 925-932Crossref PubMed Scopus (112) Google Scholar, N. S. J. Biol. 2003; 278: PubMed Scopus Google Scholar, D. T. J. PubMed Google Scholar, M.E. 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GAPDH NES, the Rev NES a nuclear mechanism and is to Biol. 1997; PubMed Scopus Google Scholar), but of NES are present with GAPDH the human protein shown to have this of NES. of CRM1 and by antibody from cellular of cells, but not cells or cells in which the GAPDH NES been that GAPDH interacts with CRM1 Moreover, CRM1 did not with the indicating the role of Lys259 in export of GAPDH. of human GAPDH insights into the of the NES and the involvement of this conserved sequence in protein-protein interactions. putative NES sequence a and a in the of human GAPDH, and this is located from the oligomerization that the is to be involved in binding of the role of the GAPDH was and its oligomerization was using This a at corresponding to the of the to that obtained with GAPDH, indicating that mutation within the putative NES does not affect oligomerization of GAPDH, and GAPDH did not molecular by of GAPDH that the of GAPDH is not by the of mutation within the putative the mutation nuclear efflux of GAPDH protein. and that of with CRM1 and not in nuclear export. were to into the mutation the of GAPDH with the of human GAPDH as a we a of the of the was with an in the St. This that the mutation the of the of the of the putative NES and the of GAPDH with the role of the putative NES sequence in protein-protein interactions and In the Lys259 is located in the of the of the putative NES of the Lys259 chain interacts with residues of and the amino of the Lys259 chain is to shown in the of the the mutation is to a chain to with which a of the of the including by mutation also a with by the the mutation to the of the of the and of the the the be to the role of the of the putative NES sequence in protein-protein interactions. with intranuclear accumulation of been or in the of thiopurine that nuclear export of GAPDH with that the intracellular of GAPDH between the cytosol and the In the of GAPDH export from the nucleus is by We the of CRM1 with GAPDH NES, but the of this binding nuclear accumulation of GAPDH, the NES has been has a complex with at least two important for of protein binding to and are by a of amino acids with sequence this NES, Lys259 is essential for proper and its mutation NES activity. GAPDH NES is from reported NES in other proteins. unlike CRM1-dependent NES, which are the GAPDH NES one is the GAPDH NES is in the C-terminal of the is a from C-terminal NES. In we that is also involved in protein-protein interactions with nuclear other than the nuclear export of this new human nuclear export signal new insights into the of that nuclear accumulation to biological activity. We the and provided by of the and of of the and of the of at St. for We for in the of the