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• W2128890789 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. 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 days. The concentrations of MP or TG were determined by spectrophotometer at 320 and 340 nm, respectively. The 96-well plates were read by a microplate spectrophotometer (Bio-Rad, Hercules, CA). The IC50 values were obtained by fitting a sigmoid Emax model to the cell viability (%) versus concentrations of drug (micromolar), determined in triplicate. Experiments were performed using an initial concentration of 1 × 105 cells/ml of media before the addition of either MP or TG. In subsequent experiments, thiopurines were added to the media in a single dose to achieve a concentration of 10 μm MP or 10 μm TG in the growth medium. The human T-lineage leukemia cell line Molt4 was obtained from ATCC (Manassas, VA); the human T-lineage leukemia P12 and B-lineage Nalm6 cell lines were obtained from the DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Cell number and viability were determined in duplicate in a Burker-Turk chamber using trypan blue exclusion. All experiments were started with an initial concentration of 0.25 × 106 cells/ml. Nuclear extracts from human acute lymphoblastic leukemia cells (Molt4, CEM, or Nalm6) were prepared according to Dignam et al. (17Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). Protein concentration was determined by Bradford dye-binding procedure using Bio-Rad protein assay. Fast Protein Liquid Chromatography—2.0 mg of GAPDH from human erythrocytes (control; Sigma, St. Louis, MO), K259N mutant GAPDH protein and total nuclear protein from ALL cells were analyzed by FPLC on Superdex 200 HR 10/30 columns (Amersham Biosciences, Piscataway, NJ) in elution buffer (50 mm Tris-HCl, pH 7.5; 100 mm KCl) at 0.3-0.4 ml/min at 4 °C. Composition of eluate was monitored by spectrophotometer at 280 nm. Fractions (0.25 ml) were collected during the FPLC separation and concentrated to 25 μl using an Ultrafree 0.5 centrifugal filter and tube (Millipore, Bedford, MA) and analyzed by Western blot. Membranes were developed with anti-GAPDH monoclonal antibody (mAb; Chemicon, Temecula, CA) (18Krynetskaia N.F. Brenner T.L. Krynetski E.Y. Du W. Panetta J.C. Ching-Hon P. Evans W.E. Mol. Pharmacol. 2003; 64: 456-465Crossref PubMed Scopus (13) Google Scholar). Glycolytic Assay—GAPDH glycolytic activity was measured by spectrophotometric assay at 340 nm. Briefly, the assay was carried out in 0.015 m sodium pyrophosphate, 0.03 m sodium arsenate (Sigma), pH 8.5, in the presence of 3.5 mm DTT, 0.26 mm NAD+, and 0.51 mm glyceraldehyde 3-phosphate (Sigma) using human GAPDH (Sigma) or cellular lysates or nuclear extracts. The reactions were performed for 10 min at 25 °C. Identification of mAb Epitope Using Pin-bound Peptides—Pin-bound peptides representing the entire GAPDH polypeptide sequence were synthesized using Multipin Peptide Synthesis kit (Chiron Technologies, San Diego, CA) in the Hartwell Center for Biotechnology (St. Jude Children's Research Hospital) using standard peptide synthesis and Sepharose Coupling. Identification of epitopes was performed according to manufacturer's instructions, using monoclonal antibody (clone 6C5; Chemicon, Temecula, CA) at a concentration of 500 ng/ml, and secondary anti-mouse HRP-conjugated antibody (Santa Cruz Biotechnology, CA) at a concentration of 50 ng/ml. A series of overlapping pin-bound 15-mer peptides was incubated with anti-GAPDH monoclonal anti-GAPDH antibody, and the IgG complex with antigen was detected by incubation with HPR-conjugated secondary antibody. Data were collected using a microplate reader at 405 nm. Affinity Chromatography—Cellular or nuclear extract from 1 × 108 cells (200 μl) was incubated with peptide 51 (DDIKKVVKQASEGPL) or peptide 52 (KPAKYDDIKKVVKQA) immobilized on Sepharose in 20 mm Tris-HCl, pH 8, 5 mm NaCl, 0.3 mm MgCl2, and 0.1 mm DTT (Buffer 1) in a 2-ml column for 30 min at room temperature. Column was washed with 15 ml of Buffer 1 and then eluted with 10 ml of Buffer 1 containing 1 m NaCl (0.3 ml/min). Fractions were collected, concentrated, and analyzed by SDS gel electrophoresis and silver staining. Mass Spectroscopy of Proteins—Proteins localized by silver staining following SDS-polyacrylamide gel electrophoresis were excised from the gel, and digested with trypsin. The peptides thus released from the gel plug were subjected to analysis by combined liquid chromatography/tandem mass spectrometry using an LCQ-Deca ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA) coupled with a capillary high-performance liquid chromatography system (Waters, Milford MA). Peptides were separated by reversed-phase chromatography using a 320-μm I.D. column packed with Waters Delta-Pak C18 stationary phase by Microtech Scientific (Santa Clara, CA). Peptides were assigned to known proteins on the basis of searches of the NCBI non-redundant protein data base performed on product ion spectra using the SEQUEST algorithm. Plasmid Preparation—GAPDH cDNA was prepared by RT-PCR from total RNA isolated from 697 human pre-B leukemia cells. The forward primer, 5′-TGTACATGGGCGGAGGCGGAGGCATGGGGAAGGTGAAGGTCG-3′, contained a NotI site, a 15-mer encoding (Gly)5 link and the first 20 nucleotides (including the initiation codon) of the coding sequence of the GAPDH cDNA. The reverse primer, 5′-GCGGCCGCTTACTCCTTGGAGGCCATGT-3′, contained a BsrG1 binding site and the last 20 nucleotides (including the termination codon) of the coding sequence of the GAPDH cDNA. The modified GAPDH cDNA was cloned into pcDNA3.1/EGFP to obtain the pcDNA3.1/GFP-(Gly)5-GAPDH fusion construct (6Schmitz H.D. Eur. J. Cell Biol. 2001; 80: 419-427Crossref PubMed Scopus (55) Google Scholar, 19Schmitz H.D. Bereiter-Hahn J. Cell Biol. Int. 2002; 26 (20): 155-164Crossref PubMed Scopus (39) Google Scholar). The portion of the pEGFP vector (Clontech, Palo Alto, CA) containing the coding region for EGFP was previously cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) using conventional methods (20Current Protocols in Molecular Biology. Vol. 1. John Wiley and Sons Inc., New York1991: 3.16.1-3.16.2Google Scholar). Truncated and mutated hybrids of the fusion constructs were prepared by site mutagenesis (Fig. 1). Sequences of all constructs were verified by DNA sequencing at the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital. Functional Characterization of the Putative NES—Fig. 1 depicts the truncated (T1, T2, and T3) or mutated (K259N and Ala mutants M1-M6) GAPDH constructs. The GFP-tagged truncated GAPDH T1 contains nucleotides 1-774, which encodes the N terminus, however, it lacks any part of the putative NES. The truncated construct, T2, contains nucleotides 1-786, which encodes the N terminus and the first four amino acids, KKVV, of the putative NES. The second truncated construct, T2, contains nucleotides 806-1008, which encodes the last three amino acids, PLK, of the putative NES and the rest of the C terminus of GAPDH. In the GFP-tagged mutated fusion constructs 1-6, amino acids, including those within the putative NES, were sequentially replaced with four alanine residues. Additionally, a mutant hybrid of the fusion construct was generated in which lysine 259 was mutated to asparagine (K259N). Transfection Assay—Transient transfection was done using LipofectAMINE Reagent 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. Expression of the GFP fusion construct in the cell lines was documented by fluorescence microscopy. Flow cytometry was used to separate transfected from non-transfected cells, and the transfected cells were grown in 1 mg/ml G418 (Clontech, Palo Alto, CA) until colonies were formed. Individual colonies were placed in a Petri dish containing 2 ml of growth medium supplemented with 1 mg/ml G418 and incubated at 37 °C and 5% CO2 until the cells achieved ∼80% confluence. The confluent cells were trypsinized and grown in a flask containing the appropriate growth medium supplemented with 10% fetal bovine serum and 1 mg/ml G418. Immunoprecipitation—Total cell lysate was prepared as recommended by Santa Cruz Biotechnology (Santa Cruz, CA), and protein concentration was determined using the PlusOne 2-D Quant kit (Amersham Biosciences, Piscataway, NJ). Immunoprecipitation was performed using a modification of the method suggested by Santa Cruz Biotechnology. Briefly, 80 μl of radioimmune precipitation assay buffer was added to 5-10 μg (in 20 μl) of total cell lysate, and the mixture was precleared by adding 0.25 μg (in 5 μl) of normal rabbit IgG (Santa Cruz Biotechnology) and 20 μl of Protein G Plus-agarose beads (Santa Cruz Biotechnology). The mixture was incubated at 4 °C for 30 min. After the incubation, the beads were pelleted by centrifugation at 2500 rpm for 5 min, and the supernatant was transferred to a fresh 1-ml microcentrifuge tube at 4 °C. 0.19 μg/μl (in 20 μl) of rabbit anti-GFP polyclonal antibody (Clontech) was added to the supernatant, and the mixture was incubated at 4 °C for 1 h. After incubation, 20 μl of Protein G Plus-agarose beads (Santa Cruz Biotechnology) were added to the mixture, followed by incubation overnight at 4 °C with constant rotation. The pellet was collected by centrifugation at 2500 rpm for 5 min, washed with radioimmune precipitation assay buffer four times, and then re-suspended in 10 μl of Nu-PAGE LDS 4× sample buffer (Invitrogen, Carlsbad, CA), vortexed, and boiled at 100 °C for 5 min. After cooling, 10 μl of running buffer and 1 μl of 1 m DTT was added to the boiled sample, and the mixture was separated on 10-12% Nu-PAGE gel (Invitrogen). Western Analysis—After immunoprecipitation and SDS-electrophoresis, the proteins were transferred to a Hybond-P membrane (Amersham Biosciences) by electrotransfer. The membrane was then incubated with mouse anti-CRM1 mAb antibody (BD Transduction Laboratories, San Diego, CA). Bound antibodies were detected using goat anti-mouse IgG HRP conjugate (Santa Cruz Biotechnology) and ECL-Plus kit (Amersham Pharmacia Biotech). The membrane was washed and re-developed with Rabbit anti-GFP polyclonal antibody (Clontech) and goat anti-rabbit IgG HRP conjugate as secondary antibody conjugate (Santa Cruz Biotechnology). Confocal Microscopy—Samples were examined using a Leica TCS NT SP confocal laser scanning microscope equipped with argon (488 nm) and krypton (568 nm) lasers. Endogenous GAPDH was stained using mouse anti-GAPDH (Chemicon) as primary antibody and goat anti-mouse IgG fluorescein isothiocyanate conjugate (Santa Cruz Biotechnology) as secondary antibody, and the nuclei were stained with propidium iodide (PI). GFP-positive cells were stained with PI. Samples were imaged with detector slit widths of 500-548 nm in the green, or fluorescein isothiocyanate channel; and 580-609 nm in the red, or PI channel, using a 100× plan apochromatic 1.4 numerical aperture oil immersion objective. Overlay images combining the green and red channels were produced using the Leica software, and the images were re-scaled and gamma-corrected in Adobe Photoshop. The nuclear and cytoplasmic fluorescence intensities were quantified using a published procedure. Single section images containing 20-40 cells were used in the analysis to determine the nuclear and cytoplasmic fluorescence (single fixed intensity × number of pixels per nucleus or cytoplasm). The nuclear area is defined by the red fluorescence due to propidium iodide. Intracellular Localization of Endogenous GAPDH and GFP-GAPDH—In untreated cells, endogenous GAPDH was localized mainly in the cytosol of colon adenocarcinoma cells (SW620 and DLD1), as revealed by immunostaining and confocal microscopy (Fig. 2A). However, following 24-48 h of thiopurine treatment (either MP or TG), GAPDH accumulated predominantly in the nucleus, with exclusion from the nucleoli. Fig. 2B shows the distribution of endogenous GAPDH in SW620 cells after 24 h of treatment with 10 μm MP. GAPDH was localized predominantly in the cytoplasm of DLD1 cells expressing the GFP-GAPDH fusion construct, similar to endogenous GAPDH, as illustrated in Fig. 2C (compared with Fig. 2A). Treatment of DLD1 cells expressing the GFP-GAPDH fusion construct with 10 μm thiopurine resulted in subcellular redistribution of GAPDH, with accumulation in the nucleus and exclusion from the nucleoli, after 48 h of thiopurine treatment (Fig. 2, E and D, compared with C). This was similar to that observed for endogenous GAPDH (Fig. 2B). Therefore, GFP-GAPDH fusion construct (Fig. 1) was used as a model to assess the effect of truncation or mutation on the localization of GAPDH. In control experiments with SW620 and DLD1 cells expressing EGFP vector, GFP was uniformly present in both cytoplasm and nucleus (data not shown). Thiopurine treatment of SW620 and DLD1 cells expressing EGFP vector had no effect on the cellular distribution of EGFP (data not shown). Identification of the Anti-GAPDH-bound Epitope within the GAPDH Polypeptide Chain—Pepscan technology was used to delineate the amino acid sequences of peptides that constitute the epitope of GAPDH recognized by anti-GAPDH mAb. Peptides 51 and 52, corresponding to overlapping segments 250KPAKYDDIKKVVKQA264 and 255DDIKKVVKQASEGPL269, respectively, gave the highest absorption at 405 nm (>2 optical units/ml) (Fig. 3A), and reaction with the other peptides resulted in negligible absorption (0.038-0.1 optical units405/ml). Treatment with monoclonal anti-PCNA antibody (a negative control) did not result in any signal, indicating the lack of nonspecific interaction with pin-bound peptides (data not shown). N-terminal sequencing verified the sequences of pin-bound peptides 51 and 52. Unbound peptides 51 and 52 inhibited immunostaining of GAPDH in Western analysis, thus confirming specific binding of anti-GAPDH MAb with epitopes encompassing amino acids 250-269 of GAPDH (data not shown). Putative NES Involved in Protein-Protein Interaction—Incubation of cellular lysate from Molt4 cells with 256DDIKKVVKQASEGPL270 covalently bound to Sepharose resulted in specific binding of GAPDH, as confirmed by SDS gel electrophoresis and mass-spectroscopy (Fig. 3B). One major and at least four minor bands were detected by silver staining of the gel. Electrospray mass spectrometry of the major band identified it as GAPDH (Fig. 3B). The identified peptide sequence within GAPDH is conserved across different species (human, Homo sapiens; rabbit, Oryctolagus cuniculis; lobster, Homarus americanus; and Palinurus versicolor) and contains invariant amino acid residues, conservative substitutions, and non-conservative changes as illustrated in Fig. 3C. This GAPDH peptide sequence is comparable to FIV Rev protein that has an atypical nuclear export signal (Fig. 3C). Oligomeric Forms and Glycolytic Activity of Nuclear GAPDH—Gel filtration FPLC chromatography depicts a typical profile of purified K259N mutant and wild-type GAPDH extracted from human erythrocytes used as control. The FPLC profile revealed that both wild-type and mutant GAPDH were eluted as tetramers with a molecular mass of 144 kDa (Fig. 4A). No peaks with molecular mass higher than 144 kDa were detected. The chromatography of nuclear extracts from ALL cells (P12, Molt4, and Nalm6) in non-denaturing conditions and subsequent Western blot analysis of the collected fractions revealed that GAPDH was present in several oligomeric forms or high molecular weight complexes in the nuclear compartment of untreated lymphoblast cells. Fig. 4B (analysis of nuclear protein from Molt4 cells) depicts a typical FPLC profile of nuclear protein distribution for ALL cells. Fractions 24-25 contained a high molecular weight protein complex exceeding the exclusion volume of the column (i.e. more than 2 × 106 Da), and fractions 35-43 contained different oligomeric forms of GAPDH, including the monomeric, dimeric, and tetrameric forms (Fig. 4, B and C). Measurement of enzymatic activity in the nuclear extracts and cellular lysates from P12, Molt4, and Nalm6 cell lines revealed that GAPDH activity in the nucleus of untreated Molt 4, P12, and Nalm6 cells was lower when compared with cellular lysates (Fig. 4D). Modification of the Putative NES Changes Intracellular Distribution of GAPDH—We performed alanine scanning using Ala tetrapeptide sequence (AAAA) across the entire length of the tested region (Fig. 1). GFP-GAPDH mutant proteins bearing Ala4 substitution in different positions of the putative NES revealed different distribution between cellular compartments. DLD1 cells stably expressing the GFP-tagged Ala mutants M1 and M4 demonstrated cytoplasmic localization of the mutant GFP-GAPDH as depicted by green GFP fluorescence (Fig. 5, A, B, G, and H, respectively). However, there is evidence of some nuclear rim association illustrated by the yellow border around the red PI-stained nucleus. GFP-tagged Ala mutant M2 was localized in both nucleus and cytoplasm of DLD1 cells (Fig. 5, C and D). In contrast, DLD1 cells stably expressing Ala-mutated GFP-tagged fusion construct M3 exhibited predominantly nuclear accumulation of the Ala mutated GAPDH fusion construct (Fig. 5, E and F). Similar distribution of GAPDH was observed in cells stably expressing the GFP-tagged Ala mutants M5 (Fig. 5, I and J) and M6 (Fig. 5, K and L). DLD1 cells stably expressing the truncated GFP-tagged fusion construct T1 exhibited predominantly nuclear accumulation of the truncated GAPDH fusion construct (Fig. 6, A and B). Cells stably expressing the GFP-tagged fusion construct T2 exhibited mainly nuclear localization of the T2 GAPDH fusion construct (Fig. 6, C and D). Similar distribution of GAPDH was observed in cells stably expressing the GFP-tagged T3 (data not shown). Stable expression of the K259N mutant fusion construct in DLD1 cells resulted in nuclear accumulation of GAPDH in the absence of thiopurine treatment (Fig. 6, G and H). The localization of K259N was similar to that exhibited by DLD1 cells stably expressing Ala-mutated GFP-tagged fusion constructs M3 (Fig. 5, E and F), M5 (Fig. 5, I and J), and M6 (Fig. 5, K and L). Effect of Thiopurine Treatment on Nuclear Accumulation of Modified GAPDH—Truncated or mutated forms of GAPDH, which accumulated in the nucleus, did not change nuclear localization of fluorescence after thiopurine treatment. Interestingly, the treatment of cells expressing the truncated fusion protein T2 with 10 μm thiopurine (TG or MP) resulted in nuclear rim association (Fig. 6, E compared with C). No further intracellular accumulation of GAPDH was observed in cells expressing the K259N mutant construct following thiopurine treatment. Similar observations were made for both SW620 and DLD1 cell lines. Leptomycin B Prevents Export of GFP-GAPDH from the Nucleus—Treatment for 2 h with 2.5 ng/ml leptomycin B (LMB), an inhibitor of CRM1-mediated nuclear export, resulted in subcellular redistribution of GAPDH from the cytoplasm to the nucleus in DLD1 cells (Fig. 7, compare A and B with C and D). Similar results were obtained when SW620 cells were treated with LMB (data not shown). Co-immunoprecipitation of GAPDH and CRM1—Western analysis using anti-GFP antibody indicated that GFP-GAPDH fusion protein was present in the cell lysate and in the anti-GFP antibody immunoprecipitate from DLD1 cells transfected with the GFP-GAPDH expression vector (Fig. 8A, lane 3, lower panel). Western analysis with anti-CRM1 antibody revealed that CRM1 was present in the immunoprecipitate, indicating that CRM1 was associated with the GFP-GAPDH fusion protein (Fig. 8A, lane 4, upper panel). In control experiments, the immunoblot of membranes obtained from DLD1 cells expressing the EGFP vector, using anti-GFP and anti-CRM1 antibodies," @default.
• W2128890789 created "2016-06-24" @default.
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• W2128890789 creator A5022553539 @default.
• W2128890789 creator A5034189317 @default.
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• W2128890789 date "2004-02-01" @default.
• W2128890789 modified "2023-10-09" @default.
• W2128890789 title "A Novel CRM1-mediated Nuclear Export Signal Governs Nuclear Accumulation of Glyceraldehyde-3-phosphate Dehydrogenase following Genotoxic Stress" @default.
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