FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1966912797> ?p ?o ?g. }

• W1966912797 endingPage "3156" @default.
• W1966912797 startingPage "3145" @default.
• W1966912797 abstract "The poly(A)-binding protein (PABP) is an important regulator of mRNA translation and stability. The cellular level of PABP is controlled by regulating its mRNA translation by a feedback mechanism. The important aspect of this mechanism is that PABP binds to an adenosine-rich cis-element at the 5′-untranslated region of its own mRNA and inhibits its translation. To assess the importance of controlling the PABP level, we studied the effect of PABP overexpression on the transcription profile using the microarray technique. In PABP-overexpressing cells, 19 mRNAs showed a reduction in cellular levels due to reduced mRNA stability, and one showed an increase due to increased mRNA stability. Among these mRNAs, the MKK-2 mRNA encodes the protein kinase activator of ERK1/2 kinase, which is involved in the phosphorylation of eukaryotic initiation factor (eIF) 4E. As a result, mRNA translation may be regulated by the cellular level of MKK-2. In this study, we show that the abundance of the MKK-2 polypeptide is reduced in PABP-overexpressing cells. In these cells, the levels of phosphorylated PABP, eIF4E, and ERK2 are also reduced. Treatment of HeLa cells with the MKK-2 inhibitor U0126 reduced PABP phosphorylation, suggesting that the phosphorylation of PABP is mediated by the MKK-2/ERK signaling pathway. Thus, a novel signaling pathway involving MKK-2 and ERK1/2 may down-regulate the activity of PABP and eIF4E by controlling their phosphorylation and compensates for the effect of excess cellular PABP. The poly(A)-binding protein (PABP) is an important regulator of mRNA translation and stability. The cellular level of PABP is controlled by regulating its mRNA translation by a feedback mechanism. The important aspect of this mechanism is that PABP binds to an adenosine-rich cis-element at the 5′-untranslated region of its own mRNA and inhibits its translation. To assess the importance of controlling the PABP level, we studied the effect of PABP overexpression on the transcription profile using the microarray technique. In PABP-overexpressing cells, 19 mRNAs showed a reduction in cellular levels due to reduced mRNA stability, and one showed an increase due to increased mRNA stability. Among these mRNAs, the MKK-2 mRNA encodes the protein kinase activator of ERK1/2 kinase, which is involved in the phosphorylation of eukaryotic initiation factor (eIF) 4E. As a result, mRNA translation may be regulated by the cellular level of MKK-2. In this study, we show that the abundance of the MKK-2 polypeptide is reduced in PABP-overexpressing cells. In these cells, the levels of phosphorylated PABP, eIF4E, and ERK2 are also reduced. Treatment of HeLa cells with the MKK-2 inhibitor U0126 reduced PABP phosphorylation, suggesting that the phosphorylation of PABP is mediated by the MKK-2/ERK signaling pathway. Thus, a novel signaling pathway involving MKK-2 and ERK1/2 may down-regulate the activity of PABP and eIF4E by controlling their phosphorylation and compensates for the effect of excess cellular PABP. Regulation of the rate of protein synthesis is important for the control of cellular growth and differentiation. Cells respond to changes in growth conditions by fine-tuning the rate of mRNA translation. Initiation of mRNA translation is the rate-limiting step and is often regulated by controlling the interaction between the 5′-cap of the mRNA and several initiation factors, including eukaryotic initiation factor (eIF) 2The abbreviations used are: eIF, eukaryotic initiation factor; PABP, poly(A)-binding protein; TIMP-1, tissue inhibitor of metalloproteinases-1; MKK-2, mitogen-activated protein kinase kinase-2; ERK, extracellular signal-regulated kinase; CMV, cytomegalovirus; UTR, untranslated region; DTT, dithiothreitol; RT, reverse transcription; ARS, autoregulatory sequence; GFP, green fluorescent protein; RNP, ribonucleoprotein; MCSF, macrophage-specific colony-stimulating factor. 4E, eIF4G, and eIF4A (1Gebauer F. Hentze M.W. Nat. Rev. Mol. Cell Biol. 2004; 5: 827-835Crossref PubMed Scopus (721) Google Scholar). A recent study has shown that the poly(A)-binding protein (PABP) also behaves like a genuine initiation factor because depletion of PABP from a cell-free extract prevents initiation of mRNA translation (2Kahvejian A. Svitki Y.V. Sukarieh R. M'Boutchou M. Sonenberg N. Genes Dev. 2005; 19: 104-113Crossref PubMed Scopus (363) Google Scholar). The 3′-poly(A) tail-bound PABP interacts with eIF4G and circularizes the translating mRNA. This process is believed to enhance mRNA translation by promoting recirculation of terminating ribosomal subunits from the 3′-end of the mRNA for another round of initiation (3Tarun Jr., S.J. Sachs A.B. EMBO J. 1996; 15: 7168-7177Crossref PubMed Scopus (581) Google Scholar, 4Imataka H. Gradi A. Sonenberg N. EMBO J. 1998; 17: 7480-7489Crossref PubMed Scopus (471) Google Scholar, 5Le H. Tanguay R.L. Balasta M.L. Wei C.C. Browning K.S. Metz A.M. Goss D.J. Gallie D.R. J. Biol. Chem. 1997; 272: 16247-16255Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 6Jacobson A. Sonenberg N. Hershey J.W.B. Mathews M.B. Poly(A) Metabolism and Translation: The Closed Loop Model. Cold Spring Harbor Press, Cold Spring Harbor, NY1996: 451-480Google Scholar). Although this model is widely accepted, it has not been proven to occur in vivo. In addition, if PABP is essential for mRNA translation, it is possible that PABP participates in mRNA translation by a yet unknown mechanism, as the presence of 3′-poly(A) (and thus circularization of mRNA) is not essential for translation initiation of capped mRNAs (7Kozak M. Gene (Amst.). 2004; 343: 41-54Crossref PubMed Scopus (38) Google Scholar, 8Both G.W. Banerjee A.K. Shatkin A.J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1189-1193Crossref PubMed Scopus (215) Google Scholar). PABP binds to the 3′-poly(A) track of eukaryotic mRNA. It also interacts with polypeptides involved in regulating the translation and stability of mRNA (9Bernstein P. Peltz S.W. Ross J. Mol. Cell. Biol. 1989; 9: 659-670Crossref PubMed Scopus (297) Google Scholar, 10Caponigro G. Parker R. Genes Dev. 1995; 9: 2421-2432Crossref PubMed Scopus (232) Google Scholar, 11Wang Z. Kiledjian M. Mol. Cell. Biol. 2000; 20: 6334-6341Crossref PubMed Scopus (86) Google Scholar, 12Mangus D.A. Evans M.C. Jacobson A. Genome Biology. 2003http://genomebiology.com/2003/4/7/223Google Scholar). Among the four highly conserved RNA-binding domains of PABP, the first two show specificity toward poly(A), whereas the third and fourth RNA-binding domains can also bind to poly(G) and poly(U) (13Burd C.G. Matunis E.L. Dreyfuss G. Mol. Cell. Biol. 1991; 11: 3419-3424Crossref PubMed Scopus (205) Google Scholar, 14Nietfeld W. Mentzel H. Pieler T. EMBO J. 1990; 9: 3699-3705Crossref PubMed Scopus (109) Google Scholar, 15Kuhn U. Pieler T. J. Mol. Biol. 1996; 256: 20-30Crossref PubMed Scopus (187) Google Scholar). The C-terminal region of PABP does not bind RNA, but can interact with other polypeptides and promotes oligomerization of PABP on poly(A) (16Melo E.D. Dhalia R. Martins de Sa C. Standart N. de Melo Neto O.P. J. Biol. Chem. 2003; 278: 46357-46368Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The C-terminal region contains a highly conserved 74-amino acid-long PABC (for PABP C-terminal) domain that binds to the eukaryotic release factor 3 and two regulators of mRNA translation, Paip1 and Paip2 (17Kozlow V. Trempe J.F. Khaleghpour K. Kalvejian A. Ekiel I. Gehring K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4409-4413Crossref PubMed Scopus (172) Google Scholar, 18Deo R.C. Bonanno J.B. Sonenberg N. Burley S.K. Cell. 1999; 98: 835-845Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). The ability of PABP to interact with eIF4G, Paip1, and Paip2 also resides within its RNA-binding domain (19Roy G. De Crescenzo G. Khaleghpour K. Kahvejian A. O'Coner-McCourt M. Sonenberg N. Mol. Cell. Biol. 2002; 22: 3769-3782Crossref PubMed Scopus (116) Google Scholar, 20Khaleghpour K. Kahvejian A. De Crescenzo G. Roy G. Svitkin Y.V. Imataka H. O'Connor-McCourt M. Sonenberg N. Mol. Cell. Biol. 2001; 21: 5200-5213Crossref PubMed Scopus (134) Google Scholar). Because of the important role of PABP in the initiation step of protein synthesis, a change in the cellular PABP level may affect not only the rate of protein synthesis, but also the protein expression profile of the cells. Therefore, control of PABP expression may be critical for cellular physiology. Accumulated evidences show that PABP expression is regulated primarily at the post-transcriptional level (21Thomas G. Thomas G. J. Cell Biol. 1986; 103: 2137-2144Crossref PubMed Scopus (89) Google Scholar, 22Adamou J. Bag J. Eur. J. Biochem. 1992; 209: 803-812Crossref PubMed Scopus (15) Google Scholar, 23Hornstein E. Git A. Nraunstein I. Avni D. Meyuhas O. J. Biol. Chem. 1999; 274: 1708-1714Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 24Wu J. Bag J. J. Biol. Chem. 1998; 273: 34535-34542Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 25Bag J. Wu J. Eur. J. Biochem. 1996; 237: 143-152Crossref PubMed Scopus (46) Google Scholar, 26de Melo Neto O.P. Standart N. Martins de Sa C. Nucleic Acids Res. 1995; 23: 2198-2205Crossref PubMed Scopus (95) Google Scholar, 27Bag J. J. Biol. Chem. 2001; 276: 47352-47360Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). However, in some instances, transcription of the PABP gene can also be regulated (28Hornstein E. Harel H. Levy G. Meyuhas O. FEBS Lett. 1999; 457: 209-213Crossref PubMed Scopus (32) Google Scholar). A number of studies have shown that regulation of cellular PABP levels is indeed important for embryonic development and growth control. For example, ectopic expression of PABP prevents maturation-specific deadenylation and translational inactivation of maternal mRNAs in Xenopus oocytes (29Wormington M. Searfoss A.M. Hurney C.A. EMBO. J. 1996; 15: 900-909Crossref PubMed Scopus (92) Google Scholar). PABP overexpression also leads to defects in cell divisions in Schizosaccharomyces pombe (30Tallada V. Daga R.R. Palomegue C. Garzon A. Jimenez J. Yeast. 2002; 19: 1139-1151Crossref PubMed Scopus (20) Google Scholar). Interestingly, in the early stages of cancer, an increase in cellular PABP levels has been observed (31Veralet M. Deregowski V. Denis G. Humblet C. Stalmans M.T. Bours V. Castronovo V. Boniver J. Defresne M.P. Biochem. Biophys. Res. Commun. 2001; 283: 12-18Crossref PubMed Scopus (16) Google Scholar), suggesting a link between growth control and PABP levels. In this work, we studied the effect of PABP overexpression on cellular mRNA levels in cultured HeLa cells. We report here that 19 mRNAs showed 2–3-fold reduced cellular levels in PABP-overexpressing cells and that the level of only one mRNA (encoding the TIMP-1 polypeptide) was increased by ∼6-fold. We show that changes in the stability of these mRNAs in PABP-overexpressing cells were responsible for the observed effects on mRNA levels. One of these mRNAs that showed reduced levels in PABP-overexpressing cells encodes MKK-2. Because MKK-2 may regulate the phosphorylation of eIF4E via the ERK1/2 signaling pathway (32Roux P.P. Blenis J. Microbiol. Mol. Biol. Rev. 2004; 168: 320-344Crossref Scopus (1936) Google Scholar, 33Richter J.D. Sonenberg N. Nature. 2005; 433: 477-480Crossref PubMed Scopus (761) Google Scholar, 34Veda T. Watanabe-Fukunaga R. Fukunaga H. Nagata S. Fukunaga R. Mol. Cell. Biol. 2004; 24: 6539-6549Crossref PubMed Scopus (411) Google Scholar), we studied how the stability of MKK-2 mRNA is affected by the cellular PABP level. We identified a novel MKK-2 mRNA stability control element that binds PABP. We show that, as a cellular response to excess PABP, the phosphorylation of PABP along with eIF4E was reduced. In addition, the immediate downstream target of MKK-2, ERK2 phosphorylation was reduced in PABP-overexpressing cells. Using a specific inhibitor of MKK-2, we show that the phosphorylation of PABP is regulated by the MKK-2/ERK1/2 kinase signaling pathway. We propose that down-regulation of MKK-2 expression to control the activity of PABP and eIF4E is a signaling mechanism of mammalian cells to override the adverse effect of excess PABP. Plasmids—A PABP cDNA clone (pHu73 plasmid) (35Grange T. Martins de Sa C. Oddos J. Pictet R. Nucleic Acids Res. 1987; 15: 4771-4787Crossref PubMed Scopus (142) Google Scholar) was digested with BamHI and SacII, which removed the first 263 nucleotides of the PABP cDNA containing the adenosine-rich autoregulatory region and released the cDNA insert from the vector (24Wu J. Bag J. J. Biol. Chem. 1998; 273: 34535-34542Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The PABP cDNA fragment was cloned at the AvrII/BamHI sites of the pCMV-SPORT-β-gal vector (Invitrogen) from which the β-galactosidase coding region was removed. This vector was named pCMVΔPABP. A second PABP expression vector was created with the FLAG epitope tag (pCMVΔPABP-FLAG) at the N-terminal end of PABP using a synthetic double-stranded oligodeoxynucleotide. β-Galactosidase reporter constructs containing different regions of the 3′-untranslated region (UTR) of MKK-2 mRNA were generated by ligating double-stranded oligodeoxynucleotides with EcoRI/NheI sticky ends to the EcoRI/BamHI fragment of the pCMV-SPORT-β-gal vector. To synthesize N-terminally His6-tagged PABP, the cDNA was amplified by PCR using pQE primers. The forward primer (5′-aaaggatccaaccccagtgcccc-3′) contained a BamHI restriction site, and the reverse primer (5′-ctaaagcttaaacagttggaacacc-3′) contained a HindIII restriction site. PCR was performed using a PfuUltra Hotstart DNA polymerase kit (Stratagene) with an initial denaturing step at 95 °C for 5 min, followed by 33 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min. The PCR product was digested with the appropriate restriction enzymes (Fermentas GmbH), purified on a 1% agarose gel using a QIAquick gel extraction kit (Qiagen Inc.), and cloned into the pQE80L prokaryotic expression vector (Qiagen Inc.). Transfection of Cells—Approximately 3 × 105 subconfluent HeLa cells grown on a 35-mm dish in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum were used for transfection. Plasmid DNA (2–3 μg) was incubated with 10 μg of Lipofectamine in 100 μl of Opti-MEMI (Invitrogen) for 30 min at room temperature before addition to the cells. Cells were incubated at 37 °C for 5 h with the DNA/liposome mixture in 1 ml of Opti-MEMI. Following incubation, 1 ml of growth medium containing 20% fetal bovine serum was added to the culture. After 12 h of incubation, the medium was replaced with fresh complete Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Analysis of RNA—RNAs from mock-, pCMVΔPABP-, or pCMVΔPABP-FLAG-transfected cells were isolated using a high pure RNA isolation kit (Roche Applied Science) according to the manufacturer's directions. Human cDNA microarrays (University Network Microarray Centre, Toronto, Ontario, Canada) were used to examine the changes in the gene expression profile during PABP overexpression in HeLa cells. An equal amount (15 μg) of RNA from either mock- or PABP-transfected cells was mixed with an oligo(dT)18 primer, a master mixture for reverse transcription (250 mm Tris-HCl (pH 8.3), 375 mm KCI, 15 mm MgCl2, 5 mm dithiothreitol (DTT), aminoallyl-dUTP, and dNTP), and 20 units of reverse transcriptase (Invitrogen). Reverse transcription proceeded for 2 h, followed by hydrolysis of the remaining RNA with NaOH, and the cDNA was purified using a PCR purification kit (Qiagen Inc). The probe was precipitated using isopropyl alcohol and resuspended in 5 μl of water. Probes were labeled using Alexa Fluor CyDye during a 1-h incubation period, and the remaining CyDye was removed using the PCR purification kit, followed again by alcohol precipitation. The probes were mixed with hybridization solution (4% each calf thymus DNA and yeast tRNA in DIG Easy Hyb (Roche Applied Science)) and incubated in a prewarmed and prehumidified chamber at 37 °C for 18 h in the dark. Microarray slides were washed with decreasing amounts of salt and SDS (first wash, 2× SSC and 0.1% SDS at 42 °C for 5 min; second wash, 1× SSC and 0.1% SDS at 25 °C for 10 min; and third wash, 0.1× SSC at 25 °C for 1 min.). The slides were dried, scanned using an Axon scanner, and analyzed using R Package software (University of California, Berkeley, CA). Each RNA sample was analyzed on three microarray slides, and three independent transfection experiments were performed. The level of a specific mRNA in the sample was determined by comparative real-time reverse transcription (RT)-PCR using Rotor-Gene 3000 (Corbett Research Australia) (36Heid C.A. Stevens J. Livak K.J. Williams P. Genome Res. 1996; 10: 986-994Crossref Scopus (5040) Google Scholar). An aliquot of total RNA (1 μg) was reverse-transcribed at 50 °C for 1 h in a total reaction volume of 25 μl using SuperScript II reverse transcriptase (Invitrogen) and 150 ng of random primers. After the reaction, 5 μl of the cDNA sample was amplified by PCR in a total reaction volume of 25 μl using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen) and 100 ng of the forward (sense) and reverse (antisense) primers specific to individual mRNAs (Table 1). Amplification was performed using an initial denaturation step at 95 °C for 4 min, followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 60 °C for 20 s, and extension at 72 °C for 20 s. The specificity of the PCR product was examined after the final cycle by generating a melting curve with a heating rate of 1 °C/s between 72 and 99 °C. The data were analyzed using Rotor-Gene 3000 software and the 2–ΔΔCt method. The relative expression values of all mRNAs were normalized to the β-actin mRNA level.TABLE 1Oligonucleotide primers for RT-PCRGene (GenBank™ accession no.)Sense oligonucleotideAntisense oligonucleotideZNF136 (BC006421)5′-CAAACTAAGGATGGTAGTCAGCG-3′ (23)aPrimer length is indicated in parentheses.5′-TGGTTCATGTCCACTGTGATCT-3′ (22)GAPDHbGAPDH, glyceraldehyde-3-phosphate dehydrogenase; CF2, coagulation factor 2; α2-AP, α2-antiplasmin; PPARA, peroxisomal proliferator-activated receptor-α; RPL27, ribosomal protein L27; FTL, ferritin light chain; RPL39L, ribosomal protein L39-like protein; MT1F, metallothionein 1F; MCC, colorectal mutant cancer protein; β-Gal, β-galactosidase. (BC083511)5′-ATGGGGAAGGTGAAGGTCG-3′ (19)5′-GGGGTCATTGATGGCAACAATA-3′ (22)CF2 (J00307)5′-GCGAGCCAACACCTTCTTG-3′ (19)5′-CCCAGAACACATCCGTAGCC-3′ (20)MCSF (M37435)5′-GCTCTCCCAGGATCTCATCAC-3′ (21)5′-TCAAAGGAACGGAGTTAAAACGG-3′ (23)BAG1 (BC001936)5′-GGCATTCCTAGCCGAGTGTG-3′ (20)5′-CCAGGGCAAAGTTTGTAGACTG-3′ (22)PRDX1 (NM_005809)5′-AATGCTAAAATTGGGCACCCT-3′ (21)5′-TGAAAGCAATGATCTCCGTGG-3′ (21)HSGRR22 (Y07846)5′-CGAGGGTCTGCACCTTTTCTC-3′ (21)5′-TTCGTAAGCTAACGGGAGTCA-3′ (21)GLUT5 (U11843)5′-AAACGTAGATGGTGAGTTCAGGA-3′ (23)5′-GCCTGTTAGAAGAGACCAGTCTG-3′ (23)ADH6 (BC039065)5′-AGCTATGGGGTCTGTGTGGTT-3′ (21)5′-AACGTCCTGAGAAGAACAACTG-3′ (22)α2-AP (BC031592)5′-ACAATCCGAACAGCTATTTGG-3′ (21)5′-GATGGCGTTGAGGAGAAGCA-3′ (19)PPARA (BC071932)5′-ACACTGTGTATGGCTGAGAAGA-3′ (22)5′-GACGGTCTCCACTGACGTG-3′ (19)RPL27 (NM_000988)5′-GGAAGACCCGGAAACTTAGGG-3′ (21)5′-GCCTGGGTGGTATTTGTCGAA-3′ (21)FTL (M11147)5′-CAGCCTGGTCAATTTGTACCT-3′ (21)5′-CCAGTTCGCGGAAGAAGTG-3′ (19)RPL39L (NM_052969)5′-TTGCAGATCGAGATTTGC-3′ (18)5′-CTCTTAGCGGAGGACAAAGG-3′ (20)MT1F (BC029453)5′-CCTGCAAGTGCAAAGAGTGC-3′ (20)5′-GGTTGTCCTGGCATCAGTCG-3′ (20)MCC (M62397)5′-CCTCAAATCCCAAAATGACC-3′ (20)5′-AGGCTCTGCTCACTCTCTGC-3′ (20)ABP1 (BC014093)5′-CCAAGTACCTGCTCTTTACC-3′ (20)5′-CTGGTGGTAGATGCTGCTGC-3′ (20)TIMP-1 (BC000866)5′-TGACATCCGGTTCGTCTACA-3′ (20)5′-TGCAGTTTTCCAGCAATGAG-3′ (20)MKK-2 (BC018645)5′-TTGCATGGAACACATGGACG-3′ (20)5′-GAGAACCGCGATGCTGACT-3′ (19)GNB5 (AF501885)5′-TGACATCAACAGTGTCCGGT-3′ (20)5′-GAGCAGAAAGCAGTCCCATC-3′ (20)β-Actin (BC013835)5′-CTCTTCCAGCCTTCCTTCCT-3′ (20)5′-CACCTTCACCGTTCCAGTTT-3′ (19)GFP (U57609)5′-TACCAGCGGTGGTTTGTTTG-3′ (20)5′-GCAGAGCGAGGTATGTAGGC-3′ (20)β-Gal (U02451)5′-CACTGGATAACGACATTG-3′ (18)5′-CAGCACCGCATCAGCAAG-3′ (18)a Primer length is indicated in parentheses.b GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CF2, coagulation factor 2; α2-AP, α2-antiplasmin; PPARA, peroxisomal proliferator-activated receptor-α; RPL27, ribosomal protein L27; FTL, ferritin light chain; RPL39L, ribosomal protein L39-like protein; MT1F, metallothionein 1F; MCC, colorectal mutant cancer protein; β-Gal, β-galactosidase. Open table in a new tab Measurement of the Effect of PABP on Cell Survival and Cell Divisions— To measure cell death, paraformaldehyde-fixed cells were stained with 4′,6-diamidino-2-phenylindole, and the nuclei were viewed under a fluorescence microscope. The cells were considered dead if the nuclei appeared fragmented and pyknotic, as described previously (37Wyttenbach A. Sauvageot O. Carmichael J. Diaz-Latoud C. Arrigo A.P. Rubinsztein D.C. Hum. Mol. Genet. 2002; 11: 1137-1151Crossref PubMed Scopus (428) Google Scholar). Approximately 200 cells were examined for each experiment. The cell-doubling time was determined by counting the number of cells in the culture after removing the cells from the plate by trypsinization at 12-h intervals. The cloning efficiency of trypsinized cells was determined by plating ∼100 cells on a 5-cm2 dish and counting the number of colonies after 4–6 days in culture. Measurement of Protein Levels—Forty-eight hours after transfection, cells were washed three times with phosphate-buffered saline and lysed in protein sample loading buffer (6% glycerol, 2% SDS, 100 mm DTT, and 0.02% bromphenol blue in 60 mm Tris-HCl (pH 6.6)) for 5 min. The polypeptides were separated by SDS-10% PAGE and electrophoretically transferred to a nitrocellulose membrane. After treating the transfer membrane for 1 h with blocking buffer (2% nonfat dry milk and 0.2% Tween 20 in phosphate-buffered saline), the membrane was incubated with a diluted primary antibody for 2 h at room temperature in blocking buffer and then incubated with an alkaline phosphatase-conjugated secondary antibody for 1 h. The bound antibody was detected with 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate as described previously (38Towbin H. Gordon J. J. Immunol. Methods. 1984; 72: 313-340Crossref PubMed Scopus (795) Google Scholar). In some experiments, a horseradish peroxidase-conjugated secondary antibody was used for detecting antigens by chemiluminescence using a Western Lightning kit (PerkinElmer Life Sciences) as described by the manufacturer. Expression and Purification of a His6-tagged Fusion Protein—Escherichia coli containing the pQE80L-PABP expression vector was grown to early log phase at 37 °C in LB medium (10 g of NaCl, 10 g of Bacto-Tryptone, and 5 g of yeast extract in 1 liter of H2O at pH 7.4) and induced for 4 h with isopropyl β-d-thiogalactopyranoside. The bacterial cells were harvested by centrifugation and lysed by incubation with 1 mg/ml lysozyme and lysis buffer (50 mm NaH2PO4, 500 mm NaCl, 30 mm imidazole, 13 mm β-mercaptoethanol, 2 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 0.5% Igepal CA-630, and 5% glycerol (pH 8.0)) at 0 °C for 30 min. The lysate was cleared by centrifugation, and the supernatant was incubated with nickel-nitrilotriacetic acid-agarose beads (Qiagen Inc.) in lysis buffer for 2 h at 0 °C. The beads were washed extensively with wash buffer (50 mm NaH2PO4, 500 mm NaCl, 50 mm imidazole, 13 mm β-mercaptoethanol, 2 mm MgCl2, 0.5% Igepal CA-630, and 5% glycerol (pH 8.0)). The bound protein was eluted with elution buffer (50 mm NaH2PO4, 500 mm NaCl, 300 mm imidazole, 13 mm β-mercaptoethanol, 2 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 2 μg/ml aprotinin, 0.5% Igepal CA-630, and 5% glycerol (pH 8.0)). The eluted fraction was equilibrated with storage buffer (10 mm HEPES-KOH (pH 7.5), 3 mm MgCl2, 140 mm KCl, 5% glycerol, 1 mm DTT, 0.02% Igepal CA-630, 0.5 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 2 μg/ml aprotinin) using a Microcon YM-30 concentration column (Millipore Corp.). In Vitro RNA Synthesis—Different regions of MKK-2 mRNA were amplified by PCR using primers containing a T7 promoter (Table 2). PCR was performed using the PfuUltra Hotstart DNA polymerase kit as described above. The gel-purified PCR product was used as a template for RNA synthesis. The accuracy of the amplicons was confirmed by sequencing.TABLE 2Primers for synthesizing different regions of MKK-2 mRNAMKK-2 geneOligonucleotideMKK-2-(1400-1417) SaS, sense oligonucleotide; AS, antisense oligonucleotide.aatagaattcgtaatacgactcactatagggcagtggccgggctccctgMKK-2-(1631-1648) ASaataggatccgtaacctaaggaagcagaMKK-2-(1525-1542) SaatagaattcgtaatacgactcactatagggcgtggcggggcgacagcgMKK-2-(1549-1566) ASAataggatccAgaggagacccccgttccMKK-2-(1631-1658) SaatagaattcgtaatacgactcactatagggtctgcttccttaggttacaaaacaaaacMKK-2-(1644-1663) ASgctttttctctccctgttttgttttgtaacMKK-2-(1400-1462) SgatccagtggccgggctccctgcgtccgctggtgacctgcccaccgtccctgtccatgccccgcccgMKK-2-(1504-1513) SaatagaattcgtaatacgactcactatagggcctcacccctMKK-2-(1473-1482) SaatagaattcgtaatacgactcactatagggggacaggctgMKK-2-(1380-1399) ASaataggatcctcacacggcggtgcgcgMKK-2-(1180-1197) ASaataggatccggcagcttaggaggtggcMKK-2-(980-997) ASaataggatccgatggggtaccttccgacMKK-2-(781-797) ASaataggatccggatgttggagggcttcMKK-2-(580-597) ASaataggatcctagaagcccacgatgtacgMKK-2-(380-397) ASaataggatccgagttcgccgaccttggcMKK-2-(181-196) ASaataggatcccggggctccgcgggccMKK-2-(1198-1215) SaatagaattcgtaatacgactcactatagggcaacggtgtgttcaccccMKK-2-(998-1115) SaatagaattcgtaatacgactcactatagggcccccgcccgacgccaaagMKK-2-(798-815) SaatagaattcgtaatacgactcactatagggtcgtgaactctagaggggMKK-2-(598-615) SaatagaattcgtaatacgactcactatagggcggggccttctacagtgaMKK-2-(398-415) SaatagaattcgtaatacgactcactatagggaaagacgatgacttcgaaMKK-2-(197-214) SaatagaattcgtaatacgactcactatagggatgctggcccggaggaagMKK-2-(1-18) SaatagaattcgtaatacgactcactataggggcggctcgctcgcctcagMKK-2-(1494-1524) SaatagaattcgtaatacgactcactatagggcaccctcctgcctcacccctgcggagagcacMKK-2-(1463-1493) SaatagaattcgtaatacgactcactatagggttccagctgaggacaggctggcgcctccaccMKK-2-(1549-1566) ASaataggatccagaggagacccccgttccMKK-2-(1525-1542) SaatagaattcgtaatacgactcactatagggcgtggcggggcgacagcgMKK-2-(1507-1524) ASaataggatccgtgctctccgcaggggtga S, sense oligonucleotide; AS, antisense oligonucleotide. Open table in a new tab Transcription was usually performed at 37 °C for 3 h in a final volume of 25 μl containing 1 μg of DNA template, 2.5 mm each NTP, and 20 units of T7 RNA polymerase (Promega) in 20 mm MgCl2, 1 mm spermidine, 0.01% Triton X-100, 20 mm DTT, and 40 mm Tris-HCl (pH 8.1). Uniformly radiolabeled RNA was synthesized under similar conditions in a final reaction volume of 25 μl containing 150 μCi of [α-32P]CTP (MP Biomedicals), and the final concentration of unlabeled CTP was reduced to 25 μmol. The contaminating nucleotides, incompletely transcribed products, and DNA template were removed by fractioning reaction mixtures on an 8% polyacrylamide gel under denaturing conditions (51Chang T.C. Yamshita A. Chen C.A. Yamshita Y. Zhu W. Durdan S. Kahuejian A. Sonenberg N. Shyu A.B. Genes Dev. 2004; 18: 2010-2023Crossref PubMed Scopus (123) Google Scholar). The amount of RNA and its specific radioactivity were determined using a spectrophotometer (1 A260 nm unit = 40 μg/ml RNA) and scintillation counter, respectively. Preparation of Cytoplasmic Extracts—HeLa cells were grown on 100-mm dishes to 30–40% confluency and transfected with 25 μg of DNA as described above. Forty-eight hours after transfection, cells were harvested to prepare the cytoplasmic protein extract as described previously (39Thomson A.M. Rogers J.T. Walker C.E. Staton J.M. Leedman P.J. BioTechniques. 1999; 27: 1032-1042Crossref PubMed Scopus (47) Google Scholar). In short, cells were grown to 90% confluency in Dulbecco's modified Eagle's medium, harvested by scraping in chilled phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, and 2 mm KH2PO4 (pH 7.4)), and collected by centrifugation at 5000 × g. The cells were lysed in hypotonic buffer (10 mm HEPES-KOH (pH 7.5), 3 mm MgCl2, 14 mm KCl, 5% glycerol, 1 mm DTT, 0.02% Igepal CA-630, 0.5 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 2 μg/μl aprotinin) by repeated passages through a 28-gauge needle. The cell lysate was centrifuged at 12,000 × g for 2 min at 4 °C to remove the nuclei and cell fragments, and the supernatant was stored at –85 °C in small aliquots. The protein concentration was estimated by the method of Bradford (40Bradford M.M. Anal. Biochem. 1976; 12: 248-254Crossref Scopus (217544) Google Scholar). UV Cross-linking Assay—For UV light-induced cross-linking assays, 60 μg of cytoplasmic extract or 1–10 ng of purified PABP was incubated with ≈2–3 ng (2 × 105 cpm) of radiolabeled RNA at 22 °C for 10 min in a total reaction volume of 30 μl in binding buffer (10 mm HEPES-KOH (pH 7.5), 3 mm MgCl2, 140 mm KCl, 5% glycerol, 1 mm DTT, 0.02% Igepal CA-630, 10 μg of E. coli tRNA, and 0.001% bromphenol blue). Heparin was added to a final concentration of 10 μg/μl, and the sample was irradiated by UV light (254 nm, 4000 microwatts/cm2) at 4 °C for 5 min. The sample was treated with RNase T1 (25 units)/RNase A (1 μg) at 37 °C for 5 min. Finally, the sample was boiled in protein sample loading buffer for 5 min and analyzed by 10% SDS-PAGE. The gels were vacuum-dried and autoradiographed. RNA Electrophoretic Mobility Shift Assay—20 μg of cytoplasmic extract was incubated with ≈1–2 ng (1 × 105 cpm) of radiolabeled RNA for 10 min at 22 °C in 18 μl of binding buffer. Subsequently, heparin was added to a final concentration of 10 μg/μl, and incubation was resumed for anoth" @default.
• W1966912797 created "2016-06-24" @default.
• W1966912797 creator A5016613196 @default.
• W1966912797 creator A5036639658 @default.
• W1966912797 creator A5090832802 @default.
• W1966912797 date "2006-02-01" @default.
• W1966912797 modified "2023-10-18" @default.
• W1966912797 title "Reduced Stability of Mitogen-activated Protein Kinase Kinase-2 mRNA and Phosphorylation of Poly(A)-binding Protein (PABP) in Cells Overexpressing PABP" @default.
• W1966912797 cites W1488522827 @default.
• W1966912797 cites W1971754292 @default.
• W1966912797 cites W1975120493 @default.
• W1966912797 cites W1975751316 @default.
• W1966912797 cites W1981668509 @default.
• W1966912797 cites W1987791514 @default.
• W1966912797 cites W1988021503 @default.
• W1966912797 cites W1992924554 @default.
• W1966912797 cites W1993424211 @default.
• W1966912797 cites W2005174488 @default.
• W1966912797 cites W2007068418 @default.
• W1966912797 cites W2023941617 @default.
• W1966912797 cites W2032436488 @default.
• W1966912797 cites W2045830695 @default.
• W1966912797 cites W2049672413 @default.
• W1966912797 cites W2049830784 @default.
• W1966912797 cites W2050270915 @default.
• W1966912797 cites W2061792951 @default.
• W1966912797 cites W2065525814 @default.
• W1966912797 cites W2065613854 @default.
• W1966912797 cites W2072105385 @default.
• W1966912797 cites W2075511135 @default.
• W1966912797 cites W2085703844 @default.
• W1966912797 cites W2086262311 @default.
• W1966912797 cites W2086939601 @default.
• W1966912797 cites W2088049733 @default.
• W1966912797 cites W2102754190 @default.
• W1966912797 cites W2102889876 @default.
• W1966912797 cites W2104065470 @default.
• W1966912797 cites W2111403104 @default.
• W1966912797 cites W2112360818 @default.
• W1966912797 cites W2116079251 @default.
• W1966912797 cites W2116153438 @default.
• W1966912797 cites W2126669980 @default.
• W1966912797 cites W2127720732 @default.
• W1966912797 cites W2129069292 @default.
• W1966912797 cites W2144099695 @default.
• W1966912797 cites W2149484524 @default.
• W1966912797 cites W2158537780 @default.
• W1966912797 cites W2160629946 @default.
• W1966912797 cites W2164121701 @default.
• W1966912797 cites W2164578725 @default.
• W1966912797 cites W2166914418 @default.
• W1966912797 cites W2167705826 @default.
• W1966912797 cites W233265278 @default.
• W1966912797 cites W233806587 @default.
• W1966912797 cites W4293247451 @default.
• W1966912797 cites W61616035 @default.
• W1966912797 doi "https://doi.org/10.1074/jbc.m508937200" @default.
• W1966912797 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16332685" @default.
• W1966912797 hasPublicationYear "2006" @default.
• W1966912797 type Work @default.
• W1966912797 sameAs 1966912797 @default.
• W1966912797 citedByCount "29" @default.
• W1966912797 countsByYear W19669127972012 @default.
• W1966912797 countsByYear W19669127972013 @default.
• W1966912797 countsByYear W19669127972015 @default.
• W1966912797 countsByYear W19669127972016 @default.
• W1966912797 countsByYear W19669127972017 @default.
• W1966912797 countsByYear W19669127972018 @default.
• W1966912797 countsByYear W19669127972019 @default.
• W1966912797 countsByYear W19669127972021 @default.
• W1966912797 crossrefType "journal-article" @default.
• W1966912797 hasAuthorship W1966912797A5016613196 @default.
• W1966912797 hasAuthorship W1966912797A5036639658 @default.
• W1966912797 hasAuthorship W1966912797A5090832802 @default.
• W1966912797 hasBestOaLocation W19669127971 @default.
• W1966912797 hasConcept C104317684 @default.
• W1966912797 hasConcept C105580179 @default.
• W1966912797 hasConcept C11960822 @default.
• W1966912797 hasConcept C124160383 @default.
• W1966912797 hasConcept C137361374 @default.
• W1966912797 hasConcept C159479382 @default.
• W1966912797 hasConcept C161238802 @default.
• W1966912797 hasConcept C184235292 @default.
• W1966912797 hasConcept C185592680 @default.
• W1966912797 hasConcept C2780887552 @default.
• W1966912797 hasConcept C41282012 @default.
• W1966912797 hasConcept C55493867 @default.
• W1966912797 hasConcept C82495950 @default.
• W1966912797 hasConcept C86803240 @default.
• W1966912797 hasConcept C90934575 @default.
• W1966912797 hasConcept C95444343 @default.
• W1966912797 hasConcept C97029542 @default.
• W1966912797 hasConcept C99405784 @default.
• W1966912797 hasConceptScore W1966912797C104317684 @default.
• W1966912797 hasConceptScore W1966912797C105580179 @default.
• W1966912797 hasConceptScore W1966912797C11960822 @default.
• W1966912797 hasConceptScore W1966912797C124160383 @default.
• W1966912797 hasConceptScore W1966912797C137361374 @default.

• next