Regulation of A + U-rich Element-directed mRNA Turnover Involving Reversible Phosphorylation of AUF1
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m305772200
ISSN1083-351X
AutoresGerald M. Wilson, Jiebo Lu, Kristina Sutphen, Yue Sun, Yung Huynh, Gary Brewer,
Tópico(s)RNA modifications and cancer
ResumoProteins binding A + U-rich elements (AREs) contribute to the rapid cytoplasmic turnover of mRNAs containing these sequences. However, this process is a regulated event and may be accelerated or inhibited by myriad signal transduction systems. For example, monocyte adherence at sites of inflammation or tissue injury is associated with inhibition of ARE-directed mRNA decay, which contributes to rapid increases in cytokine and inflammatory mediator production. Here, we show that acute exposure of THP-1 monocytic leukemia cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate mimics several features of monocyte adherence, including rapid induction and stabilization of ARE-containing mRNAs encoding interleukin-1β and tumor necrosis factor α. Additionally, TPA treatment alters the activity of cytoplasmic complexes that bind AREs, including complexes containing the ARE-specific, mRNA-destabilizing factor, AUF1. Analyses of AUF1 from control and TPA-treated cells indicated that post-translational modifications of the major cytoplasmic isoform, p40AUF1, are altered concomitant with changes in RNA binding activity and stabilization of ARE-containing mRNAs. In particular, p40AUF1 recovered from polysomes was phosphorylated on Ser83 and Ser87 in untreated cells but lost these modifications following TPA treatment. We propose that selected signal transduction pathways may regulate ARE-directed mRNA turnover by reversible phosphorylation of polysome-associated p40AUF1. Proteins binding A + U-rich elements (AREs) contribute to the rapid cytoplasmic turnover of mRNAs containing these sequences. However, this process is a regulated event and may be accelerated or inhibited by myriad signal transduction systems. For example, monocyte adherence at sites of inflammation or tissue injury is associated with inhibition of ARE-directed mRNA decay, which contributes to rapid increases in cytokine and inflammatory mediator production. Here, we show that acute exposure of THP-1 monocytic leukemia cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate mimics several features of monocyte adherence, including rapid induction and stabilization of ARE-containing mRNAs encoding interleukin-1β and tumor necrosis factor α. Additionally, TPA treatment alters the activity of cytoplasmic complexes that bind AREs, including complexes containing the ARE-specific, mRNA-destabilizing factor, AUF1. Analyses of AUF1 from control and TPA-treated cells indicated that post-translational modifications of the major cytoplasmic isoform, p40AUF1, are altered concomitant with changes in RNA binding activity and stabilization of ARE-containing mRNAs. In particular, p40AUF1 recovered from polysomes was phosphorylated on Ser83 and Ser87 in untreated cells but lost these modifications following TPA treatment. We propose that selected signal transduction pathways may regulate ARE-directed mRNA turnover by reversible phosphorylation of polysome-associated p40AUF1. In eukaryotes, cytoplasmic mRNA stability is an important checkpoint in the control of gene expression. Many mRNAs encoding regulatory proteins like cytokines, inflammatory mediators, and oncoproteins are constitutively unstable. This ensures that the steady-state levels of these mRNAs, and hence their potential for translation, remain low but also that new steady-state levels are approached quickly following changes in the rate of mRNA synthesis (reviewed in Ref. 1Ross J. Microbiol. Rev. 1995; 59: 423-450Crossref PubMed Google Scholar). In mammals, a common feature of many unstable mRNAs is the presence of an A + U-rich element (ARE) 1The abbreviations used are: ARE, A + U-rich element; ACN, acetonitrile; actD, actinomycin D; IL-1β, interleukin-1β; IMAC, immobilized metal ion affinity chromatography; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; p38MAPK, p38 mitogen-activated protein kinase; RPA, ribonuclease protection assay; TNFα, tumor necrosis factor α; TPA, 12-O-tetradecanoylphorbol-13-acetate; TTP, tristetraprolin; UTR, untranslated region. within the 3′-untranslated region (3′-UTR). These elements range from 40 to 150 nucleotides in length and exhibit significant variability in sequence composition, but they usually include one or more AUUUA motifs within a U-rich context (2Chen C.-Y.A. Shyu A.-B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1688) Google Scholar). In general, mRNA turnover mediated by AREs consists of rapid 3′ → 5′ shortening of the poly(A) tail, followed by decay of the mRNA body (3Guhaniyogi J. Brewer G. Gene. 2001; 265: 11-23Crossref PubMed Scopus (555) Google Scholar, 4Wilusz C.J. Wormington M. Peltz S.W. Nature Rev. Mol. Cell. Biol. 2001; 2: 237-246Crossref PubMed Scopus (634) Google Scholar). The regulation of mRNA decay kinetics by AREs involves their association with any of a number of cellular ARE-binding factors (reviewed in Ref. 5Wilson G.M. Brewer G. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 257-291Crossref PubMed Scopus (123) Google Scholar). One such factor, AUF1 (also referred to as heterogeneous nuclear ribonucleoprotein D), is expressed as a family of four protein isoforms resulting from alternative splicing of a common pre-mRNA (6Wagner B.J. DeMaria C.T. Sun Y. Wilson G.M. Brewer G. Genomics. 1998; 48: 195-202Crossref PubMed Scopus (239) Google Scholar). The larger isoforms, designated by their apparent molecular weights as p42AUF1 and p45AUF1, are largely nuclear (7Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (497) Google Scholar), probably due to the presence of a binding determinant for components of the nuclear scaffold (8Arao Y. Kuriyama R. Kayama F. Kato S. Arch. Biochem. Biophys. 2000; 380: 228-236Crossref PubMed Scopus (60) Google Scholar). By contrast, p37AUF1 and p40AUF1 lack this sequence determinant and, as such, may be found in both nuclear and cytoplasmic compartments. AUF1 binding to an ARE is linked to acceleration of mRNA decay, based on extensive studies correlating mRNA turnover rates with AUF1 abundance (9Buzby J.S. Lee S. van Winkle P. DeMaria C.T. Brewer G. Cairo M.S. Blood. 1996; 88: 2889-2897Crossref PubMed Google Scholar, 10Lin S. Wang W. Wilson G.M. Yang X. Brewer G. Holbrook N.J. Gorospe M. Mol. 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Association of p37AUF1 with an ARE induces the formation of protein oligomers (16Wilson G.M. Sun Y. Lu H. Brewer G. J. Biol. Chem. 1999; 274: 33374-33381Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and local conformational changes in the RNA substrate (17Wilson G.M. Sutphen K. Moutafis M. Sinha S. Brewer G. J. Biol. Chem. 2001; 276: 38400-38409Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), which in turn may recruit additional factors to generate a multisubunit, trans-acting complex on the mRNA (7Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (497) Google Scholar, 18Laroia G. Cuesta R. Brewer G. Schneider R.J. Science. 1999; 284: 499-502Crossref PubMed Scopus (348) Google Scholar). Ultimately, the mRNA is degraded by catabolic activities, which may include specific nucleases (19Dehlin E. Wormington M. Körner C.G. Wahle E. EMBO J. 2000; 19: 1079-1086Crossref PubMed Scopus (157) Google Scholar, 20Chen C.-Y. Gherzi R. Ong S.-E. Chan E.L. Raijmakers R. Pruijn G.J.M. Stoecklin G. Moroni C. Mann M. Karin M. Cell. 2001; 107: 451-464Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 21Mukherjee D. Gao M. O'Connor J.P. Raijmakers R. Pruijn G. Lutz C.S. Wilusz J. EMBO J. 2002; 21: 165-174Crossref PubMed Scopus (310) Google Scholar) or the proteasome (18Laroia G. Cuesta R. Brewer G. Schneider R.J. Science. 1999; 284: 499-502Crossref PubMed Scopus (348) Google Scholar). Besides AUF1, other RNA-binding factors have also been implicated in the regulation of mRNA decay rates through AREs. For example, association of tristetraprolin (TTP) with some ARE-containing transcripts enhances their decay (22Lai W.S. Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (638) Google Scholar, 23Ming X.-F. Stoecklin G. Lu M. Looser R. Moroni C. Mol. Cell. Biol. 2001; 21: 5778-5789Crossref PubMed Scopus (160) Google Scholar). By contrast, mammalian factors related to the Drosophila Elav (embryonic lethal abnormal vision) protein, including the ubiquitously expressed HuR and the neuron-specific Hel-N1, are thought to inhibit ARE-directed mRNA turnover (24Peng S.S.Y. Chen C.-Y.A. Xu N. Shyu A.-B. EMBO J. 1998; 17: 3461-3470Crossref PubMed Scopus (656) Google Scholar, 25Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (748) Google Scholar, 26Ford L.P. Watson J. Keene J.D. Wilusz J. Genes Dev. 1999; 13: 188-201Crossref PubMed Scopus (219) Google Scholar). Thus, associated trans-acting proteins may modulate the decay kinetics of ARE-containing mRNAs positively or negatively. Additionally, a growing number of ARE-binding proteins have been described to which no specific functions have been ascribed (27Hamilton B.J. Nagy E. Malter J.S. Arrick B.A. Rigby W.F.C. J. Biol. Chem. 1993; 268: 8881-8887Abstract Full Text PDF PubMed Google Scholar, 28Hamilton B.J. Burns C.M. Nichols R.C. Rigby W.F.C. J. Biol. Chem. 1997; 272: 28732-28741Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 29Myer V.E. Steitz J.A. RNA. 1995; 1: 171-182PubMed Google Scholar, 30Henics T. Nagy E. Oh H.J. Csermely C. von Gabain A. Subjeck J.R. J. Biol. Chem. 1999; 274: 17318-17324Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 31Wilson G.M. Sutphen K. Bolikal S. Chuang K. Brewer G. J. Biol. Chem. 2001; 276: 44450-44456Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Modulation of ARE-directed mRNA turnover has been observed in response to a plethora of stimuli. For example, the rapid decay of interleukin-3 mRNA in mast cells is inhibited by Ca2+ influx (32Stoecklin G. Hahn S. Moroni C. J. Biol. Chem. 1994; 269: 28591-28597Abstract Full Text PDF PubMed Google Scholar). Similarly, mRNAs containing AREs are stabilized during heat shock (18Laroia G. Cuesta R. Brewer G. Schneider R.J. Science. 1999; 284: 499-502Crossref PubMed Scopus (348) Google Scholar). Specific intracellular signaling pathways have also been identified, which contribute to the regulation of ARE-directed mRNA turnover. In particular, components of the p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase pathways are required for stabilization of ARE-containing mRNAs associated with the inflammatory response (33Winzen R. Kracht M. Ritter B. Wilhem A. Chen C.-Y.A. Shyu A.-B. Muller M. Gaestel M. Resch K. Holtmann H. EMBO J. 1999; 18: 4969-4980Crossref PubMed Scopus (713) Google Scholar, 34Lasa M. Mahtani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (370) Google Scholar, 35Ming X.-F. Kaiser M. Moroni C. EMBO J. 1998; 17: 6039-6048Crossref PubMed Scopus (137) Google Scholar, 36Neininger A. Kontoyiannis D. Kotlyarov A. Winzen R. Eckert R. Volk H.D. Holtmann H. Kollias G. Gaestel M. J. Biol. Chem. 2002; 277: 3065-3068Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 37Frevel M.A.E. Bakheet T. Silva A.M. Hissong J.G. Khabar K.S.A. Williams B.R.G. Mol. Cell. Biol. 2003; 23: 425-436Crossref PubMed Scopus (263) Google Scholar) and tumor cell metastasis (38Montero L. Nagamine Y. Cancer Res. 1999; 59: 5286-5293PubMed Google Scholar). Both tyrosine kinase and p38MAPK activities are required for the stabilization of interleukin-1β (IL-1β) and GRO mRNAs induced by monocyte adherence (15Sirenko O.I. Lofquist A.K. DeMaria C.T. Morris J.S. Brewer G. Haskill J.S. Mol. Cell. Biol. 1997; 17: 3898-3906Crossref PubMed Scopus (135) Google Scholar). By contrast, ARE-dependent stabilization of cyclooxygenase-2 mRNA by Gαq-coupled receptor signaling in smooth muscle is mediated by the p42/p44 MAP kinases and is independent of p38MAPK activity (39Xu K. Robida A.M. Murphy T.J. J. Biol. Chem. 2000; 275: 23012-23019Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Currently, the molecular mechanisms linking activation or inhibition of these signaling pathways to modulation of mRNA decay rates are largely unknown. One possibility is that these systems function by altering the abundance or RNA-binding properties of ARE-binding factors responsible for initiating the decay process. To test this hypothesis, we have examined the stabilization of ARE-containing mRNAs encoding the cytokine IL-1β and the inflammatory mediator tumor necrosis factor α (TNFα), using a cultured cell model that mimics key features of monocyte adherence. In particular, we show that acute exposure of THP-1 monocytic leukemia cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) rapidly induced cell adhesion, accompanied by dramatic increases in levels of IL-1β and TNFα mRNAs. Accumulation of these transcripts included significant mRNA stabilization, similar to that observed following adhesion of primary monocytes (15Sirenko O.I. Lofquist A.K. DeMaria C.T. Morris J.S. Brewer G. Haskill J.S. Mol. Cell. Biol. 1997; 17: 3898-3906Crossref PubMed Scopus (135) Google Scholar). Using this model system, we show that the distribution of cytoplasmic ARE binding activities containing AUF1 is altered following TPA treatment, coincident with changes in post-translational modifications of cytoplasmic p40AUF1. Finally, we have identified two sites of phosphorylation on p40AUF1 purified from THP-1 polysomes, which are dephosphorylated following TPA treatment, concomitant with inhibition of ARE-directed mRNA turnover. From these data, we propose that reversible phosphorylation of AUF1 may constitute a critical mechanism for regulating mRNA turnover rates through AREs. Materials—THP-1 monocytic leukemia cells were generously provided by Dr. Charles McCall. Oligoribonucleotide substrates encoding the TNFα ARE and a fragment of the rabbit β-globin coding region (Fig. 3A) were synthesized by Dharmacon (Lafayette, CO). Anti-HuR and anti-TTP antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-Hsp70 antibodies were from Stressgen Biotechnology (San Diego, CA). Radiolabeled nucleotides were from ICN (Costa Mesa, CA), and TRIzol reagent was from Invitrogen. Plasmid pGEM7Zf(+) and PolyATtract mRNA Purification Systems were purchased from Promega (Madison, WI). Actinomycin D (actD) was obtained from Calbiochem, and ampholytes were from Bio-Rad. TPA, IGEPAL-CA630, trypsin-treated l-1-tosylamido-2-phenylethyl chloromethyl ketone, carboxypeptidase Y, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid, angiotensin II, and trifluoroacetic acid were purchased from Sigma. ZipTipC4, ZipTipC18, and ZipTipMC pipette tip columns and the Montage In-Gel Digest96 Kit were obtained from Millipore, Corp. (Bedford, MA). Acetonitrile (ACN) and GaCl3 were purchased from Aldrich. Calf intestinal alkaline phosphatase was obtained from Roche Applied Science. Cell Culture—THP-1 cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum and 1 mm l-glutamine in the absence of antibiotics. Defined fetal bovine serum was lot-selected for minimal endotoxin content by HyClone (Logan, UT) and then incubated at 56 °C for 30 min to inactivate residual endotoxins prior to use. mRNA Quantitation by RNase Protection Assay (RPA)—A plasmid template for synthesis of a riboprobe complimentary to a portion of the IL-1β 3′-UTR was generously provided by Dr. Mary Vermeulen. A cDNA fragment encoding a portion of the TNFα 3′-UTR was amplified by PCR from plasmid pE4 (ATCC, Manassas, VA), and a human β-actin 3′-UTR fragment was amplified by reverse transcription-PCR from THP-1 cell total RNA. The resulting cDNA fragments were then sub-cloned into pGEM7Zf(+) to generate plasmid templates for preparation of antisense riboprobes. 32P-Labeled riboprobes used for RPAs were generated by in vitro run-off transcription as described previously (40Wilson G.M. Sun Y. Sellers J. Lu H. Penkar N. Dillard G. Brewer G. Mol. Cell. Biol. 1999; 19: 4056-4064Crossref PubMed Scopus (50) Google Scholar). Riboprobes complementary to IL-1β and TNFα mRNAs were synthesized to specific activities of 1–2 × 104 cpm/fmol, whereas β-actin antisense riboprobes were synthesized to 200 cpm/fmol. Cellular RNA samples were purified first as the total RNA fraction, extracted using TRIzol reagent according to the manufacturer's instructions, and then enriched for mRNA using the PolyATtract mRNA purification system. Specific cellular mRNAs were quantified using RPAs (8 μg of poly(A)+ RNA/lane) with RNases P1 and T1 as described previously (41Brewer G. Ross J. Methods Enzymol. 1990; 181: 202-209Crossref PubMed Scopus (78) Google Scholar). Protected RNA fragments were fractionated by denaturing gel electrophoresis and were visualized and quantified using a PhosphorImager (Amersham Biosciences). mRNA Decay Assays—Decay rates of IL-1β and TNFα mRNAs were measured by an actD time course assay. Briefly, transcription was inhibited in control or TPA-treated THP-1 cells by the addition of actD (5 μg/ml) to the culture medium, and poly(A)+ RNA samples were harvested at selected time points thereafter. The relative abundances of IL-1β and TNFα mRNAs were determined at each point by RPA and were normalized using β-actin mRNA levels. First-order decay constants (k) were solved by nonlinear regression of the percentage of IL-1β or TNFα mRNA remaining versus time of actD treatment using PRISM version 2.0 (GraphPad, San Diego, CA). Errors about regression solutions (S.E.) were calculated by the software using n–2 degrees of freedom, with replicate experiments yielding similar results. Comparisons of mRNA decay constants for control versus TPA-treated cells were performed using the unpaired t test, with differences exhibiting p < 0.05 considered significant. Fractionation of THP-1 Cells and Gel Mobility Shift Assays—Nuclear and cytoplasmic fractions of control or TPA-treated (10 nm, 1 h) THP-1 cells were prepared by resuspension (control samples) or scraping (TPA-treated samples) of PBS-washed cells in lysis buffer (10 mm Tris-HCl (pH 7.5), 150 mm KCl, 2.5 mm EDTA, and 1% IGEPAL-CA630) containing mixtures of protease inhibitors (1 μg/ml each of leupeptin and pepstatin A, 0.1 mm phenylmethylsulfonyl fluoride) and kinase/phosphatase inhibitors (50 mm sodium fluoride, 5 mm disodium pyrophosphate, 1 mm sodium orthovanadate). Lysis was performed with 7–10 strokes of a loosely fitting Dounce homogenizer and verified by phase-contrast microscopy. Nuclei were pelleted by centrifugation at 1000 × g for 10 min and resuspended directly in SDS-PAGE loading buffer for Western analyses. Protein concentrations were determined for cytoplasmic extracts (42Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217529) Google Scholar). Cytoplasmic ARE-binding activities were identified in THP-1 cytoplasmic extracts using gel mobility shift assays as described previously (43Wilson G.M. Brewer G. Methods. 1999; 17: 74-83Crossref PubMed Scopus (55) Google Scholar). Oligoribonucleotide RNA substrates were 5′-end-labeled using [γ-32P]ATP and T4 polynucleotide kinase to specific activities of 3–5 × 103 cpm/fmol as described (16Wilson G.M. Sun Y. Lu H. Brewer G. J. Biol. Chem. 1999; 274: 33374-33381Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Identical reactions were assembled for antibody supershift assays except that preimmune serum or antiserum was added to a maximum of 10% total reaction volume prior to incubation. Two-dimensional Western Analyses—Cytoplasmic extracts (50 μg of protein) from control or TPA-treated THP-1 cells (described above) were loaded onto 1.5 mm × 12-cm isoelectric focusing tube gels in 1 × SDS sample buffer (125 mm Tris-HCl (pH 6.8), 1% SDS, 5% glycerol, 10 mm dithiothreitol, 0.005% bromphenol blue) without heating. Gels contained 8 m urea, 3.5% acrylamide (30:1 acrylamide/bisacrylamide), 2% Triton X-100, 0.5% 3/10 ampholytes, 2.3% 5/7 ampholytes, and 2.3% 7/9 ampholytes, and were run between 0.1 n NaOH (cathode buffer) and 6 mm H3PO4 (anode buffer) at 400 V for 16 h and then 800 V for 2 h. Tube gels were extruded and equilibrated with 1 × SDS sample buffer prior to second dimension fractionation by SDS-PAGE, followed by electroblotting onto a polyvinylidene difluoride membrane and immunodetection of AUF1. Analyses of Polysome-associated p40AUF1 and Peptide Fragments by Mass Spectrometry—A ribosomal salt wash was prepared by first isolating polysomes from control or TPA-treated THP-1 cells by hypotonic lysis and centrifugation through 30% sucrose and then releasing polysome-associated proteins using 0.5 m potassium acetate as described previously (41Brewer G. Ross J. Methods Enzymol. 1990; 181: 202-209Crossref PubMed Scopus (78) Google Scholar). From this material, AUF1 was purified by tandem heparin and poly(U) chromatography as described (43Wilson G.M. Brewer G. Methods. 1999; 17: 74-83Crossref PubMed Scopus (55) Google Scholar). Tryptic digests were performed in SDS-PAGE gel slices using the Montage In-Gel Digest96 kit according to the manufacturer's instructions. Briefly, the SDS-PAGE band corresponding to p40AUF1 was excised, destained, and dehydrated and then digested with trypsin overnight at 30 °C. Liberated peptide fragments were lyophilized in a SpeedVac. Tryptic phosphopeptide fragments were enriched using an immobilized metal ion affinity chromatography (IMAC)-based strategy (44Posewitz M.C. Tempst P. Anal. Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (787) Google Scholar). Briefly, ZipTipMC columns were charged with 60 mm GaCl3, washed with 10% ACN containing 1% acetic acid, and equilibrated with 10% ACN containing 0.1% acetic acid. Lyophilized peptide fragments were resuspended in 0.1% acetic acid and bound to the columns through 10 application cycles. Phosphopeptides were eluted with freshly prepared 0.3 m NH4OH. Where indicated, phosphopeptide fragments selected by Ga3+-IMAC were dephosphorylated using on-target dephosphorylation reactions (45Vihinen H. Saarinen J. J. Biol. Chem. 2000; 275: 27775-27783Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Here, phosphopeptides spotted on the MALDI target plate (described below) were dissolved in 0.5 μl of 50 mm NH4HCO3 (pH 8.9) containing calf intestinal alkaline phosphatase (0.01 units). Samples were then incubated for 2 h at 37 °C in a high humidity chamber. The dephosphorylation reaction was stopped by the addition of 0.5 μl of ACN. Specific sites of phosphorylation were determined by limited carboxypeptidase Y digest (46Qian X. Zhou W. Khaledi M.G. Tomer K.B. Anal. Biochem. 1999; 274: 174-180Crossref PubMed Scopus (20) Google Scholar) of Ga3+-IMAC-selected phosphopeptide fragments. Briefly, phosphopeptides were dried and resuspended in 50 mm sodium citrate (pH 6.0). Carboxypeptidase Y was added (enzyme/peptide ratio of 2:1 by mass), and the mixture was incubated at 37 °C for 8 h. The reaction was stopped by the addition of trifluoroacetic acid to 0.5%. MALDI-TOF mass analyses of all samples were performed on a Voyager-DE PRO work station equipped with a PerkinElmer Biosystems DE-PRO mass spectrometer (PerSeptive Biosystems, Framingham, MA). Phosphopeptides isolated by Ga3+-IMAC and products from carboxypeptidase Y digests were purified and concentrated with Zip-TipC18 columns according to the manufacturer's instructions and then mixed (1:4, v/v) with a saturated α-cyano-4-hydroxycinnamic acid solution in ethanol/water/formic acid (45:45:10). Polysomal p40AUF1 purified from THP-1 cells was concentrated using ZipTipC4 columns and then mixed (1:4 v/v) with saturated sinapinic acid in 0.1% trifluoroacetic acid, 50% ACN. Samples from each mixture (2 μl) were spotted onto MALDI target plates and air-dried. The samples were analyzed by the mass spectrometer in the reflector positive or linear negative ion delayed extraction mode. Reflector positive mode was preferred for detection of phosphopeptide fragments due to its improved mass resolution (47Annan R.S. Carr S.A. Anal. Chem. 1996; 68: 3413-3421Crossref PubMed Scopus (301) Google Scholar), although this mode also contributed to increased background signals. Mass calibration was performed using angiotensin II as an external standard. The apparent molecular mass of unmodified p40AUF1 was calculated from the predicted amino acid sequence (GenBank™ accession number NM_002138) using the AAStats program of the Biology Workbench version 3.2 (San Diego Supercomputer Center; available on the World Wide Web at www.workbench.sdsc.edu). IL-1β and TNFα mRNAs Are Rapidly Induced and Stabilized in THP-1 Cells following Acute TPA Treatment—Circulating monocytes adhere at sites of infection or tissue injury. This event rapidly triggers the expression of a battery of cytokines and inflammatory mediators, involving both transcriptional and post-transcriptional mechanisms (15Sirenko O.I. Lofquist A.K. DeMaria C.T. Morris J.S. Brewer G. Haskill J.S. Mol. Cell. Biol. 1997; 17: 3898-3906Crossref PubMed Scopus (135) Google Scholar, 48Lofquist A.K. Mondal K. Morris J.S. Haskill J.S. Mol. Cell. Biol. 1995; 15: 1737-1746Crossref PubMed Google Scholar). By contrast, THP-1 monocytic leukemia cells grow constitutively in suspension but may become adherent following treatment with phorbol esters. Stimulation of THP-1 cells with phorbol esters like TPA is a popular model of monocytic differentiation to macrophage-like cells, based on the manifestation of adherence, loss of proliferation, phagocytosis, and enhanced production of proinflammatory cytokines in response to lipopolysaccharide (49Rovera G. O'Brien T.G. Diamond L. Science. 1979; 240: 868-870Crossref Scopus (453) Google Scholar, 50Schwende H. Fitzke E. Ambs P. Dieter P. J. Leukoc. Biol. 1996; 59: 555-561Crossref PubMed Scopus (450) Google Scholar, 51Lee J. Mehta K. Blick M.B. Gutterman J.U. Lopez-Berestein G. Blood. 1987; 69: 1542-1545Crossref PubMed Google Scholar, 52Rutault K. Hazzalin C.A. Mahadevan L.C. J. Biol. Chem. 2001; 276: 6666-6674Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Whereas phorbol ester treatment induces THP-1 cells to adopt many macrophage-like characteristics over a period of days, adherence per se may be observed within a mere 15-min exposure to TPA (10 nm, data not shown). To determine whether this acute TPA-induced adherence of THP-1 cells was accompanied by increased expression of cytokine and inflammatory mediator mRNAs, similar to that observed in adherent monocytes, the levels of IL-1β and TNFα mRNAs were measured across a time course of TPA treatment (Fig. 1). Increases in IL-1β mRNA levels were observed within 30–60 min and reached a maximum induction of nearly 50-fold within 8 h (Fig. 1, A and C). TNFα mRNA levels were also rapidly increased, attaining 30-fold induction within 4 h (Fig. 1, B and C). In both cases, these inductions were transient, with preinduction mRNA levels reached (TNFα) or approached (IL-1β) after 24 h of TPA treatment. During monocyte adherence, mRNA stabilization contributes to the induction of mRNAs encoding cytokines and other inflammatory mediators (15Sirenko O.I. Lofquist A.K. DeMaria C.T. Morris J.S. Brewer G. Haskill J.S. Mol. Cell. Biol. 1997; 17: 3898-3906Crossref PubMed Scopus (135) Google Scholar). To establish whether similar post-transcriptional mechanisms contributed to induction of these mRNAs in the acute TPA-treated THP-1 model, the decay kinetics of IL-1β and TNFα mRNAs were monitored prior to and following TPA-induced cell adherence by actD time course assay (Fig. 2). In all cases, mRNA decay kinetics were well approximated by first-order decay functions (Fig. 2C). Both IL-1β and TNFα mRNAs are relatively unstable in unstimulated THP-1 cells, decaying with half-lives of 29 and 8 min, respectively. However, after 1 h of TPA treatment, both mRNAs were stabilized 6–7-fold (Fig. 2C). In addition, stabilization of each mRNA was sustained as mRNA levels accumulated (Table I). Taken together, the similarities in mRNA accumulation kinetics, the transient nature of mRNA induction, and the rapid and prolonged stabilization of each mRNA following TPA treatment of THP-1 cells indicate that expression of IL-1β and TNFα mRNAs are coordinately regulated in this system, involving both transcriptional and post-transcriptional mechanisms. As such, these data suggested that acute TPA treatment of THP-1 cells mimics some features of the adhesion-dependent induction of cytokine and inflammatory mediator mRNA levels in primary monocytes.Table IDecay kinetics of IL-1β and TNFα mRNAs in THP-1 cellsmRNA10 nm T
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