Phosphorylation of p40AUF1 Regulates Binding to A + U-rich mRNA-destabilizing Elements and Protein-induced Changes in Ribonucleoprotein Structure
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m305775200
ISSN1083-351X
AutoresGerald M. Wilson, Jiebo Lu, Kristina Sutphen, Yvelisse Suarez, Smrita Sinha, Brandy Y. Brewer, Eneida C. Villanueva-Feliciano, Riza M. Ysla, Sandy Charles, Gary Brewer,
Tópico(s)RNA and protein synthesis mechanisms
ResumoMessenger RNA turnover directed by A + U-rich elements (AREs) involves selected ARE-binding proteins. Whereas several signaling systems may modulate ARE-directed mRNA decay and/or post-translationally modify specific trans-acting factors, it is unclear how these mechanisms are linked. In THP-1 monocytic leukemia cells, phorbol ester-induced stabilization of some mRNAs containing AREs was accompanied by dephosphorylation of Ser83 and Ser87 of polysome-associated p40AUF1. Here, we report that phosphorylation of p40AUF1 influences its ARE-binding affinity as well as the RNA conformational dynamics and global structure of the p40AUF1-ARE ribonucleoprotein complex. Most notably, association of unphosphorylated p40AUF1 induces a condensed RNA conformation upon ARE substrates. By contrast, phosphorylation of p40AUF1 at Ser83 and Ser87 inhibits this RNA structural transition. These data indicate that selective AUF1 phosphorylation may regulate ARE-directed mRNA turnover by remodeling local RNA structures, thus potentially altering the presentation of RNA and/or protein determinants involved in subsequent trans-factor recruitment. Messenger RNA turnover directed by A + U-rich elements (AREs) involves selected ARE-binding proteins. Whereas several signaling systems may modulate ARE-directed mRNA decay and/or post-translationally modify specific trans-acting factors, it is unclear how these mechanisms are linked. In THP-1 monocytic leukemia cells, phorbol ester-induced stabilization of some mRNAs containing AREs was accompanied by dephosphorylation of Ser83 and Ser87 of polysome-associated p40AUF1. Here, we report that phosphorylation of p40AUF1 influences its ARE-binding affinity as well as the RNA conformational dynamics and global structure of the p40AUF1-ARE ribonucleoprotein complex. Most notably, association of unphosphorylated p40AUF1 induces a condensed RNA conformation upon ARE substrates. By contrast, phosphorylation of p40AUF1 at Ser83 and Ser87 inhibits this RNA structural transition. These data indicate that selective AUF1 phosphorylation may regulate ARE-directed mRNA turnover by remodeling local RNA structures, thus potentially altering the presentation of RNA and/or protein determinants involved in subsequent trans-factor recruitment. The steady-state level of any mRNA population is a collective function of its synthetic and degradation rates. Eukaryotic mRNAs decay across a broad kinetic spectrum, discriminated largely by the presence of specific cis-acting stability determinants within each transcript (reviewed in Refs. 1Ross J. Microbiol. Rev. 1995; 59: 423-450Crossref PubMed Google Scholar and 2Guhaniyogi J. Brewer G. Gene (Amst.). 2001; 265: 11-23Crossref PubMed Scopus (559) Google Scholar). For many labile mammalian transcripts, rapid cytoplasmic mRNA turnover is directed by A + U-rich elements (AREs) 1The abbreviations used are: ARE, A + U-rich element; Cy3, cyanine 3; Fl, fluorescein; FRET, fluorescence resonance energy transfer; GMSA, gel mobility shift assay; GSK3β, glycogen synthase kinase 3β; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; PKA, protein kinase A; RNP, ribonucleoprotein; TNFα, tumor necrosis factor α. contained within their 3′-untranslated regions (reviewed in Ref. 3Chen C.-Y.A. Shyu A.-B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1703) Google Scholar). AREs constitute a diverse population of mRNA sequences that may interact with a wide variety of cellular protein factors. Binding of some factors, including AUF1 and tristetraprolin, is associated with acceleration of mRNA decay (4Lapucci A. Donnini M. Papucci L. Witort E. Tempestini A. Bevilacqua A. Niolin A. Brewer G. Schiavone N. Capaccioli S. J. Biol. Chem. 2002; 277: 16139-16146Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 5Loflin P. Chen C.-Y.A. Shyu A.-B. Genes Dev. 1999; 13: 1884-1897Crossref PubMed Scopus (264) Google Scholar, 6Sirenko 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, 7Lai 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 (643) Google Scholar), whereas factors like HuR protect ARE-containing transcripts from degradation (8Peng S.S.Y. Chen C.-Y.A. Xu N. Shyu A.-B. EMBO J. 1998; 17: 3461-3470Crossref PubMed Scopus (662) Google Scholar, 9Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (754) Google Scholar). Furthermore, many additional ARE-binding factors exist for which specific roles in mRNA metabolism have not been defined (reviewed in Ref. 10Wilson G.M. Brewer G. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 257-291Crossref PubMed Scopus (123) Google Scholar). These myriad options for trans-factor occupancy on ARE targets present opportunities for multifactoral regulation of mRNA decay kinetics through these elements, including discrimination of mRNA targets based on ARE sequence composition, and differential trans-factor activation or inhibition by specific signal transduction pathways. The latter possibility is further supported by recent findings that some ARE-binding proteins may be post-translationally modified in response to diverse stimuli. For example, phosphorylation of tristetraprolin by components of the p38 mitogen-activated protein kinase pathway may alter its ARE binding activity (11Carballo E. Cao H. Lai W.S. Kennington E.A. Campbell D. Blackshear P.J. J. Biol. Chem. 2001; 276: 42580-42587Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 12Mahtani K.R. Brook M. Dean J.L.E. Sully G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2001; 21: 6461-6469Crossref PubMed Scopus (398) Google Scholar) and/or subcellular distribution (13Johnson B.A. Stehn J.R. Yaffe M.B. Blackwell T.K. J. Biol. Chem. 2002; 277: 18029-18036Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). For HuR, phosphorylation events involving the AMP-dependent protein kinase are implicated in nuclear retention of the protein (14Wang W. Fan J. Yang X. Fürer-Galban S. Lopez de Silanes I. Von Kobbe C. Guo J. Georas S.N. Foufelle F. Hardie D.G. Carling D. Gorospe M. Mol. Cell. Biol. 2002; 22: 3425-3436Crossref PubMed Scopus (193) Google Scholar), whereas lipopolysaccharide treatment induces methylation of HuR (15Li H. Park S. Kilburn B. Jelinek M.A. Henschen-Edman A. Aswad D.W. Stallcup M.R. Laird-Offringa I.A. J. Biol. Chem. 2002; 277: 44623-44630Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Finally, AUF1 may be modified by both ubiquitination (16Laroia G. Schneider R.J. Nucleic Acids Res. 2002; 30: 3052-3058Crossref PubMed Scopus (54) Google Scholar) and phosphorylation (17Zhang 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 (504) Google Scholar, 39Wilson G.M. Lu J. Sutphen K. Sun Y. Huynh Y. Brewer G. J. Biol. Chem. 2003; 278: 33029-33038Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, whereas some potential regulatory events have been linked to these modifications, it remains unclear how they modulate the functions of ARE-binding proteins at the biochemical level, which conceivably may include alterations in the ability of each protein to interact with RNA substrates or other cellular components. In the monocytic leukemia cell line, THP-1, interleukin-1β and tumor necrosis factor α (TNFα) mRNAs are stabilized following treatment with phorbol esters. Stabilization of these mRNAs was accompanied by changes in the activity of cytoplasmic ARE-binding complexes containing AUF1 and loss of phosphate from Ser83 and Ser87 of the predominant polysome-associated AUF1 isoform, p40AUF1 (39Wilson G.M. Lu J. Sutphen K. Sun Y. Huynh Y. Brewer G. J. Biol. Chem. 2003; 278: 33029-33038Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Current evidence indicates that AUF1 selectively binds and oligomerizes on ARE substrates (18Wilson 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, 19Wilson G.M. Sutphen K. Chuang K. Brewer G. J. Biol. Chem. 2001; 276: 8695-8704Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and has the potential to remodel local RNA structure (20Wilson 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). In addition, cytoplasmic AUF1 is present in a multisubunit complex (17Zhang 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 (504) Google Scholar) containing other factors involved in the regulation of mRNA decay and translation, including the translation initiation factor eIF4G, poly(A)-binding protein, the heat shock proteins Hsp70 and Hsc70 (21Laroia G. Cuesta R. Brewer G. Schneider R.J. Science. 1999; 284: 499-502Crossref PubMed Scopus (351) Google Scholar), and lactate dehydrogenase (22Pioli P.A. Hamilton B.J. Connolly J.E. Brewer G. Rigby W.F.C. J. Biol. Chem. 2002; 277: 35738-35745Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Together, these findings suggest that AUF1 oligomers may function by recruiting additional trans-acting factors to ARE-containing mRNAs. It follows, therefore, that changes in AUF1 phosphorylation may influence mRNA decay kinetics by a number of mechanisms, including alterations in ARE-binding affinity, oligomerization potential, ribonucleoprotein (RNP) conformation, and interaction with other cytoplasmic proteins. Unlike the examples involving tristetraprolin and HuR (13Johnson B.A. Stehn J.R. Yaffe M.B. Blackwell T.K. J. Biol. Chem. 2002; 277: 18029-18036Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 14Wang W. Fan J. Yang X. Fürer-Galban S. Lopez de Silanes I. Von Kobbe C. Guo J. Georas S.N. Foufelle F. Hardie D.G. Carling D. Gorospe M. Mol. Cell. Biol. 2002; 22: 3425-3436Crossref PubMed Scopus (193) Google Scholar), phosphorylation of p40AUF1 does not appear to influence its nucleocytoplasmic distribution, since (i) both phosphorylated and nonphosphorylated p40AUF1 proteins were recovered from THP-1 cell polysomes, and (ii) no significant changes in the levels of nuclear or cytoplasmic AUF1 proteins were detected following phorbol ester treatment of these cells (39Wilson G.M. Lu J. Sutphen K. Sun Y. Huynh Y. Brewer G. J. Biol. Chem. 2003; 278: 33029-33038Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In this study, we have biochemically examined the influence of p40AUF1 phosphorylation at Ser83 and Ser87 on its interaction with the ARE from TNFα mRNA. First, we show that recombinant His6-p40AUF1 may be specifically, quantitatively, and independently phosphorylated in vitro at Ser83 by glycogen synthase kinase 3β (GSK3β) and at Ser87 by protein kinase A (PKA). Second, we employed gel mobility shift assays (GMSAs) and measurements of fluorescence anisotropy to show that phosphorylated His6-p40AUF1 retains specific ARE-binding activity but that the affinity of the p40AUF1/ARE interaction and the flexibility of the protein-bound RNA substrate varies among the different phosphorylated forms of the protein. Finally, using fluorescence resonance energy transfer (FRET), we demonstrate that the global conformation of the ARE substrate is compacted following binding of unphosphorylated, Ser83-phosphorylated, or Ser87-phosphorylated His6-p40AUF1. By contrast, His6-p40AUF1 phosphorylated at both Ser83 and Ser87 does not significantly condense the conformation of associated ARE substrates. Together, these data constitute the first biochemical evidence that post-translational modification of p40AUF1 regulates its ARE-targeting role and provide a potential mechanism for inhibition of ARE-directed mRNA turnover concomitant with loss of phosphate from polysome-associated p40AUF1. RNA Oligonucleotides—RNA oligonucleotide substrates (see Fig. 3A) encoding the ARE from TNFα mRNA (TNFα ARE) or a portion of the rabbit β-globin mRNA coding sequence (Rβ) were synthesized by Dharmacon Research (Lafayette, CO). Fl-TNFα ARE contains a 5′-fluorescein (Fl) tag, whereas TNFα ARE-Fl contains a 3′-linked Fl group. The RNA substrate Cy-TNFα ARE-Fl contains 5′-cyanine 3 (Cy3) and 3′-Fl moieties. All RNA substrates were quantified spectrophotometrically as described (20Wilson 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). Where indicated, the TNFα ARE and Rβ RNA substrates were 32P-labeled to specific activities of 3–5 × 103 cpm/fmol using [γ-32P]ATP (PerkinElmer Life Sciences) and T4 polynucleotide kinase (Promega, Madison, WI). Preparation of Recombinant His6-p40AUF1 —Plasmid pBAD/HisB-p40AUF1 was constructed by inserting the complete coding sequence of human p40AUF1 cDNA into the polylinker of pBAD/HisB (Invitrogen) using standard subcloning techniques (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The identity of the insert and continuity of the open reading frame were verified by automated DNA sequencing. Recombinant p40AUF1 was prepared as an N-terminal His6-fusion protein by arabinose induction of E. coli TOP10 cells transformed with pBAD/HisB-p40AUF1. His -p40AUF1 was then purified by Ni2+-affinity chromatography and quantified by Coomassie Blue-stained SDS-PAGE against a titration of bovine serum albumin as described previously (19Wilson G.M. Sutphen K. Chuang K. Brewer G. J. Biol. Chem. 2001; 276: 8695-8704Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Wilson G.M. Brewer G. Methods. 1999; 17: 74-83Crossref PubMed Scopus (56) Google Scholar). Where indicated, the N-terminal fragment of His6-p40AUF1 containing the His6 tag was excised using the Enterokinase Cleavage Capture Kit (Novagen, Madison, WI). In Vitro Phosphorylation of His6-p40AUF1— 32P label transfer assays consisted of His6-p40AUF1 (50 pmol) in 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 1 mm EGTA, 2 mm dithiothreitol, 0.1% Nonidet P-40, and 200 μm ATP (50-μl final volume). [γ-32P]ATP was added to yield a final specific activity of 4000 cpm/pmol ATP. After 5 min at 30 °C, reactions were initiated by adding 2500 units of either the recombinant catalytic subunit of murine PKA (Calbiochem) or recombinant rabbit GSK3β (New England Biolabs, Beverly, MA) and then returned to 30 °C. At selected time points, aliquots were removed and spotted onto P81 filters (Whatman, Clifton, NJ), which were then immediately washed three times for 2 min each in freshly made 75 mm H3PO4 and air-dried. Filter-bound 32P was quantified by liquid scintillation counting. Non-specific retention of 32P was determined by performing replicate assays in the absence of kinase. Substrate-linked 32P was then calculated as the difference in filter-retained 32P between reactions containing and lacking kinase. Additional reactions lacking the His6-p40AUF1 substrate indicated no significant retention of 32P via phosphorylation of the kinases themselves (data not shown). Phosphorylation reactions requiring both PKA and GSK3β were performed in tandem, with the PKA reaction allowed to proceed for 60 min prior to the addition of GSK3β and incubation for a further 60 min. For samples analyzed by mass spectrometry, [γ-32P]ATP was omitted from reactions, and products were desalted and concentrated using ZipTipC4 columns (Millipore, Bedford, MA) according to the manufacturer's instructions. Large scale preparation of phosphorylated His6-p40AUF1 for biochemical analyses was performed similarly, but using 5 nmol of His6-p40AUF1in a total volume of 300 μl. PKA and/or GSK3β (50,000 units) were added as necessary, with samples incubated as above. Following phosphorylation reactions, samples were diluted 10-fold in Ni2+-affinity binding buffer (50 mm sodium phosphate (pH 8.0), 500 mm NaCl, 20 mm imidazole, 5% polyethylene glycol 6000). Mock- or kinase-modified His6-p40AUF1 was then purified by Ni2+-affinity chromatography and quantified by Coomassie Blue-stained SDS-PAGE as described (19Wilson G.M. Sutphen K. Chuang K. Brewer G. J. Biol. Chem. 2001; 276: 8695-8704Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Wilson G.M. Brewer G. Methods. 1999; 17: 74-83Crossref PubMed Scopus (56) Google Scholar), except that the loaded Ni2+ column was given an additional wash with 6 column volumes of Triton washing buffer (50 mm sodium phosphate (pH 8.0), 500 mm NaCl, 20 mm imidazole, 1% Triton X-100) prior to His6-p40AUF1 elution to ensure complete removal of the kinases. Following purification, a sample from each preparation was analyzed by MALDI-TOF to verify quantitative phosphate transfer. Mass Spectrometry—In-gel tryptic digestion, immobilized metal ion affinity chromatography, alkaline phosphatase reactions, carboxypeptidase Y digests, and detection of proteins and peptide fragments by MALDI-TOF mass spectrometry were all performed exactly as described previously (39Wilson G.M. Lu J. Sutphen K. Sun Y. Huynh Y. Brewer G. J. Biol. Chem. 2003; 278: 33029-33038Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The apparent molecular weight of His6-p40AUF1 was calculated from the predicted amino acid sequence (GenBank™ accession number NM_002138 and pBAD/His vector system literature) (Invitrogen) 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). RNA-Protein Binding Assays—GMSAs using unmodified or phosphorylated His6-p40AUF1 and 32P-labeled RNA oligonucleotide substrates were performed essentially as described (18Wilson 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, 24Wilson G.M. Brewer G. Methods. 1999; 17: 74-83Crossref PubMed Scopus (56) Google Scholar), except that magnesium ions were not included in binding buffers, and heparin (5 μg/μl) and yeast tRNA (0.2 μg/μl) were included to compete for nonspecific RNA binding activities. Also, flanking regions of RNA substrates were not excised by nucleases prior to gel fractionation. Fluorescence anisotropy was employed for all quantitative assessments of RNA-protein binding equilibria. Binding reactions containing the fluorescent RNA substrate Fl-TNFα ARE and varying concentrations of unmodified or phosphorylated His6-p40AUF1 were assembled in a final volume of 100 μl containing 10 mm Tris-HCl (pH 8.0), 50 mm KCl, 2 mm dithiothreitol, 0.5 mm EDTA, 0.1 μg/μl acetylated bovine serum albumin, and 1 μg/μl heparin. Following incubation at 25 °C for 1 min, anisotropy was measured using a Beacon 2000 variable temperature fluorescence polarization system (Panvera, Madison, WI) equipped with fluorescein excitation (490-nm) and emission (535-nm) filters. Preliminary on-rate experiments verified that anisotropic equilibrium was attained within 10–20 s at this temperature for all binding equilibria described herein (data not shown). For equilibrium binding experiments, the polarimeter was operated in static mode, with each sample read as blank prior to the addition of fluorescent RNA substrates to correct for intrinsic fluorescence from other reaction components. Each anisotropy data point represents the mean of 10 measurements. Replicate experiments using enterokinase-digested His6-p40AUF1 (see Fig. 1B) yielded results similar to the undigested protein (data not shown), indicating that the His6 and flanking N-terminal sequences do not significantly influence the ARE binding activity of His6-p40AUF1. The total measured anisotropy (At) of a mixture of fluorescent species exhibiting similar fluorescence quantum yields may be interpreted based on the intrinsic anisotropy and fractional concentration of each fluorescing species, given by Ai and fi , respectively, using Equation 1 (25Jameson D.M. Sawyer W.H. Methods Enzymol. 1995; 246: 283-300Crossref PubMed Scopus (156) Google Scholar, 26Weber G. Biochem. J. 1952; 51: 145-155Crossref PubMed Scopus (510) Google Scholar, 27Lakowicz J.R. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic/Plenum, New York1999: 291-319Crossref Google Scholar). At=∑iAifi(Eq. 1) For all equilibria described in this work, total fluorescence intensity did not vary significantly as a result of protein binding (data not shown), thus validating interpretation of anisotropy by Equation 1. A sequential dimer binding mechanism yielding a tetrameric protein-RNA complex is defined by two equilibrium constants, K 1 and K 2, and presents three potential fluorescent species, R, P2R, and P4R (see Fig. 4A). Under conditions of limiting RNA (i.e. [protein]free ≈ [protein]total), measured anisotropy is thus related to the concentration of protein dimer (P2) by Equation 2 (18Wilson 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). At=AR+AP2RK1[P2]+AP4RK1K2[P2]21+K1[P2]+K1K2[P2]2(Eq. 2) Anisotropy data sets were also analyzed using a binary binding model described by Equation 3, where a single protein dimer interacting with an RNA substrate yields a single equilibrium constant (K). At=AR+AP2RK[P2]1+K[P2](Eq. 3) Application of binding algorithms to At versus [P2] data sets was performed by nonlinear least squares regression using PRISM version 2.0 (GraphPad, San Diego, CA). The appropriateness of all mathematical models was monitored by the coefficient of determination (R 2) and analysis of residual plot nonrandomness to detect any bias for data subsets (PRISM version 2.0). Where indicated, pairwise comparisons of sum-of-squares deviations between mathematical models were performed using the F test, whereas pairwise comparisons of binding or anisotropy parameters between experiments used the unpaired t test (PRISM version 2.0). In both cases, differences exhibiting p < 0.05 were considered significant. Protein off-rate experiments to measure the dynamics of RNA-protein complexes were performed as described previously (18Wilson 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, 28Wilson G.M. Sutphen K. Bolikal S. Chuang K. Brewer G. J. Biol. Chem. 2001; 276: 44450-44456Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Measurement of RNA Folding by FRET—Interpretation of RNA conformation in the P2R and P4R complexes by FRET required comparison of the protein dependence of FRET efficiency (E FRET) to the fractional concentration of each fluorescent species. Since higher concentrations of RNA (2 nm) were required for FRET experiments, the approximation that [P2]free ≈ [P2]total, used to derive Equation 2, becomes invalid. Accordingly, the fractional concentrations of free RNA, P2R, and P4R were determined by solution of an equation system relying exclusively on K 1, K 2, [RNA]total, and [P2]total. Details of this system are described in detail elsewhere (20Wilson 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). Solution sets for selected values of [P2]total with constant [RNA]total were generated using Mathematica version 4.1 (Wolfram Research, Champaign, IL). Protein-dependent changes in the scalar distance between the 5′ and 3′ termini of the Cy-TNFα ARE-Fl RNA substrate were evaluated by measuring E FRET between the fluorescent moieties as a function of protein concentration. Binding reactions were assembled as described for fluorescence anisotropy analyses (above), except that RNA substrates were included at higher concentration (2 nm) and were labeled with either (i) the FRET donor-acceptor pair (Cy-TNFα ARE-Fl) or (ii) the FRET donor alone (TNFα ARE-Fl). E FRET was calculated from the decrease in fluorescence emission of the FRET donor (λex = 490 nm, λem = 518 nm, 5-nm bandwidth) in the presence of the acceptor using Equation 4 (29Clegg R.M. Methods Enzymol. 1992; 211: 353-388Crossref PubMed Scopus (668) Google Scholar, 30Wu P. Brand L. Anal. Biochem. 1994; 218: 1-13Crossref PubMed Scopus (1176) Google Scholar). EFRET=1-(FDA/FD)(Eq. 4) F DA and F D represent the blank-corrected fluorescence of paired binding reactions containing the Cy-TNFα ARE-Fl and TNFα ARE-Fl RNA substrates, respectively. All fluorescence readings were taken using a Cary Eclipse fluorescence spectrophotomer (Varian Instruments, Walnut Creek, CA), using the Peltier multicell holder and temperature controller accessories. Where indicated, absolute scalar distances between fluorophores were calculated using Equation 5, where r represents the scalar interfluorophore distance and R 0 is the Förster distance, at which the fluorescent donor-acceptor pair yields 50% FRET efficiency. R 0 for the Cy3 and Fl linked to single-stranded DNA has been calculated as 55.7 Å (31Norman D.G. Grainger R.J. Uhrín D. Lilley D.M.J. Biochemistry. 2000; 39: 6317-6324Crossref PubMed Scopus (215) Google Scholar). EFRET=R06/(R06+r6)(Eq. 5) Recombinant p40AUF1 Is Quantitatively Phosphorylated at Ser87 by Protein Kinase A and at Ser83 by Glycogen Synthase Kinase 3β in Vitro—In unstimulated THP-1 cells, polysome-associated p40AUF1 is phosphorylated on Ser83 and Ser87 (39Wilson G.M. Lu J. Sutphen K. Sun Y. Huynh Y. Brewer G. J. Biol. Chem. 2003; 278: 33029-33038Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These modifications are lost following phorbol ester treatment of this cell line, concomitant with stabilization of some ARE-containing mRNAs and alterations in cytoplasmic ARE-binding activities containing AUF1. Three observations suggest that the effects of AUF1 phosphorylation on cytoplasmic mRNA turnover are likely to be manifested through p40AUF1. First, Ser83 and Ser87 are encoded by exon 2 of the AUF1 gene (32Wagner B.J. DeMaria C.T. Sun Y. Wilson G.M. Brewer G. Genomics. 1998; 48: 195-202Crossref PubMed Scopus (241) Google Scholar) and, due to alternative pre-mRNA splicing, are specific for the p45 and p40 isoforms of AUF1. Second, p45AUF1 is almost exclusively nuclear in THP-1 cells, whereas p40AUF1 is detected predominantly in the cytoplasm (39Wilson G.M. Lu J. Sutphen K. Sun Y. Huynh Y. Brewer G. J. Biol. Chem. 2003; 278: 33029-33038Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Finally, p40AUF1 is the major polysome-associated AUF1 isoform in this cell line. Accordingly, the next objective was to determine how phosphorylation of p40AUF1 at Ser83 and Ser87 might alter its biological function. Given that these residues are located immediately upstream of the p40AUF1 RNA-binding domain (Fig. 1A), alterations in ARE-binding activity were postulated to be a possible consequence of these phosphorylation events. In order to test this hypothesis, it was first necessary to generate purified, selectively phosphorylated forms of p40AUF1. Using the PhosphoBase version 2.0 data base (33Kreegipuu A. Blom N. Brunak S. Nucleic Acids Res. 1999; 27: 237-239Crossref PubMed Scopus (247) Google Scholar), PKA was predicted to specifically phosphorylate p40AUF1 at Ser87 (Fig. 1A). Furthermore, Ser83 presented a consensus phosphorylation site for GSK3, provided that Ser87 was previously phosphorylated. Recombinant, N-terminal His6-tagged p40AUF1 was expressed in Escherichia coli TOP10 cells and purified by Ni2+-affinity chromatography to >95% purity as described under "Experimental Procedures" (Fig. 1B). In label transfer assays with [γ-32P]ATP, both GSK3β and PKA readily and independently phosphorylated the recombinant His6-p40AUF1 substrate, approaching a stoichiometry of 1 mol of phosphate/mol of protein in each case (Fig. 1C). For both phosphorylation reactions, SDS-PAGE analysis indicated a single principal 32P-labeled product (Fig. 1D). To further confirm His6-p40AUF1 as the phosphorylated substrate, kinase reactions were repeated using unlabeled ATP and then analyzed by MALDI-TOF. A mock phosphorylation reaction (i.e. containing no enzyme) yielded a major product with an M r of 37,803 (Fig. 1E), close to 37,793, the calculated M r of unmodified His6-p40AUF1. By contrast, products identified in PKA- or GSK3β-programmed reactions gave M r values indicating increases of 80 and 79 Da, respectively, consistent with the addition of a single phosphate to His6-p40AUF1 by each kinase. Since unphosphorylated His6-p40AUF1 was not detected in either experiment, phosphorylation by each kinase was deemed to be quantitative. Finally, a reaction programmed sequentially with PKA and then GSK3β yielded a principal product with M r of 37,963. The 160-Da difference relative to the unmodified protein indicates the addition of two phosphates to His -p40AUF1 in these reactions. Having demonstrated that His6-p40AUF1 may be quantitatively phosphorylated by PKA and GSK3β, it was subsequently necessary to confirm the sites of modification on the substrate protein. To this end, PKA-, GSK3β-, and PKA + GSK3β-phosphorylated His -p40AUF1 were digested with trypsin. Released phosphopeptide fragments were then purified by immobilized metal ion affinity chromatography across a Ga3+-charged matrix and analyzed by MALDI-TOF (Fig. 2A). PKA-phosphorylated His -p40AUF1 yielded a single Ga3+-binding fragment (m/z 1051), consistent with a single phosphate linked to peptide HSEAATAQR, spanning residues 86–94 of p40AUF1. Phosphorylation with GSK3β yielded a distinct fragment (m/z 1539), characteristic of monophosphorylate
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