Annexin A2 Is a Novel RNA-binding Protein
2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês
10.1074/jbc.m311951200
ISSN1083-351X
AutoresNolan R. Filipenko, Travis J. MacLeod, Chang-Soon Yoon, David M. Waisman,
Tópico(s)RNA Research and Splicing
ResumoAnnexin A2 (ANXA2) is a Ca2+-binding protein that is up-regulated in virally transformed cell lines and in human tumors. Here, we show that ANXA2 binds directly to both ribonucleotide homopolymers and human c-myc RNA. ANXA2 was shown to bind specifically to poly(G) with high affinity (Kd = 60 nm) and not to poly(A), poly(C), or poly(U). The binding of ANXA2 to poly(G) required Ca2+ (A50% = 10 μm). The presence of RNA in the immunoprecipitates of ANXA2 isolated from HeLa cells established that ANXA2 formed a ribonucleoprotein complex in vivo. Sucrose gradient analysis showed that ANXA2 associates with ribonucleoprotein complexes and not with polyribosomes. Reverse transcriptase-PCR identified c-myc mRNA as a component of the ribonucleoprotein complex formed by ANXA2 in vivo, and binding studies confirmed a direct interaction between ANXA2 and c-myc mRNA. Transfection of LNCaP cells with the ANXA2 gene resulted in the up-regulation of c-Myc protein. These findings identify ANXA2 as a Ca2+-dependent RNA-binding protein that interacts with the mRNA of the nuclear oncogene, c-myc. Annexin A2 (ANXA2) is a Ca2+-binding protein that is up-regulated in virally transformed cell lines and in human tumors. Here, we show that ANXA2 binds directly to both ribonucleotide homopolymers and human c-myc RNA. ANXA2 was shown to bind specifically to poly(G) with high affinity (Kd = 60 nm) and not to poly(A), poly(C), or poly(U). The binding of ANXA2 to poly(G) required Ca2+ (A50% = 10 μm). The presence of RNA in the immunoprecipitates of ANXA2 isolated from HeLa cells established that ANXA2 formed a ribonucleoprotein complex in vivo. Sucrose gradient analysis showed that ANXA2 associates with ribonucleoprotein complexes and not with polyribosomes. Reverse transcriptase-PCR identified c-myc mRNA as a component of the ribonucleoprotein complex formed by ANXA2 in vivo, and binding studies confirmed a direct interaction between ANXA2 and c-myc mRNA. Transfection of LNCaP cells with the ANXA2 gene resulted in the up-regulation of c-Myc protein. These findings identify ANXA2 as a Ca2+-dependent RNA-binding protein that interacts with the mRNA of the nuclear oncogene, c-myc. The annexins are a family of more than 160 unique annexin proteins that are present in more than 65 different species ranging from fungi and protists to plants and higher vertebrates (1Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1628) Google Scholar). ANXA2 1The abbreviations used are: ANXA2, annexin A2; ATD, aminoterminal domain; CCD, carboxyl core domain; RNP, ribonucleoprotein; mRNP, messenger ribonucleoprotein; RT, reverse transcriptase; pCp, cytidine 3′,5′-bis(phosphate); TBS, Tris-buffered saline.1The abbreviations used are: ANXA2, annexin A2; ATD, aminoterminal domain; CCD, carboxyl core domain; RNP, ribonucleoprotein; mRNP, messenger ribonucleoprotein; RT, reverse transcriptase; pCp, cytidine 3′,5′-bis(phosphate); TBS, Tris-buffered saline. consists of an amino-terminal domain (ATD), which comprises the first 30 amino acid residues of the protein and the carboxyl core domain (CCD) composed of the remaining residues. The CCD of ANXA2 contains sites for binding Ca2+, phospholipid, F-actin and heparin (2Filipenko N.R. Waisman D.M. Annexins: Biological Importance and Annexin-related Pathologies. Landes Bioscience, Georgetown, TX2003: 127-156Crossref Google Scholar, 3Waisman D.M. Mol. Cell Biochem. 1995; 149: 301-322Crossref PubMed Scopus (262) Google Scholar). The ATD contains regulatory phosphorylation sites for both protein kinase C (Ser-25) and Src (Tyr-23). In fact, the first substrate discovered for Src was identified as a 36-kDa protein (ANXA2) that was phosphorylated upon transformation of cells with the Rous sarcoma virus (4Radke K. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5212-5216Crossref PubMed Scopus (153) Google Scholar). Subsequently, it was demonstrated that only about 10% of total cellular ANXA2 was phosphorylated in cells transformed by RSV (4Radke K. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5212-5216Crossref PubMed Scopus (153) Google Scholar, 5Erikson E. Erikson R.L. Cell. 1980; 21: 829-836Abstract Full Text PDF PubMed Scopus (215) Google Scholar). ANXA2 exists as three major species: a monomer, a heterodimer, or a heterotetramer (AIIt) (3Waisman D.M. Mol. Cell Biochem. 1995; 149: 301-322Crossref PubMed Scopus (262) Google Scholar). The heterodimer is composed of a single subunit of ANXA2 bound to a subunit of 3-phosphoglycerate kinase (6Jindal H.K. Chaney W.G. Anderson C.W. Davis R.G. Vishwanatha J.K. J. Biol. Chem. 1991; 266: 5169-5176Abstract Full Text PDF PubMed Google Scholar). The heterotetramer, on the other hand, comprises two subunits of ANXA2 linked together by a dimer of S100A10 (also referred to as p11), a member of the S100 family of Ca2+-binding proteins (7Erikson E. Tomasiewicz H.G. Erikson R.L. Mol. Cell. Biol. 1984; 4: 77-85Crossref PubMed Scopus (61) Google Scholar, 8Gerke V. Weber K. EMBO J. 1984; 3: 227-233Crossref PubMed Scopus (389) Google Scholar, 9Glenney Jr., J.R. Tack B.F. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7884-7888Crossref PubMed Scopus (211) Google Scholar, 10Gerke V. Weber K. EMBO J. 1985; 4: 2917-2920Crossref PubMed Scopus (191) Google Scholar). The relative amounts of heterotetrameric versus monomeric ANXA2 are variable depending on the cell or tissue examined and range from 100% heterotetrameric ANXA2 in intestinal epithelium to about 50% monomeric ANXA2 monomer in cultured fibroblasts (8Gerke V. Weber K. EMBO J. 1984; 3: 227-233Crossref PubMed Scopus (389) Google Scholar, 11Zokas L. Glenney Jr., J.R. J. Cell Biol. 1987; 105: 2111-2121Crossref PubMed Scopus (136) Google Scholar). ANXA2 consists of two functional domains. The aminoterminal regulatory domain (ATD) contains the amino-terminal 30 amino acid residues and incorporates two phosphorylation sites at Tyr-23 and Ser-25. In addition to the phosphorylation sites, the ATD also contains the site for interaction with the S100A10 dimer. The remaining CCD, encompassing residues 31–338, comprises the binding sites for Ca2+, phospholipid, heparin, and F-actin (reviewed by Refs. 1Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1628) Google Scholar, 2Filipenko N.R. Waisman D.M. Annexins: Biological Importance and Annexin-related Pathologies. Landes Bioscience, Georgetown, TX2003: 127-156Crossref Google Scholar, 3Waisman D.M. Mol. Cell Biochem. 1995; 149: 301-322Crossref PubMed Scopus (262) Google Scholar). The crystal structure of an amino-terminally truncated form of ANXA2 has been reported (12Burger A. Berendes R. Liemann S. Benz J. Hofmann A. Göttig P. Huber R. Gerke V. Thiel C. Römisch J. Weber K. J. Mol. Biol. 1996; 257: 839-847Crossref PubMed Scopus (110) Google Scholar). The protein is planar and curved with opposing convex and concave sides. The convex side faces the biological membrane and contains the Ca2+- and phospholipid-binding sites. The concave side faces the cytosol and contains both the amino and carboxyl termini. A multitude of intracellular functions have been suggested for ANXA2, including roles as a mediator of Ca2+-regulated exocytosis (13Sarafian T. Pradel L.A. Henry J.P. Aunis D. Bader M.F. J. Cell Biol. 1991; 114: 1135-1147Crossref PubMed Scopus (149) Google Scholar, 14Ali S.M. Burgoyne R.D. Cell Signal. 1990; 2: 265-276Crossref PubMed Scopus (33) Google Scholar, 15Ali S.M. Geisow M.J. Burgoyne R.D. Nature. 1989; 340: 313-315Crossref PubMed Scopus (235) Google Scholar, 16Burgoyne R.D. Nature. 1988; 331: 20Crossref PubMed Scopus (56) Google Scholar) or endocytosis (17Zeuschner D. Stoorvogel W. Gerke V. Eur. J. Cell Biol. 2001; 80: 499-507Crossref PubMed Scopus (39) Google Scholar, 18Emans N. Gorvel J.P. Walter C. Gerke V. Kellner R. Griffiths G. Gruenberg J. J. Cell Biol. 1993; 120: 1357-1369Crossref PubMed Scopus (229) Google Scholar, 19Harder T. Gerke V. J. Cell Biol. 1993; 123: 1119-1132Crossref PubMed Scopus (149) Google Scholar) as well as a role in modulating sarcolemmal phospholipid raft organization during smooth muscle cell contraction (20Babiychuk E.B. Monastyrskaya K. Burkhard F.C. Wray S. Draeger A. FASEB J. 2002; 16: 1177-1184Crossref PubMed Scopus (65) Google Scholar, 21Babiychuk E.B. Draeger A. J. Cell Biol. 2000; 150: 1113-1124Crossref PubMed Scopus (227) Google Scholar) and regulation of ion channels (22Okuse K. Malik-Hall M. Baker M.D. Poon W.Y. Kong H. Chao M.V. Wood J.N. Nature. 2002; 417: 653-656Crossref PubMed Scopus (233) Google Scholar). Since an ANXA2 knockout mouse has not been developed, it is not clear whether these reported in vitro functions represent actual physiological functions of the protein. Nevertheless, knowledge of these putative functions of ANXA2 has not provided clues as to the role that ANXA2 may play in vivo. The expression of ANXA2 is induced in various transformed cells, including v-src-, v-H-ras-, v-mos-, or SV40-transformed cells (23Ozaki T. Sakiyama S. Oncogene. 1993; 8: 1707-1710PubMed Google Scholar). Furthermore, the ANXA2 gene is growth-regulated, and its expression is stimulated by growth factors such as insulin, fibroblast growth factor, and epidermal growth factor (24Keutzer J.C. Hirschhorn R.R. Exp. Cell Res. 1990; 188: 153-159Crossref PubMed Scopus (48) Google Scholar). Up-regulated ANXA2 has also been reported in human hepatocellular carcinoma (25Frohlich M. Motte P. Galvin K. Takahashi H. Wands J. Ozturk M. Mol. Cell. Biol. 1990; 10: 3216-3223Crossref PubMed Scopus (72) Google Scholar), pancreatic adenocarcinoma (26Vishwanatha J.K. Chiang Y. Kumble K.D. Hollingsworth M.A. Pour P.M. Carcinogenesis. 1993; 14: 2575-2579Crossref PubMed Scopus (124) Google Scholar), high grade glioma (27Reeves S.A. Chavez Kappel C. Davis R. Rosenblum M. Israel M.A. Cancer Res. 1992; 52: 6871-6876PubMed Google Scholar), gastric carcinoma (28Emoto K. Sawada H. Yamada Y. Fujimoto H. Takahama Y. Ueno M. Takayama T. Uchida H. Kamada K. Naito A. Hirao S. Nakajima Y. Anticancer Res. 2001; 21: 1339-1345PubMed Google Scholar), and acute promyelocytic leukemia (29Menell J.S. Cesarman G.M. Jacovina A.T. McLaughlin M.A. Lev E.A. Hajjar K.A. N. Engl. J. Med. 1999; 340: 994-1004Crossref PubMed Scopus (319) Google Scholar). Since overexpression of the ANXA2 gene is commonly observed in both virally transformed cell lines and human tumors, it has been suspected that this up-regulated level of ANXA2 might link ANXA2 to a key step in cellular transformation. However, without a detailed knowledge of its intracellular role, it is difficult to envision the role that up-regulation of ANXA2 expression would have on cellular transformation. Typically, ANXA2 has been reported to display two distinct intracellular distributions, with the majority of the protein localized to the cytoplasmic face of the plasma membrane and a secondary diffuse cytoplasmic distribution (30Courtneidge S. Ralston R. Alitalo K. Bishop J.M. Mol. Cell. Biol. 1983; 3: 340-350Crossref PubMed Scopus (53) Google Scholar). The first indication that ANXA2 might interact with RNA was a report that utilized subcellular fractionation to show that a significant portion of ANXA2 was associated with ribonucleoprotein particles in cytoplasmic extracts of both normal and transformed cells. It was also shown that ANXA2 immunoprecipitated from UV-irradiated cultured cells associated with RNA and formed a RNA-ANXA2 cross-linked ribonucleoprotein complex. These authors also showed by biochemical fractionation experiments that about 10–15% of the total cellular ANXA2 was associated with the nucleus (31Arrigo A.P. Darlix J.L. Spahr P.F. EMBO J. 1983; 2: 309-315Crossref PubMed Scopus (19) Google Scholar). Ensuing studies showed that ANXA2 could bind to deoxyribonucleic acid structures such as Z-DNA (32Krishna P. Kennedy B.P. Waisman D.M. van de S e J.H. McGhee J.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1292-1295Crossref PubMed Scopus (25) Google Scholar) or Alu subsequences (33Boyko V. Mudrak O. Svetlova M. Negishi Y. Ariga H. Tomilin N. FEBS Lett. 1994; 345: 139-142Crossref PubMed Scopus (30) Google Scholar). Other studies have identified nuclear ANXA2 in immunoblots of nuclei and as part of a primer recognition complex that stimulates DNA polymerase α activity (6Jindal H.K. Chaney W.G. Anderson C.W. Davis R.G. Vishwanatha J.K. J. Biol. Chem. 1991; 266: 5169-5176Abstract Full Text PDF PubMed Google Scholar, 34Vishwanatha J.K. Jindal H.K. Davis R.G. J. Cell Sci. 1992; 101: 25-34Crossref PubMed Google Scholar). ANXA2 was also shown to localize with cytoskeleton-associated mRNA subpopulations (35Vedeler A. Hollas H. Biochem. J. 2000; 348: 565-572Crossref PubMed Scopus (44) Google Scholar). Most recently, it was shown that ANXA2 possessed a nuclear export sequence, and it was proposed that ANXA2 readily enters the nucleus but is rapidly exported (36Eberhard D.A. Karns L.R. VandenBerg S.R. Creutz C.E. J. Cell Sci. 2001; 114: 3155-3166Crossref PubMed Google Scholar). In the present report, we have examined HeLa cell extracts for the presence of ANXA2-binding proteins. Surprisingly, we found that several RNA-associated proteins bound to an ANXA2 affinity column, and this association was blocked by pretreatment with RNase A. We also show that in the presence of Ca2+, ANXA2 binds to ribonucleic homopolymers with a high affinity for poly(G) and in a salt-resistant manner. Subsequently, we show that ANXA2 is an RNA-binding protein that forms a messenger ribonucleoprotein (mRNP) particle. We identify c-myc RNA as a component of the ANXA2-ribonucleoprotein complex and show that ANXA2 binds directly to c-myc mRNA. Last, the expression of ANXA2 in a cell line normally devoid of this protein results in an increase in both ANXA2 and c-Myc protein. Overall, these studies identify ANXA2 as a novel RNA-binding protein that may regulate the translation of c-myc RNA. Cell Lines, DNA Vectors, and Cell Lysis—HeLa, LNCaP, and 293 HEK cells were obtained from the American Type Culture Collection (Manassas, VA) and were grown at 37 °C in 5% CO2 in Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal bovine serum (Invitrogen) and 1% antibiotic (Invitrogen). The cDNA for ANXA2 and S100A10 were PCR-amplified and ligated into pcDNA3.1/neomycin (pcDNA-S100A10) or pcDNA 3.1/Hygro (pcDNA-ANXA2) (Invitrogen). The c-myc vector (pBluescript) was a generous gift from Dr. Robert Orlowski (Chapel Hill, NC). LipofectAMINE 2000 (Invitrogen) was used as outlined in the manufacturer’s instructions to transfect 10-cm2 dishes of the human prostrate carcinoma cell line, LNCaP, with the pcDNA 3.1 vectors, pcDNA-S100A10 and pcDNA-ANXA2. Stably transfected cells were selected with 7.5 μg/ml neomycin and 5 μg/ml hygromycin, respectively. Clonal cell lines were selected by ANXA2 and S100A10 protein expression. For detergent based lysis, cells were lysed with Nonidet P-40 buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40) supplemented with protease inhibitors and clarified by centrifugation at 12, 000 × g for 10 min at 4 °C. Where indicated, cells were hypotonically lysed by resuspending cells (from one 10-cm2 dish) in 1 ml of hypotonic lysis buffer (20 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 25 mm NaCl plus protease inhibitors) and drawing the solution through a 27-gauge needle five times, followed by 25 strokes in a Dounce homogenizer. After incubating on ice for 10 min, a postnuclear supernatant was obtained by clarifying the cell lysate for 10 min at 8,000 × g. Soluble protein fractions were quantitated by BCA assay (Pierce). Immunoprecipitation and Western Blot Analysis—HeLa cell lysate (500 μg in 0.5 ml of Nonidet P-40 buffer) was precleared with 1 μg of either nonimmune mouse or rabbit IgG and 20 μl of protein G-PLUS beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at 4 °C. ANXA2 (a kind gift from Tony Hunter, La Jolla, CA) or ANXA5 (FL-319; Santa Cruz Biotechnology) antibody (1 μg) were then added to the precleared lysate, and the reactions were rocked for 1 h at 4 °C, followed by the addition of Protein G-PLUS beads (20 μl/reaction) for an additional 1 h. The immune complexes were washed three times and either boiled in SDS-PAGE sample buffer for Western blot analysis or extracted with phenol/chloroform/isoamyl alcohol (PCI 25:24:1) for RNA isolation. For Western blot analysis, proteins in sample buffer were resolved on SDS-PAGE, transferred to 0.2-μm nitrocellulose membranes, immunostained, and visualized using SuperSignal chemiluminescent substrate (Pierce). Primary antibodies (1:1000 dilution) were obtained from the following sources: Becton Dickinson/Transduction Laboratories (annexin A2 and S100A10), Santa Cruz Biotechnology (annexin A5 (FL-319)), and Cell Signaling Technology (S6 ribosomal protein). Immune Complex RNA Extraction, RNA Labeling, and RT-PCR— The ANXA2 or ANXA5 immune complexes were washed three times with RNase-free Nonidet P-40 buffer and diluted to a final volume of 200 μl with diethylpyrocarbonate-treated water. The beads were extracted with one volume of PCI and treated with 2 units of DNase I for 30 min at 37 °C. The DNase-treated RNA was then extracted with one volume of PCI, followed by ethanol precipitation using linear polyacrylamide (Sigma) as a nucleic acid carrier. The precipitated RNA was diluted to 20 μl with diethylpyrocarbonate-treated water and stored at –80 °C. The bound RNA was labeled using RNA ligase and cytidine 3′,5′-bis(phosphate) (pCp; 5′-32P-labeled; PerkinElmer Life Sciences) as previously described (37Keith G. Biochimie (Paris). 1983; 65: 367-370Crossref PubMed Scopus (14) Google Scholar). Briefly, 8 μl of the extracted RNA was mixed with 40 units of RNasin, 2 μl of Me2SO, 50 μCi of pCp, and 2 μl of RNA ligase in a final volume of 20 μl and incubated for 2 h at 37 °C, followed by PCI extraction and ethanol precipitation as described above. Incorporation of the pCp label was assessed quantitatively by scintillation counting of trichloroacetic acid precipitates. Qualitative analysis of incorporation was assessed by electrophoresis of 2 μl of labeled RNA on a 1% (w/v) agarose gel. The gel was dried under vacuum, and the labeled RNA was visualized by autoradiography. RT-PCR analysis of the bound RNA was carried out using the One-Step RT-PCR kit (Qiagen). To detect the 275-nucleotide segment at the 3′-end of the c-myc mRNA as described previously (38Chu E. Takechi T. Jones K.L. Voeller D.M. Copur S.M. Maley G.F. Maley F. Segal S. Allegra C.J. Mol. Cell. Biol. 1995; 15: 179-185Crossref PubMed Scopus (75) Google Scholar), the following primers were used: forward, 5′-GGCGAACACACAACGTCTTGGAG-3′; reverse, 5′-GCTCAGGACATTTCTGTTAGAAG-3′. Sucrose Gradient Analysis of Cell Lysates—Linear sucrose gradient was performed essentially as described (39Zalfa F. Giorgi M. Primerano B. Moro A. Di Penta A. Reis S. Oostra B. Bagni C. Cell. 2003; 112: 317-327Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Postnuclear supernatants from hypotonically lysed cells were sedimented in a 15–50% (w/v) sucrose gradient (sucrose solutions made in hypotonic lysis buffer adjusted to 100 mm NaCl) by centrifugation for 2 h at 37,000 rpm in a Beckman SW41 rotor. After centrifugation, samples were fractionated into 1-ml fractions by top displacement using a gradient fractionator (Buchler). For Western blot analysis, 20 μl of each gradient fraction was boiled for 5 min with SDS-PAGE sample buffer and analyzed as described previously. Homoribopolymer Binding Assay—Binding of cell lysates to homoribopolymers was carried out essentially as described previously with a few modifications (40Brown V. Small K. Lakkis L. Feng Y. Gunter C. Wilkinson K.D. Warren S.T. J. Biol. Chem. 1998; 273: 15521-15527Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Cell lysate (100 μg in 0.5 ml of Nonidet P-40 buffer) was incubated with a 25-μl packed volume of homoribopolymer beads (Sigma) and rotated for 30 min at 4 °C. The beads were washed three times with Nonidet P-40 buffer, boiled in 25 μl of SDS-PAGE sample buffer, and probed for ANXA2 by Western blot as described above. For purified ANXA2 binding to homoribopolymer beads, 1 μg of ANXA2 was added to 25 μl of packed homoribopolymer beads in 0.5 ml of TBS. Protein binding analysis was carried out as described above either by Western blot or by staining with Coomassie Blue where indicated. Annexin-conjugated Affinity Matrices—Purified ANXA2 and ANXA5 (10 mg each at 1 mg/ml) were coupled to 5-ml aliquots of CNBr-activated Sepharose 4B matrix according to the manufacturer’s recommendations (Amersham Biosciences). An unconjugated, control matrix was produced by incubating 5 ml of swollen gel with 10 ml of 0.1 m Tris-HCl, pH 8.5. For the isolation of ANXA2 and ANXA5 protein binding partners, 10 mg of Nonidet P-40-soluble cell lysate (2 mg/ml) was first rotated with 2 ml of the Tris-blocked matrix in the presence of either 1 mm CaCl2 or 5 mm EGTA for 2 h at 4 °C. This precleared lysate (5 mg) was then rotated with 1 ml of either the ANXA2 or ANXA5 affinity matrix for 2 h at 4 °C. The matrices were washed three times with 10 ml of Nonidet P-40 buffer, and the bound proteins were boiled for 10 min in SDS-PAGE sample buffer (0.5 ml for blocked matrix, 0.25 ml for annexin matrices). Aliquots of the eluted proteins (200 μl/lane) were resolved on 8% SDS-PAGE and stained with Coomassie Blue. To assess the contribution of either cellular RNA or DNA in binding of the proteins to the ANXA2 affinity matrix, the cell lysates were preincubated at 37 °C with either 200 units/ml RNasin (Promega), 50 units/ml DNase I (Ambion), or 500 μg/ml RNase A (Qiagen) for 30 min. After the incubation, the cell lysates were rotated in the presence of 1 mm CaCl2 with the Tris-blocked matrix followed by incubation with the ANXA2 affinity column. Bound proteins were analyzed as described above. Protein Identification by In-gel Tryptic Digestion and Mass Spectrometry—Stained bands were excised, and an automated in-gel tryptic digestion was performed on a Mass Prep Station (Micromass, UK). The gel pieces were destained, reduced (dithiothreitol), alkylated (iodo-acetamide), and digested with trypsin (Promega sequencing grade modified), and the resulting peptides were extracted from the gel and analyzed via liquid chromatography/mass spectrometry. Liquid chromatography/mass spectrometry was performed on a CapLC (Waters) high pressure liquid chromatograph and a Q-ToF-2 (Micromass) Mass Spectrometer, using a Picofrit C18 reversed-phase capillary column (New Objectives). Proteins were identified from the mass spectrometry/mass spectrometry data using MASCOT (Matrix Science, UK) and searching the NCBI data base. Expression and Purification of Recombinant ANXA2—The galactose-inducible Saccharomyces cerevisiae expression vector (pYeDP60) as well as the vector containing the cDNA for ANXA2 (pYeDP60-ANXA2) were kindly supplied by Jesus Ayala-Sanmartin (INSERM, Paris, France) and have been described previously (41Ayala-Sanmartin J. Gouache P. Henry J.P. Biochemistry. 2000; 39: 15190-15198Crossref PubMed Scopus (41) Google Scholar). The cDNA of the S100A10 protein was PCR-amplified and inserted into the pYeDP60 vector, followed by the transformation of the vectors into the protease-deficient S. cerevisiae strain FKY282 (kindly supplied by Francois Kepes, Genopole, Envy, France). The growth and induction of the yeast cultures was done as described previously (41Ayala-Sanmartin J. Gouache P. Henry J.P. Biochemistry. 2000; 39: 15190-15198Crossref PubMed Scopus (41) Google Scholar), with only slight modifications. Prior to purification, 1-liter cultures of annexin II- and S100A10-expressing yeast were mixed, resulting in the isolation of 1-mg quantities each of ANXA2 and ANXA2 heterotetramer using the purification detailed previously. The isolated proteins were purified further using gel permeation chromatography equilibrated in 40 mm Tris-HCl, 140 mm NaCl, 0.1 mm EGTA, and 0.1 mm dithiothreitol. Proteins were aliquoted and stored at –80 °C. Ultraviolet Cross-linking Assay—Full-length (1.8 kb) c-myc message was transcribed and labeled in vitro with T7 polymerase (Stratagene) with [32P]UTP. 32P-Labeled RNA probes were synthesized by in vitro transcription with the RiboProbe® system (Promega). 32P-Labeled c-myc mRNA (1.77 × 108 cpm/μg) was incubated with 1.5 μg of purified recombinant AIIt with or without unlabeled c-myc RNA, positive control template RNA, and homoribopolymers (poly(G) and poly(C)). The RNA-protein mixture binding reaction was carried out in a 20-μl reaction mixture containing 10 mm HEPES, pH 7.4, 150 mm NaCl, 2 mm MgCl2, 1 mm CaCl2, 5% glycerol, and 2 μg of yeast tRNA. The mixtures were incubated at 30 °C for 30 min, after which they were irradiated with UV on ice for 12 bursts of 30 s with a UV Stratalinker (Stratagene). RNAs were digested with 1 μl of RNase A (10 mg/ml) at 37 °C for 15 min and analyzed by 12% SDS-PAGE. Surface Plasmon Resonance—ANXA2 heterotetramer was coupled to a CM5 sensor chip in a BIAcore 3000 instrument (BIAcore, Uppsala, Sweden) using the manufacturer’s amine-coupling kit. Homoribopolymer-binding assays were conducted in 10 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm CaCl2 at 25 °C and a flow rate of 30 μl/min. A nonderivitized flow cell was used as a control for the contribution of the bulk refractive index to the surface plasmon resonance signal. After each injection, the surface was regenerated with an injection of 2 mm EGTA, 10 mm HEPES, pH 7.4, 0.15 m NaCl. An approximate equilibrium dissociation constant (Kd) was obtained by measuring the equilibrium resonance units (Req) at several poly(G) concentrations (10 nm to 1 μm) at equilibrium. Binding data were analyzed by Scatchard analysis using the BIAevaluation software according to the following relationship: Req/C = KaRmax – KaReq, where Rmax is the resonance signal at saturation, C is the concentration of free analyte, and Ka is the equilibrium association constant. ANXA2 Forms a Ribonucleoprotein Complex—As a first step in elucidation of the possible physiological function(s) of ANXA2, we attempted to isolate intracellular proteins that interacted with ANXA2. In order to isolate these binding partners, we utilized CNBr-activated Sepharose matrices conjugated with ANXA2 or a blocked, unconconjugated resin (resin control). Annexin A5 (ANXA5), which has considerable sequence and structural similarity to ANXA2, was also conjugated to the matrix as a specificity control. Cell lysates prepared from either HeLa or 293 HEK cells were first precleared with unconjugated Sepharose matrix, followed by application to either the ANXA2 or ANXA5 matrices. These cell types were chosen because of their differing levels of endogenous ANXA2; HeLa cells have an abundance of endogenous ANXA2, whereas 293 HEK cells have substantially less ANXA2 by comparison (data not shown). Since the annexins are known to require Ca2+ to bind to cellular targets, the cell lysates were incubated with the affinity matrix in the presence or absence of Ca2+. Upon completion of the binding reactions, the proteins bound to the matrices were removed by boiling in SDS-PAGE sample buffer, followed by separation on 8% polyacrylamide gels. We observed that several cellular proteins associated with the ANXA2 matrix (Fig. 1A, lane 3). These cellular proteins did not associate with the control matrix or the ANXA5 affinity matrix, and the association of these proteins with the ANXA2 matrix required Ca2+. It is interesting to note that nearly identical results were obtained using 293 HEK cell lysate as starting material (data not shown). This suggests that the bound material does not require high levels of ANXA2 expression; nor does it appear to bind to ANXA2 stoichiometrically. Having established that ANXA2 binds to a number of distinct cellular proteins in a specific and Ca2+-dependent manner, the bands were excised and digested, and the fragments were analyzed by liquid chromatography-tandem mass spectrometry. A portion of the mass spectrometry results is shown in Table I. Surprisingly, we noticed that many of the proteins identified as specific ANXA2-binding proteins were either ribosomal proteins or proteins that are known to interact with cellular RNA. For example, the major proteins that bound to the ANXA2 affinity column included poly(A)-binding protein-1 (42Wang M.Y. Cutler M. Karimpour I. Kleene K.C. Nucleic Acids Res. 1992; 203519Crossref PubMed Scopus (28) Google Scholar), ribosomal protein L4 (43Wool I.G. Chan Y.L. Gluck A. Biochem. Cell Biol. 1995; 73: 933-947Crossref PubMed Scopus (285) Google Scholar), ribosomal protein P0 (44Krowczynska A.M. Coutts M. Makrides S. Brawerman G. Nucleic Acids Res. 1989; 176408Crossref PubMed Scopus (88) Google Scholar), ribosomal protein S3a (45Kenmochi N. Kawaguchi T. Rozen S. Davis E. Goodman N. Hudson T.J. Tanaka T. Page D.C. Genome Res. 1998; 8: 509-523Crossref PubMed Scopus (132) Google Scholar) and ribosomal protein S4 (46Zinn A.R. Alagappan R.K. Brown L.G. Wool I. Page D.C. Mol. Cell. Biol. 1994; 14: 2485-2492Crossref PubMed Scopus (72) Google Scholar). This finding suggested that either RNA or RNA-binding proteins were interacting with ANXA2.Table IProteins bound to Annexin A2 Affinity MatrixSmall (40 S) ribosomal subunit proteins S2, S3a, S4, S5, S9, S13, S14, S18, S19, S25, S32Large (60 S) Ribosomal Subunit Proteins L4, L7, L10, L14, L15, L17, L19, L22, L27a, L29, L31, L34, L35Y-box-binding proteinβ-ActinMyb-binding ProteinNucleolinhnRNP KScar proteinPoly (A)-binding proteinα-Tubulinβ-TubulinRibonucleoprotein UElongation factor-1α Open table in a new tab To differentiate between these two possibilities, the ANXA2 affinity matrix binding experiments were repeated with cell lysates pretreated with RNase A, RNasin
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