annexin a2 is a novel rna-binding protein

Annexin A2 Is A Novel RNA-Binding Protein

Nolan R. Filipenko, Travis J. MacLeod, Chang-Soon Yoon and David M. Waisman*

Cancer Biology Research Group,

Departments of Biochemistry & Molecular Biology and Oncology,

University of Calgary

Calgary, Alberta, Canada T2N 4N1.

Running title: Annexin A2 is an RNA-binding protein.

*To whom correspondence should be addressed: Departments of Biochemistry & Molecular

Biology and Oncology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W.,

Calgary, Alberta T2N 4N1, Canada.

Tel.: (403) 220-3022, Fax: (403) 283-4841, E-mail: [email protected]

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 11, 2003 as Manuscript M311951200 by guest on M

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ABSTRACT

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. RT-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 upregulation 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.

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INTRODUCTION

The annexins are a family of more than 160 unique annexin proteins which are present in

more than 65 different species ranging from fungi and protists to plants and higher vertebrates

(1). ANXA2 consists of an amino-terminal domain (ATD) which comprises the first 30 amino

acid residues of the protein and the carboxyl core domain (CCD) comprised of the remaining

residues. The CCD of ANXA2 contains sites for binding Ca2+, phospholipid, F-actin and

heparin (2;3). 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 (RSV)(4). Subsequently, it was demonstrated that only about 10% of total cellular ANXA2

was phosphorylated in cell transformed by RSV (4;5).

ANXA2 exists as three major species - a monomer, a heterodimer or a heterotetramer

(AIIt) (3). The heterodimer is composed of a single subunit of ANXA2 bound to a subunit of 3-

phosphoglycerate kinase (6). 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 (7-10). 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 (8;11). ANXA2 consists of two functional domains. The amino-terminal

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regulatory domain (ATD), contains the amino-terminal thirty 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

carboxyl core domain region (CCD), encompassing residues 31-338, comprises the binding sites

for Ca2+, phospholipid, heparin and F-actin (reviewed by (1-3)). The crystal structure of an

amino-terminally truncated form of ANXA2 has been reported (12). 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 (13-16) or endocytosis (17-19) as well as a role in

modulating sarcolemmal phospholipid raft organization during smooth muscle cell contraction

(20;21) and regulation of ion channels (22). Since an ANXA2-knockout mouse has not been

developed, it is not clear if 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 in vivo.

The expression of ANXA2 is induced in various transformed cells, including v-src-, v-

H-ras-, v-mos or SV40-transformed cells (23). Furthermore, the ANXA2 gene is growth-

regulated and its expression is stimulated by growth factors such as insulin, FGF and EGF (24).

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Up-regulated ANXA2 has also been reported in human hepatocellular carcinoma (25),

pancreatic adenocarcinoma (26), high-grade glioma (27), gastric carcinoma (28) and in acute

promyelocytic leukemia (29). Since overexpression of the ANXA2 gene is commonly observed

in both virally-transformed cell lines and human tumors, it has been suspected that this

upregulated 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 upregulation 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 (30). 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 (31). Ensuing studies showed

that ANXA2 could bind to deoxyribonucleic acid structures such as Z-DNA (32) or Alu

subsequences (33). Other studies have identified nuclear ANXA2 in immunoblots of nuclei and

as part of a primer recognition complex that stimulates DNA polymerase α activity (6;34).

ANXA2 was also shown to localize with cytoskeletal-associated mRNA subpopulations (35).

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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 (36).

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. Lastly, 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.

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MATERIALS AND METHODS

Cell Lines, DNA Vectors and Cell Lysis-HeLa, LNCaP and 293 HEK cells were obtained from

American Type Culture Collection (Rockville, MD) and were grown at 37 °C in 5% CO2 in

DMEM 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 NP-40 buffer (50 mM Tris-HCl, pH 7.4,

150 mM NaCl, 1 % NP-40) supplemented with protease inhibitors and clarified by

centrifugation at 12, 000 x g for 10 minutes 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 + protease inhibitors) and drawing the solution

through a 27 guage needle five times, followed by 25 strokes in a Dounce homogenizor. After

incubating on ice for 10 minutes, a post-nuclear supernatant was obtained by clarifying the cell

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lysate for 10 minutes at 8,000 x g. Soluble protein fractions were quantitated by BCA assay

(Pierce).

Immunoprecipitation and Western Blot Analysis-HeLa cell lysate (500 µg in 0.5 ml NP-40

buffer) was pre-cleared with 1 µg of either non-immune mouse or rabbit IgG and 20 µl of

protein G-PLUS beads (Santa Cruz Biotechnology) for 1 hour 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 hour at 4 °C,

followed by addition of Protein G-PLUS beads (20 µl/reaction) for an additional 1 hour. The

immunecomplexes 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 the

SuperSignal Chemiluminescent Substrate (Pierce). Primary antibodies (1:1000 dilution) were

obtained from the following sources: Becton-Dickinson/Transduction Labs (annexin A2,

S100A10), Santa Cruz (annexin A5 (FL-319)), Cell Signaling Technology (S6 ribosomal

protein).

Immunecomplex RNA Extraction, RNA Labeling and RT-PCR-The ANXA2 or

ANXA5 immunecomplexes were washed three times with RNase-free NP-40 buffer and diluted

to a final volume of 200 µl with DEPC-treated water. The beads were extracted with one

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volume of PCI and treated with 2 units of DNase I for 30 minutes 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

DEPC-treated water and stored at –80 °C.

The bound RNA was labeled using RNA ligase and cytidine 3’,5’-bis(phosphate) (pCp –

5’-[32P], New England Nuclear) as previously described (37). Briefly, 8 µl of the extracted

RNA was mixed with 40 units of RNasin, 2 µl of DMSO, 50 µCi of pCp and 2 µl of RNA ligase

in a final volume of 20 µl and incubated for 2 hours 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 TCA 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 (38), 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 (39). Post-nuclear supernatants from hypotonically lysed cells were sedimented in a

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15-50% (w/v) sucrose gradient (sucrose solutions made in hypotonic lysis buffer adjusted to 100

mM NaCl) by centrifugation for 2 hours 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 minutes with SDS-PAGE sample buffer and analysed as described previously.

Homoribopolymer Binding Assay-Binding of cell lysates to homoribopolymers was carried out

essentially as described previously with a few modifications (40). Cell lysate (100 µg in 0.5 ml

NP-40 buffer) was incubated with 25 µl packed volume of homoribopolymer beads (Sigma) and

rotated for 30 minutes at 4 °C. The beads were washed three times with NP-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 in at 1

mg/ml) were coupled to 5 ml aliquots of CNBr-Activated Sepharose 4B matrix according to

manufacturers recommendations (Amersham Pharmacia). An unconjugatged, 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 NP-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

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CaCl2 or 5 mM EGTA for 2 hours at 4 °C. This precleared lysate (5 mg) was then rotated with 1

ml of either the ANXA2 or ANXA5 affinity matrix for 2 hours at 4 °C. The matrices were

washed three times with 10 ml of NP-40 buffer and the bound proteins were boiled for 10

minutes 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 of

RNasin (Promega), 50 units/ml of DNase I (Ambion) or 500 µg/ml RNase A (Qiagen) for 30

minutes. 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 analysed 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 de-stained, reduced (DTT), alkylated (iodoacetamide),

digested with trypsin (Promega Sequencing Grade Modified) and the resulting peptides extracted

from the gel and analyzed via LC/MS. LC/MS was performed on a CapLC (Waters) HPLC and

a Q-ToF-2 (Micromass) Mass Spectrometer, using a Picofrit C18 reversed-phase capillary

column. (New Objectives). Proteins were identified from the MS/MS data using MASCOT

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(Matrix Science, UK) and searching the NCBI database.

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 (41). 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 Saccharomyces cerevisaie strain FKY282 (kindly supplied by

Francois Kepes, Genopole, Envy, France). The growth and induction of the yeast cultures was

done as described previously (41), with only slight modifications. Prior to purification, 1 liter

cultures of annexin II and S100A10 expressing yeast were mixed, resulting in the isolation of

milligram 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 DTT. 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 X 108 cpm/ug) 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)

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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 (Stratatgene).

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 manufacturers’ amine-coupling

kit. Homoribopolymer-binding assays were conducted in 10 mM HEPES, pH 7.4, 150 mM M

NaCl, 1 mM CaCl2 at 25°C and a flow rate of 30 µL/min. A non-derivitized 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 - 1 µM) at equilibrium. Binding data were analyzed by Scatchard analysis using the BIA

evaluation 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.

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RESULTS

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

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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, digested and the fragments analysed 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 (42), ribosomal protein L4 (43), ribosomal protein P0 (44), ribosomal protein

S3a (45) and ribosomal protein S4 (46). This data suggested that either RNA or RNA-binding proteins were

interacting with ANXA2.

To differentiate between these two possibilities, the ANXA2 affinity matrix binding

experiments were repeated with cell lysates pretreated with RNase A, RNasin (an RNase

inhibitor), or DNase I. As shown in Fig. 1B, the association between ANXA2 and the cellular

proteins was abolished by pretreatment with RNase A. In contrast, the pretreatment of the

cellular extracts with either DNase I or RNasin did not affect the profile of cellular proteins

bound to the ANXA2 affinity matrix. These results therefore indicate that ANXA2 assembles

with cellular proteins to form a ribonucleoprotein (RNP) complex and intact RNA is required for

the interaction of ANXA2 with its cellular binding partners.

ANXA2 Binds RNA In Vitro. The possible interaction of ANXA2 with RNA was tested by

incubating HeLa cell lysates with agarose-conjugated RNA homopolymers. This assay system

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has been typically used to characterize the RNA binding properties of many other RNA-binding

proteins (40;47;48). HeLa cell lysates were incubated with the RNA homopolymers and bound

proteins were eluted and analyzed by Western blot using an ANXA2 monoclonal antibody. We

observed that endogenous ANXA2 bound selectively to poly(G)-agarose (Fig. 2A) and that this

interaction was dependent on Ca2+ (half-maximal at approximately 10 µM (Fig. 2B)).

Furthermore, when the bound proteins were analysed with a monoclonal antibody to S100A10,

the Western blot confirmed the presence of the S100A10 binding partner (data not shown),

suggesting that some of the ANXA2 bound to poly(G) was complexed to S100A10 as the

heterotetramer. Thus, the interaction of ANXA2 with its S100A10 binding partner did not block

its ability to interact with poly(G).

We subsequently used a battery of binding conditions to characterize the interaction of

HeLa cell lysate ANXA2 with poly(G). The binding of the endogenous ANXA2 to poly(G) was

stable in 1 mg/ml heparin and NaCl concentrations up to 0.25 M (Fig. 2C and 2D, respectively).

Furthermore, ANXA2 bound to poly(G) in the presence of as high as 1 mg/ml of yeast tRNA as a

competitor (Fig. 2E).

In order to visualize the endogenous proteins that bound to the poly(G)-agarose, the

poly(G)-binding protein fraction obtained from HeLa cell lysates was also analysed by SDS

PAGE and Coomassie blue staining. We found that a 35 kDa protein was the predominant

poly(G)-binding protein in these lysates. Mass spectrometry identified this protein band as

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ANXA2, confirming that ANXA2 was the major poly(G)-binding protein in the HeLa cell

extracts (Fig. 3).

The formation of ANXA2-containing RNP complexes suggested that either ANXA2

directly interacts with RNA or interacted via other RNA-bound protein(s). To distinguish

between these possibilities, purified ANXA2 was assayed for intrinsic RNA-binding activity

using the agarose-immobilized RNA homopolymers. We used the heterotetrameric form of

ANXA2 for these analyses, since it is the prevalent form of the protein in most cell types. As

shown in Fig. 4A, recombinant heterotetrameric ANXA2 showed specific binding to poly(G)

with very little binding to poly(A), poly(C) or poly(U). Similar results were observed with the

monomeric form of ANXA2 (data not shown). We also assessed the specificity of the poly(G)-

agarose binding by performing competition assays with unconjugated homoribopolymers.

ANXA2 was incubated with the poly(G) beads in the presence of a molar excess of free

competitor homoribopolymers. We observed that the binding of ANXA2 to the poly(G) beads

was blocked by the free poly(G) but not by free poly(A), poly(C) or poly(U) (Fig. 4B). Similarly

to the interaction of endogenous HeLa cell ANXA2 with poly(G) (Fig. 2), the binding of

recombinant ANXA2 to poly(G) was not blocked by yeast tRNA (Fig. 4C) or heparin and was

stable in the presence of physiological NaCl concentrations (data not shown).

In addition to the competition experiment described above, the interaction of unconjugated poly(G)

homopolymers with the ANXA2 heterotetramer was further characterized using two distinct methods. First, ANXA2

was conjugated to a biosensor chip and the binding to the RNA homopolymers was examined by surface plasmon

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resonance. Of the four homoribopolymers examined, ANXA2 bound only to poly(G). These experiments further

established that ANXA2 bound selectively and with high affinity (Kd of 60 nM) to poly(G) (Fig. 4D). Additionally,

only the binding of poly(G) resulted in a substantial conformational change in ANXA2 as assessed by circular

dichroism (data not shown), providing further evidence of the specificity of poly(G) binding.

Monomeric ANXA2 Harbors the RNA-Binding Site Within ANXA2 Heterotetramer – The

previous data established that poly(G) and ANXA2 form a specific and tight complex. It was

unclear if both subunits of ANXA2 heterotetramer contribute to poly(G) binding. Shown in Fig.

5A, ANXA2 binds to poly(G) whether it is complexed as the heterotetramer or as a monomer. In

contrast, the purified S100A10 subunit does not bind the homoribopolymers.

ANXA2 monomer consists of an amino-terminal domain (ATD) which comprises the

first 30 amino acid residues of the protein and a carboxyl core domain (CCD) comprised of the

remaining residues (49). The ATD is released from the molecule by proteolysis. Having

established that ANXA2 monomer contains the RNA-binding site, the RNA-binding activity of

the CCD fragment was compared with the intact ANXA2 monomer to crudely map the region of

ANXA2 responsible for binding to poly(G). As is shown in Fig. 5B, both the intact and the CCD

fragment were precipitated by poly(G) beads, demonstrating that the poly(G) binding site is

found within the CCD of ANXA2. This was unexpected as it suggested that the CCD, which is

highly conserved among the annexins, contained a key RNA-binding motif that was unique to

ANXA2. Incubation of a tissue annexin fraction containing annexins A1-A7 (50) with poly(G)-

agarose resulted in the selective binding of only a single annexin which was confirmed by

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Western blot to be ANXA2 (Fig. 5C). Additionally, a dsDNA agarose resin did not interact with

any of the annexins in the tissue fraction (Fig. 5C). These data established that the RNA-binding

activity of ANXA2 was probably not a property shared by other members of the annexin family

of proteins nor was general nucleic acid binding activity a common function of the annexin

family.

ANXA2 forms an RNP In Vivo. To determine if ANXA2 interacted with RNA in vivo, we

immunoprecipitated ANXA2 from HeLa cell lysates and analysed the content of the

immunoprecipitate. As a control, immunoprecipates of ANXA5 were also examined. The

specificity of the antibodies was confirmed by Western blot (Fig. 6A). The RNA was then

isolated from the immunoprecipitates and labeled by the pCp method (37). As shown in Fig. 6B,

the ANXA2 immunoprecipitate isolated from HeLa cell lysate was labeled with pCp and

therefore contained RNA, whereas the ANXA5 immunoprecipitate was not labeled. As an

additional control, immunoprecipitates were prepared using non-immune mouse IgG. We found

that these immunoprecipitates did not contain significant amounts of RNA, suggesting that

neither the antibodies alone nor the agarose beads used for the precipitation were responsible for

the coprecipitation of RNA with ANXA2.

To further investigate the possibility that ANXA2 formed RNP complexes in vivo, we

fractionated HeLa cell lysates on linear sucrose gradients and analysed the ANXA2 distribution

by Western blot. As a control for these experiments, ribosomal protein S6 was used as a marker

for free ribosomes and polyribosomes. The location of the free (cytosolic) protein fraction was

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monitored by assaying for tyrosine phosphatase activity using p-nitrophenylphosphate (PNPP)

as a substrate (data not shown). As shown in Fig. 7A, ANXA2 cosedimented with the RNPs in

the upper fractions of the gradient. In contrast, pretreatment of the HeLa lysate with RNase A

resulted in the appearance of ANXA2 in the free protein fraction. This result showed that

ANXA2 formed an RNP complex and that RNA was critical for the formation of this complex.

Identification of c-myc mRNA as a component of the mRNP. The ANXA2-RNP complex

resolved on the sucrose gradient was pooled and immunoprecipitated with the ANXA2 antibody

(Fig. 7B). RNA was isolated from the immunoprecipitate and subjected to RT-PCR. Since c-

Myc expression is necessary for Src transformation (51) and ANXA2 is elevated in Src-

transformed cells, we explored the possibility that ANXA2 might play a role in the regulation of

c-myc mRNA. As shown in Fig. 7C, we detected the presence of c-myc mRNA in the RNA

isolated from the ANXA2 immunoprecipitates. In contrast, we did not detect GAPDH mRNA in

the immunoprecipitates (data not shown). Our inability to detect GAPDH mRNA indicated that

ANXA2 is not a general, non-specific RNA-binding protein, but binds to a distinct subset of

cellular mRNA’s. These results establish that ANXA2 is part of a messenger RNP (mRNP)

complex in vivo which contains c-myc mRNA and possible other RNAs.

ANXA2 binds to c-myc mRNA. The presence of c-myc RNA in the ANXA2-ribonucleoprotein

complex could be due to the direct interaction of ANXA2 with c-myc mRNA or due to the

indirect interaction of ANXA2 with a c-myc-binding component of this complex. To

distinguish between these possibilities we performed a UV cross-linking assay with purified

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ANXA2 and 32P-labeled full length c-myc mRNA. As shown in Fig. 8A, ANXA2 binds

directly to c-myc mRNA. Furthermore, the interaction between ANXA2 and c-myc RNA was

blocked by either poly(G) homoribopolymer or unlabeled c-myc mRNA but not by poly(C)

homoribopolymer. In addition, the interaction between ANXA2 and c-myc mRNA was Ca2+-

dependent (Fig. 9B). To further examine the specificity of the interaction of c-myc mRNA with

ANXA2, we transcribed an irrelevant RNA (pGEM Express positive control template) and

observed that this RNA did not compete with the c-myc transcript for binding to ANXA2 (Fig.

9B, lane 3). Therefore, these data establish that the interaction between ANXA2 and c-myc

mRNA is direct, specific and Ca2+-dependent.

Expression of ANXA2 increases c-myc protein levels. The predominant form of ANXA2 in

cultured cells is complexed to its S100A10 binding partner as a heterotetramer. Previous studies

have established that although ANXA2 is a prominent protein in human prostate cell lines such

as DU-145 and PC-3, ANXA2 and its S100A10 binding partner are not present in the human

prostate, LNCaP cell line (52). We examined the c-myc protein levels in these three prostate cell

lines. As shown in Fig. 9A, the human prostatic cell line, LNCaP, is devoid of ANXA2 and also

have low expression of c-myc protein compared to the DU-145 and PC-3 cell lines (Fig. 9B).

To further explore the relationship between ANXA2 and c-myc, we transfected the LNCaP cells

with the gene for both ANXA2 and its S100A10 binding partner. Stable transfectants were

cloned and cell lines expressing both ANXA2 and S100A10 were selected. Two permanent cell

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lines, expressing both proteins were then further characterized. As shown in Fig. 9, LNCaP

clonal cell lines expressing ANXA2 (Fig. 9C) have significantly upregulated levels of c-myc

protein compared to the control cells (Fig. 9D).

.

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DISCUSSION

In the current report we establish that ANXA2 is a unique RNA-binding protein. The

absence of the well-established RNA-binding domains from the sequence of ANXA2 as well as

the selectivity of the protein for poly(G) homoribopolymers implies that ANXA2 possesses a

unique RNA-binding domain. We also demonstrate that the binding of poly(G)

homoribopolymer to ANXA2 is totally dependent on Ca2+. Thus our report is the first

demonstration of a Ca2+-dependent RNA-binding protein. This observation further establishes

that ANXA2 is a unique RNA-binding protein.

Approximately six distinct RNA-binding motifs have been identified (reviewed in (53).

These include the RNP motif, the arginine-rich motif, the RGG box, the KH motif, the double-

stranded RNA-binding motif and the zinc finger-knuckle motif. The absence of these structures

from ANXA2 implies that the RNA-binding domain of this protein is unique among the RNA-

binding proteins. Since of the seven annexins tested for RNA-binding activity, only ANXA2

bound to RNA (Fig. 5C), it is likely that the presence of an RNA-binding domain in ANXA2 is

unique to the annexin family of proteins. Direct binding studies have shown that ANXA2 is a

low affinity Ca2+-binding protein that binds Ca2+ with a Kd of about 0.5 mM. However, as

shown in Fig. 2B, the interaction of the protein with RNA occurred with a Kd (Ca2+) of about 10

µM. This suggests that the interaction of ANXA2 with RNA induces a conformational change

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resulting in a change in the architecture of the Ca2+-binding sites from low affinity to higher

affinity Ca2+-binding sites. Our observation of a Ca2+-dependent conformational change in

ANXA2 upon RNA binding, as measured by circular dichroism (data not shown), is consistent

with this suggestion. Although were unable to determine the exact RNA-binding site of

ANXA2, we did determine that the RNA-binding domain of ANXA2 is located in the carboxyl

domain of the protein.

c-Myc is a multifunctional nuclear phosphoprotein that can promote cell cycle

progression, apoptosis and cellular transformation. c-Myc regulates these activities at the

molecular level by functioning as a regulator of gene transcription, activating or repressing

specific target genes. The half-life of c-myc mRNA is regulated when cells change their growth

rates or differentiate. Two regions within c-myc mRNA determine its short half-life: one is in

the 3’-untranslated region, the other is in the coding region. A cytoplasmic RNA-binding

protein, the coding region determinant-binding protein (CRD-BP), binds to the c-myc coding

region in vitro and shields it from endonuclease digestion and thereby prolongs the mRNA half-

life (54). The 5’ untranslated region may also play a role in the translation regulation of c-myc

since the interaction of the cap binding protein, eIF4E, with this region relieves the translation

repression imposed on the c-myc mRNA by its structured 5’ UTR (55). In this work, we have

used an immunoprecipitation-RT-PCR technique to show that ANXA2 forms a RNP complex

in HeLa cells and we identify one species of mRNA in this complex as c-myc mRNA (Fig. 7C).

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Furthermore, we show that ANXA2 binds directly to c-myc mRNA (Fig. 8). Although the exact

binding site on c-myc mRNA was not identified in our report, it was observed that the

interaction of ANXA2 with c-myc mRNA results in the upregulation of c-myc protein (Fig. 9),

suggesting that the binding of ANXA2 to c-myc mRNA may have an important physiological

role in the regulation of c-myc mRNA.

In general, the RNA-binding proteins serve a number of functions including regulating

mRNA stability and the rate and efficiency of mRNA translation. In addition, the RNA-binding

proteins can participate in the specific targeting of mRNA in the cytoplasm (reviewed in (56)).

Considering that ANXA2 forms a RNP complex in vivo, and does not associate with ribosomal

subunits or polyribosomes (Fig. 7A), it is likely that ANXA2 does not directly interfere with the

translational machinery but rather directly interacts with mRNA resulting in the formation of a

mRNP complex. Therefore, our observation that ANXA2 expression results in the upregulation

of c-myc protein presents the possibility that ANXA2 may affect c-myc mRNA stability or

transport.

Our studies demonstrating that the transfection of LNCaP cells with the ANXA2 gene

results in the expression of ANXA2 protein and an enhanced expression of c-myc protein are

compatible with a model in which ANXA2 plays a role in the regulation of c-myc mRNA during

cellular transformation. Interestingly, c-myc is upregulated in many forms of cancer including

pancreatic carcinoma (57;58), acute promyelocytic leukemia (59), and glioma (60). Similarly, the

overexpression of the ANXA2 gene is commonly observed in both virally-transformed cell lines

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and human tumors. For example, the expression of ANXA2 is induced in various transformed

cells, including v-src-, v-H-ras-, v-mos or SV40-transformed cells (23). Furthermore, the

ANXA2 gene is growth-regulated and its expression is stimulated by growth factors such as insulin,

FGF and EGF (24). Up-regulated ANXA2 has also been reported in human hepatocellular

carcinoma (25), pancreatic adenocarcinoma (26), high-grade glioma (27), gastric carcinoma (28)

and in acute promyelocytic leukemia (29). Whether or not ANXA2 plays a role in the regulation

of c-myc during these events will require further studies. It is also interesting to note that the

Ca2+-dependent regulation of c-myc has been reported in HL-60 cells (61). Therefore, the

upregulation of ANXA2 or its activation by changes in Ca2+ have the potential to play a role in

the regulation of c-myc.

In conclusion, our data demonstrate that ANXA2 is a novel RNA-binding protein that

binds directly to c-myc mRNA and upregulates c-myc protein. Hypothetically, these data

provides a possible link between the Ca2+ second messenger system and the regulation of c-myc

mRNA.

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ACKNOWLEDGEMENTS

We are grateful to Lorne Burke and Paul Semchuk (Alberta Peptide Institute, University of

Alberta) for extremely efficient mass spectrometry analysis. We would also like to thank Tara

Beattie and Ivan Babic for helpful discussions throughout the preparation of the manuscript.

Additionally, we would like to acknowledge Tony Hunter (Scripps Institute, La Jolla, CA) for

the anti-annexin II antibody, Robert Orlowski (University of North Carolina, Chapel Hill, NC)

for the c-myc vector. We are also extremely grateful to both Jesus Ayala-Sanmartin (INSERM,

Paris, France) and Francois Kepes (Evry, France) for supplying materials for expression of

ANXA2 and S100A10 in S. cerevisiae. This work was supported by grants from the National

Institutes of Health (RO1CA 78639), the Alberta Heart and Stroke Foundation and the Canadian

Institutes of Health Research. N.R.F. is supported by studentships from the Alberta Heritage

Foundation for Medical Research and the Alberta Cancer Board.

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Table I. Proteins Bound To Annexin A2 Affinity Matrix

Small (40S) Ribosomal Subunit Proteins:S2, S3a, S4, S5, S9, S13, S14, S18, S19, S25, S32

Large (60S) Ribosomal Subunit Proteins:L4, L7, L10, L14, L15, L17, L19, L22, L27a, L29, L31, L34, L35

Y-box binding proteinβ-ActinMyb-Binding ProteinNucleolinhnRNP KScar proteinPoly (A)-binding proteinα-tubulinβ-tubulinRibonucleoprotein UElongation factor-1α

Protein bands were excised from Coomassie Blue stained polyacrylamide gels, digested with

trypsin followed by liquid chromatography-tandem mass spectrometry analysis of the protein

fragments.

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FIGURE LEGENDS

Figure 1. Identification of ANXA2 Binding Partners

(A) NP-40 soluble cell lysates from HeLa cells (5 mg total protein) were applied to an

unconjugated Sepharose 4B control matrix in the presence of either 1 mM Ca2+ or 5 mM EGTA

for 2 hours. The unbound material was then applied to an ANXA2 or an ANXA5 Sepharose 4B

matrix in the presence of either 1 mM Ca2+ or 5 mM EGTA. After 2 hours incubation, all

matrices were washed extensively, boiled for 5 minutes in SDS-PAGE sample buffer, followed

by Coomassie Blue staining of the proteins resolved on polyacrylamide gels. [Note: nearly

identical data was obtained using 293 HEK cell lysates as starting material]

(B) NP-40 soluble cell lysates from HeLa cells (5 mg total protein) were pretreated with either

RNase A (500 µg/ml), RNase inhibtor (200 U/ml) or DNase I (50 U/ml) for 30 minutes at 37 °C.

The treated lysates were then applied to an unconjugated Sepharose 4B control matrix in the

presence of 1 mM Ca2+ for 2 hours. The unbound material was then applied to an ANXA2

Sepharose 4B matrix in the presence of 1 mM Ca2+. After 2 hours incubation, the matrices were

washed and analysed as in (A).

Figure 2. RNA-Binding Properties of ANXA2

For (A-E), NP-40 soluble lysate from HeLa cells (100 µg/lane) were incubated with 25 µl of the

indicated homoribopolymer beads for 30 minutes (for (D-E), the Ca2+ concentration used was

100 µM). The beads were washed three times with NP-40 buffer, boiled in sample buffer and

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analysed by Western blot analysis for ANXA2 binding. In panel (F), the eluted proteins were

analysed by Coomassie Blue staining of the polyacrylamide gels.

(A) ANXA2 binds specifically to poly(G) in a Ca2+-dependent manner. HeLa cell lysates were

left either untreated or incubated with 100 µM Ca2+ or 5 mM EGTA prior to addition of the

indicated homoribopolymers.

(B) The Ca2+ requirement for poly(G) binding to ANXA2 was analysed by incubating HeLa

lysates with the indicated concentrations of Ca2+ prior to addition of the poly(G) beads.

(C) Heparin does not inhibit the interaction between ANXA2 and poly(G). Cell lysates were

treated as in (A), except heparin (1mg/ml) was added either added during the wash steps (“wash

only”) or during both the binding reaction and the wash steps (“incubation and wash”).

(D) ANXA2 binds to poly(G) in a salt-resistant manner. The concentration of salt in the HeLa

cell lysate was adjusted as indicated prior to addition to the poly(G) beads. After the 30 minute

incubation period, the beads were washed with NP-40 buffer containing the indicated

concentration of salt. (E) ANXA2 binding to poly(G) was assessed in the presence of the

indicated amounts of yeast tRNA. The tRNA was present during both the binding reaction in

addition to the wash steps.

Figure 3. ANXA2 is the major poly(G) binding protein in HeLa cells. HeLa cell lysate (250 µg)

containing 100 µM Ca2+ was added to agarose beads and rotated for 2 hours. The unbound

material was then added to poly(C)-agarose for 2 hours, followed by the addition of the unbound

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material to poly(G)-agarose for an additional 2 hours. The matrices were washed and then eluted

with 5 mM EGTA. After elution with EGTA (labeled “EGTA”), the matrices were boiled with

SDS-PAGE sample buffer (labeled “boil”), followed by analysis of all fractions on Coomassie

Blue stained polyacrylamide gels. The eluted protein migrating at ~35 kDa in the poly(G)-

agarose EGTA elution (labeled with “ * ”) was identified by mass spectrometry as ANXA2.

Figure 4. Purified ANXA2 has intrinsic poly(G) binding activity

For (A-C), 1 µg of indicated purified protein was incubated with 25 µl of homoribopolymer

beads for 30 minutes in 0.5 ml of TBS containing 100 µM Ca2+ (TBS-Ca2+). The beads were

washed three times with TBS-Ca2+, boiled in sample buffer and analysed by Western blot

analysis for ANXA2 binding.

(A) ANXA2 binds specifically to poly(G). The indicated homoribopolymer beads were

incubated with purified ANXA2 heterotetramer in the presence of 100 µM Ca2+.

(B) Unconjugatged poly(G) inhibits the binding of ANXA2 to poly(G) beads. The binding of

ANXA2 heterotetramer to poly(G) beads was assessed in the presence of a 50 molar excess of

unconjugated poly(A), poly(C), poly(G) or poly(U).

(C) ANXA2 binding to poly(G) was assessed in the presence of the indicated amounts of yeast tRNA. The tRNA

was present during both the binding reaction in addition to the wash steps.

(D) Immobilized ANXA2 binds specifically to poly(G). Unconjugated homoribopolymers (1 µM) were injected

over a CM5 biosensor chip conjugated with purified ANXA2 heterotetramer in the presence of 1

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mM Ca2+.

Figure 5. Characterization of the binding of poly(G) to ANXA2. (A) S100A10 is not responsible

for the poly(G) binding activity of ANXA2 heterotetramer. 1 µg of indicated purified protein

was incubated with 25 µl of homoribopolymer beads for 30 minutes in 0.5 ml of TBS containing

100 µM Ca2+ (TBS-Ca2+). Either ANXA2 heterotetramer, ANXA2 monomer or S100A10

were incubated with poly(G) beads in the presence of EGTA (5 mM) or Ca2+ (100 µM).

Scatchard analysis of the poly(G) binding estimated the Kd to be approximately 60 nM.

(B) The carboxy-terminal core of ANXA2 retains RNA-binding activity. Partially proteolysed ANXA2 (1 µg)

was incubated with poly(G) beads in the presence of 100 µM Ca2+ or 5 mM EGTA. (“St.” refers

to the partially proteolysed ANXA2 used for binding studies).

(C) RNA-binding activity is specific to ANXA2. A pool of annexins A1-A7 (10 µg) was

incubated with either poly(G) beads or dsDNA-agarose in the presence of either 100 µM Ca2+

or 5 mM EGTA. (“St.” refers to the pool of complete annexins A1-A7 used for binding studies).

For (B) and (C), the bound proteins were analysed by Coomassie blue staining of polyacrylamide

gels.

Figure 6. ANXA2 is part of a ribonucleoprotein complex in vivo

(A) ANXA2 and ANXA5 can be specifically immunoprecipitated from HeLa cells. HeLa cell

lysate (500 µg) was incubated for 1 hour with 1 µg of antibody followed by the addition of

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protein G-PLUS-agarose for 1 hour. Immunecomplexes were resolved by SDS-PAGE,

followed by immunostaining using antibodies specific for ANXA2 and ANXA5.

(B) ANXA2 co-immunoprecipitates with RNA in HeLa cells. Immunoprecipitation was

performed as in (A) in addition to a non-immune mouse IgG control (mIgG). Immunecomplexes

were washed and the bound RNA was extracted and 3’-labeled with [32P]-pCp. Results were

quantitated by scintillation counting or by agarose gel electrophoresis (inset).

Figure 7. Characterization of the interaction of ANXA2 with RNA. (A) ANXA2 binds to RNA in

vivo. Hypotonically lysed HeLa cells were fractionated through a linear sucrose gradient either

in the presence of an RNase inhibitor (top panels) or RNase A (bottom panels). Each gradient

was collected in ten fractions and an aliquot (20 µl/lane) of each fraction was analysed by

Western blot for ANXA2 and, as a control, ribosomal protein S6 (rS6).

(B) ANXA2 can be immunoprecipitated from sucrose gradient fractions. A 100µl aliquot (pooled

from the second and third fractions from the RNase inhibited gradient in (A)) were diluted to 1

ml with TBS and immunoprecipitated as in (Fig. 6A) for ANXA2. Non-immune mouse IgG was

used as an antibody control.

(C) ANXA2 co-immunoprecipates with c-myc mRNA. Bound RNA was extracted from the

immunoprecipitations performed in (B), followed by RT-PCR analysis for c-myc mRNA.

(Marker- 100 bp (New England Biolabs))

Figure 8. Purified recombinant ANXA2 binds to c-myc RNA. Full-length (1.8 kb) c-myc RNA labeled with 32P-

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UTP (1.77X108 cpm/µg) was incubated with purified ANXA2 heterotetramer (1.5 µg). After UV-

irradiation, samples were treated with RNase A and resolved by 12% SDS-PAGE. The arrow

depicts the position of cross-linked ANXA2. (A) Effects of homoribopolymers on c-myc

binding. Lanes 3 and 4: homoribopolymers (poly(G) and polyC) were included in the binding

mixture at 0.1 µg/µL. Lane 5 contained non-radiolabeled c-myc transcript (0.25 µg/µL) as a

competitor. (B) Calcium-dependence of c-myc binding. Lanes 1 and 2 show the binding in the

presence of either 1mM CaCl2 or 5mM EGTA. Lane 3 contained non-radiolabeled “irrelevant”

transcript as a competitor.

Figure 9. Expression of ANXA2 increases cellular levels of c-myc protein. Comparison of the

ANXA2 (A), S100A10 (A) and c-myc protein levels (B) in prostate cell lines DU145, PC-3 and

LNCaP. The levels of c-myc protein were monitored by immunoblot analysis. Expression of

ANXA2 correlates with upregulation of c-myc protein. LNCaP cells were transfected with

pcDNA3.1-ANXA2 and pcDNA3.1-S100A10 by using LIPOFECTAMINE 2000 Reagent

(Gibco BRL) and stable transfectants were cloned and clonal cell lines propagated. Two clonal

cell lines, selected for their ANXA2 and S100A10 levels, and control cells were analyzed by

immunoblotting for cellular levels of ANXA2 (C), S100A10 and c-myc (D). Tubulin

immunoreactivity is provided as a loading control.

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Nolan R. Filipenko, Travis J. MacLeod, Chang-Soon Yoon and David M. WaismanAnnexin A2 is a novel RNA-binding protein

published online December 11, 2003J. Biol. Chem.

10.1074/jbc.M311951200Access the most updated version of this article at doi:

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