Up-regulation of Nucleolin mRNA and Protein in Peripheral
Blood Mononuclear Cells by Extracellular-regulated Kinase*
Cara J.
Westmark and
James S.
Malter
From the Institute on Aging and Department of Pathology and
Laboratory Medicine, University of Wisconsin Medical School,
Madison, Wisconsin 53792
Received for publication, October 16, 2000
 |
ABSTRACT |
The signal transduction pathways regulating
nucleolin mRNA and protein production have yet to be elucidated.
Peripheral blood mononuclear cells treated with phorbol 12-myristate
13-acetate showed steady state levels of nucleolin mRNA that were
2-2.5-fold greater than untreated control cells. The up-regulation of
nucleolin mRNA was substantially repressed by U0126, a specific
inhibitor that blocks phosphorylation of extracellular-regulated kinase (ERK). Calcium ionophores A23187 and ionomycin also activated ERK and
substantially elevated nucleolin mRNA levels, demonstrating phorbol
12-myristate 13-acetate and calcium signaling converge on ERK. Drugs
that affected protein kinase C, protein kinase A, and phospholipase C
signal transduction pathways did not alter nucleolin mRNA levels
significantly. The half-life of nucleolin mRNA increased from
1.8 h in resting cells to 3.2 h with phorbol ester
activation, suggesting ERK-mediated posttranscriptional regulation.
Concomitantly, full-length nucleolin protein was increased. The higher
levels of nucleolin protein were accompanied by increased binding of a
70-kDa nucleolin fragment to the 29-base instability element in the
3'-untranslated region of amyloid precursor protein (APP) mRNA in
gel mobility shift assays. Supplementation of rabbit reticulocyte
lysate with nucleolin decreased APP mRNA stability and protein
production. These data suggest ERK up-regulates nucleolin posttranscriptionally thereby controlling APP production.
 |
INTRODUCTION |
Alzheimer's disease is characterized by the presence of senile
plaques and neurofibrillary tangles in brain tissue. The major proteinaceous material in the senile plaques is
-amyloid, a
40-42-amino acid peptide derived from the amyloid precursor protein
(APP).1 Investigation of the
molecular regulation of APP mRNA and protein levels is vital to
understanding
-amyloid accumulation and deposition in Alzheimer's
disease. Our laboratory has previously demonstrated that two
RNA-binding proteins, nucleolin and heterogeneous nuclear ribonucleoprotein C (hnRNP C), bind to a 29-base instability element in
the 3'-untranslated region (UTR) of APP mRNA (1). In rabbit reticulocyte lysate (RRL), hnRNP C binding to the 29-base element stabilized APP mRNA resulting in a 6-fold increase in APP protein production (2). The role of nucleolin was not determined in these experiments.
Nucleolin (C23) is a 110-kDa multifunctional phosphoprotein. It is an
abundant nucleolar protein (3) found in the fibrillar centers and on
organizer regions of metaphase chromosomes (4). Nucleolin plays a role
in chromatin decondensation (5), the transcription and processing of
rRNA (6-9), transcriptional regulation (10, 11), cell proliferation
(12), differentiation and maintenance of neural tissue (13), apoptosis
(14), nuclear/cytoplasmic shuttling (15), mRNA stability (16), and
mRNP assembly and masking (17). Cell surface nucleolin has been
reported to bind lipoproteins (18), laminin (13), growth factors
(19), and the complement inhibitor, factor J (20). Central
to nucleolin functions are RNA/DNA binding and helicase activities
(21).
The cDNA for nucleolin has been cloned and codes for a 707-amino
acid protein with at least three functional domains (3, 22). The
5'-flanking region and the first intron contain a high GC content
similar to the housekeeping genes. The 5' promoter has one atypical
TATA box (GTTA), one CCAAT box, three reverse complements of
CCAAT (ATTGG), two pyrimidine-rich stretches, and numerous potential
transcription factor-binding sites, whereas the 3'-UTR has five
homology blocks in a 100-base region (23).
There are several distinct features in the amino acid sequence of
nucleolin that enable this extraordinary protein to display such a vast
array of functions (3, 7). At the amino-terminal end of the molecule,
there are six (G/A/V)TP(G/A/V)KK(G/A/V)(G/A/V) repeats followed
by several glutamic/aspartic acid stretches separated by basic
sequences and four potential serine phosphorylation sites. The central
region, a putative globular domain, contains alternating hydrophobic
and hydrophilic stretches. There are four 90-residue repeats, each
containing an RNP-like consensus sequence (24). The carboxyl terminus
is glycine/arginine-rich with regularly spaced phenylalanine and
dimethylarginine residues (25). The central 40-kDa domain of nucleolin
containing the four RNA recognition motifs is responsible for the
specificity of RNA binding, and the carboxyl-terminal domain enhances
interaction but does not contribute to ligand specificity (26). The
carboxyl-terminal domain contains an ATP-dependent
duplex-unwinding activity, and phosphorylation enhances this helicase
activity (21, 27).
We were initially interested in determining how nucleolin regulation
might affect APP mRNA levels and stability. Preliminary studies
suggested cytokine-mediated signaling through protein kinases altered
nucleolin mRNA levels in PBMC. We found that activation of the
extracellular-regulated kinase (ERK)-specific mitogen-activated protein
kinase (MAPK) pathway significantly up-regulated nucleolin mRNA
levels independently of protein kinase C (PKC). Phorbol 12-myristate 13-acetate (PMA) treatment also resulted in higher levels of
full-length nucleolin protein and the disappearance of the 47-kDa
nucleolin cleavage fragment. In gel mobility shift assays, lysates from phorbol ester and ionomycin-treated peripheral blood mononuclear cells
(PBMC) contained nucleolin that bound to the 29-base instability element of APP mRNA. In RRL supplemented with nucleolin protein, APP mRNA decayed with a shortened half-life of 105 min but was indefinitely stable in RRL supplemented with globin. The loss of APP
mRNA stability resulted in decreased APP production. Therefore, ERK
activation stabilizes nucleolin mRNA resulting in increased nucleolin levels and accelerated decay of APP mRNA.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Protease inhibitor mixture (catalog number
P2714), Escherichia coli tRNA, RNase TI, 4
-phorbol, PMA,
1-oleoyl-2-acetyl-sn-glycerol (C18:1,[cis]-9/C2:0) (OAG), calcium ionophore A23187,
ionomycin calcium salt, thapsigargin, dantrolene, forskolin,
1,9-dideoxyforskolin, cAMP, (Rp)-cAMPS,
imipramine, wortmannin, 17
-estradiol, corticosterone, and
5,6-dichlorobenzimidazole riboside (DRB) were from Sigma. N-(6-Phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9),
tricyclodecan-9-yl xanthogenate (D609), and
1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18-OCH3) were from Calbiochem. Klenow enzyme, RNasin,
bis[2-aminophenylthio]butadiene (U0126), and RRL were from Promega
(Madison, WI). Lymphoprep density gradient medium and RPMI 1640 were
from Life Technologies, Inc., and TRI-reagent was purchased from
Molecular Research Center, Inc. (Cincinnati, OH). The enhanced
chemiluminescence (ECL) Western blotting detection kit,
[
-32P]dCTP and
L-[35S]methionine were from Amersham
Pharmacia Biotech; the nylon transfer membrane was from Fisher, and the
QuikHyb hybridization solution and NucTrap probe purification columns
were supplied by Stratagene (La Jolla, CA). The T7 and SP6 mMessage
mMachine in vitro transcription kits and oligo(dT)-cellulose
were purchased from Ambion, Inc. (Austin, TX).
PBMC Isolation and Stimulation, RNA Purification, and Northern
Blotting--
Peripheral blood was collected by phlebotomy from
consenting, healthy laboratory personnel and was anticoagulated with
heparin. The experimental protocol was approved by the University of
Wisconsin Hospital Human Subjects Review Committee, which meets
National Institutes of Health guidelines. PBMC were isolated by
Ficoll-Paque density centrifugation, stimulated with various kinase
effectors, and lysed in Tri-reagent as described
previously.2 RNA was
electrophoresed on formaldehyde-agarose gels, transferred to nylon
membranes, and hybridized with radiolabeled cDNA probes. The
samples were assayed in at least triplicate, normalized to a control
RNA (18 S or ribosomal protein S26 mRNA), and plotted as a
percentage of total nucleolin mRNA. Error bars depict the standard
error of the mean, and p values were calculated by the Student's t test.
Preparation of PBMC Cytoplasmic Lysates--
Cytoplasmic lysates
were prepared as described previously (29). Briefly, cultured PBMC (2 ml at 5 × 106 cells/ml) were scraped from tissue
culture wells, spun at 2,000 × g for 30 s in a
Stratagene picofuge microcentrifuge, washed three times with ice-cold
phosphate-buffered saline (PBS), and resuspended in 50 µl of ice-cold
buffer containing 25 mM Tris, pH 8, 0.1 mM
EDTA, and 1× protease inhibitor mixture. The resuspended cells were
lysed by five freeze (
80 °C)/thaw (37 °C) cycles and spun at
15,000 × g for 15 min at 4 °C. The supernatants
(cytoplasmic lysates) were transferred to fresh tubes and frozen at
80 °C. The protein concentration of the lysates was quantitatively
determined with Bio-Rad protein assay dye reagent per the
manufacturer's instructions.
Western Blot Analysis--
Lysate (10 µg) in a 12-µl volume
was mixed with 4 µl (4×) SDS reducing buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5%
-mercaptoethanol, 0.06% bromphenol blue), boiled for 5 min, and
analyzed on a 12% SDS-polyacrylamide gel. The gel was equilibrated in
Bjerrum and Schafer-Nielsen transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol, 1.3 mM SDS, pH 9.2),
and the proteins were transferred to 0.2-µm pure nitrocellulose
membrane by semi-dry electrophoretic transfer on a Bio-Rad Trans-Blot
SD apparatus at 15 V for 45 min. The nitrocellulose membrane was blocked for 1 h in 5% nonfat dry milk and stained by ECL per the manufacturer's directions. The primary antibody was anti-nucleolin (1:1000) (kindly provided by Dr. Raymond Petryshyn, Children's National Medical Center, Washington, D. C.) and the secondary antibody was anti-rabbit horseradish peroxidase (1:2000). The membrane
was exposed to x-ray film for 1 min.
Preparation of DNA Template and in Vitro
Transcription--
APP106 template was prepared by amplification of
nucleotides 2415-2520 of the plasmid
pT7APP751wt
HindIIIT90 (2) with the primers
5'-CACAATACGACTCACTATAGGGAACTTGAATTAATCCACA-3' (APP
cDNA(2415-2432)) and 5'-ACAGCTAAATTCTTTACAGT-3' (APP
cDNA(2520-2501)) by PCR (1 min at 94 °C, 1 min at
50 °C, and 10 s at 72 °C for 35 cycles). The 5' primer
included a T7 RNA polymerase promoter sequence, which is underlined.
The PCR product was extracted with Tris-saturated phenol/chloroform and
precipitated with 0.1 volume of sodium acetate and 2 volumes of
ethanol before gel purification on an 8% nondenaturing polyacrylamide
gel. Radiolabeled RNA probes were prepared according to Promega's
standard transcription protocol for T7 RNA polymerase with each
labeling reaction containing 200 ng of APP106 template and 50 µCi of
[
-32P]UTP. Reactions were incubated for 60 min at
37 °C and stopped by the addition of 2 units of RNase-free DNase I
for 15 min at 37 °C. Samples were extracted with water-saturated
phenol/chloroform, and unincorporated isotope was removed by passage
through NucTrap probe purification columns.
Gel Mobility Shift Assays--
The binding reactions were
performed similarly to the procedures used in Ref. 28. Briefly, 2 µg
of cytoplasmic lysates were incubated with 1 × 105
cpm of APP106 RNA probe in 10% glycerol, 15 mM HEPES, pH
8, 10 mM KCl, 1.0 mM dithiothreitol, 200 ng/µl E. coli tRNA, and 1 unit/µl RNasin in a total
volume of 10 µl for 10 min at 30 °C. RNase TI (20 units in 1-µl
volume) was added, and samples were digested for 30 min at 37 °C.
Reactions were cross-linked on automatic for 5 min in a UV Stratalinker
2400 from Stratagene (La Jolla, CA) on ice prior to the addition of 3.5 µl of 4× SDS reducing buffer, heat denaturation for 3 min at
100 °C, and analysis on 12% SDS-polyacrylamide gels. The gels were
dried and exposed to x-ray film or a PhosphorImager screen.
Plasmid Construction--
The construction of
pT7APP751wt
HindIIIT90 has been described (2). The plasmid
pSP
c containing the
-globin gene cloned between an SP6
promoter and a T stretch was provided by Richard Spritz (University of
Wisconsin, Madison, WI), and the nucleolin gene in the pMAM plasmid was
a gift from Meera Srivastava (Georgetown University School of Medicine,
Washington, D. C). The pMAMnucleolin plasmid was digested
with XhoI and NheI to liberate the nucleolin gene. The insert ends were blunted with Klenow and ligated with SmaI-digested pT7T90 transcription vector (provided by Jeff
Ross, University of Wisconsin, Madison, WI) to generate
pT7nuclcodingT90. The 3'-UTR of nucleolin cDNA was
amplified from Jurkat genomic DNA with the primers
5'-ACGAAGTTTGAATAGCTTCT-3' (nucleolin cDNA(2221-2240)) and 5'-GTAGGAAAAAATGGTTTTGT-3' (nucleolin
cDNA(2518-2499)) by PCR (1 min at 94 °C, 1 min at
50 °C, and 20 s at 72 °C for 35 cycles). The ends were
blunted with Klenow and ligated with SmaI-digested pUC-18
generating pUCnucl3'-UTR. pUCnucl3'-UTR was
digested with EcoRI and XbaI to liberate the
nucleolin 3'-UTR sequence, and the ends were blunted with T4 DNA
polymerase and ligated with pT7nuclcodingT90 previously
digested with EcoRI and blunted with T4 DNA polymerase. The
resulting plasmid, pT7nucleolinT90, contained the entire nucleolin coding region, a short spacer, and the complete nucleolin 3'-UTR sequence cloned between a T7 promoter and a 90-base poly(T) stretch.
mRNA Synthesis and Purification--
The plasmids
pT7APP751 wt
HindIIIT90 and pSP
c were linearized
with HindIII, and the plasmid pT7nucleolinT90 was digested with EcoRV. The digests were extracted with phenol and
chloroform, precipitated with 0.1 volume ammonium acetate and 2 volumes ethanol, resuspended in water to 0.5 µg/µl, and used as
templates (1 µg each) for in vitro transcription with
Ambion's T7 and Sp6 mMessage mMachine kits per the manufacturer's
directions for 2 h at 37 °C. The capped, polyadenylated
mRNA reactions were digested with RNase-free DNase I for 15 min at
37 °C, extracted with water-saturated phenol and chloroform, and
purified by oligo(dT)-cellulose chromatography. The oligo(dT)-cellulose
was equilibrated in high salt binding buffer containing 0.5 M NaCl, 20 mM Tris-Cl, pH 7.5, 1 mM
EDTA. The mRNA was heat-denatured for 5 min at 65 °C, chilled in
ice, adjusted to high salt binding buffer conditions, and bound to the
oligo(dT)-cellulose for 30 min at ambient temperature with mixing. The
resin was washed with high salt binding buffer followed by low salt
binding buffer (0.1 M NaCl) and finally eluted with TE (pH
7.5) (65 °C). The purity of the mRNA was checked by agarose gel
electrophoresis, and the concentration was measured by
A260.
Decay and Translation of APP751 mRNA in
Nucleolin-supplemented RRL--
RRL was programmed with 100 ng of
heat-denatured globin versus nucleolin mRNA, and
translation proceeded for 3.5 h at 30 °C per the
manufacturer's directions. Aliquots of globin- and
nucleolin-supplemented RRL were frozen at
80 °C. APP mRNA (50 ng) was incubated with 2 µl of globin- versus
nucleolin-supplemented RRL in 10-µl reactions containing 10%
glycerol, 15 mM HEPES, pH 8, 10 mM KCl, 1.0 mM dithiothreitol, and 200 ng/µl E. coli tRNA
for 10 min at 30 °C. Translation was initiated by the addition of 33 µl of fresh RRL, 0.5 µl of amino acid mixture minus methionine, 0.5 µl of amino acid mixture minus leucine, 1.4 µl of KCl, and 4.6 µl
of water. Reactions (50 µl each) were incubated at 30 °C, and
5-µl aliquots were removed at 0, 30, 60, and 120 min. RNA was
isolated with Tri-Reagent, and RNA pellets were dissolved in 25 µl of
formamide, and 5 µl of each sample was analyzed on
formaldehyde-agarose gels. For translation measurements, RRL reactions
were similar to the RNA decay reactions although
L-[35S]methionine was included. Aliquots (2.5 µl each) were removed at 0.5, 1, 2, and 3 h, diluted with 7.5 µl of water, mixed with 3.5 µl of 4× SDS reducing buffer, heated
for 20 min at 60 °C, and analyzed on 12% SDS-polyacrylamide gels.
The gels were dried and exposed to a PhosphorImager screen.
 |
RESULTS |
We examined the effect of several drugs that influence MAPK, PKC,
protein kinase A (PKA), and phospholipase C (PLC) activity as well as
calcium mobilization on the steady state level of nucleolin mRNA in
PBMC. To investigate the role of PKC, cells were cultured for 3.25 h in the presence of the phorbol ester PMA. Compared with untreated
controls, nucleolin mRNA levels increased by 2.5-fold (Fig.
1, lane 2). In all cases,
nucleolin mRNA was a single 3.0-kilobase pair transcript. The
inactive phorbol ester, 4
-phorbol, did not stimulate nucleolin
mRNA accumulation. Two additional activators of PKC, SC-9 and OAG,
that stimulate calcium/phospholipid-dependent PKC were also
tested (Fig. 1, lanes 3-4). SC-9 had no effect, whereas
OAG, a diacylglycerol analog, had a small stimulatory effect (29%) on
nucleolin mRNA levels. The PKC inhibitors bisindolylmaleimide I and
staurosporine did not block the PMA-mediated increase in nucleolin
mRNA levels indicating that PMA acts independently of PKC
(data not shown).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 1.
MAPK and calcium signaling, but not PKC,
participate in the regulation of nucleolin mRNA. PBMC were
treated with 20 ng/ml 4 -phorbol (lane 1), 20 ng/ml PMA
(lane 2), 10 µM SC-9 (lane 3), 200 µM OAG (lane 4), 0.1%
Me2SO (DMSO, lane 5), 5 µM A23187 (lane 6), 5 µM
ionomycin (lane 7), 50 nM thapsigargin
(lane 8), 10 µM dantrolene (lane
9), and 0.04% PBS (lane 10) for 3.25 h followed
by a 15-min chase with DRB. Total RNA (6 µg) was analyzed by Northern
blotting. A, representative Northern blot of the
nucleolin-specific hybridization signals. B, representative
Northern blot of the S26-specific hybridization signals. C,
histogram depicting nucleolin mRNA levels normalized to
S26 and plotted as a percentage of total nucleolin mRNA. The
error bars represent the S.E. for triplicate samples.
Student's t test results are as follows: PMA,
p = 0.0012; SC-9, p = 0.17; OAG,
p = 0.00040; A23187, p = 0.028;
ionomycin, p = 0.0028; thapsigargin, p = 0.0092; and dantrolene, p = 0.089.
|
|
The effect of several calcium mobilization drugs on nucleolin mRNA
levels was next assessed. The ionophores A23187 and ionomycin, which
transport calcium from the medium into cells, as well as thapsigargin,
which causes the release of calcium from intracellular stores, all
increased nucleolin mRNA levels 1.5-2.5-fold above unstimulated
controls (Fig. 1, lane 6-8). Dantrolene (lane 9), which blocks intracellular calcium release, had no effect on
nucleolin mRNA levels. Therefore, nucleolin mRNA levels were up-regulated in response to drugs that increase intracellular calcium
concentrations via release from intracellular stores or import from the
extracellular environment.
We examined the influence of several activators and inhibitors of PKA
and PLC signal transduction on the steady state level of nucleolin
mRNA in PBMC. Cyclic AMP, a known activator of PKA, and
(Rp)-cAMPS, an inhibitor of PKA, did not change
the steady state level of nucleolin mRNA (Fig.
2, lanes 4-5). Forskolin
activates adenylate cyclase resulting in increased cAMP levels.
1,9-Dideoxyforskolin, a negative control for forskolin, is unable to
stimulate adenylate cyclase. Activation of the adenylate cyclase
pathway with forskolin did not influence the steady state level of
nucleolin mRNA in PBMC (Fig. 2, lane 3). We did observe
a 24% increase in nucleolin mRNA levels upon treatment with
imipramine, a drug that stimulates PLC in rat brain by a
calcium-dependent process (29) (Fig.
3, lane 4). We did not observe
any significant change in nucleolin mRNA levels with D-609,
Et-18-OCH3, or wortmannin (Fig. 3, lanes 2, 3 and 5), drugs that inhibit phosphatidylcholine-specific PLC, phosphatidylinositol-specific PLC, and phosphatidylinositol 3-kinase, respectively. The steroid hormones, estrogen and corticosterone, also
did not affect nucleolin mRNA levels (Fig. 3, lanes 7 and 8). Therefore, the imipramine-mediated effects suggest
that the PLC pathway partially modulates nucleolin mRNA levels in
PBMC.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
PKA and adenylate cyclase agonists do not
affect nucleolin mRNA levels in PBMC. Cells were treated
3.5 h with 0.1% Me2SO (DMSO, lane
1), 13 µM 1,9-dideoxyforskolin (1,9-ddF,
lane 2), 13 µM forskolin (lane 3),
142 µM cAMP (lane 4), 29 µM
(Rp)-cAMPS (lane 5), 0.2% water
(lane 6), and 20 ng/ml PMA (lane 7). After a
15-min chase with DRB, total RNA was isolated and analyzed by Northern
blotting (6 µg of total RNA per lane). A, representative
Northern blot of the nucleolin-specific hybridization signals.
B, representative Northern blot of the 18 S-specific
hybridization signals. C, histogram depicting
nucleolin mRNA levels normalized to 18 S and plotted as a
percentage of total nucleolin mRNA. The error bars
represent the S.E. for triplicate samples. Student's t test
results are as follows: 1,9-dideoxyforskolin, p = 0.22;
forskolin, p = 0.29; cAMP, p = 0.96;
(Rp)-cAMPS, p = 0.065; PMA,
p = 0.063.
|
|

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 3.
PLC pathway agonists do not significantly
alter nucleolin mRNA levels in PBMC. Cells were treated 4 h with 20 ng/ml PMA (lane 1), 19 µM D609
(lane 2), 15 µM ET-18-OCH3
(lane 3), 100 µM imipramine (lane
4), 10 nM wortmannin (lane 5), 100 nM 17 -estradiol (lane 6), 1.5 µM corticosterone (lane 7), 100 nM
17 -estradiol with 1.5 µM corticosterone (est + cort, lane 8), and 0.1% PBS (lane 9)
followed by a 15-min chase with DRB. Total RNA (10 µg per lane) was
analyzed by Northern blotting. A, representative Northern
blot of the nucleolin-specific hybridization signals. B,
representative Northern blot of the S26-specific hybridization signals.
C, histogram depicting nucleolin mRNA levels
normalized to S26 and plotted as a percentage of total nucleolin
mRNA. The error bars represent the S.E. for triplicate
samples. Student's t test results are as follows: PMA,
p = 0.0029; D609, p = 1.0;
Et-18-OCH3, p = 0.11; imipramine,
p = 0.036; wortmannin, p = 0.17;
17 -estradiol, p = 0.23; corticosterone,
p = 0.68; and 17 -estradiol + corticosterone,
p = 0.16.
|
|
PMA is a known activator of the MAPK signal transduction pathway, so we
next assessed the ability of U0126 to block the PMA-mediated increase
in nucleolin mRNA levels (Fig. 4).
Cells were pretreated with 10 µM U0126 for 15 min prior
to the addition of 20 ng/ml PMA for 3 h. The cultures were spiked
with 5 µM U0126 after 1 and 2 h with PMA to maintain
anti-MEK activity. U0126 blocked the PMA-mediated increase in nucleolin
mRNA levels (Fig. 4, lane 3) pointing to the
ERK-specific MAPK pathway as a major player in regulating nucleolin
mRNA levels.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
U0126 partially blocks the PMA-mediated
increase in nucleolin mRNA levels. PBMC were treated with
0.1% Me2SO (DMSO, lane 1), 20 ng/ml
PMA (lane 2), 20 µM U0126 plus 20 ng/ml PMA
(lane 3), or 20 µM U0126 (lane 4).
The U0126, or for controls an equal quantity of Me2SO
vehicle, was added to the cells in increments to maintain activity. The
cells were pretreated for 15 min with 10 µM U0126
followed by 5 µM additions after 1 and 2 h with +/
PMA. DRB was added for 15 min, and total RNA (11 µg per lane) was
isolated and analyzed as described under "Experimental Procedures."
A, representative Northern blot of the nucleolin-specific
hybridization signals. B, representative Northern blot of
the 18 S-specific hybridization signals. C, bar
graph depicting nucleolin mRNA levels normalized to 18 S and
plotted as a percentage of total nucleolin mRNA. The error
bars represent the S.E. for triplicate samples. Student's
t test results are as follows: PMA, p = 0.016; U0126 + PMA, p = 0.0092; U0126,
p = 0.28.
|
|
Nucleolin mRNA accumulation could be secondary to increased
transcription, decreased degradation, or both. Thus, nucleolin mRNA
decay was measured in resting or PMA-treated PBMC. In resting PBMC, the
half-life of nucleolin mRNA was 1.8 h (Fig.
5), which increased almost 2-fold
(t1/2 = 3.2 h) after treatment for 3.5 h
with PMA. Therefore, nucleolin mRNA accumulation after ERK
activation can be accounted for by enhanced stability of the
message.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
PMA up-regulates and stabilizes nucleolin
mRNA. Cells were cultured in the presence of 0.002% ethanol
(lanes 1-6) or 20 ng/ml PMA (lanes 7-12) for
3.5 h. Transcription was stopped by the addition of 20 µg/ml
DRB, and after 15 min, total RNA was isolated at the indicated time
points (0, 1, 2, 4, 6, and 8 h), and 5 µg of total RNA per lane
was analyzed by Northern blotting. A, representative
Northern blot of the nucleolin-specific hybridization signals.
B, representative Northern blot of the 18 S-specific
hybridization signals. C, line graph depicting
the percentage of nucleolin mRNA versus time. The
circles represent nucleolin mRNA levels in the presence
of ethanol alone (vehicle), and the triangles represent the nucleolin
mRNA levels in the presence of PMA. The error bars
represent the S.E. for triplicate samples. Student's t test
results are as follows: at 0 h, p = 0.015; at
1 h, p = 0.00026; at 2 h, p = 0.00013; at 4 h, p = 0.00029; at 6 h,
p = 0.030; at 8 h, p = 0.00015.
|
|
As mentioned earlier, nucleolin protein has been implicated in many
functions including APP mRNA stability. Thus, we examined cell
lysates for nucleolin expression by Western blotting and nucleolin
binding activity by RNA gel mobility shift assays. There was a
time-dependent increase in nucleolin protein levels upon PMA treatment (Fig. 6). On ECL-stained
Western blots probed with a polyclonal anti-nucleolin antibody, we
observed two prominent nucleolin bands at 47 and 65 kDa in unstimulated
PBMC. After PMA stimulation for 15 min, there was an increase in
nucleolin fragments of ~70 kDa, and after 1 h, an increase in
the 80-kDa nucleolin fragment. We observed full-length nucleolin
protein (100 kDa) after 2 h of PMA treatment which continued to
increase for several hours. As full-length nucleolin increased there
was a decrease in the 47-kDa nucleolin cleavage product. RNA mobility
shifts with radiolabeled APP RNA demonstrated a 2.8-fold increase in nucleolin binding after only 20 min of PMA treatment and a 5.1-fold increase by 135 min (Fig. 7). Our
laboratory has previously demonstrated that the 70-kDa nucleolin
polypeptide is responsible for the 84-kDa nucleolin/29-base element
RNA-protein complex (1). In unstimulated cells, there were faint
nucleolin-APP RNA complexes in the 60-70-kDa range (Fig. 7, lane
1) which were previously observed only in stimulated cells (16).
These lower molecular weight complexes are not due to cell stimulation
during the PBMC isolation procedure but rather are a consequence of
including RNasin in the binding buffer. We have found an RNase A-like
activity in PBMC that is inactivated by PMA or ionophore stimulation of
the cells (data not shown). These complexes were specific for
APP mRNA since they could not be detected in gel mobility shift
assays with an APP RNA probe containing a randomized 29-base element
(data not shown).

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 6.
PMA stimulation increases the production of
nucleolin protein in PBMC. Cells were treated for 15 min with
0.002% ethanol (lane 1) or 20 ng/ml PMA for 15 min
(lane 2), 30 min (lane 3), 1 (lane 4),
2 (lane 5), 3 (lane 6), and 4 h (lane
7). Cell lysates (10 µg per lane) were analyzed by
SDS-polyacrylamide gel electrophoresis on a 12% gel, transferred to
nitrocellulose membrane, and stained by ECL with anti-nucleolin
serum.
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
PMA stimulates the ability of the 70-kDa
nucleolin polypeptide to bind to the 3'-UTR of APP mRNA. Cells
were treated for 20 min with 0.002% ethanol (lane 1) or 20 ng/ml PMA for 20 (lane 2), 45 (lane 3), 75 min
(lane 4), 105 (lane 5), 135 (lane 6),
180 (lane 7), and 255 min (lane 8). A,
representative gel mobility shift assay. The arrow denotes
the 84-kDa shift complex. B, histogram depicting
the fold increase in nucleolin binding versus stimulation
time with PMA. The error bars represent the S.E. for
triplicate samples. Student's t test results are as
follows: at 20 min, p = 0.0036; at 45 min,
p = 0.016; at 75 min, p = 0.0066; at
105 min, p = 0.0025; at 135 min, p = 0.0040; at 180 min, p = 0.0047; at 255 min,
p = 0.0065.
|
|
Cell activation with PMA, A23187, and ionomycin caused similar
increases in the 84-kDa nucleolin-APP mRNA complexes (Fig. 8), whereas PKC, PKA, or PLC activation
had no effect. Treatment of cells with U0126 prior to the addition of
PMA blocked shift formation (Fig. 8E, lane 3). Thus drugs
that activated ERK and calcium second messenger pathways increased
nucleolin mRNA, protein, and APP RNA binding activity.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 8.
PMA and ionophores stimulate the ability of
nucleolin to bind to the 3'-UTR of APP mRNA. Cells were
treated with the indicated drugs at the concentrations noted in Fig.
legends 1-3. A, gel mobility shift assays after PKC
activators. Cells were stimulated with 4 -phorbol (lane
1), PMA (lane 2), SC-9 (lane 3), and OAG
(lane 4). B, gel mobility shift assays after
calcium mobilization drugs. Cells were stimulated with
Me2SO (lane 1), A23187 (lane 2),
ionomycin (lane 3), dantrolene (lane 4), and PBS
(lane 5). C, gel mobility shift assays after
PKA/adenylate cyclase agonists. PBMC were treated with
Me2SO (lane 1), 1,9 dideoxyforskolin (lane
2), forskolin (lane 3), cAMP (lane 4),
(Rp)-cAMPS (lane 5), and water
(lane 6). D, gel mobility shift assays after PLC
effectors. Cells were treated with ethanol (lane 1), PMA
(lane 2), D609 (lane 3), ET-18-OCH3
(lane 4), imipramine (lane 5), wortmannin
(lane 6), 17 -estradiol (lane 7),
corticosterone (lane 8), 17 -estradiol with corticosterone
(lane 9), and PBS (lane 10). E, gel
mobility shift assays after ERK agonists. Cells were pretreated with 10 µM U0126 for 15 min prior to the addition of PMA for
3 h with Me2SO (lane 1), PMA (lane
2), U0126 + PMA (lane 3) and U0126 (lane 4).
The arrow denotes the 84-kDa shift complex.
|
|
Nucleolin is a well established RNA-binding protein with RNA helicase
activity (21). We assessed the role nucleolin plays in APP mRNA
stability and translation in an RRL translation system. Globin
(control) or nucleolin mRNA was translated in RRL and subsequently incubated with APP mRNA as described under "Experimental
Procedures." Fresh RRL was then transferred to the preprogrammed APP
mRNA, and translation and mRNA decay were measured by Northern
blot analysis. Preincubation of the APP mRNA template with
nucleolin increased decay of the message (t1/2 = 105 min) (Fig. 9). Nucleolin also accelerated
the decay of an APP mRNA containing a mutated 29-base element
(APPmut mRNA) and decay of globin mRNA (data not
shown). These mRNAs were all stable in RRL when preprogrammed with
globin. Therefore, although nucleolin does bind to the 29-base
instability element, it enhanced decay of APP mRNA independently of
the element.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 9.
Nucleolin destabilizes APP mRNA in an RRL
translation system. In vitro transcribed, capped,
polyadenylated nucleolin, and globin mRNAs (100 ng) were translated
in RRL for 3.5 h. A 2-µl aliquot was incubated with 50 ng of APP
mRNA for 10 min before translation was initiated by the addition of
RRL as described under "Experimental Procedures." Aliquots (5 µl)
were removed at 0, 30, 60, and 120 min followed by RNA isolation and
Northern blotting. A, representative Northern blot of
APP-specific hybridization signals. B, representative
Northern blot of 18 S-specific hybridization signals. C,
line graph depicting the percentage of APP mRNA
remaining versus time. The circles represent APP
mRNA levels after preincubation with globin, and the
triangles represent APP message levels in the presence of
nucleolin. The error bars represent the S.E. for triplicate
samples. Student's t test results are as follows: at 30 min, p = 0.021; at 60 min, p = 0.019;
at 120 min, p = 0.014.
|
|
Nucleolin is a component of translation inhibitory particles (17).
Translation of APP mRNA in RRL was measured by radioactive methionine incorporation. APP mRNA preprogrammed with nucleolin was
translated at 52% of the level as template preprogrammed with globin
(Fig. 10, 1 h). Similarly,
translation of APPmut and globin mRNAs preprogrammed
with nucleolin was also decreased (data not shown). Thus, APP
production was compromised largely by accelerated decay of its coding
mRNA.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 10.
Nucleolin decreases APP translation in a RRL
translation system. Reactions were similar to Fig. 9 except for
the addition of L-[35S]methionine. Aliquots
(2.5 µl) were removed after 0.5 (lane 1), 1 (lane
2), 2 (lane 3), and 3 h (lane 4)
followed by SDS-polyacrylamide gel electrophoresis. A, APP
translation in presence of globin. B, APP translation in
presence of nucleolin. C, line graph depicting
APP translation level versus time. These data are
representative of three experiments.
|
|
 |
DISCUSSION |
Our data indicate that activation of the MAPK or calcium second
messenger pathways in PBMC results in a substantial accumulation of
nucleolin mRNA. Drugs that activate/inhibit the PKA signal transduction pathway did not alter the steady state level of nucleolin mRNA, whereas PKC activation with OAG or PLC activation with
imipramine had only modest effects. PMA stimulation of the cells
resulted in posttranscriptional stabilization of the nucleolin message.
It was previously demonstrated that nucleolin mRNA levels increased
in response to PHA in lymphocytes (30), serum stimulation in HeLa cells
(31), vitamin A in monkey tracheobronchial epithelial cells (32),
interleukin-2 in the C30.1 T cell line (33), and during liver
regeneration in rat hepatocytes (34), whereas nucleolin mRNA levels
were down-regulated during differentiation of human neuroblastoma cells
with dibutyryl cAMP and/or retinoic acid (35) and by glucocorticoids in
murine lymphosarcoma cells (36). In this study, we report that phorbol
ester activation of ERK in PBMC increased nucleolin mRNA levels,
and this increase was predominantly due to posttranscriptional
stabilization of nucleolin mRNA. The half-life of nucleolin
mRNA in resting PBMC was 1.8 h and increased to 3.2 h in
PMA-treated cells. The cis acting domains responsible for regulated
nucleolin mRNA stability are unknown. However, a comparison of the
human, mouse, and hamster genomes reveals the presence of five homology
blocks (nucleotides 2250-2258, 2268-2289, 2296-2302, 2305-2318, and
2321-2329) in the 3'-UTR within a 100-base region immediately
following the stop codon (23). The first homology block is composed
entirely of pyrimidine residues and is found within a
uridine/cytidine-rich area. RNA-binding proteins with known
specificities for pyrimidine-rich regions, such as pyrimidine tract
binding protein, hnRNP C, or nucleolin itself, may bind to this region
and protect the mRNA from RNase attack.
ERK signaling in PBMC resulted in increased nucleolin protein levels
and binding activity to the 3'-UTR of APP mRNA. Nucleolin and hnRNP
C form multiple RNA-protein complexes with the 29-base instability
element situated ~200 bases downstream from the stop codon of APP
mRNA (1). The 70-kDa nucleolin polypeptide is a constituent of
previously identified 84-, 104-, and 140-kDa APP RNA-nucleolin
complexes. The 47- and 48-kDa nucleolin peptides constitute the 65-, 73-, 90-, and 104-kDa complexes, suggesting protein homo- and
heterodimers assemble on APP RNA (1). The 70-, 48-, and 47-kDa
polypeptides all contain the carboxyl-terminal RNP domains. Since we
observed increased binding of the 70-kDa nucleolin fragment to APP
mRNA as more full-length nucleolin was produced by PMA-stimulated
cells, and nucleolin exhibits increased susceptibility to proteolysis
upon binding nucleic acid (37), we cannot exclude the possibility that
full-length nucleolin binds to the 29-base instability element and then
undergoes proteolysis of its amino-terminal domain. The 70-kDa
nucleolin polypeptide as well as hnRNP C bind to the 5'-region of the
29-base element, whereas the smaller nucleolin fragments likely
interact with the 3'-region (1, 16). The transition from the lower
molecular weight nucleolin complexes we observe in unstimulated cells
to the predominant 84-kDa complex in PMA-treated cells coincides with
decreased stability of APP mRNA,2 suggesting that
mRNA stability is dependent on which nucleolin polypeptides bind to
the 29-base element. These data also supports a model in which there is
competition between nucleolin and hnRNP C for binding to the 5'-region
of the cis element.
Phosphorylation and cleavage likely regulate the activity of nucleolin.
Amino-terminal phosphorylation enhanced binding to histone H1 and
chromatin condensation (5). Nucleolin is phosphorylated on
serine residues by casein kinase 2 (CK2) (38, 39) during interphase,
suggesting phosphorylation plays a role in the control of rDNA
transcription, but nucleolin is phosphorylated on threonine residues by
cdc2 kinase during mitosis, which has been linked to mitotic
reorganization of nucleolar chromatin (40, 41). CK2 activity increases
during cellular growth and declines when cells reach quiescence in
parallel with the phosphorylation status of nucleolin (42) suggesting
that CK2 may be an upstream effector of nucleolin. This idea is
strengthened by the fact that the
-subunit of CK2 is physically
associated with nucleolin (43). Nucleolin is present as several
fragments of 100, 70, 60, and 50 kDa in nondividing cells (44), whereas
the major form of the protein is full-length in proliferating cells
(110 kDa). Nucleolin can be cleaved by proteases, for instance granzyme
A (45), but possesses an intrinsic self-cleaving activity (46).
Autocleavage is facilitated by phosphorylation and generates a highly
phosphorylated 30- and a 72-kDa peptide (47). Upon cell proliferation,
the self-cleaving activity is diminished due to nuclear proteolytic
inhibitor(s) that stabilize the full-length nucleolin (44, 48). These
data suggest that signaling cascades involving CK2 or cdc2 kinase
control nucleolin cleavage and activity.
Characterization of the nucleic acid-binding sites of nucleolin
revealed a large array of DNA and RNA motifs including the pre-mRNA
3' splice site sequence r(UUAG/G), the human telomeric DNA sequence
d(TTAGGG)n (49), and the 3'-UTR of poliovirus RNA (50). The
exact poliovirus 3'-UTR sequence has not been determined but may
involve nucleotides 7365-7373 (CAUUUUAGU) which are very similar to
the central portion of the APP 29-base element (16, 50). The nucleolin
recognition element is an 18-base stem-loop containing the sequence
UCCCGA (51). Mutations in this sequence prevented the specific
interaction between nucleolin and the RNA target (52). These data
suggest that nucleolin preferentially binds short stretches of C/U nucleotides.
Our laboratory has demonstrated that the proteins hnRNP C and nucleolin
bind to the 29-base instability element in the 3'-UTR of APP mRNA
(1, 16). We have also shown that hnRNP C binding to this element
stabilizes the APP message and increases protein production in RRL (2).
In PBMC, APP mRNA underwent biphasic decay upon ERK activation with
a rapid, initial fall in message quantity followed by prolonged
stability.2 We have proposed that the stable phase was a
consequence of hnRNP C binding to the 29-base instability element that
prevented RNase attack. In this paper, we demonstrate that nucleolin
mRNA was up-regulated in PBMC via PMA or calcium ionophore
treatment. The phorbol ester-mediated stabilization of nucleolin
mRNA was accompanied by accumulation and decreased processing of
nucleolin protein. The initial drop in the biphasic decay of APP
mRNA corresponds temporally with increased nucleolin production. In
RRL, APP mRNA preprogrammed with nucleolin decays with a half-life
of 105 min but is stable in globin-supplemented RRL. Declines in APP
message levels correspond with decreased translation.
The Gly-rich carboxyl-terminal domain of nucleolin contains DNA and RNA
helicase activity that is modulated by phosphorylation (21). Another
DNA and RNA helicase, G3BP, has sequence similarity with the
carboxyl-terminal portion of nucleolin and is an element of the Ras
signal transduction pathway (53). Collectively our data support a model
in which nucleolin is up-regulated in response to ERK activation and
then unwinds APP mRNA allowing RNase attack. Although nucleolin
binds to the pyrimidine-rich 29-base element, the destabilizing effect
of this protein on APP mRNA appears independent of the cis element
since APPmut mRNA also decays rapidly.
These findings are meaningful in that APP undergoes processing by
-
and
-secretases to produce
-amyloid, a 40-42-amino acid peptide
found in the senile plaques characteristic of Alzheimer's disease and
Down's syndrome. Significant levels of nucleolin have been found in
mature brain and in differentiating neuronal cells (13). Thus,
dysregulation of this multifunctional protein could play an important
role in regulating APP mRNA stability, APP levels, and
-amyloid production.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Raymond Petryshyn (Children's
National Medical Center, Washington D. C.) for anti-nucleolin
polyclonal antibody; Dr. Jeff Ross and Dr. Richard Spritz (University
of Wisconsin, Madison, WI) for the plasmids pT7T90 and pSP
c,
respectively; Dr. Meera Srivastava (Georgetown University School of
Medicine, Washington D. C.) for the plasmid pMAMnucleolin;
the nursing staff (Infusion Center, University of Wisconsin Hospital)
for drawing blood from volunteer donors; Dr. William Rehrauer
(Molecular Diagnostics Laboratory, University of Wisconsin Hospital)
for automated sequencing of plasmids; and members of the laboratory for
their thoughtful comments.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant RO1AG10675 (to J. S. M.) and NIA Research Service Award AG00213 from the National Institutes of Health (to C. J. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Wisconsin Hospital and Clinics, Rm. K4/812, 600 Highland Ave., Madison, WI 53792. Tel.: 608-263-6043; Fax: 608-265-6215; E-mail:
jsmalter@facstaff.wisc.edu.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M009435200
2
C. J. Westmark and J. S. Malter,
submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
APP, amyloid
precursor protein;
CK2, casein kinase 2;
D609, tricyclodecan-9-yl
xanthogenate;
DRB, 5,6-dichlorobenzimidazole riboside;
ECL, enhanced
chemiluminescence;
ERK, extracellular-regulated kinase;
ET-18-OCH3, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine;
hnRNP, heterogeneous nuclear ribonucleoprotein;
MAPK, mitogen-activated
protein kinase;
OAG, 1-oleoyl-2-acetyl-sn-glycerol
(C18:1,[cis]-9/C2:0);
PBMC, peripheral blood mononuclear
cells;
PBS, phosphate-buffered saline;
PKA, protein kinase A;
PKC, protein kinase C;
PLC, phospholipase C;
PMA, phorbol 12-myristate
13-acetate;
RRL, rabbit reticulocyte lysate;
SC-9, N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide;
U0126, bis[2-aminophenylthio]butadiene;
UTR, untranslated region.
 |
REFERENCES |
1.
|
Zaidi, S. H.,
and Malter, J. S.
(1995)
J. Biol. Chem.
270,
17292-17298[Abstract/Free Full Text]
|
2.
|
Rajagopalan, L. E.,
Westmark, C. J.,
Jarzembowski, J. A.,
and Malter, J. S.
(1998)
Nucleic Acids Res.
26,
3418-3423[Abstract/Free Full Text]
|
3.
|
Lapeyre, B.,
Bourbon, H.,
and Amalric, F.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
1472-1476[Abstract]
|
4.
|
Lischwe, M. A.,
Richards, R. L.,
Busch, R. K.,
and Busch, H.
(1981)
Exp. Cell Res.
136,
101-109[Medline]
[Order article via Infotrieve]
|
5.
|
Erard, M. S.,
Belenguer, P.,
Caizergues-Ferrer, M.,
Pantaloni, A.,
and Amalric, F.
(1988)
Eur. J. Biochem.
175,
525-530[Abstract]
|
6.
|
Egyhazi, E.,
Pigon, A.,
Chang, J. H.,
Ghaffari, S. H.,
Dreesen, T. D.,
Wellman, S. E.,
Case, S. T.,
and Olson, M. O.
(1988)
Exp. Cell Res.
178,
264-272[Medline]
[Order article via Infotrieve]
|
7.
|
Jordan, G.
(1987)
Nature
329,
489-490[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Ginisty, H.,
Amalric, F.,
and Bouvet, P.
(1998)
EMBO J.
17,
1476-1486[Abstract/Free Full Text]
|
9.
|
Bouvet, P.,
Diaz, J. J.,
Kindbeiter, K.,
Madjar, J. J.,
and Amalric, F.
(1998)
J. Biol. Chem.
273,
19025-19029[Abstract/Free Full Text]
|
10.
|
Belenguer, P.,
Baldin, V.,
Mathieu, C.,
Prats, H.,
Bensaid, M.,
Bouche, G.,
and Amalric, F.
(1989)
Nucleic Acids Res.
17,
6625-6636[Abstract]
|
11.
|
Yang, T. H.,
Tsai, W. H.,
Lee, Y. M.,
Lei, H. Y.,
Lai, M. Y.,
Chen, D. S.,
Yeh, N. H.,
and Lee, S. C.
(1994)
Mol. Cell. Biol.
14,
6068-6074[Abstract]
|
12.
|
Derenzini, M.,
Sirri, V.,
Trere, D.,
and Ochs, R. L.
(1995)
Lab. Invest.
73,
497-502[Medline]
[Order article via Infotrieve]
|
13.
|
Kibbey, M. C.,
Johnson, B.,
Petryshyn, R.,
Jucker, M.,
and Kleinman, H. K.
(1995)
J. Neurosci. Res.
42,
314-322[Medline]
[Order article via Infotrieve]
|
14.
|
Brockstedt, E.,
Rickers, A.,
Kostka, S.,
Laubersheimer, A.,
Dorken, B.,
Wittmann-Liebold, B.,
Bommert, K.,
and Otto, A.
(1998)
J. Biol. Chem.
273,
28057-28064[Abstract/Free Full Text]
|
15.
|
Borer, R. A.,
Lehner, C. F.,
Eppenberger, H. M.,
and Nigg, E. A.
(1989)
Cell
56,
379-390[Medline]
[Order article via Infotrieve]
|
16.
|
Zaidi, S. H.,
Denman, R.,
and Malter, J. S.
(1994)
J. Biol. Chem.
269,
24000-24006[Abstract/Free Full Text]
|
17.
|
Yurkova, M. S.,
and Murray, M. T.
(1997)
J. Biol. Chem.
272,
10870-10876[Abstract/Free Full Text]
|
18.
|
Semenkovich, C. F.,
Ostlund, R. E., Jr.,
Olson, M. O.,
and Yang, J. W.
(1990)
Biochemistry
29,
9708-9713[Medline]
[Order article via Infotrieve]
|
19.
|
Take, M.,
Tsutsui, J.,
Obama, H.,
Ozawa, M.,
Nakayama, T.,
Maruyama, I.,
Arima, T.,
and Muramatsu, T.
(1994)
J. Biochem. (Tokyo)
116,
1063-1068[Abstract]
|
20.
|
Larrucea, S.,
Gonzalez-Rubio, C.,
Cambronero, R.,
Ballou, B.,
Bonay, P.,
Lopez-Granados, E.,
Bouvet, P.,
Fontan, G.,
Fresno, M.,
and Lopez-Trascasa, M.
(1998)
J. Biol. Chem.
273,
31718-31725[Abstract/Free Full Text]
|
21.
|
Tuteja, N.,
Huang, N. W.,
Skopac, D.,
Tuteja, R.,
Hrvatic, S.,
Zhang, J.,
Pongor, S.,
Joseph, G.,
Faucher, C.,
Amalric, F.,
and Falaschi, A.
(1995)
Gene (Amst.)
160,
143-148[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Srivastava, M.,
Fleming, P. J.,
Pollard, H. B.,
and Burns, A. L.
(1989)
FEBS Lett.
250,
99-105[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Srivastava, M.,
McBride, O. W.,
Fleming, P. J.,
Pollard, H. B.,
and Burns, A. L.
(1990)
J. Biol. Chem.
265,
14922-14931[Abstract/Free Full Text]
|
24.
|
Bugler, B.,
Bourbon, H.,
Lapeyre, B.,
Wallace, M. O.,
Chang, J. H.,
Amalric, F.,
and Olson, M. O.
(1987)
J. Biol. Chem.
262,
10922-10925[Abstract/Free Full Text]
|
25.
|
Lapeyre, B.,
Amalric, F.,
Ghaffari, S. H.,
Rao, S. V.,
Dumbar, T. S.,
and Olson, M. O.
(1986)
J. Biol. Chem.
261,
9167-9173[Abstract/Free Full Text]
|
26.
|
Ghisolfi, L.,
Kharrat, A.,
Joseph, G.,
Amalric, F.,
and Erard, M.
(1992)
Eur. J. Biochem.
209,
541-548[Abstract]
|
27.
|
Ghisolfi, L.,
Joseph, G.,
Amalric, F.,
and Erard, M.
(1992)
J. Biol. Chem.
267,
2955-2959[Abstract/Free Full Text]
|
28.
|
Malter, J. S.
(1989)
Science
246,
664-666[Medline]
[Order article via Infotrieve]
|
29.
|
Fukuda, H.,
Nishida, A.,
Saito, H.,
Shimizu, M.,
and Yamawaki, S.
(1994)
Neurochem. Int.
25,
567-571[Medline]
[Order article via Infotrieve]
|
30.
|
Mehes, G.,
and Pajor, L.
(1995)
Cell Proliferation
28,
329-336[Medline]
[Order article via Infotrieve]
|
31.
|
Konishi, T.,
Karasaki, Y.,
Nomoto, M.,
Ohmori, H.,
Shibata, K.,
Abe, T.,
Shimizu, K.,
Itoh, H.,
and Higashi, K.
(1995)
J. Biochem. (Tokyo)
117,
1170-1177[Abstract]
|
32.
|
Reddy, P. M.,
An, G.,
Di, Y. P.,
Zhao, Y. H.,
and Wu, R.
(1996)
Am. J. Respir. Cell Mol. Biol.
15,
398-403[Abstract]
|
33.
|
Herblot, S.,
Chastagner, P.,
Samady, L.,
Moreau, J. L.,
Demaison, C.,
Froussard, P.,
Liu, X.,
Bonnet, J.,
and Theze, J.
(1999)
J. Immunol.
162,
3280-3288[Abstract/Free Full Text]
|
34.
|
Ohmori, H.,
Murakami, T.,
Furutani, A.,
Higashi, K.,
Hirano, H.,
Gotoh, S.,
Kuroiwa, A.,
Masui, A.,
Nakamura, T.,
and Amalric, F.
(1990)
Exp. Cell Res.
189,
227-232[Medline]
[Order article via Infotrieve]
|
35.
|
Murakami, T.,
Ohmori, H.,
Gotoh, S.,
Tsuda, T.,
Ohya, R.,
Akiya, S.,
and Higashi, K.
(1991)
J. Biochem. (Tokyo)
110,
146-150[Abstract]
|
36.
|
Meyuhas, O.,
Baldin, V.,
Bouche, G.,
and Amalric, F.
(1990)
Biochim. Biophys. Acta
1049,
38-44[Medline]
[Order article via Infotrieve]
|
37.
|
Olson, M. O.,
Kirstein, M. N.,
and Wallace, M. O.
(1990)
Biochemistry
29,
5682-5686[Medline]
[Order article via Infotrieve]
|
38.
|
Caizergues-Ferrer, M.,
Belenguer, P.,
Lapeyre, B.,
Amalric, F.,
Wallace, M. O.,
and Olson, M. O.
(1987)
Biochemistry
26,
7876-7883[Medline]
[Order article via Infotrieve]
|
39.
|
Csermely, P.,
Schnaider, T.,
Cheatham, B.,
Olson, M. O.,
and Kahn, C. R.
(1993)
J. Biol. Chem.
268,
9747-9752[Abstract/Free Full Text]
|
40.
|
Belenguer, P.,
Caizergues-Ferrer, M.,
Labbe, J. C.,
Doree, M.,
and Amalric, F.
(1990)
Mol. Cell. Biol.
10,
3607-3618[Medline]
[Order article via Infotrieve]
|
41.
|
Peter, M.,
Nakagawa, J.,
Doree, M.,
Labbe, J. C.,
and Nigg, E. A.
(1990)
Cell
60,
791-801[Medline]
[Order article via Infotrieve]
|
42.
|
Schneider, H. R.,
and Issinger, O. G.
(1989)
Biochim. Biophys. Acta
1014,
98-100[Medline]
[Order article via Infotrieve]
|
43.
|
Li, D.,
Dobrowolska, G.,
and Krebs, E. G.
(1996)
J. Biol. Chem.
271,
15662-15668[Abstract/Free Full Text]
|
44.
|
Fang, S. H.,
and Yeh, N. H.
(1993)
Exp. Cell Res.
208,
48-53[CrossRef][Medline]
[Order article via Infotrieve]
|
45.
|
Pasternack, M. S.,
Bleier, K. J.,
and McInerney, T. N.
(1991)
J. Biol. Chem.
266,
14703-14708[Abstract/Free Full Text]
|
46.
|
Bourbon, H. M.,
Bugler, B.,
Caizergues-Ferrer, M.,
Amalric, F.,
and Zalta, J. P.
(1983)
Mol. Biol. Rep.
9,
39-47[Medline]
[Order article via Infotrieve]
|
47.
|
Warrener, P.,
and Petryshyn, R.
(1991)
Biochem. Biophys. Res. Commun.
180,
716-723[Medline]
[Order article via Infotrieve]
|
48.
|
Chen, C. M.,
Chiang, S. Y.,
and Yeh, N. H.
(1991)
J. Biol. Chem.
266,
7754-7758[Abstract/Free Full Text]
|
49.
|
Ishikawa, F.,
Matunis, M. J.,
Dreyfuss, G.,
and Cech, T. R.
(1993)
Mol. Cell. Biol.
13,
4301-4310[Abstract]
|
50.
|
Waggoner, S.,
and Sarnow, P.
(1998)
J. Virol.
72,
6699-6709[Abstract/Free Full Text]
|
51.
|
Serin, G.,
Joseph, G.,
Faucher, C.,
Ghisolfi, L.,
Bouche, G.,
Amalric, F.,
and Bouvet, P.
(1996)
Biochimie (Paris)
78,
530-538[CrossRef][Medline]
[Order article via Infotrieve]
|
52.
|
Ghisolfi-Nieto, L.,
Joseph, G.,
Puvion-Dutilleul, F.,
Amalric, F.,
and Bouvet, P.
(1996)
J. Mol. Biol.
260,
34-53[CrossRef][Medline]
[Order article via Infotrieve]
|
53.
|
Costa, M.,
Ochem, A.,
Staub, A.,
and Falaschi, A.
(1999)
Nucleic Acids Res.
27,
817-821[Abstract/Free Full Text]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.