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INTRODUCTION |
T cells respond to antigen via a polypeptide complex composed of
ligand-binding T cell receptor
(TCR)1
and
chains (or
and
in 
T cells) and the CD3 subunits CD3
, CD3
,
CD3
, and CD3
(1, 2). Unlike the TCR chains, the CD3 components
have long cytoplasmic tails that associate with cytoplasmic signal
transduction molecules. This association is mediated at least in part
by a double tyrosine-based motif present in a single copy in the
CD3
, CD3
, and CD3
chains and in three copies in CD3
(3).
This motif, named immune-receptor tyrosine-based activation motif
(ITAM), becomes tyrosine phosphorylated during T cell activation by the
Src family protein-tyrosine kinases Lck and/or Fyn (4-6). Tyrosine
phosphorylated ITAM become docking sites for the Syk family
protein-tyrosine kinase ZAP70 and other signal-transducing molecules.
It is well established that antibody-mediated engagement of protein
chimeras containing the cytoplasmic tail of either CD3
or CD3
results in T cell activation (7-10). These data indicate that the
cytoplasmic tail of one of these subunits can be sufficient to induce T
cell activation. Regarding the role of CD3 subunits in T cell
activation, most of the attention has been focused on the ITAM.
However, the cytoplasmic tails of the CD3 subunits contain other
evolutionarily conserved features that suggest ITAM-independent roles
for them.
The CD3
cytoplasmic tail, highly conserved (11, 12), can be
tentatively subdivided into three regions; the N-terminal region
contains a basic amino acid cluster, the central region contains a
proline-rich sequence, and the C-terminal region contains the ITAM
(13). The proline-rich sequence contains the SH3-binding consensus
motif XPPXP, and the C-terminal region
contains the YXXLXXR endoplasmic reticulum
(ER) retention sequence, which partially overlaps the ITAM (14, 15).
Previous attempts to identify proteins that associate with the
cytoplasmic tail of CD3
have shown the specific interaction of a
nuclear protein, topoisomerase II
, and a tyrosine-phosphorylated
protein, CAST, with the N-terminal region of the CD3
tail (13,
16).
Nucleolin is a major nucleolar protein of exponentially growing
eukaryotic cells that is directly involved in the regulation of
ribosome biogenesis and maturation (17, 18). Nucleolin has a molecular
mass of 100-110 kDa and is mainly found in the fibrillar
components of the nucleoli where it associates with nascent
preribosomal RNA. Numerous reports have implicated the involvement of
nucleolin either directly or indirectly in the regulation of cell
proliferation and growth, cytokinesis, replication, embryogenesis, and
nucleogenesis (17, 18). Although predominantly localized in the
nucleolus, nucleolin has also been found in the cytoplasm and at the
plasma membrane, where it can function as a cell surface receptor for
ligands as different as coxsackie B viruses and the complement
inhibitor factor J (18-20). Because nucleolin acts as a shuttling
protein between the cytoplasm and the nucleus, it might provide a
mechanism for extracellular regulation of nuclear events.
Nucleolin activity is regulated by proteolysis, methylation,
ADP-ribosylation, and phosphorylation by casein kinase II, Cdc2, PKC,
cyclic AMP-dependent protein kinase, and ecto-protein
kinase (17, 18). Nucleolin is cleaved by a leupeptin-sensitive protease that is tightly associated with it. It has been also suggested that
nucleolin itself may possess a self-cleaving activity.
In an attempt to identify novel CD3
tail-interacting proteins, we
have utilized affinity chromatography using glutathione S-transferase (GST)
columns. In this way, we were able
to characterize nucleolin as a major CD3
-interacting protein that
associates with the central proline-rich region. We also show herein
that expression of CD3
in a heterologous cell system results in loss of both nucleolin localization in the nucleolus and redistribution to
the cytoplasm. A possible role of nucleolin/CD3
interaction in
TCR-mediated T cell activation is proposed.
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EXPERIMENTAL PROCEDURES |
Cells and Reagents--
The COS-7 African green monkey cell line
was grown in Dulbecco's modified Eagle's medium supplemented
with 5% fetal bovine serum (Sigma). The human leukemic T cell line
Jurkat was grown in RPMI 1640 medium supplemented with 5% fetal bovine serum.
The mouse monoclonal anti-human nucleolin antibody D3 (21) used in this
study was a gift from Dr. B. Ballou (University of Pittsburgh, PA). The
mouse monoclonal anti-CD8
B9.4 was donated by Dr. B. Malissen
(Center d'Immunologie, Marseille-Luminy, France). The mouse monoclonal
antibody SP34, specific for the extracellular domain of human CD3
(22), was a gift from Dr. C. Terhorst (Beth Israel Deaconess Hospital,
Boston, MA). The mouse monoclonal anti-human CD3 antibody UCHT1 was
donated by Dr. P. Beverley (The Edward Jenner Institute for Vaccine
Research, Berkshire, UK). Peptides 7 and 8, corresponding to amino
acids 170-185 and amino acids 150-166 of human CD3
respectively,
were synthesized by the N-(9-fluorenyl)methoxycarbonyl (Fmoc) method and purified by HPLC.
DNA Constructs--
To generate the GST
fusion protein, a
165-base pair cDNA fragment, corresponding to the whole cytoplasmic
tail of human CD3
(amino acids 131-185), was generated by
polymerase chain reaction. This fragment was digested and inserted into
the XhoI and NotI sites of the plasmid pGEX4T3
(Amersham Pharmacia Biotech). The truncated CD8 construct was prepared
by polymerase chain reaction by introducing a stop codon after the
second cytoplasmic amino acid of human CD8
. The polymerase chain
reaction product was cloned into the XhoI and
BamHI sites of the pSR
expression vector. The CD8/
construct expressing the extracellular and transmembrane domains of
human CD8
fused to the cytoplasmic tail of CD3
has been
previously described (23) and was a gift from Dr. C. Terhorst.
Affinity Chromatography--
To characterize proteins that
interact with the cytoplasmic tail of CD3
, a GST
column was
generated by absorbing 20 ml of GST
-producing Escherichia
coli lysate (resulting from a 1-liter culture) to 1 ml of
glutathione-Sepharose (Amersham Pharmacia Biotech). A similar preabsorb
column was prepared from GST-producing E. coli. A total of
4 × 109 Jurkat cells were lysed in 1% Nonidet P-40
lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris-HCl, pH 7.8, 10 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin). A postnuclear supernatant of the Jurkat cell lysate was serially passed first through the GST and then through the GST
columns. Both columns were washed with 100 ml of lysis buffer. GST- and
GST
-bound proteins were eluted in lysis buffer containing 10 mM glutathione (eluate 1). The eluates were dialyzed
against lysis buffer and passed through new GST and GST
columns.
These second columns were first eluted with 20 mM
triethylamine, pH 11.0 (eluate 2), and then with 10 mM
glutathione in lysis buffer (eluate 3). The three eluates were
subjected to SDS-PAGE and transferred to a polyvinylidene difluoride
membrane (Millipore). Protein bands on the membrane were visualized by
Coomassie Blue staining, excised and subjected to trypsin digestion
in situ. The resulting peptides were purified by HPLC and
were sequenced either by the Edman degradation method or by
electrospray mass spectrometry.
For affinity chromatography on peptide columns, 10 mg of peptides 7 or
8 were coupled to 1-ml Hi-Trap
N-hydroxysuccinimide-activated agarose columns
(Amersham Pharmacia Biotech). A postnuclear cell lysate from 4 × 109 Jurkat cells in lysis buffer was passed first through
the peptide 7 column and subsequently through the peptide 8 column.
After washing with 2 column volumes of lysis buffer, bound proteins were eluted in 50 mM triethylamine, pH 11.0. The eluates
were neutralized with 100 mM Tris-HCl, pH 7.4, and
subjected to SDS-PAGE.
COS Cell Transfections and Immunoprecipitation--
COS cell
transfections and immunoprecipitation were performed as previously
described (12).
Immunofluorescence and Microscopy--
COS cells were fixed and
stained for immunofluorescence as previously described (12).
Immunoselection--
CD8+ COS cells transfected with
either CD8/
or truncated CD8 were detached from the culture plates
using phosphate-buffered saline and repeated pipetting. After
incubating the cells with 4 µg/ml B9.4 antibody on ice for 1 h,
they were washed and incubated with goat anti-mouse IgG-coated magnetic
beads (Dynal) at a 3:1 bead to cell ratio for 30 min. The
CD8+ cells were isolated with a magnet (Dynal), washed with
phosphate-buffered saline, and plated on culture dishes.
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RESULTS |
Identification of Nucleolin as a Specific CD3
-binding
Protein--
To characterize proteins that specifically associate with
the cytoplasmic tail of CD3
, a construct for the expression of a
GST
fusion protein containing the whole cytoplasmic tail of CD3
was made. GST
protein purified from E. coli extracts was absorbed to glutathione-Sepharose columns. A cell lysate from the human
T cell line Jurkat was first preabsorbed to a GST column and then
passed through the GST
column. Bound proteins were eluted with
glutathione, dialyzed, and absorbed again to GST and GST
columns to
increase specificity. Proteins bound to the second columns were eluted
first with a high pH buffer and finally with glutathione. A number of
proteins were absorbed to GST
but not to GST (Fig.
1A, lanes 1 and
2). These protein bands of 110, 97, 90, 68, and 47 kDa
(indicated as arrows a, b, c,
d, and e) were excised, eluted, and fragmented,
and partial amino acid sequences were obtained. The fact that proteins
d and e reassociated with the GST
column
and resisted extraction with 50 mM triethylamine, pH 11 (Fig. 1A, lane 6), suggests that their
interaction with the cytoplasmic tail of CD3
is quite strong.
Proteins a, c, and d produced
sequences that were identical with nucleolin and products of its
partial proteolysis (Fig. 1A). Protein bands b
and e yielded no sequence. To confirm that nucleolin
associates to the cytoplasmic tail of CD3
, the eluates from the GST
and GST
columns were immunoblotted with a specific monoclonal
anti-nucleolin antibody. This antibody reacted with a 110-kDa protein
band (protein a) present in the GST
eluate but not in the
GST eluate (Fig. 1B). These results showed that nucleolin
and its partial proteolytic fragments specifically associate with the
cytoplasmic tail of CD3
in vitro.

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Fig. 1.
Identification of nucleolin as a
CD3 -binding protein. A,
affinity chromatography on GST columns. The postnuclear supernatant
of a Jurkat cell lysate was first passed through a GST column and then
through a GST column. Bound proteins were eluted with glutathione
and rerun through GST and GST columns, respectively. The second
columns were eluted first with a pH 11.0 buffer and, finally, with
glutathione. Lane 1, an aliquot of the glutathione eluate
from the first GST column; lane 2, glutathione eluate from
the first GST column; lane 3, pH 11.0 eluate from the
second GST column; lane 4, pH 11.0 eluate from the second
GST column; lane 5, glutathione eluate from the second
GST column; lane 6, glutathione eluate from the second
GST column. Molecular mass standards are indicated on the
right side. Protein bands a, b,
c, d, and e specifically binding to
the GST columns were collected and sequenced. The resulting
sequences of some tryptic fragments are indicated aligned with the
human nucleolin sequence. B, identification of nucleolin by
immunoblotting. Aliquots from the first GST and GST columns were
resolved by SDS-PAGE and immunoblotted with the anti-nucleolin
antibody.
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Nucleolin Binds to the Central Proline-rich Region of the
Cytoplasmic Tail of CD3
--
To map the binding site of nucleolin
on the cytoplasmic tail of CD3
, we used the monoclonal antibody
APA1/1, which binds a well defined sequence within this tail. Using a
GST
column to pull down CD3
-interacting proteins from
[35S]methionine-labeled Jurkat cell lysates, we detected
several major protein bands, including nucleolin and its partial
degradation product of 68 kDa, and actin (Fig.
2A, mock). Incubation of the cell lysate with APA1/1 completely inhibited the binding to GST
of
the 110- and 68-kDa nucleolin forms but not the binding of actin or
other contaminant proteins (Fig. 2A, APA1/1). A
75-kDa GST
-associated protein was also completely displaced by
APA1/1, but this protein probably represents a partial proteolysis
product of nucleolin. This result indicated that the antibody APA1/1
specifically inhibits the association of nucleolin with the cytoplasmic
tail of CD3
. In previous work (12), we mapped the binding site of APA1/1 to a 10-amino acid region in the central proline-rich region of
the tail of CD3
(Fig. 2B). This suggested that the
binding site of nucleolin maps to the central region of the tail of
CD3
. To confirm this, a competition experiment was set up using a
17-mer synthetic peptide (peptide 8) that expands the APA1/1-binding site. Peptide 8 but not a control peptide of the same length expanding the C-terminal region of CD3
(peptide 7) inhibited binding of nucleolin forms to GST
(Fig. 2A). Additional evidence
that nucleolin binds the central proline-rich region of the tail of
CD3
was the finding that nucleolin binds to a column of immobilized
peptide 8 but not of peptide 7 (Fig. 2C).

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Fig. 2.
Characterization of the nucleolin-binding
site of the cytoplasmic tail of CD3 .
A, blocking of and competition for nucleolin binding. A
postnuclear cell lysate from [35S]methionine-labeled
Jurkat cells was incubated with GST -Sepharose beads in the absence
(mock) or presence of 100 µg/ml APA1/1 antibody, 100 µg/ml peptide
7, or 100 µg/ml peptide 8. The samples were subjected to SDS-PAGE,
and the gel was dried and exposed to the PhosphorImager. The positions
of nucleolin and the 68-kDa nucleolin fragment as well as the position
of the contaminant protein actin are indicated. Compared with
mock-treated beads, incubation with APA1/1 reduced nucleolin binding by
95%. Competition by peptide 8 resulted in a 67% inhibition, whereas
peptide 7 was noninhibitory (22%). B, sequence of the
cytoplasmic tail of human CD3 indicated in the one-letter code. The
sequence deleted in mutant 9 as well as the APA1/1 binding site is
shown in bold type. The sequences corresponding to peptides
7 and 8 are also indicated. C, binding of nucleolin to
peptide columns. A postnuclear Jurkat cell lysate was incubated with
peptides 7 and 8 immobilized on agarose columns. Bound and eluted
proteins were resolved by SDS-PAGE and immunoblotted with the
anti-nucleolin antibody.
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Expression of CD3
in an Heterologous Cell System Promotes
Intracellular Redistribution of Nucleolin--
To determine whether
the observed interaction of nucleolin with the cytoplasmic tail of
CD3
resulted in a change in the intracellular distribution of these
proteins, CD3
was transfected into COS cells, and the localization
of transfected CD3
and endogenous nucleolin was assessed by
two-color immunofluorescence. Nucleolin was found in the nucleus and
the nucleolus (Fig. 3A,
red staining), whereas CD3
was detected in the cytoplasm
and nuclear membrane in an ER-characteristic pattern (Fig.
3A, green staining). Interestingly, both
stainings were mutually exclusive, i.e. nucleolin staining was not observed in CD3
-expressing cells. To determine whether the
effect of CD3
expression on nucleolin distribution correlated with
its capacity to interact with nucleolin, different CD3
mutants were assayed. Unlike wild type CD3
, transfection with a truncated (tail-less) CD3
construct did not alter nucleolin distribution (Fig.
3B). This inferred that expression of the cytoplasmic tail of CD3
was necessary for nucleolin redistribution and suggested that
the effect of CD3
on nucleolin is mediated by its ability to
interact with it. However, because the deletion of CD3
tail resulted
in a loss of its ER retention (Fig. 3B), it is also possible that the effect on nucleolin requires the localization of CD3
in the
ER rather than direct binding to nucleolin. To discriminate between
these possibilities, mutant 9, a deletion mutant that lacks 10 amino
acids of the central, proline-rich region of CD3
(Fig.
2B) was assayed. This mutant has lost the capacity to
interact with the antibody APA1/1 (12). Like wild type CD3
, mutant 9 was also located in the ER (Fig. 3B). However, mutant 9 did
not alter the nucleolar location of nucleolin, strongly suggesting that
redistribution of nucleolin requires direct binding to CD3
. Indeed,
in cells that overexpress CD3
, nucleolin was found to colocalize
with CD3
in the ER (Fig. 3B). These results indicate that
CD3
requires nucleolin binding capacity for its effect on nucleolin
distribution. Nevertheless, localization of CD3
to the ER seems to
be required as well, because transfection of two C-terminal deletion
mutants of CD3
that result in loss of ER retention (14) did not
cause CD3
redistribution (data not shown).

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Fig. 3.
Relocalization of nucleolin in
CD3 -transfected COS cells. A,
COS cells transfected with wild type CD3 were stained with the
anti-nucleolin antibody (red fluorescence) and an
anti-CD3 antibody (green fluorescence). The image shows a
0.5-µm-thick optical section taken at mid-distance from the coverslip
in the confocal microscope. Note that CD3 -expressing cells show no
staining of nucleolin. B, effect of mutations in the
cytoplasmic tail of CD3 on nucleolin distribution. COS cells were
transfected with a tail-less, truncated, CD3 mutant, with a CD3
deletion mutant lacking the nucleolin-binding site (del 9),
or with wild type CD3 . Cells were stained for nucleolin and CD3
and examined under fluorescence microscopy. Notice that the
expression of truncated (del 9) CD3 did not alter the
nuclear distribution of nucleolin, whereas expression of wild type
CD3 resulted in redistribution of nucleolin to the cytoplasm.
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Expression of a Protein Chimera Containing the Cytoplasmic Tail of
CD3
Results in Redistribution of Nucleolin--
To determine
whether expression of the cytoplasmic tail of CD3
is sufficient to
enable nucleolin relocalization, a protein chimera consisting of the
cytoplasmic tail of CD3
appended to the transmembrane and
extracellular domains of CD8
(CD8/
) was obtained (23). As a
control, a truncated mutant of CD8
lacking the cytoplasmic tail was
used (CD8t). Although the CD8/
construct is in part retained in the
ER because the cytoplasmic tail of CD3
contains an ER retention
sequence (14, 15), both CD8/
and CD8t were expressed on the cell
surface. This allowed separation of transfected COS cells from
untransfected cells by immunoselection with antibody-coated magnetic
beads. As anticipated, the expression of the CD8/
chimera in the
magnetic bead-selected COS cell population (65% CD8/
+)
led to the localization of nucleolin in the cytoplasm (Fig. 4). In contrast, in the nonselected
population (95% CD8/
) nucleolin was located in the
nucleus and nucleolus. Expression of CD8t did not alter the nuclear
localization of nucleolin (Fig. 4). This indicated that the cytoplasmic
tail of CD3
was sufficient to promote the intracellular
redistribution of nucleolin.

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Fig. 4.
Effect of CD3 tail
expression on nucleolin levels and localization. Expression of a
CD8/ chimera but not of truncated CD8 resulted in redistribution of
nucleolin to the cytoplasm. COS cells transfected with the CD8/
chimera were immunoselected. The selected and nonselected populations
were stained with anti-CD8 and anti-nucleolin antibodies and examined
under fluorescence microscopy. Notice that in the selected
CD8/ -expressing COS cells nucleolin is distributed to the cytoplasm,
whereas in the nonselected population, nucleolin is located in the
nucleus. In COS cells expressing truncated CD8 nucleolin remained in
the nucleus.
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Antibody-mediated TCR Cross-linking Increases Nucleolin Recruitment
to the TCR--
To determine whether the CD3
/nucleolin interaction
takes place in T cells and whether the interaction changes upon TCR
engagement, the human T cell line Jurkat was stimulated with the
anti-CD3 antibody UCHT1 followed by cross-linking with a secondary
antibody. Mock-stimulated and stimulated cells were lysed, and
immunoprecipitation was carried out with the anti-CD3
antibody SP34.
Immunoblotting of the SP34 immunoprecipitates with anti-nucleolin
antibody showed that nucleolin is associated to the TCR complex in
nonstimulated T cells (Fig. 5). The
association was increased in Jurkat cells stimulated with anti-CD3
antibody. These results show that the TCR complex, probably via CD3
,
interacts with nucleolin in T cells and that more nucleolin is
recruited to the TCR complex when this is cross-linked with antibodies,
suggesting that the CD3
/nucleolin interaction may have a role in T
cell activation.

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Fig. 5.
TCR engagement increases nucleolin
association to the TCR complex. Jurkat cells were stimulated with
a combination of the anti-CD3 antibody UCHT1 and a cross-linking second
antibody for 5 min (+ stimulus) or left untreated ( stimulus). The cells were then lysed and immunoprecipitation
(Ip) was carried out with anti-CD3 antibody SP34.
Immunoprecipitates were resolved by SDS-PAGE and immunoblotting was
performed with the anti-nucleolin antibody. A sample of the
total lysate was run in parallel as a control. NIS,
precipitation with nonimmune serum. H, immunoglobulin heavy
chain.
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DISCUSSION |
These results show that the cytoplasmic tail of human CD3
interacts with nucleolin in vitro. We mapped the site of
interaction to a central 10-17-amino acid proline-rich sequence within
the cytoplasmic tail. Recently, Saito's group (13, 16) has described the interaction of the cytoplasmic tail of CD3
with two other proteins, topoisomerase II
and CAST, a tyrosine phosphorylated protein. Both proteins interact with the N-terminal part of the cytoplasmic tail of CD3
, a region rich in basic amino acids. Therefore, the cytoplasmic tail of CD3
appears to interact with different proteins along its sequence: with topoisomerase II
and
CAST in the N-terminal region, with nucleolin in the central portion,
and via the ITAM (C terminus) with ZAP70 and probably other
SH2-containing proteins (23, 24).
Although at first glance it would seem unlikely that the cytoplasmic
tail of CD3
interacts with two nuclear proteins, topoisomerase II
and nucleolin, the interaction with both proteins might be facilitated
by a possible location of CD3
in the inner nuclear membrane (13).
CD3
contains a sequence at the N-terminal portion of the cytoplasmic
tail reminiscent of a nuclear localization signal. Moreover, CD3
has
a double arginine sequence in the central portion of its cytoplasmic
tail that is reminiscent of the signal sequence responsible for the
localization of glycoprotein B of human cytomegalovirus (a
transmembrane protein) in the inner nuclear membrane (25). Indeed,
CD3
has been located in the nucleus (13), although the roles of the
nuclear localization signal and the presence of a nuclear inner
membrane localization signal have not yet been demonstrated. Therefore,
the intracellular location site of CD3
for its interaction with
nucleolin, and topoisomerase II
, could conceivably be the nucleus.
A second possible location for the interaction of CD3
with nucleolin
could be the cytoplasm. Nucleolin has been shown to shuttle between the
cytoplasm and the nucleus. Indeed, the interaction of nucleolin with
several cytoplasmic proteins and even plasma membrane proteins has been
reported (18-20). We have shown in this study that nucleolin interacts
with the TCR complex in T cells, probably through CD3
. Although we
cannot discriminate whether nucleolin interacts with the TCR at the
plasma membrane or with the intracellular pool of TCR, the fact that
antibody engagement of the TCR results in increased recruitment of
nucleolin suggests that nucleolin interacts with the TCR at the plasma
membrane. In addition, our present results reveal that expression of
CD3
in COS cells results in the loss of nuclear localization of
nucleolin and, in some cases, in relocalization to the cytoplasm. This
effect is dependent on the expression of the nucleolin-interacting
sequence in the central portion of CD3
and can be transferred by
appending the CD3
tail to the extracellular and transmembrane
domains of an irrelevant protein. Thus, the correlation between
nucleolin-binding capacity and nucleolin redistribution induced by
CD3
indicates that CD3
promotes nucleolin relocalization by
directly binding nucleolin.
Similar to the effect of CD3
expression, it has been described that
infection by poliovirus causes a relocalization of nucleolin to the
cytoplasm, perhaps by binding of nucleolin to the 3' noncoding region
of poliovirus RNA (26). However, although the role of nucleolin binding
to poliovirus RNA seems to be that of promoting assembly of new
virions, the role of CD3
-induced relocalization of nucleolin is not
well understood yet. For the topoisomerase II
-CD3
interaction, it
has been proposed that it could be involved in signal transduction
because topoisomerase II inhibitors up-regulate IL-2 production and
apoptosis (13). Therefore, by binding topoisomerase II
, CD3
could
participate in TCR-induced growth arrest and apoptosis of T cells. It
has also been described that nucleolin binds specifically to a Jun
N-terminal kinase response element and that this binding is required
for interleukin-2 mRNA stabilization induced by T cell activation
signals (27). Our observation that the association of nucleolin to the
TCR is increased upon antibody-mediated cross-linking of the TCR
suggests that nucleolin/CD3
interaction may have a role in T cell
activation. Although a positive effect on T cell activation cannot be
excluded, we favor the hypothesis that recruitment of nucleolin to the
TCR through CD3
and redistribution of nucleolin to the cytoplasm may
have roles in TCR-induced growth arrest, given the important roles for
cell survival and proliferation that nucleolin plays at the nucleus
(17, 18).