Intracellular Redistribution of Nucleolin upon Interaction with the CD3epsilon Chain of the T Cell Receptor Complex*

Diana Gil, Dolores Gutiérrez, and Balbino AlarcónDagger

From the Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain

Received for publication, November 7, 2000, and in revised form, December 12, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

T cell activation through the antigen receptor (TCR) involves the cytoplasmic tails of the CD3 subunits CD3gamma , CD3delta , CD3epsilon , and CD3zeta . Whereas the biological significance of the cytoplasmic tails of these molecules is suggested, in part, by their evolutionarily conserved sequences, their interactions with signal transduction molecules are not completely understood. We used affinity chromatography columns of glutathione S-transferase fused to the CD3epsilon cytoplasmic tail to isolate proteins that specifically interact with this subunit. In this way, we identified the shuttling protein nucleolin as a specific CD3epsilon -interacting molecule. Using competition studies and affinity chromatography on peptide columns, we were able to identify a central proline-rich sequence as the nucleolin-interacting sequence in CD3epsilon . Transfection in COS cells of wild type CD3epsilon , but not of nonbinding mutants of CD3epsilon , resulted in redistribution of nucleolin from the nucleus and nucleoli to the cytoplasm. This property was transferred to a CD8 protein chimera by appending the cytoplasmic tail of CD3epsilon . We also found that nucleolin associated with the TCR complex. This association was increased upon TCR engagement, suggesting that the CD3epsilon /nucleolin interaction may have a role in T cell activation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

T cells respond to antigen via a polypeptide complex composed of ligand-binding T cell receptor (TCR)1 alpha  and beta  chains (or gamma  and delta  in gamma delta T cells) and the CD3 subunits CD3gamma , CD3delta , CD3epsilon , and CD3zeta (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 CD3gamma , CD3delta , and CD3epsilon chains and in three copies in CD3zeta (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 CD3zeta or CD3epsilon 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 CD3epsilon 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 CD3epsilon have shown the specific interaction of a nuclear protein, topoisomerase IIbeta , and a tyrosine-phosphorylated protein, CAST, with the N-terminal region of the CD3epsilon 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 CD3epsilon tail-interacting proteins, we have utilized affinity chromatography using glutathione S-transferase (GST) epsilon  columns. In this way, we were able to characterize nucleolin as a major CD3epsilon -interacting protein that associates with the central proline-rich region. We also show herein that expression of CD3epsilon 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/CD3epsilon interaction in TCR-mediated T cell activation is proposed.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-CD8alpha 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 CD3epsilon (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 CD3epsilon respectively, were synthesized by the N-(9-fluorenyl)methoxycarbonyl (Fmoc) method and purified by HPLC.

DNA Constructs-- To generate the GSTepsilon fusion protein, a 165-base pair cDNA fragment, corresponding to the whole cytoplasmic tail of human CD3epsilon (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 CD8alpha . The polymerase chain reaction product was cloned into the XhoI and BamHI sites of the pSRalpha expression vector. The CD8/epsilon construct expressing the extracellular and transmembrane domains of human CD8alpha fused to the cytoplasmic tail of CD3epsilon 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 CD3epsilon , a GSTepsilon column was generated by absorbing 20 ml of GSTepsilon -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 GSTepsilon columns. Both columns were washed with 100 ml of lysis buffer. GST- and GSTepsilon -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 GSTepsilon 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/epsilon 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Nucleolin as a Specific CD3epsilon -binding Protein-- To characterize proteins that specifically associate with the cytoplasmic tail of CD3epsilon , a construct for the expression of a GSTepsilon fusion protein containing the whole cytoplasmic tail of CD3epsilon was made. GSTepsilon 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 GSTepsilon column. Bound proteins were eluted with glutathione, dialyzed, and absorbed again to GST and GSTepsilon 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 GSTepsilon 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 GSTepsilon column and resisted extraction with 50 mM triethylamine, pH 11 (Fig. 1A, lane 6), suggests that their interaction with the cytoplasmic tail of CD3epsilon 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 CD3epsilon , the eluates from the GST and GSTepsilon columns were immunoblotted with a specific monoclonal anti-nucleolin antibody. This antibody reacted with a 110-kDa protein band (protein a) present in the GSTepsilon 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 CD3epsilon in vitro.



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Fig. 1.   Identification of nucleolin as a CD3epsilon -binding protein. A, affinity chromatography on GSTepsilon columns. The postnuclear supernatant of a Jurkat cell lysate was first passed through a GST column and then through a GSTepsilon column. Bound proteins were eluted with glutathione and rerun through GST and GSTepsilon 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 GSTepsilon column; lane 3, pH 11.0 eluate from the second GST column; lane 4, pH 11.0 eluate from the second GSTepsilon column; lane 5, glutathione eluate from the second GST column; lane 6, glutathione eluate from the second GSTepsilon column. Molecular mass standards are indicated on the right side. Protein bands a, b, c, d, and e specifically binding to the GSTepsilon 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 GSTepsilon columns were resolved by SDS-PAGE and immunoblotted with the anti-nucleolin antibody.

Nucleolin Binds to the Central Proline-rich Region of the Cytoplasmic Tail of CD3epsilon -- To map the binding site of nucleolin on the cytoplasmic tail of CD3epsilon , we used the monoclonal antibody APA1/1, which binds a well defined sequence within this tail. Using a GSTepsilon column to pull down CD3epsilon -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 GSTepsilon 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 GSTepsilon -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 CD3epsilon . 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 CD3epsilon (Fig. 2B). This suggested that the binding site of nucleolin maps to the central region of the tail of CD3epsilon . 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 CD3epsilon (peptide 7) inhibited binding of nucleolin forms to GSTepsilon (Fig. 2A). Additional evidence that nucleolin binds the central proline-rich region of the tail of CD3epsilon 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 CD3epsilon . A, blocking of and competition for nucleolin binding. A postnuclear cell lysate from [35S]methionine-labeled Jurkat cells was incubated with GSTepsilon -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 CD3epsilon 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.

Expression of CD3epsilon in an Heterologous Cell System Promotes Intracellular Redistribution of Nucleolin-- To determine whether the observed interaction of nucleolin with the cytoplasmic tail of CD3epsilon resulted in a change in the intracellular distribution of these proteins, CD3epsilon was transfected into COS cells, and the localization of transfected CD3epsilon and endogenous nucleolin was assessed by two-color immunofluorescence. Nucleolin was found in the nucleus and the nucleolus (Fig. 3A, red staining), whereas CD3epsilon 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 CD3epsilon -expressing cells. To determine whether the effect of CD3epsilon expression on nucleolin distribution correlated with its capacity to interact with nucleolin, different CD3epsilon mutants were assayed. Unlike wild type CD3epsilon , transfection with a truncated (tail-less) CD3epsilon construct did not alter nucleolin distribution (Fig. 3B). This inferred that expression of the cytoplasmic tail of CD3epsilon was necessary for nucleolin redistribution and suggested that the effect of CD3epsilon on nucleolin is mediated by its ability to interact with it. However, because the deletion of CD3epsilon tail resulted in a loss of its ER retention (Fig. 3B), it is also possible that the effect on nucleolin requires the localization of CD3epsilon 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 CD3epsilon (Fig. 2B) was assayed. This mutant has lost the capacity to interact with the antibody APA1/1 (12). Like wild type CD3epsilon , 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 CD3epsilon . Indeed, in cells that overexpress CD3epsilon , nucleolin was found to colocalize with CD3epsilon in the ER (Fig. 3B). These results indicate that CD3epsilon requires nucleolin binding capacity for its effect on nucleolin distribution. Nevertheless, localization of CD3epsilon to the ER seems to be required as well, because transfection of two C-terminal deletion mutants of CD3epsilon that result in loss of ER retention (14) did not cause CD3epsilon redistribution (data not shown).



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Fig. 3.   Relocalization of nucleolin in CD3epsilon -transfected COS cells. A, COS cells transfected with wild type CD3epsilon were stained with the anti-nucleolin antibody (red fluorescence) and an anti-CD3epsilon 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 CD3epsilon -expressing cells show no staining of nucleolin. B, effect of mutations in the cytoplasmic tail of CD3epsilon on nucleolin distribution. COS cells were transfected with a tail-less, truncated, CD3epsilon mutant, with a CD3epsilon deletion mutant lacking the nucleolin-binding site (del 9), or with wild type CD3epsilon . Cells were stained for nucleolin and CD3epsilon and examined under fluorescence microscopy. Notice that the expression of truncated (del 9) CD3epsilon did not alter the nuclear distribution of nucleolin, whereas expression of wild type CD3epsilon resulted in redistribution of nucleolin to the cytoplasm.

Expression of a Protein Chimera Containing the Cytoplasmic Tail of CD3epsilon Results in Redistribution of Nucleolin-- To determine whether expression of the cytoplasmic tail of CD3epsilon is sufficient to enable nucleolin relocalization, a protein chimera consisting of the cytoplasmic tail of CD3epsilon appended to the transmembrane and extracellular domains of CD8alpha (CD8/epsilon ) was obtained (23). As a control, a truncated mutant of CD8alpha lacking the cytoplasmic tail was used (CD8t). Although the CD8/epsilon construct is in part retained in the ER because the cytoplasmic tail of CD3epsilon contains an ER retention sequence (14, 15), both CD8/epsilon 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/epsilon chimera in the magnetic bead-selected COS cell population (65% CD8/epsilon +) led to the localization of nucleolin in the cytoplasm (Fig. 4). In contrast, in the nonselected population (95% CD8/epsilon -) 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 CD3epsilon was sufficient to promote the intracellular redistribution of nucleolin.



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Fig. 4.   Effect of CD3epsilon tail expression on nucleolin levels and localization. Expression of a CD8/epsilon chimera but not of truncated CD8 resulted in redistribution of nucleolin to the cytoplasm. COS cells transfected with the CD8/epsilon 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/epsilon -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.

Antibody-mediated TCR Cross-linking Increases Nucleolin Recruitment to the TCR-- To determine whether the CD3epsilon /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-CD3epsilon 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 CD3epsilon , 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 CD3epsilon /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-CD3epsilon 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.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These results show that the cytoplasmic tail of human CD3epsilon 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 CD3epsilon with two other proteins, topoisomerase IIbeta and CAST, a tyrosine phosphorylated protein. Both proteins interact with the N-terminal part of the cytoplasmic tail of CD3epsilon , a region rich in basic amino acids. Therefore, the cytoplasmic tail of CD3epsilon appears to interact with different proteins along its sequence: with topoisomerase IIbeta 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 CD3epsilon interacts with two nuclear proteins, topoisomerase IIbeta and nucleolin, the interaction with both proteins might be facilitated by a possible location of CD3epsilon in the inner nuclear membrane (13). CD3epsilon contains a sequence at the N-terminal portion of the cytoplasmic tail reminiscent of a nuclear localization signal. Moreover, CD3epsilon 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, CD3epsilon 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 CD3epsilon for its interaction with nucleolin, and topoisomerase IIbeta , could conceivably be the nucleus.

A second possible location for the interaction of CD3epsilon 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 CD3epsilon . 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 CD3epsilon 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 CD3epsilon and can be transferred by appending the CD3epsilon tail to the extracellular and transmembrane domains of an irrelevant protein. Thus, the correlation between nucleolin-binding capacity and nucleolin redistribution induced by CD3epsilon indicates that CD3epsilon promotes nucleolin relocalization by directly binding nucleolin.

Similar to the effect of CD3epsilon 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 CD3epsilon -induced relocalization of nucleolin is not well understood yet. For the topoisomerase IIbeta -CD3epsilon 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 IIbeta , CD3epsilon 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/CD3epsilon 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 CD3epsilon 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).


    ACKNOWLEDGEMENTS

We are grateful to Drs. D. Pappin, F. Barahona, A. Marina, and J. Vázquez for help with protein sequencing and Drs. B. Ballou, B. Malissen, and C. Terhorst for kindly providing reagents. We thank Dr. M. López-Trascasa, K. Jones, W. A. Schamel, and F. Kierszenbaum for helpful discussions and manuscript revision. We also thank Toñi Cerrato and Maite Gómez for expert technical assistance.


    FOOTNOTES

* This work was supported by Grants PM98-0132 from the Comisión Interministerial de Ciencia y Tecnología, FEDER Grant 2FD97-1436, Grant 08.3/0021/98 from the Comunidad de Madrid, and by funds from the Fundación Ramón Areces to the Centro de Biología Molecular.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.

Dagger To whom correspondence should be addressed: Centro de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain. Tel.: 34-913-97-84-58; Fax: 34-913-97-47-99; E-mail: Balarcon@cbm.uam.es.

Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M010114200


    ABBREVIATIONS

The abbreviations used are: TCR, T cell antigen receptor; ER, endoplasmic reticulum; GST, glutathione S-transferase; ITAM, immunoreceptor tyrosine activation motif; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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