©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Specific Interaction of Topoisomerase II and the CD3 Chain of the T Cell Receptor Complex (*)

(Received for publication, July 17, 1995; and in revised form, November 4, 1995)

Hiroyasu Nakano (§) Tetsuo Yamazaki Shoichiro Miyatake Naoto Nozaki (1) Akihiko Kikuchi (1) Takashi Saito (¶)

From the Division of Molecular Genetics, Center for Biomedical Science, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260, Japan Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

T cell antigen receptor (TCR)bulletCD3 complex is composed of six different subunits: TCRalpha and TCRbeta and CD3, CD3, CD3, and CD3. Antigen recognition signals are transduced from TCR to the cytoplasm through the cytoplasmic domain of the CD3 chains. To understand the downstream signal transduction pathways, we cloned genes encoding proteins capable of binding to CD3 with a probe of glutathione S-transferase fused to the cytoplasmic region of CD3. One of these clones was found to encode topoisomerase IIbeta (topoIIbeta). The binding region of CD3 is located within the N-terminal 12 amino acids containing the motif of a basic amino acid cluster. A similar motif was found in the chain of Fc receptors (FcR) but not in the CD3 chain, and indeed, FcR but not CD3 bound to topoIIbeta. The binding region of topoIIbeta was determined to be the C terminus. Since this region appears to be the regulatory region of the enzymatic activity, the binding of CD3 might affect the function of topoIIbeta. Although topoIIbeta is localized mainly in the nucleus and CD3 is a membrane protein, we demonstrated the presence of CD3 in the nuclear fraction of thymocytes, which increased upon T cell activation. The specific interaction in cells was evidenced by co-immunoprecipitation of topoIIbeta and CD3 from the nuclear fraction of T cells. The possible function of this interaction is discussed.


INTRODUCTION

The T cell antigen receptor (TCR)(^1)bulletCD3 complex is composed of six subunits: clonotypic alpha and beta chains and invariant CD3, CD3, CD3, and CD3 chains. TCRalpha and TCRbeta chains recognize antigen in association with major histocompatibility complex molecules, and the CD3 chains are responsible for transducing antigen recognition signals by TCR from the membrane to the cytoplasm. CD3, CD3, CD3, and CD3 contain a conserved amino acid sequence motif, ITAM (immunoreceptor tyrosine based activation motif) in their cytoplasmic domain(1) , which is composed of a pair of the YXXL/I sequence with a spacer of 7-8 amino acids. ITAM is also present in the cytoplasmic tail of Igalpha and Igbeta of the B cell antigen receptor complex and the chain of Fc receptors (FcR)(2). Since the TCRbulletCD3 complex does not possess any intrinsic kinase function, tyrosine kinases associated with the complex have been shown to be important for signal transduction. Fyn and Lck associate noncovalently with the TCRbulletCD3 complex and CD4/CD8, respectively(3, 4) . Upon TCR stimulation, these kinases are activated and phosphorylate several cellular substrates(5) . Two tyrosine residues within ITAM are also phosphorylated and become the binding site of the SH2 domains of ZAP-70 or Syk kinase(6, 7) . In T cells, the recruitment of ZAP-70 to CD3 or CD3 (8, 9) induces the activation of this kinase and subsequently exhibits functions such as the production of lymphokines.

Utilizing chimeric molecules such as CD8 or Tac- (the extracellular domain of CD8 or the alpha chain of the IL-2 receptor, fused to the cytoplasmic domain of or , respectively)(10, 11) , it has been demonstrated that each of the cytoplasmic domains of CD3 chains induces similar activation events to those through the TCRbulletCD3 complex, including tyrosine phosphorylation, Ca mobilization, and IL-2 production. On the other hand, there is evidence to indicate that the TCR complex is composed of two activation modules, CD3 and CD3(12, 13) . Molecules important for signaling pathways associated with each of the activation modules have to be determined. Signals mediated through the chain are required for some activation such as Thy-1(12, 13) - and CD2-mediated stimulation (14) . The relationship between signals through these two modules has not yet been clarified. In addition, it has been shown that stimulation through CD3 of immature thymocytes without undergoing rearrangement of TCR genes induces differentiation of thymocytes, indicating that signals through CD3 play important roles in T cell development(15, 16) . Furthermore, although phosphorylation-dependent signals in T cells have been extensively analyzed, the molecules associated with unphosphorylated forms of CD3 or CD3 other than Fyn have not yet been identified(3, 17) .

In order to understand the downstream signaling events through CD3, one of the TCR activation modules, we cloned genes encoding CD3-binding proteins. One of these clones was found to encode topoisomerase IIbeta (topoIIbeta)(18) . We demonstrated the presence of CD3 in the nuclear fraction upon T cell activation and showed the evidence of the specific association in vivo between CD3 and topoIIbeta in this fraction of T cells. Detailed mapping of the binding regions of both CD3 and topoIIbeta indicates that the association depends on a novel motif in CD3 and suggests that the binding might modulate the in vivo function of topoIIbeta.


MATERIALS AND METHODS

Cell Culture

The murine T cell hybridoma 2B4 (19) and DO11.10 was grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 5 mM glutamine, 50 µM 2-mercaptoethanol, and 100 µg/ml kanamycin. C57BL/6J Jcl (B6) mice were obtained from the Japanese Clea Animal Corp. (Tokyo, Japan).

Antibodies and Reagents

Anti-CD3 monoclonal antibodies (mAb), 145-2C11 and HMT3-1, were kindly provided by J. Bluestone (University of Chicago) and R. Kubo (Cytel Inc., La Jolla, CA), respectively. mAb to topoisomerase IIbeta (beta5A7) was established from the mice immunized with the full-length recombinant topoIIbeta prepared in Escherichia coli(^2)and mAb to glutathione S-transferase (GST) was a gift from Dr. Y. Tachibana (Japan Zeon Inc., Tokyo, Japan). Anti-beta-galactosidase antibody (Life Technologies, Inc.), peroxidase-conjugated anti-mouse immunoglobulin (Amersham International, Buckinghamshire, United Kingdom), anti-tubulin antibody (Seikagaku Corp., Tokyo, Japan) and a polyclonal goat anti-hamster Ab (GAH) (Organon Teknika-Cappel) were from commercial sources.

Construction of GST Fusion Proteins

GST fusion protein constructs containing the cytoplasmic region of CD3, CD3, FcR, as well as serial deletion constructs of CD3, designated ED1, ED3, ED4 and ED5, were prepared by using pGEX-2TK vector (a kind gift from Dr. E. K. Flemington, Harvard University). The following shows the names of the constructs (the corresponding amino acid (aa) regions of the constructs) and the pair of primers to prepare the insert by polymerase chain reaction (PCR): pGEX-2TK- (aa CD3135-189), E5 and E3; pGEX-2TK-ED3 (aa CD3135-169), E5 and ED3; pGEX-2TKT-ED4 (aa CD3147-189), ED4 and E3; pGEX-2TK-ED5 (aa CD3135-189), E5 and ED5; pGEX-2TK- (aa 53-164), Z5 and Z3; pGEX-2TK-c (aa 131-164), Zc5 and Z3; pGEX-2TK-FcRI (aa 59-100), G5 and G3.

All constructs were prepared by PCR with the following primers: E5, 5`-CGGGATCCAAGAATAGGAAGGCCAAG-3`; E3, 5`-CGGAATTCTCAGACTGCTCTCTGATT-3`; ED3, 5`-CGGAATTCGTCTGGGTTGGGAACAGG-3`; ED4, 5`-CGGGATCCGGAACCGGTGCTGGTAGC-3`; ED5, 5`-CGGAATTCCCGCTCCTTGTTTTGCCC-3`; Z5, 5`-CGGGATCCAGAGCAAAATTCAGCAGG-3`; Zc5, 5`-CGGGATCCGAGAGGCGGAGAGGCAAG-3`; Z3, 5`-CGGAATTCAAATGCCCTGGCTGTTAG-3`; G5, 5`-CGGGATCCCGACTCAAGATCCAGGTC-3`; G3, 5`-CGGAATTCCTGTTCTGAAGCTACTGG-3`. All PCR products were digested with BamHI and EcoRI and ligated to the BamHI/EcoRI sites of pGEX-2TK vector. pGEX-2TK-ED1 (aa CD3135-146) was made by using the internal AvaII site of CD3. pGEX-2TK- was digested with AvaII and EcoRI, blunted with a Klenow fragment, and religated. To introduce mutation(s) in the tyrosine residues of ITAM of CD3, in vitro mutagenesis was performed according to the manufacturer's instructions (Promega, Madison, WI), and pGEX-2TK-EM3 (the first tyrosine of ITAM was changed to phenylalanine) and pGEX-2TK-EM4 (both tyrosines were altered to phenylalanines) were made. pGEX-2TK-, pGEX-2TK-c, and pGEX-2TK-FcR were generated to produce GST fusion proteins containing the cytoplasmic region of , the C-terminal single ITAM of , and the cytoplamic region of FcR, respectively. Each construct was confirmed by nucleotide sequence. pGEX-2TK-fyn was kindly provided by Dr. H. Umemori (The Research Institute of Medical Science, University of Tokyo).

Deletion constructs of pGEX-topoIIbeta were prepared by using the internal EcoRI site of topoIIbeta cDNA. The cDNA insert of clone 3 was digested with EcoRI and two fragments (0.7 and 0.9 kb) were subcloned into pGEX-1 vector to generate pGEX-topoIIbeta1 (0.7 kb) and pGEX-topoIIbeta2 (0.9 kb), respectively.

These constructs were introduced into ompT-negative E. coli strain, AD202(20) . Proteins were induced with isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified as described previously(20) . After binding to glutathione-Sepharose beads, proteins were eluted with reduced glutathione (Sigma). Protein concentration was determined by the Bradford method (Bio-Rad).

Preparation of P-Labeled GST Fusion Proteins

P-Labeled GST fusion proteins were prepared as described previously(21) . Briefly, purified GST fusion proteins were adsorbed onto glutathione-Sepharose beads. The beads were washed once with a kinase buffer (20 mM Tris (pH 7.5), 100 mM NaCl, 12 mM MgCl(2)) and then resuspended in 2-3 bead volumes of the buffer containing the catalytic subunit of cAMP-dependent protein kinase (Sigma), [-P]ATP, and 1 mM dithiothreitol. The kinase reaction proceeded for 30 min and was terminated by the addition of 1 ml of a stop buffer (10 mM sodium phosphate (pH 8.0), 10 mM sodium pyrophosphate, 10 mM EDTA, 1 mg/ml bovine serum albumin). After the supernatant was removed, the beads were washed with a TNEN buffer (20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40). Labeled GST fusion proteins were eluted by agitating the beads for 15 min in 10-50 bead volumes of 20 mM reduced glutathione, 100 mM Tris (pH 8.0), 120 mM NaCl. The probes were labeled to high specific activity (approximately 5-10 times 10^5 cpm/µg of protein).

Screening of cDNA Library

A gt11 library, constructed from mRNA from an HTLV-1 transformed human T cell line, HAT109, was obtained from Dr. M. Yoshida (The Research Institute of Medical Science, University of Tokyo). A total of 1.6 times 10^6 plaques were screened with a P-labeled GST- fusion protein. After incubation of the plates at 42 °C for 4 h, the plates were overlaid with nitrocellulose filters that had been impregnated with IPTG and incubated at 37 °C overnight. The filters were then removed, washed with a TBST buffer (10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Triton X-100) at room temperature, denatured with 6 M guanidine hydrochloride in a Hepes balanced buffer (HBB) (20 mM Hepes (pH 7.5), 5 mM MgCl(2), 1 mM KCl) and renatured as described previously(21) . Thereafter, the membranes were blocked in the HBB buffer containing 5% dry milk (Yukijirushi Ltd., Hokkaido, Japan) at 4 °C for 1 h and then with 1% dry milk HBB. Labeled probes were added at approximately 5 times 10^4 cpm/ml to HBB and incubated overnight. The filters were then washed three times with phosphate-buffered saline (PBS) containing 0.2% Triton X-100 at 4 °C, dried, and exposed at -80 °C. Positive phages were subsequently isolated and the cDNA inserts were sequenced after subcloning into pBluescript.

DNA Sequencing

Double-stranded or single-stranded DNA sequencing was performed using Sequenase 2.0® (Upstate Biotechnology, Inc.) and BcaBEST® (Takara, Shiga, Japan) according to the manufacturer's instructions.

Filter Binding Experiments

The lysogens from the positive phages were prepared by standard procedure (22) for filter binding experiments. The lysogens of phages, GST-topoIIbeta1 and GST-topoIIbeta2, were induced with IPTG. The induced and uninduced proteins were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) with a transfer buffer without methanol as described previously(21) . The membranes were denatured with 6 M guanidine hydrochloride, renatured, and blocked with 5% dry milk-PBS for 1 h at room temperature. The membranes were probed with 0.1-1 µg/ml of GST- or GST alone, followed by incubation with anti-GST mAb or anti-beta galactosidase antibody in PBS-0.2% Tween 20 (PBS-T) at 4 °C overnight. After washing with PBS-T, the membranes were incubated with peroxidase conjugated anti-mouse Ig antibody at room temperature for 1 h, washed three times with PBS-T, and developed with an ECL detection system (Amersham).

In Vitro Solution Binding Experiments and Western Blots

After 5 µg of each GST fusion protein was preabsorbed on the glutathione-Sepharose by incubating at 4 °C for 1 h, the nuclear extracts of cells were added to the mixtures and further incubated at 4 °C for 2 h. The mixtures were washed three times with PBS-0.1% Nonidet P-40 (PBS-N). The lysates were subjected to SDS-PAGE, and the proteins were transferred to PVDF membranes. The membrane were blocked with Block Ace (Yukijirushi Ltd., Hokkaido, Japan) at room temperature for 1 h, incubated with anti-topoIIbeta mAb (beta5A7) for 1 h at room temperature, washed three times with a TBS-T buffer (20 mM Tris (pH 7.6), 137 mM NaCl, 0.2% Tween 20), and incubated with peroxidase-conjugated anti-mouse Ig antibody at room temperature for 1 h. After three washes, the membranes were developed with an ECL detection system.

Preparation of Nuclear Extracts

Cells were lysed in a lysis buffer (1% digitonin (Aldrich) or 1% Nonidet P-40, 50 mM Tris (pH 7.6), 150 mM NaCl, 1 mM EDTA, 0.15 unit/ml aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate, and 1 mM sodium fluoride) at 4 °C for 30 min. The lysate was centrifuged at 1500 rpm for 10 min, and the pellet was washed twice with the lysis buffer. The supernatants were used as the cytosolic fractions. The nuclear extracts were prepared as described by Drake et al.(23) with some minor modifications. Briefly, the nuclear pellets were resuspended in a nuclear extraction buffer (5 mM potassium phosphate (pH 7.0), 2 mM MgCl(2), 0.1 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, 10% glycerol, 0.5 mM iodoacetoamide). 5 M NaCl solution was added slowly to make a final concentration of 0.35 M, and nuclear fractions were extracted at 4 °C for 60 min with constant stirring. Extracts were centrifuged at 15,000 rpm for 20 min, and the supernatants were then dialyzed overnight at 4 °C against PBS containing 0.5 mM PMSF. After centrifugation at 15,000 rpm for 20 min, the supernatants were collected and used as the nuclear extracts.

Immunoprecipitation

The cell lysates or the nuclear extracts from 2B4 or DO11.10 T cell hybridoma cells were immunoprecipitated with 5 µg of anti-CD3 mAb 2C11 or anti-human CD3 mAb OKT3 as a control and protein A-Sepharose beads. The precipitates were washed with the lysis buffer containing 0.1-0.5% Nonidet P-40 for the digitonin lysate or with 1% Brij 96-containing buffer for the Brij lysate. Immunoprecipitates were analyzed on 8% SDS-PAGE.

TCR Stimulation

Stimulation was performed as described previously(24) . Briefly, 2B4 cells (4 times 10^7 cells) or murine thymocytes (7 times 10^7 cells) were incubated with 2C11 (10 µg/ml) for 30 min on ice, washed twice with RPMI 1640, and stimulated by adding prewarmed GAH (100 µg/ml) at 37 °C for 2 min. The reaction was stopped by adding ice-cold PBS, and cells were lysed for 30 min at 4 °C in a lysis buffer. Cytosolic extracts and nuclear extracts were prepared according to the same protocol as described above.

Biotinylation on Sorbent

Biotinylation on sorbent was performed as described previously(25) . Briefly, cytosolic or nuclear extracts of 2B4 hybridomas (4 times 10^7 cells) and thymocytes (7 times 10^7 cells) were immunoprecipitated with protein A-Sepharose beads coupled with anti-CD3 mAb (HMT3-1) at 4 °C for 2 h. The beads were washed three times with the lysis buffer and once with PBS, and proteins on the beads were biotinylated by incubation with 100 µg/ml biotin (Pierce) in 1 ml of the labeling buffer (0.01 M Hepes (pH 8.0), 150 mM NaCl) at 4 °C for 1 h. The beads were washed three times with the lysis buffer, and the proteins were eluted by boiling in the SDS-PAGE sample buffer and were then analyzed by nonreducing-reducing two-dimensional SDS-PAGE (12% for the first dimensional gel under nonreducing condition and 14% for the second dimensional gel under reducing condition).


RESULTS

Cloning of cDNAs Encoding the CD3-binding Proteins

A gt11 expression library prepared from a human T cell line was screened with a probe of P-labeled GST fusion protein containing the cytoplasmic domain of the CD3 chain (GST-). Eight positive clones (clones 3, 6, 10, 16, 25, 36, 43, and 46) encoding beta-galactosidase fusion proteins were picked up and subjected to further analysis. Subsequent analysis demonstrated that three (10, 43, 46) and two clones (6, 36) contained overlapping cDNA sequences, respectively. Among the resulting five cDNAs encoding CD3-binding proteins, we described the characterization of clone 3 (C3) in the present study.

Topoisomerase IIbeta Specifically Binds to CD3

To confirm the binding specificity of C3 to GST-, we performed a filter binding assay with lysogenic phage lysates. As shown in Fig. 1, the GST- probe bound to a protein of approximately 180 kDa from the IPTG-induced lysate of C3, but not from the uninduced lysate of this clone nor from the induced lysate of an irrelevant clone C1. GST alone as a control did not show any specific interaction. The 180-kDa protein from C3 also interacted with anti-beta-galactosidase antibody, confirming that this protein was a beta-galactosidase fusion protein (Fig. 1). Sequence analysis showed that C3 contained the C-terminal region of topoIIbeta. TopoIIbeta has just been cloned recently(18) , whereas its isoform, topoIIalpha, had been characterized previously. Both isoforms are considered to be involved in DNA replication and transcriptional regulation(26, 27) . However, the function of topoIIbeta and the functional difference from its isoform are not yet understood. The region of topoIIbeta corresponding to C3 was abundant in acidic charged amino acids and potential phosphorylation sites and is considered to be the regulatory region of the enzyme(18) .


Figure 1: Specific binding of GST- to induced lysate of clone 3. Uninduced (IPTG-) or IPTG-induced (IPTG+) lysates of clone 3 and an irrelevant clone C1 were separated on SDS-PAGE. The gel was stained with Coomassie Blue(1) . After the gels were transferred to the membranes, they were blotted with anti-beta-galactosidase antibody(2) , GST-(3) , or GST(4) . The arrows indicate the induced beta-galactosidase fusion proteins. The molecular size markers are indicated at the left margin.



N Terminus of the Cytoplasmic Region of CD3 Binds to TopoIIbeta

To define the precise binding region of CD3 to topoIIbeta, serial deletion mutants of CD3 were constructed. The cytoplasmic region was tentatively divided into three regions; the N-terminal region: aa 135-161; the central region, aa 162-169; and the C-terminal region, aa 170-189. The N-terminal region, the central portion, and the C-terminal region contain a basic aa cluster, a proline-rich sequence, and ITAM, respectively (Fig. 2A). We prepared serial deletion constructs by PCR and site-directed mutagenesis in the tyrosine residues of ITAM as shown in Fig. 2A. Each GST fusion protein was induced with IPTG in E. coli and purified on glutathione-Sepharose. Purified proteins were resolved in SDS-PAGE. Coomassie Blue staining showed that all constructs made the expected size of the proteins (Fig. 2B).


Figure 2: Mapping of the topoIIbeta binding region in CD3 and the specificity of the interaction. A, GST fusion constructs for the cytoplasmic domains of , various mutants of , , c, and FcR. Schematic structures of these fusion proteins were shown. B, purified GST fusion proteins described in A. GST fusion proteins were purified on glutathione-Sepharose beads, separated on SDS-PAGE, and stained with Coomassie Blue. The molecular size markers are indicated at the left margin. C, minimum binding region of CD3 to topoIIbeta and the specificity of the binding among signaling molecules. The nuclear extracts of T cell hybridomas were precipitated with each GST fusion protein and subjected to SDS-PAGE. Proteins were transferred to membranes, and the membranes were blotted with anti-topoIIbeta mAb. The arrow indicates topoIIbeta. The molecular size markers are indicated at the left margin.



The nuclear extracts prepared from 2B4 hybridoma cells were precipitated with each GST-protein prebound on glutathione beads, separated on SDS-PAGE, and transferred to a PVDF membrane. The membrane was blotted with anti-topoIIbeta mAb beta5A7. GST-, -EM3, -EM4, -ED3, -ED5, -ED1, but not GST-ED4 bound to topoIIbeta (Fig. 2C). Since GST-ED1 contains only the N-terminal 12 aa, it is likely that the binding region is located in the basic aa cluster in the N-terminal region of CD3 (Fig. 3A).


Figure 3: A, alignment of the topoIIbeta binding motif of CD3 with the cytoplasmic region of FcR. Consensus amino acid residues among each pair of sequences are boxed. Amino acid residues are represented by single letters. B, NLS-like sequences present in the cytoplasmic domains of CD3 and CD3. NLS-like sequences within CD3 and CD3 are aligned with the NLS sequences of SV40 T antigen and nucleoplasmin-like NLS, respectively. Consensus amino acid residues among each pair of sequences are boxed.



We next wanted to determine the specificity of the binding, whether the binding of topoIIbeta is specific for CD3 or whether it also binds to similar signaling molecules such as CD3 and FcR. As shown in Fig. 2C, none of GST-, GST-fyn, or GST alone bound to topoIIbeta. In contrast, GST-FcR was found to bind to topoIIbeta (Fig. 2C). Comparing the aa sequences of CD3 and FcR, we found that the homologous sequence in the 12 aa binding region of CD3 was present in FcR (Fig. 3A). This novel motif contains a basic aa cluster. The CD3 chain contains clusters of basic aa such as KKRAR and RRR, but it did not bind to topoIIbeta, demonstrating that the binding is not due to nonspecific interactions with any clusters of basic aa.

Collectively, these data demonstrate that the binding of CD3 to topoIIbeta is sequence-specific, and only 12 aa of the N terminus of the cytoplasmic region of CD3 are sufficient for the interaction.

The Most C-terminal Region of TopoIIbeta Binds to CD3

Since C3 contained a 1.6-kb fragment corresponding to the C-terminal region of topoIIbeta, we employed a filter binding assay to specify the precise binding region to CD3. The cDNA fragment was divided into two regions, and the GST fusion constructs were prepared for each region. GST-topoIIbeta1 and GST-topoIIbeta2 contained the N- and C-terminal halves of C3, respectively (Fig. 4A). Each GST protein was examined for the binding ability to CD3. IPTG-induced or uninduced lysate from the bacteria containing each construct was blotted with GST- or GST alone. As shown in Fig. 4B, only the induced lysate from GST-topoIIbeta2, but not from GST-topoIIbeta1, bound specifically to GST-. This region of topoIIbeta contains a cluster of acidic aa, suggesting that this cluster may be responsible for binding to CD3.


Figure 4: Mapping of the CD3-binding region of topoIIbeta. A, the original clone C3 was divided into two regions and GST fusion protein constructs of topoIIbeta1 and -2 were generated. The dark and striped regions represent NLS and leucin-zipper, respectively. B, total lysates of IPTG-induced (IPTG+) or uninduced (IPTG-) bacteria containing GST fusion proteins were separated on SDS-PAGE. The gel was stained with Coomassie Blue(1) . The gels were transferred to the membranes, and the membranes were blotted with either GST- (2) or GST alone(3) . The arrows indicated the IPTG-induced GST fusion proteins. The molecular size markers are indicated at the left margin.



CD3 and CD3 Are Present in the Nucleus

Unlike topoIIalpha, the expression of topoIIbeta is rather restricted, being especially high in the thymus (data not shown)(29) , suggesting that topoIIbeta might be involved in T cell function. Although CD3 binds specifically to topoIIbeta, the obvious question raised was concerning the localization of these two proteins. Whereas CD3 is a membrane protein, topoIIbeta is localized mainly in the nucleus. Therefore, we examined the possibility of whether CD3 also exists in the nucleus. One piece of evidence which may support this possibility is that both CD3 and CD3 have the consensus sequence corresponding to nuclear localizing signal (NLS). As depicted in Fig. 3B, CD3 possesses the motif homologous to the NLS of SV40 T antigen(28) , and CD3 contains the nucleoplasmin-like NLS (bipartite)(29) , respectively. Since we were unable to detect CD3 or CD3 in the nuclear fraction by Western blotting (data not shown), a highly sensitive biotinylation method was employed (30) to detect even small amounts of CD3 or CD3 in the nuclear fraction.

To this end, cytosolic and nuclear extracts from thymocytes and 2B4 hybridomas either unstimulated or stimulated by cross-linking with anti-CD3 mAb were prepared as described under ``Materials and Methods.'' These extracts were immunoprecipitated with anti-CD3 mAb HMT3-1 and protein A-Sepharose. Precipitated proteins on the beads were labeled with biotin and analyzed on two-dimensional SDS-PAGE. As shown in Fig. 5, A and B, precipitation of the cytosolic fraction showed TCRalphabeta and CD3 dimers as off-diagonal spots and CD3 as the spot slightly above the diagonal. As expected, both CD3 and CD3 were detected in the nuclear fractions of both thymocytes (Fig. 5, C and D) and 2B4 hybridomas (not shown). The chain in the nuclear fraction was observed only upon TCR stimulation, and the amount of CD3 also increased upon TCR stimulation in thymocytes. CD3 from the cytosol fraction showed two spots ( and ` in Fig. 5B). Since ` was slightly larger than and reacted with anti-phosphotyrosine mAb 4G10 by Western blotting (data not shown), ` appeared to be phosphorylated . On the other hand, most of CD3 in the nuclear fraction appeared to be the same size as `, suggesting that most of CD3 in the nuclear fraction is probably phosphorylated. TCR alpha and beta dimers were barely detected in this fraction, suggesting that the detected CD3 and CD3 in the nuclear fraction were not present as components of the whole TCRbulletCD3 complex and that the presence of CD3 and CD3 in this fraction did not merely reflect the contamination of the cytosolic fraction.


Figure 5: CD3 and CD3 are present in the nuclear fraction. 7 times 10^7 thymocytes, unstimulated (A, C) or stimulated with anti-CD3 mAb 2C11 (B, D), were lysed, and the cytosol fractions (A, B) and the nuclear fractions (C, D) were prepared as described under ``Materials and Methods.'' Each fraction was immunoprecipitated with HMT3-1 coupled with protein A-Sepharose beads. Precipitated proteins were biotinylated on the beads and subjected to two-dimensional nonreducing (12%)/reducing (14%) SDS-PAGE. The proteins were transferred onto membranes and developed with an ECL detection system. The spots corresponding to CD3 (), possibly phosphorylated (`), and CD3 homodimers () were indicated.



To confirm the latter issue, the same cytosolic and nuclear fractions used to detect CD3 were immunoprecipitated and blotted with anti-tubulin mAb, since tubulin is present exclusively in the cytosol (31) . As shown in Fig. 6, tubulin was detected only in cytosolic fraction but not at all in the nuclear fraction of our preparations. This result clearly demonstrated that the contamination of the nuclear fraction by cytosol was negligible and that CD3 is likely to be present in the nucleus.


Figure 6: Negligible carryover of the cytosol fraction into the nuclear fraction of the thymocyte preparation. The total lysates in the same cytosol (C) and nuclear fractions (N) described in the legend to Fig. 5were subjected to 14% SDS-PAGE, and the proteins were transferred to membranes. The membrane was stained with Coomassie Blue (A) or blotted with anti-tubulin mAb followed by development with an ECL detection system (B). The arrow indicates tubulin. The molecular size markers are indicated at the left margin.



These results suggest that CD3 exists in the nucleus especially after TCR activation and that it binds to topoIIbeta in the nucleus in vivo.

Specific Interaction between TopoIIbeta and CD3 in T Cells

To demonstrate the direct interaction between CD3 and topoIIbeta in vivo, the nuclear fraction was prepared from DO11.10 hybridoma cells by solubilization with either digitonin or Brij 96 and immunoprecipitated by anti-mouse CD3 (2C11) mAb and anti-human CD3 (OKT3) mAb as a control and then analyzed by Western blot with anti-topoIIbeta Ab. As shown in Fig. 7, topoIIbeta was co-precipitated from the nuclear fractions of both digitonin (Fig. 7, lanes 1 and 2)- and Brij 96 (Fig. 7, lanes 3 and 4)-lysed cells. The fact that the association was only observed in digitonin or Brij lysates and that this association was not observed when the immunoprecipitate was washed with the buffer containing a higher concentration of Nonidet P-40 than 0.3% suggested that the interaction between topoIIbeta and CD3 was not strong in vivo (data not shown).


Figure 7: Association between CD3 and topoIIbeta in T cells. 5 times 10^8 DO11.10 T hybridoma cells were lysed in the lysis buffer containing 1% digitonin or 1% Brij 96, and the nuclear fractions were extracted as described under ``Materials and Methods.'' The nuclear fraction was immunoprecipitated with anti-mouse CD3 mAb 2C11 or anti-human CD3 OKT3 as a control. The precipitates from the digitonin (lanes 1 and 2)- and Brij 96 (lanes 3 and 4)- lysates were washed with the buffer containing 0.3% Nonidet P-40 and 1% Brij 96, respectively. These samples as well as the total cell extract equivalent to 10^6 cells as a positive control for topoIIbeta (lane 5) were analyzed on 8% SDS-PAGE under reducing condition and transferred onto a PVDF membrane. The membrane was blotted with anti-topoIIbeta mAb 5A7 and developed by an ECL system. The arrow indicates the molecular weight of topoIIbeta. The smaller band in the total lysate (lane 5) appeared to be a degradation product of topoIIbeta.




DISCUSSION

We have cloned topoIIbeta as a CD3-binding molecule by west-Western screening procedure. We determined the binding regions of both CD3 and topoIIbeta as well as the specificity of the interaction. The binding region of CD3 to topoIIbeta was localized within 12 aa in the N-terminal region containing a novel motif of basic aa cluster. A similar motif was also found in FcR. Since ITAM has been thought to be the only functional domain within CD3, this is the first report that a specific binding site other than ITAM exists in the cytoplasmic region of CD3. The binding region was composed of a novel motif of a basic aa cluster. Although CD3 has other basic aa clusters, did not bind to topoIIbeta, confirming the specific binding of topoIIbeta to CD3. It has been suggested that the TCR-CD3 complex is composed of two distinct activation modules(12, 13) . Distinct signals are transduced through CD3 and CD3. The binding of , but not , to topoIIbeta may represent one of such differences in signal transduction.

In terms of the physiological interaction of these two proteins, the question of their localization then arises. TopoIIbeta is considered to be located in the nucleus whereas CD3 is a membrane protein, and this question was therefore answered by demonstrating the translocation of CD3 into the nucleus. Our finding that CD3 and CD3 have NLS-like sequences in their cytoplasmic region supports the idea of translocation of a part of CD3 and CD3 into the nucleus. There have been several reports about transmembrane-type receptors such as epidermal growth factor receptor and platelet-derived growth factor receptor, similar to CD3, translocating to the nucleus(32) . Luton et al.(33) reported that the TCRbulletCD3 complex translocated to the cytoskeleton-associated insoluble fraction upon TCR stimulation. Considering that the nuclear fraction was involved in the insoluble fraction and nuclear translocation was linked with cytoskeleton-associated proteins such as actin filament, their observation may partly reflect the translocation of the CD3 chains to the nucleus. Although we failed to detect CD3 or CD3 in the nuclear fraction by Western blotting, we eventually succeeded in detecting both CD3 and CD3 in the nuclear fractions of normal thymocytes and T hybridoma cells by labeling with a sensitive biotinylation method. Importantly, the amounts of CD3 and CD3 were increased upon TCR activation while those in the cytosolic fraction did not seem to be changed. Taken together with the result that our preparation of the nuclear fraction did not contain any detectable contamination by the cytosolic fraction, we demonstrated for the first time that CD3 and exist in the nucleus in normal T cells and increased upon T cell activation. By immunoprecipitation with anti-CD3 mAb, we clearly demonstrated the direct in vivo association between CD3 and topoIIbeta in the nuclear fraction of T cells. These data indicate that CD3 is translocated into the nucleus upon T cell activation and interacts with topoIIbeta.

In addition, there is a possibility that CD3 may also bind to topoIIbeta present in cytosol. Indeed, it has recently been observed that topoIIbeta is transiently distributed to the cytoplasm during the mitotic stage, whereas topoIIalpha is associated tightly with chromosomes constantly throughout the cell cycle. (^3)

Previous analyses of T cells expressing extensive deletion constructs of CD3 showed that ITAM is both necessary and sufficient for IL-2 production(11) . These results indicate that the topoIIbeta-binding motif of CD3 is not prerequisite for IL-2 production. However, it has recently been reported that inhibitors of topoII, quinolon derivatives, up-regulated IL-2 production upon TCR stimulation(34) . Since the CD3-binding site is the regulatory region of topoIIbeta in vitro, which was also suggested by the fact that an anti-topoIIbeta mAb specific for the C terminus region of topoIIbeta inhibited the enzymatic activity,^3 the binding of CD3 may also block the enzymatic function and result in the super induction of IL-2 production, similar to the treatment of T cells with the topoII inhibitors. Moreover, topoII inhibitors are known to induce apoptosis (35, 36) . Growth arrest and subsequent apoptosis is induced in T cells upon TCR stimulation. The binding of CD3 to the regulatory region of topoII might modulate the function similarly to the inhibitors in vivo. Although we tested this hypothesis by performing a decatenation assay with nuclear extracts, we failed to detect any significant effect of the GST- binding on the in vitro function of topoIIbeta (data not shown). We assumed that the failure of modulation in the overall decatenation assay may be due to the dominant function of topoIIalpha even under the condition that CD3 binding may modify the function of topoIIbeta protein. Analysis by the use of recombinant topoIIbeta, which is not available yet, will be required.

Collectively, CD3 appears to possess two distinct functional domains; whereas ITAM is phosphorylated and stimulates ZAP70 and the following activation pathway in the cytoplasm, the N-terminal motif of CD3 found in this study plays a functional role in the nucleus after translocation.


FOOTNOTES

*
This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, and Culture, and from the Agency for Science and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.

To whom correspondence should be addressed: Division of Molecular Genetics, Center for Biomedical Science, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260, Japan. Tel.: 81-43-226-2197; Fax: 81-43-222-1791.

(^1)
The abbreviations used are: TCR, T cell receptor; aa, amino acid; FcR, the chain of Fc receptors; GST, glutathione S-transferase; Ig, immunoglobulin; IPTG, isopropyl-beta-D-thiogalactopyranoside; ITAM, immunoreceptor tyrosine-based activation motif; mAb, monoclonal antibody; NLS, nuclear localization signal; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; topoII, topoisomerase II; HBB, Hepes balanced buffer; IL, interleukin.

(^2)
N. Nozaki and A. Kikuchi, manuscript in preparation.

(^3)
A. Kikuchi, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to Dr. H. Umemori, Tokyo University, for pGEX-2TK-fyn plasmid and Dr. M. Yoshida, Tokyo University, for a gt11 library; Dr. E. K. Flemington, Harvard University, for pGEX-2TK vector and helpful suggestions; and H. Yamaguchi for preparing the manuscript.


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