(Received for publication, July 17, 1995; and in revised form, November 4, 1995)
From the
T cell antigen receptor (TCR)CD3 complex is composed of
six different subunits: TCR
and TCR
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 II
(topoII
). 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 topoII
. The binding region of topoII
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 topoII
. Although topoII
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
topoII
and CD3
from the nuclear fraction of T cells. The
possible function of this interaction is discussed.
The T cell antigen receptor (TCR)()
CD3 complex
is composed of six subunits: clonotypic
and
chains and
invariant CD3
, CD3
, CD3
, and CD3
chains. TCR
and TCR
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 Ig
and Ig
of the B cell antigen
receptor complex and the
chain of Fc receptors (FcR
)(2).
Since the TCR
CD3 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 TCR
CD3 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
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 TCR
CD3 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 II
(topoII
)(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
topoII
in this fraction of T cells. Detailed mapping of the
binding regions of both CD3
and topoII
indicates that the
association depends on a novel motif in CD3
and suggests that the
binding might modulate the in vivo function of topoII
.
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-topoII were
prepared by using the internal EcoRI site of topoII
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-topoII
1 (0.7 kb) and pGEX-topoII
2 (0.9 kb),
respectively.
These constructs were introduced into ompT-negative E. coli strain, AD202(20) .
Proteins were induced with
isopropyl--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).
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-
-galactosidase antibody(2) , GST-
(3) ,
or GST(4) . The arrows indicate the induced
-galactosidase fusion proteins. The molecular size markers are
indicated at the left margin.
Figure 2:
Mapping of the topoII 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
topoII
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-topoII
mAb. The arrow indicates topoII
. 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-topoII mAb
5A7. GST-
, -EM3, -EM4, -ED3, -ED5, -ED1,
but not GST-ED4 bound to topoII
(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 topoII 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
topoII 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 topoII
. In contrast, GST-FcR
was found to bind
to topoII
(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 topoII
, 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 topoII
is sequence-specific, and only 12 aa of the N
terminus of the cytoplasmic region of CD3
are sufficient for the
interaction.
Figure 4:
Mapping of the CD3-binding region of
topoII
. A, the original clone C3 was divided into two
regions and GST fusion protein constructs of topoII
1 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.
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
TCR
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
and
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
TCR
CD3 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
10
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 topoII
in the nucleus in
vivo.
Figure 7:
Association between CD3 and
topoII
in T cells. 5
10
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
cells as a positive
control for topoII
(lane 5) were analyzed on 8% SDS-PAGE
under reducing condition and transferred onto a PVDF membrane. The
membrane was blotted with anti-topoII
mAb 5A7 and developed by an
ECL system. The arrow indicates the molecular weight of
topoII
. The smaller band in the total lysate (lane 5)
appeared to be a degradation product of
topoII
.
We have cloned topoII as a CD3
-binding molecule by
west-Western screening procedure. We determined the binding regions of
both CD3
and topoII
as well as the specificity of the
interaction. The binding region of CD3
to topoII
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
topoII
, confirming the specific binding of topoII
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 topoII
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. TopoII 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 TCR
CD3 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 topoII
in the nuclear
fraction of T cells. These data indicate that CD3
is translocated
into the nucleus upon T cell activation and interacts with topoII
.
In addition, there is a possibility that CD3 may also bind to
topoII
present in cytosol. Indeed, it has recently been observed
that topoII
is transiently distributed to the cytoplasm during the
mitotic stage, whereas topoII
is associated tightly with
chromosomes constantly throughout the cell cycle. (
)
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 topoII
-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 topoII
in vitro, which was also
suggested by the fact that an anti-topoII
mAb specific for the C
terminus region of topoII
inhibited the enzymatic activity,
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 topoII
(data not shown). We assumed that the
failure of modulation in the overall decatenation assay may be due to
the dominant function of topoII
even under the condition that
CD3
binding may modify the function of topoII
protein.
Analysis by the use of recombinant topoII
, 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.