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INTRODUCTION |
Human CD38 antigen is a 45-kDa type II transmembrane glycoprotein
with a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain (1, 2). It is widely expressed in different cell
types including thymocytes, activated T cells, and terminally differentiated B cells (plasma cells) (3). Other reactive cells include
natural killer cells, monocytes, macrophages, dendritic cells, and some
epithelial cells. The CD38 antigen acts as a NAD(P)+ glycohydrolase (4)
and plays a role in lymphocyte activation (3, 5). Recently it has been
identified CD31, which is mainly expressed by endothelial cells,
platelets, macrophages and a discrete subset of T cells, as a ligand
for CD38 (6). CD31 and CD38 cognate interactions are found to modulate
heterotypic adhesion as well as to induce increases in the
concentration of intracellular free Ca2+
([Ca2+]i) identical to those
obtained by means of agonistic anti-CD38
mAbs1 (6). These results
suggest that the interplay between CD38 and its ligand CD31 is an
important step in the regulation of cell life and of the migration of
leukocytes through the endothelial cell wall.
TCR/CD3-mediated signaling involves recruitment and activation of the
PTKs of the Src-family Fyn or Lck to the proximity of the TCR/CD3
complex (7-14). As a result, CD3-
and CD3-
are phosphorylated in
the two tyrosines of the immunoreceptor tyrosine-based activation motif
(ITAM), which is found three times in the CD3- chain and once in
each of the other CD3 subunits (
, 
, and ) (15). The most
widely held structural model of the TCR/CD3 complex is one comprising a
CD3-2 dimer and two CD3 pairs (, ).
Therefore, 10 ITAMs may be present within a single TCR/CD3 complex. A
view has emerged that in the TCR/CD3 complex there are at least two distinct functional units referred to as transduction modules and made
of the CD3-2 dimers and the CD3 pairs (, ),
respectively (16, 17). Although the CD3- and CD3- pairs may
have separate signaling capabilities (18), we will refer to them as the
CD3- transduction module (16). The ITAM tyrosine
phosphorylation promotes a high affinity interaction of the CD3 chains
with a second family of PTKs, the Syk/ZAP-70 family. The recruited
Syk/ZAP-70 molecules are activated by phosphorylation and contribute to
the recruitment and activation of other proteins such as LAT (linker for activation of T cells), Vav, and SLP-76. This leads to the formation of multimolecular complexes that activate several signaling cascades as the PLC-1-dependent pathway, and the ones
emanating from activation of Ras such as the MAP kinase pathway and the phosphatidylinositol 3-kinase pathway (14, 19, 20). This signaling
cascades ultimately converge on the nucleus, resulting in the changes
of gene expression that characterize T cell activation.
In a previous paper, we have shown that CD38 ligation results in
activation of at least two PTK-controlled signaling pathways, the
CD3-/ZAP-70/PLC-1-dependent cascade and the Raf-1/MAP
kinase pathway, suggesting a functional relationship between signals delivered through CD38 and the TCR (21). This assumption was strengthened by our finding that, in Jurkat T cells, CD38-mediated signaling events as increases in
[Ca2+]i or CD69 expression require
the presence of the TCR/CD3 (22). These results parallel elegant
studies done by Lund et al. (23, 24) in murine B cells,
demonstrating that co-expression of the BCR is required for
CD38-mediated signal transduction.
To further understand the molecular and functional relationship between
the CD38 receptor and the TCR in human T cells, we investigated the
participation of various components of the TCR/CD3 complex in
CD38-mediated signaling. To address these questions, we compared the
CD38- and CD3-mediated early signaling capabilities of a surface
TCR
/CD3 Jurkat T cell variant with those
signals delivered by surface TCR+/CD3+ Jurkat
cell transfectants that express either the complete TCR/CD3 complex or
a TCR/CD3 complex with impaired CD3- chain association. We have also
studied the CD38-mediated increases in protein tyrosine phosphorylation
in a TCR Jurkat T cell variant expressing either CD25-
or CD25- chimeric proteins on the cell surface. The data demonstrate
that in T cells coexpression of the TCR/CD3 complex is absolutely
required for coupling CD38 to downstream signaling events. Moreover, we
show that the CD3- signaling module is sufficient for CD38 to
induce activation of PTK- and MAP kinase-mediated signaling pathways.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
Jurkat D8 cells were obtained from wild-type
Jurkat cells (subclone E6-1, American Tissue Culture Collection (ATCC),
Rockville, MD) by the limiting dilution technique (25). JK-31-13 is a
CD38+CD3TCR variant of Jurkat T
cells lacking a functional TCR-
chain (26). This variant was
transfected by electroporation with the cDNA encoding wild-type
TCR- chain (27), or a mutant TCR- chain in which a transmembrane
tyrosine to leucine mutation was created (27). These cell lines were
made available by Dr. A. Alcover (Institut Pasteur, Paris, France), Dr.
B. Alarcón (Centro de Biologia Molecular, Consejo Superior de
Investigaciones Científicas (CSIC), Madrid, Spain), and Dr. R. Bragado (Fundación Jiménez-Díaz, Madrid, Spain).
CD25 chimeric receptor-expressing cell lines FEV20.3 (JK-CD25-+) and FEV19.5.4 (JK CD25-+)
were generated by stable transfection of a TCR-
variant of the Jurkat cell line (J.RT3-T3.5) with the cDNA encoding the complete human CD25 ecto- and transmembrane domains fused to either
the complete mouse CD3- or CD3- cytoplasmic domains (28). These
cells were kindly provided by Dr. Eric Vivier (Center d'Immunologie
INSERM-CNRS de Marseille-Luminy, Marseille, France). Cells were
cultured as described (21, 28).
Transient Transfections--
The full-length TCR- cDNA
(V6) subcloned in the pRSV.5 vector (29) was transfected into
JK-31-13 cells (30 µg of cDNA into 30 × 106
cells) by electroporation (using the BTX cell porator system (Genetronics, Inc., San Diego, CA) with a capacitance set at 975 microfarads). Twenty-four hours after transfection, dead cells were
removed by Ficoll-Paque centrifugation, and live cells were cultured
for another 24 h before performing the functional assays. Cells
were always analyzed by fluorescence-activated cell sorting for surface
expression of the transfected protein.
Antibodies and Reagents--
Purified CD3 mAb OKT3 (IgG2a) was a
gift from Dr. Goldstein (Ortho Pharmaceutical, Raritan, NJ). Anti-CD38
mAb IB4 (IgG2a) was prepared and purified by affinity chromatography on
Protein A-Sepharose and high performance liquid chromatography on
hydroxyapatite, as described (30). Affinity-purified, fluorescein
isothiocyanate-conjugated (FITC) F(ab')2 fraction of rabbit
antibody to mouse immunoglobulins (F(ab')2 FITC-R
mIg)
was purchased from Dako (Glostrup, Denmark). Affinity-purified
F(ab')2 fraction of goat antibody to mouse IgG (whole
molecule) (F(ab')2 GmIg) was purchased from Cappel
(Organon Teknika, Durham, NC). Anti-phosphotyrosine (anti-Tyr(P)) mAb
1G2 coupled to agarose beads (1G2-agarose) was obtained from Oncogene Research (Calbiochem, Cambridge, MA). Recombinant anti-Tyr(P) antibody
coupled to horseradish peroxidase (RC20-HRP), anti-Grb2 mAb antibody,
and anti-SLP76 mAb were obtained from Transduction Laboratories
(Lexington, KY). The following affinity-purified rabbit polyclonal
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA): anti-Erk-2 and anti-c-Cbl. An affinity-purified rabbit
immunoglobulins to human anti-CD3- (Dako). Anti-Zap-70 (Zap-4)
rabbit antiserum was a kind gift from Dr. S. C. Ley (Medical Research Council, London, United Kingdom) (31). Anti-CD3- antiserum 448 was a gift from Dr. B. Alarcón (Centro de Biología
Molecular, CSIC, Madrid, Spain). The anti-CD3- mAb 1D4.1 and the
anti-CD3- mAb 2F4.1 have been previously described (32, 33). The
anti-PLC-1 polyclonal antibody (C-37) was made by immunizing New
Zealand White rabbits with the synthetic peptide ADHFDSRERRAPRRTRVNGD conjugated to soluble keyhole limpet hemocyanin (Sigma-Aldrich Química, S.A., Madrid, Spain) as described previously (8). The
anti-human CD25 mAb B1.49.9 (IgG2a, mouse) was purchased from Immunotech (Marseille, France). Affinity-purified goat anti-rabbit IgG
(Fc) horseradish peroxidase (HRP) conjugate, and goat anti-mouse IgG
(H+L) HRP conjugate were from Promega (Madison, WI). Prestained SDS-PAGE standards (broad range) were from Bio-Rad. Recombinant protein
A-Sepharose was from Amersham Pharmacia Biotech. 4-Phorbol 12,13-dibutyrate (PdBu) was purchased from Sigma-Aldrich
Química, S.A.
Fluorescence-activated Cell Sorting Analysis--
Cells were
analyzed for surface expression of CD3, CD38, and CD25 by flow
cytometry as described previously (21). Samples were analyzed in a
FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Cell Stimulation, Immunoprecipitation, and Western
Blotting--
Cells were grown up to a density of 106/ml,
centrifuged, and serum-starved in RPMI + 0.5% FBS for 4 h, washed
in RPMI without serum, and resuspended at 1-2 × 107
cells/sample or otherwise indicated, in serum-free RPMI-Hepes medium,
at 4 °C. Stimulation, immunoprecipitation, and Western blotting were
performed as described in detail elsewhere (21).
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RESULTS |
In Jurkat T Cells, Cross-linking of CD38 Induces Tyrosine
Phosphorylation of Both CD3- and CD3- Subunits of the TCR/CD3
Complex--
In a previous study, we showed that, upon CD38 ligation,
ZAP-70 is tyrosine-phosphorylated and recruited by phosphorylated CD3- (21). Since tyrosine phosphorylation of both CD3- and CD3- have been identified as the biochemical hallmarks of agonistic stimulation of the TCR (34, 35), in this work we examined whether
CD3- became tyrosine-phosphorylated following CD38 engagement. To
this end, Jurkat T cells were stimulated with the anti-CD38 mAb IB4 for
various periods of time. Cells were then lysed in 1% Nonidet P-40
lysis buffer to dissociate the CD3- chain from the other CD3
subunits (36), and the CD3- and CD3- subunits were sequentially
immunoprecipitated with specific antibodies. Note that CD3- was
immunoprecipitated with an anti-human CD3- mAb, 1D4.1, that
recognizes both the 32-kDa unphosphorylated and 42-44-kDa
phosphorylated CD3- dimers (10, 21, 33). The immunoprecipitates were
resolved on SDS-PAGE under non-reducing conditions and subsequently
immunoblotted with an anti-phosphotyrosine mAb. As shown in Fig.
1A, CD38 ligation resulted in
the tyrosine phosphorylation of CD3- (lanes
8-10) with similar kinetics as of CD3- (Fig.
1B, lanes 8-10). As compared with
anti-CD3 stimulation, which was used as a positive control, the extent
and duration of anti-CD38-induced CD3-, or CD3- tyrosine
phosphorylation was significantly lower (Fig. 1, A and
B, compare lanes 5-7 with lanes 8-10). Immunoblotting with anti-CD3-
antibodies confirmed that the anti-CD3 immunoprecipitations were
specific and that equal amounts of CD3- were loaded in all lanes
(Fig. 1C, lanes 4-10). Therefore,
these results suggest that CD38 engagement with anti-CD38 mAbs fully
reproduces the distinct patterns of CD3 tyrosine phosphorylation seen
after engagement of the TCR/CD3 complex with agonistic ligands or
antibodies. Moreover, the data strongly suggest that in Jurkat T cells
both CD3- and CD3- signaling modules are involved in
CD38-mediated early signaling transduction events.
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Fig. 1.
CD38 induces tyrosine phosphorylation of both
CD3- and CD3-
subunits of the TCR/CD3 complex. 15 × 106
Jurkat cells, clone D8 (CD38+, CD3+), were
incubated for 10 min on ice with 5 µg/107 cells of
anti-CD3 mAb (OKT3) (lanes 2 and 5-7), or
anti-CD38 mAb (IB4) (lanes 3 and 8-10), or RPMI-HEPES (nonstimulated,
lanes 1 and 4). This was followed by
cross-linking with 20 µg/107 cells of the secondary
antibody F(ab')2 GmIg (lanes 3 and
8-10) for 10 min on ice. Then the time course was conducted
at 37 °C for the indicated times. Control unstimulated cells were
incubated at 37 °C for 1 min (panel B, lanes 1 and 4), or 5 min (panel A, lanes 1 and
4). A, cell lysates or anti-CD3-
immunoprecipitates (OKT3) were separated on a 12.5% SDS-PAGE gel under
non-reducing conditions and subjected to Western immunoblotting
(WB) with an anti-Tyr(P) mAb RC20-HRP (0.1 µg/ml).
Position of tyrosine-phosphorylated CD3- is indicated. B,
cell lysates or anti-CD3- immunoprecipitates (1D4.1) were resolved
as in panel A and probed with the anti-Tyr(P) mAb
RC20-HRP. Positions of the different tyrosine-phosphorylated forms of
CD3- are indicated. C, the filter shown in A
was stripped and reprobed with an affinity-purified anti-CD3- Ab. In
all panels, the molecular mass markers are indicated to the
left. All blots were developed by chemiluminescence using
the ECL detection system and then exposed to Hyperfilm-ECL (Amersham
Pharmacia Biotech). D, surface expression levels of CD3 and
CD38 in Jurkat D8 cells. Cells were stained with the anti-CD3- mAb
OKT3 (left panel) or with the anti-CD38 mAb IB4
(right panel), followed by F(ab')2
FITC-RmIg secondary Ab. Representative flow cytometric profiles are
shown (filled histograms). Negative controls
(open histograms) were obtained after staining with the
secondary Ab alone. Flow cytometric data are presented as the logarithm
of fluorescence intensity.
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CD38-induced PTK Activation Requires the Expression of a Functional
TCR/CD3 Complex on the Cell Surface--
The results on CD38-induced
CD3- and CD3- tyrosine phosphorylation suggested a functional
relationship between signals derived through CD38 and the TCR/CD3. To
address the question whether CD38 requires some of the CD3 subunits
associated with the TCR to access the intracellular signal transduction
machinery, we have used JK-31-13 cells, a
CD38+TCR/CD3 variant of the
human T cell line Jurkat (26). This cell line does not express any of
the TCR/CD3 chains on the cell surface due to a defective expression of
the TCR- gene (see phenotype in Fig.
2A). In these cells, CD38
ligation with the anti-CD38 mAb IB4 did not induce any significant
increase in protein tyrosine phosphorylation (Fig. 2B,
lane 4). Likewise and as expected, CD3 ligation
by the anti-CD3 mAb OKT3 was unable to induce increased tyrosine
phosphorylation of any substrate (Fig. 2B, lane
3). In contrast, treatment of JK-31-13 cells with the
tyrosine phosphatase inhibitor sodium pervanadate for 5 min induced a
marked increase in substrate tyrosine phosphorylation (Fig.
2C, lane 1). Overall, these results
demonstrate that the lack of responsiveness of the CD38+TCR JK-31-13 cells to CD38 stimulation
was not due to a general defect in PTK-mediated signals and strongly
suggest that expression of a functional TCR/CD3 complex on the cell
surface is required for CD38-mediated increases in protein tyrosine
phosphorylation.
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Fig. 2.
Expression of TCR/CD3 complex on the cell
surface is required for CD38-mediated increases in protein tyrosine
phosphorylation. A, the parental (JK 31-13) and
transfected (JK-B7 and JK-G6) cell lines were analyzed by flow
cytometry after staining with the same mAbs used in Fig. 1D.
Representative fluorescence histograms (filled histograms)
show the CD3 and CD38 surface expression levels in TCR JK
31-13 cells (left and right upper
panels, respectively), and in cells derived from JK-31-13
after stable TCR- chain cDNA transfection as JK B7
(middle panels) and JKG6 cells (lower
panels). B, 8 × 106
TCR JK 31-13 cells were resuspended in serum-free
RPMI-HEPES, warmed at 37 °C for 10 min. The cells were stimulated by
5 µg/107 cells of OKT3 (lane 3) or
IB4 (lane 4) or incubated with RPMI-HEPES alone
(lanes 1 and 2), at 37 °C for 3 min. This was followed by cross-linking, for 2 min at 37 °C, with 20 µg/107 cells of F(ab')2 GmIg secondary Ab
(lanes 2-4), or left in RPMI-HEPES medium alone
(lane 1). Cell lysates (250,000 cell equivalents)
were separated on 10% SDS-PAGE gel under reducing conditions and
subjected to immunoblotting with anti-Tyr(P) mAb RC20-HRP.
C, 10 × 106 Jurkat 31-13 cells were
stimulated at 37 °C by a 5-min incubation with 100 µM
sodium pervanadate. Cell lysates were resolved and probed with the
anti-Tyr(P) mAb as in panel B. D and
E, 6 × 106 JKB7 cells or 1 × 106 JKG6 cells (both TCR+/CD3+,
CD38+) were stimulated at 37 °C as described in
panel B. Cell lysates (250,000-350,000 cell
equivalents) were resolved and probed with the anti-Tyr(P) mAb as in
panel B. All blots were developed by ECL.
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To further prove that indeed the surface expression of a functional
TCR/CD3 is necessary for CD38-mediated early signaling in T cells, the
TCR JK-31-13 variant of Jurkat was transfected with the
cDNA coding for the human TCR- chain. After drug selection, cell
lines expressing the TCR/CD3 complex on their cell surface were
isolated (27), and the signal transducing capability of CD38 was
assessed. As shown in Fig. 2 (D and E), in cells
transfected with the TCR- cDNA as JK-B7 or JK-G6 cells increased
tyrosine phosphorylation of a similar number of cellular proteins was
induced in response to either CD38 or CD3 stimulation with specific
antibodies. As expected from previous experiments in untransfected
CD38+TCR+ Jurkat D8 cells (21), the extent of
increased tyrosine phosphorylation induced by both stimuli differed,
and was always significantly lower for anti-CD38 than for anti-CD3
stimulation (Fig. 2, D and E). Similar results
were obtained with another three independent TCR-+
transfectants (data not shown). To avoid potential artifacts resulting
from clonal variation, we also analyzed JK-31-13 cells transiently
transfected with the TCR- cDNA. Transient TCR- cDNA transfection of these cells by electroporation resulted in the surface
expression of the TCR/CD3 complex in about 10-20% of the cells (Fig.
3B). Despite the low number of
TCR+ cells, transfected cells responded to anti-CD38 IB4
mAb, or to anti-CD3 OKT3 mAb stimulation in a fashion equivalent to the
stable TCR-+ transfectants. Thus, both stimuli caused
increases in protein tyrosine phosphorylation (Fig. 3A,
lanes 3 and 2, respectively). Overall,
these and the above results clearly demonstrate that in T cells CD38
cannot initiate PTK-mediated signals without co-expression of a
functional TCR/CD3 complex.
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Fig. 3.
CD38-mediated increases in protein tyrosine
phosphorylation in JK 31-13 cells transiently transfected with a
TCR- chain cDNA. A,
48 h after transfection the cells were prepared and stimulated as
described in Fig. 1. Cell lysates (400,000 cell equivalents) were
separated on a 10% SDS-PAGE gel under reducing conditions and
subjected to immunoblotting with the anti-Tyr(P) mAb RC20-HRP. The
cells responded to anti-CD38 IB4 mAb or anti-CD3 OKT3 mAb stimulation,
as measured by increases in protein tyrosine phosphorylation
(lanes 3 and 2, respectively).
B, A representative fluorescence histogram (left
panel) shows the CD3 surface expression levels in JK 31-13 cells transiently transfected with the TCR- chain cDNA
(filled histogram) versus CD3 surface expression
levels in wild-type JK 31-13 cells (open histogram). In this
particular experiment, about 20% of cells were CD3+. In
the right panel (filled histogram),
CD38 surface expression levels are shown. The negative control was
obtained after staining with the secondary Ab alone (open
histogram).
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CD38-induced Cbl, PLC-1, ZAP-70, and SLP-76 Tyrosine
Phosphorylation in TCR--transfected JK-31-13 Cells--
Transient
DNA transfection in Jurkat T cels by electroporation resulted in only
about 20% of TCR+/CD3+ cells (Fig.
3B), which is within the range obtained by other authors
(37). The relative large number of cells required for immunoprecipitation experiments, and the background from noexpressing cells made difficulty the use of the transient DNA transfection approach for further functional analysis. Therefore, to circumvent this
problem, we used stably TCR-+ transfected cells in the
following experiments.
We have previously demonstrated in Jurkat T cells that Cbl, the product
of the protooncogene c-cbl, is a prominent PTK substrate, becomes tyrosine-phosphorylated upon CD38 or CD3 stimulation (21). In
addition, in T cells Cbl interacts with a number of molecules known to
be critical in signal transduction. These include PTKs such as Fyn and
ZAP-70, the adaptor molecule Grb2, and the lipid/protein kinase
phosphatidylinositol 3'-kinase (38-41). We thus investigated in the
TCR-+-transfected cells whether Cbl would become
tyrosine-phosphorylated in response to CD38 ligation. To this end,
stimulated and nonstimulated TCR+ JK-B7 cells were lysed
and subjected to immunoprecipitation with antibodies directed at Cbl
(Fig. 4A, lanes
5-8). Immunoblotting of whole lysates and Cbl
immunoprecipitates with anti-Tyr(P) mAbs demonstrated that Cbl was
readily tyrosine-phosphorylated upon CD38 ligation (Fig. 4A,
lanes 4 and 8 versus
lanes 1 and 5, respectively). Immunoblotting with anti-Cbl antibodies confirmed that the anti-Cbl immunoprecipitates were specific and that equal amounts of Cbl were
loaded in all lanes (Fig. 4B). The anti-Tyr(P) blot revealed that at least five tyrosine-phosphorylated proteins at 90, 75, 70, 65, and 60 kDa were coimmunoprecipitated with Cbl in CD38 or CD3-stimulated
cells (Fig. 4A, lanes 8 and
7, respectively). One of the associated phosphoproteins
co-migrated with ZAP-70, although there were no differences in the
total amount of ZAP-70 associated with Cbl when it was compared with
that in unstimulated or secondary antibody-stimulated cells (data not
shown).
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Fig. 4.
In the TCR+
JK-B7 cells Cbl, PLC-1, SLP-76, and
Zap70 become tyrosine-phosphorylated in response to CD38 ligation.
A, cells were stimulated as in Fig. 2B, and
lysates from 4 × 107 JKB7 cells were
immunoprecipitated with an affinity-purified anti-c-Cbl Ab. Cell
lysates (lanes 1-4) and immunoprecipitates
(lanes 5-8) were resolved by SDS-PAGE (10% gel,
reducing conditions) and transferred to PVDF membrane and subjected to
immunoblotting with anti-Tyr(P) mAb RC20-HRP. Position of
tyrosine-phosphorylated c-Cbl is indicated by an arrow.
B, the upper part of the filter shown in A was
stripped and reprobed with an affinity-purified anti-c-Cbl Ab. Position
of c-Cbl is indicated by an arrow. C-E, cells
(4 × 107 per sample) were prepared and stimulated as
described in Fig. 2B. Lysates either from unstimulated
(lane 5) or stimulated (lanes
7 and 8), or mock-stimulated (lane
6) were immunoprecipitated with the anti-Tyr(P) mAb
1G2-agarose beads (lanes 5-8). Lysates (lanes
1-4), and immunoprecipitates were resolved by SDS-PAGE (10% gel,
reducing conditions), transferred to PVDF, and the filter was cut in
parts and subjected to immunoblotting with polyclonal antibodies
anti-PLC-1 (panel C), anti-SLP76
(panel D), and anti-Zap70 (panel
E). Location of immunoprecipitated proteins is indicated by
arrows. All blots were developed by ECL.
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In wild-type CD38+TCR+ Jurkat D8 cells, an
immediate consequence of triggering CD38 is the tyrosine
phosphorylation of PLC-1 and ZAP-70 (21), and elevation of
intracellular calcium (22). It has been shown that the adaptor protein
SLP-76 is a substrate of ZAP-70, and provides an important functional
link between the TCR and activation of Ras/MAP kinase and
PLC-1/calcium pathways (42). Therefore, it was of interest to assess
in the TCR+ JK-B7 transfectant cells whether PLC-1,
ZAP-70, and SLP-76 became tyrosine-phosphorylated upon CD38 engagement.
As expected, CD38 engagement with anti-CD38 mAb for 5 min resulted in
tyrosine phosphorylation of PLC-1 (Fig. 4C) and ZAP-70
(Fig. 4E). Notably, we have also found for the first time
that SLP-76 became tyrosine-phosphorylated following CD38 stimulation
(Fig. 4D, lane 8). In contrast,
secondary antibody alone did not result in SLP-76 tyrosine
phosphorylation (Fig. 4D, lane 6). In
summary, these results clearly demonstrate that, upon transfection of
the CD38+TCR JK-31-13 cells with the TCR-
chain, TCR/CD3 expression and function, as well as CD38-mediated PTK
activation, are restored, as judged by the the receptor-induced
increases in tyrosine phosphorylation of Cbl, PLC-1, ZAP-70, and
SLP-76.
CD38-mediated MAP Kinase Activation Requires Surface Expression of
the TCR/CD3 Complex--
Activation of p21ras
leads to recruitment and activation of Raf-1, which then activates
MEK-1 and MEK-2, which subsequently activate the MAP kinases Erk-1 and
Erk-2 (43). Activation of MAP kinases occurs through phosphorylation of
threonine 202 and tyrosine 204 of human MAP kinase (Erk-1) at the
sequence TEY by MEK (44). We have previously demonstrated that, in
Jurkat T cells, CD38-mediated MAP kinase Erk2 activation is both PTK-
and protein kinase C-dependent (21). To examine whether
CD38-mediated Erk-2 activation required surface expression of the
TCR/CD3 complex, we assessed in both the TCR JK-31-13 and
the TCR+ JK-B7 cells the levels of Erk-2 tyrosine
phosphorylation and its mobility shift following CD38 engagement with
the agonistic antibody IB4. Tyrosine phosphorylation and reduced
mobility on SDS-PAGE of Erk-2 (attributable to threonine
phosphorylation) have been associated with activation of this enzyme by
MEK (21, 45-47).
In untransfected JK-31-13 cells, which did not express the TCR/CD3
complex on the cell surface, CD38 or CD3 ligation did not result in
Erk-2 tyrosine phosphorylation (Fig.
5A, lanes
9 and 8, respectively), nor was mobility shift
observed (Fig. 5A, lanes 4 and
3, respectively). In contrast, Erk-2 became
tyrosine-phosphorylated upon 10-min incubation with the phorbol ester
PdBu (Fig. 5A, lane 10). Moreover, in
PdBu-treated cells, the mobility of Erk-2 on SDS-PAGE was retarded as
compared with those treated with the other stimuli (Fig. 5A,
lane 5), indicating activation of this enzyme
(21, 47, 48).
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Fig. 5.
CD38-mediated MAP kinase activation depends
on the cell surface expression of the TCR/CD3 complex.
TCR JK-31-13 cells (A) and TCR+
JKB7 cells (C) were prepared and stimulated as described in
Fig. 2B. Lysates (3 × 107 cell
equivalents) either from unstimulated (lane 6),
or stimulated (lanes 8-10), or mock-stimulated
with F(ab')2 GmIg secondary Ab (lane
7), were immunoprecipitated with an affinity-purified
anti-Erk-2 Ab. Lysates (lanes 1-5) and
immunoprecipitates (lanes 6-10) were resolved by
SDS-PAGE (10% gel, nonreducing conditions), transferred to PVDF, and
subjected to immunoblotting. The right panels
were probed with anti-Tyr(P) mAb RC20-HRP. In each panel the position
of tyrosine-phosphorylated Erk-2 is indicated. The left
panels were probed with an anti-Erk-2 Ab, and the position
of total Erk-2 is indicated. B and D, the filters
shown on the right in A or C were
stripped and reprobed with an affinity-purified anti-Erk-2 Ab, showing
equal levels of immunoprecipitated Erk-2 protein. All blots were
developed by ECL.
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In TCR--transfected JK-B7 cells, which expressed the complete
TCR/CD3 complex on the cell surface, Erk-2 became
tyrosine-phosphorylated upon CD38 cross-linking with IB4 mAb (Fig.
5C, lane 9), or upon CD3 cross-inking
with OKT3 mAb (Fig. 5C, lane 8), or
PdBu treatment (Fig. 5C, lane 10).
Moreover, in whole cell lysates from OKT3, IB4, and PdBu-treated JK-B7
cells, it was observed a reduced electrophoretic mobility of Erk-2
(Fig. 5C, lanes 3-5) as compared with
that in control unstimulated or secondary antibody-stimulated cells
(Fig. 5C, lanes 1 and 2,
respectively). Overall, these results demonstrate that
CD38-mediated activation of the Ras/MAP kinase signaling pathway
strongly depends on the surface expression of the TCR/CD3 complex.
Association of the CD3- Signaling Module with the TCR Is
Sufficient to Mediate Anti-CD38 Induction of Protein Tyrosine
Phosphorylation and MAP Kinase Activation--
To ascertain which
component of the TCR/CD3 complex is required for CD38-mediated
signaling, we used JK-31-13 cells transfected with the cDNA coding
for the TCR-, but harboring a point mutation of a tyrosine residue
to leucine in the transmembrane domain (named Y11L). This point
mutation impairs the association of the CD3- signaling module with
the TCR (27). Despite this defect, cell lines as JK-C2
TCR-+-mut expressing the TCR on the cell surface were
isolated (Fig. 6A). In these
cells, the TCR is tightly associated with the CD3- signaling
module (27). Moreover, these cells were able to transduce CD3-mediated
signals as assessed by the ability of anti-CD3 mAbs to induce increases
in protein tyrosine phosphorylation (Fig. 6B,
lanes 3 and 4), demonstrating that the
TCR devoid of CD3- was functionally active. Thus, with these
mutants, we can address the question whether the association of CD3-
to the TCR is required for CD38-mediated signaling events and whether
the other TCR-associated signaling module, CD3-, can
compensate for CD3- function.
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Fig. 6.
The
CD3- signaling
module of the TCR is sufficient to allow CD38-mediated PTK
activation. A, CD3 and CD38 surface expression in JKC2
TCR+mut cells. Cells were stained with the same mAbs
used in Fig. 1D. B, patterns of CD38- and
CD3-induced increases in protein tyrosine phosphorylation. Cells were
prepared and stimulated for the indicated times as described in Fig. 1.
Cell lysates (250,000 cell equivalents) from unstimulated
(lane 1) or stimulated with OKT3 mAb
(lanes 3 and 4), or with IB4+
F(ab')2 GmIg secondary Ab (lanes 5 and 6), or mock-stimulated with F(ab')2 GmIg
secondary Ab (lane 2) were separated on 10%
SDS-PAGE gel under reducing conditions and subjected to immunoblotting
with anti-Tyr(P) mAb RC20-HRP. C, CD38- and CD3-induced
CD3- tyrosine phosphorylation. JKC2 TCR+mut cells
(15 × 106) were prepared and stimulated for the
indicated times as described in Fig. 1. Cell lysates (lanes
1-3) or anti-CD3- immunoprecipitates (lanes
4-8) were separated on a 12.5% SDS-PAGE gel (non-reducing
conditions) and subjected to immunoblotting with anti-Tyr(P) mAb
RC20-HRP. Position of tyrosine-phosphorylated CD3- is indicated.
D, the filter shown in C was stripped and
reprobed with an affinity-purified anti-CD3-. Position of CD3- is
indicated. E, CD38-mediated deficient induction of CD3-
tyrosine phosphorylation in JKC2 TCR+mut cells. Cells
(15 × 106) were prepared and stimulated for the
indicated times as described in Fig. 1. Cell lysates (lanes
1-3) or anti-CD3- immunoprecipitates (anti-CD3- 1D4.1
mAb) (lanes 4-8) were separated on a 12.5%
SDS-PAGE gel (non-reducing conditions) and subjected to immunoblotting
with anti-Tyr(P) mAb RC20-HRP. F, the filter shown in
E was stripped and reprobed with an anti-CD3- antiserum
448. Position of tyrosine-phosphorylated 42-44-kDa CD3- dimers is
indicated in panels E and F. All blots
were developed by ECL.
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First, to examine whether CD38 engagement induced PTK activation, the
JK-C2 TCR-+-mut cells were stimulated with the anti-CD38
mAb IB4 and whole lysates were subjected to SDS-PAGE followed by
anti-Tyr(P) immunoblotting. As shown in Fig. 6B
(lanes 5 and 6), in these cells CD38
ligation resulted in tyrosine phosphorylation of a variety of
endogeneous cellular proteins. Since the mutation in the transmembrane
domain of TCR- resulted in impaired association of CD3-, it was
compelling to examine whether the CD3 subunits, CD3- and CD3-,
became tyrosine-phosphorylated upon CD38 cross-linking. Analysis of the
anti-Tyr(P) blots of CD3- immunoprecipitates from
anti-CD38-stimulated cells showed that, indeed, CD3- became
tyrosine-phosphorylated(Fig. 6C, lanes 7 and 8).
CD3- was immunoprecipitated from lysates of stimulated and control
cells with an anti-human CD3- mAb, 1D4.1, followed by nonreducing
SDS-PAGE and immunoblotting using either an anti-Tyr(P) mAb (Fig.
6E) or the anti-CD3- antiserum, 448 (Fig. 6F).
In contrast to CD3-, CD3- was barely tyrosine-phosphorylated
following CD38 ligation (Fig. 6E, lanes
7 and 8), with little appearance of the high
molecular weight forms of phosphorylated CD3- (Fig. 6F, lanes 7 and 8). The failure of the
anti-CD38 mAb to induce an increase in CD3- tyrosine phosphorylation
was not due to the presence in unstimulated cells of a low molecular
weight form of tyrosine-phosphorylated CD3- (Fig. 6E,
lane 4), because CD3 ligation with the
anti-CD3- mAb OKT3 induced a significant increase in CD3-
tyrosine phosphorylation (Fig. 6E, lanes
5 and 6), with the appearance of 42-44-kDa
CD3- dimers (Fig. 6F, lanes 5 and 6). Note that these 42-44-kDa phosphorylated CD3- dimers
could also be seen in whole lysates blotted with the anti-CD3-
antiserum 448 (Fig. 6F, lane 2 versus lane 3). Since ITAM tyrosine
phosphorylation is critical for ITAM-mediated signaling functions,
these data indicate that association of CD3- to the TCR is not
necessary for CD38 to induce PTK activation. Moreover, the predominant
CD3- tyrosine phosphorylation with little phosphorylation of CD3- strongly suggests that, in these cells, the signals originated from
CD38 converge on CD3-.
Assessment of MAP kinase activation in mutant cells upon CD38 ligation
was performed as described above (Fig. 5). In JK-C2 TCR-+-mut cells, increased tyrosine phosphorylation and
mobility shift of Erk-2 was clearly detectable after 5 min of CD38, or
CD3 stimulation with specific antibodies (Fig.
7A). These results prove that
CD38-mediated MAP kinase activation does not require the association of
the CD3- chain with the TCR. Collectively, these results imply that, in these cells, the CD3- module couples CD38 to the signal transduction machinery.
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Fig. 7.
CD38-mediated MAP kinase activation occurs in
a Jurkat cell mutant with a defective CD3-
association to the TCR/CD3 complex. A, JKC2
TCR+mut cells (15 × 106 cells/point)
were prepared and stimulated for the indicated times as described in
Fig. 1. Cell lysates (lanes 1-3) or anti- Erk-2
immunoprecipitates (lanes 4-8) were separated on
a 10% SDS-PAGE gel (reducing conditions) and subjected to
immunoblotting with anti-Tyr(P) mAb RC20-HRP. Position of
tyrosine-phosphorylated Erk-2 is indicated. B, the filter
shown in A was stripped and reprobed with an
affinity-purified anti-Erk-2 Ab, showing equal levels of
immunoprecipitated Erk-2 protein (lanes 4-8). In
total cell lysates, a significant Erk-2 mobility shift (lane
3) was induced by CD38 ligation, similar to that induced by
CD3 engagement (lane 2). All blots were developed
by ECL.
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Deficient Coupling of CD38 to Signaling in CD25- and CD25-
Chimeric Receptor-expressing Cells--
The relatively long
cytoplasmic domains of both CD2 and CD3- are required for
CD2-mediated signaling (49, 50), and it has been proposed that CD4 is
recruited to the TCR/CD3 complex by interaction of the Lck Src homology
2 domain with TCR/CD3-associated tyrosine-phosphorylated ZAP-70 (51), a
process that requires CD3- surface expression (7, 8). These data
imply that the relevant biochemical components for CD2, or CD4 to
interact with the CD3 subunits are intracellular structures. Given that
CD38 signaling function seems to be linked to the CD3- and
CD3- transduction modules, we analyzed whether the
intracellular domains of either CD3- or CD3- were sufficient to
couple CD38 to PTK and MAP kinase activation. To test these
possibilities, we used two CD25 chimeric receptor-expressing cell lines
FEV20.3 (JK-CD25-+), and FEV19.5.4
(JK-CD25-+). These cells were generated by stable
transfection of a TCR- variant of the Jurkat cell line
(J.RT3-T3.5) with the cDNA encoding the complete human CD25 ecto-
and transmembrane domains fused to either the complete mouse CD3-,
or CD3- cytoplasmic domains (28) (Fig.
8A, left
panels). As demonstrated earlier, these cells do not express
the TCR/CD3 complex on the cell surface, and the chimeric molecules do
not associate with endogenous CD3 subunits and therefore act as
physically independent signaling molecules (28). Both cell lines
expressed CD38 on the cell surface (Fig. 8A,
right panels). CD38 cross-linking resulted in
very weak induction of protein tyrosine phosphorylation in
JK-CD25-+ cells and non-detectable induction in
JK-CD25-+ cells (Fig. 8B, lanes
4 and 9, respectively). By contrast, in theses
cells direct cross-linking of either the CD25- or the CD25-
chimeric molecules with an anti-CD25 mAb resulted in significant increases in protein tyrosine phosphorylation (Fig. 8B,
lanes 3 and 8, respectively), which
was still detectable at a 10-fold lower dose of anti-CD25 mAb (Fig.
8B, lanes 5 and 10,
respectively).
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Fig. 8.
Deficient coupling of CD38 to PTK activation
in CD25 chimeric receptor-expressing cells. A, surface
expression of the CD25/ and CD25/ chimeric molecules
(left panels) and CD38 (right
panels) in FEV20.3 (JK-CD25-+), and FEV19.5.4
(JK-CD25-+) cells. Cells were stained with the
anti-human CD25 mAb (B1.49.9), or the anti-CD38 (IB4), followed by
F(ab')2 FITC-RmIg secondary Ab. Negative controls were
stained with the secondary Ab alone. B, pattern of
receptor-mediated increases in protein tyrosine phosphorylation.
JK-CD25- and JK-CD25- cells (2 × 107
cells/point) were prepared as described in Fig. 1. Then cells were
incubated for 10 min on ice with 10 µg/107 cells of
anti-CD38 mAb (IB4) (lanes 4 and 9),
or with 10 µg/107 cells anti-human CD25 mAb (B1.49.9)
(lanes 3 and 8), or with 1 µg/107 cells of the anti-human CD25 mAb (lanes
5 and 10), or left in RPMI-HEPES (nonstimulated,
lanes 1, 2, 6, and
7). This was followed by cross-linking with 30 µg/107 cells (lanes 2-4 and
7-9) or 3 µg/107 cells (lanes
5 and 10) of the secondary antibody
F(ab')2 GmIg for 10 min on ice. Then the stimulation was
conducted for 5 min at 37° C. Cell lysates (300,000 cell
equivalents) were separated on 10% SDS-PAGE gel (reducing conditions)
and subjected to immunoblotting with anti-Tyr(P) mAb RC20-HRP. All
blots were developed by ECL.
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We next examined whether CD25- or CD25- themselves, both of which
contain cytoplasmic ITAMs, were tyrosine-phosphorylated when CD38 was
cross-linked with the anti-CD38 mAb IB4. This was determined by
immunoprecipitation of the chimeric proteins from control and
stimulated cells, followed by immunoblotting with an anti-Tyr(P) mAb.
Whereas in JK-CD25-+ cells CD25- was weakly
phosphorylated after CD38 ligation (Fig. 9A, lane
3), in JK-CD25-+ cells no signal could be
detected for CD25- (Fig. 9B, lane
3). In contrast, in JK-CD25-+ cells CD25-
was strongly tyrosine-phosphorylated when directly cross-linked with an
anti-CD25 mAb (Fig. 9A, lanes 2 and
4). As reported earlier in TCR BW 5147 cells
stably transfected with CD25- chimeras (52), in
JK-CD25-+ cells CD25- resulted in very weak (if any)
tyrosine phosphorylation after CD25 ligation (Fig. 9B,
lanes 2 and 4). This could due to the
presence of a single ITAM in its cytoplasmic domain.
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Fig. 9.
Deficient induction of CD38-mediated chimeric
tyrosine phosphorylation and MAP kinase activation. JK-CD25-
and JK-CD25- cells were prepared, stimulated and lysed as described
in Fig. 8. A, the CD25- protein was immunoprecipitated
from JK-CD25- cell lysates with the anti-CD3- mAb 1D4.1.
Immunoprecipitates were run on 12.5% SDS-PAGE (nonreducing conditions)
and subjected to immunoblotting with anti-Tyr(P) mAb RC20-HRP
(lanes 1-4) or anti-CD3- antiserum 448 (lanes 5-8). In both panels position of
tyrosine-phosphorylated CD25- is indicated. B, CD25-
chimeric protein was immunoprecipitated from JK-CD25- cell-lysates
with the anti-CD3- mAb 2F4.1. Immunoprecipitates were run on 12.5%
SDS-PAGE (nonreducing conditions) and subjected to immunoblotting with
the anti-Tyr(P) mAb (lanes 1-4), or with an
affinity-purified anti-CD3- Ab (lanes 5-8).
Position of CD25- is indicated. C, Erk-2 protein was
immunoprecipitated from JK-CD25- cell lysates with the anti-Erk2 Ab.
Immunoprecipitates were run on 10% SDS-PAGE (nonreducing conditions)
and subjected to immunoblotting with the anti-Tyr(P) mAb
(lanes 1-4) or an affinity-purified anti-Erk2 Ab
(lanes 5-8). D, Erk-2 was
immunoprecipitated from JK-CD25- cell lysates with the anti-Erk2 Ab.
Immunoprecipitates were separated as in panel C
and subjected to immunoblotting with the anti-Tyr(P) mAb
(lanes 1-4) or with the anti-Erk2 Ab
(lanes 5-8). The position of Erk2 and
phosphotyrosine Erk2 are indicated in panels C
and D. All blots were developed by ECL.
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To examine whether the MAP kinase Erk-2 was activated upon CD38 or
chimera cross-linking, we monitored receptor-induced increases in Erk-2
tyrosine phosphorylation and changes in its electrophoretic mobility.
To assess Erk-2 tyrosine phosphorylation, Erk-2 was immunoprecipitated
from lysates of unstimulated or stimulated cells by using an anti-Erk-2
antibody and then immunoblotted with an anti-Tyr(P) mAb (Fig. 9,
C and D, right panels).
Whereas CD38 ligation only induced a faint increase in Erk-2 tyrosine
phosphorylation in CD25-+ cells (Fig. 9C,
lane 7), the same stimulus did not induce
detectable Erk-2 tyrosine phosphorylation in JK-CD25-+
cells (Fig. 9D, lane 7). In contrast,
cross-linking of the respective chimeric proteins with an anti-CD25 mAb
led to a significant increase in Erk-2 tyrosine phosphorylation (Fig.
9, C and D, lane 6), even when a 10-fold lower dose of anti-CD25 mAb were used (Fig. 9, C and D, lane 8). To
monitor changes in Erk-2 electrophoretic mobility, we performed in
whole cell lysates from unstimulated or stimulated cells Western blot
analysis with an anti-Erk-2-specific antibody (Fig. 9, C and
D, left panels). No mobility shift of Erk2 was observed in cell lysates from anti-CD38-treated
JK-CD25-+ and JK-CD25-+ cells (Fig.
9C, lane 3; Fig. 9D,
lane 3), whereas a reduced electrophoretic mobility of Erk2 was readily detected in cell lysates from
anti-CD25-treated cells (Fig. 9, C and D,
lanes 2 and 4). These results provided evidence that the cytoplasmic domains of either CD3- or CD3- alone are not sufficient to mediate CD38-induced signaling events, despite the fact that they are independently capable of signal transduction, leading to PTK and MAP kinase activation.
| |
DISCUSSION |
The results show that CD38 ligation leads to both PTK and MAP
kinase activation, in TCR+ T cells that express the whole
set of CD3 subunits (i.e. the CD3-- homodimer and the
CD3-, and CD3-, heterodimers), or in TCR+ cells
with a defective CD3- association. Since the CD3-- homodimer contributes with 6 out of 10 ITAMs within a given TCR/CD3 complex, these data highlight the importance of the so-called CD3-
signaling module, identifying it as a necessary and sufficient
component of the TCR/CD3 complex involved in T cell activation through
CD38. The lack of PTK and MAP kinase activation in cells expressing chimerical CD25- or CD25- receptors in response to CD38
stimulation strongly suggest that either the extracellular, or the
transmembrane domain, of one, or more CD3 subunits are required for
CD38-mediated signaling.
How does CD38 cross-linking lead to activation of Src family PTKs and
tyrosine phosphorylation of CD3- and CD3- ITAMs? That CD3- and
CD3- subunits become tyrosine-phosphorylated upon CD38 engagement
could be explained by a model in which CD38 ligation by agonistic
antibodies would induce CD38 association with the TCR/CD3 complex. In
this model, a fraction of Lck could directly associate with CD38. The
recruitment of CD38 to the proximity of the TCR/CD3 complex would allow
the putative CD38-associated PTK (e.g. Lck) to reach a
relative high concentration for the effective tyrosine phosphorylation
of the CD3 chains. So far, there is little evidence to suggest that Lck
is constitutively associated with CD38, but overall the results suggest
that signals delivered by CD38 must be integrated at or near the T cell
surface membrane that is in contact with both Lck and the TCR/CD3
complex. If so, CD38 could act as an authentic co-receptor bringing the associated PTK in the proximity of the CD3 chains to promote an augmented T cell response.
It is important to note that resident PTKs are associated with the
TCR/CD3 complex in nonactivated T cells. These include Fyn that was
found associated with the TCR/CD3 complex at a low stoichiometry (53),
and Lck, demonstrated to be present in a complex that also includes CD4
or CD8 and CD5 at the cell surface of nonactivated T cells (54-56). In
a variation of the model for CD38 signaling described above, we
envision that the CD3 chains may function as docking molecules to link
CD38 to the TCR/CD3-associated PTKs. In this scenario CD38 would be
unable by itself to deliver PTK-dependent activating
signals and CD38 signaling capabilities would be regulated exclusively
by the ability of CD38 ligands to bring CD38 in close proximity to the
TCR/CD3 complex.
In its simplest interpretation, therefore, CD38-mediated signaling
capacity is determined by the efficiency of ligand-induced CD38/TCR/CD3
co-aggregation. However, we cannot rule out that conformational changes
in either CD38, the TCR/CD3 complex, or both are required for optimal
signaling. It is interesting that the two epitopic sites (the amino
acid sequences of 220-241 and 273-285) recognized by all known
agonistic anti-human CD38 mAbs, including IB4, are distinct to the site
recognized by the nonagonistic anti-CD38 mAb OKT10 (amino acid sequence
of 280-298) at the C-terminal portion of human CD38 (57). Despite the
closeness of the sites recognized by IB4 and OKT10, the biological
effects exerted by these antibodies are quite different. Thus, both IB4
and OKT10 mAbs induce the TCR/CD3 complex to co-cap with CD38 (30),
suggesting that both antibodies are able to promote an association
between CD38 and the TCR/CD3 complex. However, IB4, unlike OKT10,
induces increases in PLC-1 tyrosine phosphorylation,
Ca2+ mobilization, long term down-modulation of the TCR/CD3
complex, and apoptosis (this paper and Ref. 22). Therefore, these
results suggest that a transient TCR/CD3 re-localization induced by
anti-CD38 mAbs is not sufficient to stimulate CD38-mediated signaling.
We favor the interpretation that there may be preferred conformations of CD38 and the TCR/CD3 complex that allow stable interactions between
these molecules and that are optimal for signal transduction.
Evidence that the extracellular or transmembrane domains of the CD3
chains are crucial for their functional interaction with CD38 is
indirectly provided by the functional data on a TCR
Jurkat variant transfected with CD25- or CD25- chimeras. The expression of these chimeras at the cell surface, without other TCR/CD3
components, is not sufficient to rescue CD38-mediated responses (Figs.
8 and 9). In contrast, it is apparent from the analysis of the
TCR+ transfectants with a deficient CD3- chain
association that the CD3- signaling module is sufficient for
CD38-mediated signaling (Figs. 6 and 7). The most obvious structural
differences between the CD3- signaling module and the
CD25-chimerical proteins rely in the number of ITAMs, and in the
presence or not of the CD3 extracellular and transmembrane domains.
Thus, in a single CD3- module, four ITAMs are present,
corresponding to the , and pairs, versus three
ITAMs in CD25- and a single ITAM in CD25-. Since the different
CD3 ITAMs may function in a quantitative manner (58), the lack of
response by CD38-stimulated CD25-- or CD25--expressing cells may
indicate that more than three ITAMs are necessary for efficient
CD38-mediated signaling and therefore, both CD25- and CD25-
chimeric proteins are unable to reach the threshold of activation of
any signaling pathway emanating from CD38. This is unlikely, because in
those cells ligation of the CD25- or CD25- chimeras with
anti-CD25 mAbs results in increases in protein tyrosine phosphorylation
and Erk-2 activation (Figs. 8 and 9), with efficacies similar to those
elicited by cross-linking the CD3- module in the CD3-
deficient cells (Figs. 6 and 7). We speculate that the organization of
the CD3 chains in dimers, including CD3-, CD3-, and
CD3--, could facilitate the proper architecture to oligomerize
with other receptors, whereas the chimeric receptors made up of single
chains as CD25- or CD25- are less efficient in that respect. It
is, therefore, reasonable to assume that the extracellular, or
transmembrane domains of the CD3 chains may play a role in the
TCR/CD3/CD38 interaction.
While the distinct signaling pattern induced by CD38 in the
TCR+ transfectants with a deficient CD3- chain
association underscore the importance of the CD3- module in
mediating CD38 signaling, we cannot exclude a functional role for
CD3- in this process. In fact, the observation that in wild-type
Jurkat T cells CD38 ligation results in both CD3- tyrosine
phosphorylation (Fig. 1B) and recruitment of ZAP-70 by
CD3- (21) may reflect the participation of CD3- in the CD38
signaling pathway. It is likely that a convergence, or synergism of
CD3-- and CD3--mediated signals may occur at certain critical
points downstream of CD3 tyrosine phosphorylation and ZAP-70
activation. In this sense, SLP-76, which becomes
tyrosine-phosphorylated upon CD38 cross-linking (Fig. 4D),
may play such a role as it is suggested to operate in CD3-mediated
signals (59).
We speculate that a specific pairing of CD38 with a particular CD3
subunit may endow CD38 with distinct signaling capabilities. This could
allow different signaling responses depending on how a particular
ligand (e.g. CD31, agonistic versus nonagonistic anti-CD38 mAbs, etc.) would affect the CD38/CD3 interaction. This may
have some relevance to the observation that different CD38 antibodies
and ligands can induce qualitatively different signals (6, 22, 57).
Alternatively, the modular architecture of the TCR/CD3 complex permits
the occurrence of multiple TCR "isoforms" made of distinct
polypeptide combinations (60-62). These isoforms may coexist and b
Transducing Module Mediates CD38-induced
Protein-tyrosine Kinase and Mitogen-activated Protein Kinase Activation
in Jurkat T Cells*
§,
,
TOP
ABSTRACT
INTRODUCTION
