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-Chain Signaling Motifs1
,*
Departments of
*
Medicine, and Microbiology and Immunology, and
Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; and
Laboratory of Mammalian Genes and Development, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß TCR is a multimeric protein complex comprising
ligand-binding and signal-transducing subunits. The signal transduction
processes are mediated by the immunoreceptor tyrosine-based activation
motifs (ITAMs), and up to 10 ITAMs are present within a single TCR
complex. This multiplicity may allow for signal amplification and/or
the formation of qualitatively distinct intracellular signals. Notably,
the TCR-
subunit contains three ITAMs, and exists as a
disulfide-linked homodimer in the TCR complex. In normal murine
thymocytes and peripheral T cells, a proportion of TCR- molecules is
constitutively tyrosine phosphorylated and associated with the ZAP-70
protein tyrosine kinase. We examined the contribution of the different
TCR- ITAMs in regulating the constitutive phosphorylation of the
TCR- subunit in thymocytes by analyzing TCR--deficient mice that
had been reconstituted with either full-length or single
ITAM-containing TCR- subunits. We report in this work that in the
absence of a full-length TCR- subunit, there is no apparent
constitutive phosphorylation of the remaining TCR/CD3 ITAMs. Following
TCR ligation, all of the CD3 ITAMs become inducibly phosphorylated and
associate with the ZAP-70 protein tyrosine kinase. Regardless of the
number of TCR- ITAMs present in the TCR complex, we report that a
number of molecules involved in downstream signaling events, such as
ZAP-70, SLP-76, and pp36, are all inducibly tyrosine phosphorylated
following TCR ligation. These results support the notion that the
different TCR ITAMs function in a quantitative rather than qualitative
manner. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
TCR is assembled as an oligomeric complex consisting of six different
transmembrane proteins (1). The six proteins include the Ag-binding
subunits (TCR-ß) that are noncovalently associated with the
signal-transducing chains (CD3-
, CD3-
, CD3-
, and TCR-
and/or TCR-
). These signaling chains all contain a common
sequence (YxxLx(6, 7, 8)YxxL), termed the
immunoreceptor tyrosine-based activation motif
(ITAM)4 (2). The ITAM motif
is present as a single copy in the CD3-, CD3-, and CD3-
subunits, and as three copies in the TCR- subunit (3). In T cell
lines and clones, one of the initial events following TCR engagement is
the phosphorylation of tyrosine residues in the ITAMs, a process
regulated by the Src-family PTKs, Lck or Fyn (4, 5, 6, 7). The
phosphorylation of the two tyrosines within an ITAM promotes a high
affinity interaction with a second family of PTKs, the Syk/ZAP-70
family. This interaction is mediated by the two SH2 domains of
Syk/ZAP-70 and the two phosphotyrosines in the ITAM (8, 9, 10). The
recruited Syk/ZAP-70 molecules are activated by phosphorylation and
contribute to a cascade of downstream signals that are crucial for the
initiation of cellular responses.
The most widely held structural model of the TCR is one comprising a
dimer and two CD3 pairs (, ). Therefore, 10 ITAMs may
be present within a single TCR complex. It remains to be determined how
10 ITAMs contribute to Ag receptor signal transduction. The presence of
multiple ITAMs within the TCR complex may allow for signal
amplification and/or the formation of qualitatively distinct
intracellular signals. These questions have, in part, been addressed in
both cell lines and in mice by the use of chimeric proteins containing
the cytoplasmic domains of one or more TCR ITAMs. In the Jurkat T cell
line, stimulation of such chimeric receptor complexes comprising one,
two, or three copies of a single TCR- ITAM resulted in the induction
of identical patterns of tyrosine-phosphorylated proteins, with the
triplication of one ITAM resulting in enhanced signaling (11). In
transgenic mice expressing chimeric constructs encoding the cytoplasmic
domain of either the TCR- or CD3- subunit, cross-linking the
chimeric receptor expressed on thymocytes induced a greater degree of
proliferation with TCR- than CD3- (12). These results are
consistent with the idea that three ITAMs (TCR-) provide
quantitatively stronger signals than a single ITAM (CD3-). However,
one cannot rule out the possibility in these systems that TCR- ITAMs
activated qualitatively distinct pathways that enhanced proliferation
relative to CD3-.
The suggestion that the 10 TCR ITAMs function in a quantitative manner
is also consistent with studies on mice expressing TCR complexes
lacking one or more ITAMs. Mice deficient in the TCR -chain
(TCR--/-) have substantially reduced numbers of both
CD4+CD8+ thymocytes and mature single-positive
T cells (13, 14, 15, 16). Reconstituting these TCR--/- mice
with transgenes encoding TCR- subunits lacking all three ITAMs
restored T cell development (17, 18). This indicates that the
developmental defects in the -deficient mice are primarily a
consequence of impaired TCR surface expression, with the TCR- ITAMs
primarily augmenting the efficiency of T cell development (17). This is
especially evident in TCR-ß transgenic mice bearing TCRs of known
specificity, in which a reduction in the number of TCR- ITAMs can
dramatically reduce the efficiency of both positive and negative
selection (18). Second, T cells isolated from TCR--deficient mice,
when reconstituted with the TCR- subunit, exhibited a significant
level of self-reactivity in vitro (19). These data suggest that
multiple ITAMs are involved in signal amplification and can
influence T cell repertoire selection.
In addition to their contribution to signal amplification, the 10 ITAMs
in the TCR complex may also have qualitatively distinct functions. For
example, in certain cell lines, stimulation of chimeric proteins
comprising the cytoplasmic domains of either TCR- or CD3- can
result in qualitatively different patterns of induced phosphoproteins
(20). Different TCR ITAMs differ both qualitatively and quantitatively
in their ability to couple to programmed cell death, with the first
ITAM of TCR- being particularly effective in inducing apoptosis
(21). Finally, the third ITAM of TCR- and the CD3- ITAM may also
be required for activation-dependent interactions with the actin
cytoskeleton (22, 23). These qualitative differences may be explained
by the sequence heterogeneity in the different ITAMs (24). Thus,
although all doubly phosphorylated ITAMs can interact with the ZAP-70
PTK, the phosphorylated ITAMs have differential abilities to interact
with distinct SH2-containing proteins such as Fyn, Shc, Grb2, and SHIP
(SH2-containing inositol phosphatase) (25, 26, 27). For example, Shc has
been reported to interact with phosphorylated TCR-, but not CD3-
(28). Moreover, in in vitro binding studies, Shc appears to complex a
doubly phosphorylated CD3- or CD3- ITAM, or the third ITAM of
(3) much more effectively than the first or second
ITAMs (1 or 2), while Grb2 appears to
bind more effectively to the phosphorylated 1, CD3-,
and CD3- ITAMs than the other TCR ITAMs.
It is interesting to note that the TCR -chain is constitutively
tyrosine phosphorylated and associates with the ZAP-70 PTK when
isolated from normal murine thymocytes and peripheral lymph node T
cells (29). The constitutive tyrosine phosphorylation appears specific
to TCR-, since the other CD3 ITAMs are not easily detected in a
constitutively phosphorylated form, but can be inducibly phosphorylated
(29, 30). The constitutive phosphorylation of the TCR -chain in
murine thymocytes is regulated primarily by the Lck PTK (7, 29). It
remains unclear which TCR- ITAMs are required for this constitutive
tyrosine phosphorylation and how this influences TCR signal
transduction.
To address the contribution of the TCR- ITAMs in regulating the
constitutive phosphorylation of the TCR- subunit, and to examine
qualitative signaling differences with the various ITAMs, we analyzed
mice deficient in the TCR- subunit that had been reconstituted with
full-length -chain or TCR -chain subunits containing single
ITAMs. We report in this work that in the absence of a full-length
TCR- subunit containing three ITAMs, there is no apparent
constitutive phosphorylation of the remaining TCR/CD3 ITAMs. Following
TCR ligation, all of the CD3 ITAMs become inducibly phosphorylated and
associate with the ZAP-70 PTK. Regardless of the number of TCR-
ITAMs present in the TCR complex, we report that various substrates,
including ZAP-70, SLP-76, and pp36, were all inducibly tyrosine
phosphorylated. These results support the notion that the different TCR
ITAMs function in a quantitative manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were purchased from The Jackson Laboratory (Bar
Harbor, ME) and maintained in Animal Care Facility at University of
California, San Francisco (UCSF). The TCR--deficient mice
reconstituted with full-length TCR-, the third ITAM of
(D66–114), the first ITAM of (D108–150), or TCR- molecules
lacking all three ITAMs (D67–150) have been described in detail
elsewhere (13, 17, 18). All of the mice are maintained in microisolator
cages at UCSF. Murine thymocytes, lymph node T cells, and spleen cells
were isolated as previously described (29).
Abs and antisera
The Abs used for flow cytometry, immunoprecipitations, and
Western blotting are as follows: fluorescein-conjugated
anti-CD8, phycoerythrin-conjugated anti-CD4, and rabbit
anti-p44 erk were purchased from Caltag (South San
Francisco, CA). 145-2C11, CD3- (American Type Culture Collection,
Rockville, MD); 4G10, phosphotyrosine (Upstate Biotechnology, Lake
Placid, NY); 6B10.2, TCR- (31) were used as indicated. Anti-ZAP-70
antisera were generated against a peptide corresponding to the human
sequence from amino acids 282–307. Anti-SLP-76 was kindly provided by
Dr. G. Koretzky (University of Iowa, Iowa City, IA). Horseradish
peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit
IgG were obtained from Southern Biotechnology (Birmingham, AL);
alkaline phosphatase-conjugated goat anti-rabbit Ig and goat
anti-mouse Ig were purchased from Bio-Rad (Hercules, CA).
Stimulation, precipitation, and immunoblotting
Thymocyte and lymph node T cells were washed several times in PBS and were resuspended in PBS at a concentration of 1 to 2 x 108 cells/ml. The stimulations, precipitations, and Western blotting was performed as described in detail elsewhere (29).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit requires more than the first or third TCR- ITAMs
We have reported previously that in both murine thymocytes and
lymph node T cells, a proportion of TCR- subunits is constitutively
tyrosine phosphorylated and associated with the ZAP-70 PTK (29). Since
the TCR- subunit comprises three ITAMs, we assessed the contribution
of the different TCR- ITAMs in regulating the constitutive and
inducible phosphorylation of the TCR/CD3 subunits and the inducible
phosphorylation of ZAP-70. For this purpose, we analyzed
TCR--deficient mice that had been reconstituted with TCR-
transgenes lacking one or more ITAMs (13, 17). Thymocytes were
isolated from normal mice and TCR--deficient mice reconstituted with
transgenes encoding TCR- molecules without any ITAMs (D67–150), the
third ITAM of (D66–114), the first ITAM of (D108–150), or the
full-length TCR- subunit. As described in detail elsewhere, and
shown in this study for comparative purposes, thymocytes from the
D67–150 and D66–114 mice have relatively normal numbers of
CD4+CD8+ thymocytes that are capable of
maturing into both the CD4-CD8+ and
CD4+CD8- single-positive populations (Fig. 1
) (17). It should be noted that as a
consequence of the transgene expression, the TCR density is elevated
relative to wild-type mice (Fig. 1).
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-chain under
conditions that maintain the association of the TCR- subunit. The
precipitates were resolved on SDS-PAGE and subsequently immunoblotted
with anti-phosphotyrosine Abs. As shown in Figure 2A, a proportion of TCR-
molecules is constitutively tyrosine phosphorylated (21 kDa) when
isolated from control thymocytes (Fig. 2A, lane
1). TCR ligation resulted in the tyrosine phosphorylation
of CD3- (lane 2) and a weak induction of
ZAP-70 phosphorylation that was revealed following more prolonged
enhanced chemoluminescence exposures. In mice reconstituted with
TCR- molecules lacking any TCR- ITAMs (D67–150), there was no
detectable phosphoprotein migrating near 10 kDa, a size corresponding
to the mutated subunit (lane 3). Moreover,
none of the CD3 chains demonstrated any appreciable constitutive
phosphorylation in these mice (D67–150). Following TCR ligation, there
was a marked increase in the tyrosine phosphorylation of CD3-,
CD3-, and CD3-, and ZAP-70 in the thymocytes from the D67–150
mice (lane 4 vs lane 3) (32). In
the -/- mice reconstituted with the third ITAM of
TCR- (D66–114), the TCR -chain was also not constitutively
tyrosine phosphorylated since no phosphoprotein was detected migrating
near 12 kDa (lane 5). TCR stimulation resulted
in the tyrosine phosphorylation of the CD3 subunits and ZAP-70
(lane 6). However, the third ITAM of was
not easily detected as a phosphorylated protein even after TCR
stimulation. Only prolonged enhanced chemoluminescence exposures
revealed the presence of some phosphorylated from the D66–114 mice
(data not shown). We have also determined that thymocytes from the
TCR--/- mice reconstituted with the first ITAM of
TCR- (D108–150) also exhibited very little constitutive or
inducible phosphorylation of TCR- (data not shown).
TCR--/- mice reconstituted with the full-length
TCR- responded in a manner similar to wild-type mice
(lanes 7–8 vs lanes 1–2).
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ITAM (D66–114, D108–150) subunit exhibited limited
constitutive or inducible phosphorylation when prepared from murine
thymocytes, it was possible that the truncated TCR- subunits were
not coprecipitating with the TCR complex and/or were not preferred
substrates following TCR ligation. We did not have any anti-TCR-
antisera or mAbs that could precipitate all of the truncated TCR-
construct. Instead, we directly immunoprecipitated ZAP-70, as ZAP-70
can associate with any of the doubly phosphorylated ITAMs (9). As
reported elsewhere and shown in this study for comparative purposes,
ZAP-70 is constitutively associated with the tyrosine-phosphorylated
TCR- subunit (21 kDa) when isolated from control unstimulated
thymocytes (Fig. 2
B, lane 1). In mice
reconstituted with TCR- molecules lacking any TCR- ITAMs
(D67–150), there was no detectable 10-kDa phosphoprotein that
coprecipitated with ZAP-70. In addition, none of the CD3 subunits
appeared to be associated with ZAP-70, consistent with our results with
the anti-CD3- immunoprecipitates (Fig. 2, A and
B). Following TCR ligation, there was a marked
increase in the tyrosine phosphorylation of CD3-, CD3-, and
CD3-, which coprecipitated with tyrosine-phosphorylated ZAP-70
(lane 4 vs lane 3) (32). It also
should be noted that we were unable to detect any constitutively or
inducibly tyrosine-phosphorylated 12-kDa TCR- (D66–114) molecules
coprecipitating with ZAP-70 before or after TCR ligation in the
-/- mice reconstituted with the third ITAM of TCR-
(D66–114) (lanes 5 and 6).
TCR--/- mice reconstituted with the full-length
TCR- responded in a manner similar to wild-type mice
(lanes 7–8 vs lanes 1–2). Taken
together, the aforementioned results suggested that the single ITAM
(D66–114) subunit was not a preferred substrate following TCR
ligation.
To further examine the possibility that the D66–114 TCR- subunit
was not normally phosphorylated following receptor engagement,
thymocytes from the different mice were treated with the protein
tyrosine phosphatase inhibitor pervanadate. As shown in Figure 3, pervanadate treatment of normal
thymocytes leads to a significant increase in phosphorylation
(lane 2). In the absence of any ITAMs,
pervanadate treatment resulted in a substantial increase in the
phosphorylation of the CD3 subunits (lane 4 vs
lane 3). Importantly, when thymocytes from the
D66–114 mice were stimulated in this manner, a large increase in the
phosphorylation of the third ITAM of TCR- that coprecipitated with
CD3- was found (lane 6). These results
suggest that TCR- containing only the third ITAM of TCR- is
stably associated with the TCR complex, but is not a preferred
substrate in vivo or in vitro following anti-TCR stimulation. We
have also confirmed these findings by directly precipitating the
TCR- subunit with anti-phosphotyrosine mAbs, and found very
little constitutive or inducible phosphorylation of the truncated
TCR- subunits (data not shown). In summary, the results demonstrate
that even in the absence of any constitutive or inducible
phosphorylation of the truncated TCR- constructs, the CD3-,
CD3-, and CD3- subunits are effectively tyrosine phosphorylated
and can associate with ZAP-70 following TCR engagement. In fact, the
degree of tyrosine phosphorylation of the CD3 subunits appears much
more pronounced in mice reconstituted with -chains lacking ITAMs
than in wild-type mice. However, the variability noted in the TCR
density for the various mice precludes a conclusive statement
regarding this possibility. Second, the constitutive and inducible
tyrosine phosphorylation of the TCR -chain detected in murine
thymocytes requires more than the first or third ITAM of
TCR-.
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ITAMs
Several reports have indicated that both quantitative and
qualitative differences exist between the various TCR/CD3 ITAMs and
their ability to couple to cytosolic effector molecules and promote T
cell apoptosis (20, 21). Since differences in the constitutive and
inducible phosphorylation of the TCR/CD3 ITAMs were noted between the
normal and the D67–150 and D66–114 transgenic mice, it was possible
that the various effector molecules could be differentially regulated.
To examine this issue, we initially compared the patterns of tyrosine
phosphoproteins induced following TCR ligation. Ligation of the TCR
expressed on thymocytes from normal mice results in the induced or
increased tyrosine phosphorylation of a number of proteins with m.w. of
110, 95, 80, 70, 36, and 21 kDa (Fig. 4).
Notably, with the exception of the TCR- subunit, most of these
phosphoproteins were also induced in the cells from D67–150, D66–114,
and transgenic mice (Fig. 4, lanes 4, 6, and
8). Differences in the intensity of the
phosphoproteins shown in the blots may reflect the variability seen in
the TCR density and were not a consistent finding (Fig. 1). Kinetic
analyses from 1 to 30 min after TCR stimulation failed to reveal any
significant differences (data not shown).
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, TCR ligation of thymocytes from normal
mice results in the phosphorylation of SLP-76 (Fig. 5, lane
2 vs lane 1). Notably, two phosphoproteins of 36
and 130 kDa are also coprecipitated with SLP-76 in the thymocytes. In
the absence of any functional TCR- ITAMs, or in mice expressing a
single TCR- ITAM, SLP-76 is inducibly phosphorylated and
coprecipitates with p36 and p130, as detected by
anti-phosphotyrosine immunoblotting (Fig. 5, lanes 2,
4, and 6). Precipitates formed with normal
rabbit sera served as negative controls in these experiments
(lanes 7 and 8). To further confirm
that no differences in the patterns of substrates that are inducibly
phosphorylated exist between the various mice, we also used GST-Grb2
fusion proteins to coprecipitate inducibly phosphorylated proteins. As
shown in Figure 6, in normal mice, a
number of phosphoproteins can be precipitated with GST-Grb2 fusion
following TCR ligation (lane 2 vs lane
1). Regardless of whether the TCR complex lacks all three
TCR- ITAMs, or comprises one TCR- ITAM, similar phosphoproteins
are found to precipitate with GST-Grb2 (Fig. 6, lanes
3–8). We have also directly immunoprecipitated specific
signaling molecules, including vav, Pyk2, and
erk, and found that these proteins are inducibly tyrosine
phosphorylated following TCR stimulation in all of the TCR- ITAM
mice examined (data not shown). In summary, these results demonstrate
that a number of TCR-induced substrates, including SLP-76, p36, and
p130, are induced even in situations in which the TCR has no TCR-
ITAMs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ITAMs on TCR-mediated
signal transduction in thymocytes and peripheral lymph node T cells, we
analyzed TCR--deficient mice that had been reconstituted with
TCR- constructs lacking one or more TCR- ITAMs.
We have reported previously that in thymocytes and peripheral T cells,
the TCR- subunit is constitutively tyrosine phosphorylated and
associated with the ZAP-70 PTK (29). The constitutive phosphorylation
of TCR- is, in part, but not entirely a consequence of TCR
interactions with MHC molecules in both the thymus and peripheral
lymphoid organs (29). In this study, we show that there is no apparent
constitutive phosphorylation of TCR- subunits containing only the
first or third ITAM. These results suggest that the constitutive
phosphorylation of the TCR- subunit requires at least two, and
perhaps all three, ITAMs. In fact, it is proposed that one of the roles
of tandem-phosphorylated ITAMs is to facilitate the binding and
autophosphorylation of ZAP-70 (34). The binding of ZAP-70 to multiple
phosphorylated ITAMs may also protect the ITAMs from protein tyrosine
phosphatases (35). Alternatively, the constitutive phosphorylation of
TCR- may reside in the second ITAM. It is also possible that the
truncation of the TCR -chains in the transgenic mice decreases the
accessibility of the Lck PTK, since Lck regulates the constitutive TCR
-chain phosphorylation in murine thymocytes (7). This seems less
likely since any of the three TCR- ITAMs, when expressed
independently as chimeric proteins, are readily phosphorylated
following chimera receptor cross-linking (11). Additional TCR-
transgenic mice would be needed to address these issues.
The importance of the constitutive tyrosine phosphorylation of the
full-length TCR- subunit remains unclear at present. Since the
ZAP-70 PTK is already complexed to the pool of tyrosine-phosphorylated
TCR-, the population of ZAP-70/phosphorylated TCR- complexes may
represent cells undergoing signaling events (29). Alternatively, the
TCR complex may be poised or "armed" to respond to TCR
interactions, leading to activation of ZAP-70, which is already
localized at the plasma membrane. It also has been suggested that the
phosphorylated TCR- molecules may be blocked in their capacity to
respond to TCR stimulations, unless the
CD4--associated Lck PTK is co-engaged with the TCR
(36, 37). Regardless of which interpretation is correct, our results
suggest that a composite of TCR- ITAMs is required for the
constitutive tyrosine phosphorylation of TCR-.
In this study, we also show that none of the CD3 subunits appears to
undergo any constitutive tyrosine phosphorylation even in the absence
of any TCR- ITAMs. However, following TCR ligation, there is a
dramatic increase in the induction of CD3 subunit phosphorylation, as
well as the recruitment and phosphorylation of ZAP-70. This occurs with
all of the CD3 ITAMs, , , and (32). This finding is
consistent with the findings of Malissen and colleagues, who showed
that the CD3-, CD3-, and CD3- can form an independent
signaling module (38). Even in the situation in which a single ITAM of
TCR- is present as part of the TCR complex, the ITAMs from the CD3
subunits are preferentially phosphorylated relative to TCR-
following TCR cross-linking. The simplest interpretation of these
results is that in the absence of full-length TCR-, the CD3 subunits
become more accessible to Lck.
The TCR- subunit is important for T cell development by promoting
surface expression of the TCR (17). Notably, the capacity of TCR- to
facilitate thymopoeisis does not require ITAM-mediated signals
(17). This suggests that the remaining CD3 ITAMs (4/TCR complex) in
TCR--/- mice reconstituted with ITAM-less TCR-
transgenes are either compensating for and/or functioning in the
absence of TCR-. Our results are consistent with these
interpretations since, in the absence of TCR- ITAMs, the CD3
subunits are inducibly phosphorylated and associated with the ZAP-70
PTK. Moreover, thymocytes expressing low avidity TCRs require more
TCR- ITAMs for their selection (18). Taken together, the
aforementioned data support the concept that the TCR- ITAMs function
primarily as signal amplifiers during signaling and repertoire
selection in the thymus.
One unresolved issue concerning the different TCR ITAMs is whether they
couple to distinct signaling pathways or substrates. For example,
previous studies have revealed that the TCR- ITAMs and the CD3-
ITAMs, when expressed as chimeric molecules in cell lines and
cross-linked, resulted in the induction of distinct phosphoproteins
(20). Yet, the same chimeric constructs, when expressed as transgenes
in mice, mediated the phosphorylation of identical substrates (12). In
a related signaling system, the ITAM of the FcRIß serves as a
signal amplifier by recruiting the Lyn PTK, which in turn
phosphorylates the ITAM of the FcRI (39). Importantly, Lyn and
not Syk appears to interact with the phosphorylated FcRIß ITAM,
suggesting some SH2 specificity. However, it should be noted that the
distance between the YxxL sequences is only six amino acids, a distance
that may not be adequate for Syk-SH2 binding. The idea that different
SH2-containing signaling molecules can couple differentially to
different phosphorylated ITAMs has been reported in studies mainly
involving in vitro binding assays (25, 26, 27, 40). However, the affinities
between these SH2-containing proteins and phosphorylated ITAMs are 10-
to 100-fold less than that between ZAP-70/Syk and the doubly
phosphorylated ITAMs. In our experiments, the induction of a number of
phosphoproteins, including SLP-76, vav, Pyk2, ZAP-70, p36,
and p130, occurs with TCR complexes comprising only CD3 ITAMs or
complexes, including the third ITAM of TCR-. These results would
indicate a substantial amount of redundancy exists between the
different ITAMs. This is also supported by the reports that both CD3
- and TCR -chain promote programmed cell death in thymocytes, and
induce double-positive development in a Rag- mice (12).
The implication of these findings is that the ITAMs function primarily
to couple to the ZAP-70/Syk family of PTKs. It is possible that the
different ITAMs may still interact with distinct substrates; however,
the importance of such interactions will require further study.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology, The University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75235-9140.
3 Address correspondence and reprint requests to Dr. A. Weiss, Howard Hughes Medical Institute, U-330, 3rd and Parnassus Avenues, UCSF, San Francisco, CA 94143-0724.
4 Abbreviations used in this paper: ITAM, immunotyrosine-based activation motif; GST, glutathione-S-transferase; PTK, protein tyrosine kinase; PVDF, polyvinylidene fluoride; SH2, Src homology 2.
Received for publication June 16, 1997. Accepted for publication September 23, 1997.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain. J. Exp. Med. 177:1093. and CD3 cytoplasmic domains can independently generate signals for T cell development and function. Immunity 2:401.[Medline]
chain of the T cell antigen receptor complex. Science 261:918. chains. EMBO J. 12:4357.[Medline]
/ gene. EMBO J. 12:4347.[Medline]
-/- mice and identification of a novel T cell population in the intestine. EMBO J. 12:4863.[Medline]
chain in T cell development and selection. Science 266:1047. chain signaling motifs in selection of the T cell repertoire. J. Exp. Med. 185:893./-/- mice overtly react with self-major histocompatibility complex molecules upon restoration of normal surface density of T cell receptor-CD3 complex. J. Exp. Med. 185:707.. Science 255:79. chain to mature T cell apoptosis. J. Exp. Med. 183:2109. chain associates with the actin cytoskeleton upon activation of mature T lymphocytes. Immunity 3:623.[Medline]
chain is associated with the cytoskeleton. Proc. Natl. Acad. Sci. USA 92:4768. chain tyrosine-based activation motif. J. Biol. Chem. 270:13981. subunits and the CD3 , , and chains. Eur. J. Immunol. 26:1063.[Medline]
in murine thymocytes and lymph node T cells. Immunity 1:675.[Medline]
subunit. J. Exp. Med. 182:1585. and enhances its autophosphorylation and generates specific binding sites for SH2 domain containing proteins. Mol. Cell. Biol. 15:3171.[Abstract]
proteins in immature CD4+CD8+ thymocytes. Proc. Natl. Acad. Sci. USA 90:2438.RIß subunit functions as an amplifier of FcRI-mediated cell activation signals. Cell 85:985.[Medline]
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W.-J. Chae, H.-K. Lee, J.-H. Han, S.-W. V. Kim, A. L.M. Bothwell, T. Morio, and S.-K. Lee Qualitatively differential regulation of T cell activation and apoptosis by T cell receptor {zeta} chain ITAMs and their tyrosine residues Int. Immunol., September 1, 2004; 16(9): 1225 - 1236. [Abstract] [Full Text] [PDF]
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 L. A. Pitcher, P. S. Ohashi, and N. S. C. van Oers T Cell Antagonism is Functionally Uncoupled from the 21- and 23-kDa Tyrosine-Phosphorylated TCR {zeta} Subunits J. Immunol., July 15, 2003; 171(2): 845 - 852. [Abstract] [Full Text] [PDF]
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 W. Y. Lin and M. R. Roberts Developmental dissociation of T cells from B, NK, and myeloid cells revealed by MHC class II-specific chimeric immune receptors bearing TCR-zeta or FcR-gamma chain signaling domains Blood, September 26, 2002; 100(8): 3045 - 3048. [Abstract] [Full Text] [PDF]
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M. C. Haks, E. Pepin, J. H.N. van den Brakel, S. A.A. Smeele, S. M. Belkowski, H. W.H.G. Kessels, P. Krimpenfort, and A. M. Kruisbeek Contributions of the T Cell Receptor-associated CD3{gamma}-ITAM to Thymocyte Selection J. Exp. Med., July 1, 2002; 196(1): 1 - 13. [Abstract] [Full Text] [PDF]
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L. M. Spain and P. Liu TCR{beta} Transmembrane Tyrosines Are Required for Pre-TCR Function J. Immunol., January 1, 2002; 168(1): 127 - 133. [Abstract] [Full Text] [PDF]
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A. L. Mora, S. Stanley, W. Armistead, A. C. Chan, and M. Boothby Inefficient ZAP-70 Phosphorylation and Decreased Thymic Selection In Vivo Result from Inhibition of NF-{kappa}B/Rel J. Immunol., November 15, 2001; 167(10): 5628 - 5635. [Abstract] [Full Text] [PDF]
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H. S. Azzam, J. B. DeJarnette, K. Huang, R. Emmons, C.-S. Park, C. L. Sommers, D. El-Khoury, E. W. Shores, and P. E. Love Fine Tuning of TCR Signaling by CD5 J. Immunol., May 1, 2001; 166(9): 5464 - 5472. [Abstract] [Full Text] [PDF]
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M. C. Haks, T. A. Cordaro, J. H. N. van den Brakel, J. B. A. G. Haanen, E. F. R. de Vries, J. Borst, P. Krimpenfort, and A. M. Kruisbeek A Redundant Role of the CD3{{gamma}}-Immunoreceptor Tyrosine-Based Activation Motif in Mature T Cell Function J. Immunol., February 15, 2001; 166(4): 2576 - 2588. [Abstract] [Full Text] [PDF]
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C. , J. , , , , E. , and P. Function of CD3{epsilon}-mediated Signals in T Cell J. Exp. Med., September 18, 2000; 192(6): 913 - 920. [Abstract] [Full Text] [PDF]
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P. E. Love, J. Lee, and E. W. Shores Critical Relationship Between TCR Signaling Potential and TCR Affinity During Thymocyte Selection J. Immunol., September 15, 2000; 165(6): 3080 - 3087. [Abstract] [Full Text] [PDF]
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 K. F. Nolan, C.-O. Yun, Y. Akamatsu, J. C. Murphy, S.-O. Leung, E. J. Beecham, and R. P. Junghans Bypassing Immunization: Optimized Design of ""Designer T Cells"" against Carcinoembryonic Antigen (CEA)-expressing Tumors, and Lack of Suppression by Soluble CEA Clin. Cancer Res., December 1, 1999; 5(12): 3928 - 3941. [Abstract] [Full Text] [PDF]
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M. Carleton, N. R. Ruetsch, M. A. Berger, M. Rhodes, S. Kaptik, and D. L. Wiest Signals Transduced by CD3{epsilon}, But Not by Surface Pre-TCR Complexes, Are Able to Induce Maturation of an Early Thymic Lymphoma In Vitro J. Immunol., September 1, 1999; 163(5): 2576 - 2585. [Abstract] [Full Text] [PDF]
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M. Zubiaur, M. Guirado, C. Terhorst, F. Malavasi, and J. Sancho The CD3-gamma delta epsilon Transducing Module Mediates CD38-induced Protein-tyrosine Kinase and Mitogen-activated Protein Kinase Activation in Jurkat T Cells J. Biol. Chem., July 16, 1999; 274(29): 20633 - 20642. [Abstract] [Full Text] [PDF]
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H. Liu and D. A. A. Vignali Differential CD3{zeta} Phosphorylation Is Not Required for the Induction of T Cell Antagonism by Altered Peptide Ligands J. Immunol., July 15, 1999; 163(2): 599 - 602. [Abstract] [Full Text] [PDF]
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T. L. Geiger, D. Leitenberg, and R. A. Flavell The TCR {zeta}-Chain Immunoreceptor Tyrosine-Based Activation Motifs Are Sufficient for the Activation and Differentiation of Primary T Lymphocytes J. Immunol., May 15, 1999; 162(10): 5931 - 5939. [Abstract] [Full Text] [PDF]
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F. Grassi, E. Barbier, S. Porcellini, H. von Boehmer, and P.-A. Cazenave Surface Expression and Functional Competence of CD3-Independent TCR {zeta}-Chains in Immature Thymocytes J. Immunol., March 1, 1999; 162(5): 2589 - 2596. [Abstract] [Full Text] [PDF]
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B. Johansson, E. Palmer, and L. Bolliger The Extracellular Domain of the {zeta}-Chain Is Essential for TCR Function J. Immunol., January 15, 1999; 162(2): 878 - 885. [Abstract] [Full Text] [PDF]
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 E. N. Kersh, A. S. Shaw, and P. M. Allen Fidelity of T Cell Activation Through Multistep T Cell Receptor  PhosphorylationScience, July 24, 1998; 281(5376): 572 - 575. [Abstract] [Full Text]
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