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*
Department of Pathology, University of Western Australia, Queen Elizabeth II Medical Center, Nedlands, Western Australia, Australia;
Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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chain in c-Cbl-deficient
thymocytes, which results in a higher level of -chain-associated
ZAP-70 that is initially accessible for activation. Thus, more ZAP-70
is activated and more of its substrates (LAT and SLP-76) become
tyrosine-phosphorylated after TCR stimulation. However, an additional
mechanism of ZAP-70 regulation is evident at a later time
poststimulation. At this time, ZAP-70 from both normal and
c-Cbl-/- thymocytes becomes hyperphosphorylated; however,
only in normal thymocytes does this correlate with ZAP-70
down-regulation and a diminished ability to phosphorylate LAT and
SLP-76. In contrast, c-Cbl-deficient thymocytes display altered
phosphorylation kinetics, for which LAT phosphorylation is increased
and SLP-76 phosphorylation is sustained. Thus, the ability to
down-regulate the phosphorylation of two ZAP-70 substrates is impaired
in c-Cbl-/- thymocytes. These findings provide evidence
that c-Cbl is involved in the negative regulation of the
phosphorylation of LAT and SLP-76 by ZAP-70. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß subunits, which are
noncovalently associated with the TCR chains and the 
, 
, and
chains of the CD3 complex. In thymocytes, engagement of these
receptors with cross-linking Ab leads to activation of the Fyn protein
tyrosine kinase and to the subsequent phosphorylation of a limited
number of intracellular substrates, of which the c-Cbl protein is the
most prominent (3, 4). However, this signal fails to initiate
downstream signaling events, and it is only following the aggregation
of the TCR with coreceptor molecules such as CD4 that phosphorylation
of multiple downstream substrates and calcium mobilization occurs (3).
A key event in initiating this signaling cascade is the activation of
the CD4-associated kinase Lck which, in turn, phosphorylates and
activates the ZAP-70 tyrosine kinase (3, 5). ZAP-70 is a
-chain-associated kinase that is essential for the development of
normal T cells (6, 7, 8) and phosphorylates key mediators of T cell
activation such as SLP-76 and LAT (9, 10, 11, 12, 13, 14, 15).
The regulation of ZAP-70 has been extensively studied in recent years
and initially involves the activation of its kinase domain by Lck
through the phosphorylation of tyrosine 493 (16, 17). ZAP-70
subsequently becomes hyperphosphorylated by the autokinase activity or
transkinase activity of ZAP-70 molecules on adjacent -chains (2).
These phosphorylation sites include tyrosines 292, 492, 597, and 598;
substitution with phenylalanine at any of these sites results in
prominent gain-of-function phenotypes as demonstrated by lymphokine
promoter activation (18, 19, 20). From these experiments, it has been
postulated that these tyrosines are required to recruit regulatory
proteins that suppress ZAP-70 function and inhibit further
phosphorylation of substrates. Evidence from a number of studies now
indicates that c-Cbl may be one of these negative regulators of ZAP-70
through its ability to bind phosphorylated tyrosine 292 in ZAP-70 via
its novel Src homology 2 (SH2)3
domain (4, 21, 22, 23, 24, 25).
The original clue that c-Cbl may function as a negative regulator of tyrosine kinases came from genetic studies in Caenorhabditis elegans that identified the Cbl homologue Sli-1 (26). More recent studies in mammalian cells have also provided evidence that c-Cbl can regulate the activity of protein tyrosine kinases. Overexpression of c-Cbl inhibits Syk kinase activity and suppresses the release of serotonin from mast cells stimulated through the high-affinity receptor for IgE (27). In Jurkat T cells, c-Cbl overexpression has been found to reduce Ras-dependent AP-1 activation following Ag receptor stimulation (28), and the treatment of cells with antisense c-Cbl enhances the activation of the Janus kinase-STAT pathway (29). Compelling evidence for mammalian c-Cbl functioning as a negative regulator of tyrosine kinase activity has also come from recent studies of c-Cbl-deficient mice (4, 24). Analysis of TCR and CD4 signaling revealed that c-Cbl-/- thymocytes have a large enhancement in the amount of tyrosine-phosphorylated ZAP-70 and numerous unidentified proteins compared with wild-type (wt) thymocytes.
At present, the mechanism of how c-Cbl depletion can enhance the
phosphorylation of ZAP-70 and other tyrosine kinase substrates is not
known. However, a crucial factor in this analysis is the increased
level of cell surface TCRß, CD3, and CD4 in c-Cbl-/-
thymocytes (4, 24). Because the activation of ZAP-70 in thymocytes is
dependent upon both its association with the TCR chain and the
amount of available CD4-associated Lck (3), it is likely that more of
these components exist in c-Cbl-/- thymocytes, resulting
in more activated ZAP-70 and increased phosphorylation of its
substrates. In this study, we show that this is the explanation for the
enhanced phosphorylation of ZAP-70 following the stimulation of
c-Cbl-deficient thymocytes. However, we also observed a sustained
phosphorylation of LAT and SLP-76 in c-Cbl-deficient thymocytes at a
time when ZAP-70 is normally down-regulated and no longer
phosphorylating these proteins to a high level. Thus, the
phosphorylation kinetics of ZAP-70 substrates are markedly perturbed in
c-Cbl-/- thymocytes, indicating an additional level of
deregulation that is independent of effects due to differences in
receptor levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of c-Cbl-deficient mice by gene targeting of W9.5 embryonic stem cells has been described previously (4). Mouse stocks were maintained by matings of C57BL/6 x SJLSv intercrosses that were homozygous for either wt c-Cbl or the c-Cbl mutation.
Thymocyte stimulation by Ab cross-linking
Single-cell suspensions of thymocytes were prepared from 6- to
7-wk-old mice at 5 x 107 cells/ml in RPMI 1640
supplemented with 5% FCS (RPMI/5% FCS). Biotinylated hamster
anti-CD3 (500A2) and anti-CD4 (GK1.5) Abs (PharMingen, San
Diego, CA) were added to the cells at 10 µg/ml and incubated on ice
for 10 min before the cells were washed once in RPMI/5% FCS.
Cross-linking was conducted by adding 40 µg/ml streptavidin in RPMI
1640, and the cells were stimulated by incubation on ice or at 37°C
for various times before one wash in ice-cold PBS and lysis.
Immunoprecipitations and immunoblotting
Stimulated thymocytes were lysed at 3–5 x 107
cells/ml in ice-cold Nonidet P-40 lysis buffer (50 mM Tris-HCl (pH
7.4), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, and 1%
Nonidet P-40) supplemented with 10 µg/ml aprotinin, 10 mM NaF, and 1
µg/ml each of chymostatin, leupeptin, and pepstatin. After incubating
for 10 min on ice, lysates were cleared by centrifugation at 4000
x g for 8 min. Cleared lysates (1-ml aliquots) were then
analyzed by immunoprecipitation and immunoblotting as described
previously (30). Anti-Lck Abs were purchased from Zymed (San Francisco,
CA) for immunoprecipitation and from Santa Cruz Biotechnology (Santa
Cruz, CA) for blotting, anti-SLP-76 Abs (monoclonal H3) were
provided by Dr. A. Chan (Washington University School of Medicine, St.
Louis, MO) and by Antibody Solutions (Palo Alto, CA), anti-ZAP-70
(1213) Abs were provided by Dr. L. Samelson (National Institutes of
Health. Bethesda, MD) for immunoprecipitation and by Transduction
Laboratories (Lexington, KY) for blotting, anti-LAT Abs were
provided by Dr. L. Samelson, anti-TCR Abs were obtained from
Zymed for immunoprecipitation and from Dr. A. Tsygankov (Temple
University School of Medicine, Philadelphia, PA) for blotting,
anti-CD4 (RM4-4) Abs were provided by PharMingen, anti-c-Cbl
Abs were obtained from Transduction Laboratories, and
antiphosphotyrosine (4G10) Abs were provided by Dr. B. Druker (Oregon
Health Sciences University, Portland, OR). Quantitation of p16 and
phosphorylated p21 TCR protein levels by densitometric scanning was
performed using the Computing Densitometer and ImageQuant software from
Molecular Dynamics (Sunnyvale, CA).
Immune complex kinase assays
Anti-Lck or anti-ZAP-70 immunoprecipitates from unstimulated
or anti-CD3 plus CD4-stimulated thymocytes were washed three times
in Nonidet P-40 lysis buffer and once in kinase buffer (20 mM MOPS
buffer (pH 7.0), 5 mM MgCl2, and 5 mM MnCl2).
Anti-Lck immunoprecipitates were incubated with 25 µl of kinase
buffer containing 12.5 µCi [-32P]ATP (4000 Ci/mmol,
Bresatec, Adelaide, Australia) for 10 min at room temperature with
occasional mixing. The kinase reaction was stopped by the addition of 1
ml of ice-cold modified RIPA buffer (20 mM MOPS (pH 7.0), 150 mM NaCl,
1 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS)
and centrifuged briefly; the supernatant containing unincorporated
radioisotope was discarded. Immunoprecipitates were washed an
additional three times in RIPA buffer, by which time minimal
radioactivity was detected in the discarded supernatant. The
immunoprecipitates were resuspended in 75 µl of 1x Laemmli sample
buffer, incubated at room temperature for 10 min, and subsequently
boiled for 3 min; next, the supernatant was transferred to a fresh
tube. ZAP-70 immune complex kinase assays were performed as described
above, but in the presence of 5 µM of cold ATP and with an addition
of purified erythrocyte band 3 protein (cdb3) (from Drs. R. Wange
(National Institute on Aging, Baltimore, MD) and L. Samelson) as an
exogenous substrate (1 µg/immunoprecipitation) followed by analysis
of the supernatants. Samples were separated by electrophoresis through
a 10% SDS-PAGE gel, dried at 80°C under vacuum for 30 min, and
analyzed by autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and CD4. Thus, more thymocytes have a
higher density of TCR/CD3 and CD4, which could enhance signal
transduction. Consistent with this, we and others reported recently
that the stimulation of c-Cbl-/- thymocytes with
anti-CD3 plus CD4 Abs resulted in an increase in the amount of
tyrosine-phosphorylated ZAP-70 and many other proteins compared with wt
thymocytes (4, 24). To determine the reasons for these effects, we
compared thymocytes from c-Cbl+/+ and
c-Cbl-/- mice for TCR chain levels and for the degree
of association between ZAP-70 and TCR.
Increased association between ZAP-70 and TCR in c-Cbl-deficient
thymocytes
Thymocytes from 7-wk-old mice were left unstimulated or incubated
with biotin-labeled anti-CD3 and anti-CD4 Abs followed by
cross-linking with avidin. Next, the thymocytes were incubated on ice
for an additional 15 min or placed at 37°C for 5 min before lysis and
analysis by immunoprecipitation and immunoblotting. Preliminary
experiments had found that these two timepoints were optimal for
capturing ZAP-70 in its hypophosphorylated and hyperphosphorylated
forms, respectively (data not shown and
Figs. 3–5
).
Immunoprecipitation with anti-TCR Abs and immunoblotting with
anti-TCR or antiphosphotyrosine revealed that the
c-Cbl-/- thymocytes express a markedly higher level of
p16 -chain compared with wt thymocytes (Fig. 1A). Quantitation by
densitometric scanning showed this increase to be 
3.8-fold.
Interestingly, this increase is accompanied by a disproportionately
larger increase (9.5-fold) in the amount of tyrosine-phosphorylated
p21 and p23 -chain isoforms in c-Cbl-/- thymocytes
compared with wt thymocytes (Fig. 1, A and B).
This finding indicates that c-Cbl depletion not only increases the
absolute level of TCR but also further enhances its tyrosine
phosphorylation. We also observed a decrease in the amount of
detectable TCR following stimulation of both c-Cbl+/+
and c-Cbl-/- thymocytes. A likely cause of this decrease
is the ubiquitination of TCR chains that occurs following TCR
activation, which may mask epitopes recognized by TCR Abs (31).
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immunoprecipitates with ZAP-70 Abs clearly
demonstrated that the increased amount of tyrosine-phosphorylated
-chain allowed for a greater level of ZAP-70 association (Fig. 1
C). Thus, c-Cbl-/- thymocytes have more
TCR chain-associated ZAP-70; therefore, more ZAP-70 is available for
activation, even though total levels of ZAP-70 are equivalent between
wt and c-Cbl-/- thymocytes (Fig. 1D). Increase in CD4-associated Lck in c-Cbl-deficient thymocytes
In thymocytes, Lck is required for the regulation of TCR chain
tyrosine phosphorylation (5), and ZAP-70 activation is dependent upon
the amount of available CD4-associated Lck (3). Therefore, it was of
interest to examine the relative amounts of Lck and CD4-associated Lck
in c-Cbl+/+ and c-Cbl-/- thymocytes.
Immunoblotting of total lysates showed that there is a moderate
increase in the amount of Lck in c-Cbl-/- thymocytes
(Fig. 2A) and an even greater
increase in the amount of CD4-associated Lck (Fig. 2B),
presumably because of higher levels of CD4 on c-Cbl-/-
thymocytes (4, 24). Consistent with these findings, an immune complex
kinase assay showed that a larger pool of Lck is activated in
c-Cbl-/- thymocytes following receptor cross-linking
(Fig. 2C). These findings suggest that Lck is not limiting
in its availability to activate the increased amount of TCR
chain-associated ZAP-70.
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A crucial observation to emerge from these studies was the finding
that the kinetics of ZAP-70 phosphorylation did not differ between wt
and c-Cbl-/- thymocytes. This was done by comparing the
progression in ZAP-70 phosphorylation at a very early point after
stimulation (i.e., cells maintained on ice for 15 min following
receptor cross-linking) with cells at a later timepoint (i.e., cells
incubated at 37°C for 5 min) (Fig. 3).
By comparing a 1x exposure for antiphosphotyrosine immunoblots of
ZAP-70 from c-Cbl-/- thymocytes (lanes
5 and 6) with a 5x exposure from c-Cbl+/+
thymocytes (lanes 2 and 3), it can be seen
that there is an equivalent increase in ZAP-70 phosphorylation between
15 min on ice and 5 min at 37°C. Thus, even though there is markedly
more phosphorylated ZAP-70 in c-Cbl-/- thymocytes, the
kinetics of its activation and subsequent hyperphosphorylation appear
unaltered in the c-Cbl-deficient thymocytes. This indicates that the
effect on ZAP-70 activation is quantitative (i.e., a consequence of
more ZAP-70 being available for phosphorylation and activation).
Sustained tyrosine phosphorylation of SLP-76 and LAT in c-Cbl-deficient thymocytes
An examination of tyrosine-phosphorylated proteins following
anti-CD3 plus CD4 stimulation revealed that, unlike ZAP-70, the
phosphorylation of substrates SLP-76 and LAT showed contrasting
kinetics between c-Cbl+/+ and c-Cbl-/-
thymocytes. An examination of lysates from normal thymocytes shown in
Fig. 4A revealed that at an
early timepoint poststimulation (i.e., 15 min on ice, when there is a
low level of ZAP-70 phosphorylation), the phosphorylation of SLP-76 is
high. However, at the later time of 5 min at 37°C, the increase in
ZAP-70 phosphorylation corresponds with a large decrease in SLP-76
phosphorylation. A similar effect is seen with LAT; its phosphorylation
pattern is also the opposite of ZAP-70, because it peaks at 15 min on
ice but is markedly reduced by 5 min of stimulation at 37°C. This
effect on LAT in c-Cbl+/+ thymocytes is clearly seen with a
longer exposure of the antiphosphotyrosine blot of Fig. 4A.
In contrast, our analysis of c-Cbl-deficient thymocytes showed that
during the transition from stimulation on ice to stimulation at 37°C,
there is no decrease in SLP-76 phosphorylation, and remarkably the
phosphorylation of LAT is increased, even though ZAP-70 phosphorylation
is elevated (Fig. 4A). This contrasting pattern of tyrosine
phosphorylation between c-Cbl+/+ and c-Cbl-/-
thymocytes is clearly illustrated when long and short exposures are
compared for ZAP-70 and SLP-76 (Fig. 4C).
Immunoprecipitations of ZAP-70, SLP-76, and LAT and immunoblotting with
antiphosphotyrosine confirmed these effects (Fig. 4D), which
demonstrate sustained hyperphosphorylation of SLP-76 and LAT in
c-Cbl-deficient thymocytes. This is in marked contrast to normal
thymocytes, which exhibit a regulatory pattern that involves a rapid
rise and fall in SLP-76 and LAT tyrosine phosphorylation.
Fig. 4A also shows that the sustained phosphorylation of
SLP-76 and LAT in c-Cbl-/- thymocytes declines by 15 min
after stimulation at 37°C, suggesting that the absence of c-Cbl is
affecting an early regulatory event that is ultimately overcome by
additional mechanisms involved in the negative regulation of ZAP-70
and/or the dephosphorylation of SLP-76 and LAT. Importantly, these
regulatory mechanisms do not involve altered protein levels of SLP-76,
LAT, or ZAP-70 (Fig. 4, B and D).
The effect on ZAP-70 activity was also examined by an immune complex
kinase assay using purified erythrocyte band 3 protein (cdb3) as an
exogenous substrate (Fig. 5). Consistent
with in vivo studies, the ZAP-70 isolated from c-Cbl+/+
thymocytes is most active at phosphorylating cdb3 at an early point
after stimulation (i.e., 15 min on ice) and markedly less active after
5 min of stimulation at 37°C. This trend provides additional proof
that these stimulation procedures are capturing ZAP-70 during its
transition from an active to an inactive kinase. Also consistent with
the in vivo studies is the observation that ZAP-70 from
c-Cbl-/- thymocytes can phosphorylate more cdb3 (because
there is more activated ZAP-70) and retains the ability to
phosphorylate cdb3 to a high level at the later timepoint (Fig. 5, 5
min 37°C). However, this effect is not sustained indefinitely. We
found that between 5 and 15 min of stimulation at 37°C, there is a
marked drop in the in vitro phosphorylation of cdb3 by ZAP-70, although
it remains significantly higher in the c-Cbl-/-
thymocytes compared with the wt thymocytes (data not shown). This
decreased ZAP-70 activity is consistent with the drop in LAT and SLP-76
tyrosine phosphorylation shown in Fig. 4A.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and CD4 (4). Therefore, the
increase in the amount of tyrosine-phosphorylated ZAP-70 may be an
indirect effect, because there is likely to be a larger pool of
"armed" TCR chain-associated ZAP-70 and CD4-associated Lck in
c-Cbl-deficient thymocytes, which would result in the marked increase
in the amount of tyrosine-phosphorylated ZAP-70. In this study, we have
shown that these are the reasons for the increased level of ZAP-70
phosphorylation following T cell stimulation. First, there is more
TCR chain; as a consequence, there is an equivalent increase in both
tyrosine-phosphorylated -chain and ZAP-70 associated with the
-chain (Fig. 1, A–C), even though total ZAP-70 levels
are unaltered (Fig. 1D). In addition, we found that
c-Cbl-/- thymocytes have slightly higher levels of Lck
and even higher levels of CD4-associated Lck (Fig. 2, A and
B). The combination of these imbalances clearly favors an
increase in the amount of ZAP-70 that can be activated (Fig. 3).
A key factor in our analysis of ZAP-70 and the phosphorylation of its
substrates (SLP-76 and LAT) was the procedure of incubating thymocytes
on ice following receptor cross-linking, which allowed us to examine
signaling events at a very early point after stimulation. This method
has been used previously to study the kinetics of c-Cbl tyrosine
phosphorylation and ubiquitination in CSF-1-stimulated macrophages
(32). Importantly, this allowed us to capture ZAP-70 when it is highly
active and able to phosphorylate SLP-76, LAT, and cdb3 to a high level
(Figs. 4 and 5). This activity also correlated with ZAP-70 being
hypophosphorylated, which presumably represents a predominance of
ZAP-70 molecules that are singly phosphorylated on the activating
tyrosine 493 (16, 17). At a later timepoint poststimulation, we found
that there was a very large increase in ZAP-70 phosphorylation in both
c-Cbl+/+ and c-Cbl-/- thymocytes, and that
the relative increase was equivalent between both populations (Fig. 3).
Thus, although more ZAP-70 is available for activation in
c-Cbl-/- thymocytes, its sequential pattern of increasing
phosphorylation over time appears unaltered.
However, at this later time it was apparent that there was a
perturbation in the function of ZAP-70 from c-Cbl-/-
thymocytes that appeared unrelated to the quantitative changes
described above. In c-Cbl+/+ thymocytes, the increased
phosphorylation of ZAP-70 coincided with a large decrease in the
phosphorylation of its substrates, SLP-76 and LAT (Fig. 4). This
observation is consistent with the phosphorylation of additional
tyrosine residues (i.e., 292, 492, 597, and 598), which are involved in
the negative regulation of ZAP-70 (18, 19, 20). In contrast, it was clear
that at this later time after stimulation, the activity of
ZAP-70 from c-Cbl-/- thymocytes was markedly altered with
respect to its phosphorylation of SLP-76 and LAT such that SLP-76
phosphorylation remained constant and LAT
phosphorylation increased (Fig. 4). Thus, the regulation of
ZAP-70 is perturbed in c-Cbl-/- thymocytes.
How c-Cbl is involved in the regulation of the phosphorylation of SLP-76 and LAT by ZAP-70 is unknown, because c-Cbl itself does not possess a known catalytic activity. However, c-Cbl has been found to associate with ZAP-70 in Jurkat T cells (21), and this interaction appears to be through its divergent SH2 domain and the negative regulatory tyrosine 292 of ZAP-70 (23). Importantly, the crystal structure of this interaction has been resolved recently (25). In this study however, we were unable to detect an in vivo interaction between c-Cbl and ZAP-70 in wt thymocytes, suggesting that the interaction may be weak or transient. Interestingly, two studies that investigated the function of tyrosine 292 in ZAP-70 both predicted that this site would interact with an inhibitory molecule to negatively regulate ZAP-70 function (18, 19). It is noteworthy that a point mutation in the SH2 domain that prevents c-Cbl binding to phosphorylated tyrosine 292 of ZAP-70 correspondingly abolishes the negative regulatory activity of the C. elegans homologue Sli-1 (22, 25, 26). Thus, the properties of c-Cbl and its C. elegans homologue are consistent with the predictions regarding this inhibitory molecule, and the c-Cbl depletion investigated here may be preventing the induction of this aspect of ZAP-70 regulation. The mechanism of how an interaction of the SH2 domain of c-Cbl with tyrosine 292 affects ZAP-70 or its substrates remains to be resolved, as does a determination of the roles of the extensive SH3 and SH2 domain-binding regions and RING finger motif of c-Cbl. It is possible that recruitment of c-Cbl binding proteins into this complex is essential for the regulatory process that involves phosphorylated tyrosine 292. The negative regulation of ZAP-70 by its tyrosine phosphorylation has also been shown to be affected by the SH2-containing protein tyrosine phosphatase-1 (SHP-1) (33); however, we have found no evidence of a c-Cbl interaction with SHP-1 (data not shown). SHP-1 can directly decrease the amount of tyrosine phosphorylated ZAP-70, which in turn reduces the ability of ZAP-70 to phosphorylate substrates. Our findings indicate that the negative regulation of ZAP-70 by c-Cbl is distinct from that of SHP-1 because it does not directly affect ZAP-70 phosphorylation; however, like SHP-1, it does alter the ability of ZAP-70 to phosphorylate substrates. How these two proteins act to coordinate the activity of ZAP-70 will be an important aspect to consider in future studies.
Our study of the kinetics of tyrosine phosphorylation also revealed
that the phosphorylation of c-Cbl peaks at a later time than either
SLP-76 or LAT and follows more closely the hyperphosphorylation of
ZAP-70 (Fig. 4A). Therefore, this timing fits closely with
c-Cbl being phosphorylated when ZAP-70 is being down-regulated and is
consistent with its role as a negative regulator of T cell activation.
In addition, c-Cbl is phosphorylated independently of ZAP-70, SLP-76,
and LAT because it is phosphorylated by the Src kinase Fyn (34) which,
unlike ZAP-70, SLP-76, and LAT, can be activated by TCR/CD3 stimulation
alone (4). Although the precise role of Fyn in TCR-mediated signaling
in thymocytes has yet to be resolved, the predominance of c-Cbl as a
substrate suggests c-Cbl phosphorylation is a major function for this
kinase.
In summary, this study provides biochemical evidence that c-Cbl is a negative regulator of ZAP-70. Furthermore, these findings help to provide a mechanism to explain the enhanced positive selection seen in the thymii of c-Cbl knockout mice that express an MHC class II-restricted transgenic TCR (24). Because signaling via ZAP-70 is essential for the progression from double-positive to single-positive thymocytes (6, 7, 8), it is probable that weak signals in the c-Cbl-deficient thymocytes are enhanced; this allows for the selection of thymocytes that would have otherwise been eliminated through "neglect". However, intriguing paradoxes relating to the c-Cbl knockout thymocytes still remain. First, we would have predicted that with enhanced phosphorylation of activators of T cell signaling (i.e., SLP-76 and LAT) there would be a compensation to suppress this signal by reducing receptor levels. However, we and others have shown that the opposite of this expectation occurs, with increased TCR, CD3, and CD4 expression in c-Cbl-/- thymocytes (4, 24). The explanation for this effect has yet to be addressed; however, it is possible that c-Cbl may be involved in TCR processing, because an overexpression of c-Cbl has been found recently to enhance platelet-derived growth factor and epidermal growth factor receptor ubiquitination and degradation (35, 36). Second, we have found that c-Cbl-/- thymocytes do not have a proliferative advantage over normal thymocytes when cultured with anti-TCR Ab (4), nor do they make more IL-2 (W.Y.L., unpublished observations). In view of the biochemical data, these are surprising findings; however, mounting evidence that c-Cbl is involved in many signaling events that can affect T cell anergy (37), phosphatidylinositol 3-kinase complex formation (38, 39, 40, 41, 42, 43, 44, 45), cell spreading and migration (46), actin rearrangement (47), integrin signaling (46, 48), and receptor ubiquitination (35, 36) highlights the complexity of predicting how depletion may ultimately affect a cell’s response to stimuli, which involves many signaling pathways.
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Acknowledgments |
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Abs, Dr. Brian Druker for
antiphosphotyrosine Abs, and Drs. Ronald Wange and Larry Samelson for
purified cdb3 protein. We also thank Drs. Larry Samelson, Jeroen van
Leeuwen, Robin Scaife, and Tammy Morshead for helpful comments in the
preparation of this manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Wallace Y. Langdon, Department of Pathology, University of Western Australia, Queen Elizabeth II Medical Center, Nedlands, Western Australia 6907, Australia. E-mail address:
3 Abbreviations used in this paper: SH2, Src homology 2; wt, wild type; SHP-1, SH2-containing protein tyrosine phosphatase-1.
Received for publication February 3, 1999. Accepted for publication April 5, 1999.
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1 in an SLP-76-deficient T cell. Science 281:413.. Proc. Natl. Acad. Sci. USA 95:7927.This article has been cited by other articles:
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  L. Balagopalan, V. A. Barr, C. L. Sommers, M. Barda-Saad, A. Goyal, M. S. Isakowitz, and L. E. Samelson c-Cbl-Mediated Regulation of LAT-Nucleated Signaling Complexes Mol. Cell. Biol., December 15, 2007; 27(24): 8622 - 8636. [Abstract] [Full Text] [PDF]
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 S. E. Gustin, C. B. F. Thien, and W. Y. Langdon Cbl-b Is a Negative Regulator of Inflammatory Cytokines Produced by IgE-Activated Mast Cells J. Immunol., November 1, 2006; 177(9): 5980 - 5989. [Abstract] [Full Text] [PDF]
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J. Zhang, Y. J. Chiang, R. J. Hodes, and R. P. Siraganian Inactivation of c-Cbl or Cbl-b Differentially Affects Signaling from the High Affinity IgE Receptor J. Immunol., August 1, 2004; 173(3): 1811 - 1818. [Abstract] [Full Text] [PDF]
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 Y. J. Chiang, C. L. Sommers, M. S. Jordan, H. Gu, L. E. Samelson, G. A. Koretzky, and R. J. Hodes Inactivation of c-Cbl Reverses Neonatal Lethality and T Cell Developmental Arrest of SLP-76-deficient Mice J. Exp. Med., July 6, 2004; 200(1): 25 - 34. [Abstract] [Full Text] [PDF]
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C. B.F. Thien, R. M. Scaife, J. M. Papadimitriou, M. A. Murphy, D. D.L. Bowtell, and W. Y. Langdon A Mouse with a Loss-of-function Mutation in the c-Cbl TKB Domain Shows Perturbed Thymocyte Signaling without Enhancing the Activity of the ZAP-70 Tyrosine Kinase J. Exp. Med., February 17, 2003; 197(4): 503 - 513. [Abstract] [Full Text] [PDF]
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C. Dumont, N. Blanchard, V. Di Bartolo, N. Lezot, E. Dufour, S. Jauliac, and C. Hivroz TCR/CD3 Down-Modulation and {zeta} Degradation Are Regulated by ZAP-70 J. Immunol., August 15, 2002; 169(4): 1705 - 1712. [Abstract] [Full Text] [PDF]
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T. Yasuda, T. Tezuka, A. Maeda, T. Inazu, Y. Yamanashi, H. Gu, T. Kurosaki, and T. Yamamoto Cbl-b Positively Regulates Btk-mediated Activation of Phospholipase C-{gamma}2 in B Cells J. Exp. Med., July 1, 2002; 196(1): 51 - 63. [Abstract] [Full Text] [PDF]
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M. P. Loreto, D. M. Berry, and C. J. McGlade Functional Cooperation between c-Cbl and Src-Like Adaptor Protein 2 in the Negative Regulation of T-Cell Receptor Signaling Mol. Cell. Biol., June 15, 2002; 22(12): 4241 - 4255. [Abstract] [Full Text] [PDF]
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N. Rao, I. Dodge, and H. Band The Cbl family of ubiquitin ligases: critical negative regulators of tyrosine kinase signaling in the immune system J. Leukoc. Biol., May 1, 2002; 71(5): 753 - 763. [Abstract] [Full Text] [PDF]
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 A. Freywald, N. Sharfe, and C. M. Roifman The Kinase-null EphB6 Receptor Undergoes Transphosphorylation in a Complex with EphB1 J. Biol. Chem., February 1, 2002; 277(6): 3823 - 3828. [Abstract] [Full Text] [PDF]
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A. Magnan, V. Di Bartolo, A.-M. Mura, C. Boyer, M. Richelme, Y.-L. Lin, A. Roure, A. Gillet, C. Arrieumerlou, O. Acuto, et al. T Cell Development and T Cell Responses in Mice with Mutations Affecting Tyrosines 292 or 315 of the ZAP-70 Protein Tyrosine Kinase J. Exp. Med., August 20, 2001; 194(4): 491 - 506. [Abstract] [Full Text] [PDF]
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 A. A. de Melker, G. van der Horst, J. Calafat, H. Jansen, and J. Borst c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route J. Cell Sci., January 6, 2001; 114(11): 2167 - 2178. [Abstract] [Full Text] [PDF]
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Y. Yi, M. McNerney, and S. K. Datta Regulatory Defects in Cbl and Mitogen-Activated Protein Kinase (Extracellular Signal-Related Kinase) Pathways Cause Persistent Hyperexpression of CD40 Ligand in Human Lupus T Cells J. Immunol., December 1, 2000; 165(11): 6627 - 6634. [Abstract] [Full Text] [PDF]
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N. Rao, M. L. Lupher Jr., S. Ota, K. A. Reedquist, B. J. Druker, and H. Band The Linker Phosphorylation Site Tyr292 Mediates the Negative Regulatory Effect of Cbl on ZAP-70 in T Cells J. Immunol., May 1, 2000; 164(9): 4616 - 4626. [Abstract] [Full Text] [PDF]
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