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INTRODUCTION |
Binding of antigenic peptides presented by major
histocompatibility complex molecules to the T cell receptor
(TCR)1/CD3 complex induces a
rapid increase in the activities of two families of nonreceptor PTKs,
i.e. the Src (Fyn and Lck) and Syk (Syk and Zap-70) families
(1, 2). Activation of Lck and/or Fyn leads to tyrosine phosphorylation
of immunoreceptor tyrosine-based activation motifs present in the
intracellular domains of CD3 and
subunits, resulting in the
subsequent recruitment and activation of Zap-70 and Syk. The activated
PTKs in turn propagate activation signals by phosphorylating multiple
intracellular proteins, eventually leading to T cell activation,
lymphokine production, and proliferation. However, many aspects of the
underlying signaling cascades remain unclear.
The c-cbl (Casitas B-lineage
lymphoma) proto-oncogene was originally isolated as a
cellular homologue of v-cbl, a part of the transforming gene
of the Cas NS-1 murine leukemia retrovirus (3, 4). The 120-kDa product
of c-cbl, Cbl, consists of an amino-terminal v-Cbl
homologous region, a Ring finger, and a carboxyl-terminal proline-rich
domain containing several potential tyrosine phosphorylation sites. Cbl
is rapidly phosphorylated on tyrosine residues in response to
stimulation of various cell surface receptors, including the TCR
(reviewed in Ref. 5), suggesting a critical role for Cbl in signal
transduction pathways. The importance of Cbl in intracellular signal
transduction is further emphasized by the observation that it
associates with several other signaling molecules: e.g. the constitutive binding to Grb2 (6-9); phosphotyrosine
(Tyr(P))-dependent interaction with phosphatidylinositol
3-kinase (PI3-K) (7, 8, 10-12), Crk-L (13-20), and Vav (21); and
phosphoserine-dependent interaction with 14-3-3 proteins
(22, 23).
The rapid tyrosine phosphorylation of Cbl induced by TCR engagement is
complemented by the observation of the physical interaction of Cbl with
upstream PTKs such as Syk/Zap-70 and Fyn. Both Src family kinases such
as Fyn (6, 8, 24-26) and Syk (25, 27) or Zap-70 (24, 28) have been
reported to associate with Cbl. The association of Zap-70 with Cbl is
activation-dependent and is mediated by a Tyr(P)-binding
(PTB) domain in the amino-terminal region of Cbl (28). More recently, a
N(D)XY sequence was mapped in Zap-70 (Tyr-292) (29) and Syk
(Y316) (30), respectively, as the Cbl PTB-binding motif. Interestingly,
the Tyr-292 of Zap-70 was reported to negatively regulate T cell
activation and was postulated to be a binding site for a negative
regulatory protein (31, 32). Consistent with this notion is the
observation that Cbl binds and inhibits the kinase activity of Syk
(33).
In contrast to Cbl, a mutated form of Cbl (70Z) with a 17-amino acid
deletion near the amino-terminal region of a Ring finger domain is
transforming in NIH3T3 fibroblasts (34, 35). Recently, it was shown
that 70Z binds and enhances the kinase activity of the epidermal growth
factor (EGF) receptor (36) and the platelet-derived growth factor
(PDGF) receptor (37). We presented data showing that 70Z is
constitutively active in the transactivation of NFAT promoter (38), a
critical component of the interleukin-2 gene and other cytokine genes
(39, 40). The 70Z-induced NFAT activation synergizes with ionomycin
treatment and is dependent on a functional Ras (38). However, the exact
mechanism underlying the 70Z-mediated signal leading to NFAT activation
remains unclear. Exploration of this mechanism will be critical in
understanding the functional role of Cbl and its oncogenic mutants in T
cell activation and leukemogenesis.
Our hypothesis is that 70Z-induced NFAT transactivation is mediated via
complex(es) formation of 70Z with its binding partners. To test this
hypothesis, we systematically constructed a series of point-mutated 70Z
mutants at an amino-terminal loss of function (Gly-306) site and at
carboxyl-terminal potential Tyr(P) or phosphoserine sites and analyzed
their interactions with respective binding partners and effects on NFAT
activation. We demonstrated that expression of the G306E mutant, which
disrupted the interaction with Zap-70, abolished 70Z-induced NFAT
activation under both basal and ionomycin-stimulated conditions.
However, mutations at Tyr-700, Tyr-731, and Tyr-774, but not at other
single tyrosine or serine residues, enhanced the basal and
ionomycin-treated activation of NFAT. Our results suggest that the
interaction of the amino-terminal PTB domain of 70Z with Zap-70 plays a
positive role and that the interactions of its carboxyl-terminal Tyr(P)
residues with potential binding partners such as PI3-K and Crk-L can
play a negative role in this event.
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MATERIALS AND METHODS |
Antibodies--
Polyclonal rabbit anti-Cbl (c-15), anti-Crk-L,
anti-TCR
chain, and anti-Zap-70 antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-Tyr(P) monoclonal antibody 4G10,
anti-Vav monoclonal antibody, and anti-p85 subunit of PI3-K polyclonal antibody were from Upstate Biotechnology (Lake Placid, NY).
Anti-hemagglutinin (HA) monoclonal antibody (12CA5) was from Boehringer
Mannheim. Anti-14-3-3 monoclonal antibody was described previously (22, 41). An anti-CD3
(OKT3) was purified from ascites fluid by protein
A-Sepharose affinity chromatography. Horseradish peroxidase-conjugated F(ab')2 fragments of donkey anti-rabbit IgG or sheep
anti-mouse IgG were from Amersham.
Plasmids--
cDNAs encoding HA-tagged wild-type Cbl, 70Z,
in pEFneo (42) were reported (38). HA-tagged 70Z subcloned into pGEM11Z
was used for point mutations with a site-directed mutagenesis kit (QuickChange; Stratagene) with a pair of sense and antisense primers of
20-23 mer around the targeting site. The mutated sequences were
verified by direct DNA sequencing. The following mutations were
constructed (Fig. 1): 1) 70Z point
tyrosine to phenylalanine mutations including the potential Tyr(P)
residues in its carboxyl terminus (Tyr-552, Tyr-674, Tyr-700, Tyr-731,
Tyr-735, and Tyr-774, single site; Tyr-700/774 and Tyr-869/871, double
site) or a mutant with all eight tyrosine residues mutated (Y8F); 2)
point serine to alanine mutation at the Ser-619 single site and the
Ser-619/639 double site, which are within the 14-3-3 binding sites
(23); and 3) a loss of function glycine to glutamic acid mutation at Gly-306. cDNAs encoding mutated 70Z constructs were subcloned back
into pEFneo. The NFAT-luciferase reporter construct was provided by Dr.
G. Crabtree.

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Fig. 1.
Schematic representations of 70Z and its
mutant derivatives. All constructs were tagged with a HA epitope
at the amino terminus. Shown are the PTB domain, Ring finger
(RING), the 17-amino acid deletion, the proline-rich domain
(PRD), the leucine zipper (LZ), and the positions
of amino acids for point mutations. Asterisks indicate the
positions of mutated residue(s).
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Cell Culture, Transfection, and Stimulation--
Simian virus 40 T antigen (TAg)-transfected human leukemic Jurkat T cells (Jurkat-TAg)
were grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented
with 10% fetal bovine serum and antibiotics. For protein expression in
Jurkat-TAg T cells, cells were transfected with the appropriate amount
of plasmids (usually 5-10 µg, total) by electroporation as described
previously (23). Cells were resuspended (2 × 107
cells/ml) in 0.5 ml of medium, equilibrated at 37 °C for 5 min, and
activated with OKT3 (4 µg/ml) for 5 min. Stimulation was terminated by adding 0.5 ml of 2× Nonidet P-40 lysis buffer (2% Nonidet P-40, 40 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM EDTA, 10 mM sodium pyrophosphate, 10 mM NaF, 4 mM Na3VO4,
and 20 µg/ml each of aprotinin and leupeptin). Cells were lysed for
10 min at 4 °C, and insoluble materials were removed by
centrifugation at 15,000 × g (4 °C for 10 min). For
luciferase assays, cells were washed, resuspended in RPMI 1640 medium
containing 0.2% fetal calf serum, and incubated for 4-6 h in 24-well
plates. The cells were then left unstimulated or stimulated with either
OKT3 ascites (1:500) or purified OKT3 (3 µg/ml), ionomycin (100 ng/ml), or both ionomycin and phorbol myristate acetate (50 ng/ml) for
another 8-10 h.
Immunoprecipitation and Immunoblotting--
Lysates (1 × 107 cells) were mixed with antibodies for 2 h,
followed by the addition of 40 µl of protein A/G Plus-Sepharose beads
(Santa Cruz Biotechnology) for an additional hour at 4 °C. Immunoprecipitates were washed four times with 1× Nonidet P-40 lysis
buffer and boiled in 30 µl of 2× Laemmli's buffer. Samples were
subjected to SDS-10% polyacrylamide gel electrophoresis analysis and
electrotransferred onto polyvinylidene difluoride membranes (Millipore). Membranes were immunoblotted with the indicated primary antibodies (usually 1 µg/ml), followed by horseradish
peroxidase-conjugated secondary antibodies. Membranes were washed and
visualized with an ECL detection system (Amersham). When necessary,
membranes were stripped by incubation in 62.5 mM Tris-HCl,
pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS for 1 h
at 70 °C with constant agitation, washed, and then reprobed with
other antibodies as indicated.
Luciferase Assay--
The luciferase assay to determine the
activation of reporter genes was described previously (38). Luciferase
activity was determined in triplicate and expressed as arbitrary units
(AU). The standard deviation among triplicates was
10%, and each
experiment was repeated at least three times.
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RESULTS |
Expression of Y700F, Y731F, Y774F, or Y700/774F Enhanced
70Z-mediated NFAT Activation--
We previously showed that 70Z
induces the transactivation of NFAT in synergy with a
Ca2+ signal (38). However, the mechanism underlying this
effect is unknown. To understand this mechanism, we first made several Tyr to Phe mutations at Tyr-700, Tyr-731, Tyr-774, or Tyr-700/774, which are shown to be the binding sites for Vav, PI3-K, and Crk-L (both
Tyr-700 and Tyr-774), respectively (16, 18, 21, 38), and reconfirmed
their respective roles for protein-protein interaction. As shown in
Fig. 2A, the Y700F or Y774F
single mutation had only a partial effect on Crk-L interaction.
However, a Y700/774F double mutation completely abolished its
interaction with Crk-L, indicating that both tyrosine residues are
required for the Crk-L interaction (18). A Y731F mutation disrupted the
interaction with p85, consistent with our previous observation (38).
Under the same conditions, we did not detect Vav/70Z interaction as
reported previously for Vav/Cbl interaction (21), suggesting that a
Vav/70Z interaction is relatively weaker than that of 70Z with Crk-L or
PI3-K.

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Fig. 2.
Effects of 70Z Tyr to Phe mutants on NFAT
activation. A, Jurkat-TAg T cells were transfected with
plasmids containing HA-tagged 70Z or its mutants including Y700F,
Y731F, Y774F, or Y700/774F (3 µg of each). Thirty-six h later,
lysates from 1 × 107 unstimulated ( ) or
OKT3-stimulated (+) T cells were immunoprecipitated with anti-HA. The
immunoprecipitates were subjected to SDS-polyacrylamide gel
electrophoresis, transferred to a polyvinylidene difluoride membrane,
and immunoblotted with anti-p85 or anti-Crk-L as indicated. The
membrane was reprobed sequentially with anti-Tyr(P) and anti-HA.
Molecular weight markers are indicated on the left.
B, Jurkat-TAg cells were transfected with empty vector or
the indicted 70Z plasmids (3 µg of each) plus 3 µg of NFAT-luc
reporter plasmid. Twenty-four h later, cells were left unstimulated
(Control), or stimulated with OKT3 ascites (1:500) or
ionomycin (Iono; 100 ng/ml) for 6-10 h. Cells were then
lysed, and the luciferase activity was determined. Bars
represent the mean of triplicate samples. Standard deviations are
depicted by the error bars. B represents five
separate experiments. C, Jurkat-TAg cells were transfected
with plasmids encoding HA-tagged 70Z or its mutants as indicated. Cells
were analyzed as described in A. D, effects of
70Z mutants on NFAT activation. The experiment was performed as
described in B. Bars represent the mean ± S.D. of triplicate samples. D represents four separate
experiments.
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Next we examined whether these mutants affect 70Z-mediated NFAT
transactivation. Plasmids containing 70Z or its mutants were cotransfected into Jurkat-TAg cells with the NFAT-luc reporter gene.
Cells were left unstimulated or stimulated with OKT3 or ionomycin and
assayed for luciferase activity. As shown in Fig. 2B, 70Z
induced NFAT activation under resting conditions, which synergized with
ionomycin treatment, consistent with our previously published result
(38). However, OKT3 stimulation did not enhance but rather reduced the
luciferase activity in comparison with cells transfected with the empty
vector under the same conditions, suggesting that OKT3-stimulated
tyrosine phosphorylation of 70Z and its recruitment of other binding
molecules may play a negative role in the TCR-mediated NFAT activation.
This suggestion is supported by the observation from the point-mutated
70Z proteins. Increases in the NFAT-driven luciferase activity, albeit
to slightly different degrees, were observed in cells transfected with
Y700F, Y731F, Y774F, or Y700/774F under unstimulated conditions or
OKT3- or ionomycin-stimulated conditions as compared with unmutated
70Z. The increases in NFAT activation were more obvious in
ionomycin-treated samples. These results suggest that these tyrosine
residues are not responsible for the observed ability of 70Z in NFAT
transactivation. Rather, interactions of 70Z with its binding partners
such as PI3-K or Crk-L via these residues may play a negative role in this event.
Although the tyrosine residues at 700, 731, and 774 are known to be
responsible for the protein-protein interactions, as also shown above,
there are other potential tyrosine residues whose nature of tyrosine
phosphorylation or potential role in protein-protein interactions are
unclear. Therefore, we made additional point mutations at Tyr-552,
Tyr-674, and Tyr-735, a double mutant at Tyr-869 and Tyr-871, or a
mutant with all eight tyrosine residues mutated to phenylalanine (Y8F).
These mutants were expressed in Jurkat-TAg cells and analyzed for their
interactions with PI3-K and Crk-L. As shown in Fig. 2C,
Y552F, Y735F, or Y869/871F did not have any effect on their
interactions with PI3-K and Crk-L. However, Y8F completely disrupted
the interactions with PI3-K and Crk-L. A single Y674F mutant did not
express well and was not included in those experiments. These mutants
were then analyzed for their ability to induce NFAT transactivation. As
shown in Fig. 2D, Y552F, Y735F, and Y869/831F showed only
slightly enhancing effects on NFAT transactivation as compared with 70Z
under unstimulated conditions or OKT3- or ionomycin-stimulated
conditions. OKT3. However, Y8F, a mutant with all eight
carboxyl-terminal tyrosine residues mutated, markedly enhanced its
ability to activate NFAT under unstimulated or stimulated conditions.
This result suggests that tyrosine residues at 552, 735, 869, and 871 are not critical for 70Z-mediated NFAT transactivation. The observed
effect of Y8F may represent a synergy of the Y700F, Y731F, and Y774F mutations.
Effects of 70Z Mutants Deficient in 14-3-3 Binding on NFAT
Activation--
We previously demonstrated that Cbl interacts with
14-3-3 in an activation-dependent manner via two
phosphoserine-containing motifs in Cbl (22, 23). Of these motifs,
Ser-619 and Ser-639 have been predicated to be the serine
phosphorylation sites responsible for the binding (23, 43). We then
constructed a S619A single mutant and a S619/639A double mutant in 70Z
and analyzed their interaction with 14-3-3. As shown in Fig.
3A, a mutation at Ser-619 reduced both basal and stimulation-induced interaction with 14-3-3; mutation at both the Ser-619 and Ser-639 sites abolished
stimulation-induced 14-3-3 binding, although a residual basal level of
70Z/14-3-3 association remained. Next, we examined the effects of these
two mutants on NFAT activation. Mutations at either single site (S619A) or double sites (S619/639A) showed only a slightly higher activation of
NFAT as compared with 70Z under either unstimulated conditions or OKT3-
or ionomycin-stimulated conditions (Fig. 3B). Analysis of
cell lysates with anti-HA revealed comparable amounts of proteins among
all the samples (Fig. 3C). Although 14-3-3 may still have some effects on 70Z in these mutants because of remaining basal level
interaction, this result suggests that an OKT3-stimulated, enhanced
70Z/14-3-3 interaction is not critical in 70Z-induced NFAT
activation.

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Fig. 3.
Effects of 70Z mutants deficient in 14-3-3 binding on NFAT activation. A, Jurkat-TAg T cells
transfected with empty vector or with plasmids containing 70Z, S619A,
or S619/639A were left unstimulated ( ) or stimulated with OKT3 (+).
Cell lysates were immunoprecipitated with anti-HA. The
immunoprecipitates were analyzed with anti-14-3-3 and reprobed with
anti-HA. B, Jurkat-TAg T cells transfected with the
indicated plasmids (3 µg of each) plus NFAT-luc reporter plasmid (3 µg) were left unstimulated or stimulated as indicated. Cell lysates
were determined for luciferase activity. Bars represent the
mean ± S.D. of triplicate samples. C, aliquots of the
same cell lysates were analyzed with anti-HA. B and
C are representative of three separate experiments.
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A Loss of Function G306E Mutant Was Critical for 70Z-mediated NFAT
Activation--
Previous studies have shown that a point mutation
(G306E) in v-Cbl, which corresponds to a loss of function mutation in
Sli-1, a Caenorhabditis elegans Cbl homologue, disrupts its
interaction with PTKs including Zap-70 (28) and ablates v-Cbl-induced
cell transformation (37, 44). Subsequent studies, including our own,
demonstrated that this region contains a PTB domain that interacts with
a N(D)XY motif in Zap-70 (29) and Syk (30). These
observations suggest that this Cbl/PTK binding can play an important
role in Cbl-mediated signaling. In support of this suggestion is the
observation that Cbl inhibits Syk activity by directly binding the
latter (33). To understand whether 70Z-mediated NFAT activation
involves a similar mechanism, we made a G306E mutation in 70Z and
transfected this mutant plasmid plus NFAT-luc into Jurkat-TAg cells.
G306E completely abrogated 70Z-induced NFAT activation under resting
conditions (Fig. 4). OKT3 stimulation of
the cells overexpressing G306E induced further inhibition of NFAT
activation by G306E as compared with 70Z. More importantly, this mutant
also abolished ionomycin-induced NFAT activation by 70Z. This result
provides further evidence for an evolutionarily conserved mechanism by
which 70Z exerts its biological function via the interaction of its
amino-terminal PTB domain with upstream PTKs, most likely Syk/Zap-70.
In addition, the increased inhibition of NFAT activation by G306E under
OKT3 stimulation could be explained by a Tyr(P)-mediated negative
signaling, in agreement with the aforementioned observations (Fig.
2).

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Fig. 4.
Effect of 70Z G306E mutant on NFAT
activation. A, Jurkat-TAg cells were transfected with empty
plasmid or with plasmids containing 70Z or G306E mutant (3 µg of
each) plus 3 µg of NFAT-luc reporter construct. Lysates were prepared
from cells treated as indicated, and luciferase activity was
determined. Bars represent the mean ± S.D. of
triplicate samples. B, lysates from A were
immunoblotted with anti-HA. A and B are
representative of six separate experiments.
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The G306E Mutant Showed Reduced Tyrosine Phosphorylation and
Disrupted the 70Z/Zap-70 Interaction--
We have previously shown
that the interaction of the Cbl PTB domain with the Syk Tyr-316 residue
is required for maximal tyrosine phosphorylation of Cbl (30). To
further explore the mechanism underlying the G306E-mediated effect on
NFAT activation, we first examined whether the G306E mutation affects
its tyrosine phosphorylation. Lysates prepared from cells transfected
with empty vector, HA-tagged 70Z, or G306E left unstimulated or
stimulated with OKT3 were immunoblotted with anti-Tyr(P). As compared
with 70Z, G306E showed a markedly reduced tyrosine phosphorylation
(Fig. 5A, top
panel). Reprobing of the membrane with anti-HA indicated that both
70Z and G306E were expressed at a similar level (bottom
panel).

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Fig. 5.
Tyrosine phosphorylation of G306E and its
interaction with Zap-70. A, plasmids encoding HA-tagged 70Z
or G306E were transfected into Jurkat-TAg cells. Lysates from 1 × 107 unstimulated ( ) or OKT3-stimulated (+) cells were
analyzed with anti-Tyr(P). Arrow indicates the position of
the HA-tagged 70Z proteins. The same membrane was reprobed with
anti-HA. B, the same lysates were immunoprecipitated with
anti-HA and immunoblotted with anti-Tyr(P) (top panel).
Arrow indicates the position of Zap-70. The membrane was
stripped and reprobed with anti-HA (bottom panel).
C, interaction of 70Z with Zap-70 under ionomycin-stimulated
conditions. Jurkat-Tag cells were cotransfected with Zap-70 plus 70Z or
G306E. Lysates prepared from unstimulated cells ( ) or cells
stimulated with ionomycin (100 ng/ml; 5 min) were analyzed with
anti-Tyr(P) and then reprobed with anti-HA. The position of 70Z was
indicated. Lysates were immunoprecipitated with anti-HA, immunoblotted
with anti-Tyr(P), and then reprobed with anti-HA. The position of
Zap-70 or 70Z was indicated. D, effect of overexpression of
70Z or G306E on the tyrosine phosphorylation of Zap-70. Jurkat-TAg
cells transfected with empty vector or with plasmids containing 70Z or
G306E (3 µg of each) were left unstimulated ( ) or stimulated with
OKT3 (+). Cell lysates were immunoprecipitated with anti-Zap-70 and
immunoblotted with anti-Tyr(P). The position of Zap-70 (top
panel) or phospho- (middle panel) is indicated by
arrows. The same membrane was reprobed with anti-Zap-70
(bottom panel).
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We then examined whether the reduced tyrosine phosphorylation of G306E
resulted from a disruption of its interaction with Zap-70. The same
cell lysates were then immunoprecipitated with anti-HA and
immunoblotted with anti-Tyr(P). As shown in Fig. 5B, top panel, a ~70-kDa Tyr(P)-containing protein was
coimmunoprecipitated with anti-HA from cells overexpressing HA-tagged
70Z under OKT3-stimulated conditions, which comigrated with Zap-70.
Reprobing the same membrane with anti-Zap-70 failed to reveal Zap-70,
suggesting that the nature of the 70Z/Zap-70 interaction is relatively
weak. This weak interaction was consistent with the observations
reported by other groups (24, 29). Importantly, the same
Tyr(P)-containing protein, most likely Zap-70, was not detected or was
only weakly detected in anti-HA immunoprecipitates from cells
overexpressing HA-tagged G306E under the same conditions. This result
suggests that an evolutionarily conserved loss of function mutation in 70Z disrupts its interaction with Zap-70, which is required for the
maximal tyrosine phosphorylation of 70Z.
Next we examined whether the G306E mutation has any effect on tyrosine
phosphorylation and its interaction with Zap-70 under ionomycin-stimulated conditions. To this end, we coexpressed Zap-70 with 70Z or G306E in Jurkat-TAg cells. As shown in Fig. 5C,
coexpression of Zap-70 with 70Z induced the tyrosine phosphorylation of
70Z under resting or ionomycin-stimulated conditions. Ionomycin
stimulation caused only a slight increase in the tyrosine
phosphorylation of 70Z. However, the tyrosine phosphorylation of G306E
was not detectable under the same conditions. We then analyzed the
interaction of 70Z or G306E with Zap-70 under unstimulated or
ionomycin-stimulated conditions. A ~70 kDa Tyr(P)-containing protein
comigrating with Zap-70 was coimmunoprecipitated with anti-HA
from cells overexpressing HA-tagged 70Z and Zap-70. However, the
interaction of G306E with Zap-70 was markedly impaired under either
resting or ionomycin-stimulated conditions.
Previous studies have shown that 70Z interacts with and enhances the
tyrosine phosphorylation and the kinase activity of the EGF and PDGF
receptors, which is proposed to be responsible for 70Z-induced cell
transformation in fibroblasts (36, 37). To examine whether 70Z-induced
NFAT transactivation is mediated by a similar mechanism (namely, the
activation of Zap-70), we compared the tyrosine phosphorylation of
Zap-70 among cells transfected with empty vector, 70Z, or G306E by
analyzing anti-Zap-70 immunoprecipitates with anti-Tyr(P). As shown in
Fig. 5D, top panel, almost no difference was
observed among all the samples under OKT3-stimulated conditions. This
result suggests that 70Z does not significantly affect the tyrosine
phosphorylation and probably the kinase activity of Zap-70. In
agreement with this suggestion is the observation that overexpression of 70Z or G306E did not change the amount of the tyrosine
phosphorylated TCR
chain coimmunoprecipitated by anti-Zap-70 (Fig.
5D, middle panel), suggesting that 70Z did not
affect the ability of Zap-70 to bind to TCR. Equivalent amounts of
Zap-70 were detected in all the samples (Fig. 5D,
bottom panel).
Functional Role of Wild-Type Cbl in NFAT Transactivation--
Cbl
has been demonstrated to be a negative regulator in several mammalian
systems. Recently, it was shown that it is also a negative regulator in
TCR-induced AP-1 activation (45). In principle, Cbl can also be a
negative regulator in TCR-mediated NFAT activation because the NFAT
promoter used in our studies consists of a NFAT binding site and an
AP-1 binding site and requires both a Ca2+ signal and a
Ras-dependent signal for its activation (39, 40). We have
previously shown that wild-type Cbl has no effect on basal and
ionomycin-stimulated NFAT activation (38). To examine whether wild-type
Cbl has any effect on TCR-mediated NFAT activation, we transfected
Jurkat-TAg cells with empty vector, 70Z, or wild-type Cbl plus NFAT-luc
reporter plasmid and analyzed the NFAT-driven luciferase activity. As
shown in Fig. 6, wild-type Cbl inhibited TCR-induced NFAT by 90%, which was stronger than 70Z-mediated inhibition under the same conditions. This inhibitory role of wild-type
Cbl in TCR-induced NFAT transactivation is specific, because the
overexpression of Cbl had no effect or only a subtle effect on
ionomycin- (Fig. 6) or ionomycin plus phorbol myristate acetate-induced
NFAT activation (data not shown). This result indicates that Cbl is a
negative regulator in TCR-induced NFAT activation.

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Fig. 6.
Effect of wild-type Cbl on NFAT activation.
A, Jurkat-TAg cells were transfected with empty plasmid or
with plasmids containing 70Z or wild-type Cbl (3 µg of each) plus 3 µg of NFAT-luc reporter construct. Lysates prepared from cells
treated as indicated were determined for luciferase activity.
Bars represent the mean ± S.D. of triplicate samples.
B, lysates from A were immunoblotted with
anti-HA. A and B are representative of five
separate experiments.
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DISCUSSION |
We previously showed that oncogenic Cbl mutant 70Z induces NFAT
activation in T cells in synergy with a Ca2+ signal and in
a functional Ras-dependent manner (38). Dissecting 70Z-mediated signaling using our established system could shed light on
the biological function of Cbl proteins in T cell activation and
leukemogenesis. Here we analyzed the biological role of an amino-terminal loss of function (Gly-306) site, the carboxyl-terminal potential Tyr(P), or phosphoserine sites of 70Z in the induction of
NFAT, a critical component of interleukin-2 and other cytokine genes.
We demonstrate that a mutation at Gly-306 abrogated 70Z-induced NFAT
transactivation under both basal and ionomycin-treated conditions. However, mutations at Tyr-700, Tyr-731, Tyr-774, and Tyr-700/774, but
not other Tyr to Phe or Ser to Ala mutations, increased both basal and
ionomycin-treated NFAT activation. Our results suggest that 70Z plays
both a positive and a negative role via its interaction with different
targeting molecules.
In the present study, we clearly demonstrate that 70Z-mediated NFAT
activation is mediated by its interaction with upstream PTKs, most
likely Zap-70, because a loss of function mutation at Gly-306, which
disrupts the interaction with Zap-70, almost completely abolished its
ability to induce NFAT activation under both basal and
ionomycin-stimulated conditions. This observation is consistent with
the following previous findings: a corresponding mutation in Sli-1, a
Cbl homologue in C. elegans, ablates Sli-1-mediated negative
signaling (46); and a G306E mutant of v-Cbl disrupts its interaction
with Zap-70 (28) or with EGF and PDGF receptors and abolishes its
transforming ability in NIH3T3 cells (37, 44). Indeed, it was shown
that by binding to EGF and PDGF receptors, 70Z enhances their tyrosine
phosphorylation and kinase activity, which provides a mechanistic
insight into 70Z-mediated cell transformation (36, 37). The observed
NFAT activation by 70Z could use a similar mechanism, i.e.
the activation of Zap-70 in T cells. However, 70Z does not have a
significant effect on the tyrosine phosphorylation of Zap-70. Our
result is consistent with the following observation in basophilic
cells: cotransfection of 70Z with Syk did not affect the tyrosine
phosphorylation and kinase activity of Syk (33). Although the
interaction of 70Z with upstream PTKs induces a positive signal, it is
possible that it exerts a different ability by binding different PTKs
(Syk/Zap-70 versus EGF/PDGF receptor kinases) and/or in a
different cellular context.
Recently, a N(D)XY motif in Zap-70 (Tyr-292) (29) and Syk
(Tyr-316) (30) was identified to be responsible for the interaction with Cbl. Interestingly, Zap-70 Tyr-292 is a negative regulatory site;
the Y292F mutant exhibits constitutive activation toward NFAT
activation (31, 32). Interestingly, Y292F does not show any difference
in terms of tyrosine phosphorylation and kinase activity from wild-type
Zap-70 (31, 32). We recently observed the same result with Syk Y316F
mutant.2 Our results suggest
that by binding to the same site in Zap-70 or Syk, 70Z can induce a
positive signal. It seems unlikely that the positive signal induced by
70Z/Zap-70 interaction results from a direct activation of the latter;
rather, it results from an indirect mechanism, e.g. by
indirectly affecting the tyrosine phosphorylation of other Zap-70
substrates such as Vav, phospholipase C
1, or LAT, which may function
as positive mediators for 70Z-mediated NFAT activation. However, the
possibility cannot be excluded that a weak activation of Zap-70 by 70Z,
although not detectable by current biochemical methods, is sufficient
for NFAT activation. In this regard, the Zap-70 Y292F mutant is not
active under resting conditions, but it induces NFAT activation (31,
32).
In the present study, we also demonstrate that 70Z mutants Y700F,
Y731F, and Y774F enhance both basal and ionomycin-treated NFAT
activation, suggesting that Tyr-700, Tyr-731, and Tyr-774 play a
negative role in this event, most likely via an interaction with their
binding partners such as PI3-K or Crk-L. This notion is supported by
the following findings: 1) overexpression of constitutively active
PI3-K inhibited TCR-mediated NFAT activation (47); 2) a Crk-L-C3G-Rap-1
signaling cascade is proposed to be a negative mediator for Cbl in
anergic T cells (48); and 3) as shown in the present study, TCR
cross-linking of 70Z-overexpressing T cells causes tyrosine
phosphorylation and reduces NFAT transactivation by 70Z. In addition,
the functional consequences of these mutations are specific, because
mutations at other tyrosine or serine residues do not change the
ability of 70Z to induce NFAT activation. Specifically, a mutation at
Tyr-735, which is only four amino acids from Tyr-731, exhibits the same
effect as unmutated 70Z on NFAT activation. These results suggest that
a negative regulatory function of Tyr-700, Tyr-731, or Tyr-774 results
from their interactions with downstream targeting molecules,
e.g. PI3-K or Crk-L, not from structural changes from the
Tyr to Phe mutation. However, it cannot be excluded that these tyrosine
residues may bind other SH2-containing proteins, which are mediators
for the observed OKT3-induced inhibition of NFAT by 70Z. Other
possibilities such as the removal of 70Z from competing intracellular
interactions, which may also enhance NFAT transactivation, cannot be
ruled out either. In any case, it can be concluded that these tyrosine
residues (Tyr-700, Tyr-731, or Tyr-774) play a negative role in
70Z-induced NFAT activation.
Taken together, our results clearly suggest a dual regulatory mechanism
by which 70Z participates in TCR-mediated signaling: one by
up-regulating the upstream PTKs (Syk/Zap-70) that it binds, and another
by down-regulating downstream signaling via
Tyr(P)-dependent interactions with its binding partners
such as PI3-K and/or Crk-L (Fig. 7). The
positive signal induced by 70Z/Zap-70 interaction is critical in
ionomycin-stimulated NFAT activation. Mutation of the 70Z/Zap-70
interaction such as G306E disrupts this positive signal and thus
abolishes ionomycin-induced NFAT activation. In the absence of a
70Z/Zap-70-induced positive signal, a Tyr(P)-dependent negative signal becomes dominant, leading to an enhanced inhibition of
OKT3-mediated NFAT activation. This model may also have significant implications for the biological function of wild-type Cbl. In the case
of wild-type Cbl, an amino terminus-mediated interaction of wild-type
Cbl with PTKs contributes to a negative regulatory function of Cbl.
This model is consistent with several recent studies; for example, Cbl
is a negative regulator of Syk (33) via direct physical association
between the two proteins, or a Cbl-mediated Crk-L-C3G-Rap1 signaling
pathway is responsible for the defect in interleukin-2 production in
anergic T cells (48). However, it is not known at present how the
interaction of PTKs with the amino-terminal PTB domain of 70Z induces a
positive signal, whereas the interaction with that of wild-type Cbl
induces a negative one. Clearly, additional studies are needed to
elucidate the molecular mechanism by which Cbl family proteins regulate
the upstream Syk/Zap-70 family kinases with which they associate. These
studies will be critical to understand the involvement of Cbl proteins
in the regulation of TCR- or other cell surface receptor-mediated
signaling pathways.