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INTRODUCTION |
The proto-oncogene product c-Cbl is a ubiquitously expressed
complex adapter protein that associates with numerous signaling molecules in a variety of cell types (reviewed in Ref. 1). It was
originally identified as a viral oncogene product (v-Cbl) that causes
B-lymphomas and myeloid leukemias in mice (2). Cloning of its cellular
homolog revealed that c-Cbl is a 906 amino acid protein that lacks any
obvious catalytic domain (3). It contains a N-terminal phosphotyrosine
binding (PTB)1 domain (4, 5),
a C3HC4 ring finger motif (6), a proline-rich region that includes a
binding site for the Grb2 SH3 domain (7), and a C-terminal region that
includes several tyrosine residues located within consensus binding
sequences for the SH2 domain containing Crk and p85 PI3K adapter
proteins (8). A Cbl-related molecule called Cbl-b has been cloned in
humans (9), and Cbl homologs have been identified in Drosophila
melanogaster (D-Cbl) (10, 11) and Caenorhabditis
elegans (Sli-1) (12).
Cbl is rapidly tyrosine-phosphorylated in response to engagement of
numerous receptors that activate protein-tyrosine kinases, including
immunoreceptors, receptor protein-tyrosine kinases, hematopoietic
growth factor receptors, and integrins, as well as oncogenic tyrosine
kinases. Tyrosine-phosphorylated Cbl associates with the SH2 domains of
the adapter proteins Crk(L) and/or p85 PI3K upon engagement of many of
these receptors (reviewed in Ref. 1). Similar to Grb2, Crk family
adapter proteins are composed almost exclusively of SH2 and SH3
domains. CrkI and CrkII are alternatively spliced products of a single
gene that contain a N-terminal SH2 domain followed by one or two SH3
domains, respectively (13). CrkL is the product of a different gene; it
has a structural organization similar to CrkII (14). Several studies
have indicated that Crk(L) proteins may promote transformation, either
when expressed as truncated isoforms or when overexpressed (13, 15,
16). Moreover, a multimolecular complex of Abl, CrkL, Cbl, and,
possibly, p85 PI3K has been postulated to mediate, at least in part,
the transforming ability of oncogenic Abl (16-19). Interestingly,
Crk(L) proteins associate through their N-terminal SH3 domain with C3G (20), a guanine nucleotide exchange factor for the small GTPase Rap1
(21, 22). In various model systems, overexpression of Rap1 can
antagonize Ras signaling, presumably through competitive inhibition of
Ras-GTP binding to downstream effector molecules (Ref. 23 and
references therein). Thus, it is possible that the interaction of Cbl
with Crk(L) regulates Ras signaling through C3G and Rap1 and that Cbl
serves as a docking protein to recruit the Crk(L)-C3G complex (24).
This is analogous to the well described role of activated receptor
tyrosine kinases, which recruit the Grb2-Sos complex to the plasma
membrane to activate membrane-bound Ras (25). Elucidation of the
functional significance of the Cbl-Crk(L)/p85 PI3K interaction for
normal as well as oncogenic protein-tyrosine kinase signaling pathways
is therefore of considerable interest.
Several lines of evidence indicate that Cbl functions as a negative
regulator of protein-tyrosine kinase signaling pathways. First, in the
flatworm C. elegans, the G315E loss-of-function allele of
Sli-1 rescues vulval development induced by a reduction-of-function allele of the Let23 epidermal growth factor receptor homolog (12). Second, in D. melanogaster, overexpression of D-Cbl in
transgenic flies inhibits the sevenless protein-tyrosine kinase-induced
development of the R7 photoreceptor neuron (11). Finally, in RBL 2H3
mast cells, overexpression of mammalian Cbl inhibits Fc
RI-induced Syk-tyrosine kinase activity and serotonin release (26). Given the
strong evolutionary conservation of the N-terminal half of the Cbl
protein and the observation that the Cbl G306E mutation, which
corresponds to the Sli-1 G315E loss-of-function mutation, inactivates
its PTB domain (4, 5), it is possible that the negative regulation of
protein-tyrosine kinase signaling pathways by mammalian Cbl requires
direct interaction of its PTB domain with protein-tyrosine kinases.
Several oncogenic variants of the c-Cbl proto-oncogene product have
been identified. The c/v-Cbl oncoprotein (also referred to as Cbl-N)
consists of the N-terminal 357 amino acids of the c-Cbl protein, which
includes the PTB domain (2, 3). Introduction of the G306E mutation into
c/v-Cbl blocks its association with activated (receptor) tyrosine
kinases in vivo (5, 27, 28), as well as its transforming
activity in vitro (27). Thus, it was proposed that
transformation caused by overexpression of c/v-Cbl results from
competitive inhibition of endogenous Cbl binding to phosphotyrosine
residues on activated protein-tyrosine kinases, thereby blocking the
negative regulatory role of Cbl on tyrosine kinases (27).
Interestingly, the oncogenic 70Z Cbl mutant protein, which contains a
17-amino acid internal deletion including the N-terminal cysteine
residue of the Ring finger, shows increased baseline tyrosine
phosphorylation in fibroblasts (28-30) and enhances NFAT transcription
in Jurkat T cells (31). Here, we tested two alternative models that
have been suggested to explain 70Z Cbl-mediated transformation and
activation of NFAT in Jurkat T cells. In the first model, 70Z Cbl acts
through increased tyrosine phosphorylation and association with Crk(L)
and p85 PI3K adapter proteins, which may lead to deregulation of Rap1
and, perhaps, Ras activation. In the second model, 70Z Cbl acts through
its PTB domain, possibly to competitively inhibit the PTB domain
dependent regulatory function of endogenous Cbl on protein-tyrosine
kinases. Our study demonstrates that 70Z Cbl-induced NFAT/AP1
activation requires an intact PTB domain but not Crk(L) or p85 PI3K
interaction. Our findings are most consistent with the possibility that
70Z Cbl acts as a dominant negative to inhibit the negative regulatory
role of endogenous Cbl on protein-tyrosine kinases.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Antibodies--
Jurkat E6.1 and Jurkat-TAg (32)
cell lines were maintained in RPMI medium supplemented with 10% fetal
bovine serum (FBS) at a cell density of 0.1 to 1 × 106 cells/ml. HuTK
and CV-1 cells were
maintained in Dulbecco's modified Eagle's medium/10% FBS. The
following antibodies (Abs) were used in this study: 4G10
anti-phosphotyrosine and anti-p85 PI3K from Upstate Biotechnology;
anti-Crk from Transduction Laboratories; anti-Erk2, anti-CrkII,
anti-CrkL, and anti-Cbl (C15) from Santa Cruz; anti-active MAPK from
Promega; and anti-hemagglutinin (HA) (12CA5) from Boehringer Mannheim
and anti-Myc (9E10) ascites.
cDNA Constructs--
pSX SR
, pSX HA Cbl, and pSX HA 70Z
were described previously (7). pSX HA Cbl Y700F, Y774F and
Y700F/Y731F/Y774F were made by subcloning a 3.0-kilobase
BamHI fragment from the corresponding pAlter constructs
(gifts from Wallace Y. Langdon) (33) into the BglII site of
pSX SR
. pSX HA Cbl Y700F/Y774F was made by site directed mutagenesis
of pSX HA Cbl Y774F using the Quick Change site-directed
mutagenesis kit (Stratagene) and oligos
5'-TGAAGAGGACACAGAATTCATGACTCCCTCTTC-3' (sense) and
5'-GAAGAGGGAGTCATGAATTCTGTGTCCTCTTCA-3' (antisense), thereby creating a
diagnostic EcoRI site. The construct was verified by
sequencing. pSX HA 70Z Y700F, Y774F, Y700F/Y774F, and Y700F/ Y731F/Y774F were made by replacing coding sequences 3' of the unique
BglII site from pSX HA 70Z with the same fragments from the
corresponding pSX HA Cbl mutant constructs. pSX HA 70Z G306E was made
using the Quick Change site-directed mutagenesis kit (Stratagene)
and oligos 5'-CAGTGGGCTATTGAGTATGTTACTGC-3' and
5'-GCAGTAACATACTCAATAGCCCACTG-3'. The construct was verified by
sequencing. pSC65, pSC65 HA Cbl, and pSC65 HA 70Z were described
previously (34). pSC65 HA Cbl Y700F and Y774F were made by subcloning a
3.0-kilobase BamHI fragment from pALTER HA Cbl Y700F or
Y774F (gifts from W. Langdon) (33) into the BglII site of
pSC65. pSC65 HA Cbl Y700F/Y774F and Y700F/Y731F/Y774F, pSC65 HA 70Z
Y700F, and Y774F, Y700F/Y774F, and Y700F/Y731F/Y774F were made by
replacing the coding sequences 3' of the unique BglII site
with the corresponding fragments from pSX HA Cbl constructs carrying
the appropriate tyrosine to phenylalanine mutations. An N-terminal
9-amino acid Myc epitope-tag was inserted into the SalI-NcoI sites of pAK10 CrkI, CrkI R38V, CrkI
W169L, and CrkII (gifts from M. Matsuda) (35) using oligos
5'-TCGACATGGAGCAGAAGCTGATCAGCGAGGAGGACTTGGCCATG-3' (sense) and
5'-GATCCATGGCCAAGTCCTCCTCGCTGATCAGCTTCTGCTCCATG-3' (antisense). The con- structs were verified by sequencing.
pSC65 Myc CrkI, Myc CrkI W169L, and Myc CrkII were made by subcloning the SalI-KpnI fragment of the corresponding pAK10
constructs into the SalI-KpnI sites of pSC65.
pSC65 Myc CrkI R38V was made by replacing the 915-base pair
NcoI fragment from pSC65 Myc CrkI with that from the
corresponding pAK10 Myc CrkI R38V construct.
Expression of Recombinant Vaccinia Virus--
Recombinant
vaccinia virus was made by standard procedures. Briefly, near confluent
CV-1 cells were infected in 25-cm2 flasks for 2 h with
wild-type WR' strain TK+ vaccinia virus at a multiplicity
of infection of 0.25 and transfected overnight with 20 µg of the
appropriate constructs using Lipofectin in Opti-MEM medium (Life
Technologies, Inc.) followed by an additional 24 h of culture in
Dulbecco's modified Eagle's medium/10% FBS. Infected/transfected
cells were harvested by centrifugation and lysed by repeated cycles of
freeze-thawing and sonication. Blue recombinant TK
plaques were purified by three rounds of plaque purification on
confluent HuTK
in 1% low melting agarose/1× basal
medium Eagle (Life Technologies, Inc.)/5% FBS and three rounds of
amplification in Dulbecco's modified Eagle's medium/10% FBS in the
continuous presence of 25 µg/ml BrdUrd (Sigma). Crude viral stocks
were titered on HuTK
cells and used to infect Jurkat T
cells at a multiplicity of infection of 5. After 15 h, infected
Jurkat T cells were harvested. Cell viability was routinely determined
by trypan blue exclusion and always exceeded 95%.
OKT3 Stimulation, Immunoprecipitation, SDS-PAGE, and
Immunoblotting--
Jurkat T cells were washed once in ice-cold RPMI
medium without FBS and resuspended at 1 × 108
cells/ml. Generally, 1-2 × 107 cells were
preincubated at 37 °C for 5 min, before cross-linking CD3 by
addition of OKT3 ascites (1:100). Cells were incubated at 37 °C for
the indicated time periods and solubilized for 30 min on ice in lysis
buffer containing 150 mM NaCl, 25 mM Tris, pH
7.5, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 mg/ml aprotonin and leupeptin, and
1% Brij97 or another detergent as indicated. Immunoprecipitation of
postnuclear lysates, denaturing SDS-PAGE, and immunoblotting were
performed according to standard procedures.
Transient Tranfection and SEAP Reporter Gene
Assays--
Secreted alkaline phosphatase (SEAP) reporter gene
constructs composed of multimers of the NFAT or AP1 response elements
(NFAT-SEAP and AP1-SEAP) were kindly provided by G. Crabtree (32). In
general, 1 × 107 Jurkat-TAg cells were transfected
with 5 µg of reporter construct and 10 µg of test construct by
electroporation using the Bio-Rad gene pulser (310 kV, 200 ohms, 960 microfarad). Transfected Jurkat-TAg cells were cultured in bulk for
24 h and subsequently stimulated in duplicate with immobilized
OKT3 (1 µl of ascites/well), PMA (Sigma) at 10 ng/ml, ionomycin
(Calbiochem) at 1 µg/ml or PMA plus ionomycin in 1 ml of phenol
red-deficient RPMI/10% FBS at a density of 3 × 106
transfected cells/ml. After stimulation for 15 h, cell cultures were incubated for 1 h at 65 °C to inactivate endogenous
phosphatases, and supernatants were assayed in duplicate at 37 °C
for SEAP activity using p-nitrophenyl phosphate (Sigma) at
1.8 mg/ml in diethanolamine bicarbonate, pH 10.0, as a substrate.
Absorbance at 405 nm was determined using a MR 5000 microtiter plate
reader (Dynatech), usually between 6 and 12 h of incubation.
Presented data are representative of at least three independent experiments.
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RESULTS |
70Z Cbl Shows Increased Basal and Activation-induced Tyrosine
Phosphorylation and Association with Crk(L) and p85 PI3K in Jurkat T
Cells--
The oncogenic 70Z/3 mutant form of Cbl undergoes increased
baseline tyrosine phosphorylation in fibroblasts (28-30). To determine whether increased tyrosine phosphorylation of 70Z Cbl could also be
observed in T cells, Jurkat T cells were infected with recombinant vaccinia virus expressing HA-tagged wt or 70Z Cbl proteins. Infected cells were stimulated with anti-CD3
mAb, and tyrosine
phosphorylation of HA immunoprecipitates was evaluated by
immunoblotting. As shown in Fig.
1A, 70Z Cbl displays increased
basal and activation-induced tyrosine phosphorylation relative to wt
Cbl (compare lanes 3 and 4 with lanes
5 and 6). In contrast to the observed differences in
their phosphotyrosine content, wt and 70Z Cbl showed similar kinetics
of tyrosine phosphorylation with maximal levels obtained after 2 min of
stimulation and returning to baseline levels after 20-40 min of
stimulation (Fig. 1B). These kinetics closely resembled those of endogenous Cbl (data not shown). These results suggest that
increased tyrosine phosphorylation of 70Z Cbl is a more general phenomenon, as it occurs in cell types as different as fibroblasts and
T cells.

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Fig. 1.
70Z Cbl shows increased basal and CD3-induced
tyrosine phosphorylation in Jurkat T cells. A, Jurkat T
cells were infected for 15 h with recombinant vaccinia virus
generated with vector alone (pSC65) or vector encoding HA-tagged wt Cbl
(HA Cbl) or 70Z Cbl (HA 70Z). Cells were
stimulated for 2 min in the presence or absence of OKT3 mAb and
solubilized in 1% Brij97 lysis buffer, and postnuclear lysates were
immunoprecipitated (IP) with anti-HA mAb, followed by
SDS-PAGE and sequential immunoblotting (IB) with
anti-phosphotyrosine (4G10) and anti-HA mAbs as indicated.
The band detected in the anti-HA immunoblot (lower panel) of
pSC65 infected cells that migrates just above Cbl is a background band
caused by HA blotting and does not represent Cbl, as it is not detected
by anti-Cbl blotting (see also Fig. 3). B, after infection
with either HA Cbl or HA 70Z, Jurkat T cells were stimulated with OKT3
mAb for the indicated time periods and lysed in 1% Brij97 lysis
buffer, and postnuclear lysates were immunoprecipitated (IP)
with anti-HA mAb followed by SDS-PAGE and sequential immunoblotting
(IB) with anti-phosphotyrosine (4G10) and
anti-Cbl (C15) Abs as indicated.
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Activation-induced tyrosine phosphorylation of endogenous Cbl leads to
its association with Crk(L) and p85 PI3K adapter proteins (reviewed in
Ref. 1). In order to determine whether increased tyrosine
phosphorylation of 70Z Cbl leads to increased association with these
adapter proteins, Jurkat T cells were infected with recombinant
vaccinia virus encoding HA-tagged wt or 70Z Cbl and stimulated with
anti-CD3
mAb, and lysates were immunoprecipitated with anti-CrkII or
anti-CrkL Abs followed by anti-HA immunoblotting. Both CrkII (Fig.
2, top two panels) and CrkL
(Fig. 2, third and fourth panels from top) showed
increased basal and activation-induced association with 70Z relative to
wt Cbl (compare lanes 3 and 4 with lanes
5 and 6). This was not due to differences in the level of Cbl protein expression, as anti-HA immunoblotting revealed that wt
and 70Z Cbl were expressed at similar levels (Fig. 2, bottom
panel). Myc epitope-tagged CrkI (see Fig. 4) as well as p85 PI3K
(see Fig. 5) also showed increased association with 70Z relative to wt
Cbl in unstimulated and anti-CD3-stimulated Jurkat T cells. Taken
together, these findings demonstrate that increased basal and
activation-induced tyrosine phosphorylation of 70Z relative to wt Cbl
leads to increased association with Crk(L) and p85 PI3K adapter
proteins.

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Fig. 2.
70Z Cbl proteins show increased basal and
CD3-induced association with Crk(L) adapter proteins. Jurkat T
cells were infected, stimulated with OKT3, and solubilized as in Fig.
1A. Postnuclear lysates were immunoprecipitated
(IP) with anti-CrkII, anti-CrkL, or anti-HA Abs followed by
SDS-PAGE and (sequential) immunoblotting (IB) with anti-HA
and anti-Crk, anti-HA and anti-CrkL, or anti-HA, respectively.
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Activation-induced Tyrosine Phosphorylation of Cbl Is Not
Restricted to Phosphotyrosine Residues 700, 731, and 774--
To
further characterize the tyrosine phosphorylation and interaction of wt
and 70Z Cbl proteins with Crk(L) and p85 PI3K in vivo, we
generated recombinant vaccinia viruses encoding wt or 70Z Cbl carrying
tyrosine to phenylalanine mutations in Tyr-700, Tyr-731, and/or
Tyr-774. Tyr-700 and Tyr-774 are located within a consensus sequence
for binding to the SH2 domains of Crk, whereas Tyr-731 is predicted to
bind the p85 PI3K SH2 domain (8). Furthermore, in vitro
analysis has revealed that the Crk SH2 domain binds to the
phosphotyrosine residue 774 (24, 36). However, in Abl transformed
cells, CrkL association with wt Cbl is decreased but not absent when
either Y700F or Y774F point mutations are introduced into Cbl (33),
suggesting that the CrkL SH2 domain associates with both
phosphotyrosine residues. p85 PI3K association with Cbl has been
previously mapped to phosphotyrosine 731 in vitro (24) and
in vivo (31, 37). To determine whether simultaneous mutation
of Tyr-700, Tyr-731, and Tyr-774 to phenylalanine leads to loss of wt
and 70Z Cbl tyrosine phosphorylation, Jurkat T cells were infected with
recombinant vaccinia virus encoding various Cbl constructs and tyrosine
phosphorylation of Cbl proteins evaluated in the absence or presence of
anti-CD3 stimulation. Interestingly, wt and 70Z Cbl carrying the
Y700F/Y774F and, more importantly, the Y700F/Y731F/Y774F mutations
showed anti-CD3-induced tyrosine phosphorylation (Fig.
3), indicating that a tyrosine(s) other than Tyr-700, Tyr-731, and Tyr-774 undergoes activation-induced phosphorylation. Moreover, as seen for Tyr-700, Tyr-731, and Tyr-774, at least one of these additional tyrosines undergoes increased phosphorylation in 70Z relative to wt Cbl (Fig. 3). As we have been
unable to detect tyrosine phosphorylation of Cbl 1-655 truncation constructs (Ref. 34 and data not shown), it seems that these additional
tyrosine residues are located in the C-terminal region (amino acids
655-906) of Cbl. Consistent with this idea, Cbl contains four
additional tyrosine residues in that region, i.e. Tyr-674, Tyr-735, Tyr-869, and Tyr-871 (3).

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Fig. 3.
Wild-type and 70Z Cbl proteins undergo
activation-induced phosphorylation on tyrosines other than Tyr-700,
Tyr-731, and Tyr-774. Jurkat T cells were infected with
recombinant vaccinia virus as indicated and stimulated for 2 min in the
presence or absence of OKT3, and postnuclear Brij97 lysates were
immunoprecipitated (IP) with anti-HA mAbs followed by
SDS-PAGE and sequential immunoblotting (IB) with
anti-phosphotyrosine (4G10) and anti-Cbl (C15)
Abs.
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Disruption of Crk(L) and p85 PI3K Association with wt and 70Z
Cbl Carrying the Y700F/Y731F/Y774F Triple Mutation--
To further
characterize the molecular basis for the interaction between Cbl and
Crk(L)/p85 PI3K proteins in vivo, we analyzed the
interaction of Myc epitope-tagged CrkI, as well as endogenous CrkII and
CrkL with wt or 70Z Cbl carrying Y700F, Y731F, and/or Y774F mutations.
Recombinant vaccina virus carrying Myc epitope-tagged CrkI was
generated for this purpose. As previously mentioned, Myc CrkI displayed
increased association with 70Z relative to wt Cbl in both unstimulated
and anti-CD3-stimulated Jurkat T cells (Fig.
4A, compare lanes 3 and 4 with lanes 9 and 10).
Furthermore, both Y700F and Y774F single point mutations reduced, but
did not eliminate, binding of CrkI to wt and 70Z Cbl (Fig.
4A, compare lanes 5-8 with lanes 3 and 4 and lanes 11-14 with lanes 9 and 10). Identical results were obtained also for
coprecipitation of wt and 70Z Cbl with endogenous CrkII and CrkL (data
not shown). In contrast, wt and 70Z Cbl carrying the double
(Y700F/Y774F) or triple (Y700F/Y731F/Y774F) mutation failed to show
basal and activation-induced association with CrkI (Fig. 4B,
compare lanes 5-8 with lanes 3 and 4 and lanes 11-14 with lanes 9 and 10),
as well as with endogenous CrkII and CrkL (data not shown). Additional experiments using Myc epitope-tagged Crk proteins that contain a
mutation that inactivates its SH2 (R38V) or SH3 (W169L) domain confirmed that the interaction between Cbl and Crk is mediated through
the SH2 but not the SH3 domain of Crk in vivo (data not shown).

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Fig. 4.
Crk(L) adapter proteins do not associate with
wt or 70Z Cbl carrying the Y700F/Y774F mutation. A,
Jurkat T cells were co-infected with recombinant vaccinia virus as
indicated and stimulated for 2 min in the presence or absence of OKT3
mAb, and postnuclear Brij97 lysates were immunoprecipitated
(IP) with anti-Myc mAb. Because Myc CrkI exactly co-migrates
with the immunoglobulin light chain of the immunoprecipitating Ab,
equal amounts were run in parallel in reducing and nonreducing SDS-PAGE
followed by immunoblotting (IB) with anti-HA (top
panel) or anti-Myc (middle panel) mAbs, respectively.
In addition, whole cell lysates (WCL) were immunoblotted
with anti-HA mAb to determine the expression levels of Cbl proteins
(bottom panel). B, Jurkat T cells were
co-infected with recombinant vaccinia virus as indicated and further
treated as described in A.
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We also analyzed the interaction of p85 PI3K with various wt and 70Z
Cbl constructs. Initial experiments revealed that following solubilization in Brij97, p85 coprecipitated via the OKT3 mAb with the
activated T cell receptor/CD3 complex (data not shown). To exclude the
possibility that p85 PI3K could be detected in anti-HA
immunoprecipitates as a consequence of its association with the
activated TCR/CD3 complex rather than Cbl proteins, we instead used
Triton X-100 to solubilize the cells. As shown in Fig.
5A, we could not detect
activation-induced coprecipitation of p85 PI3K in anti-HA
immunoprecipitates of vector infected Jurkat T cells under these
conditions (Fig. 5A, lanes 1 and 2). We did detect a faint background band in both unstimulated and
anti-CD3-stimulated vector-infected Jurkat T cells that comigrated with
p85 PI3K. Nevertheless, we clearly observed increased p85 PI3K binding
to wt Cbl in anti-CD3-stimulated relative to unstimulated HA Cbl infected Jurkat T cells (Fig. 5A, compare lanes 3 and 4 with lanes 1 and 2), as well as
increased basal and activation-induced p85 PI3K association with 70Z
relative to wt Cbl (Fig. 5A, compare lanes 7 and
8 with lanes 3 and 4). Significantly,
anti-HA immunoblotting of anti-p85 PI3K immunoprecipitates more clearly
revealed increased association of p85 PI3K with 70Z relative to wt Cbl
when compared with anti-p85 PI3K immunoblotting of anti-HA
immunoprecipitates (compare Fig. 5B and Fig. 5A).
Even though p85 PI3K was readily observed in anti-HA immunoprecipitates
of HA Cbl expressing cells (Fig. 5A, lanes 3 and
4), activation-induced coprecipitation of HA-tagged Cbl with
p85 PI3K could barely be detected (Fig. 5B, lanes 3 and
4). These findings are consistent with the possibility that
overexpressed HA Cbl associates with a small fraction of total p85
PI3K, whereas the overexpressed and heavily tyrosine-phosphorylated 70Z
Cbl protein associates with a large fraction of total p85 PI3K.
Importantly, and consistent with published data (24, 31, 37), p85 PI3K
did not associate with wt or 70Z Cbl carrying the Y700F/Y731F/Y774F
triple mutation (Figs. 5, A and B, compare lanes 5 and 6 with lanes 3 and
4, and lanes 9 and 10 with lanes 7 and 8). Taken together, our findings demonstrate that
Crk(L) proteins interact through their SH2 domains with both
phosphotyrosine residues 700 and 774 in wt and 70Z Cbl proteins
in vivo, and that simultaneous mutation of Tyr-700, Tyr-731,
and Tyr-774 disrupts interaction of Crk(L) and p85 PI3K with wt and 70Z
Cbl.

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Fig. 5.
p85 PI3K does not associate with wt or 70Z
Cbl proteins carrying the Y700F/Y731F/Y774F mutation.
A, Jurkat T cells were infected with recombinant vaccinia as
indicated and stimulated for 2 min in the presence or absence of OKT3,
and postnuclear Triton X-100 lysates were immunoprecipitated with
anti-HA followed by SDS-PAGE and sequential immunoblotting with
anti-p85 PI3K and anti-HA Abs. Anti-p85 PI3K immunoblotting generated a
background signal (as seen in pSC65-infected cells) that comigrated
with endogenous p85. Whole cell lysates (WCL) were also
immunoblotted with anti-HA to document equal expression of the
HA-tagged wt and 70Z Cbl constructs (bottom panel).
B, Jurkat T cells were infected, stimulated, and
lysed as in A, and lysates were immunoprecipitated
(IP) with anti-p85 PI3K mAb followed by SDS-PAGE and
sequential immunoblotting (IB) with anti-HA and anti-p85
PI3K Abs. The top panel was overexposed to visualize the
presence of small amounts of HA Cbl in p85 immunoprecipitates. Shorter
exposures revealed differences in p85 PI3K association with 70Z Cbl in
unstimulated and stimulated cells. Whole cell lysates (WCL)
were immunoblotted with anti-HA mAb to document similar expression
levels of Cbl proteins.
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Disruption of Crk(L)/p85 PI3K Interaction with wt and 70Z Cbl Does
Not Affect Erk MAPK Activation--
Crk(L) proteins associate through
their N-terminal SH3 domain with C3G (20), a guanine nucleotide
exchange factor for the small G protein Rap1 (21). Overexpression of
Crk(L) and C3G has been reported to activate Rap1 (38). In various
model systems, overexpression of Rap1 can antagonize Ras signaling,
presumably through competitive inhibition of Ras-GTP binding to
downstream effector molecules (Ref. 23 and references therein). In
Jurkat T cells, activation of the Ras effector protein Raf results in activation of the dual specificity MAP kinase kinases MEK1/2 that, in
turn, activate the MAP kinases Erk1/2 (39). In order to determine whether interaction of Crk(L) with wt or 70Z Cbl had any effect on Ras
signaling, we analyzed the effect of overexpression of various Cbl
constructs on the activation of MAP kinases Erk1 and Erk2. Thus, Jurkat
T cells were infected with recombinant vaccinia virus expressing the
various wt and 70Z Cbl proteins, and activation of Erk1/2 was evaluated
by immunoblotting whole cell lysates with anti-active MAPK Ab. Neither
overexpression of 70Z Cbl nor overexpression of the Y700F/Y774F or
Y700F/Y731F/Y774F mutant derivatives of wt and 70Z Cbl showed any
significant and reproducible effect on basal or anti-CD3-induced
activation of Erk1/2 relative to wt Cbl or the vector control (Fig.
6). Moreover, none of the wt or 70Z Cbl
proteins had any effect on basal or anti-CD3-induced Erk activation
upon prolonged stimulation (data not shown), excluding the possibility
that 70Z Cbl induces prolonged Erk activation relative to wt Cbl or the
vector control. These results demonstrate that increased basal and
activation-induced association of 70Z Cbl with Crk(L) and p85 PI3K does
not detectably affect Erk1/2 activation, suggesting that the Cbl-Crk(L)
and Cbl-p85 PI3K interactions do not regulate Ras signaling in Jurkat T
cells.

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Fig. 6.
Interaction between Cbl and Crk(L) or p85
PI3K does not affect basal or CD3-induced Erk1/2 activation.
Jurkat T cells were infected with recombinant vaccinia virus as
indicated, stimulated for 5 min in the absence or presence of OKT3, and
lysed in 1% Triton X-100 lysis buffer, and whole cell lysates
(WCL) were immunoblotted with anti-active MAPK or anti-HA
Abs.
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70Z Cbl Retains Its Ability to Induce NFAT and AP1 in the Absence
of Crk(L)/p85 PI3K Association--
As 70Z Cbl is known to activate
the NFAT transcription factor in Jurkat T cells (31), we analyzed the
effect of the Cbl-Crk(L) and Cbl-p85 PI3K interactions on
transcriptional activation of NFAT and AP1 transcription factors using
SEAP reporter gene assays (32). Jurkat-TAg cells were transiently
transfected with HA-tagged wt or 70Z Cbl expression constructs together
with the appropriate reporter gene construct. Transfected cells were
left unstimulated or stimulated with immobilized anti-CD3 mAb, PMA,
ionomycin or PMA plus ionomycin, and supernatants were assayed for SEAP
reporter gene activity. Results were expressed relative to the response obtained after stimulation with PMA plus ionomycin, which served as an
internal control for the SEAP responsiveness between different groups
of transfected cells. It should be noted that we did not observe any
reproducible effect of overexpressing various Cbl proteins on the
absolute response induced by PMA plus ionomycin (data not shown).
Overexpression of wt Cbl did not reproducibly and significantly affect
NFAT activity under any conditions relative to the vector control (Fig.
7A), nor did overexpression of
wt Cbl affect AP1 driven reporter gene activity (Fig. 7B).
In contrast, overexpression of 70Z Cbl led to a significant and
reproducible increase in NFAT reporter activity in unstimulated cells
relative to wt Cbl or the vector control but caused no significant and reproducible changes in response to anti-CD3 mAb (Fig. 7A).
Indeed, titration of the anti-CD3 mAb over a 100-fold range did not
reveal any significant effect of 70Z relative to wt Cbl on
anti-CD3-induced NFAT activation (data not shown). 70Z Cbl also
up-regulated AP1 activity in unstimulated cells, although this increase
was less pronounced (2-3-fold induction over the vector control)
compared with the increase in NFAT activity (5-10-fold induction over
the vector control) (Fig. 7B). Overexpression of wt or 70Z
Cbl Y700F/Y774F double or Y700F/Y731F/Y774F triple mutants did not lead
to significant changes in NFAT or AP1 activity relative to their
unmutated counterparts (Fig. 7, A and B). It
should be noted that the relatively small increase in NFAT activation
induced by 70Z Y700F/Y774F and 70Z Y700F/Y731F/Y774F relative to 70Z
Cbl observed in this experiment was not consistently observed. In
contrast to the reported cooperation of 70Z Cbl with ionomycin to
induce NFAT (31), our results demonstrated that SEAP reporter gene
activity in cells stimulated with ionomycin (Fig. 7, A and
B) or PMA alone (data not shown) was similar to that
observed in unstimulated cells. It should be noted that the observed
effects of oncogenic 70Z Cbl proteins on NFAT and AP1 activation were
not due to differences in expression levels of Cbl proteins, as
evidenced by anti-Cbl immunoblotting of whole cell lysates (Fig.
7C). Taken together, our results demonstrate that (i) 70Z
Cbl up-regulates NFAT and AP1 activity in unstimulated Jurkat T cells,
(ii) 70Z Cbl does not cooperate with ionomycin to induce NFAT or AP1
activity, and (iii) the 70Z Cbl-induced activation of NFAT and AP1 is
not mediated through its interaction with Crk(L) and p85 PI3K adapter
proteins.

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Fig. 7.
Activation of NFAT and AP1 by oncogenic 70Z
Cbl in unstimulated Jurkat T cells does not require interaction with
Crk(L) and p85 PI3K. Jurkat-TAg cells were transiently transfected
with vector (pSX SR ) or the indicated Cbl expression constructs
together with the NFAT (A) or AP1 (B) reporter
gene construct and either left unstimulated or stimulated for 15 h
with immobilized OKT3, ionomycin (1 µg/ml), or PMA (10 ng/ml) plus
ionomycin. SEAP reporter activity was measured and plotted relative to
the response induced by PMA plus ionomycin. C, whole cell
lysates (WCL) of the experiments shown in A and
B were immunoblotted (IB) for Cbl expression
using anti-Cbl (C15) Abs.
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Disruption of the PTB Domain Blocks 70Z Cbl-induced NFAT/AP1
Activation--
Thien and Langdon (27, 30) have hypothesized that the
mechanisms underlying 70Z Cbl- and v-Cbl-induced transformation are
distinct. Specifically, they hypothesized that 70Z Cbl induced transformation results from a positive signal, perhaps related to its
increased tyrosine phosphorylation (29, 30), whereas v-Cbl appears to
act as a dominant negative by competing with endogenous Cbl for
phosphotyrosine residues on activated tyrosine kinases, thereby
blocking the putative negative regulatory role of Cbl (27). Such a
model appears to be supported by the observations that 70Z Cbl, but not
v-Cbl, enhances NFAT activation in Jurkat T cells (Ref. 31 and data not
shown), as well as epidermal growth factor receptor kinase activity in
unstimulated NIH3T3 cells (30). Furthermore, v-Cbl-induced
transformation requires higher levels of expression as compared with
70Z Cbl-induced transformation (30). However, evidence that the
molecular mechanism(s) underlying 70Z Cbl- and v-Cbl-induced
transformation are different from each other is presently not
available. As v-Cbl-induced transformation is blocked by the G306E
mutation (27), we analyzed the effect of the G306E mutation on NFAT and
AP1 activation induced by 70Z Cbl oncoproteins. Jurkat TAg cells were
transiently transfected with HA-tagged 70Z Cbl in either the absence or
the presence of the G306E mutation. Activation of NFAT and AP1
transcription factors was assessed in unstimulated Jurkat T cells.
Most importantly, the 70Z Cbl oncoprotein but not its G306E mutant
derivative up-regulated NFAT and AP1 activity (Fig.
8, A and B), even
though 70Z Cbl and its G306E mutant derivative were expressed at
similar levels (Fig. 8C). As the PTB domain of Cbl is known
to bind to the ZAP70 phosphotyrosine residue 292 in vitro
and upon coexpression in Cos cells (4, 5), we next evaluated whether
the G306E mutation might also affect tyrosine phosphorylation of the
70Z Cbl oncoprotein. As illustrated in Fig. 8D, the
increased tyrosine phosphorylation of 70Z Cbl that is observed in
unstimulated and OKT3-stimulated Jurkat T cells is blocked by the G306E
mutation and is comparable to that observed in wt Cbl. Taken together,
our findings confirm earlier reports that c/v-Cbl does not up-regulate
NFAT and AP1 activity (31) and demonstrate that increased tyrosine
phosphorylation of 70Z Cbl-induced as well as 70Z Cbl-induced NFAT and
AP1 activation requires the presence of an intact PTB domain.

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Fig. 8.
Activation of NFAT and AP1 by oncogenic 70Z
Cbl requires an intact PTB domain. Jurkat TAg cells were
transiently transfected with vector or the indicated Cbl expression
constructs together with NFAT (A) or AP1 (B)
reporter gene constructs and either left unstimulated or stimulated for
15 h in the presence of PMA (10 ng/ml) plus ionomycin (1 µg/ml).
C, whole cell lysates of the experiments shown in
A and B were immunoblotted with anti-Cbl (C15)
Abs to verify expression of transfected Cbl proteins. D,
Jurkat T cells were infected with recombinant vaccinia virus as
indicated and further treated as in Fig. 1A.
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DISCUSSION |
The Cbl proto-oncogene product is a ubiquitously expressed complex
adapter protein that functions as a negative regulator of
protein-tyrosine kinases (1, 11, 12, 26). Cbl is rapidly tyrosine-phosphorylated and associates with Crk(L) and p85 PI3K adapter
proteins upon engagement of numerous protein-tyrosine kinase-linked
receptors in a variety of cell types (reviewed in Ref. 1).
Interestingly, the 70Z Cbl oncoprotein shows increased tyrosine
phosphorylation in fibroblasts (28-30) and activates NFAT-mediated transcription in Jurkat T cells (Ref. 31 and this study), but the
molecular mechanisms underlying 70Z Cbl-induced NFAT activation and
transformation have not been previously identified. Here we demonstrate
that 70Z Cbl shows increased basal and CD3-induced tyrosine
phosphorylation, leading to increased association with Crk(L) and p85
PI3K adapter proteins in Jurkat T cells. However, disruption of Crk(L)
and p85 PI3K association with oncogenic 70Z Cbl did not block NFAT and
AP1 activation. In contrast, 70Z Cbl-induced NFAT/AP1 activation was
completely blocked by the G306E mutation, indicating that 70Z Cbl
requires an intact PTB domain for NFAT/AP1 activation in Jurkat T cells.
Our studies confirm the previous finding (31) that oncogenic 70Z Cbl
but not wt or c/v-Cbl activates NFAT and further extends these findings
to show that 70Z Cbl also enhances AP1 activity. Our study differs from
that of Liu et al. (31) in that we did not detect
cooperation of 70Z Cbl with ionomycin to induce NFAT activation, even
when Jurkat T cells were stimulated after serum starvation or when
using lower concentrations of ionomycin (data not shown). We also did
not confirm the data from Rellahan et al. (40), who reported
a 3-fold reduction in AP1-mediated reporter activity in cells
overexpressing Cbl relative to the vector control. The cause of the
discrepancies between our study and these other studies is not clear at
present, but we note that we used PMA plus ionomycin stimulation as an
internal control to normalize for reporter gene activity between
different groups of transfected cells.
The 70Z Cbl oncoprotein shows increased baseline tyrosine
phosphorylation in fibroblasts (28-30) and, as demonstrated in this study, in Jurkat T cells. However, the tyrosine residues in 70Z Cbl
that undergo increased phosphorylation relative to wt Cbl have not been
identified thus far. In vitro studies have previously identified Tyr(P)-774 and Tyr(P)-731 in wt Cbl as binding sites for the
SH2 domains of Crk and p85 PI3K, respectively (24, 36). In
vivo studies have demonstrated a role for both Tyr(P)-700 and Tyr(P)-774 in CrkL binding in Abl transformed cells (33) and for
Tyr(P)-731 in p85 PI3K binding (31, 37). Our studies have confirmed and
further extended these findings in determining that CrkI, CrkII, and
CrkL associate with both Tyr(P)-700 and Tyr(P)-774 in vivo
and that increased phosphorylation of Tyr-700, Tyr-731, and Tyr-774 in
70Z Cbl results in increased recruitment of Crk(L) and p85 PI3K adapter
proteins. Interestingly, increased tyrosine phosphorylation of 70Z is
not restricted to these three residues, as increased basal and
activation-induced phosphorylation was also observed in 70Z
Y700F/Y731F/Y774F relative to wt Y700F/Y731F/Y774F Cbl. Our findings
apparently contrast with those recently reported by Feshchenko et
al. (41), who did not detect appreciable tyrosine phosphorylation
of the wt Cbl Y700F/Y731F/Y774F triple mutant in response to
pervanadate treatment of transiently transfected Jurkat T cells. We
suggest that this difference may be due to the higher sensitivity of
the recombinant vaccinia virus expression system. Taken together with
the general finding that the Cbl 1-655 truncation mutant is not
appreciably tyrosine-phosphorylated (Refs. 34 and 41 and data not
shown), we tentatively conclude that there is at least one other
tyrosine in the C-terminal (amino acids 655-906) region that shows
increased basal and activation-induced phosphorylation in 70Z
versus wt Cbl.
The molecular mechanisms underlying 70Z Cbl-induced transformation and
NFAT/AP1 activation have not been previously identified. In theory, the
70Z Cbl oncoprotein may itself act as a positive signal transducer or,
alternatively, it may inhibit a negative regulator that prevents or
down-regulates an activating signal. Consistent with the former
possibility, several groups have previously suggested that 70Z
Cbl-induced transformation may, at least in part, be due to its
increased phosphotyrosine content and association with Crk(L) adapter
proteins (1, 28-30), perhaps through regulation of Ras signaling via
the C3G-Rap1-Ras pathway. Indeed, our initial results demonstrated
increased basal and activation-induced association of 70Z Cbl with
Crk(L) and p85 PI3K adapter proteins. However, neither increased
association of Crk(L) and p85 PI3K adapter proteins with 70Z relative
to wt Cbl nor disruption of Crk(L)/p85 PI3K association with wt or 70Z
Cbl detectably affects basal or anti-CD3-induced Erk1/2 activation.
Consistent with these findings, Thien and Langdon (30) have been unable
to detect an effect of 70Z Cbl overexpression on Erk activation in
fibroblasts. As Ras activation is both necessary and sufficient to
activate Erk (Ref. 39 and references therein), these findings suggest
that the Cbl-Crk(L) and Cbl-p85 PI3K interactions do not affect Ras
signaling. Moreover, our study clearly demonstrates that the 70Z Cbl
oncoprotein retains its ability to activate NFAT and AP1 in the absence
of Crk(L) and p85 PI3K binding, indicating that 70Z-induced NFAT/AP1
activation is not mediated through increased association with these
adapter molecules.
Although we can exclude the possibility that 70Z Cbl-mediated NFAT
activation is mediated through increased phosphorylation of Tyr-700,
Tyr-731, and Tyr-774, our findings demonstrate increased phosphorylation of 70Z Cbl on additional tyrosine residues. Therefore, it remains possible that increased tyrosine phosphorylation of 70Z Cbl
on these additional tyrosine residues contributes to its oncogenic and
NFAT/AP1 activating properties. Our finding that 70Z Cbl requires its
PTB domain for induction of NFAT and AP1 is consistent with the model
that mutation of the Ring finger domain of 70Z Cbl activates or exposes
its N-terminal PTB domain, which would allow increased recruitment to
activated protein-tyrosine kinases. Although it is not known whether
the reported enhanced association of 70Z Cbl with the epidermal growth
factor and platelet-derived growth factor receptor tyrosine kinases
(27, 28) depends on its PTB domain, introduction of the G306E mutation
into c/v-Cbl abrogates its association with activated (receptor)
tyrosine kinases in vivo as well as its transforming
activity in vitro (4, 5, 27, 28). Indeed, our findings also
demonstrate that the G306E mutation blocks increased tyrosine
phosphorylation of 70Z Cbl, suggesting that increased tyrosine
phosphorylation of 70Z Cbl results from increased or prolonged
recruitment to activated tyrosine kinases via its PTB domain.
Considering this model, however, it seems paradoxical that the PTB
domain of Cbl associates with phosphotyrosine residue 292 of ZAP70 both
in vitro and in vivo (4, 5), as mutation of this
tyrosine residue disrupts interaction with the Cbl PTB domain (5) yet
up-regulates NFAT activation in unstimulated Jurkat T cells (42).
As discussed above, 70Z Cbl-induced transformation and NFAT/AP1
activation may also result from inhibition of a negative regulator that
prevents or down-regulates an activating signal. As studies in D. melanogaster (11), C. elegans (12), and mammalian RBL 2H3 mast cells (26) indicate that Cbl functions as an evolutionary conserved negative regulator of protein-tyrosine kinases, it is possible that 70Z Cbl acts as a dominant negative by blocking the
negative regulatory role of endogenous Cbl on protein-tyrosine kinase
signaling pathways. Significantly, c/v-Cbl has previously been proposed
to act as a dominant negative by competing with endogenous Cbl for
phosphotyrosine binding sites on activated (receptor) tyrosine kinases
(27, 30). Most important, our study demonstrates that 70Z Cbl-induced
NFAT/AP1 activation in unstimulated Jurkat T cells is completely
blocked by the G306E mutation. This observation is consistent with a
dominant negative effect of 70Z Cbl on the evolutionary conserved PTB
domain-dependent negative regulatory role of endogenous Cbl
on protein-tyrosine kinase signaling pathways. In this model, the
increased tyrosine phosphorylation of 70Z Cbl, which depends on its PTB
domain, may be the consequence of increased recruitment to an activated
tyrosine kinase via its PTB domain and/or the inability of endogenous
Cbl to inhibit or down-regulate activated protein-tyrosine kinases in
the presence of the 70Z Cbl PTB domain. Importantly, competitive inhibition of endogenous Cbl binding to the ZAP70 phosphotyrosine residue 292 by the 70Z Cbl PTB domain is consistent with a negative regulatory role for the interaction of endogenous Cbl with the ZAP70
Tyr-292 residue (4, 5, 42). Whether wt or 70Z Cbl interacts with ZAP70
following T cell receptor activation and whether such an interaction
plays any biologically significant role in T cell receptor signal
transduction remains to be determined. If this model is correct, then
it remains to be determined why 70Z Cbl, but not v-Cbl, is able to
activate NFAT in Jurkat T cells and up-regulate epidermal growth factor
receptor kinase activity in fibroblasts.
In summary, we have determined the molecular basis for NFAT and AP1
induction by oncogenic 70Z Cbl in unstimulated Jurkat T cells. Our
results demonstrate that NFAT/AP1 activation by 70Z Cbl is not mediated
through increased interaction with Crk(L) and p85 PI3K adapter proteins
but instead depends on an intact PTB domain. These findings are most
consistent with a dominant negative action of the 70Z Cbl PTB domain on
the negative regulatory role of endogenous Cbl on protein-tyrosine kinases.