(Received for publication, June 13, 1995; and in revised form, January 2, 1996)
From the
We and others have recently identified Cbl, the protein product
of the c-cbl protooncogene, as an early tyrosine kinase
substrate upon T cell activation and have shown that Cbl forms in
vivo complexes with Src family tyrosine kinases, Grb2 adaptor
protein, and the p85 subunit of PI-3 kinase. Here we show that Cbl
associates with all three forms of the human Crk protein, predominantly
CrkL, following T cell receptor activation of Jurkat T cells.
Association between Cbl and Crk proteins was confirmed in normal human
peripheral blood-derived T cells. In vitro, Cbl was able to
interact with the Crk SH2 domain but not the SH3 domain. A
phosphopeptide corresponding to a potential Crk SH2 domain-binding
motif in Cbl (pYDVP) specifically inhibited binding between Cbl and Crk
SH2 domain. Anti-Cbl antibody completely immunodepleted the
CrkL-associated 120kDa phosphotyrosyl polypeptide, suggesting that the
recently described p130-related Crk-associated
p116 of T cells may be Cbl. Consistent with this possibility, the 4F4
antibody used to characterize the p116 polypeptide cross-reacted with
Cbl protein when it was resolved on one- or two-dimensional gels. CrkL
was constitutively associated with a substantial amount of the guanine
nucleotide exchange protein C3G, and a fraction of the C3G protein was
coimmunoprecipitated with Cbl in activated Jurkat T cells. These
results suggest the possibility that Cbl may participate in a signaling
pathway that regulates guanine nucleotide exchange on small G-proteins
in T cells.
Tyrosine phosphorylation of intracellular substrates is an early
and obligatory event in T cell receptor (TCR)-mediated ()lymphocyte activation. The TCR-CD3 components themselves
lack intrinsic tyrosine kinase activity but physically associate with
two Src family protein tyrosine kinases, p59
(Fyn) and p56
(Lck)(1, 2) . Fyn associates with the CD3
/
and TCR
/
chains of the TCR-CD3
complex(3) , while Lck associates with the CD4 and CD8
coreceptors(4, 5) . Accumulating evidence suggests
that these two kinases play critical roles in T cell development and
activation(1, 2) . A third cytoplasmic tyrosine
kinase, ZAP-70, associates with the
and
chains of the
TCR-CD3 complex following their tyrosine phosphorylation and is
required for propagating signals downstream of the Src
kinases(6, 7, 8) . All subsequent events,
including phosphatidylinositol metabolism, mobilization of
intracellular calcium, Ras activation, and eventual transcriptional
modulation and mitogenesis are dependent upon initial tyrosine kinase
activity(1, 2) . Identification and functional
characterization of tyrosine kinase substrates in activated T cells are
therefore critical to our understanding of TCR signaling.
Recently,
we described a Fyn/Lck SH3 domain-binding protein, p120, that served as
one of the earliest tyrosine kinase substrates upon TCR
stimulation(9) . Subsequently, we and others have identified
this protein as p120 (Cbl), the product of the
c-cbl protooncogene(10, 11) . Cbl was
initially identified as the cellular homolog of the Cas NS-1 murine
leukemia retroviral oncogene v-cbl, which induces pre-B and
myeloid leukemias in infected mice and acutely transforms NIH 3T3
fibroblasts(12, 13) . Another mutant form of Cbl with
a 17-amino acid internal deletion has been isolated from a murine pre-B
lymphoma 70/Z3 and is also transforming (13, 14) .
However, the mechanisms of oncogenic transformation, as well as the
physiological role(s) of Cbl in signaling pathways are unknown.
Therefore, identification of Cbl as an intermediate in a tyrosine
kinase-dependent signaling pathway provided an impetus to elucidate its
biochemical function. Moreover, Cbl tyrosine phosphorylation has also
been demonstrated upon stimulation through the Fc
receptor(15) , the B cell antigen
receptor(16, 17) , and the granulocyte-macrophage
colony-stimulating factor and erythropoietin receptors(18) ,
suggesting that Cbl plays a role in multiple antigen receptor- and
mitogen receptor-associated tyrosine kinase activation pathways in
hematopoietic cells.
The primary structure of Cbl reveals a lack of any obvious catalytic domains. However, Cbl possesses multiple potential tyrosine phosphorylation sites and proline-rich motifs, which could mediate concurrent association with SH2 and SH3 domain-containing polypeptides, respectively(19, 20, 21) . Cbl also has a carboxyl-terminal leucine zipper, a motif known to promote homo- and heterodimerization of other proteins (22) . Thus, Cbl is well-suited for a potential role in assembling intracellular signaling complexes. In support of this model, we have previously demonstrated the in vivo association of Cbl with two adaptor proteins, Grb2 and the p85 subunit of PI 3-kinase; Grb2 association was exclusively mediated by SH3 domains, while p85 associated with Cbl primarily through its SH2 domains(11) . Another adaptor protein, Nck, has also been shown to bind to Cbl via SH3 domains(23) .
Here we show that among a panel of isolated
SH2 domains, that of Crk displays the highest in vitro binding
to Cbl. This binding was specifically abrogated by a Cbl-derived
phosphotyrosyl peptide incorporating a consensus Crk SH2-binding motif. In vivo, all three cellular forms of Crk (CrkI, CrkII, and
CrkL) bound to Cbl following TCR stimulation. We also show that the
recently described p130-related Crk-associated
p116 of T cells (24) is Cbl. Finally, we show that C3G, the
guanine nucleotide-releasing factor for a small G-protein
Rap1(25, 26, 27) , was constitutively
associated with Crk proteins and became associated with Cbl upon T cell
activation. These results suggest a potential role of Cbl in connecting
Src-family tyrosine kinase activation pathways with guanine nucleotide
exchange on small G-proteins.
For immunodepletion studies, cell lysates were incubated with two serial aliquots of antibodies together with protein A-Sepharose beads, and the resulting precleared supernatant was used for further immunoprecipitations.
For GST fusion protein binding reactions, 5-10 µg of fusion protein noncovalently immobilized on glutathione-Sepharose beads was incubated for 1 h with cell lysate and washed 6 times with lysis buffer, and bound polypeptides were eluted in sample buffer. For peptide competition, both lysate and fusion protein were preincubated with peptides for 30 min prior to the binding assay, as described(11) .
For two-dimensional gel analysis, proteins were resolved by isoelectric focusing (IEF) in pH 3.5-10.0 ampholines (Pharmacia) in the first dimension, followed by SDS-PAGE and Western blotting, as described previously(11) .
GST fusion proteins of Fyn (Fig. 1A, upper panel, lanes 5 and 6) and Lck SH2 domains (lanes 7 and 8), but not the GST alone (lanes 3 and 4) bound to Cbl, primarily in lysates of anti-CD3-stimulated Jurkat cells, as previously reported (11) . Cbl binding to Abl SH2 (lanes 9 and 10) was comparable with that of Fyn and Lck SH2 but was barely detectable in GTPase-activating protein SH2-N binding reactions (lanes 11 and 12). Notably, the strongest binding to Cbl was seen with Crk SH2 (lanes 13 and 14); substantial binding was observed in lysates of unstimulated cells and increased further upon T cell activation. A 20-fold shorter exposure of this blot emphasizes the markedly higher binding of Cbl to Crk SH2 (Fig. 1A, middle panel). Equal Cbl signals were present in lysates of unstimulated and stimulated cells (upper panel, lanes 1 and 2), indicating that the SH2 domain binding to Cbl was activation-dependent. Anti-Tyr(P) reprobing of the blot showed activation-induced tyrosine phosphorylation of Cbl, which correlated with its capacity to bind to various SH2 domains (Fig. 1A, lower panel).
Figure 1:
Preferential binding of Cbl to GST
fusion protein of the Crk SH2 domain and selective inhibition of
binding by Cbl-derived phosphotyrosyl peptide incorporating a potential
Crk SH2-specific motif. A, binding of Cbl to Crk SH2 compared
to other SH2 domains. Binding reactions were carried out by incubating
lysate from 2.5 10
unstimulated(-) or
anti-CD3 (SPV-T3b)-stimulated (+) Jurkat cells (in a 2-ml volume)
with 10 µg of the indicated GST fusion proteins noncovalently
immobilized on glutathione-Sepharose beads (5 µl of packed beads)
for 1 h. Whole cell lysate (10
cells) or binding reactions
were subjected to SDS-PAGE, transferred to polyvinylidene difluoride
membranes, and blotted with anti-Cbl (upper and middle
panels) or anti-Tyr(P) antibody (lower panel), followed
by horseradish peroxidase-conjugated second step antibodies and
detection by ECL. The middle panel represents a 20-fold lower
exposure of the membrane shown in the top panel, emphasizing
the markedly higher binding of Cbl to Crk SH2 as compared with other
SH2 domains. The lower panel represents anti-Tyr(P) reprobing
of the same membrane shown in the upper panels. B,
specific competition of Cbl binding to GST-Crk SH2 domain by Cbl
Tyr(P)
peptide. Competing peptides were separately added
to bead-bound fusion proteins and cell lysate at the indicated
concentrations (shown in µM; 0, no peptide). After 30 min,
beads and lysate were mixed, and binding reactions and immunoblotting
with anti-Cbl antibody were carried out as in Fig. 1A.
Peptides were: EDDGpYDVPKPPV (pYDVP; Cbl amino acids 770-881);
EDDGYDVPKPPV (YDVP); HSDpYMNMTPR (pYMNM; p85 SH2-specific); HSDYMNMTPR
(YMNM). C, lack of Cbl binding to Crk SH3 domain. Binding
reactions with the indicated GST fusion proteins and anti-Cbl
immunoblotting were carried out as in Fig. 1A.
As noted above, a
substantial amount of Cbl bound to Crk SH2 even in lysates of
unstimulated cells, where overall tyrosine phosphorylation was
relatively low. In order to establish that Cbl-Crk SH2 interaction was
mediated by phosphotyrosyl peptide binding, we carried out competition
experiments with phosphotyrosyl peptides. Cbl amino acids 774-777
(YDVP) (13) correspond to a consensus high affinity
phosphotyrosyl motif for Crk SH2 domain, which was shown to select the
pYDXP motif from a degenerate phosphopeptide
library(36) . As shown in Fig. 1B, a
concentration-dependent inhibition of Cbl binding to GST-Crk SH2 was
observed when Cbl Tyr phosphopeptide was included in the
binding reactions (lanes 3-7). The requirement for
tyrosine phosphorylation of the peptide was demonstrated by the lack of
competition with the unphosphorylated peptide (lane 8). The
specificity of peptide competition was demonstrated by the inability of
the Cbl Tyr
peptide to inhibit Cbl binding to PI 3-kinase
p85 SH2 domains (lanes 14-15). Conversely,
phosphorylated CD28 Tyr
peptide was without effect on Crk
SH2-Cbl binding (lane 9) but completely abrogated p85 SH2-Cbl
binding (lane 12), as expected(11) . These results
demonstrate that the Crk SH2 domain binding to Cbl is mediated via its
interaction with a phosphotyrosyl peptide motif on Cbl.
In contrast to prominent binding of Cbl to Crk SH2 domain, no Cbl binding to Crk SH3 domain was observed under conditions that revealed a substantial binding of Cbl to Fyn SH3 fusion protein (Fig. 1C). These results are consistent with our earlier studies, which failed to detect a 120-kDa phosphotyrosyl protein in Crk SH3-binding reactions from T cells(9) .
Figure 2:
Activation-dependent association of Cbl
with Crk proteins in Jurkat T cells. Immunoprecipitations (I.P.) from lysates of 5 10
unstimulated
(-) or anti-CD3 (SPV-T3b)-stimulated (+) Jurkat cells were
carried out with the antibodies shown on top and subjected to
immunoblotting with anti-Tyr(P) (upper panel) followed by
anti-Cbl antibody (lower panel). Tyrosine-phosphorylated Cbl
and p36/38 are indicated by brackets on the left. Ig, immunoglobulin heavy chain.
Some basal tyrosine phosphorylation was observed on directly immunoprecipitated Cbl, with a substantial increase in Tyr(P) signal upon T cell activation (Fig. 2, upper panel, lanes 3 and 4). While little phosphotyrosine signal was detected in immunoprecipitates of unstimulated Jurkat cells carried out with anti-Crk (which immunoprecipitates both CrkI and CrkII) (lane 5) or anti-CrkII antibody (lane 7), a prominent 120-kDa phosphotyrosyl polypeptide was observed with each of these antibodies following anti-CD3-stimulation (lanes 6 and 8). A low but easily detectable p120 signal was observed in anti-Tyr(P) immunoblotting of anti-CrkL immunoprecipitate from unstimulated cells with a marked increase in signals upon activation of cells (lanes 9 and 10). Among the three antibodies used, that against CrkL showed the highest p120 signals. In each case, Crk-associated p120 comigrated with Cbl. Minor unidentified tyrosine-phosphorylated species of 100, 75, and 36-40 kDa were also observed in anti-CrkL immunoprecipitates of activated T cells, particularly upon longer exposures (lane 10 and data not shown).
To assess if the Crk-associated tyrosine-phosphorylated p120 was Cbl, we reprobed the blot with an anti-Cbl antibody (Fig. 2, lower panel). This analysis demonstrated that each of the three antibodies against Crk proteins co-immunoprecipitated Cbl. Cbl coimmunoprecipitation with anti-Crk and anti-CrkII antibodies was observed in lysates of anti-CD3-stimulated but not unstimulated Jurkat cells (compare lanes 5 and 7 with lanes 6 and 8). Small amounts of Cbl were observed in anti-CrkL immunoprecipitates prior to activation and increased substantially upon anti-CD3 stimulation of cells, correlating with the levels of Cbl tyrosine phosphorylation seen by anti-Tyr(P) immunoblotting (lanes 9 and 10). Overlay of anti-Tyr(P) and anti-Cbl blots showed that these bands coincided exactly (not shown).
While the above results clearly demonstrated Cbl association with CrkII and CrkL, we could not directly assess this for CrkI. To address this question, cell lysates were first precleared with either a control antibody (NRS) or with anti-CrkII antibody followed by immunoprecipitation with anti-Crk (CrkI- and CrkII-reactive) or anti-CrkII antibody. Anti-Crk immunoblotting (Fig. 3A, lower panel) revealed that only a small amount of CrkII could be immunoprecipitated by anti-Crk (compare lanes 1 and 2 with lanes 5 and 6) or anti-CrkII antibody (compare lanes 3 and 4 with lanes 7 and 8) in anti-CrkII-immunodepleted lysates compared with control (NRS-precleared) lysates. Furthermore, anti-Crk antibody immunoprecipitated equal amounts of CrkI protein in control and anti-CrkII-precleared lysates (compare lanes 1 and 2 with lanes 5 and 6). Anti-Tyr(P) immunoblotting (Fig. 3A, upper panel) demonstrated that anti-CrkII preclearing completely removed the p120 band in anti-CrkII immunoprecipitates (compare lanes 4 and 8), whereas p120 was still observed in anti-Crk antibody immunoprecipitate (compare lanes 2 and 6). Anti-Cbl immunoblotting confirmed that p120 was Cbl (not shown). These results show that CrkI protein also associates with Cbl, thereby strongly indicating that all three forms of Crk associate with Cbl in activated T cells.
Figure 3:
Immunodepletion analysis of Cbl-Crk
association in Jurkat T cells. A, association of Cbl with CrkI
demonstrated by immunodepletion. Lysates of unstimulated or anti-CD3
(SPV-T3b)-stimulated Jurkat T cells were precleared twice with NRS or
anti-CrkII antibody. Precleared lysates from 5 10
cells were then subjected to immunoprecipitation with anti-Crk
(reactive with CrkI and CrkII) or anti-CrkII antibody, followed by
serial immunoblotting with anti-Tyr(P) (upper panel) and
anti-Crk (lower panel) antibodies. Immunoprecipitated species
and Ig heavy chain are shown on the left. B,
immunodepletion of Jurkat lysates with anti-Cbl antibody eliminates the
CrkL-associated 120-kDa Tyr(P) polypeptide. Lysates of
unstimulated(-) or anti-CD3 (SPV-T3b)-stimulated Jurkat cells
were precleared twice with NRS or anti-Cbl antibody. Precleared lysates
from 5
10
cells were immunoprecipitated with
anti-Cbl or anti-CrkL antibody and subjected to immunoblotting with
anti-Tyr(P) (upper panel) and anti-Cbl (lower panel)
antibodies. Immunoprecipitated species and Ig heavy chain are shown on
the left. Lower Cbl signals in unstimulated lanes (lanes 1 and 5) reflect unequal amounts of
lysates.
To assess if Cbl was the only 120-kDa Tyr(P) polypeptide associated with Crk proteins, we immunodepleted Cbl from cell lysates and then carried out anti-CrkL immunoprecipitations. Anti-Cbl immunoprecipitation followed by anti-Tyr(P) and anti-Cbl immunoblotting (Fig. 3B) demonstrated a marked decrease (although not a complete loss) of Cbl protein in anti-Cbl-immunodepleted lysates (compare lanes 1 and 2 with lanes 5 and 6). Significantly, anti-CrkL immunoprecipitation from Cbl-depleted lysates showed undetectable levels of Cbl and, concurrently, an essentially complete loss of the p120 band in anti-Tyr(P) blot (compare lanes 4 and 8). These results indicate that Cbl is the major, and perhaps the only, Crk-associated 120-kDa Tyr(P) polypeptide in T cells.
Anti-Tyr(P) immunoblotting of anti-Cbl immunoprecipitates showed a prominent activation-dependent tyrosine phosphorylation of Cbl (Fig. 4, upper panel, lanes 1 and 2); anti-Cbl immunoblotting revealed equal amounts of Cbl in both lanes (lower panel). Significantly, a 120 Tyr(P) polypeptide comigrating with Cbl was observed in anti-Crk immunoprecipitate from activated cells (lane 6). Easily detectable 120-kDa Tyr(P) polypeptide signal was observed in anti-CrkL immunoprecipitate from unstimulated cells but increased markedly upon activation (upper panel, lanes 7 and 8). Anti-Cbl blotting demonstrated the p120 Tyr(P) polypeptide to be Cbl. Thus, Cbl shows an activation-dependent association with Crk proteins in normal T cells. Interestingly, anti-Crk and anti-CrkL antibodies also showed prominent activation-dependent co-immunoprecipitation of unidentified 75-80-kDa (upper panel) and 36-40-kDa (not shown) Tyr(P) polypeptides, similar in size to minor bands seen in anti-CrkL immunoprecipitations from activated Jurkat T cells.
Figure 4:
Association of Cbl with Crk proteins
following activation of peripheral blood-derived normal human T cells.
Normal human T cells were deprived of IL-2 for 1 day and either left
unstimulated(-) or were stimulated with an IgM anti-CD3 antibody
2Ad2. Lysates from 5 10
cells were
immunoprecipitated with the indicated antibodies and subjected to
immunoblotting with anti-Tyr(P) (upper panel) followed by
anti-Cbl (lower panel) antibody. Cbl and Ig are indicated.
Anti-HLA (W6/32 antibody against HLA class I) is used as a negative
control.
The 4F4 antibody immunoblotted with a 120-kDa protein in anti-CrkL (Fig. 5A, upper panel, lanes 3 and 4), anti-Tyr(P) (lanes 7 and 8), and 4F4 (lanes 9 and 10) immunoprecipitates of anti-CD3-stimulated Jurkat cell lysates with little or no reactivity in immunoprecipitates from unstimulated cells, as expected. Surprisingly, the 4F4 antibody prominently immunoblotted with the directly immunoprecipitated Cbl protein from activated Jurkat cells with only low reactivity to that from unstimulated cells (lanes 5 and 6). Additionally, the 4F4 antibody specifically reacted with a number of other proteins in anti-Tyr(P) immunoprecipitates (lane 8), some of which were also detected in 4F4 immunoprecipitates (lane 10), suggesting that the 4F4 epitope is induced on a number of proteins following T cell activation. This result is consistent with an earlier observation that 4F4 antibody reacts with several phosphotyrosyl proteins and that this reactivity is inhibited by phosphotyrosine(41) .
Figure 5:
Anti-p130 antibody 4F4
reacts with Cbl in activated Jurkat cells. A,
immunoprecipitates of 5
10
unstimulated(-) or
anti-CD3 (SPV-T3b)-stimulated (+) Jurkat T cells were subjected to
immunoblotting with 4F4 (upper panel) followed by anti-Cbl
antibody (lower panel). Cbl (and the comigrating
CrkL-associated p120) and Ig are indicated by brackets on the left. B, immunodepletion of CrkL-associated 120-kDa
Tyr(P) polypeptide by anti-Cbl antibody. The filter used in Fig. 3B was reprobed here with the 4F4 antibody. C, 4F4 antibody recognizes the major species of Cbl protein
resolved by two-dimensional gel electrophoresis. Anti-Cbl
immunoprecipitation from 10
anti-CD3 (SPV-T3b)-stimulated
Jurkat cells was eluted in IEF sample buffer, resolved by IEF using pH
3.5-10.0 ampholines in the first dimension, and by SDS-PAGE in
the second dimension. Proteins were transferred to polyvinylidene
difluoride and immunoblotted with 4F4 (upper panel) followed
by anti-Cbl antibody (lower panel). An aliquot of
immunoprecipitated Cbl was resolved by SDS-PAGE on the same gel to
allow identification of the IEF-resolved polypeptides as Cbl (not
shown). Anti-Cbl antibody-reactive polypeptides (lower panel)
are indicated by letters A-D. Spot D corresponds to protein that did not fully enter the IEF
gel.
Reprobing of the filter with anti-Cbl antibody (Fig. 5A, lower panel) showed equivalent amounts of Cbl in anti-Cbl immunoprecipitations from unstimulated and activated lysates (compare lanes 5 and 6), suggesting that the 4F4-reactive epitope was induced on Cbl upon T cell activation. The reactivity of the 4F4 antibody with the 120-kDa species in anti-CrkL (lanes 3 and 4) and anti-Tyr(P) immunoprecipitates (lanes 7 and 8) correlated with their coimmunoprecipitation of Cbl. Low levels of Cbl were detectable by anti-Cbl immunoblotting in 4F4 immunoprecipitates from activated Jurkat cells (compare lanes 9 and 10, lower panel); although the amount of coimmunoprecipitated Cbl was relatively small, it was specific, as no Cbl was detected in control lanes (lanes 1 and 2).
Separately, we reprobed the filter shown in Fig. 3B with the 4F4 antibody (Fig. 5B). This analysis showed that immunodepletion with anti-Cbl eliminated the 4F4-reactive p120 band in anti-CrkL immunoprecipitates.
To further confirm that 4F4 antibody
reacts with Cbl, we subjected anti-Cbl immunoprecipitates from
activated Jurkat cell lysates to two-dimensional gel analysis (IEF,
followed by SDS-PAGE), and serial immunoblotting with 4F4 and anti-Cbl
antibodies (Fig. 5C). The 4F4 antibody recognized two
distinct spots of approximately 120 kDa and a third spot of similar
size near the origin of the IEF gel. Anti-Cbl reprobing of the filter
showed that the 4F4-reactive spots aligned exactly with spots
A, C, and D identified in the anti-Cbl blot.
Collectively, the results presented above strongly suggest that
p130-related Crk-associated p116 (24) is Cbl.
First, we examined whether SOS and/or C3G polypeptides were associated with CrkL protein. Lysates of unstimulated and anti-CD3-stimulated Jurkat cells were immunoprecipitated with various antibodies and immunoblotted with anti-SOS or anti-C3G antibody (Fig. 6A). A small but readily detectable pool of SOS was observed in association with Grb2 in unstimulated lysates (lower panel, lane 5); a substantial increase in this association was observed upon anti-CD3 activation (lane 6), confirming previous results(11, 44) . In contrast, SOS was not detectable in anti-CrkL immunoprecipitates (lanes 7 and 8), except after prolonged exposure of the blots (not shown). A relatively large fraction of C3G was constitutively associated with CrkL, and this association remained unchanged upon activation (Fig. 6A, upper panel, lanes 7 and 8). Notably, C3G was not detected in ant-Grb2 immunoprecipitates (lanes 5 and 6) except after overexposure of the blot (data not shown). These analyses indicated that C3G is preferentially associated with CrkL as compared with Grb2 in T cells and suggested that the C3G-CrkL complex may associate with Cbl upon T cell activation.
Figure 6:
Association of Cbl with the guanine
nucleotide exchange protein C3G in activated T cells. A, whole
cell lysates (10 cells) or immunoprecipitates from 5
10
unstimulated(-) or anti-CD3
(SPV-T3b)-stimulated (+) Jurkat cells with the indicated
antibodies (shown on top) were subjected to serial
immunoblotting with anti-C3G (upper panel) and anti-SOS (lower panel) antibodies. B, whole cell lysates
(10
cells) or immunoprecipitates from 5
10
unstimulated(-) or anti-CD3 (SPV-T3b)-stimulated (+)
JMC-HA-Cbl cells with the indicated antibodies (shown on top)
were subjected to immunoblotting with anti-C3G antibody. JMC-HA-Cbl is
a transfectant of Jurkat that expresses HA-tagged Cbl at approximately
5-fold higher levels compared with endogenous Cbl (see
``Experimental Procedures''). Anti-HLA class I antibody is
used as a negative control. A background band is observed in anti-HLA
and anti-Cbl lanes just above the specific C3G band, which is indicated
on the left.
In initial experiments using Jurkat cells, a small amount of C3G could be observed to associate with Cbl but was detectable only after long exposures of gels (data not shown). To facilitate the analysis of this complex, we derived a transfectant of the Jurkat-JMC cell line, JMC-HA-Cbl (see ``Experimental Procedures''), which expresses about 5-fold higher levels of Cbl protein as compared with parental cells (data not shown). Similar to parental Jurkat cells (Fig. 2), anti-CD3 stimulation of JMC-HA-Cbl induced tyrosine phosphorylation of a number of cellular polypeptides including the transfected Cbl (data not shown), and CrkL immunoprecipitates from these cells showed a substantial level of constitutively associated C3G (Fig. 6B, lanes 7 and 8). Significantly, a small amount of C3G was specifically observed in anti-Cbl immunoprecipitations from activated cells (lane 6). These results indicate that a Cbl-CrkL-C3G ternary complex is induced by T cell activation.
In contrast to the receptor tyrosine kinases, whose autophosphorylation creates multiple distinct phosphopeptide motifs as docking sites for SH2 domain-containing signaling proteins(19, 21, 29, 45) , the tyrosine phosphorylation motifs associated with the lymphocyte antigen receptors lack significant diversity(1, 2) , and are therefore unlikely to directly recruit multiple signaling proteins. Identification and characterization of polypeptides that fulfill such a role is likely to enhance our understanding of lymphocyte antigen receptor signal transduction.
Recently, we identified one such substrate, p120, which interacts with Fyn and Lck SH3 domains via proline-rich peptide motifs and with Fyn and Lck SH2 domains via phosphotyrosyl interactions(9) . We and others have identified p120 as Cbl, the product of the c-cbl protooncogene, and have shown it to associate with PI 3-kinase by binding to SH2 domains of the p85 subunit and with Grb2 by binding to its SH3 domains(10, 11, 46) . The SH2/SH3 domain-containing adaptor protein Nck has also been shown to associate with Cbl via its SH3 domains(23) , although we have been unable to detect this association in T lymphocytes (data not shown). Together, these observations are consistent with a potential role of Cbl in assembling multiple T cell signaling proteins into complexes, as suggested by the presence of an extended proline-rich region and multiple potential tyrosine phosphorylation sites throughout Cbl(13) .
Here we report the association of
tyrosine-phosphorylated Cbl with the Crk family of SH2/SH3 domain
adaptor proteins in activated T lymphocytes. Such an interaction was
suggested by a remarkably strong in vitro binding of v-Crk SH2
domain to Cbl and the failure of Crk SH3 domain to bind to this
polypeptide. Cbl amino acid residues 774-777 (YDVP) represented a
potential Crk SH2 domain-binding motif since Crk SH2 was shown to
select a pYDXP motif from degenerate phosphopeptide
libraries(36) . Indeed, a Tyr(P) peptide incorporating the Cbl
YDVP motif specifically abrogated Crk SH2-Cbl binding, while it had no
effect on PI 3-kinase p85-Cbl binding (Fig. 1B). In
vivo, Cbl became associated with all three Crk isoforms (CrkI,
CrkII, and CrkL) in an activation-dependent manner; however,
association with CrkL was most prominent ( Fig. 2and Fig. 3). Cbl tyrosine phosphorylation and its prominent
association with Crk proteins, in particular CrkL, were also observed
upon TCR stimulation of normal peripheral blood-derived T lymphocytes (Fig. 4), indicating that this association is not a peculiarity
of transformed cells such as Jurkat. Whether preferential association
of Cbl with CrkL reflects a higher abundance of CrkL protein in T
cells, a higher affinity of CrkL SH2 domain for Cbl or the possible
influence of surrounding non-SH2 sequences of CrkL remains unknown at
present. Notably, the affinity of Crk SH2 for phosphotyrosyl
p130 was shown to be increased by the N-terminal 32
residues of the v-Crk protein(47) .
Our analyses show that Cbl is the major, and most likely the only 120-kDa Tyr(P) polypeptide associated with Crk proteins. This was supported by comigration of Crk-associated p120 with Cbl, its immunoreactivity with anti-Cbl antibodies, and an essentially complete immunodepletion of this band by preclearing with anti-Cbl antibody. Furthermore, Cbl was the major Crk-associated Tyr(P) polypeptide in Jurkat T cells, although minor 100-, 75-80-, and 36-40-kDa Tyr(P) polypeptides were also observed upon longer exposures, particularly with anti-CrkL antibody ( Fig. 2and Fig. 3and data not shown). Interestingly, analysis of normal T cells revealed that 75-80-kDa (Fig. 4) and 36-40-kDa Tyr(P) polypeptides (data not shown) were prominently associated with Crk proteins. Although the identity of these polypeptides is unknown, these results suggest that Crk adaptor proteins may be involved in multiple signaling pathways in primary T cells.
In view of our findings that Cbl interacts with Crk
proteins in activated T cells and a complete immunodepletion of the
Crk-associated 120-kDa Tyr(P) polypeptide by anti-Cbl antibody, we were
intrigued by a recent report (24) of a Crk-associated 116-kDa
Tyr(P) polypeptide in activated T cells. This protein was shown to
cross-react with an antibody, 4F4, raised against the Src substrate
p130 (39) and recently identified as Crk-associated
p130(40) . The primary structure of Cbl does not
show any significant homology with p130
, suggesting that
the 4F4-reactive, putative p130
-related T cell
polypeptide may be tyrosine-phosphorylated Cbl. Consistent with this
possibility, another study had shown that 4F4-reactivity of a
Fyn-associated T cell p116 polypeptide was sensitive to phosphotyrosine
competition(41) . Immunoblotting and removal of the
Crk-associated 4F4-reactive p120 band by anti-Cbl antibody, together
with the reactivity of 4F4 antibody with Cbl spots resolved on
two-dimensional gels, demonstrated that p130
-related p116
and Cbl are identical. Notably, however, only a small quantity of Cbl
was observed in 4F4 immunoprecipitates (Fig. 5A). This
likely represents an inefficient immunoprecipitation by this IgM
subclass antibody and/or its selective immunoprecipitation of only
tyrosine-phosphorylated Cbl. Indeed, 4F4 immunoblot showed stronger
p120 signals in anti-Cbl compared with 4F4 immunoprecipitations (Fig. 5A). Consistent with the previous suggestion that
4F4 antibody recognizes selected phosphotyrosyl peptide
motifs(41) , this antibody specifically blotted with a number
of additional T cell proteins immunoprecipitated by anti-Tyr(P)
antibody (Fig. 5A).
In addition to its rapid
tyrosine phosphorylation following TCR ligation, Cbl is a major target
of tyrosine kinases coupled to the B cell antigen
receptor(16, 17) , granulocyte-macrophage
colony-stimulating factor, and EPO receptors on hematopoietic cells (18) and the Fc receptor on myelomonocytic
cells(15) . It will be of interest to know whether Crk is a
major Cbl-associating protein in these other signaling pathways as
well. In this regard, both CrkL and Cbl are major
tyrosine-phosphorylated substrates of BCR/Abl in Philadelphia-positive
leukemic cells, and CrkL-Cbl complexes have been demonstrated in these
cells(14, 48, 49, 50) .
The prominent association of Cbl with Crk proteins suggested the possibility that Crk-mediated interactions may be involved in Cbl function. Previous studies have demonstrated that Crk interacts via its N-terminal SH3 domain with the guanine nucleotide exchange factors SOS and C3G(24, 25, 26, 42, 43) , suggesting that Crk proteins activate Ras and/or other small G-protein signaling pathways. This possibility was consistent with the requirement of the SH3 domain for the transforming ability of V-Crk(34) . Indeed, overexpression of Crk was shown to induce Ras-dependent differentiation of PC12 neuronal cells; functional SH2 and SH3 domains were required for this effect (42) . Recent experiments have shown that the preferred target for C3G is Rap1 rather than Ras, suggesting that Crk proteins may link activated receptors to multiple small G-proteins. We observed a strong constitutive association of CrkL with the guanine nucleotide exchange factor C3G in Jurkat T cells (Fig. 6). Importantly, a fraction of C3G was co-immunoprecipitated with Cbl in activated cells. Thus, Cbl, through its interaction with Crk proteins, becomes coupled to a guanine nucleotide exchange factor. These findings strongly suggest the possibility that tyrosine phosphorylation of Cbl may provide one mechanism to link upstream tyrosine kinases to small G-protein regulation in T cells.
Grb2 showed an easily detectable association with SOS and this association increased further upon T cell activation (Fig. 6A), as reported earlier(11, 44) . In contrast, very little C3G was associated with Grb2 in T cells. Conversely, little SOS was associated with CrkL. Thus, in T cells, the Crk proteins and Grb2 appear to preferentially couple to distinct guanine nucleotide releasing factors that regulate Rap1 and Ras, respectively. Given that Cbl and SOS both bind to Grb2 SH3 domains and provide its alternate ligands(11, 46) , Cbl-Crk-C3G complex formation may provide a mechanism for coupling tyrosine phosphorylation with small G-protein regulation in T cells, distinct from the previously examined Shc-Grb2-SOS and p36/38-Grb2-SOS complexes implicated in Ras regulation(44, 51, 52, 53, 54) . Consistent with this scheme, SOS was not detected in association with Cbl before or after T cell activation(11, 46) .
In view of the results discussed above, the activation-dependent recruitment of the PI 3-kinase to tyrosine-phosphorylated Cbl (11, 46) is quite intriguing. PI 3-kinase is thought to play a role as either a regulator or effector of Ras function(55, 56, 57) . Distinct Tyr(P) motifs favorable for binding to SH2 domains of PI 3-kinase p85 (e.g. Cbl residues 371-374 (YCEM) and 731-734 (YEAM)) and Crk (pYDVP; Cbl residues 774-777, Fig. 1B) (19, 36) are found in Cbl(13) . Thus, Cbl might simultaneously recruit multiple modulators of small G-proteins in the vicinity of activated T cell receptors, perhaps as a signal amplification mechanism.
In conclusion, we demonstrate a T-cell activation-dependent SH2 domain-mediated association of Crk proteins, in particular CrkL, with tyrosine-phosphorylated Cbl, and show that activation induces a complex of Cbl with the guanine nucleotide exchange protein C3G. Together with the known oncogenicity of altered forms of Cbl as well as Crk, these findings suggest that Crk-Cbl complexes may play a role to mediate a growth-related signal downstream of the lymphocyte antigen receptors.