(Received for publication, March 16, 1995; and in revised form, May 11, 1995)
From the
Previously, we have identified p120 as a Fyn/Lck SH3 and SH2
domain-binding protein that is tyrosine phosphorylated rapidly after T
cell receptor triggering. Here, we used direct protein purification,
amino acid sequence analysis, reactivity with antibodies, and
two-dimensional gel analyses to identify p120 as the human c-cbl protooncogene product. We demonstrate in vivo complexes
of p120 with Fyn tyrosine kinase, the adaptor
protein Grb2, and the p85 subunit of phosphatidylinositol (PI)
3-kinase. The association of p120
with Fyn and
the p85 subunit of PI 3-kinase (together with PI 3-kinase activity) was
markedly increased by T cell activation, consistent with in vitro binding of p120
to their SH2 as well as SH3
domains. In contrast, a large fraction of p120
was associated with Grb2 prior to activation, and this
association did not change upon T cell activation. In vitro,
p120
interacted with Grb2 exclusively through
its SH3 domains. These results demonstrate a novel
Grb2-p120
signaling complex in T cells, distinct
from the previously analyzed Grb2-Sos complex. The association of
p120
with ubiquitous signaling proteins strongly
suggests a general signal transducing function for this enigmatic
protooncogene with established leukemogenic potential but unknown
physiological function.
The engagement of the T cell receptor (TCR) ()by
major histocompatibility complex-bound antigenic peptides leads to T
cell activation, a prerequisite for effective immune responses. One of
the earliest and obligatory biochemical steps in T cell activation is
the tyrosyl phosphorylation of cellular proteins including the receptor
components themselves(1, 2) . Unlike growth factor
receptors with intrinsic tyrosine kinase domains(3) , the TCR
components signal through noncovalently associated cytoplasmic tyrosine
kinases(1, 2) . In particular two Src family kinases,
p59
(Fyn) and p56
(Lck),
have been demonstrated to play critical and apparently nonoverlapping
roles in T cell activation. Fyn interacts physically with the
cytoplasmic signaling domains of the TCR
/
and CD3
and
chains(4) , whereas Lck interacts with the cytoplasmic
tails of the CD4 and CD8 coreceptors(5, 6) . In
addition, Lck plays a role in T cell activation apparently independent
of its CD4/8 association(1, 2) . A distinct
cytoplasmic tyrosine kinase ZAP-70 has also been demonstrated to be
critical for T cell activation and apparently functions downstream of
the Src family kinases(1) .
Similar to the TCR, Src kinase-mediated tyrosine phosphorylation is an early and a critical event downstream of other surface receptors that lack intrinsic tyrosine kinase domains, such as the B cell antigen receptor(1) , Fc receptors(7) , and certain cytokine receptors(8) . Thus, elucidation of tyrosine phosphorylation-dependent signaling events downstream of the TCR is likely to provide insights of general significance.
The mechanisms by which early phosphorylation substrates are recruited to TCR-coupled tyrosine kinases are poorly understood. Studies with receptor tyrosine kinases such as epidermal growth factor receptor have elucidated the critical roles of the Src homology domains (SH2 and SH3) in assembling signaling complexes(3, 9, 10) . The SH2 domains bind to phosphotyrosyl (pY) peptide motifs (11, 12) and mediate activation-induced phosphorylation-dependent interactions between signaling proteins(3, 9, 10) . In contrast, the SH3 domains bind to small proline-rich peptide motifs(13, 14, 15, 16, 17, 18) , thus providing a basis for protein-protein interactions prior to receptor activation.
In an attempt to define the role of SH3 domain-mediated binding to recruit cellular proteins to T cell tyrosine kinases, we recently identified a Fyn/Lck SH3 domain-binding protein p120(19) . Notably, p120 was one of the earliest tyrosine phosphorylation substrates upon triggering through the TCR. Preliminary evidence indicated that p120 was present in vivo as preformed complexes with Fyn and Lck and served as a substrate for these tyrosine kinases in vitro(19) . Subsequently, an unidentified protein of similar size (116 kDa), which was also tyrosine phosphorylated rapidly by stimulation of Jurkat T cells through their TCR, was shown to interact in vitro with Grb2-SH3 fusion proteins and to bind to Grb2-SH2-specific phosphopeptide matrices, indirectly suggesting that this protein was present as an in vivo complex with Grb2 (20) . More recently, it was reported that a 120-kDa TCR-induced tyrosine-phosphorylated protein of Jurkat T cells was reactive with antibodies to the c-cbl protooncogene product, and in vitro binding experiments demonstrated that Cbl protein in cell lysates was able to bind to Grb2 fusion proteins(21) .
In the present study we have characterized further the in vivo interactions of the Fyn/Lck SH3 domain-binding protein, p120, with other T cell signaling proteins that possess SH2 and SH3 domains. Unlike earlier studies(20, 21) , we use coimmunoprecipitation analyses to show in vivo complexes of p120 with SH2/SH3-bearing T cell signaling proteins, the Src family tyrosine kinase Fyn and adaptor protein Grb2; in addition, we demonstrate that the Grb2-associated fraction of p120 is a target of early TCR-elicited tyrosine phosphorylation. Significantly, we also demonstrate a predominantly SH2 domaindependent interaction of p120 with the PI 3-kinase p85, which results in the recruitment of this enzyme into p120-containing protein complexes in an activation-dependent manner.
We used the in vivo Grb2-p120 association to immunoaffinity
purify the p120 polypeptide. The determined amino acid sequences of
three distinct tryptic peptides revealed that p120 is identical to the
human p120 protooncogene(22) , and this
was established further by immunochemical and two-dimensional gel
analyses. These analyses independently confirm and extend the recent
results showing that the Cbl protein is a target of TCR-mediated
tyrosine phosphorylation in Jurkat cells(21) . Together, these
results strongly suggest that p120
serves as a
multifunctional SH2 and SH3 domain-binding protein in the tyrosine
kinase-mediated signal transduction cascade downstream of the TCR.
Given the preferential expression of c-Cbl in hematopoietic
cells(23) , induction of pre-B and myeloid cell leukemias by
its viral form(24) , and its in vivo complexes with
ubiquitous signaling proteins (this study), it is likely that c-Cbl
also participates in signaling downstream of other hematopoietic cell
receptors.
The second-step reagents for immunoblotting were: I-labeled protein A (Amersham Corp.); horseradish
peroxidase (HRPO)-conjugated protein A and sheep anti-mouse IgG
(Cappel-Organon Technika, Durham, NC); and donkey anti-rabbit Ig-HRPO
(Amersham Corp.).
Fusion proteins were affinity purified on glutathione-Sepharose
beads (Pharmacia) using a Triton X-100-soluble fraction of the
isopropyl-1-thio--
-galactopyranoside-induced Escherichia
coli (DH5
strain), as described(19, 32) .
Proteins were quantitated by Bradford assay (Bio-Rad) against a bovine
serum albumin standard and analyzed on Coomassie gels to confirm
quantitation and to assess purity (usually more than 95%).
To purify p120 from HSB2 cells, we took
advantage of its ability to form stable in vivo complexes with
Grb2 adaptor protein (described below). To facilitate immunoaffinity
purification of p120, we generated a transfectant cell line
(HSB2-Grb2myc) that expressed high levels of a Myc epitope-tagged Grb2
protein. This is seen as a 28-kDa species in anti-Grb2 and anti-Myc
immunoprecipitates of HSB2-Grb2myc cells but not in the HSB2-puro control cells (Fig.1A). Anti-pY immunoblotting
revealed the association of p120 with transfected Grb2myc protein (Fig.1B). Thus, HSB2-Grb2myc transfectant provided a
suitable reagent for immunoaffinity purification of p120.
Figure 1:
Expression of
the Myc epitope-tagged Grb2 in HSB2 cells and its interaction with
p120. Transfectants were derived by retroviral infection with a control
vector (HSB2-puro) or a Grb2myc vector (HSB2-Grb2myc).
Antibodies: anti-TCR
1, control (Cont.), anti-Myc epitope
9E10 (
myc), or rabbit anti-Grb2 (
Grb2). Panel A, Grb2myc expression in HSB2-Grb2myc cells. Triton
X-100 lysates of 2
10
cells, metabolically labeled
with [
S]methionine and cysteine, were
immunoprecipitated with the indicated antibodies (shown on top), and immunoprecipitated species (see arrows)
were resolved by SDS-PAGE and visualized by fluorography. Panel
B, association of Grb2myc with p120. Whole cell lysate (10
cells) or immunoprecipitations from 2
10
cells with indicated antibodies (I.P., shown on top) were subjected to immunoblotting with anti-pY mAb (upper panel) followed by sheep anti-mouse-HRPO and ECL
detection. The lower portion of the same filter was probed
with anti-Grb2 followed by donkey anti-rabbit-HRPO and ECL
detection.
The determined sequences of three distinct peptides are shown in Table1. The eight determined amino acids of peptide 1 showed complete identity with amino acids 117-124 of the human c-cbl protooncogene which are preceded by a potential trypsin cleavage site (Arg-116)(22) . All of the six determined residues of peptide 2 matched amino acids 129-134 of the human c-Cbl protein; a glutamine at c-Cbl position 128 corresponded to the unidentified first residue in peptide 2 and was preceded by a tryptic cleavage site (lysine). Seven of 10 unambiguous and 3 of 4 probable residues of peptide 3 matched those within amino acids 837-850 of the human c-Cbl without any gaps. Again, a tryptic cleavage site (lysine) preceded this region in c-Cbl. The four mismatches included three unambiguous residues; the reason(s) for this discrepancy is not known. Since a single functional c-cbl gene has been identified in the human genome(22, 41, 42) , the determined peptide sequences indicate that p120 is identical to the human c-Cbl protein.
To confirm further the identity between p120 and c-Cbl, we examined
whether p120 (operationally defined as the Fyn/Lck SH3 domain-binding
protein) (19) was recognized by an anti-Cbl antibody. Anti-pY
immunoblotting demonstrated expected binding of p120 to wild type Fyn
SH2 and SH3 domains but not to their nonbinding point mutants or to Abl
SH3 (Fig.2). Anti-Cbl antibody selectively immunoblotted the
fusion protein-bound p120. Furthermore, p120 detected in fusion protein
binding reactions and anti-pY immunoprecipitates comigrated with
directly immunoprecipitated p120 (data not
shown).
Figure 2:
p120 is recognized by anti-Cbl antibodies.
Whole cell lysates (10 cells) or GST fusion protein binding
reactions from anti-CD3 (2Ad2)-stimulated Jurkat T cells (5
10
cells) were resolved by SDS-PAGE and subjected to
immunoblotting with anti-pY antibody 4G10 followed by detection with
I-protein A (top). The filter was reprobed with
anti-Cbl antibody and detected with HRPO-conjugated anti-rabbit
antibody using ECL (bottom).
Finally, we compared p120 and c-Cbl proteins by two-dimensional gel analysis to demonstrate their biochemical identity. Fyn SH3-bound material, anti-Cbl immunoprecipitates or their mixture was resolved by IEF followed by SDS-PAGE, and resolved species were visualized by anti-pY immunoblotting (Fig.3). The cell lysates were derived from anti-CD3-stimulated Jurkat cells. Anti-pY immunoprecipitates resolved into a number of distinct spots or arrays of spots, corresponding to major pY proteins observed in one-dimensional analysis (run on the side). Only one array of these spots was observed in Fyn-SH3-bound material and corresponded to p120 band in the SH3 binding reaction subjected to direct SDS-PAGE on the same gel; an additional spot near the origin of the IEF gel may reflect incomplete entry of the protein. An identical series of spots was observed in anti-Cbl immunoprecipitate, and a mixture of Fyn-SH3-bound material and anti-Cbl immunoprecipitate produced a pattern identical to each of the individual components. Similar two-dimensional gel analysis of Grb2-associated p120 also demonstrated it to be identical to c-Cbl protein (not shown). Together, the peptide sequences, reactivity with anti-Cbl antibody, and identical two-dimensional gel patterns demonstrate unambiguously that p120 is identical to the human c-cbl protooncogene product. Comparison of anti-pY with other two-dimensional gel patterns (Fig.3) suggests that additional tyrosine-phosphorylated polypeptides in the 120-kDa size range exist in T cells, but these are distinct from c-Cbl.
Figure 3:
Biochemical identity between Fyn
SH3-binding p120 and c-Cbl proteins demonstrated by two-dimensional gel
analyses. GST Fyn-SH3 binding reactions or immunoprecipitations
(-pY or
-Cbl) were carried out from Triton X-100 lysates of
10
anti-CD3 (2Ad2)-stimulated Jurkat cells and proteins
were eluted into IEF sample buffer with SDS. Individual samples or
their mixtures (indicated on top) were subjected to IEF on a
pH 3.5-10 Ampholine in the first dimension (IEF; from alkali to
acid) and SDS-PAGE in the second dimension. The resolved proteins were
transferred to PVDF membrane and subjected to anti-pY immunoblotting,
followed by HRPO-protein A and ECL detection. An aliquot of anti-pY
immunoprecipitate and Fyn-SH3 binding reaction was resolved during
SDS-PAGE on each gel to identify p120 unambiguously. Two-dimensional
standards were included with samples and visualized by Coomassie
staining of filters or immunoblotting with anti-actin and
anti-myoglobin antibodies. The identity of p120 and c-Cbl is revealed
by their identical migration when resolved as a mixture (middle
panel on left). NRS, normal rabbit serum.
Figure 4:
In vivo association of p120 with Fyn, Grb2, and PI
3-kinase p85 in Jurkat T cells. Immunoprecipitations from Triton X-100
lysates of 5
10
unstimulated(-) or anti-CD3
(2Ad2)-stimulated (+) Jurkat cells with indicated antibodies (I.P., shown on top) or whole cell lysates (from
10
cells) were resolved on SDS, 10% PAGE, transferred to
PVDF membranes, and subjected to immunoblotting with antibodies
indicated on the right, followed by HRPO-conjugates
(anti-mouse-HRPO for anti-pY blot; anti-rabbit-HRPO for anti-Cbl blot;
and HRPO-protein A for anti-PI 3-kinase p85 and anti-Grb2 blots) and
ECL detection. Immunoprecipitated species are indicated by arrows on the left; Ig, immunoglobulin heavy chain. Two unmarked solid arrows indicate the positions of 100- and
75-kDa polypeptides (referred to as p100 and p75 in the text). The open arrow indicates a 90-kDa phospholipase C
1-associated
polypeptide. Normal rabbit serum (NRS) and
CD8 are used
as negative controls for polyclonal rabbit and monoclonal mouse
antibodies, respectively. Each immunoblot represents a reprobing of the
same filter.
Anti-pY
immunoblotting demonstrated an activation-dependent increase in pY
content on p120 (lanes 9 and 10). Conversely, anti-Cbl immunoblotting showed that the
amount of p120
in anti-pY immunoprecipitates
increased with anti-CD3 stimulation (lanes 13 and 14). Anti-Fyn immunoprecipitates revealed an associated
120-kDa protein reactive with both the anti-pY and anti-Cbl antibodies (lane 7), and a higher signal was detected by each antibody in
anti-CD3-stimulated cells (lane 8); the enhancement of
Fyn-p120
association upon T cell activation
extends our previous results that p120 can concurrently bind to Fyn SH3
and SH2 domains in vitro(19) .
Significantly, a
similar amount of p120 was associated with Grb2
in unstimulated and anti-CD3-stimulated Jurkat cells; however,
Grb2-associated Cbl showed an increased pY signal after anti-CD3
stimulation (lanes 3 and 4; also see Fig.5below for time course). p120
was
also detected in immunoprecipitations of the PI 3-kinase p85; increased
p120
signals were observed after anti-CD3
stimulation using anti-Cbl as well as anti-pY immunoblotting (lanes
5 and 6).
Figure 5:
Time course of tyrosine phosphorylation of
Grb2-associated p120 upon T cell activation.
Anti-Grb2 immunoprecipitations from 5
10
unstimulated (-) or anti-CD3 (2Ad2)-stimulated (+)
Jurkat cells were resolved by SDS-PAGE and subjected to anti-pY (top panel) or anti-Grb2 (bottom panel)
immunoblotting, followed by detection with
I-protein A.
The time of anti-CD3 stimulation is shown in seconds (s) or
minutes (m). The top and bottom panels represent upper and lower parts of a single
filter.
In contrast to its association with Grb2 and
PI 3-kinase p85, p120 was not detected in
immunoprecipitates of phospholipase C
1, although phospholipase
C
1 was immunoprecipitated efficiently (confirmed by
immunoblotting; data not shown) and showed activation-dependent
tyrosine phosphorylation (lanes 11 and 12). Thus,
p120
forms in vivo complexes with
certain SH2/SH3 domain-containing T cell signaling proteins but not
with others. Each of these interactions was confirmed by stable
(Myc-tagged Grb2) or transient (HA-tagged p85 and untagged Fyn)
transfection into Jurkat T cells (data not shown). The identity of
Grb2- and p85-associated p120 protein as Cbl was also confirmed by
two-dimensional gel analysis (data not shown).
The above analyses
also yielded two additional findings. First, anti-PI 3-kinase p85
immunoprecipitations revealed a coimmunoprecipitated 36-38-kDa
inducibly tyrosine-phosphorylated polypeptide identical in mobility to
p36/38 which associates with Grb2 and phospholipase C1 (compare lanes 4, 6, and 12)(43, 44) . Second, both Grb2 and PI
3-kinase p85 coimmunoprecipitated unidentified polypeptides of 100 and
75 kDa which underwent activation-dependent tyrosine phosphorylation.
These polypeptides are likely to mediate interactions between Grb2 and
p85, both of which lack tyrosine phosphorylation (data not shown), yet
coimmunoprecipitate with each other (Fig.4, lanes
3-6) and are incorporated into pY complexes (Fig.4, lane 13 versus 14) in an activation-dependent manner. It is
likely that the 75-kDa polypeptide corresponds to the recently
identified Grb2 SH3 domain-binding SLYP-76 protein(45) .
Figure 6:
Association of the PI 3-kinase activity
with p120. Immunoprecipitations carried out with
the indicated antibodies (I.P., shown on top) from 5
10
unstimulated(-) or anti-CD3
(2Ad2)-stimulated (+) Jurkat cell lysates were subjected to lipid
kinase assays as described under ``Materials and Methods.''
The reaction products were subjected to TLC and visualized by
autoradiography. Lane 11 shows PI products generated with
anti-p85 immunoprecipitates from A431 cells which overexpress epidermal
growth factor receptor. Note that PI species in the middle lanes migrated slower for technical reasons; the phosphatidylinositol
trisphosphate species (PIP3) is the third major spot after the
origin.
The major tyrosine-phosphorylated species associated
with Grb2 in vivo, namely p120, p100,
p75, and p36/38, were observed in wild type GST-Grb2 binding reactions (Fig.7A). Binding to p120
was
essentially abolished when the N-terminal SH3 domain alone was mutated
and was decreased but still substantial when the C-terminal SH3 domain
alone was mutated. p120
did not bind to double
SH3 mutants of Grb2. Anti-Cbl blots revealed an identical binding
pattern. In contrast to p120
binding, binding to
p36/38 was retained in single or double SH3 domain mutants, whereas it
was abolished by the SH2 domain mutation. In addition, a fusion protein
with only the Grb2-SH2 domain failed to bind to
p120
, whereas it did bind to p36/38. These
results demonstrate that p120
binds to Grb2
exclusively through its SH3 domains; the exclusive binding of p36/38 to
SH2 domain confirms previous reports(43, 44) .
Figure 7:
Binding of p120 to
GST fusion proteins of Grb2 is mediated through SH3-proline peptide
interactions. Panel A, mutations in Grb2 SH3 domains abrogate
p120
binding, but the SH2 mutation does not.
Cell lysates were incubated with GST fusion proteins noncovalently
immobilized on glutathione-Sepharose beads (5-µl packed beads;
total volume 1 ml) for 1 h, and bound proteins were solubilized in
sample buffer. Whole cell lysate (10
cells) or binding
reactions of the indicated GST fusion proteins (10 µg each; shown
on top) with lysates of 2.5
10
anti-CD3
(SPV-T3b)-stimulated Jurkat cells were subjected to anti-pY
immunoblotting, followed by HRPO-protein A and ECL detection. 3-2-3 refers to N-terminal SH3, SH2, and C-terminal SH3 domains of Grb2. Asterisks denote mutated domains. Mutated residues were:
N-terminal SH3, P49L; SH2, R86K; C-terminal SH3, P206L. Lanes 11 and 12 are from a separate experiment. The filter was
stripped and immunoblotted with anti-Cbl antibody (lower
panel). Panel B, the p120
binding
to GST-Grb2 is abrogated by competing proline-rich peptides, whereas
p36/38 binding is retained. Competing peptides were added separately to
bead-bound fusion proteins and cell lysate at the indicated
concentrations (shown in µM). After 15 min, beads and
lysate were mixed, and binding reactions and immunoblotting were
carried out as in panel A. Peptides were: PI 3-kinase p85
amino acids 83-101, Sos1 amino acids 1147-1162, and dynamin
amino acids 786-806. -, no peptide. Panel C, the
p120
binding is retained in the presence of
competing pY peptides, whereas the p36/38 binding is abrogated. Binding
reactions and immunoblotting were as in panel B. Peptides
were: EPQpYEEIPIYL (pYEEI); EPQYEEIPIYL (YEEI);
PSpYVNVQNL (pYVNV); PSpYVAVQNL (pYVA*V). -, no
peptide.
For phosphopeptide competition, we
used a Grb2 SH2-specific Shc-derived peptide (pYVNV motif), a mutant
Shc peptide in which the critical Arg has been changed to Ala, and a
Src family SH2-specific peptide (pYEEI
motif)(11, 12) . Twenty µM pYEEI (but not
YEEI) markedly reduced pY protein binding to Fyn SH2, with nearly
complete inhibition of p120 binding; pYVNV had
relatively little effect (Fig.7C). Whereas pYVNV
produced a dose-dependent inhibition of Grb2 binding to p36/38, binding
of p120
was retained; the decrease in
p120
(and p75) signal seen even at the lowest
concentration (0.8 µM) was not dose-related. The
specificity of the pYVNV peptide was demonstrated by the inability of
either the pYVAV or the pYEEI peptide to inhibit pY protein binding to
Grb2. Together, the peptide competition and mutational data demonstrate
that p120
interacts with Grb2 exclusively
through the SH3 domain.
Figure 8:
Binding of p120 to
GST fusion proteins of the PI 3-kinase p85. Panel A, both the
SH2 domains and the SH3 domain of PI 3-kinase p85 are capable of
binding to p120
. Binding reactions and anti-pY
immunoblotting were carried out as in Fig.7A. p120 is
indicated. The particular domains incorporated in GST fusion proteins
are shown on top. Panel B, binding of
p120
to GST-p85-SH2(N)/SH3 is mediated
predominantly through SH2-pY peptide interactions. Peptide competitions
were performed as in Fig.7B. Peptides were: pYEEI;
HSDpYMNMTPR (pYMNM); HSDYMNMTPR (YMNM); PI 3-kinase
p85
amino acids 83-101 (p85). -, no peptide.
Note that pYMNM alone nearly completely competes out the p120 binding
to p85-SH2(N)/SH3 fusion protein.
Interestingly, 20 µM phosphopeptide markedly reduced the binding of p120 to SH3/SH2(N) fusion protein. Consistent with this result,
100 µM p85 peptide produced a relatively minor inhibition
of p120
binding. A combination of phosphopeptide
and proline-rich peptide produced an essentially complete inhibition.
Thus, although both the SH2 and SH3 domains of PI 3-kinase p85 can bind
to p120
, SH2 binding predominates when both
domains are present in a single fusion protein.
Figure 9:
Lack of complex formation between
p120 and Sos proteins. Immunoprecipitations with
indicated antibodies (I.P., shown on top) were
subjected to immunoblotting with anti-Sos antibody followed by sheep
anti-mouse Ig-HRPO and ECL detection. This blot represents a reprobing
of the membrane used in Fig.4.
We employed a
direct protein purification approach to determine the identity of p120.
Two of the three peptides showed a complete match with the human
p120 sequences(22, 41, 42) . The third
peptide, whose sequence had some ambiguities, also corresponded to a
Cbl sequence but with four mismatches out of 14. Although the reasons
for mismatch (e.g. incorrect sequence, signals derived from a
comigrating contaminant peptide, or an alternative mRNA transcript in
HSB2 cells) are unknown, collectively the sequence data provided direct
evidence for the identity of p120 with the c-cbl protooncogene
product. Further support for this conclusion was provided by
immunochemical cross-reactivity and demonstration of identical
two-dimensional gel profiles of c-Cbl and p120. Given that only a
single functional c-cbl gene is known to be present in the
human genome(22, 41, 42) , these results
establish conclusively the identity of the SH3 domain-binding p120
polypeptide (19) as the human c-cbl protooncogene
product. Our direct biochemical analyses confirm and extend the recent
finding that c-Cbl protein is tyrosine phosphorylated upon TCR
stimulation (21) . Interestingly, c-Cbl was also identified by
expression cloning with GST fusion proteins of Nck protein, which
contains three SH3 domains(50) .
It is noteworthy that the
only well characterized protein known to interact with Grb2 in T cells
is the Ras guanine nucleotide exchanger
Sos(43, 44, 52, 53) . The Ras
pathway is essential for T cell activation(54, 55) ,
underscoring the importance of Grb2-Sos complexes. Our studies
demonstrate that in T cells, Grb2 also forms a stable complex with a
non-Sos protooncogene, p120, and that the
Grb2-associated Cbl is an early target of TCR-coupled tyrosine kinases.
These findings define a distinct Grb2-mediated signaling pathway
downstream of the TCR. It is unlikely that Grb2 concurrently binds to
Sos and p120
, linking these proteins in series
in a single signaling cascade. First, the structural requirements for
Grb2 binding to p120
and Sos were identical,
with an essential role of the N-terminal SH3 domain and a smaller
contribution of the C-terminal SH3
domain(27, 36, 46) . Second, although both
p120
and Sos were readily detectable in Grb2
immunoprecipitates, no Sos-p120
complexes could
be detected ( Fig.4and Fig. 9). Since Grb2 is known to
form Shc-mediated complexes with phosphorylated TCR
, a
Grb2-p120
complex may also interact with TCR
, establishing a signaling pathway parallel to Grb2-Sos.
Significantly, the amount of PI 3-kinase activity in anti-Cbl
immunoprecipitates far exceeded that in anti-Fyn or anti-TCR
immunoprecipitates. In addition, PI 3-kinase p85 protein was difficult
to detect in association with Src family kinases (data not shown),
whereas PI 3-kinase p85-Cbl interaction was readily demonstrable. These
data lead us to suggest that p120
may play a
prominent role in coupling the PI 3-kinase enzyme with the TCR
signaling machinery. It is likely that other tyrosyl phosphoproteins
found to associate with PI 3-kinase p85 (e.g. p36/38, p75,
p100) also contribute to this pathway. The
p120
-mediated PI 3-kinase recruitment may
complement other previously described mechanisms, such as the Fyn SH3
binding to proline-rich regions of the PI 3-kinase
p85(35, 56) .
Given
the widespread signaling roles of the proteins that we have shown to
interact with Cbl, together with its interaction with another SH3
domain-containing adaptor protein Nck(50) , our findings
suggest that p120 is likely to function in
signal transduction downstream of TCR as well as other related
receptors. Consistent with this suggestion, tyrosine phosphorylation of
the Cbl protein was observed recently in HL60 myelomonocytic cell line
upon triggering through Fc
receptors(60) . Interestingly,
v-cbl is known to induce pre-B and myeloid leukemias in mice,
and human c-cbl on chromosome 11q23 is closely linked to
breakpoints involved in translocations [t(4;11) or t(11;14)]
found in B cell, myeloid, and T cell
leukemias(24, 61) . Notably, we have observed
p120
to be one of the earliest tyrosine
phosphorylation substrates upon triggering through the B cell receptor
and have detected Grb2-Cbl and PI 3-kinase p85-Cbl complexes in B
cells.
Thus, it is likely that oncogenicity of the aberrant
forms of cbl results from a constitutive activation of the
signaling machinery in which it physiologically participates.
Consistent with this suggestion, recent analyses have shown that
oncogenic point mutants of p120
are
constitutively tyrosine phosphorylated, and that Cbl protein interacts
with the BCR-abl and v-abl oncogenes (42 and data not
shown).
Demonstration of p120 as an
SH3-binding protein strongly implicates its proline-rich region in
binding to SH3 domains. Examination of the proline-rich regions of
p120
reveals multiple potential binding motifs,
including consensus sequences preferred by Src family and PI 3-kinase
p85 SH3 domains (e.g. RPLPCTP (amino acids 563-569) and
RPIPKVP (amino acids
593-599))(14, 15, 16, 17, 18) .
Given the substantial length of this region and multiplicity of
potential SH3-binding motifs, it will be of interest to determine
whether SH3 domains of different signaling proteins bind the same or
distinct sites. The latter would suggest the interesting possibility
that p120
may concurrently tether multiple SH3
domain-containing signaling proteins.
In conclusion, we demonstrate
that the Src family SH3 domain-binding protein p120 is identical to the
c-cbl protooncogene product and forms in vivo complexes with the Fyn tyrosine kinase, Grb2 adaptor protein, and
PI 3-kinase p85. These studies identify p120 as
a multifunctional SH2 and SH3 domain-binding protein and strongly
suggest signal transduction functions for this protooncogene with known
oncogenic potential but with no previously known physiological
function.