(Received for publication, July 19, 1995; and in revised form, August 30, 1995)
From the
The proto-oncogene product, Cbl, is a 120-kDa protein present in
lymphocytes that contains numerous PXXP motifs in its
COOH-terminal region and constitutively binds the SH3-containing
adaptor protein Grb2. Cross-linking of CD3 and CD4 receptors in Jurkat
T cells causes tyrosine phosphorylation of Cbl and its association with
phosphatidylinositol 3`-kinase (Meisner, H., Conway, B., Hartley, D.,
and Czech, M. P.(1995) Mol. Cell. Biol. 15, 3571-3578).
Here we demonstrate that Cbl is also present in nonlymphoid cells, and
that epidermal growth factor (EGF) elicits its rapid tyrosine
phosphorylation in human embryonic 293 cells. Immunoprecipitates of Cbl
from lysates of these cells contain Grb2 in the basal state, while EGF
stimulation causes co-precipitation of tyrosine-phosphorylated EGF
receptors. Similarly, EGF receptor immunoprecipitates from EGF-treated
293 cells contain Cbl and Grb2. Both Grb2 and EGF receptors are
released from Cbl in the presence of a proline-rich peptide that binds
the NH-terminal SH3 domain of Grb2. These results indicate
that autophosphorylated EGF receptors associate with the SH2 domain of
Grb2, which is complexed through its SH3 domain with proline-rich
regions of Cbl. Such recruitment of Cbl to EGF receptors may reflect an
important mechanism for its tyrosine phosphorylation and for assembling
signaling components that mediate or modulate EGF actions.
Following stimulation, receptor and non-receptor tyrosine
kinases initiate signaling mechanisms by phosphorylation of specific
sets of cellular proteins. Such tyrosine phosphorylation can regulate
enzyme activity (1, 2) or serve to recruit signaling
proteins containing Src homology (SH2) ()or phosphotyrosine
interacting domains(3, 4, 5) . In
T-lymphocytes, a major phosphoprotein appearing in response to T-cell
receptor cross-linking is the 120-kDa product of the c-cbl proto-oncogene(6, 7) . Cbl is located in cytosol
and cytoskeleton and contains numerous proline-rich PXXP
motifs, as well as several tyrosine residues that serve as possible
binding sites for SH3 and SH2 domains, respectively(8) . The
viral v-cbl oncogene product is a severely truncated,
transforming form of c-Cbl found in the nucleus and
cytosol(9) . We recently demonstrated that a population of Cbl
is stably associated with the adaptor protein Grb2, and that activation
of T-lymphocytes by cross-linking of the T-cell antigen receptor
recruits PI 3-kinase into Cbl complexes(6) . These data suggest
that Cbl may play an important role in facilitating signaling
mechanisms in T-cells.
Although the possible role of Cbl in
hematopoietic cell signaling has been emphasized
previously(6, 7, 10) , the presence of Cbl in
HeLa cells (9) prompted us to evaluate the role of this protein
in other signaling systems. Of particular interest is the association
of Cbl with Grb2, which is distributed widely among cell types. Grb2 is
composed of one SH2 and two SH3 domains that link proteins containing
phosphotyrosine to cellular proteins with proline-rich
regions(11, 12) . For example, Grb2 is known to bind
via its SH2 domain to phosphotyrosine residues in the EGF receptor (13, 14, 15) and to insulin receptor
substrate 1 (IRS-1), which serves as a docking protein for SH2 domains
in insulin-sensitive cells(16) . The NH-terminal
SH3 domain binds stably to Cbl in hematopoietic
cells(6, 7) , as well as to dynamin (17) and
C3G (18) in other cell types. Grb2 also has a major role in the
control of Ras signaling by binding to the guanine nucleotide-releasing
factor Sos (19, 20, 21) . Both Sos and Cbl
possess the identical proline-rich sequence PPVPPPR that has been
identified as the region in Sos that binds to the Grb2 SH3
domain(19) . In this report we show that Cbl is indeed present
in all cell types examined, and that addition of EGF to cultured human
kidney cells causes the recruitment of Cbl into a complex containing
the EGF receptor and its rapid tyrosine phosphorylation. Our data
indicate that such association of Cbl with the EGF receptor may be
mediated through Grb2.
We previously identified a major tyrosine-phosphorylated 120-kDa protein in T-cell receptor-activated Jurkat T-cells as the product of the Cbl proto-oncogene(6) . Although this protein has been found primarily in hematopoietic cells, Fig. 1shows that Cbl is present in Nonidet P-40 lysates from several other cells and tissues. In addition to Jurkat cells, high levels were found in two kidney cell lines (COS and 293), while CHO cells and 3T3-L1 preadipocytes expressed approximately 10% of this level. In differentiated 3T3-L1 adipocytes or rat adipose tissue, the concentration of Cbl was about 5% that of Jurkat cells. The low expression of Cbl in adipose cells is in agreement with a decrease of Cbl RNA during differentiation(24) . The two bands seen in 3T3-L1 lysates (right lane) represent Cbl with different stoichiometries of tyrosine phosphorylation, as defined by anti-phosphotyrosine blotting (not shown).
Figure 1: Distribution of the proto-oncogene product c-Cbl among various cell types. Lysates of cultured cells were prepared as described under ``Experimental Procedures.'' Proteins were solubilized in SDS-PAGE sample buffer, and 20 µg was loaded per lane. Following electrophoresis and transfer to nitrocellulose, proteins were blotted against Cbl antiserum and visualized by chemiluminescence.
The abundance of Cbl in 293 human kidney cells, coupled with the sensitivity of these cells to EGF(19) , led us to determine whether EGF receptor activation results in tyrosine phosphorylation of this protein. Fig. 2(lanes 1-5) reveals that stimulation of 293 cells with EGF for 2 min led to the appearance of a tyrosine-phosphorylated 120-kDa protein in total lysates (lane 2) and in anti-Cbl immunoprecipitates (lane 5) that blotted with Cbl antiserum (lower panel). EGF action also resulted in the appearance of other phosphotyrosine bands in anti-Cbl precipitates, most noticeably at 170 kDa. In Jurkat T-lymphocytes, cross-linking of the CD3/CD4 receptors (Fig. 2, lanes 6-10) increased tyrosine phosphorylation of Cbl and led to the co-precipitation of bands at 100, 83, 72, and 60 kDa, but not at 170 kDa, as detected with 4G10 antibody. As depicted in the lower panel of Fig. 2, the amount of Cbl that was immunoprecipitated with anti-Cbl antibody is similar in stimulated versus unstimulated 293 or Jurkat cells. These data demonstrate that EGF action causes marked tyrosine phosphorylation of Cbl in 293 cells. Moreover, EGF also enhances either the association of Cbl with tyrosine-phosphorylated proteins or the tyrosine phosphorylation of proteins constitutively bound to Cbl.
Figure 2: Cbl is tyrosine-phosphorylated upon activation of 293 cells with EGF. Jurkat and 293 cells were serum-starved for 4 h and activated either with EGF (62 ng/ml; lanes 1-5) or with OKT3 plus OKT4 mAbs (lanes 6-10) for 2 min at 37 °C. Lysates were precleared with normal rabbit serum before immunoprecipitation with normal (lanes 3 and 8) or anti-Cbl (lanes 4, 5, 9, and 10) serum. Lanes 1, 2, 6, and 7 are total lysates containing 20 µg of protein. After electrophoresis and transfer to nitrocellulose, samples were probed with 4G10 (top) or Cbl (bottom) antisera and visualized by chemiluminescence. Molecular mass markers (kDa) are denoted at right. The solid arrow points to the 170-kDa protein in lane 5.
The co-immunoprecipitation of a 170-kDa tyrosine-phosphorylated protein with Cbl in EGF-treated 293 cells but not activated Jurkat cells suggested that this protein might be the EGF receptor itself. Immunoprecipitation of 293 cell lysates with Cbl antiserum adsorbed a 170-kDa tyrosine-phosphorylated band (Fig. 3, upper panel, lane 8) that blotted faintly to EGF receptor antiserum from activated cells, but not with normal serum or from nonactivated cells (middle panel, lanes 5-8). The low signal may be due to the weaker recognition of tyrosine-phosphorylated receptors compared to nonphosphorylated receptors by the EGF receptor antibody on Western blots (compare lanes 4 and 5). EGF receptor immunoprecipitates showed a phosphotyrosine band at 120 kDa in activated (lane 5) but not in serum-starved cells (lane 4). Blotting with Cbl antibody confirmed that the 120-kDa band in EGF receptor immunoprecipitates is Cbl (lanes 3-6, lower panel). The absence of EGF receptor protein bound to anti-Cbl antibody and of Cbl protein in anti-EGF receptor immunoprecipitates of lysates of untreated 293 cells demonstrates EGF-mediated recruitment of Cbl to tyrosine-phosphorylated EGF receptors. Comparison by densitometry of the amount of the EGF receptor species in anti-Cbl immunoprecipitates versus EGF receptor immunoprecipitates indicates that approximately 30% of the total cellular EGF receptors associate with Cbl. This same value is obtained by analysis of either anti-EGF receptor or anti-tyrosine phosphorylation immunoblots (lanes 5 and 8). The large fraction of Cbl bound to the EGF receptor upon activation suggests an important functional relevance.
Figure 3: Activation of 293 cells with EGF induces the association of the EGF receptor with Grb2 and Cbl. Total lysates from serum-starved(-) or activated (+) cells are shown in lanes 1 and 2. Lysates were precleared with normal serum and immunoprecipitated with normal (lanes 3 and 6), anti-Cbl (lanes 7 and 8), or anti-EGF receptor antibody (lanes 4 and 5). Following electrophoresis and transfer to nitrocellulose, filters were probed with the antiphosphotyrosine antibody 4G10 (top), anti-EGF receptor, anti-Cbl, or anti-Grb2 (bottom), and visualized by chemiluminescence. Molecular mass markers (kDa) are shown at right.
In EGF-activated
fibroblasts, a 170-kDa protein appearing in Grb2 and Sos
immunoprecipitates has been identified as the EGF
receptor(5, 19, 25, 26) , and it has
been proposed that a complex of Sos, Grb2, and the EGF receptor is
formed which leads to increased membrane localization and subsequent
Ras activation. We therefore tested whether Grb2-Cbl complexes might be
recruited to EGF receptors in an analogous manner. Blotting of the
resolved proteins present in the anti-Cbl and anti-EGF receptor
immunocomplexes shown in Fig. 3with Grb2 mAb (bottom
panel) revealed that Grb2 was bound to the EGF receptor in cell
lysates from EGF-activated cells (lane 5), but not from
unstimulated cells (lane 4). Cbl immunoprecipitates, on the
other hand, contained Grb2 in the basal state (lane 7), and
EGF modestly increased the amount bound (lane 8). An increase
in the Grb2/Cbl ratio was seen in several experiments (see Fig. 4). A constitutive association with Cbl via the
NH-terminal SH3 domain of Grb2 has also been found in
Jurkat cells(6, 7) . Taken together, the data in Fig. 3support the hypothesis that EGF receptor tyrosine
phosphates recruit the SH2 domain of Grb2 proteins that are bound to
Cbl through their NH
-terminal SH3 domain. In addition, the
activation-dependent increase of Grb2 complexed to Cbl supports our
previous observation that the SH2 domain of Grb2 proteins can also bind
directly to tyrosine-phosphorylated Cbl, despite the lack of a
consensus YXNX motif(6, 15, 27) . It is not clear from
our data whether a single Grb2 protein can bind Cbl with both its SH2
and SH3 domains or whether some Grb2 proteins bind through SH2 and some
through their SH3 domain.
Figure 4: The EGF receptor and Grb2 are dissociated from Cbl by a proline-rich peptide. Cells were activated with EGF, and lysates reacted with Cbl antiserum, plus 0.05, 0.2, or 0.5 mM proline-rich peptide (lanes 4-6), at 4 °C for 2 h. Nitrocellulose filters containing electrophoresed proteins were blotted with anti-Tyr(P) (top), anti-Cbl (middle), or anti-Grb2 (bottom) antisera, and visualized by chemiluminescence.
In order to test more definitively whether the proline-rich regions of Cbl are required for binding Grb2 and the EGF receptor, peptide-mediated dissociation of Cbl-Grb2 and Cbl-EGF receptor complexes was attempted. A peptide containing the proline-rich sequence (PPVPPR) corresponding to amino acids 494-499 of Cbl was added to lysates from EGF-activated 293 cells, followed by immunoprecipitation with Cbl antiserum. Fig. 4shows that the EGF receptor (top panel) and Grb2 (bottom panel) were both dissociated from Cbl to the same extent as the peptide concentration was increased. A Western blot (Fig. 4, middle panel) confirms that the same amount of Cbl was immunoprecipitated at all peptide concentrations. Quantitation by densitometry of the relative amount of Grb2 and EGF receptor bound to Cbl in the presence of the proline-rich peptide (Fig. 5, closed symbols) revealed that the approximate half-maximal dissociation of the EGF receptor and Grb2 from Cbl occurs at 50 µM peptide. When a nonrelevant peptide was added (open symbols), no dissociation of Grb2 or EGF receptor from Cbl was observed. These results are consistent with EGF-induced coupling of the EGF receptor to Cbl via the SH2 and SH3 domains of Grb2. However, we cannot exclude the possibility that some other SH3-containing adaptor molecule may link the EGF receptor to Cbl in this system.
Figure 5: Dissociation of the EGF receptor and Grb2 from Cbl by proline-rich peptide. The autoradiograms depicted in Fig. 4were scanned by densitometry. Values are shown as a percentage of the density of Grb2 (squares) and EGF receptor (circles) in EGF-activated cells, incubated with either a proline-rich peptide (solid symbols) or a nonrelevant peptide (open symbols).
A simple hypothesis that explains
the data presented here is that Grb2-Cbl complexes are recruited to
tyrosine phosphorylation sites on the activated EGF receptor, and that
tyrosine phosphorylation of Cbl results from its juxtaposition with the
receptor kinase. This hypothesis predicts that Cbl may be recruited to
complexes that include other proteins that bind the EGF receptor, such
as Shc proteins. Shc SH2 and phosphotyrosine interacting domains bind
EGF receptor tyrosine phosphates, and Shc itself becomes
tyrosine-phosphorylated(3, 4, 20, 28) .
Consistent with the above prediction, we observe Shc protein in
immunoprecipitates of anti-Cbl antibody when lysates from EGF-treated
293 cells are used. ()Nonetheless, further experiments will
be necessary to unequivocally test whether Cbl recruitment to EGF
receptors is actually necessary for its tyrosine phosphorylation. Cells
expressing mutant EGF receptors missing autophosphorylation sites fail
to show a pronounced block in signaling (29) . Li et
al. (29) have thus proposed that the EGF receptor may
signal like the insulin receptor, in that SH2 domain-containing
proteins are not directly bound, but rather receptor
autophosphorylation causes the tyrosine phosphorylation of the docking
proteins IRS-1 and Shc, which then serve as binding sites for SH2
proteins. For example, following insulin stimulation, Shc becomes
rapidly tyrosine-phosphorylated, leading to Grb2-SH2 domain binding (30) . It will be important in future studies to test whether
tyrosine phosphorylation of Cbl is normal in response to EGF receptors
with ablated tyrosine phosphorylation sites.
The multiple potential
tyrosine phosphorylation sites within the protein sequence of Cbl
suggest it might serve to recruit multiple signaling proteins
containing SH2 or phosphotyrosine interacting domains. In this sense,
it may function similarly to IRS-1, which becomes
tyrosine-phosphorylated at many sites in response to insulin and binds
SH2 domain-containing proteins(16) . Upon growth factor
stimulation, both proteins become transiently associated with higher
concentrations of SH3 and SH2 domain proteins, including Grb2, Nck, and
PI 3-kinase(6, 7, 10, 16) .
Interestingly, we observe several tyrosine-phosphorylated proteins that
co-immunoprecipitated with Cbl in both Jurkat and 293 cells (Fig. 2, lanes 5, 9, and 10). One of
these appears to be the tyrosine kinase Fyn, which binds in vitro to a 120-kDa tyrosine-phosphorylated protein through
SH2 and SH3 domains (31, 32) . Evidence suggests that
the formation of transient complexes of Cbl with SH2 domain proteins
regulates cell growth(9) . This may be achieved by allowing
localized increases in the activity of those proteins that bind via
SH2-phosphotyrosine interaction, analogous to PI 3-kinase and Syp
phosphatase interactions with
phosphopeptides(14, 33) . Such localized increases in
specific activity may be sufficient to promote signaling pathways. In
support of this, we have noted a 4-fold increase in PI 3-kinase bound
to Cbl upon T-cell receptor cross-linking in Jurkat
T-cells(6) . It is not clear whether PI 3-kinase is recruited
to Cbl in 293 cells because activity of this enzyme is very low in this
model system(34) .
In any case, the findings
presented here showing recruitment of Cbl to EGF receptor complexes and
its marked tyrosine phosphorylation in response to EGF suggest that Cbl
may play a key role in EGF receptor signaling.
Note Added in Proof-After the preparation of this report, two laboratories have demonstrated that Cbl becomes tyrosine-phosphorylated by EGF in fibroblasts overexpressing the EGF receptor(35, 36) . Galisteo et al. (36) conclude that Cbl is not complexed to Grb2 and binds directly to the EGF receptor in HER14 cells. The results presented here and elsewhere (6, 7) document a stable Grb2-Cbl complex in Jurkat and 293 cells, suggesting that two mechanisms may operate to couple EGF receptors to Cbl.