(Received for publication, August 21, 1995; and in revised form, December 13, 1995)
From the
Several recent studies have demonstrated that Grb2, composed entirely of SH2 and SH3 domains, serves as an adaptor protein in tyrosine kinase signaling pathways. Cbl, the protein product of c-cbl proto-oncogene, has been reported to be phosphorylated on tyrosine residues upon T cell receptor (TCR) engagement. Here we show that in unstimulated Jurkat cells Cbl is co-immunoprecipitated with monoclonal antibody against Grb2. However, in lymphocytes activated through the TCR, Cbl loses its ability to bind to Grb2 precipitated either with anti-Grb2 antibody or with an immobilized tyrosine phosphopeptide, Y1068-P, derived from the epidermal growth factor receptor. In vitro studies confirm that the ability of Cbl to bind to both SH3 domains of Grb2 is strongly reduced in activated T lymphocytes. Investigation of the time course of Cbl dissociation from Grb2 reveals that it is transient and correlates with the kinetics of tyrosine phosphorylation of Cbl. Moreover, Cbl is co-immunoprecipitated with Crk, another SH2/SH3 domain-containing protein, upon TCR stimulation. Tyrosine-phosphorylated Cbl binds exclusively to the SH2 domain of Crk. These results suggest that different adaptor proteins may have different roles in the regulation of c-cbl proto-oncogene product.
Recent studies by a number of laboratories have characterized
the mechanism by which growth factors activate the Ras signaling
pathway. This mechanism involves formation of complexes of the Sos
guanine nucleotide exchange protein, and Grb2, an SH2 ()and
SH3 domain-containing adaptor protein with autophosphorylated growth
factor receptors(1, 2, 3) . The SH3 domains
of Grb2 bind to the carboxyl-terminal proline-rich domain of
Sos(2) , whereas the SH2 domain binds to
tyrosine-phosphorylated sites (1, 2, 3) .
Complex formation of Sos with autophosphorylated receptor tyrosine
kinases via Grb2 results in the translocation of Sos from the cytosol
to the plasma membrane, where its substrate Ras is localized (1) .
The Crk protein, a homologue of the product of the
v-crk oncogene, is an SH2 and SH3 domain-containing adaptor
protein related to Grb2 and Nck(4) . Two forms of cellular Crk
proteins have been found: Crk II has the domain structure SH2-SH3-SH3,
while the shorter Crk I consists of one SH2 and one SH3
domains(5, 6) . Recently, a third member of the Crk
family has been cloned from chronic myelogenous leukemia cells and
referred to as Crk-L(7) . It has been shown that Crk proteins
associate with two guanine nucleotide exchange proteins for Ras, Sos,
and C3G(8, 9, 10) . In addition, they
interact via their SH2 domains with both phosphorylated paxillin and
p130(11, 12) .
Stimulation of T lymphocytes via their TCR results in phosphorylation of multiple intracellular proteins on tyrosine residues(13, 14) . We and others have shown that in the lysates of activated T cells Grb2 associates with several phosphotyrosine proteins(15, 16, 17, 18) , including proteins with molecular masses of 36, 52, 75, and 120 kDa. The 36-kDa membrane-bound phosphoprotein binds to the SH2 domain of Grb2. In UCHT1-stimulated T cells, p36 was shown to be associated with the complex of Sos and Grb2 and thus implicated in Ras activation(15, 16) . The 52-kDa tyrosine phosphoprotein is identical with Shc adaptor protein(19) . The 75-kDa tyrosine kinase substrate, that has been recently cloned and referred to as SLP-76, interacts with the SH3 domains of Grb2(17, 20) .
It has been shown that a 116-kDa tyrosine phosphoprotein implicated in the TCR signaling pathway binds to the amino-terminal SH3 domain of Grb2(18) . Recently, a 120-kDa tyrosine phosphoprotein which in vitro binds to SH3 domains of Fyn, Lck, and Grb2, has been identified as Cbl(21) . The v-cbl oncogene is the transforming gene of the murine Cas NS-1 retrovirus which induces pre-B cell lymphomas and myeloid leukemias(22) . The homologue of v-cbl in mammalian cells is the c-cbl proto-oncogene that encodes Cbl, a 120-kDa protein predominantly localized in the cytoplasm(23) . Furthermore, in vivo association of Nck, another SH2 and SH3 domain-containing adaptor protein with Cbl, was also demonstrated(24) .
In this paper, we characterize the in vivo interaction of Cbl with two adaptor proteins, Grb2 and Crk. We report that, upon T cell activation, Cbl rapidly and transiently dissociates from Grb2. Tyrosine-phosphorylated Cbl then binds to the SH2 domain of Crk. We provide evidence that Grb2 and Crk have a critical role in the regulation of Cbl proto-oncogene product.
c-DNA encoding full-length
GST fusion proteins c-Crk II and c-Crk II SH2 (SH2-deleted mutant)
were donated by Dr. H. Hanafusa(25) . The SH2 fragment of c-Crk
(amino-terminal 146 amino acids) was isolated by BamHI/BglII digestion of full-length c-Crk II and
subsequent ligation into BamHI-digested pGEX2TK (Pharmacia
Biotech Inc.). The GST fusion proteins encoding both SH3 domains of
Grb2 have been described previously(17) . The Y1068
phosphopeptide derived from the autophosphorylated EGF receptor was
synthesized with the sequence PVPEY(phos)INQS. The unphosphorylated
peptide, PVPEYINQS, was also made(1, 15) .
We have recently used an immobilized tyrosine phosphopeptide derived from the Y1068 autophosphorylation site of the EGF receptor, to precipitate the complex of Grb2 and Sos from the lysates of T lymphocytes(15) . It has been demonstrated that the phosphopeptide, Y1068-P binds to the SH2 domain of Grb2 with high affinity, whereas the SH3 domains of Grb2 are available for protein-protein interaction(1, 15) . To test if the phosphopeptide could precipitate proteins other than Sos in complex with Grb2, both the phosphorylated and unphosphorylated forms of Y1068 peptide immobilized on beads were incubated with lysates from untreated and TCR cross-linked Jurkat cells. Probing of Y1068-P precipitates with anti-phosphotyrosine antibody, 4G10, demonstrates that a 120-kDa phosphoprotein is present in the lysate of unstimulated cells (Fig. 1). Interestingly, upon T cell activation, the amount of this phosphoprotein is strongly decreased in the Y1068-P precipitate, while the appearance of a 75-kDa tyrosine phosphoprotein was detected in the same precipitate. Immunoblotting with an antibody against the 120 kDa c-cbl proto-oncogene product shows the presence of Cbl in the Y1068-P precipitates from unstimulated cells and a decreased amount of Cbl in the precipitates from UCHT1-stimulated cells (Fig. 1). These data suggest that the 120-kDa tyrosine phosphoprotein observed in our experiment is identical with Cbl or at least Cbl is a component of the 120-kDa tyrosine phosphoprotein band. The Y1068-P phosphopeptide precipitates equal amounts of Grb2 from either unstimulated or activated T lymphocytes(15) ; thus, the decreased amount of Cbl in the precipitates indicates that the association of Grb2 with Cbl is strongly inhibited in the lysates of activated cells. Identical results were obtained using human peripheral blood T lymphoblasts (data not shown). It is noteworthy that while Cbl disappeared from the Grb2 complex, in the 4G10 blot a relatively large amount of a 120-kDa tyrosine phosphoprotein still bound to Grb2 even in stimulated cells. It is likely that another tyrosine phosphoprotein exists in stimulated T cells with a molecular mass of 115-120 kDa. Analysis of the partial amino acid sequence of an unidentified 116-kDa phosphoprotein, that can also bind to Grb2, suggests that it is different from Cbl(20) .
Figure 1: Binding of Cbl to the complex of the phosphopeptide Y1068-P and Grb2 is inhibited by TCR stimulation. Tyrosine phosphopeptide Y1068-P and its unphosphorylated form, Y1068, were immobilized on Affi-Gel 10 and used for protein precipitation from Jurkat cells stimulated for 2 min or left untreated. Proteins were separated by SDS-7.5% PAGE then immunoblotted with anti-phosphotyrosine (anti-PTyr) or anti-Cbl antibodies.
To prove the interaction of Grb2 with Cbl in another experimental system, immunoprecipitations with anti-Grb2 antibody covalently bound to Sepharose beads were performed. Jurkat cells were stimulated with UCHT1 antibody for 2 min or left untreated, proteins were immunoprecipitated with anti-Grb2, and then probed with anti-Cbl antibody. Fig. 2demonstrates that in the lysate of resting cells Cbl co-immunoprecipitates with Grb2. In contrast, in the lysate of activated cells, Cbl is not associated with Grb2. Immunoblotting of whole cell lysates from quiescent or activated T cells with anti-Cbl antibody shows equal quantities of immunoreactive bands (Fig. 2). This excludes the possibility that the disappearance of Cbl from the Grb2 immunoprecipitates is due to the proteolytic degradation of the protein. For control immunoprecipitates, an antipeptide antibody raised against the guanine nucleotide releasing factor (GRF) that is exclusively present in neuron cells was used (Fig. 2)(26) .
Figure 2: Co-immunoprecipitation of Cbl with Grb2 is inhibited upon T cell activation. Jurkat cells were stimulated with UCHT1 antibody for 2 min or left untreated. Immunoprecipitates of Grb2 were then subjected to immunoblotting with anti-Cbl antibody. For control immunoprecipitation, anti-GRF antibody was used. Immunoblot of whole cell lysates of Jurkat cells with anti-Cbl antibody is also shown.
T cell stimulation results in a rapid and significant phosphorylation of Cbl detected with anti-phosphotyrosine antibody(21) . Donovan et al.(21) have demonstrated Cbl phosphorylation only in whole cell lysates of Jurkat cells. Therefore, we immunoprecipitated Cbl and then performed immunoblotting with 4G10. Fig. 3demonstrates that Cbl has a certain level of basal phosphorylation in quiescent cells. However, in response to TCR activation, Cbl is rapidly phosphorylated on tyrosine residues. Maximal phosphorylation is seen after 5 min of stimulation, with a reduction to background levels by 45 min.
Figure 3: Time course of Cbl phosphorylation on tyrosine residues in response to CD3 activation. Jurkat cells were treated with UCHT1 antibody for the indicated times. Cbl immunoprecipitates from cell lysates were resolved by SDS-7.5% PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-phosphotyrosine antibody 4G10.
We also investigated the kinetics of Cbl dissociation from Grb2 upon T cell activation. Jurkat cells were treated with UCHT1 antibody for 0, 5, 15, and 45 min; then Cbl was immunoprecipitated with anti-Grb2 antibody. Fig. 4shows that after 5 min of stimulation Cbl is not co-immunoprecipitated with Grb2. However, after 15 min of UCHT1 treatment, the amount of Cbl is slightly increased in the Grb2 immunoprecipitate and returns to the level seen in unstimulated cells after 45 min. These data demonstrate a marked correlation between the kinetics of Cbl phosphorylation (Fig. 3) and the kinetics of dissociation of Cbl from Grb2 (Fig. 4A). To ensure that the loss of Cbl from Grb2 immunoprecipitates is not an artifact due to different amounts of Grb2 present in the immunocomplexes, Grb2 immunoprecipitates with monoclonal anti-Grb2 antibody were probed. The result clearly demonstrates that Grb2 precipitates contain equal immunoreactive bands (Fig. 4B). Grb2 was not found to undergo any phosphorylation on either tyrosine or serine/threonine residues in T lymphocytes activated via their TCR(15) . Therefore, it is possible that the inability of phosphorylated Cbl to bind to Grb2 in T cells activated for a short time is due to conformational changes in the structure of Cbl upon increased tyrosine phosphorylation, although at present definitive proof of this hypothesis is not available.
Figure 4: Time course of Cbl dissociation from Grb2 in activated T lymphocytes. Jurkat cells were stimulated with UCHT1 antibody for the indicated times. A, Grb2 immunoprecipitates were then subjected to SDS-7.5% PAGE, transferred to nitrocellulose membrane, and probed with anti-Cbl antibody. B, proteins were immunoprecipitated with anti-Grb2 monoclonal antibody, separated by SDS-12.5% PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-Grb2 antibody.
It has been reported that only the amino-terminal SH3 domain of Grb2 can bind Cbl(21) . We investigated the in vitro binding of Cbl to GST fusion proteins of Grb2. Lysates of quiescent and activated Jurkat cells were mixed with both SH3 domains of GST-Grb2 immobilized on beads, and then protein precipitates were immunoblotted with anti-Cbl antibody. As shown in Fig. 5, both SH3 domains are able to bind to Cbl in lysates of unstimulated cells, although based on our several experiments, Cbl has an in vitro preference for the amino-terminal SH3 domain of Grb2. In addition, Fig. 5demonstrates that in UCHT1-treated cells the amount of Cbl associated with both SH3 domains of Grb2 is decreased. Donovan et al.(21) also reported that the amino-terminal SH3 domain of Grb2 binds less Cbl from the lysate of vanadate-treated Jurkat cells than from the lysate of unstimulated cells(21) . These data suggest that in activated Jurkat cells the affinity of tyrosine-phosphorylated Cbl for the SH3 domains of Grb2 is strongly reduced (Fig. 5).
Figure 5: In vitro binding of Cbl to GST fusion proteins of carboxyl- and amino-terminal SH3 domains of Grb2. Jurkat cells were stimulated with UCHT1 antibody for 2 min or left untreated. Lysates were then subjected to affinity purification with the indicated GST fusion proteins (4 µg/point) immobilized on agarose beads. Bound proteins were then subjected to SDS-7.5% PAGE, transferred to nitrocellulose, and immunoblotted with anti-Cbl antibody.
It has been reported recently that Crk
adaptor proteins associate via their SH2 domain with a
tyrosine-phosphorylated 116-kDa protein and thus implicated in TCR
signaling(27) . One of the possible candidate proteins of this
size is Cbl. Therefore, we probed immunoprecipitates of Crk with
anti-phosphotyrosine antibody, 4G10, and anti-Cbl. As shown in Fig. 6, although some amount of Cbl is co-immunoprecipitated
with Crk from the lysates of unstimulated cells, activation of Jurkat
cells induces increased complex formation of Cbl with Crk. Immunoblot
analysis with anti-phosphotyrosine antibody confirms the increased
association of Crk with the 120-kDa phosphoprotein (Fig. 6).
Using different GST fusion proteins of Crk II, we have investigated
which domain of Crk II interacts with Cbl. Lysates of unstimulated and
activated Jurkat cells were mixed with fusion proteins immobilized on
glutathione-agarose beads, and then protein precipitates were probed
with anti-Cbl antibody. Consistent with the in vivo data, the
full-length Crk II protein was able to precipitate a small amount of
Cbl from unstimulated cells. Following TCR activation, we could detect
an increased association of Crk II GST fusion protein with Cbl (Fig. 7). The SH2 domain of Crk interacted in a manner similar
to the full-length protein with Cbl. Although we added equal amounts (4
µg) of GST fusion proteins to cell lysates, the experiment shows
that in activated cells the SH2 domain alone can bind more Cbl than the
full-length protein. The affinity of the single SH2 domain of Crk for
Cbl could be higher than that of the full-length Crk. Finally, the
SH2-deleted Crk II fusion protein (SH2) was unable to bind to Cbl
either from the lysate of activated or unstimulated T cells (Fig. 7). Using full-length GST protein of Crk-L, we have
detected inducible association of Crk-L with Cbl upon T cell activation
(data not shown), suggesting that all members of the Crk family are
able to form complexes with Cbl via their SH2 domains.
Figure 6: Cbl associates with Crk in intact Jurkat cells. T cells were stimulated with anti-CD3 antibody UCHT1 for 2 min. Proteins were then immunoprecipitated with anti-Crk antibody. Following SDS-7.5% PAGE and transfer to nitrocellulose, samples were analyzed by anti-Cbl antibody and anti-phosphotyrosine antibody 4G10. For control immunoprecipitation, anti-GRF antibody was used.
Figure 7: Crk SH2 domain interacts with Cbl from activated T cell lysates. T cells were activated with UCHT1 antibody for 2 min. GST, GST-Crk II, GST-Crk SH2, and GST-Crk II SH2 mutant fusion proteins bound to glutathione-agarose beads were added to cell lysates. Proteins bound to the beads were separated by SDS-7.5% PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-Cbl antibody.
Cbl, the protein product of the c-cbl proto-oncogene, has recently been implicated in several tyrosine kinase signaling pathways(21, 28, 29) . It has also been reported that Cbl can bind to two adaptor proteins, Grb2 and Nck, via their SH3 domains in T lymphocytes and HL60 cells, respectively(21, 24) . Our data are consistent with these reports in that Cbl associates with Grb2 in unstimulated T cells in vitro and in vivo, in an SH3 domain-dependent manner. However, upon CD3 activation of Jurkat cells, Cbl rapidly and transiently dissociates from Grb2 (Fig. 1, Fig. 2, and Fig. 4A). Agonist-induced dissociation of a proline-rich domain-containing protein from Grb2 has not previously been reported for any protein in any cell type. Donovan et al.(21) have shown that Cbl undergoes a rapid tyrosine phosphorylation in response to TCR stimulation. We have demonstrated that the kinetics of Cbl phosphorylation correlate remarkably with the kinetics of Grb2/Cbl dissociation.
The mechanism by which Cbl dissociates from Grb2 upon T cell activation is unknown but may relate to tyrosine phosphorylation of Cbl. Phosphorylation of Sos exchange protein on serine/threonine residues has been reported (30, 31) to result in the disassembly of Sos from Grb2. This finding suggests that conformational changes in the structure of a proline-rich domain-containing protein due to phosphorylation may cause its dissociation from Grb2. In the case of Sos phosphorylation either in fibroblasts or in T cells, this mechanism seems to be involved in the negative feedback regulation of Ras signaling pathway(30, 31, 32) . By contrast, we show here that a TCR agonist induces a rapid and transient phosphorylation of Cbl and its dissociation from Grb2. Therefore, functionally the dissociation of Cbl from Grb2 is completely different from the disassembly of Sos from Grb2.
While this manuscript was in preparation, association of Cbl with Grb2 and phosphatidylinositol 3`-kinase in Jurkat cells has been reported (33) . In contrast with our results, Cbl and Grb2 have been shown to form a constitutive complex regardless of the activation state of Jurkat cells. One possible explanation is that we stimulated the cells via their TCR, while Meisner et al.(33) used co-stimulation of the TCR and CD4 receptors. Cross-linking of both the TCR and CD4 receptors may result in a more intensive phosphorylation of Cbl, or alternatively, phosphorylation may occur on different sites. Based on in vitro data with SH2 and SH3 domains of Grb2 GST fusion proteins, tyrosine-phosphorylated Cbl can bind to both SH2 and SH3 domains of Grb2 in fully activated cells(33) . In CD3-activated Jurkat cells, we have not detected binding of Cbl to the SH2 domain of Grb2 (data not shown).
In addition, we show that
tyrosine-phosphorylated Cbl binds to another SH2/SH3 domain-containing
adaptor protein, Crk, via its SH2 domain. Sawasdikosol et al.(27) have reported that in activated T lymphocytes a
phosphotyrosine protein with a molecular mass of 116 kDa binds to the
SH2 domain of Crk. Therefore, it is highly likely that this 116-kDa
phosphotyrosine protein is identical with Cbl. Using SH2 mutant Crk for
protein precipitation, we failed to detect the association of Cbl with
the Crk SH3 domains. This suggests that the mechanism by which Cbl
binds to Crk is completely different from the association of Cbl with
Grb2 and Nck. At present, the role of Crk in regulation of Cbl is
unclear. Two other phosphotyrosine proteins have been shown to be
associated with Crk, in a similar SH2-dependent manner, paxillin and
the Crk-associated substrate
p130(11, 12) . In addition, Crk proteins
can be associated, via their SH3 domains, with two Ras-specific guanine
nucleotide exchange proteins, Sos and
C3G(8, 9, 10) . Therefore, phosphorylated Cbl
in complex with Crk/Sos or Crk/C3G might be involved in Ras signaling
pathways in T cells.
It has been suggested that Cbl is a nuclear
protein which may function as a transcripton factor(23) : it
contains a possible nuclear localization sequence, a putative leucine
zipper at the carboxyl terminus, and a zinc finger-related protein
motif(29) . Therefore, Cbl or tyrosine-phosphorylated Cbl could
be transported into the nucleus. This is supported by the fact that the
truncated form of Cbl, the protein product of v-cbl oncogene,
can enter the nucleus and bind DNA(23) . Moreover, it has been
recently suggested that the carboxyl terminus of Cbl is involved in the
retention of Cbl in the cytoplasm and the inhibition of DNA
binding(23) . The truncation in the sequence of v-Cbl occurs at
the carboxyl-terminal domain which contains several proline-rich
motifs. These motifs are likely to be responsible for the interaction
with the SH3 domains of Grb2. Taken together, this suggests that Grb2
might have a role in the regulation of Cbl in quiescent cells by acting
as a retention factor. However, every effort so far to detect Cbl in
the nucleus has been unsuccessful. Tanaka et al.(28) have very recently reported that in macrophages
stimulated via their Fc receptor or in HER14 cells activated with
EGF, Cbl was found to translocate from the cytoplasm to the trans-Golgi region of the cells. Further studies will
therefore elucidate the possible role of Cbl in complexes with
different adaptor proteins in tyrosine kinase signaling pathways.