The role of cbl-b in signaling by the epidermal growth factor receptor (EGFR) was studied and compared with c-cbl. We demonstrate in vivo, that cbl-b, like c-cbl, is phosphorylated and recruited to the EGFR upon EGF stimulation and both cbl proteins can bind to the Grb2 adaptor protein. To investigate the functional role of cbl proteins in EGFR signaling, we transfected cbl-b or c-cbl into 32D cells overexpressing the EGFR (32D/EGFR). This cell line is absolutely dependent on exogenous IL-3 or EGF for sustained growth. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls. This growth inhibition by cbl-b was the result of a dramatic increase in the number of cells undergoing apoptosis. Consistent with this finding, cbl-b overexpression markedly decreased the amplitude and duration of AKT activation upon EGF stimulation compared to either vector controls or c-cbl overexpressing cells. In addition, the duration of EGF mediated MAP kinase and Jun kinase activation in cells overexpressing cbl-b is shortened. These data demonstrate that cbl-b inhibits EGF-induced cell growth and that cbl-b and c-cbl have distinct roles in EGF mediated signaling.
The c-cbl proto-oncogene is the cellular homolog of the v-cbl oncogene, the transforming gene of the Cas NS-1 murine retrovirus, which causes pre B cell lymphomas and myelogenous leukemia in mice and transforms NIH3T3 cells (Blake et al., 1993; Langdon et al., 1989). The transforming v-cbl protein is a gag-v-cbl fusion protein containing only the N-terminal 40% of c-cbl (Blake et al., 1993). It is believed that the transforming cbl protein acts as a dominant inhibitor of the normal cbl protein function (Miyake et al., 1997). The c-cbl protein is phosphorylated upon activation of a variety of receptors which signal via protein tyrosine kinases (PTK) including the EGF, B-Cell, CSF-1, Fcγ, c-Kit, PDGF and T-Cell receptors (reviewed in Miyake et al., 1997; Smit and Borst, 1997). c-cbl also interacts with the activated receptors and this interaction is mediated both by association with the adaptor protein Grb2 and by direct binding of a unique phosphotyrosine binding (PTB) domain in the N-terminus of c-cbl (reviewed in Miyake et al., 1997; Smit and Borst, 1997). The c-cbl protein has also been shown to associate with a variety of SH2 and SH3 proteins involved in signal transduction including CRK, Fyn, Lck, NCK, PI-3-Kinase and Shc (Miyake et al., 1997; Smit and Borst, 1997). Thus, c-cbl appears to be an important molecule involved in many signal transduction pathways.
Developmental studies in C. Elegans and Drosophila melanogaster have demonstrated that the cbl family proteins are able to act as inhibitors of epidermal growth factor receptor (EGFR) mediated development (Hime et al., 1997; Meisner et al., 1997; Yoon et al., 1995). Genetic experiments in C. elegans have indicated that the function of the cbl protein is at the level of the receptor and the Sem5 protein, placing the cbl proteins at an early point in the signal transduction cascade (Jongeward et al., 1995). In addition, one oncogenic form of c-cbl, 70Z-c-cbl, has been shown to stimulate the kinase activity of both resting and stimulated EGFR (Thien and Langdon, 1997). There are conflicting data in the literature that mammalian c-cbl protein may directly inhibit EGFR autophosphorylation (Thien and Langdon, 1997; Ueno et al., 1997). The biological role of the normal c-cbl protein in EGF signaling remains to be elucidated.
We recently cloned human cbl-b, a homolog of the c-cbl proto-oncogene (Keane et al., 1995). cbl-b shares several structural similarities with c-cbl, most notably in the N-terminal PTB domain, the C3HC4 zinc finger and the proline rich domain (Keane et al., 1995). We have previously shown that cbl-b, like c-cbl, binds to a variety of signaling proteins in vitro via SH3 interactions (Keane et al., 1995). Here we compare the role of these two mammalian cbl family members in signaling by the EGFR.
Interaction of cbl-b and c-cbl with the EGFR
Other investigators have previously shown that c-cbl is phosphorylated and recruited to the EGFR upon EGF stimulation (Bowtell and Langdon, 1995; Fukazawa et al., 1996; Galisteo et al., 1995; Khwaja et al., 1996; Levkowitz et al., 1996; Meisner and Czech, 1995; Odai et al., 1995a; Soltoff and Cantley, 1996; Tanaka et al., 1995; Ueno et al., 1997). To compare the interaction of cbl-b and c-cbl with the EGFR, HA-epitope tagged cbl-b or HA-epitope tagged c-cbl was transfected along with the EGFR into human embryonic kidney cells expressing the SV40 large T-antigen (293T). The EGFR was also transfected alone for comparison. The cells were then starved and stimulated with EGF and the resultant lysates used to compare the interactions between the EGFR and each cbl protein. These lysates were immunoblotted and probed with anti-phosphotyrosine (anti-pty), anti-cbl-b, anti-c-cbl, anti-EGFR and anti-HA (Figure 1a). Lysates from cells transfected with either cbl-b and the EGFR or c-cbl and the EGFR each showed prominent EGF-induced tyrosine phosphoproteins at ∼180 kDa and ∼120 kDa corresponding to the positions of the EGFR and the cbl proteins, respectively (Figure 1a, top panel). The unique phosphoproteins seen in the c-cbl and cbl-b transfected cells represent degradation products of the respective cbl proteins. This was determined by sequentially reprobing the blot with antibodies for c-cbl or cbl-b which recognize epitopes at the N and C termini of the respective proteins (data not shown). In contrast, the cells expressing the EGFR alone showed prominent phosphorylation of the EGFR but did not have the prominent 120 kDa corresponding to the cbl proteins (longer exposure did show a phosphorylated 120 kDa band corresponding to the endogenous cbl proteins). Cells overexpressing either cbl-b or c-cbl did not have consistent significant differences in the phosphorylation levels of the stimulated EGFR compared to each other or compared to cells overexpressing the EGFR alone (Figure 1a). Interestingly, 293T cells transfected with the EGFR alone consistently had significantly higher levels of phosphorylation of the unstimulated receptor compared to cells expressing the EGFR and either c-cbl or cbl-b (Figure 1a).
Several other observations may be made from the data in this figure. First, the anti-HA antibody shows that the transfected proteins are expressed at easily detectable levels. Second, cbl-b migrates at a slightly higher position than c-cbl. Finally, the lower band seen with the anti- cbl-b antibody is likely to be degraded protein since it is not seen with the anti-HA antibody (the HA epitope is on the C-terminus of the protein) nor when the protein is immunoprecipitated (see Figure 1b).
The lower panels in Figure 1a also suggest that the anti-cbl-b and anti-c-cbl antibodies specifically identify their respective proteins on immunoblots. However, longer exposure of the blots probed for c-cbl or cbl-b revealed expression of the endogenous c-cbl and cbl-b respectively in 293 cells at levels ∼20 – 30-fold less than the transfected proteins (data not shown). This raised the possibility that the antibodies did have some cross reactivity. To confirm the specificity of the two antibodies for both immunoprecipitation and immunoblotting, cbl-b and c-cbl were cotransfected into 293T cells and lysates were immunoprecipitated with each antibody and with non-immune rabbit serum (nrs) (Figure 1b). Equal amounts of the precipitates were run on two parallel gels and then immunoblotted with anti-cbl-b and anti-c-cbl antibodies. The data shown indicate that both antisera are specific for both immunoprecipitation and immunoblotting. These data also indicate that there is no significant interaction between cbl-b and c-cbl since there is no evidence of co-immunoprecipitation of one with the other.
To further compare the interactions of cbl-b and c-cbl with the EGFR, we immunoprecipitated the EGFR from EGF stimulated 293T lysates containing either cbl-b or c-cbl (Figure 2, top panels). Both cbl-b and c-cbl (heavy arrows) were co-precipitated with the EGFR (light arrows) in the stimulated cells but not in the unstimulated cells. There was no difference in the kinetics of the recruitment to the EGFR at the time points analysed. When these immunoprecipitates were probed with an anti-phosphotyrosine (anti-pty) antibody, phosphorylated proteins were seen that migrated at the sizes of EGFR and cbl proteins. To confirm that cbl-b and c-cbl were phosphorylated upon EGF stimulation, the transfected cbl proteins were immunoprecipitated with the anti-HA antibody and probed with the anti-pty antibody (Figure 2, bottom panels). Both cbl proteins are phosphorylated upon EGF stimulation. A phosphoprotein that migrates at the size of the EGFR is also seen when the cbl proteins are immunoprecipitated. Together these data demonstrate that both cbl-b and c-cbl are similarly phosphorylated and recruited to the EGFR upon EGF stimulation.
c-cbl is recruited to the activated EGFR predominantly through its interaction with the adaptor protein Grb2 (reviewed in Miyake et al., 1997; Smit and Borst, 1997). To test whether cbl-b also interacts with Grb2, 293T cells were transfected with the EGFR, cbl-b and the EGFR, or c-cbl and the EGFR and then the cells were starved or stimulated with EGF. The respective cbl proteins were immunoprecipitated with the anti-HA antibody and the precipitates were probed for Grb2. Grb2 co-precipitated with cbl-b and c-cbl from both starved and EGF stimulated lysates (Figure 3, top panel). Only slight enhanced binding of Grb2 was seen in the cbl-b precipitate from EGF stimulated cells and no EGF enhanced binding was seen between Grb2 and c-cbl. The anti-pty blot demonstrated that the precipitated cbl proteins became heavily phosphorylated upon EGF stimulation and that they associated with the EGFR as demonstrated below (Figure 3, middle panel). The cbl proteins were equally precipitated by the anti-HA antibody (Figure 3, bottom panel) and the lack of Grb2 in the precipitates from the cells transfected with the EGFR alone indicates that the precipitation of Grb2 was the result of an interaction between the cbl protein and Grb2. Using a Gst-Grb2 fusion protein to precipitate in vitro translated deletion mutants of cbl-b we have localized the binding of Grb2 to the proline rich C-terminus half of the cbl-b protein (data not shown). The N-terminus of c-cbl contains a unique PTB which has been shown to directly bind to tyrosine phosphorylated proteins (Lupher et al., 1996, 1997). A construct of cbl-b containing only the first 349 amino acids (including the conserved PTB) was able to bind to the activated EGFR but was unable to interact with the Gst-Grb2 fusion protein (data not shown). Thus, like c-cbl, cbl-b is able to bind to the EGFR through both an adaptor mediated and PTB domain mediated mechanism.
Since both cbl-2 and c-cbl are phosphorylated and recruited to the EGFR upon stimulation and interact with the receptor through similar mechanisms, we investigated whether the two proteins compete for their interaction with the EGFR. 293T cells were transfected with c-cbl and the EGFR in the presence or absence of an excess of cbl-b (Figure 4a) or with cbl-b and the EGFR in the presence or absence of an excess of c-cbl (Figure 4b) and then the cells were starved and then stimulated with EGF. Lysates from each transfection were immunoprecipitated with the anti-EGFR antibody. Less c-cbl co-precipitates with the EGFR in the presence of excess cbl-b than in the absence of cbl-b (Figure 4a) and similarly, less cbl-b co-precipitates with the EGFR in the presence of excess c-cbl than in the absence of c-cbl (Figure 4b). The excess of competing cbl protein was demonstrated by probing the lysates with the anti-HA antibody which detects both proteins (Figure 4, lysate panels). These data suggest that cbl-b and c-cbl compete for binding to the EGFR.
Interestingly, when lysates containing both cbl proteins were probed with anti-pty the level of EGF induced phosphorylation of the 120 kDa protein was not significantly increased compared to cells expressing only one or the other of the cbl proteins (Figure 4a and b, lysate panels). Immunoprecipitation of c-cbl from the lysates in Figure 4a or cbl-b from the lysates in Figure 4b revealed that in the presence of excess of the other cbl protein, phosphorylation of the precipitated protein was decreased (data not shown). This suggests that the cbl proteins are phosphorylated by the same kinases and compete with one another as substrates.
Effects of cbl-b and c-cbl on EGF induced cell growth of 32D/EGFR cells
In order to investigate the functional role of the interaction of the cbl proteins with the EGFR, we transfected cbl-b or c-cbl into 32D cells which overexpress the EGFR. The 32D cell line is a murine hematopoietic cell line which is absolutely dependent on exogenous IL-3 for sustained growth and it rapidly undergoes apoptosis in the absence of IL-3 (Greenberger et al., 1983; Ihle et al., 1981, 1982; Prystowsky et al., 1982). 32D cells do not normally express any endogenous EGFR (or other members of the EGFR family) and do not grow in EGF (Alimandi et al., 1997). In contrast, 32D cells overexpressing the EGFR (32D/EGFR) can be grown in the presence of either IL-3 or EGF (Pierce et al., 1988). These 32D/EGFR cells, used in our experiments, were maintained in growth media supplemented with 5% conditioned medium containing IL-3 (which will be referred to as IL-3). 32D/EGFR cells were transfected with either cbl-b, c-cbl, or a vector control and stable clones were selected which were able to grow in medium containing IL-3 and G418. These clones were then analysed for their ability to grow in EGF or IL-3. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls (Figure 5a and b), while only slight inhibition of growth of cbl-b clones was observed in IL-3. In contrast, both c-cbl and vector clones grew well in either EGF or IL-3. Even at lower concentrations of IL-3, which only stimulated growth to the same degree as EGF, there was no significant difference between the clones expressing cbl-b and those overexpressing c-cbl or the vector controls. Thus the inhibition is specific for the EGFR pathway. The expression of cbl-b, c-cbl and the EGFR in representative clones is shown in Figure 5c. Both cbl-b and c-cbl were expressed at levels five- to tenfold above that of the endogenous protein. Reprobing the blots with the anti-HA antibody demonstrated that cbl-b and c-cbl were expressed at similar levels (data not shown). There was no significant difference between the clones in the total expression (Figure 5c) or in the cell surface expression of the EGFR protein determined by flow cytometry (data not shown).
Cells overexpressing cbl-b cultured in EGF were shrunken, refractile cells with pyknotic nuclei and were similar to those seen when cultured in the absence of growth factor (Figure 6a). TUNEL assays, which detect DNA breaks characteristic of apoptosis in situ (Ben-Sasson et al., 1995), revealed that the cbl-b overexpressing cells cultured in the absence of growth factor and those cultured in EGF were undergoing apoptosis (Figure 6a, top panels). In contrast, the cbl-b clones grew well in IL-3 with little or no evidence of apoptosis. Cells overexpressing c-cbl underwent apoptosis when grown in the absence of growth factor but grew well in either EGF or IL-3 with little or no evidence of apoptosis when grown in either growth factor (Figure 6b, bottom panels). The vector control cells behaved like the cells overexpressing c-cbl (data not shown). In order to quantitate the fraction of cells undergoing apoptosis, cells were stained with propidium iodide and the fraction of cells in the sub-G1 peak was determined. A high proportion of cells in the cbl-b clones cultured in EGF were undergoing apoptosis when compared to those grown in IL-3 (Figure 6b). In contrast, only a small fraction of the cells in the c-cbl clones and vector controls undergoes apoptosis when grown in the presence of either growth factor (Figure 6b). The proportion of apoptotic cells in the cbl-b clones grown in EGF increased with time in culture but this increase was not as rapid as seen in cells grown in the absence of growth factor (Table 1).
Cell cycle analysis revealed that the large increase in apoptotic cells in cbl-b clones grown in EGF was the only clear difference between cbl-b clones and the c-cbl clones and vector controls. There was no evidence of cell cycle arrest in the cbl-b clones cultured in EGF at early (within the first 16 h in culture) or late (after several days in culture) time points. In contrast, cells cultured in the absence of growth factor showed clear evidence of G1 arrest within the first 16 h in culture. Consistent with the lack of cell cycle arrest in the cbl-b clones cultured in EGF, thymidine incorporation by the cbl-b clones cultured in EGF for 1 – 2 days was similar to that of c-cbl clones and vector controls (data not shown).
Both cbl-b and c-cbl were phosphorylated and recruited to the EGFR in the 32D/EGFR clones overexpressing these proteins (data not shown). In order to investigate the effects of cbl-b and c-cbl on EGFR signaling, we measured the EGF induced activation of MAP kinase (MAPK) and Jun kinase (JNK) in clones overexpressing cbl-b, c-cbl, or the vector control (Figure 7a and b). EGF stimulation of the 32D/EGFR cells overexpressing cbl-b induced rapid stimulation (within 5 min) of both MAPK and JNK activities and this stimulation was similar to that seen in c-cbl overexpressing cells or the vector controls. However, the MAPK and JNK activities in the cbl-b clones returned towards the baseline earlier (e.g. 30 min) than the activities in the c-cbl and vector controls.
The activation of the serine threonine kinase AKT by growth factor receptors has been shown to inhibit apoptosis (Franke et al., 1997). This activation results from the specific phosphorylation of AKT on serine 473 and threonine 308 in response to growth factor stimulation (Alessi et al., 1996). Because of the marked increase in apoptosis we observed in cbl-b clones cultured in EGF, we assayed the activation of AKT in clones overexpressing cbl-b, c-cbl, or the vector controls (Figure 8). The EGF induced activation of AKT was markedly decreased in the clones overexpressing cbl-b compared to cells overexpressing c-cbl or the vector controls. In addition, as seen with MAPK and JNK above, the duration of the activation of AKT was shortened in cells overexpressing cbl-b compared to cells overexpressing c-cbl or the vector controls.
AKT is activated downstream of phosphatidylinositol 3-kinase (PI 3-kinase) (Franke et al., 1997) and c-cbl has been shown to associate with the 85 kDa regulatory subunit (p85) of PI 3-kinase upon activation of the EGFR (Miyake et al., 1997; Smit and Borst, 1997). To test whether cbl-b interacts with the 85 kDa subunit of PI 3-kinase, 293T cells were transfected with the EGFR or cbl-b and the EGFR. The cbl-b protein was immunoprecipitated with the anti-HA antibody and the precipitates were probed for the p85 subunit of PI 3-kinase (Figure 9). The p85 subunit of PI 3-kinase co-precipitated with the heavily phosphorylated cbl-b from the EGF stimulated lysates. The lack of the p85 subunit of PI 3-kinase in the precipitates from the cells transfected with the EGFR alone indicates that the precipitation of p85 was the result of an interaction between the cbl-b protein and p85 (Figure 9, bottom panel). We were unable, however, to demonstrate any EGF induced association between either cbl-b or c-cbl and the p85 subunit of PI 3-kinase in the 32D/EGFR clones overexpressing cbl-b or c-cbl respectively (data not shown).
The cbl family of proteins is found in metazoans from nematodes to vertebrates and the proteins have several highly conserved domains including a novel N-terminal PTB motif and a zinc finger (Blake et al., 1991; Hime et al., 1997; Keane et al., 1995; Lupher et al., 1996, 1997; Meisner et al., 1997; Yoon et al., 1995). A role for the cbl proteins in EGFR signaling was first demonstrated in C. elegans by genetic studies that show that sli-1 (the cbl homolog) is a negative regulator of the Let-23 receptor tyrosine kinase (the EGFR homolog) in vulva development (Jongeward et al., 1995; Yoon et al., 1995). These developmental effects have been extended to Drosophila where the cbl homolog has been shown to associate with the Drosophila EGFR and overexpression of Drosophila cbl in the eye of Drosophila embryos inhibits EGFR dependent photoreceptor cell development (Hime et al., 1997; Meisner et al., 1997). In mammalian cells, several studies have shown that c-cbl becomes phosphorylated and recruited to the EGFR upon stimulation (Bowtell and Langdon, 1995; Fukazawa et al., 1996; Galisteo et al., 1995; Khwaja et al, 1996; Levkowitz et al., 1996; Meisner and Czech, 1995; Odai et al., 1995a; Soltoff and Cantley, 1996; Tanaka et al., 1995; Ueno et al., 1997). However, biological consequences of this interaction have not been demonstrated.
The data presented here show that cbl-b, but not c-cbl, inhibits EGFR induced growth in 32D/EGFR cells (Figure 5). These cells are absolutely dependent on growth factor (either EGF or IL-3) and rapidly undergo apoptosis in the absence of exogenous growth factor (Greenberger et al., 1983; Ihle et al., 1981, 1982; Pierce et al., 1988; Prystowsky et al., 1982). The inhibition of growth in the cbl-b clones is not associated with any cell cycle arrest but it is associated with a dramatic increase in the fraction of cells undergoing apoptosis (Figure 6). Recent findings have established that cell growth in response to growth factor stimulation requires active inhibition of apoptosis via the activation of AKT in addition to stimulation of cell cycle progression (Ahmed et al., 1997; Dudek et al., 1997; Franke et al., 1995, 1997; Hemmings, 1997; Kulik et al., 1997). EGF stimulated cells overexpressing cbl-b had a markedly decreased activation of AKT and the duration of AKT activation was foreshortened compared to cells overexpressing c-cbl or the vector controls (Figure 8). This result is consistent with the increased proportion of cells undergoing apoptosis.
The inhibition of EGFR stimulated growth in the 32D/EGFR cells by cbl-b and not by c-cbl clearly demonstrates distinct biological roles for cbl-b and c-cbl. The precise molecular mechanism by which cbl-b exerts its inhibitory effect is not yet known. In both transiently transfected cells (293T cells) and in stable clones (32D/EGFR cells), cbl-b and c-cbl are phosphorylated and recruited to the EGFR upon activation of the receptor. Both cbl proteins interacted with the Grb2 adaptor protein in the unstimulated cells and we saw a slight increase in the association between cbl-b and Grb2 upon stimulation but not between c-cbl and Grb2 (Figure 3). While some investigators have shown modest increases in the interaction of c-cbl with Grb2 upon EGF stimulation (Fukazawa et al., 1996; Khwaja et al., 1996; Meisner and Czech, 1995), others have found a constitutive interaction between c-cbl and Grb2 (Levkowitz et al., 1996; Odai et al., 1995b). The binding of the proline rich C-terminus of in vitro translated cbl-b to a Gst-Grb2 fusion protein is consistent with an SH3 mediated interaction. We also have found that the N-terminus of cbl-b, like the N-terminus of c-cbl (Miyake et al., 1997; Smit and Borst, 1997), is able to associated with the EGFR (not shown). The similarities of cbl-b and c-cbl in binding to the EGFR and Grb2 (Figures 2 and 3), and the ability of each cbl protein to decrease the binding of the other to the EGFR (Figure 4) suggest that both interact with the receptor through a common site and/or mechanism. The binding site of the cbl proteins on the EGFR has not yet been identified. There was no inhibition of EGF induced phosphorylation of the EGFR (Figure 1) suggesting that binding of cbl-b (or c-cbl) to the EGFR does not directly affect its activation by EGF. This is consistent with prior observations that c-cbl does not directly alter the phosphorylation or kinase activity of the EGFR (Thien and Langdon, 1997). However, there are some published data which suggest that c-cbl can inhibit phosphorylation of the EGFR (Ueno et al., 1997) and the reason for these differing results is unknown. Cotransfection of the cbl proteins did decrease the phosphorylation of the unstimulated EGFR when the EGFR was overexpressed in 293T cells indicating that the cbl proteins may indeed inhibit activation of the EGFR. No such high levels of phosphorylated unstimulated EGFR were seen in the vector controls or parental cell line of the 32D/EGFR cells. This suggests that the high level of phosphorylated unstimulated EGFR may be unique to 293T cells or a consequence of the high levels of expression of the EGFR obtained upon transient transfection. The significance of the decrease in phosphorylation of the unstimulated EGFR by cbl-b and c-cbl in the 293T cells remains to be determined.
Overexpression of cbl-b inhibited activation of downstream MAPK, JNK and AKT pathways (Figures 7 and 8). cbl-b has been shown to inhibit JNK activation by Vav (Bustelo et al., 1997) but there are no published data addressing the effects of cbl-b on MAPK or AKT. Previous reports do not show inhibition of MAPK activation by c-cbl in NIH3T3 cells (Bowtell and Langdon, 1995; Thien and Langdon, 1997; Ueno et al., 1997). AKT activation was more profoundly affected than MAPK activation and this is consistent with the increase in apoptosis and absence of cell cycle arrest. AKT activation is downstream of growth factor induced activation of PI 3-kinase (Franke et al., 1997). c-cbl has been shown to associate with the 85 kDa regulatory subunit of PI 3-kinase upon activation of a variety of receptors, including the EGFR (Miyake et al., 1997; Smit and Borst, 1997) and c-cbl has been demonstrated to enhance IL-4 induced PI 3-kinase activity and mitogenic and survival signals in Ba/F3 cells (Ueno et al., 1998). We have demonstrated an EGF induced association between cbl-b and the p85 regulatory subunit of PI 3-kinase in transiently transfected 293T cells that overexpress cbl-b (Figure 9). cbl-b has one consensus binding site for the SH2 domain of PI 3-kinase (Y363CEM) which is conserved between all of the cbl family of proteins. However, cbl-b lacks the second binding site found in c-cbl (Y731 EAM) which is believed to be the site at which PI 3-kinase binds to c-cbl (Liu et al., 1997). We were unable to demonstrate any interaction of either cbl-b or c-cbl with the p85 subunit of PI 3-kinase in 32D/EGFR cells. We were also unable to demonstrate any direct effect of cbl-b on the binding of c-cbl to p85 in the transiently transfected 293T cells (unpublished observation). Thus, the mechanism by which cbl-b inhibits AKT activation in the transfected 32D/EGFR cells remains to be determined. The recruitment of cbl-b to the EGFR upon activation and the inhibition of activation of multiple downstream kinases suggests that cbl-b functions at a step in the pathway close to the receptor. The foreshortening of MAPK, JNK and AKT activation by cbl-b suggests that cbl-b may enhance feedback inhibition of the EGFR.
Overall, our data demonstrate that a mammalian cbl protein, cbl-b is able to inhibit EGFR-induced growth and this inhibition is due to a failure to activate anti-apoptotic pathways. These data further demonstrate that while these proteins share some structural and biochemical similarities, there are major functional differences between the cbl-b and c-cbl proteins.
Materials and methods
Rabbit polyclonal anti-cbl-b (H121; Santa Cruz Biotechnology) and anti-c-cbl (C-15; Santa Cruz Biotechnology) were used for both immunoblotting and immunoprecipitation. Mouse monoclonal anti-HA (12CA5; Boehringer Mannheim) and anti-EGFR (Ab3; Oncogene Science) were used for immunoprecipitation and rabbit polyclonal anti-HA antibody (Y-11, Santa Cruz Biotechnology), anti-EGFR (1005, Santa Cruz Biotechnology), anti-p85 subunit of PI 3-kinase (06-195; Upstate Biotechnology), and anti-Grb2 (C-23; Santa Cruz Biotechnology) were used for immunoblotting. Horseradish peroxidase linked anti-phosphotyrosine (4G10; Upstate Biotechnology Inc.) was used for immunoblotting. Horseradish peroxidase linked donkey anti-rabbit Ig (Amersham) was used along with ECL detection reagent (Super Signal; Pierce) to visualize immunoblots. Anti-ERK antibody (SC-154, Santa Cruz Biotechnology) was used to immunoprecipitate active ERK1 and ERK2 for the immunocomplex MAPK assay.
A nine amino acid epitope tag from the influenza virus hemagglutin protein (HA) (Wilson et al., 1984) was added to the C-terminal of the full length human cbl-b open reading frame (Keane et al., 1995) by PCR and the cDNA was cloned into pCEFL, a mammalian expression vector with the elongation factor promoter and a neomycin selectable marker (provided by Dr Silvio Gutkind). The construct was sequenced to verify that there were no mutations introduced. HA-tagged c-cbl was provided by Dr Wallace Langdon (Andoniou et al., 1996). This construct was also cloned into the pCEFL vector. The GST-cjun79 fusion construct used as a substrate in the JNK assay was provided by Dr Silvio Gutkind. The fusion protein was purified from bacterial lysates as previously described (Coso et al., 1995).
Immunoblotting and immunoprecipitation
Immunoblotting was performed as previously described (Ausubel et al., 1994) and detection by chemiluminescence was performed using ECL (Amersham) according to the instructions provided. Briefly, for immunoprecipitation protein from total cell lysate was incubated for 30 min on ice with antibody. Immune complexes were recovered by incubation with protein A/G+ agarose beads (Santa Cruz Biotechnology) at 4°C for 1 h with tumbling. Immune complexes were washed five times in cold lysis buffer, resuspended in 2× loading buffer (Promega), boiled for 5 min, and then resolved by 10% SDS – PAGE. The gels were transferred to nitrocellulose membranes (Schleicher and Schuell) or to PVDF membranes (Immobilon P, Millipore).
293 cells transfected with the SV40 large T antigen (293T) (provided by Mike Erdos) were maintained in culture in DMEM supplemented with 10% fetal calf serum and 1% Penicillin-Streptomycin (Pen-Strep) and were transfected with various constructs using calcium phosphate (5 Prime→3 Prime, Inc.) according to the protocol included with the reagents. To measure the effects of EGF stimulation, 293T cells were grown to 70% confluence and serum starved in DMEM supplemented with 0.1% bovine serum albumin (BSA) and 1% Pen-Strep for 4 h. One hundred ng/ml of recombinant human EGF (Collaborative Biomedical Products) was added for the times indicated, the cells were washed two times in ice-cold PBS containing 0.2 mM sodium orthovanadate and the cells were lysed in ice-cold lysis buffer (10 mM Tris HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X 100, 10% Glycerol, 1 mM 4-(2 aminoethyl) benzenesulfonyl fluoride (AEBSF), 20 μg/ml Leupeptin, 20 μg/ml Aprotinin, 10 μg/ml Pepstatin, 2 mM sodium orthovanadate). The lysates were cleared of debris by centrifugation at 16 000 g for 15 min at 4°C.
32D/EGFR cells were maintained in culture in RPMI 1640 supplemented with 15% fetal calf serum, 5% WEHI 3B conditioned medium and 1% Pen-Strep. The WEHI 3B cell line was maintained in RPMI 1640 supplemented with 15% fetal calf serum and 1% Pen-Strep. WEHI 3B conditioned media containing IL-3 was produced by culturing the WEHI 3B cells to a high density and then harvesting the supernatants. 32D/EGFR cells were transfected by electroporation as previously described (Pierce et al., 1988) and stable clones were selected and maintained in growth media supplemented with 750 μg/ml G418 (GIBCO – BRL). To assess the growth in different conditions, cells were pelleted, resuspended in RPMI 1640 containing 15% FCS, 1% Pen-Strep, but no growth factor. Cells were seeded in 24 well plates at 1×104 cells/well. Either EGF (10 ng/ml), IL-3 (5% conditioned media) or no growth factor was added. The cells were incubated for the indicated time and then cells were harvested and counted using trypan blue to assess viability. Each data point was done in triplicate.
TUNEL assays to detect fragmented DNA in situ (Ben-Sasson et al., 1995) were peformed on cell cytospins using the In Situ Death Detection Kit (Boehringer Mannheim).
MAPK was assayed as previously described (Crespo et al., 1994). Briefly, cells were stimulated with EGF (100 ng/ml) for the indicated time, washed in ice-cold PBS containing 0.2 mM sodium orthovanadate, and the cells were lysed in ice-cold lysis buffer (20 mM HEPES, pH 7.5, 2.5 mM MgCl2, 10 mM EGTA, 40 mM β-glycerophosphate, 1.0% Nonidet P-40, 1 mM DTT, 1 mM AEBSF, 20 μg/ml Leupeptin, 20 μg/ml Aprotinin, 2 mM sodium orthovanadate). Cleared lysates were incubated at 4°C with tumbling with anti-Erk antibody and protein A/G+ agarose beads for 1 h. The beads were washed three times with PBS containing 1% Nonidet P-40 and 2 mM sodium orthovanadate, once with 100 mM Tris (pH 7.5) containing 0.5 M LiCl, and once with kinase reaction buffer (12.5 mM MOPS, pH 7.5, 7.5 mM MgCl2, 3.3 μM DTT, 12.5 mM β-glycerophosphate, 0.5 mM EGTA, 0.5 mM NaF, 0.5 mM sodium orthovanadate). The beads were resuspended in 30 μl of kinase reaction buffer containing 10 μCi [γ-32P]ATP (3000 Ci/mmol; Amersham), 20 μM cold ATP and 1.5 mg/ml Myelin Basic Proein (MBP) as a substrate. The reactions were incubated at 30°C for 20 min and terminated by the addition of 15 μl of 4×Laemmli buffer. The samples were heated to 95°C for 5 min and analysed by SDS – PAGE on 12% gels. The gels were dried and the phosphorylated MBP was assessed by autoradiography using AR film and an intensifying screen (Kodak).
JNK was assayed as previously described (Coso et al., 1995; Crespo et al., 1994). Briefly, cells were stimulated with EGF (100 ng/ml) for the indicated time, washed in ice-cold PBS containing 0.2 mM sodium orthovanadate and the cells were lysed in ice-cold lysis buffer (25 mM HEPES, pH 7.5, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 20 mM β-glycerophosphate, 0.1% Triton X 100, 1 mM AEBSF, 20 μg/ml Leupeptin, 0.1 mM sodium orthovanadate). Cleared lysates were incubated at 4°C with tumbling with 1 μg of GST-cjun79 protein bound to glutathione-agarose beads for 3 – 4 h. The beads were washed three times with PBS containing 1% Nonidet P-40 and 1 mM sodium orthovanadate, once with 100 mM Tris (pH 7.5) containing 0.5 M LiCl, and once with kinase reaction buffer (25 mM HEPES pH 7.5, 20 mM MgCl2, 2 mM DTT, 20 mM β-glycerophosphate, 0.1 mM sodium orthovanadate). The beads were resuspended in 30 μl of kinase reaction buffer containing 1 μCi [γ-33P]ATP (3000 Ci/mmol; Amersham) and 50 μM cold ATP, incubated at 30°C for 20 min, and the reactions were terminated by the addition of 15 μl of 4×Laemmli buffer. The samples were heated to 95°C for 5 min and analysed by SDS – PAGE on 12% gels. The gels were dried and the phosphorylated GST-cjun79 was assessed by autoradiography using Biomax MR film (Kodak).
AKT activation was assessed by the specific phosphorylation of AKT on serine 473 using the Phosphoplus AKT (Ser473) antibody kit (New England Biolabs) according to the method provided with the kit.
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We would like to thank Silvio Gutkind for his advice, discussion and critical review of this manuscript. We would also like to thank Nelson Ellmore and Veena Kapoor for assistance with cell culture and flow cytometry.
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