Oncogene (1997) 15, 2909 ± 2919 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Tyrosine kinase activity of the EGF receptor is enhanced by the expression of oncogenic 70Z-Cbl Christine BF Thien and Wallace Y Langdon Department of Pathology, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6907, Australia The 120 kD product of the c-Cbl oncogene is a signal transduction across the cell membrane into the prominent substrate of protein tyrosine kinases that cytoplasm. Furthermore, Cbl has been found to lacks a known catalytic activity but possesses an array associate constitutively or inductively with many of binding sites for cytoplasmic signalling proteins. An signalling proteins such as the Grb2, Crk and Nck oncogenic form of Cbl was recently identi®ed in the 70Z/ adaptor proteins, members of the Src, Abl and Syk 3 pre-B cell lymphoma which has a small deletion at the tyrosine kinase families, the p85 regulatory subunit of N-terminus of the Ring ®nger domain. This form of Cbl, PI 3-kinase and 14-3-3 proteins (Donovan et al., 1994; termed 70Z-Cbl, exhibits an enhanced level of tyrosine de Jong et al., 1995; Buday et al., 1996; Rivero- phosphorylation compared with c-Cbl. Here we demon- Lezcano et al., 1994; Ribon et al., 1996; Andoniou et strate that the expression of 70Z-Cbl induces a tenfold al., 1994, 1996; Smit et al., 1996; Tanaka et al., 1996; enhancement in the kinase activity of the EGF receptor Tsygankov et al., 1996; Fournel et al., 1996; Ota et al., in serum-starved and EGF-stimulated cells. In serum- 1996; Kim et al., 1995; Meisner et al., 1995; Solto and starved cells this results in EGF receptor autopho- Cantley, 1996; Liu et al., 1996). To date however a sphorylation and the recruitment of Grb2, Shc and Sos1 de®nite function for Cbl has not emerged from these but does not induce a corresponding increase in MAP studies. The most revealing clue about the function of kinase activity. Furthermore the expression of 70Z-Cbl Cbl has come from genetic studies in C. elegans where greatly enhances EGF-induced tyrosine phosphorylation the Cbl homologue, Sli-1, has been identi®ed as a of the protein tyrosine phosphatase SHP-2. We also negative regulator of the Let-23 receptor tyrosine show that the Cbl/EGF receptor complex is predomi- kinase (Jongeward et al., 1995; Yoon et al., 1995). nantly associated with CrkII and is distinct to the Grb2/ Importantly these experiments have demonstrated that Shc/Sos1 complex that associates with the EGF Sli-1 acts at the level of Let-23 and the Sem5 adaptor receptor. These ®ndings therefore demonstrate a bio- (Jongeward et al., 1995), a ®nding consistent with chemical eect of an oncogenic Cbl protein and support mammalian studies that place Cbl at an initiating point predictions from C. elegans that Cbl functions as in tyrosine kinase mediated signal transduction. regulator of receptor tyrosine kinases. Studies of oncogenic forms of Cbl have also provided clues about Cbl function. Cbl can be Keywords: growth factor; oncogene; receptor; signal converted to an oncogenic protein either by a large transduction; tyrosine kinase carboxy truncation that generated v-Cbl, or by a small internal deletion at the amino terminus of the Ring ®nger that was identi®ed in a mutant allele of Cbl from the mouse pre-B cell lymphoma line, 70Z/3 (Figure 1a) Introduction (Langdon et al., 1989; Blake et al., 1991; Andoniou et al., 1994). The v-Cbl truncation removes a large The product of the c-Cbl proto-oncogene has been proline-rich SH3-binding region and the Ring ®nger identi®ed as a ubiquitous substrate of protein tyrosine domain to reveal a novel phosphotyrosine binding kinases. Cbl lacks a de®ned catalytic domain but is (PTB) domain that forms a direct association with the rapidly phosphorylated on tyrosine residues following ZAP-70 tyrosine kinase and the EGF receptor (Lupher the stimulation of a wide range of cell surface receptors et al., 1996; Thien and Langdon, 1997). These studies which include growth factor receptors, immunoglobu- suggest that v-Cbl competes with c-Cbl for binding lin receptors, antigen receptors and integrin receptors sites on activated receptor complexes, and that (Donovan et al., 1994; Tanaka et al., 1995; Galisteo et transformation could involve a dominant negative al., 1995; Bowtell and Langdon, 1995; Odai et al., mechanism that blocks the putative regulatory role of 1995; Marcilla et al., 1995; Wang et al., 1996; Cory et c-Cbl. Indeed we have found that v-Cbl transformation al., 1995; Panchamoorthy et al., 1996; Kontani et al., is dependent on the expression of this protein at high 1996; Ota et al., 1996; Ojaniemi et al., 1997). Indeed in levels (WL, unpublished). In contrast, transformation many cell types Cbl appears to be one of the most by 70Z-Cbl appears to involve a positive signalling prominent and rapidly phosphorylated substrates of mechanism. This protein exhibits a markedly enhanced protein tyrosine kinases (Andoniou et al., 1994; level of tyrosine phosphorylation under conditions of Donovan et al., 1994; Panchamoorthy et al., 1996). minimal growth factor stimulation, and has the This suggests a pivotal role in early events following capability to transform ®broblasts at protein levels where v-Cbl transformation is not evident (Andoniou et al., 1994; Bowtell and Langdon, 1995). Recently the Correspondence: WY Langdon eects of 70Z-Cbl on the transcriptonal activation of Received 2 June1997; revised 1 August 1997; accepted 1 August 1997 nuclear factor of activated T cells (NFAT) were 70Z-Cbl enhancement of EGF receptor kinase activity CBF Thien and WY Langdon 2910 The mechanism by which 70Z-Cbl mediates fibro- a blast transformation and Ras-dependent activation of NFAT remains to be resolved. However the elevated level of tyrosine phosphorylation of 70Z-Cbl protein compared to c-Cbl has provided direction for further investigation. We originally hypothesized that the enhanced tyrosine phosphorylation was due to an intrinsic property of 70Z-Cbl which increased its accessibility to tyrosine kinases (Andoniou et al., 1994). In this study we have further investigated the mechanism of 70Z-Cbl tyrosine phosphorylation using b NIH3T3 ®broblasts expressing the human epidermal growth factor receptor (EGFR). The ®ndings presented here show that the 70Z-Cbl protein markedly enhances the kinase activity of the EGFR and that this results in an increase in the tyrosine phosphorylation of the EGFR, Shc, Cbl and SHP-2. Results Tyrosine phosphorylation of 70Z-Cbl in serum-starved cells To determine the mechanism for the enhanced tyrosine phosphorylation of 70Z-Cbl we examined NIH3T3 cells that co-express the EGFR and HA-tagged constructs of c-Cbl, 70Z-Cbl or v-Cbl (Figure 1a). The morphology of these cells is shown in Figure 1b c d where cells with no introduced Cbl (a), or cells overexpressing HA-c-Cbl (b) exhibit a ¯at morphol- -- c-Cbl 70Z-Cbl v-Cbl -- c-Cbl 70Z-Cbl v-Cbl -- c-Cbl 70Z-Cbl v-Cbl -- c-Cbl 70Z-Cbl v-Cbl ogy, and cells expressing HA-70Z-Cbl (c) and HA-v- EGF : – – – – + + + + EGF : – – – – + + + + Cbl (d) are refractile and rounded in appearance 202 – – EGFR consistent with a transformed phenotype. These cells were grown to *90% con¯uency in complete media – c-Cbl – c-Cbl 103 – before replacement with media containing 0.5% FCS for 24 h. The cells were either left quiescent or were 68 – stimulated with 1 ng/ml of EGF for 2 min at 378C before lysis and immunoprecipitation with anti-HA antibodies. The immunoprecipitated proteins were 44 – – v-Cbl separated by SDS polyacrylamide gel electrophoresis and immunoblotted with anti-phosphotyrosine or anti- I.P. : Anti-HA I.P. : Anti-HA Blot : Anti-HA Blot : Anti-P-Tyr HA antibodies. The anti-HA immunoblot in Figure 1c shows the expression of the tagged Cbl proteins and Figure 1 (a) Diagrammatic representation of the HA-tagged c- reveals that 70Z-Cbl has the lowest level of protein Cbl, 70Z-Cbl and v-Cbl constructs used in this study. (b) Morphology of NIH3T3 cells expressing the human EGFR with expression. The anti-phosphotyrosine blot of these (a) no introduced Cbl; (b) HA-c-Cbl; (c) HA-70Z-Cbl and (d) HA- samples shows that in quiescent cells (Figure 1d, v-Cbl. (c) and (d) Enhanced tyrosine phosphorylation of 70Z-Cbl EGF7) the 70Z-Cbl protein has a markedly higher in serum-starved cells. NIH3T3 cells expressing the human EGFR level of tyrosine phosphorylation compared to c-Cbl and HA-tagged c-Cbl, 70Z-Cbl or v-Cbl constructs were grown in media containing 0.5% FCS for 24 h. Cells were left quiescent protein, even though it is expressed at a lower level, (EGF7) or were stimulated with 1 ng/ml of EGF for 2 min at and that it is associated with tyrosine phosphorylated 378C (EGF+) before lysis and immunoprecipitation with anti-HA EGFR. These results are consistent with our previous antibodies. The immunoprecipitated proteins were separated by reports that have shown a high level of 70Z-Cbl SDS polyacrylamide gel electrophoresis and immunoblotted with tyrosine phosphorylation in minimally stimulated cells anti-HA antibodies (c) or anti-phosphotyrosine antibodies (d). NIH3T3 cells expressing the human EGFR but with no (Andoniou et al., 1994; Bowtell and Langdon, 1995). introduced form of Cbl is represented by ± Following EGF stimulation there is a large increase in the amount of tyrosine phosphorylated c-Cbl protein and this protein is recruited to the activated EGFR (Figure 1d,EGF+). There is also a slight increase in investigated in Jurkat T cells. Transient expression of 70Z-Cbl tyrosine phosphorylation, however the 70Z-Cbl, but not c-Cbl or v-Cbl, was found to induce amount of immunoprecipitated protein was markedly an increase in the basal activity of NFAT which was reduced after EGF stimulation since it was not further enhanced by calcium ionophore (Liu et al., detected with anti-HA antibodies.