The ability of activated growth factor RTKs to induce cellular proliferation and differentiation is dependent on their capacity to associate with and activate a number of substrates. We have demonstrated that Neu-mediated transformation is dependent on at least four independent tyrosine autophosphorylation sites that can functionally substitute for each other (9
). Two of these functionally redundant sites (YB and YD) have been shown to specifically couple to either the Grb2 or Shc adapter protein 9
). Here we have demonstrated that YB and YD are involved in activation of the Ras/Erk signaling pathway. Although both of these phosphorylation sites are capable of recruiting the Grb2 adapter protein, only the YB phosphorylation site is dependent on a functional Grb2 protein. To further explore the biological properties of these Neu-coupled signaling pathways, we have generated transgenic mice that express activated versions of Neu coupled specifically to either the Grb2 (YB) or Shc (YD) adapter protein or a Neu receptor lacking the major Neu tyrosine autophosphorylation sites (NYPD) in the mammary epithelium. The results of these analyses revealed that both the YB and YD strains were capable of efficiently inducing mammary tumors, whereas the NYPD strains developed focal mammary tumors only after a long latency period. However, only the YB-induced mammary tumors were able to metastasize efficiently to distal sites. Taken together, these observations argue that the YB and YD phosphorylation mutants are involved in activating both distinct and common effector pathways.
The transforming potential of the different neu
add-back mutants correlated with their capacity to stimulate the Erk kinase cascade (Fig. C). Although all add-back neu
mutants were capable of efficiently activating the Erk kinase pathway, the transforming activity of three of these mutants (YB, YD, and YE) was inhibited by Rap1A, which acts as a competitive inhibitor of Ras (20
). In contrast, the YC mutant appeared refractory to Rap1A-mediated inhibition. One potential explanation for this observation is that the YC phosphorylation site recruits a signaling pathway that operates independently of the Ras signaling pathway. Indeed, it has been demonstrated that the Crk adapter protein recruits the Rap1A-specific C3G exchange factor (16
), and this in turn results in stimulation of MAP kinase pathway (22
). Given the observation that Crk specifically interacts with site C in vitro (Fig. ), it is likely that site C is actually involved in the activation of Rap1A and stimulation of the Erk kinases, thus explaining its resistance to Rap1A inhibition. Further evidence supporting the contention that site C funnels through a signaling pathway distinct from the other transforming add-back mutants stems from recent observations that reovirus infection selectively kills Ras-transformed cells through lytic virus production (8
). We and our collaborators have recently demonstrated that reovirus infection of YB-, YD-, and YE-expressing cells results in virus-mediated cell lysis but is incapable of killing cells transformed by the YC mutant (P. Lee, D. Dankort, and W. J. Muller, unpublished observations). Taken together, these data again argue that both Ras-dependent and Ras-independent pathways are involved in Neu-mediated transformation.
We have conducted further detailed mutagenesis analysis of the Grb2 and Shc binding sites to determine that the YB and YD phosphorylation sites are highly specific binding sites for these adapter proteins. The results of these analyses demonstrate that the transforming potential of these mutants is directly correlated with ability of these sites to recruit Grb2 and Shc, respectively (not shown). Although YB and YD mutants are capable of either directly or indirectly recruiting the Grb2 protein, only the YB Neu mutant is dependent on Grb2 function to stimulate DNA synthesis (Fig. ). These observations argue that Shc has the ability to induce DNA synthesis in a Grb2-independent manner. Consistent with this view, it has recently been demonstrated that tyrosines 239 and 240 within Shc are capable of stimulating mitogenesis and cell survival independent of the Grb2/Ras signaling pathway (16
). Although the Shc-associated proteins have yet to be identified, several unidentified proteins specifically associate with Shc through these tyrosine phosphorylation sites (49
). Identification of these novel proteins associated with Shc will provide important insight into the role of Shc in Neu-mediated proliferation.
Further evidence supporting the contention that Shc and Grb2 utilize distinct effector pathways stems from studies of transgenic mice expressing the YB and YD Neu mutants in the mammary epithelium. Mammary epithelial expression of either YB or YD efficiently induced mammary tumors in these strains after a latency period ranging from 102 to 152 days (Fig. ). In contrast, a maximum of 50% (depending on the NYPD line) of NYPD female transgenic mice developed focal mammary tumors after a 351-day observation period. These observations argue that coupling of Neu to either the Grb2 (YB strains) or Shc (YD strains) adapter protein is able to efficiently promote transformation of mammary epithelial cells. In fact, the 100% of the mammary tumor phenotype is higher than the 80% penetrance phenotype observed with the comparable activated neu
). The difference in biological behavior of these strains may reflect the fact that the latter activated neu
strains still carry the negative regulatory tyrosine residue (tyrosine 1028) whereas the YB and YD strains do not (9
The potent transforming activity of YD and YB strains was associated with their capacity to couple to both the Erk and Akt kinase cascades. In addition, mammary tumor progression in the YB- and YD-induced mammary tumors was also associated with a dramatic upregulation of the ErbB-3 RTK and activation of the associated PI-3′ kinase signaling pathway. Thus, stimulation of both Ras and PI-3′ kinase-dependent pathways was sufficient to promote efficient mammary tumor induction. In contrast to these observations, the NYPD-induced mammary tumors failed to exhibit significant activation of either Erk or Akt kinase, suggesting that mammary tumorigenesis in this model system occurs through signaling pathways independent of either Ras or PI-3′ kinase. Indeed, we have recently demonstrated that the NYPD mutant still retains the capacity to couple to the Src family kinases and itself retains wild-type kinase activity in vitro (Dankort and Muller, unpublished). Conceivably, stimulation of the Src family kinases may be responsible for the residual transforming activity exhibited by the NYPD neu
mutant. Indeed, it has been demonstrated that mammary epithelial expression of activated Src is capable of inducing focal mammary tumors after a long latency period (53
). Another possible mechanism by which the NYPD neu
mutant is capable of inducing mammary tumors is through reversion of specific Neu autophosphorylation sites. Indeed, previous studies have shown that 10% of the mammary tumors arising in transgenic mice expressing a mutant polyomavirus middle T oncogene decoupled from the Shc adapter molecule possess reversion mutations that restore the ability of Shc to efficiently couple to polyomavirus middle T antigen (54
). Finally, it is also possible that the NYPD mutant induces tumorigenesis through the formation of specific heterodimers with other members of the epidermal growth factor receptor family. In this regard, we have noted that tumors derived from NYPD strains express elevated levels of ErbB-3 (Fig. ). Future studies with these NYPD strains should provide important insight into the contribution of each of these coupled signaling pathways in Neu-mediated tumorigenesis and metastasis.
Although mammary epithelial expression of either the YB or YD mutant efficiently induced mammary tumors, histological examination of these Neu-induced mammary tumors have revealed that they have distinct morphologies (Fig. and ). In particular, the YB tumors displayed papillary morphology, whereas the YD-induced tumors developed a nodular phenotype similar to that displayed by the parental activated neu
strains. The differences in the morphologies may reflect differences in the repertoire of signaling proteins coupled to Shc and Grb2. Indeed, it has been demonstrated that in addition to Grb2, Shc couples to several unidentified proteins (49
). Moreover, in certain cell types Shc can signal independent of Grb2/Ras activation through a pathway involving the upregulation of the c-Myc transcription (16
). Taken together, these observations argue that YB and YD neu
mutants function through both common and distinct signaling pathways.
Another potentially important phenotypic difference between the YB and YD strains is that only the YB strains efficiently develop metastatic lesions. The difference in the metastatic properties of these neu
mutants is not due to difference in rate of tumor development since the nonmetastatic YD tumors actually develop a much greater tumor load. One potential explanation for the differential metastatic properties of the YB-induced tumors is that Neu is coupled to signaling pathways that confer enhanced metastatic properties to these cells. Although the molecular basis for this difference in metastatic phenotype is unclear, we have previously shown that the dosage of Grb2 can have a profound effect on tumor induction in transgenic mice (7
). In addition to affecting tumor induction, reduction of Grb2 levels also impairs ductal outgrowth of the mammary epithelium (7
), suggesting that it may be involved in promoting epithelial cell migration. Direct recruitment of Grb2 to the Met receptor appears to confer metastatic potential to engineered cell lines (15
), and there appears to be a direct link with Grb2 to sustain myoblast proliferation and/or survival during migration from the somites in Met receptor knock-in mice (28
). Given the importance of migration in metastatic disease, it is conceivable that the YB mutant, through its direct interaction with Grb2, promotes metastasis through stimulation of cell migration and or survival of migrating cells. In this regard, it has been demonstrated that phosphospecific antibodies to Neu tyrosine phosphorylation site 1253 (YE) appear to detect a minority of ErbB-2 human breast cancers that exhibit the invasive phenotype (11
). It is conceivable that other ErbB-2 phosphorylation sites such as YB (tyrosine 1144) may also predict particularly aggressive human breast cancer phenotypes. Future studies with these mutant Neu strains and conditional ablation of Grb2 in mice should provide important insights into the molecular basis of metastatic progression.