A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Rasdependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites.The neu/c-ErbB-2 gene encodes a 185-kDa transmembrane receptor tyrosine kinase that is a member of the epidermal growth factor receptor (EGFR) family (4,15,76). Elevated expression of the EGFR family members has been implicated in the etiology of human ovarian and breast cancers (reviewed in references 31 and 41). In particular, amplification and consequent overexpression of the neu receptor tyrosine kinase have been observed in a large proportion of primary human breast cancers (58,64,65). Consistent with these observations, expression of either wild-type neu or a constitutively active mutant (V664E, neuNT [3,5]) in the mammary epithelia of transgenic mice results in the induction of metastatic mammary tumors (11,27,47). The induction of mammary tumors in the wild-type neu transgenic mice correlates with elevated

The Grb2 and Shc adapter proteins play critical roles in coupling activated growth factor receptors to several cellular signaling pathways. To assess the role of these molecules in mammary epithelial development and tumorigenesis, we have generated transgenic mice which individually express the Grb2 and Shc proteins in the mammary epithelium. Although mammary epithelial cell-specific expression of Grb2 or Shc accelerated ductal morphogenesis, mammary tumors were rarely observed in these strains. To explore the potential role of these adapter proteins in mammary tumorigenesis, mice coexpressing either Shc or Grb2 and a mutant form of polyomavirus middle T (PyV mT) antigen in the mammary epithelium were generated. Coexpression of either Shc or Grb2 with the mutant PyV mT antigen resulted in a dramatic acceleration of mammary tumorigenesis compared to parental mutant PyV mT strain. The increased rate of tumor formation observed in these mice was correlated with activation of the epidermal growth factor receptor family and mitogen-activated protein kinase pathway. These observations suggest that elevated levels of the Grb2 or Shc adapter protein can accelerate mammary tumor progression by sensitizing the mammary epithelial cell to growth factor receptor signaling.The murine mammary gland represents a unique system to study the responsiveness of cells to diverse signals stimulating cell death, survival, proliferation, and differentiation. The control of mammary epithelial proliferation and differentiation is ultimately regulated by hormonal and peptide factors that exert their biological action through a variety of receptor molecules. Elevated expression of growth factors or their cognate receptors can result in deregulated mammary epithelial cell proliferation, which can ultimately progress to the malignant phenotype. For example, elevated expression of the ErbB-2/ Neu receptor tyrosine kinase has been implicated in the genesis of a large proportion of human breast cancers (39,40). Consistent with these observations, mammary epithelial expression of ErbB-2 in transgenic mice results in the efficient induction of mammary tumors (4,14,16,25,35). Whereas it is clear that oncogenes such as erbB-2 induce malignancy, the precise molecular mechanism by which this occurs is unclear.One potential mechanism by which receptor tyrosine kinases (RTKs) can induce proliferation is through interaction with a number of Src homology 2 (SH2)-or protein tyrosine binding domain (PTB)-containing adapter proteins (27). Although adapter proteins such as Shc (Src homology and collagen) and Grb2 (growth factor receptor-bound protein 2) lack intrinsic enzymatic activity, they play an important role in connecting growth factors to specific signaling pathways (23,28,29,32). Grb2 is a 25-kDa protein which contains a central SH2 domain flanked by two SH3 domains. Activation of RTKs can result in the direct recruitment of Grb2 via its SH2 domain to specific tyrosine-phosphorylated residues within the receptor. Subsequent recruitment of the guanine...