Cell-surface receptors frequently employ scaffold proteins to recruit cytoplasmic targets, but the rationale for this is uncertain. Activated receptor tyrosine kinases, for example, engage scaffolds such as Shc1 that contain phosphotyrosine (pTyr) binding (PTB) domains. Using quantitative mass spectrometry, we find that Shc1 responds to epidermal growth factor (EGF) stimulation through multiple waves of distinct phosphorylation events and protein interactions. Following stimulation, Shc1 rapidly binds a group of proteins that activate pro-mitogenic/survival pathways dependent on recruitment of the Grb2 adaptor to Shc1 pTyr sites. Akt-mediated feedback phosphorylation of Shc1 Ser29 then recruits the Ptpn12 tyrosine phosphatase. This is followed by a sub-network of proteins involved in cytoskeletal reorganization, trafficking and signal termination that binds Shc1 with delayed kinetics, largely through the SgK269 pseudokinase/adaptor protein. Ptpn12 acts as a switch to convert Shc1 from pTyr/Grb2-based signaling to SgK269-mediated pathways that regulate cell invasion and morphogenesis. The Shc1 scaffold therefore directs the temporal flow of signaling information following EGF stimulation.

To explore the in vivo significance of ShcA during mammary tumorigenesis, we used mice expressing several phosphotyrosine-deficient ShcA alleles under the control of their endogenous promoter. We show that all three ShcA tyrosine phosphorylation sites are involved in the early stages of mammary tumour progression, including loss of the myoepithelial cell layer surrounding hyperplasias and during progression to carcinoma. We have determined that signals emanating from Y313 are important for tumour cell survival, whereas Y239/240 transduce signals promoting tumour vascularization. We further demonstrate that loss of ShcA expression in mammary epithelial cells abrogates tumour development. This study is the first to directly demonstrate that signalling downstream from the ShcA adaptor protein is critical for breast cancer development.

Amplification of the Neu/c-erbB-2 receptor tyrosine kinase has been implicated as an important event in the genesis of human breast cancer. Indeed, transgenic mice bearing either an activated form of neu or the wild-type proto-oncogene under the transcriptional control of the mouse mammary tumor virus promoterenhancer frequently develop mammary carcinomas (L. Bouchard, L. Lamarre, P. J. Tremblay, and P. Jolicoeur, Cell 57:931-936, 1989 Leder, Cell 54:105-115, 1988). Induction of mammary tumors in transgenic mice expressing the wild-type Neu receptor is associated with activation of the receptor's intrinsic tyrosine kinase activity (Guy et al., Proc. Natl. Acad. Sci. USA 89:10578-10582, 1992). Here, we demonstrate that activation of Neu in these transgenic mice occurs through somatic mutations located within the transgene itself. Sequence analyses of these mutations revealed that they contain in-frame deletions of 7 to 12 amino acids in the extracellular region proximal to the transmembrane domain. Introduction of these mutations into a wild-type neu cDNA results in an increased transforming ability of the altered Neu tyrosine kinase. These observations suggest that oncogenic activation of Neu in mammary tumorigenesis frequently occurs by somatic mutation.The neu (c-erbB-2 or HER2) proto-oncogene encodes a receptor tyrosine kinase belonging to the epidermal growth factor receptor family (2, 9). Oncogenic activation of Neu can occur through multiple molecular mechanisms, including a point mutation in its transmembrane domain (3) and deletion of the extracellular domain (4). Amplification and consequent overexpression of neulc-erbB-2 have also been observed in a significant proportion of human breast and ovarian cancers (14,24,27). In fact, several studies have shown that the degree of amplification is inversely correlated to a poor clinical outcome for breast cancer patients in whom the cancer has not spread to the lymph nodes (11,22). Although elevated levels of neulc-erbB-2 are observed in these human cancers, no comparable mutations have been detected in these tumor samples (15).Consistent with these clinical observations, expression of activated or wild-type neu targeted to the mammary epithelia of transgenic mice results in the efficient induction of mammary tumors (5,12,19). In mice expressing elevated levels of the wild-type Neu protein, the appearance of mammary tumors is associated with a high incidence of metastatic disease (12). Mammary gland-specific expression of Neu results in the appearance of focal mammary tumors after a variable latency period (12). Both the focal nature and long latency period suggest that additional genetic events are needed to transform the mammary epithelium. Mammary tumorigenesis in these transgenic strains is correlated with elevated intrinsic Neu tyrosine kinase activity and the de novo tyrosine phosphorylation of several cellular proteins (12). The RNA and protein (12).While it is clear that activation of Neu plays an important role in the induction of mammary tumors...