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...

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The neu (c-erbB-2, Her-2) protooncogene is amplified and overexpressed in 20-30% of human breast cancers. Although transgenic mouse models have illustrated the role of Neu in the induction of mammary tumors, Neu expression in these models is driven by a strong viral promoter of questionable relevance to the human disease. To ascertain whether expression of activated Neu under the control of the endogenous promoter in the mammary gland could induce mammary tumors we have generated mice that conditionally express activated Neu under the transcriptional control of the intact endogenous Neu promoter. Expression of oncogenic neu in the mammary gland resulted in accelerated lobulo-alveolar development and formation of focal mammary tumors after a long latency period. However, expression of activated Neu under the normal transcriptional control of the endogenous promoter was not sufficient for the initiation of mammary carcinogenesis. Strikingly, all mammary tumors bear amplified copies (2-22 copies) of the activated neu allele relative to the wild-type allele and express highly elevated levels of neu transcript and protein. Thus, like human erbB-2-positive breast tumors, mammary tumorigenesis in this mouse model requires the amplification and commensurate elevated expression of the neu gene.

To investigate the function of the Rb-related p107 gene, a null mutation in p107 was introduced into the germ line of mice and bred into a BALB/cJ genetic background. Mice lacking p107 were viable and fertile but displayed impaired growth, reaching about 50% of normal weight by 21 days of age. Mutant mice exhibited a diathetic myeloproliferative disorder characterized by ectopic myeloid hyperplasia in the spleen and liver. Embryonic p107 ؊/؊ fibroblasts and primary myoblasts isolated from adult p107 ؊/؊ mice displayed a striking twofold acceleration in doubling time. However, cell sort analysis indicated that the fraction of cells in G 1 , S, and G 2 was unaltered, suggesting that the different phases of the cell cycle in p107 ؊/؊ cells was uniformly reduced by a factor of 2. Western analysis of cyclin expression in synchronized p107 ؊/؊ fibroblasts revealed that expression of cyclins E and A preceded that of D1. Mutant embryos expressed approximately twice the normal level of Rb, whereas p130 levels were unaltered. Lastly, mutant mice reverted to a wild-type phenotype following a single backcross with C57BL/6J mice, suggesting the existence of modifier genes that have potentially epistatic relationships with p107. Therefore, we conclude that p107 is an important player in negatively regulating the rate of progression of the cell cycle, but in a strain-dependent manner.The Rb family of structurally related nuclear phosphoproteins, consisting of Rb, p107, and p130, is believed to play important roles in regulating cell proliferation and differentiation (42). A central function of the Rb family is to negatively regulate the activity of E2F transcription factors that control the transcription of many cell cycle-regulated genes (41). Cyclin-dependent kinases (cdks) differentially regulate the phosphorylation of Rb, p107, and p130 during the cell cycle. Consequently, different Rb family members are hypophosphorylated during different phases of the cell cycle, allowing the formation of complexes that contain specific E2F transcription factors (10-13, 21, 55).The E2F family of transcription factors is encoded by multiple genes (at least six E2Fs and three DP-type members) and can regulate the transcription of many different genes that are putatively activated or repressed by specific E2F:DP heterodimers (26). Rb family-E2F1-5:DP complexes are believed to bind promoters at E2F sites and inhibit transcription by binding HDAC1, a histone deacetylase, to repress gene expression via chromatin remodeling (8,36,37) or, alternatively, to interfere with functional interactions between transactivation domains and components of the basal transcriptional machinery (9, 53). Thus, different E2F-regulated genes can be either activated or repressed depending on whether E2F:DP or an Rb family-E2F:DP complex is bound. Presumably, it is the cyclic activation and repression of E2F-regulated genes that controls progression through the cell cycle (41, 62).The phenotype of mice carrying targeted mutations in Rb supports the assertion that Rb...

Signaling mediated by ErbB2 is thought to play a critical role in numerous developmental processes. However, due to the embryonic lethality associated with the germ line inactivation of erbB2, its role in adult tissues remains largely obscure. Given the expression of ErbB2 at the neuromuscular junction, we have created a muscle-specific knockout to assess its role there. This resulted in viable mice with a progressive defect in proprioception due to loss of muscle spindles. Interestingly, a partial reduction of ErbB2 levels also reduced the number of muscle spindles. Although histological analysis of the muscle revealed an otherwise normal architecture, induction of muscle injury revealed a defect in muscle regeneration. Consistent with these observations, primary myoblasts lacking ErbB2 exhibit extensive apoptosis upon differentiation into myofibers. Taken together, these results illustrate a dual role for ErbB2 in both muscle spindle maintenance and survival of myoblasts.ErbB2 (also known as Neu and HER2) is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (7,13,23). This family is comprised of EGFR (48), ErbB2 (4,12,36,39,48,51), ErbB3 (27), and ErbB4 (35). Gene targeting experiments have revealed that each of these EGFR family members plays a critical role in regulating embryonic development. For example, germ line elimination of erbB2 or erbB4 results in embryonic lethality at day 10.5 of embryogenesis due to defects in cardiac and neural development (19, 28). Although inactivation of erbB3 function has a less severe impact on cardiac development, embryonic lethality is also observed due to defects in neural and Schwann cell differentiation (5,14,38). Interestingly, elimination of EGFR receptor function results in a strain-dependent perinatal lethality (21,41,46).Given the importance of EGFR family members in embryonic development, it has been difficult to elucidate the relative contribution of this family to the maintenance and development of adult tissues. The embryonic lethality associated with inactivation of erbB2 has previously been rescued by myocardial expression of an erbB2 transgene (29,33,49). However, these mice die at birth due to loss of motor neurons and defects in Schwann cell development. To avoid this perinatal lethality, specific deletions may be created to address the role of ErbB2 in various adult tissues through the use of the Cre/ LOXP1 recombinase system. Indeed this approach has been used to generate a peripheral-nerve-specific deletion of erbB2 that resulted in the extensive demyelination of the nerves (17, 18).In addition to its role in Schwann cell development, ErbB2 is expressed in skeletal muscle and is concentrated at the neuromuscular junction (NMJ) along with ErbB3 and ErbB4 EGFR family members (1, 34, 52). Indeed, EGFR family members have been implicated as important functional components of the NMJ (24). Neuregulins serve as ligands for ErbB3 and ErbB4 and are released from the motor neuron end plate, where they are thought to act...

Changes in protein-protein interactions may allow polypeptides to perform unexpected regulatory functions. Mammalian ShcA docking proteins have amino-terminal phosphotyrosine (pTyr) binding (PTB) and carboxyl-terminal Src homology 2 (SH2) domains, which recognize specific pTyr sites on activated receptors, and a central region with two phosphorylated tyrosine-X-asparagine (pYXN) motifs (where X represents any amino acid) that each bind the growth factor receptor-bound protein 2 (Grb2) adaptor. Phylogenetic analysis indicates that ShcA may signal through both pYXN-dependent and -independent pathways. We show that, in mice, cardiomyocyte-expressed ShcA directs mid-gestational heart development by a PTB-dependent mechanism that does not require the pYXN motifs. In contrast, the pYXN motifs are required with PTB and SH2 domains in the same ShcA molecule for the formation of muscle spindles, skeletal muscle sensory organs that regulate motor behavior. Thus, combinatorial differences in ShcA docking interactions may yield multiple signaling mechanisms to support diversity in tissue morphogenesis.