E-cadherin is an essential adhesion protein as well as a tumor suppressor that is silenced in many cancers. Its adhesion-dependent regulation of signaling has not been elucidated. We report that E-cadherin can negatively regulate, in an adhesion-dependent manner, the ligand-dependent activation of divergent classes of receptor tyrosine kinases (RTKs), by inhibiting their ligand-dependent activation in association with decreases in receptor mobility and in ligand-binding affinity. E-cadherin did not regulate a constitutively active mutant RTK (Neu*) or the liganddependent activation of LPA receptors or muscarinic receptors, which are two classes of G protein-coupled receptors. EGFR regulation by E-cadherin was associated with complex formation between EGFR and E-cadherin that depended on the extracellular domain of E-cadherin but was independent of b-catenin binding or p120-catenin binding. Transfection of E-cadherin conferred negative RTK regulation to human melanoma and breast cancer lines with downregulated endogenous E-cadherin. Abrogation of E-cadherin regulation may contribute to the frequent ligand-dependent activation of RTK in tumors.

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The three deleted in liver cancer genes (DLC1-3) encode RhoGTPase-activating proteins (RhoGAPs) whose expression is frequently down-regulated or silenced in a variety of human malignancies. The RhoGAP activity is required for full DLC-dependent tumor suppressor activity. Here we report that DLC1 and DLC3 bind to human tensin1 and its chicken homolog. The binding has been mapped to the tensin Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains at the C terminus of tensin proteins. Distinct DLC1 sequences are required for SH2 and PTB binding. DCL binding to both domains is constitutive under basal conditions. The SH2 binding depends on a tyrosine in DCL1 (Y442) but is phosphotyrosine-independent, a highly unusual feature for SH2 binding. DLC1 competed with the binding of other proteins to the tensin C terminus, including ␤3-integrin binding to the PTB domain. Point mutation of a critical tyrosine residue (Y442F) in DLC1 rendered the protein deficient for binding the tensin SH2 domain and binding full-length tensin. The Y442F protein was diffusely cytoplasmic, in contrast to the localization of wild-type DLC1 to focal adhesions, but it retained the ability to reduce the intracellular levels of Rho-GTP. The Y442F mutant displayed markedly reduced biological activity, as did a mutant that was RhoGAP-deficient. The results suggest that DLC1 is a multifunctional protein whose biological activity depends on cooperation between its tensin binding and RhoGAP activities, although neither activity depends on the other. DLC1 ͉ Src homology 2 and phosphotyrosine binding domains ͉ tumor suppressor gene

SummaryCell migration requires coordination between integrin-mediated cell adhesion to the extracellular matrix and force applied to adhesion sites. Talin plays a key role in coupling integrin receptors to the actomyosin contractile machinery, while deleted in liver cancer 1 (DLC1) is a Rho GAP that binds talin and regulates Rho, and therefore actomyosin contractility. We show that the LD motif of DLC1 forms a helix that binds to the four-helix bundle of the talin R8 domain in a canonical triple-helix arrangement. We demonstrate that the same R8 surface interacts with the paxillin LD1 and LD2 motifs. We identify key charged residues that stabilize the R8 interactions with LD motifs and demonstrate their importance in vitro and in cells. Our results suggest a network of competitive interactions in adhesion complexes that involve LD motifs, and identify mutations that can be used to analyze the biological roles of specific protein-protein interactions in cell migration.

Previously, using an inbred strain screen and QTL mapping strategies, we demonstrated the presence of loci in the mouse genome that significantly influenced the ability of a transgene-induced mammary tumor to metastasize to the lung. Here we present data supporting the signal transduction molecule, Sipa1, as a candidate for the metastasis efficiency modifier locus Mtes1. Sequence analysis of genes in a candidate haplotype block revealed a non-synonymous animo acid polymorphism in the Sipa1 PDZ protein-protein interaction domain. Biochemical analysis indicates that the missense substitution had a significant effect on the Sipa1 RapGAP function. Spontaneous metastasis assays using cells expressing ectopic Sipa1 or Sipa1 shRNA to modulate the expression of Sipa1demonstrate that the metastatic capacity of a highly aggressive mouse mammary tumor cell line is correlated with cellular Sipa1 levels. Examination of human gene expression data is consistent with the role of Sipa1 concentration in metastatic progression. Together these data suggest that the PDZ domain polymorphism is likely to be at least one of the underlying genetic polymorphisms responsible for the Mtes1 locus. This is also, to the best of our knowledge, the first demonstration of a constitutional genetic polymorphism having a significant impact on tumor metastasis.

The deleted in liver cancer 1 (DLC1) tumor suppressor gene, which is frequently inactivated in cancer, encodes a Rho-GAP (GTPase activating protein) focal adhesion protein whose negative regulation of Rho-GTPases is necessary but not sufficient for its full tumor suppressor activity. Here, we report that DLC1 forms a complex with two prooncogenic focal adhesion proteins, talin and the focal adhesion kinase (FAK). We identified an 8-aa sequence (residues 469LDDILYHV476) in DLC1 and designated it an LD-like motif, because it shares homology with the LD motifs of paxillin. This motif was necessary for DLC1 binding to talin and FAK, because a DLC1 mutant, from which six of the residues have been deleted, and another mutant carrying amino acid substitutions in three of the residues are deficient for binding both proteins and localization of DLC1 to focal adhesions. FAK binding was independent of talin and vice versa. In bioassays, both DLC1 mutants were less active than wild-type (WT) DLC1, although the ability of the mutants to negatively regulate overall Rho-GTP was not impaired. We conclude that the LD-like motif, which binds talin and FAK, is required for the full tumor suppressor activity of DLC1 and contributes to the association of DLC1 with focal adhesions. C ancer develops as a multifactorial process that may include the activation of oncogenes and antiapoptotic genes as well as the inactivation of tumor suppressor genes and proapoptotic genes (1). Deleted in liver cancer 1 (DCL1) is a tumor suppressor gene that is inactivated by deletion, mutation, or methylation in a variety of tumors, including lung, breast, and prostate cancer (2, 3). It is the prototypic member of a multigene family, and the two other genes, DLC2 and DLC3, encode closely related proteins. DLC2 and DLC3 have been studied less intensively than DLC1, but the available data suggest that they are frequently down-regulated in tumors (2).The functional basis for the frequent inactivation of DLC1 in cancer has not been explored in detail. The DLC1 protein localizes to focal adhesions, which can regulate normal and neoplastic cell movement and signaling through mechanisms that are incompletely understood (4). DLC1 contains several motifs, including a Rho-GAP catalytic domain that negatively regulates Rho-GTPases by accelerating their intrinsic GTPase activity. Rho-A and Rho-C may be up-regulated in human tumors (5), and studies of cultured cells have shown that the Rho-GAP activity of DLC1 participates in its inhibition of cell migration and anchorage-independent growth (6). However, although the Rho-GAP function of the DLC proteins seems to be necessary for their full biological activity, it is not sufficient. For example, tumor-associated loss of function mutants have been described that seem to have WT Rho-GAP activity (7), and other Rho-GAPs, such as p190 Rho-GAP, do not seem to be frequently inactivated in cancer.These observations suggest that DLC1-3 may possess additional activities that contribute to their frequent inactivation in cancer...

We have shown that members of the erbB family undergo homodimer and heterodimer formation. The rat p185c-and the epidermal growth factor receptor (EGFR) can associate into an active heterodimeric tyrosine kinase.Overexpression of these two receptors also results in a transformed phenotype. We now show that mutant Neu proteins resulting from a point mutation at the ATP-binding site (N757) or cytoplasmic domain deletions (N691stop) are still able to undergo EGF-induced heterodimerization with EGFR. Analysis of heterodimer formation between EGFR and truncated Neu proteins revealed that heterodimerization is preferred over homodimerization of EGFR. N757 can be transphosphorylated by assiated EGFR upon EGF stimulation. However, the heterodimer composed of EGFR and N69lstop is kinase inactive. These results provided evidence that the Neu ectodomain is sufficent to associate with EGFR physically, and the cytoplasmic domain interaction is required for heterodimeric kinase activation, indicating that Neu/c-erbB2 is not just a simple substrate for EGFR but a transactivator as well.The protooncogene, rat neu or human ERBB2, encodes a 185-kDa transmembrane glycoprotein pl8Sc-neu or ERBB2, respectively. The protein products are found in adult secretory epithelial cells of the lung, salivary gland, breast, pancreas, ovary, gastrointestinal tract, and skin (1-3). Oncogenic neu, initially identified in rat neuroglioblastomas (4), was found to be activated by a point mutation generating a single amino acid substitution in the transmembrane region (5). This alteration results in constitutive activity of its intrinsic kinase and in malignant transformation of the cells (6) possibly due to a shift in the molecular equilibrium from monomeric to dimeric forms (7).Neu/c-erbB2 is highly homologous with, but distinct from, epidermal growth factor receptor (EGFR) (4,8). Numerous studies (9-12) indicated that EGFR and Neu/c-erbB2 are able to interact (13). We have demonstrated that heterodimers of EGFR and p185c-ncu were readily detected and that the intermolecular association of EGFR and pl85c-neu appeared to up-regulate EGFR function (14). Heterodimers can be detected in the absence of chemical cross-linkers, and these receptors associate into an active kinase complex both in vivo and in vitro (15). Heterodimeric association of EGFR and ERBB2 has also been found in human breast cancer cell lines (SK-Br-3) (16) and transfected cells (17).In

Targeted disruption of both alleles of mouse sos1, which encodes a Ras-specific exchange factor, conferred mid-gestational embryonic lethality that was secondary to impaired placental development and was associated with very low placental ERK activity. The trophoblastic layers of sos1 -/-embryos were poorly developed, correlating with high sos1 expression in wild-type trophoblasts. A sos1 -/-cell line, which expressed readily detectable levels of the closely related Sos2 protein, formed complexes between Sos2, epidermal growth factor receptor (EGFR) and Shc efficiently, gave normal Ras·GTP and ERK responses when treated with EGF for ഛ10 min and was transformed readily by activated Ras. However, the sos1 -/-cells were resistant to transformation by v-Src or by overexpressed EGFR and continuous EGF treatment, unlike sos1 ⍣/-or wildtype cells. This correlated with Sos2 binding less efficiently than Sos1 to EGFR and Shc in cells treated with EGF for ജ90 min or to v-Src and Shc in v-Srcexpressing cells, and with less ERK activity. We conclude that Sos1 participates in both short-and longterm signaling, while Sos2-dependent signals are predominantly short-term.