Tumor-suppressor gene products in cell contacts: the cadherin-APC-armadillo connection.

Various structural components of intercellular junctions have recently been found to represent (or be related to) products of tumor-suppressor genes. The tumor-suppressor gene product adenomatous polyposis coli (APC) binds to beta 2-catenin (homologous to the product of Drosophila armadillo), which is cytoplasmically associated with the cell adhesion molecule E-cadherin.

Dominant and recessive genes involved in tumor cell invasion.

The past year has been the discovery and further analysis of several genes and protein products that are critically involved in the generation of invasive and metastatic tumor cells. Like oncogenes and tumor suppressor genes, the genes responsible for invasive and metastatic phenotypes can function in a dominant or recessive fashion. In this review, particular emphasis will be given to the dominantly acting genes encoding the cell adhesion molecule CD44 and the motility factor scatter factor, and the recessively acting genes encoding the cell adhesion molecule E-cadherin and nm23.

On the mechanism of ATP-induced shape changes in human erythrocyte membranes. II. The role of ATP.

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.

The Met receptor tyrosine kinase transduces motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells.

Depending on the target cells and culture conditions, scatter factor/hepatocyte growth factor (SF/HGF) mediates several distinct activities, i.e., cell motility, proliferation, invasiveness, tubular morphogenesis, angiogenesis, or cytotoxicity. A small isoform of SF/HGF encoded by a natural splice variant, which consists of the NH2-terminal hairpin structure and the first two kringle domains but not the protease homology region, induces cell motility but not mitogenesis. Two types of SF/HGF receptors have recently been discovered in epithelial cells, the high affinity c-Met receptor tyrosine kinase, and low affinity/high capacity binding sites, which are probably located on heparan sulfate proteoglycans. In the present study, we have addressed the question whether the various biological activities of SF/HGF are transduced into cells by a single type of receptor. We have here examined MDCK epithelial cells transfected with a hybrid cDNA encoding the ligand binding domain of the nerve growth factor (NGF) receptor and the membrane-spanning and tyrosine kinase domains of the Met receptor. We demonstrate that all biological effects of SF/HGF upon epithelial cells such as the induction of cell motility, proliferation, invasiveness, and tubular morphogenesis can now be triggered by the addition of NGF. Thus, it is likely that all known biological signals of SF/HGF are transduced through the receptor tyrosine kinase encoded by the c-Met protooncogene.

E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton.

beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E-cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression.

Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to uvomorulin.

It has previously been shown that the monoclonal antibody anti-Arc-1 dissociates Madin-Darby canine kidney (MDCK) epithelial cells and changes their morphology in vitro (Imhof, B.A., H.P. Vollmers, S.L. Goodman, and W. Birchmeier, 1983, Cell, 35:667-675). In this article we demonstrate that the anti-Arc-1 antibody recognizes an uvomorulin-like molecule on MDCK cells, i.e., it immunoprecipitates an 84-kD protein fragment from a tryptic digest of cell surfaces in the presence of Ca2+ (as does anti-uvomorulin antiserum). Furthermore, anti-uvomorulin antiserum prevents the binding of anti-Arc-1 to MDCK cells. The distribution of the Arc-1 antigen is also quite similar to that of uvomorulin: it is enriched at the cell-cell contacts both of MDCK cells and of cells in various canine tissues. In the intestinal epithelium the antigen could be further localized in the region of the junctional complex. To study the mechanism of action of the dissociating antibody, MDCK cells grown on Nuclepore filters in Boyden chambers were exposed to anti-Arc-1 from either the upper or lower compartment. It could be shown that the antibody interfered with cell adhesion only from the basolateral but not from the apical cell surface. Antibody action was inhibited in the presence of colchicine but not cytochalasin B. Furthermore, cell dissociation was prevented when the cellular cAMP level was raised. These findings indicate that the anti-Arc-1 antibody acts on a target below the tight junctions (possibly on the antigen located in the junctional complex), and they confirm that cytoskeleton and metabolic factors are actively involved in the maintenance of junctional integrity.

Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells.

The generation of invasiveness in transformed cells represents an essential step of tumor progression. We have previously shown that MDCK epithelial cells, which are deprived of intracellular adhesion by the addition of anti-Arc-1/uvomorulin antibodies, become invasive for collagen gels and embryonal heart tissue (Behrens, J., M. M. Mareel, F. M. Van Roy, and W. Birchmeier. 1989. J. Cell Biol. 108: 2435-2447.). Here we examined whether invasiveness is also induced by scatter factor, which is known to dissociate epithelial cells (Stoker, M., E. Gherardi, M. Perryman, and J. Gray. 1987. Nature (Lond.). 327:239-242.). Scatter factor was purified to homogeneity from conditioned medium of human fibroblasts by heparin-Sepharose chromatography, followed by cation exchange chromatography, gel filtration, or preparative SDS gel electrophoresis. We found that scatter factor represents a 92,000 mol wt glycoprotein which, apparently, is converted by limited proteolysis into disulfide-linked 62,000 and 34/32,000 mol wt subunits. Reversed phase HPLC and sequence analysis of tryptic peptides confirmed the suggested molecular structure, and revealed further that scatter factor exhibits sequence similarities to hepatocyte growth factor and to plasminogen. Purified scatter factor in fact induces the invasiveness into collagen matrices of MDCK epithelial cells, and induces or promotes the invasiveness of a number of human carcinoma cell lines. Apparently, the effect on the human cells depends on their respective degree of differentiation, i.e., cell lines with a less pronounced epithelial phenotype were more susceptible to the factor. Scatter factor does not seem to influence synthesis, steady-state level, and phosphorylation of the cell adhesion molecule Arc-1/uvomorulin. Thus, scatter factor represents a clearly defined molecular species which induces, in vitro, the progression of epithelial cells to a more motile, i.e., invasive phenotype.

Hepatocyte growth factor/scatter factor induces a variety of tissue-specific morphogenic programs in epithelial cells.

Hepatocyte growth factor/scatter factor (HGF/SF) is the mesenchymal ligand of the epithelial tyrosine kinase receptor c-Met. In vitro, HGF/SF has morphogenic properties, e.g., induces kidney epithelial cells to form branching ducts in collagen gels. Mutation of the HGF/SF gene in mice results in embryonic lethality due to severe liver and placenta defects. Here, we have evaluated the morphogenic activity of HGF/SF with a large variety of epithelial cells grown in three-dimensional collagen matrices. We found that HGF/SF induces SW 1222 colon carcinoma cells to form crypt-like structures. In these organoids, cells exhibit apical/basolateral polarity and build a well-developed brush border towards the lumen. Capan 2 pancreas carcinoma cells, upon addition of HGF/SF, develop large hollow spheroids lined with a tight layer of polarized cells. Collagen inside the cysts is digested and the cells show features of pancreatic ducts. HGF/SF induces EpH4 mammary epithelial cells to form long branches with end-buds that resemble developing mammary ducts. pRNS-1-1 prostate epithelial cells in the presence of HGF/SF develop long ducts with distal branching as found in the prostate. Finally, HGF/SF simulates alveolar differentiation in LX-1 lung carcinoma cells. Expression of transfected HGF/SF cDNA in LX-1 lung carcinoma and EpH4 mammary epithelial cells induce morphogenesis in an autocrine manner. In the cell lines tested, HGF/SF activated the Met receptor by phosphorylation of tyrosine residues. These data show that HGF/SF induces intrinsic, tissue-specific morphogenic activities in a wide variety of epithelial cells. Apparently, HGF/SF triggers respective endogenous programs and is thus an inductive, not an instructive, mesenchymal effector for epithelial morphogenesis.

V-src kinase shifts the cadherin-based cell adhesion from the strong to the weak state and beta catenin is not required for the shift.

The elevation of tyrosine phosphorylation level is thought to induce the dysfunction of cadherin through the tyrosine phosphorylation of beta catenin. We evaluated this assumption using two cell lines. First, using temperature-sensitive v-src-transfected MDCK cells, we analyzed the modulation of cadherin-based cell adhesion by tyrosine phosphorylation. Cell aggregation and dissociation assays at nonpermissive and permissive temperatures indicated that elevation of the tyrosine phosphorylation does not totally affect the cell adhesion ability of cadherin but shifts it from a strong to a weak state. The tyrosine phosphorylation levels of beta catenin, ZO-1, ERM (ezrin/radixin/moesin), but not alpha catenin, vinculin, and alpha-actinin, were elevated in the weak state. To evaluate the involvement of the tyrosine phosphorylation of beta catenin in this shift of cadherin-based cell adhesion, we introduced v-src kinase into L fibroblasts expressing the cadherin-alpha catenin fusion protein, in which beta catenin is not involved in cell adhesion. The introduction of v-src kinase in these cells shifted their adhesion from a strong to a weak state. These findings indicated that the tyrosine phosphorylation of beta catenin is not required for the strong-to-weak state shift of cadherin-based cell adhesion, but that the tyrosine phosphorylation of other junctional proteins, ERM, ZO-1 or unidentified proteins is involved.

Requirement for beta-catenin in anterior-posterior axis formation in mice.

The anterior-posterior axis of the mouse embryo is defined before formation of the primitive streak, and axis specification and subsequent anterior development involves signaling from both embryonic ectoderm and visceral endoderm. Tauhe Wnt signaling pathway is essential for various developmental processes, but a role in anterior-posterior axis formation in the mouse has not been previously established. Beta-catenin is a central player in the Wnt pathway and in cadherin-mediated cell adhesion. We generated beta-catenin-deficient mouse embryos and observed a defect in anterior-posterior axis formation at embryonic day 5.5, as visualized by the absence of Hex and Hesx1 and the mislocation of cerberus-like and Lim1 expression. Subsequently, no mesoderm and head structures are generated. Intercellular adhesion is maintained since plakoglobin substitutes for beta-catenin. Our data demonstrate that beta-catenin function is essential in anterior-posterior axis formation in the mouse, and experiments with chimeric embryos show that this function is required in the embryonic ectoderm.

Reconstitution of mammary gland development in vitro: requirement of c-met and c-erbB2 signaling for branching and alveolar morphogenesis.

We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met-mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.

Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell-cell adhesion.

The generation of invasiveness in transformed cells represents an essential step of tumor progression. We show here, first, that nontransformed Madin-Darby canine kidney (MDCK) epithelial cells acquire invasive properties when intercellular adhesion is specifically inhibited by the addition of antibodies against the cell adhesion molecule uvomorulin; the separated cells then invade collagen gels and embryonal heart tissue. Second, MDCK cells transformed with Harvey and Moloney sarcoma viruses are constitutively invasive, and they were found not to express uvomorulin at their cell surface. These data suggest that the loss of adhesive function of uvomorulin (which is identical to E-cadherin and homologous to L-CAM) is a critical step in the promotion of epithelial cells to a more malignant, i.e., invasive, phenotype. Similar modulation of intercellular adhesion might also occur during invasion of carcinoma cells in vivo.

Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses.

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met-specific branching morphogenesis. It associates directly with c-Met via the c-Met-binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met-binding site is localized to a 13-amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met-binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1-specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.

Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene.

Loss of histotypic organization of epithelial cells is a common feature in normal development as well as in the invasion of carcinomas. Here we show that the v-src oncogene is a potent effector of epithelial differentiation and invasiveness. MDCK epithelial cells transformed with a temperature-sensitive mutant of v-src exhibit a strictly epithelial phenotype at the nonpermissive temperature for pp60v-src activity (40.5 degrees C) but rapidly loose cell-to-cell contacts and acquire a fibroblast-like morphology after culture at the permissive temperature (35 degrees C). Furthermore, the invasiveness of the cells into collagen gels or into chick heart fragments was increased at the permissive temperature. The profound effects of v-src on intercellular adhesion were not linked to changes in the levels of expression of the epithelial cell adhesion molecule E-cadherin. Rather, we observed an increase in tyrosine phosphorylation of E-cadherin and, in particular, of the associated protein beta-catenin. These results suggest a mechanism by which v-src counteracts junctional assembly and thereby promotes invasiveness and dedifferentiation of epithelial cells through phosphorylation of the E-cadherin/catenin complex.

Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland.

We have examined the role of two mesenchymal ligands of epithelial tyrosine kinase receptors in mouse mammary gland morphogenesis. In organ cultures of mammary glands, hepatocyte growth factor (HGF, scatter factor) promoted branching of the ductal trees but inhibited the production of secretory proteins. Neuregulin (NRG, neu differentiation factor) stimulated lobulo-alveolar budding and the production of milk proteins. These functional effects are paralleled by the expression of the two factors in vivo: HGF is produced in mesenchymal cells during ductal branching in the virgin animal; NRG is expressed in the mesenchyme during lobulo-alveolar development at pregnancy. The receptors of HGF and NRG (c-met, c-erbB3, and c-erbB4), which are expressed in the epithelial cells, are not regulated. In organ culture, branching morphogenesis and lobulo-alveolar differentiation of the mammary gland could be abolished by blocking expression of endogenous HGF and NRG by the respective antisense oligonucleotides; in antisense oligonucleotide-treated glands, morphogenesis could again be induced by the addition of recombinant HGF and NRG. We thus show that two major postnatal morphogenic periods of mammary gland development are dependent on sequential mesenchymal-epithelial interactions mediated by HGF and NRG.

Motogenic and morphogenic activity of epithelial receptor tyrosine kinases.

Receptor tyrosine kinases play essential roles in morphogenesis and differentiation of epithelia. Here we examined various tyrosine kinase receptors, which are preferentially expressed in epithelia (c-met, c-ros, c-neu, and the keratin growth factor [KGF] receptor), for their capacity to induce cell motility and branching morphogenesis of epithelial cells. We exchanged the ligand-binding domain of these receptors by the ectodomain of trkA and could thus control signaling by the new ligand, NGF. We demonstrate here that the tyrosine kinases of c-met, c-ros, c-neu, the KGF receptor, and trkA, but not the insulin receptor, induced scattering and increased motility of kidney epithelial cells in tissue culture. Mutational analysis suggests that SHC binding is essential for scattering and increased cell motility induced by trkA. The induction of motility in epithelial cells is thus an important feature of various receptor tyrosine kinases, which in vivo play a role in embryogenesis and metastasis. In contrast, only the c-met receptor promoted branching morphogenesis of kidney epithelial cells in three-dimensional matrices, which resemble the formation of tubular epithelia in development. Interestingly, the ability of c-met to induce morphogenesis could be transferred to trkA, when in a novel receptor hybrid COOH-terminal sequences of c-met (including Y14 to Y16) were fused to the trkA kinase domain. These data demonstrate that tubulogenesis of epithelia is a restricted activity of tyrosine kinases, as yet only demonstrated for the c-met receptor. We predict the existence of specific substrates that mediate this morphogenesis signal.

E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells.

The ability of carcinomas to invade and to metastasize largely depends on the degree of epithelial differentiation within the tumors, i.e., poorly differentiated being more invasive than well-differentiated carcinomas. Here we confirmed this correlation by examining various human cell lines derived from bladder, breast, lung, and pancreas carcinomas. We found that carcinoma cell lines with an epithelioid phenotype were noninvasive and expressed the epithelium-specific cell-cell adhesion molecule E-cadherin (also known as Arc-1, uvomorulin, and cell-CAM 120/80), as visualized by immunofluorescence microscopy and by Western and Northern blotting, whereas carcinoma cell lines with a fibroblastoid phenotype were invasive and had lost E-cadherin expression. Invasiveness of these latter cells could be prevented by transfection with E-cadherin cDNA and was again induced by treatment of the transfected cells with anti-E-cadherin mAbs. These findings indicate that the selective loss of E-cadherin expression can generate dedifferentiation and invasiveness of human carcinoma cells, and they suggest further that E-cadherin acts as an invasion suppressor.

Novel p62dok family members, dok-4 and dok-5, are substrates of the c-Ret receptor tyrosine kinase and mediate neuronal differentiation.

Docking proteins are substrates of tyrosine kinases and function in the recruitment and assembly of specific signal transduction molecules. Here we found that p62dok family members act as substrates for the c-Ret receptor tyrosine kinase. In addition to dok-1, dok-2, and dok-3, we identified two new family members, dok-4 and dok-5, that can directly associate with Y1062 of c-Ret. Dok-4 and dok-5 constitute a subgroup of dok family members that is coexpressed with c-Ret in various neuronal tissues. Activated c-Ret promotes neurite outgrowth of PC12 cells; for this activity, Y1062 in c-Ret is essential. c-Ret/dok fusion proteins, in which Y1062 of c-Ret is deleted and replaced by the sequences of dok-4 or dok-5, induce ligand-dependent axonal outgrowth of PC12 cells, whereas a c-Ret fusion containing dok-2 sequences does not elicit this response. Dok-4 and dok-5 do not associate with rasGAP or Nck, in contrast to p62dok and dok-2. Moreover, dok-4 and dok-5 enhance c-Ret-dependent activation of mitogen-activated protein kinase. Thus, we have identified a subclass of p62dok proteins that are putative links with downstream effectors of c-Ret in neuronal differentiation.

Essential role of Gab1 for signaling by the c-Met receptor in vivo.

The docking protein Gab1 binds phosphorylated c-Met receptor tyrosine kinase directly and mediates signals of c-Met in cell culture. Gab1 is phosphorylated by c-Met and by other receptor and nonreceptor tyrosine kinases. Here, we report the functional analysis of Gab1 by targeted mutagenesis in the mouse, and compare the phenotypes of the Gab1 and c-Met mutations. Gab1 is essential for several steps in development: migration of myogenic precursor cells into the limb anlage is impaired in Gab1-/- embryos. As a consequence, extensor muscle groups of the forelimbs are virtually absent, and the flexor muscles reach less far. Fewer hindlimb muscles exist, which are smaller and disorganized. Muscles in the diaphragm, which also originate from migratory precursors, are missing. Moreover, Gab1-/- embryos die in a broad time window between E13.5 and E18.5, and display reduced liver size and placental defects. The labyrinth layer, but not the spongiotrophoblast layer, of the placenta is severely reduced, resulting in impaired communication between maternal and fetal circulation. Thus, extensive similarities between the phenotypes of c-Met and HGF/SF mutant mice exist, and the muscle migration phenotype is even more pronounced in Gab1-/-:c-Met+/- embryos. This is genetic evidence that Gab1 is essential for c-Met signaling in vivo. Analogy exists to signal transmission by insulin receptors, which require IRS1 and IRS2 as specific docking proteins.

Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1.

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.