Antibodies against a synthetic peptide as a probe for the kinase activity of the avian EGF receptor and v-erbB protein

The transforming protein v-erbB of avian erythroblastosis virus (AEV) displays extensive sequence homology with the presumptive protein-tyrosine kinase domain of the human EGF receptor and with the src protein-tyrosine kinase family of oncogenes. However, no kinase activity has previously been demonstrated for the v-erbB protein. Here antibodies generated against a synthetic peptide from the C terminus of human EGF receptor are shown to immunoprecipitate the EGF receptor from human and avian cells, as well as the v-erbB proteins from AEV-transformed cells that become phosphorylated on tyrosine residues upon the addition of gamma-32P-ATP. The immunoprecipitates are also able to phosphorylate exogenous tyrosine-containing substrates. Hence, it is likely that both avian EGF receptor and v-erbB proteins are protein tyrosine-specific protein kinases. Since the kinase activity of v-erbB protein cannot be regulated by EGF, it is proposed that the tyrosine protein kinase function of v-erbB may be constitutively activated.     

Analysis of the role of the Shc and Grb2 proteins in signal transduction by the v-ErbB protein.

The epidermal growth factor receptor, EGFR, has been implicated in cell transformation in both mammalian and avian species. The v-ErbB oncoprotein is an oncogenic form of the chicken EGFR. The tyrosine kinase activity of this oncoprotein is required for transformation, but no transformation-specific cellular substrates have been described to date. Recently activation of the ras signal transduction pathway by the EGFR has been shown to involve the Shc and Grb2 proteins. In this communication, we demonstrate that the Shc proteins are phosphorylated on tyrosine residues and are complexed with Grb2 and the chicken EGFR following ligand activation of this receptor. In fibroblasts and erythroid cells transformed by the avian erythroblastosis virus (AEV) strains H and ES4, the Shc proteins are found to be constitutively phosphorylated on tyrosine residues. The tyrosine-phosphorylated forms of the AEV strain H v-ErbB protein are found in a complex with Shc and Grb2, but the Shc proteins do not bind to the AEV strain ES4 v-ErbB protein. Mutant forms of the v-ErbB protein (in which several of the tyrosines that become autophosphorylated have been deleted by truncation) are unable to transform erythroid cells but can still transform fibroblasts. Analysis of cells transformed by one of these mutants revealed that the truncated v-ErbB protein could no longer bind to either Shc or Grb2, but this oncoprotein still gave rise to tyrosine-phosphorylated Shc proteins that complexed with Grb2 and led to activation of mitogen-activated protein (MAP) kinase. The results suggest that stable binding of Grb2 and Shc to the v-ErbB protein is not necessary to activate this signal transduction pathway and assuming that the mutant activate MAP kinase in erythroid cells in a manner similar to that of fibroblasts, that activation of this pathway is not sufficient to transform erythroid cells.     

Cooperation of v-jun and v-erbB oncogenes in embryo fibroblast transformation in vitro and in vivo.

Retroviral vectors carrying either the v-jun and v-erbB sequences or the v-jun gene linked to the neomycin resistance gene were constructed on the basis of the structural genome organization of avian erythroblastosis virus (AEV). These viruses, called JB and JN, respectively, were rescued as Rous-associated virus-1 pseudotypes, and they were shown to successfully transform chicken embryo fibroblasts in vitro. However, in agar, colonies developed from JB-infected fibroblasts were three to five times larger than those obtained after infection with JN or with AEV Pst124 carrying only a functional v-erbB gene. In vivo, on chorioallantoic membrane (CAM) assays, JB produced fibrosarcomas that were more rapidly growing and much larger than those induced by JN or AEV Pst124. Moreover, in chickens infected in ovo with JB, multiple fibrosarcomas arose in different organs a few days after birth, whereas no tumor could be detected in parallel experiments in either JN- or AEV Pst124-infected animals. These results demonstrate that in embryo fibroblast cells, v-jun and v-erbB can act synergistically to enhance the transformation potential of either oncogene alone both in vitro and in vivo.     

Tyrosine kinase activity may be necessary but is not sufficient for c-erbB1-mediated tissue-specific tumorigenicity.

Expression of mutant avian c-erbB1 genes results in tissue-specific transformation in chickens. Site-directed mutagenesis was used to generate kinase-defective mutants of several tissue-specific v-erbB transforming mutants by replacement of the ATP-binding lysine residue in the kinase domain with an arginine residue. These kinase-defective v-erbB mutants were analyzed for their in vitro and in vivo transforming potentials. Specifically, kinase-defective mutants of erythroleukemogenic, hemangioma-inducing, and sarcomagenic v-erbB genes were assessed for their oncogenic potential. In vitro transformation potential was assessed by soft-agar colony formation in primary cultures of chick embryo fibroblasts (CEF). In vivo transformation potential was determined by infection of 1-day-old line 0 chicks with concentrated recombinant retrovirus and then monitoring of birds for tumor formation. These transformation assays demonstrate that kinase activity is absolutely essential for transformation by tissue-specific transforming mutants of the avian c-erbB1 gene. Since all of the tissue-specific v-erbB mutants characterized to date exhibit tyrosine kinase activity in vitro but do not transform all tissues in which they are expressed, we conclude that v-erbB-associated tyrosine kinase activity may be necessary but is not sufficient to induce tumor formation.     

Temperature-sensitive mutants of avian erythroblastosis virus: surface expression of the erbB product correlates with transformation

The v-erbB gene of avian erythroblastosis virus (AEV) codes for an integral plasma membrane glycoprotein, gp74erbB. Expression of gp74erbB and its intracellular precursors, gp66erbB and gp68erbB, has been studied in cells transformed by two temperature-sensitive mutants of AEV. After shift to 42 degrees C, the processing of gp68erbB is blocked in tsAEV-transformed, but not in wtAEV-transformed, erythroblasts and fibroblasts. In addition, gp74erbB disappears from the surface of tsAEV cells within 12 hr after shift. Thus tsAEV mutants probably bear a lesion in v-erbB that affects the maturation and subcellular localization of gp74erbB. The tsAEV erythroblasts, when "committed" to differentiation by a pulse-shift to 42 degrees C, reexpress gp74erbB during terminal differentiation at 36 degrees C. This suggests that tsAEV erythroblasts become insensitive to the transforming functions of gp74erbB at a certain stage of differentiation.     

Tyrosine phosphorylation of a 120-kilodalton pp60src substrate upon epidermal growth factor and platelet-derived growth factor receptor stimulation and in polyomavirus middle-T-antigen-transformed cells.

The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.     

Structural domains of the avian erythroblastosis virus erbB protein required for fibroblast transformation: dissection by in-frame insertional mutagenesis.

Avian erythroblastosis virus (AEV) induces erythroblastosis and fibrosarcomas. The viral erbB protein is required for AEV-mediated oncogenesis. To explore the structural aspects of the v-erbB polypeptide necessary for its oncogenic function, we created a series of small in-frame insertions in different domains of the v-erbB oncogene. AEV genomes bearing lesions within the v-erbB kinase domain demonstrated a drastically decreased ability to transform avian fibroblasts, establishing a functional role for this structurally conserved oncogene domain. In contrast, mutations in the extracellular domain, between the transmembrane region and the kinase domain, or at the extreme C terminus of the v-erbB protein had no effect on AEV-mediated fibroblast transformation. One lesion within the v-erbB kinase domain, a 10-amino acid insertion, produced a temperature-sensitive mutant capable of fibroblast transformation at 36 degrees C but not at 41 degrees C, suggesting that small in-frame insertions have general utility for the in vitro creation of conditional mutants.     

Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells.

The cytoskeletal protein talin was found to undergo enhanced phosphorylation at tyrosine residues in chicken embryo fibroblasts following transformation by Rous sarcoma virus. An increase in the tyrosine phosphorylation of talin was also observed within 6 h in cells infected by the temperature-sensitive mutant tsNY68 after a shift from the nonpermissive to the permissive temperature. The overall extent of phosphorylation was 0.07 mol of phosphate per mol of talin and was not appreciably altered by transformation. In uninfected cells talin was shown to be phosphorylated at multiple sites by tryptic peptide mapping. Following transformation most of these sites remained phosphorylated, to the same or to a lesser extent, while novel, phosphotyrosine-containing phosphopeptides appeared. Talin was phosphorylated at tyrosine in cells infected by Rous sarcoma virus mutants which induce altered or partial transformation morphologies; thus the increased phosphorylation of talin at tyrosine occurred irrespective of the morphology induced. Transformation by Y73 also induced elevated levels of phosphotyrosine in talin, whereas transformation by the avian erythroblastosis and Fujinami sarcoma viruses did not.     

Mutation of a protein kinase C phosphorylation site in the erbB protein of avian erythroblastosis virus.

Tumor promoter-stimulated phosphorylation of threonine 98 of the erbB protein of avian erythroblastosis virus (AEV) correlates with inhibition of erbB-dependent mitogenesis. To more clearly define the role of phosphorylation of this residue in regulation of the activity of the erbB protein, we have constructed erbB mutations which encode alanine (Ala-98), tyrosine (Tyr-98), or serine (Ser-98) at position 98. The biosynthesis and stability of the three mutant proteins were similar to those of the wild-type erbB protein, and all three retained the ability to transform chicken embryo fibroblasts. Treatment of transformed CEF with 12-tetradecanoylphorbol-13-acetate (TPA) stimulated incorporation of 32Pi into wild-type and mutant erbB proteins and resulted in a slight decrease in the electrophoretic mobilities of all the erbB proteins. Tryptic maps of erbB phosphopeptides showed no endogenous or TPA-stimulated phosphorylation of alanine 98 or tyrosine 98 in cells transformed by the Ala-98 and Tyr-98 mutants. Analysis of tryptic phosphopeptides by high-pressure liquid chromatography revealed that TPA treatment of cells stimulated phosphorylation of other sites of the erbB protein in addition to threonine 98. A high endogenous level of phosphorylation of serine 98 of the Ser-98 mutant protein was found, and TPA treatment of cells did not result in further phosphorylation of this residue. Cells transformed by wild-type and mutant AEV were equally sensitive to TPA-dependent inhibition of growth in soft agar and TPA-dependent inhibition of [3H]thymidine incorporation. TPA treatment inhibited tyrosine phosphorylation to a similar extent in cells transformed by wild-type or Ala-98 AEV. These data indicate that phosphorylation of threonine 98 of the erbB protein is not responsible for TPA-dependent inhibition of growth of AEV-transformed cells or TPA-induced inhibition of erbB-dependent tyrosine phosphorylation. TPA-stimulated phosphorylation of the erbB protein at other sites may mediate these effects. The data also show that subtle changes in a phosphorylation site (i.e., changing threonine to serine) can drastically alter recognition by protein kinases.     

Cells transformed by Rous sarcoma virus release transforming growth factors

Chicken embryo fibroblasts and hamster BHK cells transformed by Rous sarcoma virus (RSV) release in their culture media growth factors which enhance markedly anchorage-independent colony formation in gelified medium, at the restrictive temperature (41 degrees 5 C), of chicken embryo fibroblasts (CEF) infected by RSV mutants with a ts mutation of the src gene. This action is not observed with uninfected CEF, and, therefore, appears to require some expression of the viral src gene in the target cells. The enhancing factors are proteins related to the family of the transforming growth factors (TGFs) by their molecular weight (about 20 kd), their heat and acid resistance, and their sensitivity to dithiothreitol. They do not compete with 125I EGF for binding on the EGF receptors of the membrane of A431 cells. As chicken embryo fibroblasts are devoid of EGF receptors, their activity is not potentiated by EGF.     

Analysis of a tyrosine-specific protein kinase activity associated with the retroviral erbB oncogene product

The transforming protein erbB of avian erythroblastosis virus (AEV) has considerable sequence homology with the epidermal growth factor (EGF) and appears to represent a truncated form of this receptor. The sequence of the erbB gene is furthermore related to that of other viral transforming genes such as src, fps, yes or abl. The transforming proteins of these src-related oncogenes as well as receptors for EGF, platelet-derived growth factor (PDGF), and insulin are associated with tyrosine-specific protein kinases. It has been difficult to demonstrate this activity for the erbB protein. To analyze the erbB gene product, we prepared polyclonal antibodies against a bacterially expressed erbB DNA restriction fragment (BamHI/BamHI). The antiserum is shown to immunoprecipitate the erbB protein from AEV-transformed chicken fibroblasts and also recognizes the EGF receptor protein. Both proteins become phosphorylated in vitro on tyrosine residues upon the addition of [gamma-32P]ATP. The protein kinase activity is low compared to other oncogene-specific kinases. This is not due to kinase blocking by the serum, because erbB carboxyterminal synthetic peptide antibodies give rise to low levels of protein kinase activity as well indicating that this may be a characteristic property of erbB in vitro.     

Host range mutants of v-src: alterations in kinase activity and substrate interactions.

Host range mutants of Schmidt-Ruppin v-src that transform chicken embryo fibroblasts (CEF) but not Rat-2 cells were generated previously by linker insertion-deletion mutagenesis (J. E. DeClue and G. S. Martin, J. Virol. 63:542-554, 1989). One of these mutants, SRX5, in which Tyr-416 is substituted by the sequence Ser Arg Asp, retained high levels of kinase activity in vitro and in vivo, both in CEF and in Rat-2 cells. Phosphorylation of p36 (the calpactin I heavy chain) was drastically reduced in cells expressing SRX5 src, suggesting that the phenotype of SRX5 results from an alteration in substrate recognition by the src kinase. Three mutants, SPX1, SHX13, and XD6, containing linker insertions or small deletions within the src homology 2 (SH2) region, induced reduced levels of kinase activity in both CEF and Rat-2 cells. However, the residual levels of kinase activity in Rat-2 cells were above the threshold at which wild-type pp60v-src transforms Rat-2 cells, indicating that the reduction in kinase activity was not sufficient to account for the failure to transform. Cells infected by these mutants exhibited reduced levels of phosphorylation of 120- and 62-kDa proteins. We have reported elsewhere (M. F. Moran, C. A. Koch, D. Anderson, C. Ellis, L. England, G. S. Martin, and T. Pawson, Proc. Natl. Acad. Sci. USA 87:8622-8626, 1990) that ras GTPase-activating protein GAP and associated protein p62 are not tyrosine phosphorylated in Rat-2 cells expressing SHX13 or XD6. The transformation defect in Rat-2 cells may result from the failure to phosphorylate those proteins. The fifth mutant, XD4, contains a deletion which removes all of the src homology 3 (SH3) and most of the SH2 sequences of src. The protein encoded by XD4 is active as a kinase when expressed in CEF, indicating that in CEF the SH2 and SH3 regions of v-src are not necessary for kinase activity and transformation. The XD4 src product is not tyrosine phosphorylated and is inactive as a kinase when expressed in Rat-2 cells. Thus, host cell factors can affect the tyrosine phosphorylation and activity of the v-src kinase in the absence of the SH2 and SH3 regions. These results indicate that the host-dependent transformation phenotype results from alterations in src kinase activity and substrate specificity.     

Evidence that the tyrosine kinase domain of a small fraction of epidermal growth factor receptor molecules is exposed on the outer surface of A431 cells

Intact A431 cells were labeled with [gamma-32P]ATP. The major phosphorylation product of the ecto-kinase activity of A431 cells had the molecular mass of 170 kd and was identified as EGF receptor by specific immunoprecipitation. This phosphorylation was not stimulated by EGF added to the reaction buffer, but replacement of MgCl2 by MnCl2 in the buffer remarkably stimulated phosphorylation. An exogenous protein substrate, alpha-casein, was also phosphorylated by intact A431 cells. The analyses for phospho-amino acids of both EGF receptor and alpha-casein revealed that phosphorylation occurred mainly at phosphotyrosine residues. Tryptic phospho-peptides of the EGF receptor of intact A431 cells labeled with [gamma-32P]ATP were fractionated by HPLC. The elution patterns were essentially the same as that of the autophosphorylated EGF receptor, indicating that the phosphorylation sites of EGF receptor labeled in vivo with [gamma-32P]ATP are located in three tyrosine residues in the carboxyl terminus. These results indicate that the carboxyl-terminal tyrosine kinase domain of a small fraction of the EGF receptor molecules of an A431 cell is exposed on the outer surface of the cells.     

Functional heterogeneity of proto-oncogene tyrosine kinases: the C terminus of the human epidermal growth factor receptor facilitates cell proliferation.

Previous reports have indicated that the C termini of the membrane-associated tyrosine kinases encoded by c-src and c-fms proto-oncogenes have a negative effect on their biological activity and that this effect is mediated by their C-terminal tyrosine residue. To determine whether this was true for the human epidermal growth factor (EGF) receptor, which is also a membrane-associated tyrosine kinase proto-oncogene, we have constructed two premature termination mutants, dc19 and dc63, that delete the C-terminal 19 and 63 amino acids, respectively, from the human full-length receptor (hEGFR). The smaller deletion removes the C-terminal tyrosine residue, while the larger deletion removes the two most C-terminal tyrosines; similar deletions are found in v-erbB. As previously shown for the gene encoding the full-length EGF receptor, the two C-terminal mutants induced EGF-dependent focal transformation and anchorage-independent growth of NIH 3T3 cells. However, both dc19 and dc63 were quantitatively less efficient than the gene encoding the full-length receptor, with dc63 being less active than dc19. Although the C-terminal mutants displayed lower biological activity than the gene encoding the full-length receptor, the mutant receptors were found to be similar in several respects to the full-length receptor. These parameters included receptor localization, stability in the absence of EGF, receptor half-life in the presence of EGF, EGF binding, extent of EGF-dependent autophosphorylation in vitro, and EGF-dependent phosphorylation of an exogenous substrate in vitro. Therefore, the C-terminal 63 amino acids of the human receptor have no detectable influence on EGF-dependent early events. We conclude that in contrast     

Constitutively activated neu oncoprotein tyrosine kinase interferes with growth factor-induced signals for gene activation.

The neu receptor oncoprotein tyrosine kinase, capable of transforming cultured fibroblasts and causing mammary carcinomas in transgenic mice, carries a point mutation in its transmembrane domain and shows a constitutive tyrosine kinase activity. We analyzed the neu tyrosine kinase and its substrates in transfected NIH 3T3 fibroblasts by phosphotyrosine immunoblotting. Tyrosine phosphorylated proteins were similar but not identical in epidermal growth factor (EGF)-stimulated cells expressing the human EGF receptor (EGFR) or a chimeric EGFR/neu receptor but differed from phosphotyrosyl proteins constitutively expressed in neu oncogene-transformed cells. The neu oncoprotein in the latter cells was phosphorylated in tyrosine in a ligand-independent manner and had a shortened half-life in comparison with the normal neu protein. Tumor promoter pretreatment inhibited ligand-induced receptor tyrosine phosphorylation and decreased tyrosine phosphorylated neu oncoprotein. Prolonged pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also prevented the induction of immediate early growth factor-regulated genes in response to neu activation. Expression of the neu oncogene but not the protooncogene in NIH 3T3 cells was associated with enhanced levels of the jun and fos oncoproteins and loss of serum growth factor induction of immediate early mRNA responses. The constitutively activated neu oncoprotein tyrosine kinase thus deregulates cellular genomic responses to growth factors.     

Activation of c-erbB in avian leukosis virus-induced erythroblastosis leads to the expression of a truncated EGF receptor kinase.

Chicken erythroblastosis caused by avian leukosis virus (ALV) is thought to be mediated by activation of the c-erbB/EGF receptor oncogene by a promoter-insertion mechanism. Here we study the proteins expressed by two ALV-induced leukemias and compare them with the avian EGF receptor and with the oncogene product of avian erythroblastosis virus (v-erbB) which was shown to be a truncated EGF receptor. It appears that the two leukemias express truncated EGF receptors of slightly different sizes with intrinsic tyrosine kinase activity. Hence, acute and chronic retroviruses utilize a common pathway for transformation. Moreover, the proteins expressed in the leukemias are similar to the avian EGF receptor with respect to their phosphopeptide maps, suggesting that they do not carry the C-terminal deletion characteristic of v-erbB.     

The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity

Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine-specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine-phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells.     

Transformation by the v-fms oncogene product: an analog of the CSF-1 receptor

The product of the c-fms proto-oncogene is related to, and possibly identical with, the receptor for the macrophage colony-stimulating factor, M-CSF (CSF-1). Unlike the product of the v-erbB oncogene, which is a truncated version of the EGF receptor, the glycoprotein encoded by the v-fms oncogene retains an intact extracellular ligand-binding domain so that cells transformed by v-fms express CSF-1 receptors at their surface. Although fibroblasts susceptible to transformation by v-fms generally produce CSF-1, v-fms-mediated transformation does not depend on an exogenous source of the growth factor, and neutralizing antibodies to CSF-1 do not affect the transformed phenotype. An alteration of the v-fms gene product at its extreme carboxyl-terminus represents the major structural difference between it and the c-fms-coded glycoprotein and may affect the tyrosine kinase activity of the v-fms-coded receptor. Consistent with this interpretation, tyrosine phosphorylation of the v-fms products in membranes was observed in the absence of CSF-1 and was not enhanced by addition of the murine growth factor. Cells transformed by v-fms have a constitutively elevated specific activity of a guanine nucleotide-dependent, phosphatidylinositol-4,5-diphosphate-specific phospholipase C. We speculate that the tyrosine kinase activity of the v-fms/c-fms gene products may be coupled to this phospholipase C, possibly through a G regulatory protein, thereby increasing phosphatidylinositol turnover and generating the intracellular second messengers diacylglycerol and inositol triphosphate.     

Tissue- and transformation-specific phosphotyrosyl proteins in v-erbB-transformed cells.

To understand the mechanism of tissue-specific and transformation-specific signaling by the v-ErbB oncoprotein, we have investigated signaling pathways downstream of this transmembrane tyrosine kinase. In this report, we describe tissue-specific patterns of phosphotyrosyl proteins in three distinct cell types transformed by the v-erbB oncogene: fibroblasts, erythroblasts, and endothelial cells. In addition, we describe transformation-specific tyrosine phosphorylation events and signal complex formation in v-erbB-transformed fibroblasts. Two patterns of phosphotyrosyl proteins have been detected in v-erbB-transformed cells. The first is a fibroblast-specific pattern which includes unique phosphotyrosyl proteins of 170 kDa (c-ErbB1), 158 kDa, and 120 kDa (the catenin-like protein p120cas). The second is an erythroblast/endothelial cell-specific pattern which includes a prominent unidentified phosphotyrosyl protein of 120 kDa. Evaluation of the phosphotyrosyl proteins p120cas and SHC in chicken embryo fibroblasts infected with transforming and nontransforming v-erbB mutants reveals transformation-specific patterns of tyrosine phosphorylation. One corollary of these phosphorylation events in v-erbB-transformed fibroblasts is the formation of a complex involving SHC, growth factor receptor-bound protein 2, and a novel 75-kDa phosphotyrosyl protein. The results of these studies suggest that the v-ErbB oncoprotein can couple to multiple signal transduction pathways, that these pathways are tissue specific, and that v-erbB-mediated transformation involves specific tyrosine phosphorylation events.