Epidermal growth factor (EGF) modulation of feline sarcoma virus fms tyrosine kinase activity, internalization, degradation, and transforming potential in an EGF receptor/v-fms chimera.

The feline sarcoma virus oncogene v-fms has significantly contributed to the dissection of peptide growth factor action since it encodes the transmembrane tyrosine kinase gp140v-fms, a transforming version of colony-stimulating factor 1 receptor, a member of the growth factor receptor tyrosine kinase family. In this study, the functional significance of structural differences between distinct tyrosine kinase types, in particular between cellular receptors and viral transforming proteins of distinct structural types, has been further investigated, and their functional compatibility has been addressed. For this purpose, major functional domains of three structurally distinct tyrosine kinases were combined into two chimeric receptors. The cytoplasmic gp140v-fms kinase domain and the kinase domain of Rous sarcoma virus pp60v-src were each fused to the extracellular ligand-binding domain of the epidermal growth factor (EGF) receptor to create chimeras EFR and ESR, respectively, which were studied upon stable expression in NIH 3T3 fibroblasts. Both chimeras were faithfully synthesized and routed to the cell surface, where they displayed EGF-specific, low-affinity ligand-binding domains in contrast to the high- and low-affinity EGF-binding sites of normal EGF receptors. While the EFR kinase was EGF controlled for autophosphorylation and substrate phosphorylation in vitro, in vivo, and in digitonin-treated cells, the ESR kinase was not responsive to EGF. While ESR appeared to recycle to the cell surface upon endocytosis, EGF induced efficient EFR internalization and degradation, and phorbol esters stimulated protein kinase C-mediated downmodulation of EFR. Despite its ligand-inducible kinase activity, EFR was partly EGF independent in mediating mitogenesis and cell transformation, while ESR appeared biologically inactive.     

A specific combination of substrates is involved in signal transduction by the kit-encoded receptor.

The kit protooncogene encodes a transmembrane tyrosine kinase related to the receptors for the platelet derived growth factor (PDGF-R) and the macrophage growth factor (CSF1-R), and was very recently shown to bind a stem cell factor. To compare signal transduction by the kit kinase with signaling by homologous receptors we constructed a chimeric protein composed of the extracellular domain of the epidermal growth factor receptor (EGF-R) and the transmembrane and cytoplasmic domains of kit. We have previously shown that the chimeric receptor transmits potent mitogenic and transforming signals in response to the heterologous ligand. Here we demonstrate that upon ligand binding, the ligand-receptor complex undergoes endocytosis and degradation and induces short- and long-term cellular effects. Examination of the signal transduction pathway revealed that the activated kit kinase strongly associates with phosphatidylinositol 3'-kinase activity and a phosphoprotein of 85 kd. In addition, the ligand-stimulated kit kinase is coupled to modifications of phospholipase C gamma and the Raf1 protein kinase. However, it does not lead to a significant change in the production of inositol phosphate. Comparison of our results with the known signaling pathways of PDGF-R and CSF1-R suggests that each receptor is coupled to a specific combination of signal transducers.     

Substrate phosphorylation specificity of the human c-kit receptor tyrosine kinase.

The chimeric EK-receptor (EK-R), consisting of the epidermal growth factor receptor (EGF-R) extracellular binding domain and p145c-kit cytoplasmic signal-generating sequences, was fully functional in forming high and low affinity EGF binding sites and in ligand-regulated receptor and substrate phosphorylation activities. Relative to EGF-R, EK-R activation stimulated kit-characteristic phosphorylation of human 293 fibroblast substrate polypeptides. Transient coexpression of EK-R with candidate substrates resulted in ligand-induced phosphorylation of phospholipase C gamma and guanosine triphosphatase-activating polypeptide. The RAF-1 serine/threonine kinase was shown to be associated with activated EK-R, but no tyrosine phosphorylation could be detected. The faithfulness of EK-R substrate phosphorylation specificity was confirmed with stem cell factor-stimulated p145c-kit.     

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.     

A Melanophore-Based Screening Assay for Erythropoietin Receptors

A rapid, functional assay in frog melanophore cells for the erythropoietin receptor (EPOR), a member of the cytokine receptor family, is demonstrated. A chimeric receptor that comprised the extracellular portion of the murine EPOR and the transmembrane and intracellular domains of the human epidermal growth factor receptor (EGFR) was subcloned into the expression vector pJG3.6. When the full-length EGFR was expressed in melanophores, EGF but not EPO mediated pigment dispersion in a time- and dose-dependent manner with an EC50 of 12.6 6 2.9 pM. However, when the chimeric EPOR/EGFR was expressed, EPO but not EGF stimulated pigment dispersion in a time- and dose-dependent manner with an EC50 of 380 6 107 pM. Neither EGF nor EPO had any effect on pigment dispersion in wild-type melanophores. EGF- and EPO-mediated pigment dispersion was blocked by the bis-indolylmaleimide protein kinase C inhibitor Ro 31-8220. This study extends the use of the melanophore-based bioassay to include cytokine receptors in addition to G protein- and tyrosine kinase-coupled receptors. It represents a potentially powerful method for screening of combinatorial libraries to identify novel small molecule agonists and antagonists to this clinically important class of binding sites as well as performing studies of functional ligand-receptor interactions.     

Epidermal growth factor: the receptor and its function

Epidermal growth factor (EGF) is a small polypeptide hormone with mitogenic properties in vivo and in vitro. EGF elicits biologic responses by binding to a cell surface receptor which is a transmembrane glycoprotein containing a cytoplasmic protein tyrosine kinase. EGF responses are mediated by ligand binding and activation of this intrinsic protein kinase. The receptor can be phosphorylated by other protein kinases, and this may regulate receptor function. Stimulation of the receptor tyrosine kinase activity by ligand binding must regulate the activity of an as yet undefined molecule(s) responsible for transmitting a mitogenic signal to the nucleus.     

Structural analysis of the transmembrane domain of the epidermal growth factor receptor

The ligand-binding domain of the epidermal growth factor (EGF) receptor is separated from the cytoplasmic protein tyrosine kinase domain by a predicted single transmembrane segment. Antipeptide antibodies prepared against the outer portion of the predicted transmembrane segment confirmed this area was exposed only when cells were treated with permeabilizing agents. To investigate structural requirements for signal transduction by the transmembrane domain, three types of mutant EGF receptor were prepared. The first type was designed to shorten the transmembrane domain, the second to place proline substitutions within this domain, and the third to make amino acid substitutions analogous to those present in the transforming c-erbB2/neu oncoprotein. Mutant human receptors were expressed in null recipient mouse B82L and Chinese hamster ovary cells. All receptors bound EGF and exhibited EGF-stimulated protein tyrosine kinase activity in vivo as assayed using a 125I-labeled monoclonal anti-phosphotyrosine antibody. EGF stimulated growth of cells expressing each mutant receptor with similar dose-response characteristics. In contrast to other growth factor receptors, the transmembrane domain of the EGF receptor is tolerant to a variety of changes which neither mimic EGF action by constitutive activation nor interfere with ligand-induced signal transduction.     

Biological and biochemical activities of a chimeric epidermal growth factor-Elk receptor tyrosine kinase.

Eph, Elk, and Eck are prototypes of a large family of transmembrane protein-tyrosine kinases, which are characterized by a highly conserved cysteine-rich domain and two fibronectin type III repeats in their extracellular regions. Despite the extent of the Eph family, no extracellular ligands for any family member have been identified, and hence, little is known about the biological and biochemical properties of these receptor-like tyrosine kinases. In the absence of a physiological ligand for the Elk receptor, we constructed chimeric receptor molecules, in which the extracellular region of the Elk receptor is replaced by the extracellular, ligand-binding domain of the epidermal growth factor (EGF) receptor. These chimeric receptors were expressed in NIH 3T3 cells that lack endogenous EGF receptors to analyze their signaling properties. The chimeric EGF-Elk receptors became glycosylated, were correctly localized to the plasma membrane, and bound EGF with high affinity. The chimeric receptors underwent autophosphorylation and induced the tyrosine phosphorylation of a specific set of cellular proteins in response to EGF. EGF stimulation also induced DNA synthesis in fibroblasts stably expressing the EGF-Elk receptors. In contrast, EGF stimulation of these cells did not lead to visible changes in cellular morphology, nor did it induce loss of contact inhibition in confluent monolayers or growth in semisolid media. The Elk cytoplasmic domain is therefore able to induce tyrosine phosphorylation and DNA synthesis in response to an extracellular ligand, suggesting that Elk and related polypeptides function as ligand-dependent receptor tyrosine kinases.     

A chimeric EGF-R-neu proto-oncogene allows EGF to regulate neu tyrosine kinase and cell transformation.

The neu oncogene, characterized by Weinberg and colleagues, is a transforming gene found in ethylnitrosourea-induced rat neuro/glioblastomas; its human proto-oncogene homologue has been termed erbB2 or HER2 because of its close homology with the epidermal growth factor receptor (EGF-R) gene (c-erbB1). Expression of the rat neu oncogene is sufficient for transformation of mouse NIH 3T3 fibroblasts in culture and for the development of mammary carcinomas in transgenic mice, but the neu proto-oncogene has not been associated with cell transformation. We constructed a vector for expression of a chimeric cDNA and hybrid protein consisting of the EGF-R extracellular, transmembrane and protein kinase C-substrate domains linked to the intracellular tyrosine kinase and carboxyl terminal domain of the rat neu cDNA. Upon transfection with the construct, NIH 3T3 cells gave rise to EGF-R antigen-positive cell clones with varying amounts of specific EGF binding. Immunofluorescence and immunoprecipitation using neu- and EGF-receptor specific antibodies demonstrated a correctly oriented and positioned chimeric EGF-R-neu protein of the expected apparent mol. wt on the surface of these cells. EGF or TGF alpha induced tyrosine phosphorylation of the chimeric receptor protein, stimulated DNA synthesis of EGF-R-neu expressing cells and led to a transformed cell morphology and growth in soft agar. In contrast, the neu proto-oncogene did not show kinase activity or transforming properties when expressed at similar levels in NIH 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Insulin-EGF receptor chimerae mediate tyrosine transphosphorylation and serine/threonine phosphorylation of kinase-deficient EGF receptors.

To study cross-talk between unoccupied epidermal growth factor (EGF) receptors and activated EGF receptor kinases, we have used double-transfected cells, IHE2 cells, expressing both an enzymatically active insulin-EGF chimeric receptor and an inactive kinase EGF receptor mutant. Using immunoaffinity-purified receptors, we show that insulin increased phosphorylation of the insulin-EGF chimeric beta subunit and of the kinase-deficient EGF receptor. Stimulation of intact IHE2 cells with insulin leads to a rapid tyrosine autophosphorylation of the insulin-EGF chimeric beta subunit and to tyrosine phosphorylation of the unoccupied kinase-deficient EGF receptor. Insulin-stimulated transphosphorylation of the kinase-deficient EGF receptor yields the same pattern of tryptic phosphopeptides as those in EGF-induced autophosphorylation of the wild-type human EGF receptor. We conclude that insulin, through activation of the insulin-EGF chimeric receptor, mediates transphosphorylation of the kinase-deficient EGF receptor, further confirming that EGF receptor autophosphorylation may proceed by an intermolecular mechanism. In addition to receptor tyrosine phosphorylation, we find that exposure of cells to insulin results in enhanced phosphorylation on serine and threonine residues of the unoccupied kinase-deficient EGF receptor. These results suggest that insulin-EGF chimeric receptor activation stimulates at least one serine/threonine kinase, which in turn phosphorylates the kinase-deficient EGF receptor. Finally, we show that transphosphorylation and coexpression of an active kinase cause a decrease in the number of cell surface kinase-deficient EGF receptors without increasing their degradation rate.     

Epidermal growth factor stimulates phosphorylation of RAF-1 independently of receptor autophosphorylation and internalization.

Phosphorylation of the RAF-1 protooncogene product and activation of its associated serine/threonine kinase are common features of the response of cells to peptide growth factors. We have used wild-type and mutant epidermal growth factor (EGF) receptors to investigate mechanisms of RAF-1 phosphorylation. In vivo EGF treatment rapidly stimulated phosphorylation of RAF-1 exclusively on serine residues. Stimulation of RAF-1 phosphorylation occurred at 37 degrees C but not at 4 degrees C and persisted after dissociation of EGF from its receptor. EGF-induced RAF-1 serine phosphorylation required the intrinsic tyrosine kinase activity of the EGF receptor but was independent of EGF receptor self-phosphorylation and of ligand-induced receptor internalization. Down-regulation of protein kinase C did not affect the EGF-induced increase in RAF-1 phosphorylation. These data suggest that the activated tyrosine kinase activity of the EGF receptor enhances serine phosphorylation of RAF-1 via an intermediary molecule(s).     

Intrapeptide autophosphorylation of the epidermal growth factor receptor: regulation of kinase catalytic function by receptor dimerization

The epidermal growth factor (EGF) receptor is a transmembrane polypeptide of 170 000 daltons (Da) with a cytoplasmically facing protein kinase domain. The regulation of the tyrosine kinase activity of the EGF receptor by added EGF and by receptor association state was studied in an in vitro system. The rate of autophosphorylation of the solubilized and purified EGF receptor was found to be independent of receptor concentration. To determine whether the zero-order kinetics observed point to intrapeptide phosphorylation, we measured the sedimentation characteristics of the undenatured solubilized receptor. The receptor was found to exist in two association-dissociation states-a monomeric 7.7S form and a dimeric 12S form. The 7.7S form is an active tyrosine kinase; it has high basal activity, and the activity is not further stimulated by EGF; it appears to be an EGF-independent form of the receptor kinase. The 12S form is devoid of catalytic activity, but in the presence of EGF it dissociates into the active monomeric form. Freshly purified receptor preparations contain mainly the monomeric receptor, have high basal kinase activity, and show low EGF stimulatability (less than 1.3-fold). Aging of the receptor results in progressive dimerization and decay of EGF-independent kinase activity (and increase in EGF stimulatability). All of these processes are reversed in the presence of EGF or dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential inhibitory effects of TGF-beta on EGF-, PDGF-, and HBGF-1-stimulated MG63 human osteosarcoma cell growth: possible involvement of growth factor interactions at the receptor and postreceptor levels

The growth of MG63 human osteosarcoma cell line in 5% serum is stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or heparin-binding growth factor-1 (HBGF-1). The mitogenic effect of EGF and PDGF is completely blocked by TFG-beta at 1 ng per ml and the effect of HBGF-1 is attenuated by 75-80%. Treatment of MG63 cells with TGF-beta reduces HBGF-1 receptor binding affinity from 1.24 x 10(-11) M to 3.51 x 10(-11) M with no change on the receptor number (1.1 x 10(3) per cell). The receptor-binding affinity of EGF and PDGF is not altered by TGF-beta treatment; however, the number of EGF receptor is increased by 25%. Both EGF and PDGF stimulate MG63 cellular tyrosine kinase activity, and such stimulation is inhibited by TGF-beta pretreatment. No change in the cellular protein tyrosine phosphorylation pattern can be detected in HBGF-1-stimulated cells with and without TGF-beta pretreatment. These data suggest that TGF-beta inhibits EGF and PDGF mitogenicity by blocking EGF- and PDGF-stimulated tyrosine kinase activity and attenuates HBGF-1 mitogenicity by decreasing its receptor affinity.     

Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.     

Alteration of epidermal growth factor receptor activity by mutation of its primary carboxyl-terminal site of tyrosine self-phosphorylation

The epidermal growth factor (EGF) receptor, which exhibits intrinsic protein tyrosine kinase activity, undergoes a rapid, intramolecular self-phosphorylation reaction following EGF activation. The primary sites of tyrosine self-phosphorylation in vivo are located in the extreme carboxyl-terminal region of the molecule, principally Tyr-1173. To test the biological and biochemical consequences of this EGF receptor self-phosphorylation, we made the mutation Tyr----Phe-1173. Membranes containing the mutated receptor exhibited an ED50 for EGF activation of tyrosine kinase activity equivalent to control receptor at both high and low substrate levels, but exhibited reduced basal and EGF-stimulated tyrosine kinase activity at low, non-saturating substrate levels. The Tyr----Phe-1173 mutant possessed high affinity EGF binding and could still self-phosphorylate other tyrosine sites in an intramolecular fashion with a low Km for ATP (200 nM), suggesting that this alteration did not grossly change receptor structure. When EGF-dependent growth of Chinese hamster ovary cells expressing comparable levels of control or mutant EGF receptor was measured, the ability of the mutant receptor to mediate cell growth in response to EGF was reduced by approximately 50%, yet both receptors exhibited a similar affinity and ED50 for EGF. These results support the concept that this self-phosphorylation site can act as a competitive/alternate substrate for the EGF receptor, and that this region of the molecule is important in modulating its maximal biological activity.     

Differential heat stress stability of epidermal growth factor receptor and erbB-2 receptor tyrosine kinase activities

The epidermal growth factor (EGF) and erbB-2 receptors are structurally related membrane-bound tyrosine kinases. While these proteins exhibit close sequence homology, 50% overall and 80% in the tyrosine kinase domains, they respond very differently to heat stress. In NIH-3T3 or NR6 cells transfected with wild-type EGF-R and incubated at 37°C or heat shocked at 46°C, EGF binds to its receptor and stimulates receptor autophosphorylation to equivalent extents. At 46°C, however, the basal tyrosine kinase activity of the wild-type erbB-2 receptor is rapidly lost. When cells containing chimeric receptors composed of the EGF-R extracellular domain and intracellular domain of erbB-2 were heat stressed, ¹²⁵I-EGF bound to the receptors, but did not stimulate receptor autophosphorylation. The decline in EGF-stimulated chimeric erbB-2 receptor autophosphorylation is dependent on the length of heat shock, with nearly 100% of the kinase activity lost after 60 min at 46°C. The loss of chimeric receptor erbB-2 kinase activity is not due to degradation of receptor protein, nor is it attributable to a specific transmembrane domain from either the EGF or erbB-2 receptors. Sensitivity of erbB-2 to heat stress is also not a result of denaturation of this receptor's carboxy-terminal domain. Insertion of the erbB-2 tyrosine kinase domain into the EGF-R confers heat stress sensitivity to the resultant chimeric receptor. Thus, although the EGF-R and erbB-2 kinase domains show a high degree of homology, the secondary/tertiary structures of these domains would seem to be stabilized in distinct manners. © 1993 Wiley-Liss, Inc.     

Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells.

In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.     

EGF-stimulated tyrosine phosphorylation of p185neu: a potential model for receptor interactions.

p185neu is a receptor-like protein encoded by the neu/erbB-2 proto-oncogene. This protein is closely related to the epidermal growth factor (EGF) receptor, but does not bind EGF. We report here that incubation of Rat-1 cells with EGF stimulates tyrosine phosphorylation of p185. This effect is specific to EGF since neither platelet derived growth factor (PDGF) nor insulin, which also bind to receptors with ligand-stimulated tyrosine kinase activity, induced tyrosine phosphorylation of p185. The EGF-stimulated tyrosine phosphorylation of p185 and of the EGF receptor occurred with similar kinetics and EGF dose-responses, and both phosphorylations were prevented by down-regulation of the EGF receptor with EGF. Since p185 does not bind EGF, these results suggested that p185 is a substrate for the EGF receptor kinase. Incubation of cells with EGF before lysis stimulated the tyrosine phosphorylation of p185 in immune complexes. This suggested that EGF, acting through the EGF receptor, can regulate the intrinsic kinase activity of p185.