Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.     

The T cell antigen receptor zeta chain is tyrosine phosphorylated upon activation

The T cell antigen receptor is composed of at least seven chains derived from six different gene products. Upon stimulation, several chains can be phosphorylated. Two of these, CD3-gamma and CD3-epsilon are phosphorylated on serine residues. In addition, a 21-kDa nonglycosylated receptor component is phosphorylated, upon activation, on tyrosine residues. We have referred to this phosphoprotein as p21 because we have previously not been able to assign the tyrosine phosphorylation to any of the described receptor subunits (Samelson, L. E., Patel, M. D., Weissman, A. M., Harford, J. B., and Klausner, R. D. (1986) Cell 46, 1083-1090). In this paper, we demonstrate that it is the 16-kDa zeta chain which is the tyrosine phosphorylated subunit, and thus the p21 nomenclature can be replaced. This phosphorylation results in a shift of the apparent Mr of zeta to 21 kDa. Proof that p21 is tyrosine phosphorylated zeta was afforded by a number of approaches. Specific anti-zeta antibodies directly precipitated phospho-p21. Metabolically labeled protein corresponding to p21 could only be observed after activation. When this 21-kDa band was isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reanalyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment with alkaline phosphatase, its migration was identical with that of zeta. Furthermore, peptide mapping of metabolically labeled p21 (after gel isolation and dephosphorylation) showed it to be indistinguishable from p21. Thus, one of the early events of T cell activation is the tyrosine phosphorylation of the zeta chain of the T cell antigen receptor.     

Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization.

Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptor's intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin-coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affinity sites and are required for EGF-induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein-tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein-tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v-abl kinase region transforms Rat-I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor.     

TGF-alpha-driven tumor growth is inhibited by an EGF receptor tyrosine kinase inhibitor.

The simultaneous presence of the EGFR and its ligand TGF-alpha in human tumor tissues suggests that autocrine TGF-alpha stimulation drives tumor growth. Here we show that autocrine TGF-alpha stimulation does cause increased tumor growth in vivo, an effect that was proven to be mediated via EGFR activation, and that this TGF-alpha/EGFR autocrine loop was accessible to an EGFR specific tyrosine kinase inhibitor. Clones of the EGFR expressing glioma cell line U-1242 MG were transfected with TGF-alpha cDNA using a tetracycline-inhibitory system for gene expression. TGF-alpha expression was inhibited by the presence of tetracycline, and subcutaneous tumors forming from cell lines injected into nude mice could be inhibited by feeding mice tetracycline. We confirmed that TGF-alpha mRNA and protein were present in these tumors and that, subsequently, the endogenous EGFR was activated. Tumor growth could be inhibited by an EGFR specific tyrosine kinase inhibitor of the type 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline, administered daily by intraperitoneal injection, thereby interrupting the autocrine loop.     

EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling

Binding of EGF to cells expressing human EGF receptor stimulated rapid tyrosine phosphorylation of phospholipase C-II (PLC-II), as revealed by immunoblotting analysis with phosphotyrosine-specific antibodies. Tyrosine phosphorylation of PLC-II was stimulated by low physiological concentrations of EGF (1 nM), was quantitative, and was already maximal after a 30 sec incubation with 50 nM EGF at 37 degrees C. Interestingly, antibodies specific for PLC-II were able to coimmunoprecipitate the EGF receptor and antibodies against EGF receptor also coimmunoprecipitated PLC-II. According to this analysis, approximately 1% of EGF receptor molecules were associated with PLC-II molecules. The protein tyrosine kinase inhibitor tyrphostin RG50864, which blocks EGF-dependent cell proliferation, blocked EGF-induced tyrosine phosphorylation of PLC-II, its association with EGF receptor, and EGF-induced Ca2+ release. Hence, EGF-induced tyrosine phosphorylation of PLC-II may be a regulatory event linking the tyrosine kinase activity of EGF receptor to the PIP2 hydrolysis signaling pathway.     

The insulin-like growth factor II receptor is phosphorylated by a tyrosine kinase in adipocyte plasma membranes

Incorporation of 32P from [gamma-32P]ATP into tyrosine residues of the insulin-like growth factor (IGF)-II receptor was observed in a Triton X-100-insoluble fraction of rat adipocyte plasma membranes. IGF-II receptor phosphorylation proceeded to a stoichiometry of approximately 0.5 mol of phosphate/IGF-II binding site after 10 min of incubation at 4 degrees C. A Km for ATP of 6 microM was calculated for this phosphorylation reaction. Addition of IGF-II caused an approximately 2-fold increase in tyrosine phosphorylation of the IGF-II receptor in this preparation. In contrast, phosphorylation of angiotensin II by the Triton X-100 washed membranes was not stimulated by IGF-II. Incubation of purified receptor immobilized on IGF-II agarose or of receptor-enriched low density microsomal membranes with [gamma-32P]ATP did not result in appreciable incorporation of [32P]phosphate into the IGF-II receptor nor into exogenous substrates. These data suggest that the IGF-II receptor is not a tyrosine protein kinase capable of autophosphorylation but that it is a substrate for a tyrosine protein kinase endogenous to the adipocyte plasma membrane. The stimulatory effect of IGF-II on the tyrosine phosphorylation of its receptor may be due to a conformational change which converts the receptor to a better substrate for this tyrosine kinase.     

EGF receptor clustering is induced by a 0.4 mT power frequency magnetic field and blocked by the EGF receptor tyrosine kinase inhibitor PD153035

Atomic force microscopy (AFM), transmission electron microscopy (TEM), and confocal laser scanning microscopy were used to investigate the effects of a 50 Hz 0.4 mT magnetic field (MF) on the clustering of purified epidermal growth factor receptors (EGFRs) and EGFRs in Chinese hamster lung (CHL) cell membrane. The results demonstrate that exposing purified EGFRs to the MF for 30 min induces receptor clustering. The peak height of apparent clusters increased from 1.42 +/- 0.18 (sham-exposed) to 3.08 +/- 0.38 nm (exposed) while the mean half-width increased from 21.7 +/- 2.2 to 33.0 +/- 4.0 nm. A similar effect was also observed by TEM. Treatment of purified EGFR with PD153035 (PD), an EGFR-specific tyrosine kinase (TK) inhibitor, inhibited the MF-induced EGFR clustering of the purified proteins, an effect also observed for the receptors in cell membrane in the absence of EGF. These results strongly suggest that the 50 Hz 0.4 mT MF interferes with the EGFR signaling pathway, most likely by interacting with the cytoplasmic TK domain.     

The insulin receptor tyrosine kinase domain in a chimaeric epidermal growth factor-insulin receptor generates Ca2+ signals through the PLC-gamma1 pathway.

The receptors for insulin (IR) and epidermal growth factor (EGFR) are members of the tyrosine kinase receptor (TKR) family. Despite homology of their cytosolic TK domains, both receptors induce different cellular responses. Tyrosine phosphorylation of insulin receptor substrate (IRS) molecules is a specific IR post-receptor response. The EGFR specifically activates phospholipase C-gamma1 (PLC-gamma1). Recruitment of substrate molecules with Src homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains to phosphotyrosines in the receptor is one of the factors creating substrate specificity. In addition, it has been shown that the TK domains of the IR and EGFR show preferences to phosphorylate distinct peptides in vitro, suggesting additional mechanisms of substrate recognition. We have examined to what extent the substrate preference of the TK domain contributes to the specificity of the receptor in vivo. For this purpose we determined whether the IR TK domain, in situ, is able to tyrosine-phosphorylate substrates normally used by the EGFR. A chimaeric receptor, consisting of an EGFR in which the juxtamembrane and tyrosine kinase domains were exchanged by their IR counterparts, was expressed in CHO-09 cells lacking endogenous EGFR. This receptor was found to activate PLC-gamma1, indicating that the IR TK domain, in situ, is able to tyrosine phosphorylate substrates normally used by the EGFR. These findings suggest that the IR TK domain, in situ, has a low specificity for selection and phosphorylation of non-cognate substrates.     

The trkB tyrosine protein kinase is a receptor for neurotrophin-4.

Neurotrophin-4 is a novel member of the nerve growth factor family of neurotrophins recently isolated from Xenopus and viper DNA. We now report that the Xenopus NT-4 protein (XNT-4) can mediate some of its biological properties through gp145trkB, a murine tyrosine protein kinase previously identified as a primary receptor for the related brain-derived neurotrophic factor (BDNF). XNT-4 displaces 125I-labeled BDNF from binding to cells expressing gp145trkB receptors, induces their rapid phosphorylation on tyrosine residues, and causes the morphologic transformation of NIH 3T3 cells when coexpressed with gp145trkB. Moreover, XNT-4 induces the differentiation of PC12 cells into sympathetic-like neurons only if they ectopically express gp145trkB receptors. None of these biochemical or biological effects could be observed when XNT-4 was added to cells expressing the related receptors. Replacement of one of the extracellular cysteines (Cys-345) of gp145trkB by a serine residue prevents its activation by XNT-4 but not by BDNF. Therefore, XNT-4 and BDNF may interact with at least partially distinct domains within the gp145trkB receptor.     

Tec kinase associates with c-kit and is tyrosine phosphorylated and activated following stem cell factor binding.

Stem cell factor (SCF) plays a crucial role in hematopoiesis through its interaction with the receptor tyrosine kinase c-kit. However, the signaling events that are activated by this interaction and involved in the control of growth or differentiation are not completely understood. We demonstrate here that Tec, a cytoplasmic, src-related kinase, physically associates with c-kit through a region that contains a proline-rich motif, amino terminal of the SH3 domain. Following SCF binding, Tec is tyrosine phosphorylated and its in vitro kinase activity is increased. Tyrosine phosphorylation of Tec is not detected in the response to other cytokines controlling hematopoiesis, including colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). Conversely, the cytoplasmic kinase JAK2 is activated by IL-3 but not by SCF stimulation. The activation of distinct cytoplasmic kinases may account for the synergy seen in the actions of SCF and IL-3 on hematopoietic stem cells.     

SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.

Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Location of the epidermal growth factor binding site on the EGF receptor. A resonance energy transfer study

As a first step toward developing a structural map of key sites on the epidermal growth factor (EGF) receptor, we have used resonance energy transfer to measure the distance of closest approach between the receptor-bound growth factor molecule and lipid molecules at the surface of the plasma membrane. EGF, specifically labeled at its amino terminus with fluorescein 5-isothiocyanate, was used as an energy donor in these experiments, while either octadecylrhodamine B or octadecylrhodamine 101, inserted into plasma membranes isolated from human epidermoid carcinoma (A431) cells, served as the energy acceptors. The energy transfer measurements indicate that the amino terminus of the bound growth factor is about 67 A away from the plasma membrane. On the basis of the dimensions of the EGF molecule, this suggests that EGF binds to a site on its receptor that is a considerable distance (52-82 A) from the surface of these cells. Identical results were obtained under conditions where the receptor functions as an active tyrosine kinase, suggesting that the relative juxtaposition of the EGF binding domain to the membrane surface does not change with receptor autophosphorylation or with the activation of the receptor tyrosine kinase activity.     

The human fibroblast receptor for gp86 of human cytomegalovirus is a phosphorylated glycoprotein.

A human embryonic lung (HEL) cell receptor for gp86 of human cytomegalovirus that functions in virus-cell fusion was further characterized. Anti-idiotype antibodies that mimic gp86 were used to immunoprecipitate the 92.5-kDa fibroblast membrane receptor for gp86, which was preincubated with various endoglycosidases. The receptor, which has a pI ranging from 5.3 to 5.6, appears to be a glycoprotein with primarily N-linked sugar residues, some of which have high concentrations of mannose and some of which are complex oligosaccharides. Western blots (immunoblots) of electrophoretically transferred receptor incubated with various biotinylated lectins confirmed the presence of sugar moieties, including N-acetylglucosamine, glucose or mannose, and galactose, but not fucose or N-acetylgalactosamine. This gp86 receptor from uninfected HEL cells also incorporated radiolabeled phosphate from orthophosphoric acid, indicating that it is a constitutively phosphorylated receptor.     

Association of the tyrosine phosphorylated epidermal growth factor receptor with a 55-kD tyrosine phosphorylated protein at the cell surface and in endosomes

After the intraportal injection of EGF, the EGF receptor (EGFR) is rapidly internalized into hepatic endosomes where it remains largely receptor bound (Lai et al., 1989. J. Cell Biol. 109:2751-2760). In the present study, we evaluated the phosphotyrosine content of EGFRs at the cell surface and in endosomes in order to assess the consequences of internalization. Quantitative estimates of specific radioactivity of the EGFR in these two compartments revealed that tyrosine phosphorylation of the EGFR was observed at the cell surface within 30 s of ligand administration. However, the EGFR was also highly phosphorylated in endosomes reaching levels of tyrosine phosphorylation significantly higher than those of the cell surface receptor at 5 and 15 min after EGF injection. A 55-kD tyrosine phosphorylated polypeptide (pyp55) was observed in association with the EGFR at the cell surface within 30 s of EGF injection. The protein was also found in association with the EGFR in endosomes as evidenced by coprecipitation studies using a mAb to the EGFR as well as by coelution with the EGR in gel permeation chromatography. Limited proteolysis of isolated endosomes indicated that the tyrosine phosphorylated domains of the EGFR and associated pyp55 were cytosolically oriented while internalized EGF was intraluminal. The identification of pyp55 in association with EGFR in both hepatic plasma membranes and endosomes may be relevant to EGFR function and/or trafficking of the EGFR.     

The BBXB motif of RANTES is the principal site for heparin binding and controls receptor selectivity

The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, (44)RKNR(47) and (55)KKWVR(59). The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the (44)AANA(47) mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the (55)AAWVA(59) mutant retained full binding capacity. Mutation of the (44)RKNR(47) site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as well as their ability to induce chemotaxis in vitro. In all assays the single point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant showed a 80-fold reduction in affinity for CCR1, despite normal binding to CCR5. It was only able to induce monocyte chemotaxis at micromolar concentrations. The triple 40s mutant was also able to inhibit HIV-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations.     

Evidence that the antiestrogen binding site is a histamine or histamine-like receptor

N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine X HCl (DPPE), a compound selective for the antiestrogen binding site, is structurally similar to the aminoethyl ether group of antihistamines. Our studies now reveal that H1-, but not H2-antagonists, also compete for this site in the order: DPPE = hydroxyzine = perchlorperazine greater than phenyltoloxamine greater than pyrilamine greater than diphenhydramine. The affinity of these compounds for the antiestrogen binding site correlates with their in vitro cytotoxicity against MCF-7 and EVSA-T human breast cancer cells. Tamoxifen, DPPE and hydroxyzine also bind to H1 receptors present in digitonin-solubilized rat liver microsomes, but with less affinity than pyrilamine, which is selective for this site; the ratio of H1 to antiestrogen binding sites in this preparation is 4:1. The data suggest that the antiestrogen binding site may be, in whole or in part, a receptor for histamine different from H1 and H2.     

Design and synthesis of a novel photoaffinity probe for labelling EGF receptor tyrosine kinases

The epidermal growth factor receptor (EGFR) and HER2 are two important tyrosine kinases that play crucial roles in signal transduction pathways that regulate numerous cellular functions including proliferation, differentiation, migration, and angiogenesis. In the past 20?years, many proteomic methods have emerged as powerful methods to evaluate proteins in biological processes and human disease states. Among them, activity-based protein profiling (ABPP) is one useful approach for the functional analysis of proteins. In this study, a novel photoaffinity probe 11 was designed and synthesised to assess the target profiling of the reactive group in the photoaffinity probe 11. Biological evaluation was performed, and the results showed that the novel photoaffinity probe binds to EGFR and HER2 proteins and it hits targets by the reactive group.     

A hydrophobic patch in ecotropic murine leukemia virus envelope protein is the putative binding site for a critical tyrosine residue on the cellular receptor

In the receptor for ecotropic murine leukemia viruses, tyrosine 235 contributes a critical hydrophobic side chain to the virus-receptor interaction (14). Here we report that tryptophan 142 in ecotropic Moloney murine leukemia virus envelope protein is essential to virus binding and infection. Replacement of tryptophan 142 by alanine or serine resulted in misfolding. However, replacement by methionine (W142M) allowed correct folding of the majority of glycoprotein molecules. W142M virus showed a marked reduction in virus binding and was almost noninfectious, suggesting that tryptophan 142 is involved in receptor binding. In contrast, W142Y virus containing a replacement of tryptophan 142 with an aromatic residue (tyrosine) was as efficient as wild-type virus in infection and binding of cells expressing the wild-type receptor. However, W142Y virus was 100-fold less efficient than wild-type virus in infection of cells expressing a mutant receptor containing tryptophan instead of the critical tyrosine. These results strongly support tryptophan 142 being an essential residue on the virus envelope protein that interacts directly with the critical hydrophobic residue at position 235 of the ecotropic receptor. Tryptophan 142 forms one side of a shallow hydrophobic pocket on the surface of the envelope protein, suggesting that it might comprise the complete putative binding site for tyrosine 235. We discuss the implications of our findings with respect to two models of the envelope protein trimer. Interestingly, both models place tryptophan 142 at the interface between adjacent subunits of the trimer.     

Loss of cytotoxic effect of epidermal growth factor (EGF) on EGF receptor overexpressing cells is associated with attenuation of EGF receptor tyrosine kinase activity

The biological activity of epidermal growth factor (EGF) is mediated through the intrinsic tyrosine kinase activity of the EGF receptor (EGFR). In numerous cell types, binding of EGF to the EGFR stimulates the tyrosine kinase activity of the receptor eventually leading to cell proliferation. In tumor-derived cell lines, which overexpress the EGFR, however, growth inhibition is often seen in response to EGF. The mechanism for growth inhibition is unclear. To study the relationship between growth inhibition and EGFR kinase activity, we have used a cell line (PC-10) derived from a human squamous cell carcinoma that overexpresses EGFR. When exposed to 25 ng/ml EGF at low cell densities (1,300 cells/cm²), PC-10 cells exhibit cell death. In contrast, if EGF is added to high density cultures, no EGF mediated cell death is seen. When PC-10 cells were maintained at confluency in the presence of 25 ng/ml EGF for a period of 1 month, they were subsequently found competent to proliferate at low density in the presence of EGF. We designate these cells APC-10. The APC-10 cells exhibited a unique response to EGF, and no concentration of EGF tested could produce cell death. By ¹²⁵I-EGF binding analysis and [³⁵S]methionine labeling of EGFR, it was found that the total number of EGFR on the cell surface of APC-10 was not decreased relative to PC-10. No difference between PC-10 and APC-10 was seen in EGF binding affinity to the EGFR. Significantly, EGF stimulated autophosphorylation of the EGFR of APC-10 was 8–10-fold lower than that of PC-10. This reduced kinase activity was also seen in vitro in membrane preparations for EGFR autophosphorylation as well as phosphorylation of an exogenously added substrate. No difference between PC-10 and APC-10 in the overall pattern of EGFR phosphorylation in the presence or absence of EGF was detectable. However, the serine and threonine phosphorylation of the EGFR of APC-10 cells was consistently 2–3-fold lower than that seen in PC-10 cells. These results suggest a novel mechanism for EGFR overexpressing cells to survive EGF exposure, one that involves an attenuation of the tyrosine kinase activity of the EGFR in the absence of a change in receptor levels or receptor affinity. © 1994 Wiley-Liss, Inc.