Anti-pp60src antibodies are substrates for EGF-stimulated protein kinase

Epidermal growth factor (EGF) stimulates phosphorylation of its own receptor at a tyrosine residue. Similarly, the viral gene product pp60src, which is responsible for cellular transformation by avian sarcoma virus (ASV), phosphorylates itself and immunoglobulin directed against pp60src at tyrosine residues. This unusual site of phosphorylation catalysed by two membrane-associated protein kinases involved in growth control prompted us to study the immunological relatedness of the EGF-stimulated protein kinase and the pp60src. Using anti-pp60src antisera, we attempted to immunoprecipitate the EGF-stimulated protein kinase solubilized from plasma membranes. We report here that neither the EGF-stimulated kinase nor the EGF receptor were immunoprecipitable by anti-pp60src sera. However, anti-pp60src IgG served as a specific substrate for the EGF-stimulated kinase, suggesting a close similarity between the EGF-stimulated kinase and pp60src.     

Purified EGF receptor-kinase interacts specifically with antibodies to Rous sarcoma virus transforming protein

Transformation by several RNA tumour viruses seems to be mediated by virally coded protein kinases which specifically phosphorylate tyrosine. A tyrosine-specific protein kinase also seems to be involved in the mitogenic action of epidermal growth factor (EGF). This EGF-stimulated kinase activity is closely associated with the EGF receptor, with which it copurifies during EGF-affinity chromatography. Because both the virus- and EGF-stimulated tyrosine kinases may be involved in stimulation of cell growth, and because the viral kinases may be antigenically related to normal cell proteins, we examined the interaction of antibodies to viral tyrosine kinases with the affinity-purified EGF receptor-kinase preparation. We report here that the receptor-kinase specifically phosphorylates antibodies directed against the transforming protein kinase pp60src of Rous sarcoma virus. However, none of these antibodies, including those which cross-react with the normal cellular homologue of pp60src (pp60sarc), precipitate the receptor-kinase. These results suggest that the EGF receptor-kinase is related to, but probably not identical with, pp60sarc.     

Epidermal growth factor-dependent phosphorylation of lipocortin

Lipocortin-like proteins are a family of steroid-induced inhibitors of phospholipase activity with potential anti-inflammatory activity. Related proteins have been detected in a variety of tissues and species. The best characterized form is a protein of relative molecular mass (Mr) approximately 40,000 (40K), which is phosphorylated in vivo by protein tyrosine kinases and by protein serine-threonine kinases. It has been proposed that the phospholipase inhibitory activity of lipocortin can be regulated by its phosphorylation. In the A431 cell line, a protein of approximately 35K is phosphorylated by the protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Here we report that human lipocortin is phosphorylated near its amino terminus by the EGF receptor/kinase. By peptide mapping and immunological analyses, we show that lipocortin and the endogenous 35K substrate for the EGF receptor/kinase from A431 cells are the same protein.     

Autophosphorylation sites on the epidermal growth factor receptor

The epidermal growth factor (EGF) receptor is a tyrosine-specific protein kinase with autophosphorylating activity. A 300 amino acid-long region of the receptor's cytoplasmic domain matches (35-90% homology) sequences of transforming proteins from the src family and includes a putative nucleotide binding site. Several of the src transforming proteins have tyrosine kinase activity, but v-erb-B, which appears to be a truncated EGF receptor, is virtually identical to the receptor over this region and yet lacks detectable kinase activity. To locate possible acceptor sites in the v-erb-B protein, we have mapped these sites in the human EGF receptor. We report here that three tyrosine sites near the C-terminus are phosphorylated in vitro. In intact cells, we find that EGF stimulates phosphorylation of several sites, the tyrosine 14 residues from the C-terminus being modified the most extensively. The equivalent site is absent in the v-erb-B protein of avian erythroblastosis virus (AEV) and may influence tyrosine kinase activity.     

Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts

Fibroblastic cultures derived from normal human tissues undergo a finite number of population doublings when serially subcultivated in vitro (see refs 1, 2 for reviews). Epidermal growth factor (EGF) serves as a mitogen for early doubling level cultures of the human fetal lung-derived cell strain, WI-38, under serum-free conditions. The ability of cells from late doubling level cultures to respond mitogenically to EGF is lost, however, despite undiminished binding of EGF throughout the replicative lifespan. The ultimate effects of EGF, that is DNA synthesis and mitosis (see ref. 4 for review), occur after a sequence of events initiated by binding of ligand to specific cellular receptors. The receptor for EGF has been characterized as a 145,000-165,000 (145 K-165 K) molecular weight doublet, and, like the receptors for platelet-derived growth factor and insulin, and the transforming proteins of certain of the RNA tumour viruses, is a tyrosine-specific protein kinase with autophosphorylating activity. Moreover, several of the cellular target molecules of tyrosine phosphorylation have been found to be substrates for two or more of these kinases. The hypothesis that tyrosine phosphorylation underlies a common mechanism of growth control prompted us to ask whether the loss of responsiveness to EGF by late doubling level WI-38 cells is accompanied by altered expression of the EGF receptor, and specifically whether changes occur in the ability of receptors from populations of cells of various in vitro ages to catalyse tyrosine autophosphorylation. We show here that autophosphorylating activity is absent from the EGF receptor of cells which have lost their mitogenic responsiveness to EGF.     

A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor

The cell surface receptors for insulin and epidermal growth factor (EGF) appear to share a common evolutionary origin, as suggested by structural similarity of cysteine-rich regions in their extracellular domains and a highly conserved tyrosine-specific protein kinase domain. Only minor similarity is found outside this catalytic domain, as expected for receptors that have different ligand specificities and generate different biological signals. The EGF receptor is a single polypeptide chain but the insulin receptor consists of distinct alpha and beta subunits that function as an alpha 2 beta 2 heterotetrameric receptor complex. Provoked by this major structural difference in two receptors that carry out parallel functions, we have designed a chimaeric receptor molecule comprising the extracellular portion of the insulin receptor joined to the transmembrane and intracellular domains of the EGF receptor to investigate whether one ligand will activate the tyrosine kinase domain of the receptor for the other ligand. We show here that the EGF receptor kinase domain of the chimaeric protein, expressed transiently in simian cells, is activated by insulin binding. This strongly suggests that insulin and EGF receptors employ closely related or identical mechanisms for signal transduction across the plasma membrane.     

Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane

The receptor for epidermal growth factor (EGF) is a 170,000-180,000 molecular weight single-chain glycoprotein of 1,186 amino acids. Its sequence suggests that it has an external EGF-binding domain, formed by the NH2-terminal 621 amino acids, linked to a cytoplasmic region by a single membrane-spanning segment. In the cytoplasmic portion, starting 50 residues from the membrane, there is a 250-residue stretch similar to the catalytic domain of the src gene family of retroviral tyrosine protein kinases, and, indeed, a tyrosine-specific protein kinase activity intrinsic to the receptor is stimulated when EGF is bound. Increased tyrosine phosphorylation of cellular proteins, detected in A431 cells following EGF binding, may be important in the mitogenic signal pathway. Tumour promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), counteract this increase, as well as causing loss of a high affinity class of EGF binding sites. The major receptor for TPA has been identified as the serine/threonine-specific Ca2+/phospholipid-dependent diacylglycerol-activated protein kinase, protein kinase C. By substituting for diacylglycerol, TPA stimulates protein kinase C. Protein kinase C phosphorylates purified EGF receptor at specific sites, and this reduces EGF-stimulated tyrosine protein kinase activity. TPA treatment of A431 cells increases serine and threonine phosphorylation of the EGF receptor at the same sites, which suggests that the reduction of EGF receptor kinase activity in TPA-treated cells is a consequence of the receptor's phosphorylation by the kinase. We have attempted to identify these phosphorylation sites and show here that protein kinase C phosphorylates threonine 654 in the human EGF receptor. This threonine is in a very basic sequence nine residues from the cytoplasmic face of the plasma membrane in the region before the protein kinase domain; it is thus in a position to modulate signalling between this internal domain and the external EGF-binding domain.     

Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells

The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precursor, deduced from cDNA clones derived from placental and A431 carcinoma cells, reveals close similarity between the entire predicted v-erb-B mRNA oncogene product and the receptor transmembrane and cytoplasmic domains. A single transmembrane region of 23 amino acids separates the extracellular EGF binding and cytoplasmic domains. The receptor gene is amplified and apparently rearranged in A431 cells, generating a truncated 2.8-kilobase mRNA which encodes only the extracellular EGF binding domain.     

Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin

Epidermal growth factor (EGF), through interaction with specific cell surface receptors, generates a pleiotropic response that, by a poorly defined mechanism, can induce proliferation of target cells. Subversion of the EGF mitogenic signal through expression of a truncated receptor may be involved in transformation by the avian erythroblastosis virus (AEV) oncogene v-erb-B, suggesting that similar EGF receptor defects may be found in human neoplasias. Overexpression of EGF receptors has been reported on the epidermoid carcinoma cell line A431, in various primary brain tumours and in squamous carcinomas. In A431 cells the receptor gene is amplified. Here we show that 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes. Amplified receptor genes were not detected in the other brain tumours examined. Further analysis of EGF receptor defects may show that such altered expression and amplification is a particular feature of certain human tumours.     

The neu oncogene encodes an epidermal growth factor receptor-related protein

The neu oncogene is repeatedly activated in neuro- and glioblastomas derived by transplacental mutagenesis of the BDIX strain of rat with ethylnitrosourea. Foci induced by the DNAs from such tumours on NIH 3T3 cells contain the neu oncogene and an associated phosphoprotein of relative molecular mass 185,000 (p185). Previous work has shown that the neu gene is related to, but distinct from, the gene encoding the EGF receptor (c-erb-B). Here we describe a neu complementary DNA clone isolated from a cell line transformed by this oncogene; the clone has biological activity in a focus-forming assay. The nucleotide sequence of this clone predicts a 1,260-amino-acid transmembrane protein product similar in overall structure to the EGF receptor. We found that 50% of the predicted amino acids of neu and the EGF receptor are identical; greater than 80% of the amino acids in the tyrosine kinase domain are identical. Our results suggest strongly that the neu gene encodes the receptor for an as yet unidentified growth factor.     

Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate

The generation of second messengers from the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdInsP2) by phosphoinositidase C has been implicated in the mediation of cellular responses to a variety of growth factors and oncogene products. The first step in the production of PtdInsP2 from phosphatidylinositol (PtdIns) is catalysed by PtdIns kinase. A PtdIns kinase activity has been found to associate specifically with several oncogene products, as well as with the platelet-derived growth factor (PDGF) receptor. We have previously identified two biochemically distinct PtdIns kinases in fibroblasts, and have found that only one of these, designated type I, specifically associates with activated tyrosine kinases. We have now characterized the site on the inositol ring phosphorylated by type I PtdIns kinase, and find that this kinase specifically phosphorylates the D-3 ring position to generate a novel phospholipid, phosphatidylinositol-3-phosphate (PtdIns(3)P). In contrast, the main PtdIns kinase in fibroblasts, designated type II, specifically phosphorylates the D-4 position to produce phosphatidylinositol-4-phosphate (PtdIns(4)P), previously considered to be the only form of PtdInsP. We have also tentatively identified PtdIns(3)P as a minor component of total PtdInsP in intact fibroblasts. We propose that type I PtdIns kinase is responsible for the generation of PtdIns(3)P in intact cells, and that this novel phosphoinositide could be important in the transduction of mitogenic and oncogenic signals.     

ATP-stimulated interaction between epidermal growth factor receptor and supercoiled DNA

The receptor for epidermal growth factor (EGF) has been identified as a transmembrane glycoprotein that has tyrosine-specific kinase activity. The kinase activity of the receptor is enhanced in the presence of EGF (or related peptides), and the phosphorylation of a number of substrates, as well as autophosphorylation of the receptor, has been reported. Analogous findings have been described for the insulin receptor and the receptor for platelet-derived growth factor (PDGF). Thus, a number of hormone receptors and several viral transforming proteins appear to share the highly unusual property of tyrosine-specific kinase activity. Nevertheless, the specific relationship between tyrosine kinase activity and the control of cell growth and replication is unknown. It is known that after the initial binding of EGF to the plasma membrane, the hormone together with its receptor is rapidly internalized in endocytic vesicles and the hormone is eventually degraded in lysosomes. It is possible that the function of EGF is simply to stimulate internalization of its receptor, and that as a result of its altered location the receptor is able to phosphorylate a cytoplasmic component or even interact directly with a nuclear component. We now report that the purified receptor for EGF is able to interact with and nick supercoiled double-stranded DNA in an ATP-stimulated manner.     

Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors

The primary structure of the receptor for platelet-derived growth factor (PDGF), determined by means of cloning a cDNA that encodes the murine pre-PDGF receptor, is closely related to that of the v-kit oncogene product and the receptor for macrophage colony stimulating factor (CSF-1). Common structural features include the presence of long sequences that interrupt the tyrosine-specific protein kinase domains of each molecule. The PDGF and CSF-1 receptors also share a characteristic distribution of extracellular cysteine residues. Ubiquitin is covalently bound to the purified PDGF receptor, the human gene for which is on chromosome 5.     

Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases.

The critical pathways through which protein-tyrosine kinases induce cellular proliferation and malignant transformation are not well defined. As microinjection of antibodies against p21ras can block the biological effects of both normal and oncogenic tyrosine kinases, it is likely that they require functional p21ras to transmit their mitogenic signals. No biochemical link has been established, however, between tyrosine kinases and p21ras. We have identified a non-catalytic domain of cytoplasmic tyrosine kinases, SH2, that regulates the activity and specificity of the kinase domain. The presence of two adjacent SH2 domains in the p21ras GTPase-activating protein (GAP) indicates that GAP might interact directly with tyrosine kinases. Here we show that GAP, and two co-precipitating proteins of relative molecular masses 62,000 and 190,000 (p62 and p190) are phosphorylated on tyrosine in cells that have been transformed by cytoplasmic and receptor-like tyrosine kinases. The phosphorylation of these polypeptides correlates with transformation in cells expressing inducible forms of the v-src or v-fps encoded tyrosine kinases. Furthermore, GAP, p62 and p190 are also rapidly phosphorylated on tyrosine in fibroblasts stimulated with epidermal growth factor. Our results suggest a mechanism by which tyrosine kinases might modify p21ras function, and implicate GAP and its associated proteins as targets of both oncoproteins and normal growth factor receptors with tyrosine kinase activity. These data support the idea that SH2 sequences direct the interactions of cytoplasmic proteins involved in signal transduction.