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.     

Phorbol ester and diacylglycerol induce protein phosphorylation at tyrosine

The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is an efficient tumour promoter in vivo. In vitro, TPA activates the phospholipid- and Ca2+-dependent protein kinase, kinase C. This activation is believed to reflect the structural similarity between TPA and diacylglycerol, the endogenous protein kinase C activator which is produced in vivo by hydrolysis of phosphatidylinositol (reviewed in ref. 3). Protein kinase C phosphorylates protein substrates at serine and threonine residues in vitro. The effects of TPA on cultured fibroblasts--including enhanced hexose uptake, disruption of actin stress fibres and growth stimulation--are very similar to those induced by certain retrovirus transforming proteins and by peptide growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and multiplication-stimulating activity (MSA). These transforming proteins and mitogenic agents seem to act by inducing tyrosine-specific protein phosphorylation. Such observations suggested that some of the effects of TPA in vivo may be mediated by protein phosphorylation at tyrosine residues. A 42,000-molecular weight (42 K) polypeptide was previously shown to be phosphorylated at tyrosine in cells transformed by avian sarcoma viruses and in cells stimulated by EGF, PDGF or MSA (J. Cooper, personal communication and refs 11 and 12; this polypeptide was originally designated 43 K or spot n in ref. 10). We show here that this polypeptide also becomes phosphorylated at tyrosine in cells treated with TPA. Furthermore, exogenously added diacylglycerol likewise stimulates the phosphorylation of this protein at tyrosine.     

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.     

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.     

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.     

Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor

Epidermal growth factor (EGF) and transforming growth factor type I (TGF) are polypeptides of 53 and 50 amino acid residues, respectively. Both bind to EGF receptor, a 1,200-residue transmembranous glycoprotein, leading to phosphorylation of the receptor, enhancement of its tyrosine-specific kinase activity and ultimately to stimulation of cell growth. We report here that a 140-residue polypeptide encoded by one of the early genes of vaccinia virus (VV) is related closely to EGF and TGF. The presence of putative signal and transmembranous sequences further suggests that the viral protein might be an integral membrane protein, but that, as in the case of EGF itself, the membrane-associated form may be the precursor of a soluble growth factor. Production of EGF-like growth factors by virally infected cells could account for the proliferative diseases associated with members of the poxvirus family such as Shope fibroma virus, Yaba tumour virus, and molluscum contagiosum virus (MCV).     

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.     

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.     

Involvement of the epidermal growth factor receptor in the invasion of cultured mammalian cells by Salmonella typhimurium.

Salmonella infection continues to be a major world-wide health problem. One essential pathogenic feature common to all Salmonella is their ability to penetrate the cells of the intestinal epithelium which are normally non-phagocytic. The internalization of Salmonella into mammalian cells is thought to be a receptor-mediated phenomenon and the invasion of cultured epithelial cells depends on several Salmonella genes, but nothing is known about the host determinants participating in this interaction. Protein tyrosine phosphorylation follows stimulation of many cell-surface receptors to initiate signal transduction pathways that stimulate cellular responses. We report here that invasion of cultured Henle-407 cells by Salmonella typhimurium induces the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. In contrast, an isogenic strain of S. typhimurium that is defective in invasion owing to a mutation in the invA gene is unable to induce such phosphorylation. Addition of EGF to cultured Henle-407 cells allowed the internalization of the invasion-defective S. typhimurium invA mutant although it did not cause the internalization of an adherent, but non-invasive, strain of Escherichia coli. This result indicates that stimulation of the EGF receptor is involved in the invasion of cultured Henle-407 cells by S. typhimurium.     

Increased phosphorylation of ribosomal protein S6 following microinjection of insulin receptor-kinase into Xenopus oocytes

The protein products of several transforming retroviruses as well as the receptors for several hormones and growth factors, including insulin, have been shown to possess a protein kinase activity in vitro specific for tyrosine residues in protein substrates, including themselves. In the case of pp60src and the insulin receptor, autophosphorylation activates the tyrosine kinase activity towards exogenous substrates. Experiments indicate that, in vivo, many of these viruses or growth factors induce an increase in cellular phosphotyrosine, as well as an increase in the phosphorylation of serine residues on proteins, including ribosomal protein S6. It seems likely that some of the effects of insulin might be mediated by phosphorylation of intracellular substrates by its receptor. As the beta subunit of the receptor is a transmembrane protein, such phosphorylation could occur either while the receptor is still in the membrane or after its internalization. In various cell systems, internalized receptors are degraded, reshuttled back to the plasmalemma or maintained in a separate compartment before reinsertion in the membrane; shuttling of the insulin receptor could provide the opportunity for it to phosphorylate various intracellular components as part of its mechanism of signal transduction. To approach directly the question of whether the receptor can elicit a signal while acting at an intracellular location, we have microinjected Xenopus oocytes with the insulin receptor kinase. The results indicate that an S6 protein-serine kinase is stimulated or an S6 protein-serine phosphatase inhibited by the activity of the insulin receptor, supporting the concept that the insulin receptor acting within the cell can elicit a biological response.     

Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells.

Normal cells in culture have membrane receptors for epidermal growth factor (EGF); EGF stimulates cells to divide by binding to these receptors. Cells transformed by murine and feline sarcoma viruses rapidly lose the ability to bind EGF, whereas cells transformed by the DNA tumour viruses, polyoma and SV40, or infected with non-transforming RNA tumour viruses have normal levels of functional EGF receptors. The results suggest that a product of the sarcoma virus genome specifically changes cell EGF receptors; the sarcoma gene product may, then, be functionally related to EGF.     

Transforming potential of the c-fms proto-oncogene (CSF-1 receptor)

The c-fms proto-oncogene encodes a transmembrane glycoprotein that is probably identical to the receptor for the macrophage colony stimulating factor, CSF-1. Forty C-terminal amino acids of the normal receptor are replaced by 11 unrelated residues in the feline v-fms oncogene product, deleting a C-terminal tyrosine residue (Tyr969) whose phosphorylation might negatively regulate the receptor kinase activity. We show that the human c-fms gene stimulates growth of mouse NIH 3T3 cells in agar in response to human recombinant CSF-1, indicating that receptor transduction is sufficient to induce a CSF-1 responsive phenotype. Although cells transfected with c-fms genes containing either Tyr969 or Phe969 were not transformed, cotransfection of these genes with CSF-1 complementary DNA induced transformation, with c-fms(Phe969) showing significantly more activity than c-fms(Tyr969). In the absence of CSF-1, chimaeric v-fms/c-fms genes encoding the wild-type c-fms C terminus were poorly transforming, whereas chimaeras bearing Phe969 were as transforming as v-fms. Thus, the Phe969 mutation, although not in itself sufficient to induce transformation, activates the oncogenic potential of c-fms in association with an endogenous ligand or in conjunction with mutations elsewhere in the c-fms gene that confer ligand-independent signals for growth.     

Abelson murine leukaemia virus transformation involves loss of epidermal growth factor-binding sites

Malignant transformation by mammalian RNA sarcoma viruses has previously been shown to involve a reduction in receptor sites for a well characterized 6,000-molecular weight (MW) growth-promoting substance, designated epidermal growth factor (EGF). Although Abelson murine leukaemia virus (AbLV) resembles sarcoma viruses in its ability to transform embryo fibroblasts in cell culture, AbLV induces a rapid B-cell lymphoid leukaemia rather than fibrosarcomas in vivo. The major translational product of AbLV is a highly phosphorylated polyprotein of MW 120,000 which exhibits an associated tyrosine-specific protein kinase activity and probable transforming function. We show here that AbLV transformation resembles transformation by RNA sarcoma viruses with respect to the abolition of EGF-binding sites. EGF binding is restored to control levels following loss of polyprotein expression in morphological revertants of AbLV-transformed clones and remains uninfluenced in cell lines infected with transformation-defective (td) AbLV mutants encoding polyproteins deficient in protein kinase activity. These findings indicate that AbLV transformation involves a polyprotein-associated, tyrosine-specific protein kinase activity which mediates its effect through a mechanism resulting directly or indirectly in the abolition of EGF-binding sites.     

Enhancement of polyoma virus middle T antigen tyrosine phosphorylation by epidermal growth factor

Polyoma virus codes for three proteins involved in host cell transformation: the large, middle and small T antigens. Middle T antigen is a major transforming protein which is responsible for the induction of the phenotype of transformed cells and, without it, transformation does not occur (reviewed in refs 1-4). Middle T antigen alone can transform established cell lines, although large, and possibly small, T antigens are also required for the full expression of the phenotype of transformed cells in media with a low concentration of serum. A subfraction of middle T antigen is associated with a protein kinase activity which phosphorylates middle T antigen in vitro on tyrosine. There is a strong correlation between the level of this kinase activity and the degree of expression of the phenotype of transformed cells. We report here that epidermal growth factor (EGF) stimulates tyrosine phosphorylation of middle T antigen, suggesting the possibility that mitogenic growth factor(s) regulates this phosphorylation activity.     

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.     

Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences

Each of six peptides derived from the human epidermal growth factor (EGF) receptor very closely matches a part of the deduced sequence of the v-erb-B transforming protein of avian erythroblastosis virus (AEV). In all, the peptides contain 83 amino acid residues, 74 of which are shared with v-erb-B. The AEV progenitor may have acquired the cellular gene sequences of a truncated EGF receptor (or closely related protein) lacking the external EGF-binding domain but retaining the transmembrane domain and a domain involved in stimulating cell proliferation. Transformation of cells by AEV may result, in part, from the inappropriate acquisition of a truncated EGF receptor from the c-erb-B gene.     

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.     

High tyrosine kinase activity in normal nonproliferating cells

Protein phosphorylation at serine and threonine residues has been implicated in the regulation of many cellular processes. More recently, tyrosine residue phosphorylation has been shown to be associated with stimulation of cell proliferation, including viral transformation and stimulation by epidermal growth factors (EGF), platelet-derived growth factor (PDGF) and other compounds related to cellular growth such as insulin and dimethyl sulphoxide. To compare protein kinases and phosphoproteins of normal and leukaemic human haematopoietic cells in vivo and in vitro, we first have investigated the percentages of phosphoserine, phosphothreonine and phosphotyrosine obtained after hydrolysis of proteins from different blood cell fractions phosphorylated in vitro. We report here that phosphotyrosine formed less than 1% of the soluble fractions from polymorphonuclear cells, mononuclear cells (80% circulating lymphocytes, 20% monocytes), blood platelets and red blood cells (not shown). Surprisingly, high percentages of phosphorylated tyrosine were found only in the particulate fractions from non-proliferating anuclear cells, platelets and red blood cells.     

Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation

The embryonic pyruvate kinase M2 (PKM2) isoform is highly expressed in human cancer. In contrast to the established role of PKM2 in aerobic glycolysis or the Warburg effect, its non-metabolic functions remain elusive. Here we demonstrate, in human cancer cells, that epidermal growth factor receptor (EGFR) activation induces translocation of PKM2, but not PKM1, into the nucleus, where K433 of PKM2 binds to c-Src-phosphorylated Y333 of β-catenin. This interaction is required for both proteins to be recruited to the CCND1 promoter, leading to HDAC3 removal from the promoter, histone H3 acetylation and cyclin D1 expression. PKM2-dependent β-catenin transactivation is instrumental in EGFR-promoted tumour cell proliferation and brain tumour development. In addition, positive correlations have been identified between c-Src activity, β-catenin Y333 phosphorylation and PKM2 nuclear accumulation in human glioblastoma specimens. Furthermore, levels of β-catenin phosphorylation and nuclear PKM2 have been correlated with grades of glioma malignancy and prognosis. These findings reveal that EGF induces β-catenin transactivation via a mechanism distinct from that induced by Wnt/Wingless and highlight the essential non-metabolic functions of PKM2 in EGFR-promoted β-catenin transactivation, cell proliferation and tumorigenesis.