Showing papers on "Receptor tyrosine kinase published in 1983"

Detection and quantification of phosphotyrosine in proteins.

Abstract: Publisher Summary This chapter discusses the detection and quantification of phosphotyrosine in proteins. Protein kinases can be differentiated according to their amino acid specificity. Casein kinases of type II and the cAMP-dependent kinases phosphorylate serine, while casein kinases of type I phosphorylate threonine and, to a lesser extent, serine. Another group of kinases with strict specificity for tyrosine is described in the chapter. Most tyrosine-specific protein kinase activities detected to date are implicated in cell growth control. They include the kinases associated with the cell surface receptors for two polypeptide growth factors, epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), and with the transforming proteins of at least five genetically distinct groups of retroviruses. The activity of a tyrosine protein kinase in the cell is apparent from an increase in the gross level of phosphotyrosine in cell proteins. The induction of a cellular tyrosine protein kinase by growth factors or the introduction of a viral tyrosine protein kinase by infection can raise the proportion of acid-stable protein bound phosphate present as phosphotyrosine 5–10 times. Phosphatases specific for tyrosine are as yet poorly characterized, but may be inhibited by traces of Zn 2+ or by adding free phosphotyrosine or analogs. The simplest strategy is to lyse cells directly into a denaturing solution, or to stop an in vitro reaction with denaturing agents.

Three glycolytic enzymes are phosphorylated at tyrosine in cells transformed by Rous sarcoma virus.

Abstract: Isozymes of enolase, phosphoglycerate mutase and lactate dehydrogenase are phosphorylated at tyrosine in cells transformed by viruses whose transforming proteins are tyrosine protein kinases. The role of these structural modifications in the regulation of glycolysis in such cells is not known.

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

Abstract: 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 conditions3. 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 lifespan3. 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 receptors5,6. The receptor for EGF has been characterized as a 145,000–165,000 (145 K–165 K) molecular weight doublet7,8, and, like the receptors for platelet-derived growth factor9 and insulin10,11, and the transforming proteins of certain of the RNA tumour viruses12–17, is a tyrosine-specific protein kinase with autophosphorylating activity8,18,19. Moreover, several of the cellular target molecules of tyrosine phosphorylation have been found to be substrates for two or more of these kinases20,21. 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.

Phosphorylation of gastrin-17 by epidermal growth factor-stimulated tyrosine kinase.

Abstract: Tyrosine phosphorylation seems to be a key event in the control of cellular growth. Several viral transforming proteins, including the src protein of Rous sarcoma virus1, the p120 protein of Abelson leukaemia virus2 and the middle T antigen of polyoma virus3, are phosphorylated by associated tyrosine kinases4–6. The levels of kinase activity correlate with the transforming efficiency of the virus6–8. The receptors for epidermal growth factor (EGF)9,10, platelet-derived growth factor (PDGF)11 and insulin12 are also phosphorylated by associated tyrosine kinase activities, which are stimulated by EGF, PDGF and insulin, respectively. The EGF-stimulated kinase13,14 and the src protein14,15 share similar substrate specificity for tyrosines immediately C-terminal to a sequence of acidic amino acids. Such a sequence is also found adjacent to the phosphotyrosine of middle T antigen16,17, and in the homologous region18 of the hormone gastrin, adjacent to a tyrosine which is sulphated in approximately half the gastrin isolated from gastric mucosa19. Reports that gastrin acts as a growth factor for cells of the gastrointestinal tract20 suggested that phosphorylation of this tyrosine might be physiologically more relevant than sulphation. We report here that synthetic human gastrin 17 is phosphorylated by the EGF-stimulated tyrosine kinase of A431 cell membranes. The Km values of 53–87 and 223–547 µM obtained in the presence and absence of EGF, respectively, are the lowest reported so far for this enzyme.

Two different protein kinase activities are associated with the insulin receptor.

Abstract: In intact rat hepatocytes insulin stimulates the phosphorylation of the beta-subunit of its receptor exclusively on serine residues, which are also phosphorylated in the absence of insulin. In contrast, in partially purified insulin receptors derived from these same cells and in highly purified insulin receptors obtained by immunoprecipitation with anti-receptor antibodies, the receptor beta-subunit is phosphorylated solely on tyrosine residues. For both cell-free systems, insulin's stimulatory action on receptor phosphorylation leads to an increase in phosphotyrosine. When partially purified receptors were used to phosphorylate two exogenous substrates, casein and histone, insulin was found to stimulate the phosphorylation of both tyrosine and serine. However, the basal and insulin-stimulated kinase activity of immunoprecipitated receptors was only tyrosine-specific. From these observations we propose that the insulin-receptor complex consists of two different insulin-stimulatable kinase activities: (1) a tyrosine-specific kinase, which is a constituent of the insulin-receptor structure and whose activation is likely to be the first post-binding event in insulin action; and (2) a serine-specific kinase, which is closely associated with the receptor in the cell membrane.

Phosphorylation of human growth hormone by the epidermal growth factor-stimulated tyrosine kinase.

Abstract: In the present study, we have demonstrated that human growth hormone (hGH) can be phosphorylated by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Phosphotyrosine was the predominant phosphoamino acid released from phosphorylated hGH on partial acid hydrolysis. All five tyrosine-containing tryptic peptides of hGH are also phosphorylated by the EGF-stimulated tyrosine kinase. The highest phosphate incorporation was found for peptide T4 (residues 20-38), which is distinguished by a high frequency of acidic amino acids. The phosphorylated peptides have been characterized by HPLC and two-dimensional mapping on paper. Comparison with the labeled peptides obtained on tryptic digestion of phosphorylated hGH suggests that tyrosine phosphorylation is restricted to two tryptic peptides, T4 (tyrosine-28 or -35) and T6 (tyrosine-42). It is suggested that the absence of early insulin-like activity in the naturally occurring Mr 20,000 variant of hGH, which has an internal deletion spanning residues 32-46, may be a consequence of the loss of the tyrosine phosphorylation sites at residues 35 and 42.

Role of tyrosine phosphorylation in malignant transformation by viruses and in cellular growth control.

Abstract: Publisher Summary This chapter discusses the role of tyrosine phosphorylation in malignant transformation by viruses and in cellular growth control. Tyrosine protein-kinases appear to be essential cellular gene products. The regulated phosphorylation of tyrosine in proteins has been implicated in growth control and the organization of cytoskeleton in normal cells. The molecular basis for transformation by some acutely transforming retroviruses may involve unscheduled phosphorylation of cellular proteins on tyrosine. One indication of this is that cells transformed by these viruses have phosphotyrosine levels in protein five to ten fold higher than the parental untransformed cells. Moreover, the transforming proteins of these viruses have an associated tyrosine-specific protein-kinase activity. All acutely transforming retroviruses contain in their genomes sequences recognizable as having been derived from the genome of a normal cell. These cellular sequences are responsible for the oncogenic potential of these viruses, encoding at least part of their transforming proteins. The group of viruses whose transforming proteins have associated tyrosine protein-kinase activity can be subdivided into five classes representing five distinguishable types of cellular sequence. The onc genes corresponding to these cellular sequences are src, fps or fes, yes, ros, and abl . In addition to these five genes, evidence is mounting that the genes encoding certain growth-factor receptors may also specify tyrosine protein-kinases. There are strong indications that the epidermal growth factor receptor has intrinsic tyrosine protein-kinase activity.

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

Abstract: 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 lines5, 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 serum6. A subfraction of middle T antigen is associated with a protein kinase activity which phosphorylates middle T antigen in vitro on tyrosine7–11. There is a strong correlation between the level of this kinase activity and the degree of expression of the phenotype of transformed cells7–9,12. 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.

The receptor for epidermal growth factor functions as a tyrosyl-specific kinase.

Abstract: Publisher Summary Studies show that a number of protein kinases is associated with tumor viruses phosphorylate tyrosine residues. Following these studies, this chapter determines that the epidermal growth factor (EGF)-activated kinase reaction is specific for tyrosine residues. The finding that the protein kinase activity associated with the EGF receptor shares with several viral transforming proteins the highly unusual property of phosphorylating tyrosine suggested the possibility that these proteins might be structurally related. Therefore, the chapter examines the interaction of antibodies to viral tyrosine kinases with the EGF-receptor–kinase preparation. In this examination, it was found that the affinity-purified EGF-receptor-kinase specifically phosphorylates antibodies directed against the transforming protein kinase pp60 src of Rous sarcoma virus. However, none of these antibodies, including those that cross-react with the normal cellular homolog of pp60 src , recipitate the receptor-kinase. These results suggest that the EGF-receptor-kinase is related to, but not identical with, pp60 c-src .

Chapter 22. Tyrosine-Specific Protein Kinases

Abstract: Publisher Summary The enzymes that comprise tyrosine kinases can be distinguished from other protein kinases by a number of criteria, most notably, their specificity for phosphorylation of tyrosine residues The importance of these phosphotransferases is not merely a consequence of their tyrosine-specificity, but the evidence that all of the enzymes in this class appear to be associated with the regulation of cellular morphology and growth control At first, tyrosine phosphorylation is identified in association with the transforming proteins of several oncogenic viruses “Transforming” proteins are those polypeptides that are responsible for the initiation and maintenance of the transformed phenotype A second group of tyrosine-specific protein kinases has been identified in normal cells This group contains the membrane receptors for many cellular growth factors, including epidermal growth factor (EGF), insulin, and platelet derived growth factor (PDGF) Interaction of each of these factors with their respective cellular receptor results in activation of tyrosine-specific kinase activity associated with the receptor molecule The precise mechanism, whereby these tyrosine kinases interact with other cellular proteins to exert their biological effects, is presently unknown This review discusses the current state of knowledge on these two groups of tyrosine kinases

Protein Kinases Specific for Tyrosine Residues and the Role of Tyrosine Phosphorylation of Proteins in Cell Transformation

Abstract: Retroviruses are the etiological agents of various types of neoplasms (sarcomas, carcinomas and leukemias) in many avian and mammalian species (reviewed in ref. 1). On the basis of their pathogenicity in laboratory animals, oncogenic retroviruses have been broadly divided into two classes. Acute viruses induce tumours 15 to 30 days after injection in a susceptible host and are able to transform adequate target cells in vitro. Chronic viruses do not induce morphological transformation in vitro and require long latency periods (months to years depending on the virus-host considered) to induce tumours. This difference in pathogenicity is the result of the presence in the RNA genome of acute retroviruses of specific nucleotide sequences encoding protein unnecessary for viral replication but required for induction and maintenance of in vitro transformation and in vivo oncogenic potential. These transformation-specific sequences are referred to as v-onc genes. Retroviruses v-onc genes show strong sequence homology to specific cellular loci (c-onc). The c-onc genes are evolutionary conserved and are expressed in normal cells, sometimes in a tissue-specific manner.