Eleanor Erikson

Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II

Ribosomal protein S6 is a component of the eukaryotic 40S ribosomal subunit that becomes phosphorylated on multiple serine residues in response to a variety of mitogens, including insulin, growth factors, and transforming proteins of many oncogenic viruses. Recently, an activated S6 kinase (S6 KII) has been purified to homogeneity from Xenopus eggs1, and characterized immunologically2 and at the molecular level3. Purified S6 KII can be deactivated in vitro by incubation with either protein phosphatase 1 or protein phosphatase 2A. Reactivation and phosphorylation of S6 KII occurs in vitro with an insulin-stimulated micro-tubule-associated protein-2 (MAP-2) protein kinase which is itself a phosphoprotein that can be deactivated by protein phosphatase 2A. These studies suggest that a step in insulin signalling involves sequential activation by phosphorylation of at least two serine/threonine protein kinases.

The specific interaction of the Rous sarcoma virus transforming protein, pp60src, with two cellular proteins

Abstract Sera from rabbits bearing tumors induced by Rous sarcoma virus (RSV) were previously found to contain antibody to the RSV transforming protein, pp60 src . Two additional transformation-specific phosphoproteins from RSV-transformed avian cells are immunoprecipitated with these sera. These proteins, having molecular weights of 90,000 (pp90) and 50,000 (pp50), are not precipitated from uninfected or transformation-defective virus-infected cells and are not related to any RSV structural proteins. Neither pp50 nor pp90 shares any partial or complete proteolytic cleavage peptides with pp60 src , suggesting that pp90 and pp50 do not represent either a precursor or a cleavage product of pp60 src . Sedimentation analysis of RSV-transformed cell lysates on glycerol gradients revealed that the RSV pp60 src protein is present as two forms, one of which represents the majority (95%) of pp60 src and sediments as a monomer, 60,000 molecular weight protein and the other of which sediments with pp90 and pp50 as an apparent 200,000 molecular weight complex. Lysates from cells transformed by viruses containing a temperature-sensitive defect in the src gene contain a greater percentage of pp60 src associated with pp90 and pp50 under both permissive (35°C) and nonpermissive (41°C) conditions compared to wild-type virus-infected cell lysates. Phosphoserine and phosphotyrosine were found associated with pp60 src molecules that sedimented as a monomer, whereas pp60 src molecules that are complexed with pp90 and pp50 contain phosphoserine and greatly reduced amounts of phosphotyrosine. Only the monomer form of pp60 src is capable of phosphorylating IgG in the immune complex phosphotransferase reaction. Normal uninfected chicken cells contain a protein that shares identical partial proteolytic cleavage peptides with the pp90 protein immunoprecipitated from RSV-transformed cells. This pp90 protein is one of the major cytoplasmic proteins in uninfected cells. Antibody directed against pp90 also immunoprecipitates pp60 src and pp50 from lysates of RSV-transformed chicken cells.

Phosphorylated and nonphosphorylated forms of avian sarcoma virus polypeptide p19.

Abstract The major avian sarcoma virus phosphoprotein, p19, was found in virions in a phosphorylated and nonphosphorylated form. Both forms of p19 were immunoprecipitated with antiserum from hamsters bearing ASV-induced tumors, supporting previous claims that p19 is a viral RNA-encoded polypeptide. In addition, both forms contained the same methionine-labeled tryptic peptides. Antiserum raised against p27 was used to specifically immunoprecipitate [ 32 P]- or [ 32 S]methionine-labeled polypeptides from extracts of cells. A phosphoprotein which was specifically precipitated from extracts of infected cells comigrated with a similarly precipitated [ 35 S]methionine-labeled polypeptide with a molecular weight of approximately 76,000. Moreover, the 76,000 MW phosphoprotein contains the same tryptic phosphopeptide as p19. The significance of these observations is discussed.

Antibody to virion structural proteins in mammals bearing avian sarcoma virus-induced tumors.

Abstract The production of antibody to virion structural proteins was examined in rabbits and hamsters bearing tumors induced by avian sarcoma virus (ASV). Antibody activity was analyzed by the immunoprecipitation of polypeptides from preparations of radiolabeled ASV virions. Antipolymerase and antiglycoprotein activities were monitored by inhibition of the enzyme activity of virion polymerase and by neutralization of focus formation by group D ASV, respectively. All sera from hamsters bearing primary ASV-induced tumors had antibody against the viral gs antigens, but no antipolymerase or antiglycoprotein activity was detectable. Most sera from hamsters bearing tumors induced by injection of cloned hamster tumor cells displayed anti-gs activity, and in addition, some sera exhibited anti-polymerase activity as well. All sera from rabbits bearing primary tumors contained antibody against the gs antigens and the virion polymerase, and some of these sera were shown to have antibody against the virion glycoprotein, gp85, as well.

Studies on the Structure and Function of the Avian Sarcoma Virus Transforming Gene Product

RNA tumor viruses quickly and efficiently transform cells and are therefore extremely useful agents for the study of the molecular events in oncogenesis. It is well established that the product(s) of a single avian sarcoma virus (ASV) gene (src) is responsible for the induction and maintenance of cell transformation in vitro and tumor production in infected animals [6]. We have carried out experiments designed to identify this product by techniques that require no assumptions concerning its mechanism of action. This approach was necessary because we anticipated that the src protein would be present at relatively low levels in the transformed cells, therefore making direct identification impossible. For example, the precursor to the major virion structural proteins is present at such low levels it can be identified only by immunoprecipitation [5]. Furthermore, since there are such a variety of biochemical changes reported to occur in transformed cells, it is difficult to predict at what level the src gene product might disrupt normal cellular processes. This, in turn, makes an accurate forecast concerning its function unlikely.

The origin of 7 S RNA in virions of avian sarcoma viruses

Abstract Duck and chick embryo fibroblasts were labeled with 32 PO 4 3− and cellular 7S RNA was purified. Ribonuclease T 1 digests were prepared and the resulting oligonucleotides were fractionated by two-dimensional electrophoresis. Duck 7 S RNA lacked one large oligonucleotide present in chicken 7 S RNA, thus establishing that the two species contain 7 S RNAs of different sequence. The Prague C strain of Rous sarcoma virus was grown in each type of host and the 7 S RNA found in progeny virus was examined. It was found to be identical to that of the host which produced the virus.