Characterization of 40,000- and 25,000-dalton intermediate precursors to Rauscher murine leukemia virus gag gene products.

Under steady-state labeling conditions, Rauscher murine leukemia virus-infected NIH Swiss mouse cells contain at least three major polyproteins derived from the viral gag gene. They have molecular weights of 65,000, 40,000, and 25,000. They have been termed pPr65gag, Pr40gag, and pPr25gag. pPr65gag has been shown by a number of laboratories to be composed of all four core proteins (p15, pp12, p30, and p10). In this paper, Pr40gag was found to contain p30 and p10 antigenic determinants and peptide sequences, whereas pPr25gag was found to contain p15 and pp12. Pr40gag and pPr25gag are rapidly labeled precursor proteins that were detectable early in pulse-chase experiments. Both precursors disappeared during the later stages of the chase period concurrent with the appearance of the mature viral core proteins. pPr65gag and pPr25gag were found to be phosphorylated, pPr25 having a higher specific activity of 32P than pPr65. In spite of this, peptide mapping studies, as well as the identification of the phosphorylated amino acid residues of pPr65, and pPr25, and pp12, indicated that the same sites are phosphorylated regardless of whether the precursors or the mature pp12 are examined.     

RNA tumor virus phosphoproteins: primary structural analysis and identification of phosphopeptides.

Two-dimensional tryptic peptide mapping was used to compare the peptide sequences of the phosphoprotein (pp12) of cloned ecotropic and amphotropic wild mouse leukemia viruses, strains 1504 and 292. The maps of two ecotropic isolates were very similar to one another, as were the maps of two amphotropic isolates. There was also extensive similarity between the maps of this protein from ecotropic and amphotropic viruses, although characteristic peptide differences were readily recognized. These differences were consistent with the general type specificity of oncovirus phosphoproteins. The pp12 of the field isoalte of 292 virus contained five phosphopeptides, and the non-phosphorylated and variously phosphorylated species of this pp12 showed identical peptide maps, indicating differential phosphorylation of a single polypeptide.     

Detection of phosphorylated forms of Moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis.

We detected phosphorylation of the major Moloney murine leukemia virus (M-MuLV) capsid polypeptide, p30, by using 32Pi-labeled virions. This was observed both on two-dimensional polyacrylamide gels directly or on one-dimensional gels of viral lysates that had been immunoprecipitated with monospecific goat anti-p30 serum. The phosphorylation event had been difficult to detect because pp12 the major virion phosphoprotein incorporates almost all of the 32P label added to infected cells (Y. Yoshinaka and R. B. Luftig, Virology 116:181-195, 1982). When immunoprecipitates from M-MuLV lysates labeled with 32Pi were compared with those labeled with [35S]methionine, it was calculated that the degree of phosphorylation at the p30 domain of Pr65gag was only 0.22 to 0.54% relative to phosphorylation at the p12 domain. Two-dimensional gel electrophoresis of the 32P-labeled p30 immunoprecipitates showed that there were three phosphorylated p30 forms with isoelectric points (pIs) of 5.7, 5.8, and 6.0. These forms were generally more acidic than the [35S]methionine-labeled p30 forms, which had pIs of 6.0, 6.1, 6.3 (the major constituent with greater than 80% of the label), and 6.6. The predominant phosphoamino acid of the major phosphorylated p30 form (pI 5.8) was phosphoserine. Further, tryptic peptide analysis of this p30 form showed that only one peptide was predominantly phosphorylated. Based on a comparison of specific labeling of p30 tryptic peptides with [14C]serine, [35S]methionine, and 32Pi, we tentatively assigned the phosphorylation site to a 2.4-kilodalton NH2-terminal peptide containing triple tandem serines spanning the region from amino acids 4 to 24.     

Translational products encoded by newly acquired sequences of independently derived feline sarcoma virus isolates are structurally related

Polyproteins encoded by several independent isolates of feline sarcoma virus (FeSV) were analyzed with respect to molecular weight, extent of phosphorylation, and tryptic peptide composition. As previously reported, cells nonproductively transformed by the Gardner strain of FeSV express a polyprotein which has a molecular weight of approximately 115,000 and contains feline leukemia virus p15, p12, and minor portion of p30. In addition, a major 72,000-dalton possible cleavage product can be identified. Snyder-Theilen FeSV-transformed cells express a major polyprotein of approximately 115,000 daltons and a second highly related 80,000-dalton protein. The p12 structural component of Gardner FeSV P115, but not Snyder-Theilen FeSV 115, corresponds to feline leukemia virus subgroup A with respect to immunological type specificity, a finding consistent with the independent origin of these viruses. Tryptic peptide analysis revealed five methionine-containing peptides specific to the nonstructural portion of Gardner FeSV 115, three of which were also represented in Snyder-Theilen FeSV P115, three of which were also represented in Snyder-Theilen FeSV P115. None of these [35S]methionine-labeled tryptic peptides were present in translational products representative of the complete feline leukemia virus subgroup A genome, including Pr180gag-pol, Pr65gag, and Pr82env. Similarly phosphorylated tryptic peptides within the structural (p12) and nonstructural components of Gardner FeSV P115 and Snyder-Theilen FeSV P115 Are highly related. These findings support the possibility that acquired sequences of two independently derived isolates of FeSV encode structurally related proteins.     

Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene.

Post-translational modifications of retrovirus gag gene-encoded polyproteins include proteolytic cleavage, phosphorylation, and glycosylation. To study the sequence of these events, we labeled JLS-V9 cells chronically infected with Rauscher murine leukemia virus in pulse-chase experiments with the radioactive precursors [35S]methionine, [14C]mannose, [3H]glucosamine, and [32P]phosphate. Newly synthesized gag polyproteins which incorporated label, and the modified products derived from them, were identified by immunoprecipitation of cell lysates with anti-p30 rabbit serum, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Pulse-chase experiments were carried out in the presence as well as in the absence of tunicamycin, an inhibitor of glycosylation. Among the three major polyproteins synthesized in the absence of tunicamycin, two were found to be glycosylated but not phosphorylated. These were designated gPr80gag and gP94gag. Both shared identical [35S]methionine peptides with Pr65gag and p30. Of the two nonglycosylated precursors, Pr65gag and Pr75gag, only Pr65gag was found to be detectably phosphorylated, and Pr75gag could be readily identified only when glycosylation was inhibited. On the basis of these results, a scheme for the post-translational modification of gag polyproteins is proposed. According to this scheme the gag gene-encoded polyproteins are processed from a common precursor, Pr75gag, by two divergent pathways: one leading through the intermediate Pr65gag to internal virion components via cleavage and phosphorylation and the other via tunicamycin-sensitive mannosylation to the intermediate gPr80gag, which is further glycosylated to yield cell surface polyprotein gP94gag.     

Polyprotein Precursors to Mouse Mammary Tumor Virus Proteins

Mouse mammary tumor virus (MMTV) derived from the culture medium of GR cells contained seven proteins, identified as gp55, gp33, p25, pp20, p16, p12, and p10. The major viral phosphoprotein was the 20,000-molecular-weight protein, pp20. Immunoprecipitation of cytoplasmic extracts from pulse-labeled GR cells identified three MMTV gag-specific proteins, termed Pr78(gag), Pr110(gag), and Pr180(gag+). These intracellular polyproteins were precipitable from cytoplasmic extracts by antisera to virions p25 and p12 but not by antisera to gp55. The major intracellular gag-specific precursor polyprotein, Pr78(gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, p10, and presumably pp20. This precursor is presumably derived from nascent chain cleavage or rapid posttranslational cleavage of the larger intracellular precursor-like protein, designated Pr110(gag). Pr110(gag) contained all but one of the leucine-containing tryptic peptides of Pr78(gag), plus several additional peptides. In addition to Pr78(gag) and Pr110(gag), monospecific antisera to virion p12 and p25 were also capable of precipitating from pulse-labeled cells a small amount of a 180,000-molecular-weight precursor-like protein, designated Pr180(gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78(gag) and Pr110(gag) plus several additional peptides. By analogy to type C viral systems, Pr180(gag+) is presumed to represent a gag-pol common precursor which is the major pathway for synthesis of MMTV polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two env-specific proteins, designated gPr76(env) and gP79(env). The major env precursor, gPr76(env), could be labeled with radioactive glucosamine and was shown to contain antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A minor glycoprotein, gP79(env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79(env) represents fucosylated gPr76(env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.     

Phosphorylation of murine mammary tumor virus precursor polypeptides.

Phosphorylation of the murine mammary tumor virus (MuMTV) structural proteins was studied in an MuMTV-infected epithelial cell line derived from a BALB/cf C3H mouse mammary tumor. Immunoprecipitation of 32P-labeled cell extracts with monospecific anti-p27 serum revealed that phosphorylation occurred at the stage of the core-protein polyprotein precursor prp75. Two forms of phosphorylated prp75 were found: one migrating with an apparent molecular weight of 80,000, and the other with a molecular weight of 76,000. The 80,000-molecular-weight species was found to be the most heavily phosphorylated. In addition, a relatively stable phosphorylated processing intermediate of 34,000 molecular weight was observed as well. Tryptic peptide mapping analysis of the 32P-labeled viral proteins indicated a precursor product relationship between the intracellular phosphorylated, high-molecular-weight peptides and the mature MuMTV phosphoproteins p23 and p27. Phosphopeptide analysis also suggested that phosphorylation of the viral proteins occurred in discrete steps and that the attached phosphate groups were conserved throughout the processing steps.     

Translation of bovine leukemia virus virion RNAs in heterologous protein-synthesizing systems.

Bovine leukemia virus 60 to 70S RNA was heat denatured, the polyadenylic acid-containing species were separated by velocity sedimentation, and several size classes were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The major RNA species sedimented at 38S and migrated as a single component of molecular weight 2.95 x 10(6) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The predominant polypeptides of the in vitro translation of bovine leukemia virus 38S RNA were products with molecular weights of 70,000 and 45,000; minor components with molecular weights of 145,000 and 18,000 were also observed. Two lines of evidence indicate that the 70,000- and 45,000-molecular weight polypeptides represent translation products of the gag gene of the bovine leukemia virus genome (Pr70gag and Pr45gag). First, they are specifically precipitated by a monospecific antiserum to the major internal protein, p24, and second, they are synthesized and correctly processed into virion proteins p24, p15, and p10 in Xenopus laevis oocytes microinjected with bovine leukemia virus 38S RNA. The 145,000-molecular weight polypeptide was immunoprecipitated by the anti-p24 serum and not by an antiserum to the major envelope glycoprotein, gp60. It contained all the tryptic peptides of Pr70gag and additional peptides unique to it, and thus represents in elongation product of Pr70gag in an adjacent gene, presumably the pol gene. The 18,000-molecular weight product was antigenically unrelated to p24 and gp60 and shared no peptides in common with Pr70gag, Pr45gag, or the 145,000-molecular weight polypeptide. It was maximally synthesized on a polyadenylic acid-containing virion 16 to 18S RNA, and we present evidence that this RNA is a 3' end-derived subgenomic fragment of the bovine leukemia virus genome rather than a contaminating cellular RNA.     

Impaired Maturation of Mouse Mammary Tumor Virus Precursor Polypeptides in Lymphoid Leukemia Cells, Producing Intracytoplasmic A Particles and No Extracellular B-Type Virions

Processing of polypeptides of the mouse mammary tumor virus, a type B retrovirus, was investigated in a transplanted thymic lymphoma cell line of the GR strain (GRSL). This cell line was maintained in vivo in ascites form and in vitro as a suspension culture. GRSL cells produce clusters of intracytoplasmic A particles and are virtually deficient in the production of mature extracellular B-type particles. As control, a mammary tumor cell line of the same mouse strain capable of complete virion synthesis was used. The kinetics of viral polypeptide synthesis were studied by pulse labeling with various isotopes (including (35)S and (32)P), followed by immunoprecipitation of cell lysates with monospecific antisera to the major mouse mammary tumor virus gag and env proteins, p27 and gp52, respectively. Both the primary gag and env precursor polypeptides were synthesized in the GRSL cells, but their conversion into viral proteins was impaired. The major gag precursor, Pr73(gag), was stable over a period of 8 h, and mature viral core polypeptides could not be detected. Also, the highly phosphorylated intermediates in the proteolytic processing of Pr73(gag) in virus-producing cells were absent in GRSL cells. By immunoprecipitation, Pr73(gag) was detected in a GRSL particle fraction with the density of intracytoplasmic A particles. The precursor for envelope proteins, Pr73(env), was turned over without the generation of mature viral envelope components gp52 and gp36. The in vivo-transplanted ascites GRSL cells, however, were shown to express gp52 on the cell surface together with a 73,000-dalton polypeptide, as indicated by cell surface iodination and immunoprecipitation.     

Characterization of the pp70 protein phosphorylation in the extract of rice young panicle

The endogenous phosphorylation pattern of the extract prepared from rice young panicle has been examined. The extract was phosphorylated in vitro by incubating with [gamma-32p] ATP, and then analyzed by SDS-PAGE and autoradiography. At least eight major phosphoproteins with apparent molecular weights of 70, 60, 52, 40, 33, 30, 20, 16 and 15 kd were detected in the crude extract of rice young panicle. The pp70 which represents the major phosphoprotein in the crude extract of young panicle of rice is a cytosolic protein. Photoaffinity labeling with 8-azido-ATP revealed that a major protein with molecular weight 42,000 dalton but not pp70 can be specifically bound ATP. This suggests that pp70 is not a protein kinase itself, but a substrate of protein kinase. The in vitro phosphorylation of the pp70 occurs at a serine or threonine residue and is time and temperature dependent. The phosphorylation of pp70 does not depend by exogenous Ca++ or cAMP, suggesting that pp70 is a major substrate of an Ca++ or cAMP independent protein kinase in rice young panicle.     

Primate retroviruses: intracistronic mapping of type D viral gag gene by use of nonconditional replication mutants.

Nonconditional replication mutants of squirrel monkey retrovirus (SMRV), an endogenous type D virus of primates, are shown to be defective in post-translational processing of nonglycosylated virus-coded structural proteins. Utilizing such mutants, in combination with sensitive radioimmunological assays, we demonstrate the existence of a 72,000-molecular-weight precursor polyprotein (Pr72gag) encoded by a region of the SMRV genome designated gag. Post-translational cleavage of this precursor polyprotein gives rise to virion structural proteins of 35,000 (p35), 16,000 (p16), 12,000 (p12), and 9,000 (p9) molecular weight. Three of these viral proteins, p35, p16, and p9, are shown to be phosphorylated. Analysis of viral antigen expression in cell lines nonproductively infected with either of two replication-defective SMRV mutants or mink cells productively infected with wild-type SMRV resulted in the detection of several SMRV Pr72gag intermediate cleavage products. Adjacent proteins within such intermediates are identified by use of specific competition immunoassays, and the intracistropic order of individual structural proteins with SMRV Pr72gag was tentatively deduced as NH2-p16-p12-p35-p9-COOH.     

Formyl-Met-Leu-Phe-dependent serine kinase for a 64,000 molecular weight protein of polymorphonuclear leukocytes in a cell-lysate system

In the Triton X-100-treated polymorphonuclear leukocytes (PMN), which were stimulated with formyl-Met-Leu-Phe (FMLP) for 1 min, a 64,000 molecular weight protein (p64) was preferentially phosphorylated by the incubation with [gamma-32P]ATP in the presence of Mg2+, but not in the presence of Ca2+. Phosphoamino acid analysis of pp64 revealed that the p64-kinase was a serine-specific protein kinase. The p64 was maximally phosphorylated in the first minute, suggesting that the rapid phosphorylation was related to the initial reaction for activation of the FMLP-stimulated PMN functions. The FMLP-stimulated phosphorylation of p64 was slightly inhibited by the addition of cGMP in the reaction mixture. However, addition of cAMP, the cyclic nucleotide-dependent kinase inhibitor (H-8), protein kinase C-inhibitor (H-7) or Ca/calmodulin-dependent kinase inhibitor (W-7), showed no effect on the phosphorylation. These data suggest that phosphorylation of p64 seems to be a novel protein kinase specific to p64.     

Identification of a Rapidly Labelled 350K Histidine-Rich Protein in Neonatal Mouse Epidermis

Abstract. The major histidine-rich protein (HRP) found in the stratum corneum of neonatal mouse epidermis (band 2 protein, molecular weight 27,000) is a relatively late product of epidermal differentiation and incorporates labelled amino acids in vivo only after a 6–9 h lag period. A number of putative precursor HRPs in the 70–300 K molecular weight range were initially identified using short pulse labelling times and our previously described methods for isolation of epidermis and extraction of proteins. However, when steps were taken to minimise proteolysis during preparation, a single species of approximately 350 K molecular weight was the most strongly labelled protein following a 1 h in vivo pulse of [³H]-histidine. This protein was stable in sodium dodecyl sulphate dithiothreitol at 100°C and in 4 M urea, suggesting a single covalently linked polypeptide. The kinetics of labelling and the localisation of the 350 K HRP in the lower granular layers suggest that it is a precursor of the stratum corneum HRP. The processing of the 350 K HRP to the stratum corneum species appears to involve a complex series of specific cleavage steps which give rise to a number of HRPs of intermediate molecular weight.     

Identification of a rapidly labelled 350K histidine-rich protein in neonatal mouse epidermis

The major histidine-rich protein (HRP) found in the stratum corneum of neonatal mouse epidermis (band 2 protein, molecular weight 27,000) is a relatively late product of epidermal differentiation and incorporates labelled amino acids in vivo only after a 6-9 h lag period. A number of putative precursor HRPs in the 70-300 K molecular weight range were initially identified using short pulse labeling times and our previously described methods for isolation of epidermis and extraction of proteins. However, when steps were taken to minimise proteolysis during preparation, a single species of approximately 350 K molecular weight was the most strongly labelled protein following a 1 h in vivo pulse of [3H]-histidine. This protein was stable in sodium dodecyl sulphate dithiothreitol at 100 degrees C and in 4 M urea, suggesting a single covalently linked polypeptide. The kinetics of labelling and the localisation of the 350 K HRP in the lower granular layers suggest that it is a precursor of the stratum corneum HRP. The processing of the 350 K HRP to the stratum corneum species appears to involve a complex series of specific cleavage steps which give rise to a number of HRPs of intermediate molecular weight.     

Identification and characterization of the nonphosphorylated precursor of pp17, a phosphoprotein associated with phorbol ester induction of growth arrest and monocytic differentiation in HL-60 promyelocytic leukemia cells

Treatment of HL-60 promyelocytic leukemia cells with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), causes rapid phosphorylation and dephosphorylation of pp17, a 17-20-kDa, pI 5.5 cytosolic protein, as an early event in a response sequence leading to growth arrest and terminal differentiation into monocytes (Feuerstein, N., and Cooper, H. L., (1984) J. Biol. Chem. 259, 2782-2788). In the present study, we have identified the nonphosphorylated precursor to pp17 by tryptic peptide mapping of single proteins recovered from two-dimensional gels. The pI of the precursor, p17, was 5.9, and the apparent Mr of both p17 and pp17 was 18,400 by SDS-polyacrylamide gel electrophoresis (one dimension). p17 was shown to be a major cytosolic protein, comprising about 0.5% of steady state labeled protein in that fraction. Both p17 and pp17 were found exclusively in the cytosol (detergent-released SOL) and were not detected in membranes, cytoskeleton, or nuclei. In untreated cells, about 90% of the protein was present in the nonphosphorylated form. Upon TPA treatment, pre-existing p17 was rapidly phosphorylated to pp17. After 15 min, the two forms were nearly equal in quantity. This corresponds to phosphorylation, within that period, of about 0.2% of total cytosolic protein, represented by a single species. The maximum level of pp17 was reached within 1 h, with pp17 exceeding p17 by about 25%. Quantitatively, therefore, the phosphorylation of p17 to pp17 is one of the most prominent early biochemical responses to TPA treatment. Available data indicate that p17 predominates in rapidly proliferating cells, while phosphorylation to pp17 occurs where cell growth is modified by TPA or other agents. Thus, the p17/pp17 system is potentially a major mechanism for intracellular propagation of growth regulatory signals. We propose the name, prosolin, for this prominent cytosolic protein.     

Identification of a cellular protein substrate phosphorylated by the avian sarcoma virus-transforming gene product

The avian sarcoma virus-transforming gene product (pp60src) appears potentially able to mediate cell transformation via phosphorylation since it is tightly associated with a protein kinase activity. We have searched for and have been able to identify a normal cellular protein that appears to be a substrate of pp60src. The phosphorylation of this protein (34K) in transformation-specific in ASV-transformed cells of both avian and mammalian origin. Moreover, the 34K polypeptide serves as a substrate for the pp60src phosphotransferase activity in vitro and is phosphorylated at a site identical to the major site of phosphorylation in vivo. These data suggest that upon transformation the 34,000-dalton protein is phosphorylated directly as a result of pp60src activity.