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B M Sefton

Publications and source records attributed to B M Sefton.

At least 73 records · Page 4Linked to original sources
PubMed1986

Cytoskeletal organization, vinculin-phosphorylation, and fibronectin expression in transformed fibroblasts with different cell morphologies.

Neoplastic transformation of fibroblasts results in widely different cell morphologies. We have attempted to correlate cell morphology with cytoskeletal organization and fibronectin expression in murine and avian fibroblasts transformed by a diverse group of viral and chemical agents. The distribution of vinculin, alpha-actinin, actin, and surface fibronectin was studied, and, where appropriate, also the extent of phosphotyrosine modification of vinculin. Irrespective of the transforming agent we found that increased cell rounding was generally correlated with a reduction in vinculin-containing focal adhesions, a dissolution of microfilament bundles, and a reduction of extracellular fibronectin. In contrast, spindle-shaped fibroblasts expressed relatively high levels of surface fibronectin. Reorganization of vinculin, actin, and alpha-actinin into rosette-like structures was observed in polygonal or rounded cells transformed by viruses encoding tyrosine kinases, but was not seen in fibroblasts transformed by agents without associated tyrosine kinase activity or in spindle-shaped cells. No correlation was found between the extent of phosphotyrosine modification of vinculin and the extent of cell rounding. Irrespective of cell morphology, the extent of tyrosine phosphorylation of vinculin was high in all cells transformed by viruses carrying the src gene, but low in those transformed by viruses expressing the fps gene. Our results indicate that the morphology of a transformed cell is determined by a combination of several factors which are affected to different extents by different transforming agents.

Actin Cytoskeleton↗
PubMed1986

The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity.

We have constructed two point mutants of Rous sarcoma virus in which the amino-terminal glycine residue of the transforming protein, p60src, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60src proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p60src proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60src proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60src was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p60src proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p60src apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur.

Animals↗
PubMed1986

Phosphorylation of the transforming protein of Rous sarcoma virus: direct demonstration of phosphorylation of serine 17 and identification of an additional site of tyrosine phosphorylation in p60v-src of Prague Rous sarcoma virus.

We provide direct evidence that serine 17 is the major site of serine phosphorylation in p60v-src, the transforming protein of Rous sarcoma virus, and in its cellular homolog, p60c-src. The amino acid composition of the tryptic peptide containing the major site of serine phosphorylation in p60v-src was deduced by peptide map analysis of the protein labeled biosynthetically with a variety of radioactive amino acids. Manual Edman degradation revealed that the phosphorylated serine in this peptide was the amino terminal residue. These data are consistent only with the phosphorylation of serine 17. The major site of serine phosphorylation in chicken p60c-src, the cellular homolog of p60v-src, is contained in a tryptic peptide identical to that containing serine 17 in p60v-src of Schmidt Ruppin Rous sarcoma virus of subgroup A. Serine 17 is therefore also phosphorylated in p60c-src. The p60v-src protein encoded by Prague Rous sarcoma virus was found to contain two sites of tyrosine phosphorylation. The previously unrecognized site of tyrosine phosphorylation may be tyrosine 205 or possibly tyrosine 208. Treatment of Prague Rous sarcoma virus-infected cells with vanadyl ions stimulated the protein kinase activity of p60v-src and increased the phosphorylation of tyrosine 416 but not the phosphorylation of the additional site of tyrosine phosphorylation.

Amino Acid Sequence↗
PubMed1986

Direct identification of palmitic acid as the lipid attached to p21ras.

p21v-H-ras, the transforming protein of Harvey murine sarcoma virus, contains a covalently attached lipid. Using thin-layer chromatography, we identified the acyl group as the 16-carbon saturated fatty acid palmitic acid. No myristic acid was detected in fatty acids released from in vivo-labeled p21v-H-ras. The p21v-K-ras protein encoded by Kirsten sarcoma virus was also palmitylated. The processing and acylation of p21v-K-ras however differed from that of p21v-H-ras. Three forms of [3H]palmitic acid-labeled p21ras proteins were detected in Kirsten sarcoma virus-transformed cells. This contrasted with Harvey sarcoma virus, in which two forms of p21v-H-ras contained palmitic acid. Analysis by partial proteolysis of p21v-H-ras labeled with [3H]palmitic acid suggested that all of the lipid found in intact p21v-H-ras was located in the C-terminal region. On sodium dodecyl sulfate-polyacrylamide gels, p21v-H-ras labeled with [3H]palmitic acid migrated slightly ahead of the majority of p21v-H-ras. Of the mature forms of p21v-H-ras, apparently only a subpopulation contains palmitic acid.

Animals↗
PubMed1986

Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src.

All 15 protein kinases whose amino acid sequence is known contain a lysine residue at a position homologous to that of lysine-295 in p60src, the transforming protein of Rous sarcoma virus. The ATP analog p-fluorosulfonyl 5'-benzoyl adenosine inactivates both p60src and the catalytic subunit of the cyclic AMP-dependent protein kinase by modification of this lysine. We used oligonucleotide-directed mutagenesis to examine the possible functions of this residue. Lysine-295 in p60src was replaced with a glutamic acid, an arginine, or a histidine residue, and mutant p60src proteins were characterized in chicken cells infected by mutant viruses. None of these three mutant p60src proteins had tyrosine protein kinase activity in vitro, and none induced morphological transformation of infected cells. Since neither a histidine nor an arginine residue can replace the function of lysine-295, we suggest that it carries out the specialized function of proton transfer in the phosphotransferase reaction. All three mutant viruses underwent reversion to wild type during passage in tissue culture. Because the rate with which this occurred differed significantly among the mutants, reversion appears to have resulted from errors in transcription, rather than from recombination with the cellular src gene.

Amino Acid Sequence↗
PubMed1986

From c-src to v-src, or the case of the missing C terminus.

The acutely-transforming avian virus, Rous sarcoma virus, arose as the result of the transduction of the c-src gene of the chicken by a relatively benign avian leucosis virus. In the viral genome, the src gene is expressed in the form of a 60 kDa phosphoprotein, pp60v-src, which is a protein-tyrosine kinase. Cellular transformation results from the excessive or chronic phosphorylation of cellular proteins by pp60v-src. The c-src gene has been present in the genome of eukaryotes, at least since the evolutionary emergence of Drosophila. Like its viral descendant, it encodes a 60 kDa protein-tyrosine kinase, termed pp60c-src. Both pp60v-src and pp60c-src are bound to membranes. pp60c-src is expressed at low levels in most cell types but it is found in high amounts in neural tissues and in platelets. pp60c-src might therefore participate in vesicle-mediated secretion. pp60c-src is less active as a protein-tyrosine kinase then pp60v-src and does not induce cellular transformation, even when expressed at levels comparable to those of pp60v-src. The potency of pp60v-src apparently results from the fact that the region of the c-src gene encoding the C terminus of pp60c-src was lost during the genesis of the v-src gene. This region of pp60c-src contains a site of tyrosine phosphorylation whose occupancy apparently leads to diminished enzymatic activity. The deletion of this site may abolish the normal regulation of the protein kinase activity. If so, transformation could simply be the consequence of the inability of the cell to regulate the activity of pp60v-src.

Animals↗
PubMed1985

Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation.

p60src, the transforming protein kinase of Rous sarcoma virus, contains the 14-carbon saturated fatty acid, myristic acid, linked through an amide bond to the alpha-amino group of its NH2-terminal glycine residue. Myristic acid is known to be attached to four other eukaryotic proteins. In each case the fatty acid is also linked through an amide bond to an NH2-terminal glycine. We have used oligonucleotide-directed mutagenesis to examine the amino acid specificity of the enzyme that myristoylates the NH2 terminus of these proteins. Replacement of the NH2-terminal glycine in p60src with either alanine or glutamic acid prevented myristoylation completely. This indicates that the myristoylating enzyme may have an absolute specificity for glycine. Strikingly, neither nonmyristoylated mutant src protein induced morphological transformation of infected cells, even though wild-type levels of phosphorylation of cellular proteins on tyrosine were observed in these cells. Since conversion of the NH2-terminal residue from glycine to alanine should have little effect on the conformation of p60src, the inability of this mutant p60src protein to induce morphological transformation suggests that the myristoyl moiety is essential for the transforming activity of the protein.

Animals↗
PubMed1985

Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog.

The lipid bound to p60src, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [3H]myristic acid or [3H]palmitic acid. Incorporation of [3H]myristic acid was noticeably greater than incorporation of [3H]palmitic acid. All of the [3H]myristic acid-derived label in p60src was present as myristic acid. In contrast, none of the radioactivity derived from [3H]palmitic acid was recovered as palmitic acid. Instead, all 3H incorporated into p60src from [3H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-src also contains myristic acid. By comparison of the extent of myristylation of p60v-src with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, we estimate that greater than 80% of the molecules of p60v-src contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-src contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [3H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [3H]myristic acid-labeled p60src in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60src present in cells.

Animals↗
PubMed1984

Coronavirus multiplication: locations of genes for virion proteins on the avian infectious bronchitis virus genome.

Six overlapping viral RNAs are synthesized in cells infected with the avian coronavirus infectious bronchitis virus (IBV). These RNAs contain a 3'-coterminal nested sequence set and were assumed to be viral mRNAs. The seven major IBV virion proteins are all produced by processing of three polypeptides of ca. 23, 51, and 115 kilodaltons. These are the core polypeptides of the small membrane proteins, the nucleocapsid protein, and the 155-kilodalton precursor to the large membrane proteins GP90 and GP84, respectively. To determine which mRNAs specify these polypeptides, we isolated RNA from infected cells and translated it in a messenger-dependent rabbit reticulocyte lysate. Proteins of 23, 51, and 110 kilodaltons were produced. Two-dimensional tryptic peptide mapping demonstrated that these proteins were closely related to the major virion proteins. Fractionation of the RNA before cell-free translation permitted the correlation of messenger activities for synthesis of the proteins with the presence of specific mRNAs. We found that the smallest RNA, RNA A, directs the synthesis of P51, the nucleocapsid protein. RNA C, which contains the sequences of RNA A, directs the synthesis of the small membrane protein P23. RNA E directs the synthesis of the large virion glycoproteins. These results supported a model in which only the unique 5'-terminal domain of each IBV mRNA is active in translation and enabled us to localize genes for virion proteins on the IBV genome.

Capsid↗
PubMed1984

Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells.

Incubation of quiescent chicken embryo cells with platelet-derived growth factor, epidermal growth factor, or serum was found to stimulate phosphorylation of two proteins of ca. 42,000 daltons on tyrosine. These proteins are structurally related to each other and to two proteins phosphorylated on tyrosine under similar conditions in mitogen-treated mouse fibroblasts. Three other very different mitogenic agents, the protease trypsin and the chemically unrelated tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and teleocidin, stimulated phosphorylation of the same proteins. In all cases, phosphotyrosine was detected in these phosphoproteins. Although additional changes in protein phosphorylation were evident, no other proteins were observed by two-dimensional gel electrophoresis which contained increased amounts of phosphotyrosine in mitogen-treated chicken embryo cells. One of these 42,000-dalton proteins was shown previously to be phosphorylated on tyrosine in chicken embryo cells transformed with various retroviruses whose transforming proteins possess tyrosine protein kinase activity. Phosphorylation of the 42,000-dalton proteins could be important in the regulation of cell division.

Animals↗
PubMed1984

Myristic acid is attached to the transforming protein of Rous sarcoma virus during or immediately after synthesis and is present in both soluble and membrane-bound forms of the protein.

Myristic acid, a minor component of cellular fatty acids, has been shown previously to be covalently bound to most molecules of p60src, the transforming protein of Rous sarcoma virus. We have now determined at what time during the life cycle of p60src, and where within the cell, this lipid becomes attached to the protein. p60src was found to acquire myristic acid at only one time, during or immediately after its synthesis. p60src is known to be synthesized on free polysomes and appears at the cytoplasmic face of the plasma membrane after a lag of 10 min. The addition of myristic acid to p60src therefore precedes the binding of the protein to the plasma membrane. The lipid attached to p60src is a permanent, metabolically stable part of the protein; we found no evidence for turnover of the myristyl moiety. However, we did find myristate attached to various soluble forms of p60src and to a large number of cytosolic cellular proteins as well. This demonstrates that the attachment of myristic acid to a protein is not in itself sufficient to convert a soluble protein into a membrane-bound protein.

Animals↗
PubMed1984

Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells.

The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid.

Adenosine Triphosphate↗
PubMed1983

Some lymphoid cell lines transformed by Abelson murine leukemia virus lack a major 36,000-dalton tyrosine protein kinase substrate.

Fibroblasts transformed by Abelson murine leukemia virus differ from normal fibroblasts in that they contain several cellular proteins, including one of 29 and one of 36 kilodaltons, which are phosphorylated at tyrosine residues. Since it has been shown before that these proteins also become phosphorylated at tyrosine after transformation of fibroblasts by a number of other retroviruses, their phosphorylation may play an important role in the transformation of these cells. In contrast, the 36-kilodalton phosphoprotein was not detectable in three of the four lines of Abelson virus-transformed B lymphoma cell lines studied here. These three cell lines, RAW307.1.1, 18-48, and 18-81, and a B lymphoma induced by mineral oil, WEHI 279, were all found to lack both the phosphorylated and unphosphorylated forms of the 36-kilodalton protein. It thus appears that expression of this major cell protein is not essential for the survival of B lymphoma cells in culture and that the phosphorylation of the 36-kilodalton protein at tyrosine is not essential for transformation of pre-B lymphocytes by Abelson virus.

Abelson murine leukemia virus↗
PubMed1982

The transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus contain tightly bound lipid.

We have found that the transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus all contain tightly bound lipid. This modification could play a role in the binding of these proteins to cellular membranes. The lipid associated with p60src, the transforming protein of Rous sarcoma virus, is located in the NH2-terminal domain of the polypeptide. This is the region of the protein that has been shown previously to participate in binding the protein to membranes. Two mature forms of p21, the transforming protein of Harvey sarcoma virus, contain lipid. Lipid is not, however, associated with newly synthesized p21. While mature p60src and p21 are bound to cellular membranes, the newly synthesized forms of these proteins are not. The posttranslational addition of lipid may therefore be the means by which these proteins acquire an affinity for membranes.

Abelson murine leukemia virus↗
PubMed1982

Immunofluorescent localization of the transforming protein of Rous sarcoma virus with antibodies against a synthetic src peptide.

Antisera were raised against a synthetic peptide (src-c) containing the six COOH-terminal amino acids of p60src, the transforming protein of Rous sarcoma virus (RSV). Antibodies specific for the src-c peptide were purified by affinity chromatography and then used to study the location of p60src in transformed cells. The distribution of p60src was compared to that of vinculin, a candidate cytoskeletal substrate of p60src, by indirect double immunofluorescence microscopy. In RSV-transformed rat, mouse, and chicken cells, an extensive codistribution of p60src with vinculin was observed. Both proteins were concentrated in the few remaining focal adhesion plaques, in transformation-induced rosette clusters at the ventral cell surface, and in cell-cell contact areas. In addition, antibodies to both proteins stained the cytoplasm diffusely. In all cells examined, the immunofluorescent staining patterns produced by antibodies to the src-c peptide were indistinguishable from those obtained by immunolabeling of p60src with sera from RSV-infected tumor-bearing rabbits. The excellent agreement of the results obtained with two completely independent antibody preparations indicates strongly that the observed immunolabeling patterns correctly define the intracellular distribution of p60src. The significance of the intracellular location of p60src to the transforming activities of the protein is discussed.

Animals↗
PubMed1982

Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation.

We have identified the single phosphorylated tyrosine in p60src, the transforming protein of Rous sarcoma virus, as part of the sequence. NH2-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg-COOH. Therefore, this is a sequence that is recognized efficiently by a tyrosine protein kinase in vivo. Phosphorylation of tyrosine in cellular proteins appears to play a role in malignant transformation by four classes of genetically distinct RNA tumor viruses. Phosphorylated tyrosines in several other proteins resemble of the tyrosine in p60src in that they are located 7 residues to the COOH-terminal side of a basic amino acid and either 4 residues to the COOH-terminal side of, or in close proximity to, a glutamic acid residue. Therefore it is possible that these features play a role in the selection of sites of phosphorylation by some tyrosine protein kinases. However, several clear exceptions to this rule exist.

Abelson murine leukemia virus↗