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A G Papageorge

Publications and source records attributed to A G Papageorge.

31 records · Page 2Linked to original sources
PubMed1986

A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

Animals↗
PubMed1986

Mutational analysis of a ras catalytic domain.

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.

Arginine↗
PubMed1985

Transfection of v-rasH DNA into MCF-7 human breast cancer cells bypasses dependence on estrogen for tumorigenicity.

The natural history of estrogen-responsive breast cancers often involves a phenotypic change to an estrogen-unresponsive, more aggressive tumor. The human breast cancer cell line, MCF-7, which requires estradiol for tumor formation in vivo and shows growth stimulation in response to estradiol in vitro, is a model for hormone-responsive tumors. The v-rasH onc gene was transfected into MCF-7 cells. The cloned MCF-7ras transfectants, which expressed the v-rasH messenger RNA and v-rasH p21 protein (21,000 daltons), were characterized. In contrast to the parental cell line, MCF-7ras cells no longer responded to exogenous estrogen in culture and their growth was minimally inhibited by exogenous antiestrogens. When tested in the nude mouse, the MCF-7ras cells were fully tumorigenic in the absence of estrogen supplementation. Thus, cells acquiring an activated onc gene can bypass the hormonal regulatory signals that trigger the neoplastic growth of a human breast cancer cell line.

Animals↗
PubMed1985

Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains.

The mammalian p21 ras proteins contain a 20-amino acid region that is highly divergent, in contrast to the strong sequence conservation that is common to other regions of these proteins. This major variable region is located near the C terminus just upstream from a conserved cysteine residue that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor the protein in the membrane.

Amino Acid Sequence↗
PubMed1984

Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus.

Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha-MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane localization. We have now further characterized the post-translational processing of these mutants and have also studied two C-terminal v-rasH point mutants: one encodes serine in place of cysteine-186, the other threonine for valine-187. The Thr-187 mutant was transformation-competent, and its p21 protein was processed normally, as was the p21 encoded by a transformation-competent deletion mutant from which amino acids 166-175 had been deleted. The Ser-186 mutant was defective for transformation. The p21s encoded by the Ser-186 mutant and by the previously described transformation-defective mutants did not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell-encoded ras proteins also contain this cysteine, we conclude that this amino acid residue is required for all ras proteins.

Animals↗
PubMed1984

Saccharomyces cerevisiae synthesizes proteins related to the p21 gene product of ras genes found in mammals.

A family of normal vertebrate genes and oncogenes has been called the ras gene family. The name ras was assigned to this gene family based on the species of origin of the viral oncogenes of the rat-derived Harvey and Kirsten murine sarcoma viruses. There are now three known functional members of the ras gene family, and genes homologous to ras genes have been detected in the DNA of a wide variety of mammals and in Drosophila melanogaster. Prior experiments have detected proteins coded for by ras genes in a large number of normal cells, cell lines, and tumors. We report here the detection of ras-related proteins in D. melanogaster, a result predicted by the earlier detection of ras-related genes in the Drosophila genome. We also report for the first time the detection of ras-related proteins in a single-cell eucaryocyte, Saccharomyces cerevisiae. These proteins, approximately 30K in size, are recognized by both a monoclonal antibody which binds to the p21 coded for by mammalian ras genes and a polyclonal rat serum made by transplanting a v-Ha-ras-induced tumor in Osborne-Mendel rats. The p21 of v-Ha-ras and the 30K proteins from S. cerevisiae share methionine-labeled peptides as detected by two-dimensional tryptic peptide maps. The results indicate that S. cerevisiae synthesizes ras-related proteins. A genetic analysis of the function of these proteins for yeast cells may now be possible.

Animals↗
PubMed1982

Mechanism of activation of a human oncogene.

The oncogene of the human EJ bladder carcinoma cell lines arose via alteration of a cellular proto-oncogene. Experiments are presented that localize the genetic lesion that led to activation of the oncogene. The lesion has no affect on levels of expression of the oncogene. Instead, it affects the structure of the oncogene-encoded protein.

Base Sequence↗
PubMed1980

Guanine nucleotide-binding and autophosphorylating activities associated with the p21src protein of Harvey murine sarcoma virus.

The purified p21src protein of Harvey sarcoma virus shows a guanine nucleotide-binding activity and, in addition, at elevated temperature an autophosphorylating activity at a threonine residue using as phosphoryl donor GTP or dGTP but not ATP or dATP. These biochemical activities are unique among those associated with transforming proteins of RNA-containing or DNA-containing tumour viruses.

Animals↗
PubMed1979

Guanine nucleotide-binding activity as an assay for src protein of rat-derived murine sarcoma viruses.

We have recently identified a 21,000-dalton protein, p21, coded for by Kirsten or Harvey murine sarcoma virus. On the basis of the results obtained with the p21 of a mutant of Kirsten sarcoma virus, temperature sensitive for the maintenance of transformation, we concluded that the p21 was required for the maintenance of transformation induced by either virus. We report herein that when extracts from cells transformed by Kirsten or Harvey sarcoma virus are incubated with [(3)H]GDP or [alpha-(32)P]GTP, picomole quantities of guanine nucleotide can be immunoprecipitated with antisera that contain antibodies to the p21. Previously we have shown that the immunoprecipitability of [(35)S]methionine-labeled p21 of the temperature-sensitive mutant of Kirsten sarcoma virus is thermolabile. The binding of guanine nucleotide is shown herein also to be thermolabile in extracts of cells transformed by the same mutant. However, the immunoprecipitability of the [(35)S]methionine-labeled p21 in such extracts of the temperature-sensitive mutant can be preserved if the extracts containing labeled p21 are incubated with added GDP or GTP prior to heating. The results suggest an interaction between p21 and certain guanine nucleotides, and the possible roles of guanine nucleotides and p21 in the maintenance of transformation are discussed.

Animals↗
PubMedDate not supplied

The p21 ras C-terminus is required for transformation and membrane association.

The Harvey murine sarcoma virus (Ha-MuSV) transforming gene, v-rasH, encodes a 21,000 molecular weight protein (p21) that is closely related to the p21 proteins encoded by the cellular transforming genes of the ras gene family. The primary translation product (prop21), which is found in the cytosol, undergoes posttranslational modification and the mature protein subsequently becomes associated with the inner surface of the plasma membrane and binds lipid tightly. The p21 proteins have the capacity to bind guanine nucleotides non-covalently in vitro. To assess the biological relevance of these biochemical features of the protein, we have now studied a series of deletion mutants located at or near the C-terminus of the viral p21 protein. Our tissue culture studies indicate that amino acids located at or near the C-terminus are required for cellular transformation, membrane association and lipid binding.

Animals↗