RESUMEN
We examined the interaction of Abelson murine leukemia virus protein P120 with other cellular components after extraction with the nonionic detergent Triton X-100. Most of the Abelson murine leukemia virus P120-associated kinase activity was found in the detergent-insoluble matrix in both lymphoid and fibroblast cell lines. The P120 labeled during a short exposure of cells to [35S]-methionine was mainly in the detergent-insoluble matrix (lymphoid cells) or equally distributed in the detergent-insoluble matrix and the soluble fraction (fibroblasts). Steady-state-labeled P120 was distributed equally in the two fractions (lymphoid cells) or mostly in the soluble portion (fibroblasts). Thus, there was an apparent movement of P120 from the detergent-insoluble matrix to the detergent-soluble fraction and a concomitant loss of enzymatic activity. When the detergent-insoluble matrix was incubated with [32P]ATP in situ, phosphorylation of tyrosine residues of P120 was observed. We found an 80,000-molecular-weight fragment of P120 (designated F80) after extraction of fibroblast cells with detergent. F80 was not found in extracted lymphoid cells, but mixing labeled lymphoid cells and unlabeled fibroblasts before extraction produced the fragment. F80 contained the gag determinants of P120 but did not react with Abelson-specific serum. These data allowed us to assign various features of the protein to regions of the P120 molecule and to localize the Abelson-specific antigenic determinants to the C-terminal region of the molecule.
Asunto(s)
Virus de la Leucemia Murina de Abelson/análisis , Virus de la Leucemia Murina/análisis , Proteínas Quinasas/análisis , Fracciones Subcelulares/análisis , Proteínas Virales/análisis , Virus de la Leucemia Murina de Abelson/inmunología , Animales , Línea Celular , Epítopos , Fibroblastos , Linfocitos , Ratones , Octoxinol , Fragmentos de Péptidos/inmunología , Fosforilación , Polietilenglicoles , Proteínas Virales/metabolismoRESUMEN
The transforming protein of Rous sarcoma virus, p60src, the Abelson virus protein, p120, and the Y73 virus protein, p90, all have associated tyrosine protein kinase activities in vitro. Possible structural homology between these functionally related proteins was investigated by two-dimensional analysis of both methionine-containing and phosphate-containing tryptic peptides derived from biosynthetically labeled proteins. Marked differences were found between the maps of both [35S]methionine-labeled and 32P-labeled tryptic peptides. This suggests that the transforming gene of Rous sarcoma virus and the putative transforming genes of Abelson virus and Y73 virus are different. In addition, each of these genes has been shown previously to be unrelated to the putative transforming gene of Fujinami sarcoma virus, another virus which encodes a protein with associated tyrosine protein kinase activity. Therefore, it appears that there exist at least four distinct classes of functionally related RNA tumor viruses. Analysis of phosphorylated tryptic peptides did, however, reveal homology between one of the two phosphotyrosine-containing tryptic peptides of p90 of Y73 virus and the single phosphotyrosine-containing tryptic peptide of p60src of Rous sarcoma virus. Comigration of these two peptides in several different buffers and the identical mobility of their phosphorylated cleavage products after secondary digestion with protease V8 of Staphylococcus aureus indicated that p60src and p90 contain an identical site of tyrosine phosphorylation in vivo. The results are discussed with respect to the evolution of RNA tumor viruses which encode proteins with associated tyrosine protein kinase activities and the limitations of analysis of detecting homology between genes by both molecular hybridization and peptide mapping.
Asunto(s)
Transformación Celular Viral , Proteínas Quinasas/metabolismo , Retroviridae/análisis , Proteínas Virales/análisis , Virus de la Leucemia Murina de Abelson/análisis , Virus del Sarcoma Aviar/análisis , Metionina/análisis , Péptidos/análisis , Proteínas Tirosina Quinasas , Proteínas Virales/fisiologíaAsunto(s)
Virus de la Leucemia Murina de Abelson/análisis , Virus de la Leucemia Murina/análisis , Proteínas Quinasas/metabolismo , Retroviridae/análisis , Proteínas Virales/análisis , Virus de la Leucemia Murina de Abelson/metabolismo , Animales , Gatos , Transformación Celular Neoplásica , Transformación Celular Viral , Ratones , Fosforilación , Biosíntesis de Proteínas , Retroviridae/metabolismo , Proteínas Virales/metabolismoRESUMEN
A previously described 120,000 dalton polyprotein, P120, encoded by the Abelson strain of murine leukemia virus (AbLV) is compared to translational products representing the entire Moloney murine leukemia virus (MuLV) genome. Each of three [35S]-methionine tryptic present in Moloney-MuLV Pr65gag are also represented in Pr180gag-pol. Of these, one peptide corresponding to Moloney-MuLV p12, but neither of two p30 specific peptides are present in AbLV P120. None of the twelve remaining methionine containing peptides present in AbLV P120 appear to correspond to those of either Moloney-MuLV Pr82env. AbLV P120 and a 110,000 dalton polyprotein encoded by a second transforming isolate of mouse origin, designated AK-T8, are both shown to be highly phosphorylated. Sites of phosphorylation included known phosphorylated structural (p12) components, as well as components encoded by acquired cellular sequences. Immunoprecipitates of AbLV P120 obtained from either cells or pseudotype virions are shown to contain protein kinase activity which recognizes AbLv P120 as substrate. This activity may represent an intrinsic property of the polyprotein itself or represent a cellular enzyme associated with AbLV P120 in the form of an enzyme-substrate complex.
Asunto(s)
Virus de la Leucemia Murina de Abelson/análisis , Virus de la Leucemia Murina/análisis , Fosfopéptidos/análisis , Proteínas Virales/análisis , Animales , Línea Celular , Ratones , Peso Molecular , Virus de la Leucemia Murina de Moloney/análisis , Proteínas Quinasas/análisis , Precursores de Proteínas/análisisRESUMEN
Abelson murine leukemia virus (A-MuLV) is a replication-defective virus that transforms both fibroblasts and hematopoietic cells in vitro. The virus encodes a 120,000-molecular-weight protein (P120) that is composed of Moloney murine leukemia virus-derived gag gene sequences and A-MuLV--specific sequences. This protein is the only A-MuLV--encoded protein that has been detected, and thus P120 is a candidate for the transforming protein of A-MuLV. We now report isolation and characterization of three new A-MuLV isolates that do not synthesize P120 but do produce analogous proteins of larger (160,000 molecular weight) and smaller (100,000 and 90,000 molecular weight) size. All of these A-MuLV isolates transform fibroblasts and lymphoid cells in vitro. Because the different A-MuLV proteins vary in the A-MuLV--specific region of the molecule, these variants may set a maximum limit on the size of the A-MuLV transforming protein.
Asunto(s)
Virus de la Leucemia Murina de Abelson/genética , Virus de la Leucemia Murina/genética , Mutación , Proteínas Virales/genética , Virus de la Leucemia Murina de Abelson/análisis , Animales , Línea Celular , Transformación Celular Neoplásica , Transformación Celular Viral , Ratones , Peso Molecular , Ratas , Proteínas Virales/análisisRESUMEN
Virions produced from cells transformed by A-MuLV contain a 30S, 5.6 kb RNA that can be translated in a cell-free system to form the characteristic A-MuLV protein. This RNA was mapped by heteroduplex methods using DNA probes from M-MuLV, the presumed parent of A-MuLV. The overall organization of the RNA was determined by using full-length M-MuLV reverse transcribed DNA and visualizing the heteroduplexes in the electron microscope. This showed that A-MuLV and M-MuLV have homologous sequences at both ends of their RNAs but that the central portion of the A-MuLV genome is not homologous to sequences in M-MuLV RNA. A precise measure of the lengths of the shared regions was obtained by using S1 nuclease to digest hybrids between 32P-labeled M-MuLV DNA and A-MuLV RNA; the resulting fragments were analyzed for their length by electrophoresis. The regions of homology were shown to be 1320 nucleotides long at the 5' end and 730 nucleotides long at the 3' end. Thus approximately 6200 nucleotides of the approximately 8300 in M-MuLV RNA were deleted when the A-MuLV genome was formed, but an insert of 3600 nucleotides, presumably derived from the normal murine genome, was inserted in place of the deleted region.