Viral Harvey ras p21 expressed in Escherichia coli purifies as a binary one-to-one complex with GDP

The ras oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton protein, p21, which mediates transformation. Viral Harvey ras p21, cloned into Escherichia coli HB101 lambda/pRAS1, has been purified to homogeneity by a three-step procedure. The purified E. coli p21 can be bound by a monoclonal antibody to viral Harvey ras p21 and has an amino acid composition consistent with that predicted from its DNA sequence. Purified E. coli p21 has been shown by HPLC analysis on an ion-exchange column to contain near stoichiometric amounts of GDP. This noncovalently associated GDP is seen in the UV absorption spectrum of the purified protein. The noncovalently associated GDP acts as a competitive inhibitor of the interaction of added guanine nucleotides with p21.     

Photoaffinity labeling with GTP of viral p21 ras protein expressed in Escherichia coli

The v-ras oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton protein, p21, which mediates transformation produced by that virus. Previous work has shown that both p21v-rasH and the cellular homolog p21c-rasH appear to bind guanine nucleotides. We report here the expression in Escherichia coli of v-rasH to produce a biochemically active p21 fusion protein which retains both guanine nucleotide binding and autophosphorylating activity. Furthermore, direct interaction of this protein with GTP is unequivocally demonstrated by photoaffinity labeling it with [alpha-32P]GTP.     

Elevated levels of a specific class of nuclear phosphoproteins in cells transformed with v-ras and v-mos oncogenes and by cotransfection with c-myc and polyoma middle T genes

Transformation of a rat thyroid epithelial cell line (FRTL5-C12) with Kirsten and Harvey murine sarcoma viruses (carrying the ras oncogenes) results in elevated levels of three perchloric acid-soluble nuclear phosphoproteins. These three proteins are also induced to high levels in the PC-C13 thyroid epithelial cell line when transformed by the myeloproliferative sarcoma virus (carrying the v-mos oncogene) and when transformed by transfection with the c-myc proto-oncogene followed by infection with the polyoma leukaemia virus (PyMuLV) carry the polyoma middle T antigen gene. Neither c-myc or PyMuLV alone induced high levels of the three nuclear proteins. Untransformed thyroid fibroblasts have high levels of two of the three proteins and can be transformed by PyMuLV alone resulting in the appearance of the third protein. Transformation with Harvey sarcoma virus also results in the induction of the third protein. The three phosphoproteins have been purified by h.p.l.c. and shown to be related to the HeLa protein HMGI already described. The results of these studies indicate that elevated levels of these HMGI-like proteins are associated with neoplastic transformation and/or with an undifferentiated phenotype.     

Molecular cloning, genomic analysis, and biological properties of rat leukemia virus and the onc sequences of Rasheed rat sarcoma virus.

Rasheed rat sarcoma virus (RaSV) has been shown to code for a protein of 29,000 Mr not present in replication-competent rat type C helper virus (RaLV)-infected cells. This protein is a fused gene product consisting of a portion of the RaLV p15 gag protein and the transformation-specific 21,000 Mr (p21) ras protein, which is also found in Harvey murine sarcoma virus. We now report the molecular cloning of both the SD-1 (Sprague-Dawley) strain of RaLV and the transforming ras sequences of RaSV. Heteroduplex analysis of these cloned DNAs demonstrated that the RaSV ras gene (v-Ra-ras) was inserted into the rat type C viral genome with a small deletion of RaLV genetic information in the 5' region of the gag gene and that the v-Ra-ras gene (0.72 kilobase pair) is homologous to and colinear with the p21 ras gene of Harvey murine sarcoma virus (v-Ha-ras). Restriction enzyme mapping confirmed the homology demonstrated by heteroduplex mapping, showing strong site conservation of restriction endonucleases known to cleave v-Ha-ras. Cloned v-Ra-ras DNA transformed NIH 3T3 cells, inducing the synthesis of the p29 RaSVgag-ras protein.     

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.     

Comparative biochemical properties of p21 ras molecules coded for by viral and cellular ras genes.

In earlier studies, we molecularly cloned a normal cellular gene, c-rasH-1, homologous to the v-ras oncogene of Harvey murine sarcoma virus (v-rasH). By ligating a type c retroviral promotor to c-rasH-1, we could transform NIH 3T3 cells with the c-rasH-1 gene. The transformed cells contained high levels of a p21 protein coded for by the c-rasH-1 gene. In the current studies, we have purified extensively both v-rasH p21 and c-rasH p21 and compared the in vivo and in vitro biochemical properties of both these p21 molecules. The p21 proteins coded for by v-rasH and c-rasH-1 shared certain properties: each protein was synthesized as a precursor protein which subsequently became bound to the inner surface of the plasma membrane; each protein was associated with guanine nucleotide-binding activity, a property which copurified with p21 molecules on a high-pressure liquid chromatography molecular sizing column. In some other properties, the v-rasH and c-rasH p21 proteins differed. In vivo, approximately 20 to 30% of v-rasH p21 molecules were in the form of phosphothreonine-containing pp21 molecules, whereas in vivo only a minute fraction of c-rasH-1 p21 contained phosphate, and this phosphate was found on a serine residue. v-rasH pp21 molecules with an authentic phosphothreonine peptide could be synthesized in vitro in an autophosphorylation reaction in which the gamma phosphate of GTP was transferred to v-rasH p21. No autophosphorylating activity was associated with purified c-rasH-1 p21 in vitro. The results indicate a major qualitative difference between the p21 proteins coded for by v-rasH and c-rasH-1. The p21 coded for by a mouse-derived oncogenic virus, BALB murine sarcoma virus, resembled the p21 coded for by c-rasH-1 in that it bound guanine nucleotides but did not label appreciably with 32Pi. The forms of p21 coded for by other members of the ras gene family were compared, and the results indicate that the guanine nucleotide-binding activity is common to p21 molecules coded for by all known members of the ras gene family.     

Biologic and molecular characterization of two newly isolated ras-containing murine leukemia viruses.

A murine sarcoma virus (MSV) was recovered from an (NFS X NS.C58v-1) F1 mouse which developed splenic sarcoma and erythroleukemia 6 months after inoculation with a mink cell focus-inducing murine leukemia virus (MuLV) isolated from an NFS mouse infected with a wild mouse ecotropic MuLV. The MSV, designated NS.C58 MSV-1, induced foci of transformation in mouse and rat fibroblasts, and inoculation of mice of various strains 2 weeks of age or younger resulted in erythroleukemia and sarcomatous lesions in spleen, lymph node, and brain. The MSV provirus was molecularly cloned from a genomic library prepared from transformed non-producer rat cells. The 8.8-kilobase proviral DNA contained a 1.0-kilobase p21 ras coding segment which replaced most of the gp70-encoding portion of an MuLV, most likely the endogenous C58v-1 ecotropic virus. The ras oncogene is closely related to v-Ha-ras by hybridization, expression of p21 protein, and nucleotide sequence. It is nearly identical in sequence to v-bas, the only previously described transduced, activated mouse c-ras. At position 12 in the p21 coding region, arginine is substituted for the naturally occurring glycine present in c-ras. A second MSV isolate is described which is similar to NS.C58 MSV-1 except for a 100- to 200-base-pair deletion in the noncoding region of the ras-containing insert.     

Harvey sarcoma virus genome contains no extensive sequences unrelated to those of other retroviruses except ras.

The Harvey murine sarcoma virus genome contains two rat-derived sets of genetic information recombined with the Moloney mouse leukemia virus. The rat sequences represent a ras oncogene and a rat VL30 element. The VL30 sequences have several discrete regions of similarity with retroviral sequences which were detected by searching a protein database for similarities with predicted polypeptide sequences from the VL30 regions. On the 5' side, the most similar sequences were those of feline sarcoma viruses; on the 3' side, murine leukemia viruses were the most similar. Some of the regions of similarity could also be detected directly by searching a nucleic acid sequence database with the viral DNA sequences. The most extensive region of similarity was that which corresponded to the endonuclease in the pol gene of a murine leukemia virus. The majority of the rat-derived sequences present in the Harvey sarcoma virus genome can now be attributed exclusively to ras or retrovirus- or retrotransposon-related sequences.     

Development and analysis of a transformation-defective mutant of Harvey murine sarcoma tk virus and its gene product.

The Harvey murine sarcoma virus has been cloned and induces focus formation on NIH 3T3 cells. Recombinants of this virus have been constructed which include the thymidine kinase gene of herpes simplex virus type 1 in a downstream linkage with the p21 ras gene of Harvey murine sarcoma virus. Harvey murine sarcoma tk virus rescued from cells transfected with this construct is both thymidine kinase positive and focus inducing in in vitro transmission studies. The hypoxanthine-aminopterin-thymidine selectability of the thymidine kinase gene carried by this virus has been exploited to develop three mutants defective in the p21 ras sequence. All three are focus negative and thymidine kinase positive when transmitted to suitable cells. Of these, only one encodes a p22 that is immunologically related to p21. This mutant has been used to explore the relationship between the known characteristics of p21 and cellular transformation. Data presented herein indicate that the p21 of Harvey murine sarcoma virus consists of at least two domains, one which specifies the guanine nucleotide-binding activity of p21 and the other which is involved in p21-membrane association in transformed cells.     

Characterization of the phosphorylation sites and the surrounding amino acid sequences of the p21 transforming proteins coded for by the Harvey and Kirsten strains of murine sarcoma viruses

The transforming protein coded for by the onc gene (v-rasHa) of Harvey murine sarcoma virus (Ha-MuSV) is the 21,000-dalton protein (p21) which is the immediate agent responsible for the virus-induced malignant transformation of normal cells. The p21 proteins of Ha-MuSV and the closely related Kirsten murine sarcoma virus are heavily phosphorylated in vivo. In the partially purified Ha-MuSV p21, the protein shows a guanine nucleotide-binding activity and, in addition, a very unique autophosphorylating activity at a threonine residue using as phosphoryl donor GTP but not ATP. In the present study, we compared the tryptic peptide maps of the Ha-MuSV p21 phosphorylated in vivo and in vitro. The results show that the major phosphorylation site is identical. Since the GTP-specific phosphorylation is very unique and distinct from all other known protein kinases, the present observation suggests that the in vitro enzymatic activity is responsible for the p21 phosphorylation in vivo. We have analyzed the amino acid sequence surrounding the major phosphorylation site of the Ha-MuSV p21 by automated Edman degradations of the tryptic phosphopeptides. Threonine residue 59 from the initiator methionine residue 1 of the p21 protein is the phosphorylated amino acid residue, and the surrounding amino acid sequence is NH2...-Thr-Cys-Leu-Leu-Asp-Ile-Leu-Asp-Thr-Thr(P)-Gly-Gln-Glu-Glu-Tyr-...COOH. The p21 proteins of both the Ha-MuSV and the closely related Kirsten murine sarcoma virus share the same phosphopeptide. The amino acid sequence of the phosphorylation site is distinct from all other known protein kinases.     

Markedly elevated levels of an endogenous sarc protein in a hemopoietic precursor cell line.

The src gene product of Harvey murine sarcoma virus is a 21,000-dalton guanine nucleotide-binding protein. We have recently shown that a wide variety of vertebrate cell strains and cell lines express much lower levels of an endogenous p21 immunologically related to the Harvey murine sarcoma virus-coded p21. In this report, we have examined the levels of endogenous p21 in a unique hemopoietic precursor cell line, 416B, which was originally described as a continuous cell line of a hemopoietic stem cell, CFU-S. The currently available 416B cells express markedly elevated levels of endogenous p21. The level of endogenous p21 in the 416B cells is 5- to 10-fold higher than the level of p21 in Harvey murine sarcoma virus-infected cells and more than 100 times higher than the level of endogenous p21 that we have observed in a variety of other fresh or cultured cells. The results indicate that marked regulation of the levels of an endogenous sarc gene product can occur, and speculation about a possible role for endogenous p21 in normal hemopoietic stem cells is discussed.     

Nucleotide sequence of the two rat cellular rasH genes.

We present the nucleotide sequence of the coding region of the rat c-rasH-1 gene and a partial sequence analysis of the rat c-rasH-2 gene. By comparing these sequences with the Harvey murine sarcoma virus ras gene, we predict that the p21 protein encoded by the Harvey virus differs from the cellular c-rasH-1-encoded p21 at only two amino acids; those at positions 12 and 59. Alterations at each of these positions may play a role in activating the viral p21 protein. The c-rasH-2 gene is likely to be a nonfunctional pseudogene because it lacks introns, cannot be activated to transform NIH 3T3 cells, and differs in sequence from both c-rasH-1 and v-rasH at several base pair positions.     

Insulin stimulates the phosphorylation level of v-Ha-ras protein in membrane fraction

Insulin was found to stimulate the phosphorylation of the 21,000-dalton protein encoded by the ras oncogene of Harvey murine sarcoma virus in membrane fraction both in vivo and in vitro. When the human ras proteins expressed in E. coli were reconstituted with purified human insulin receptor, GTPase activity of normal or its mutated oncogenic ras protein was not stimulated by the addition of insulin. Likewise, tyrosine kinase activity or insulin binding capacity of the receptor was not influenced when assayed in the presence of the ras proteins. These results suggest that ras proteins may be coupled with the insulin receptor system through some unidentified membrane factors.     

Further genetic localization of the transforming sequences of the p21 v-ras gene of Harvey murine sarcoma virus.

The sequences encoding the 21-kilodalton transforming protein (p21 ras) of Harvey murine sarcoma virus have previously been localized genetically to a 1.3-kilobase segment of the viral DNA (E. H. Chang, R. W. Ellis, E. M. Scolnick, and D. R. Lowy, Science 210:1249-1251, 1980). Within this segment, DNA sequence analysis has found a single open reading frame large enough to encode the viral p21 (R. Dhar, R. W. Ellis, T. Y. Shih, S. Oroszlan, B. Shapiro, J. Maizel, D. Lowy, and E. M. Scolnick, Science 217:934-937, 1982). There are three potential in-frame ATG initiation codons at the 5' end of this open reading frame. By constructing a mutant of Harvey murine sarcoma virus DNA from which the first two ATG codons of this open reading frame have been deleted, we now show by transfection of the mutant viral DNA into NIH 3T3 cells that only the third ATG codon is necessary and sufficient for synthesis of the viral p21 and for cellular transformation.     

Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family.

We have isolated eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV). These antibodies specifically immunoprecipitate both phosphorylated and non-phosphorylated forms of p21 from lysates of cells transformed by Ha-MuSV. All eight react with the products of closely related ras genes expressed in cells transformed by two additional sarcoma viruses (rat sarcoma virus and BALB sarcoma virus) or by a cellular Harvey-ras gene placed under the control of a viral promoter. Three of the antibodies also react strongly with the p21 encoded by the v-ras gene of Kirsten MuSV. These same three antibodies immunoprecipitate the predominant p21 species synthesized normally in a variety of rodent cell lines, including the p21 produced at high levels in 416B murine hemopoietic cells. This suggests that an endogenous gene closely related to Kirsten-ras is expressed in these cells. The monoclonal antibodies have been used to confirm two properties associated with p21; localization at the inner surface of the membrane of Ha-MuSV-transformed cells, assayed by immunofluorescence microscopy, and binding of guanine nucleotides.     

An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors.

The genetic organization of the 5' genomic RNA domain of the highly oncogenic Harvey murine sarcoma virus appears to be unusual in that a multifunctional untranslated leader precedes the v-ras oncogene. This 5' leader is 1,076 nucleotides in length and is formed of independent regions involved in key steps of the viral life cycle: (i) the Moloney murine leukemia virus 5' repeat, untranslated 5' region, and primer binding site sequences necessary for the first steps of proviral DNA synthesis, (ii) the virus-like 30S (VL30)-derived sequence containing a functional dimerization-packaging signal (E/DLS) directing viral RNA dimerization and packaging into MLV virions, and (iii) an Alu-like sequence preceding the 5' untranslated sequence of v-rasH which contains the initiation codon of the p21ras oncoprotein. These functional features, the unusual length of this leader (1,076 nucleotides), and the presence of stable secondary structures between the cap and the v-ras initiation codon might well cause a premature stop of the scanning ribosomes and thus inhibit v-ras translation. In order to understand how Harvey murine sarcoma virus achieves a high level of expression of the ras oncogene, we asked whether the rat VL30 sequence, 5' to v-ras, could contribute to an efficient synthesis of the ras oncoprotein. The implications of the VL30 sequence in the translation initiation of Ha-ras were investigated in the rabbit reticulocyte lysate system and in murine cells. Results show that the rat VL30 sequence allows a cap-independent translation of a downstream reporter gene both in vitro and in murine cells. Additional experiments performed with dicistronic neo.VL30.lacZ mRNAs indicate that the 5' VL30 sequence (positions 380 to 794) contains an internal ribosomal entry signal. This finding led us to construct a new dicistronic retroviral vector with which the rat VL30 sequence was able to direct the efficient expression of a 3' cistron and packaging of recombinant dicistronic RNA into murine leukemia virus virions.     

Comparative analysis of p21 proteins from various cell types by two-dimensional gel electrophoresis

The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF-4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins.     

Nucleotide sequence analysis of the BALB/c murine sarcoma virus transforming gene.

We determined the nucleotide sequence of the v-H-ras-related oncogene of BALB/c murine sarcoma virus. This oncogene contains an open reading frame of 189 amino acids that initiates and terminates entirely within the mouse cell-derived ras sequence. The protein encoded by this open reading frame matches the sequence predicted for the T24 human bladder carcinoma oncogene product, p21, in all but two positions. The presence of a lysine residue in position 12 of BALB/c murine sarcoma virus p21 likely accounts for its oncogenic properties.     

Rat sarcoma virus: further analysis of individual viral isolates and the gene product.

Rasheed rat sarcoma virus, derived by in vitro cocultivation of two rat cell lines (Rasheed et al., Proc. Natl. Acad. Sci. U.S.A. 75:2972-2976, 1978), has been reported to code for a protein of 29,000 Mr, immunologically related to the 21,000 Mr src gene product of Harvey and Kirsten sarcoma viruses. Rat sarcoma virus p29 was thought to contain at least part of a rat type C virus structural protein, since antiserum prepared against whole rat virus was able to immunoprecipitate rat sarcoma virus p29 but not Harvey or Kirsten sarcoma virus p21 (Young et al., Proc. Natl. Acad. Sci. U.S.A. 76:3523-3527, 1979). We now report that antiserum directed against rat type C virus p15, but not viral p12, p10, or p27, immunoprecipitated rat sarcoma virus p29. The p15 antiserum was also able to immunoprecipitate both denatured p29 and a peptide derived by V-8 protease cleavage of p29, indicating that this antiserum contains antibodies directed against primary amino acid determinants. Finally, five separate isolates of rat sarcoma virus were found to code for p29, which indicates that a highly specific site of recombination is involved in the generation of sarcoma viruses in rat cells.