Abstract
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.
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Selected References
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- Bolanowski M. A., Earles B. J., Lennarz W. J. Fatty acylation of proteins during development of sea urchin embryos. J Biol Chem. 1984 Apr 25;259(8):4934–4940. [PubMed] [Google Scholar]
- Capon D. J., Chen E. Y., Levinson A. D., Seeburg P. H., Goeddel D. V. Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature. 1983 Mar 3;302(5903):33–37. doi: 10.1038/302033a0. [DOI] [PubMed] [Google Scholar]
- Courtneidge S. A., Levinson A. D., Bishop J. M. The protein encoded by the transforming gene of avian sarcoma virus (pp60src) and a homologous protein in normal cells (pp60proto-src) are associated with the plasma membrane. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3783–3787. doi: 10.1073/pnas.77.7.3783. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DeFeo D., Gonda M. A., Young H. A., Chang E. H., Lowy D. R., Scolnick E. M., Ellis R. W. Analysis of two divergent rat genomic clones homologous to the transforming gene of Harvey murine sarcoma virus. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3328–3332. doi: 10.1073/pnas.78.6.3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dhar R., Ellis R. W., Shih T. Y., Oroszlan S., Shapiro B., Maizel J., Lowy D., Scolnick E. Nucleotide sequence of the p21 transforming protein of Harvey murine sarcoma virus. Science. 1982 Sep 3;217(4563):934–936. doi: 10.1126/science.6287572. [DOI] [PubMed] [Google Scholar]
- Dhar R., Nieto A., Koller R., DeFeo-Jones D., Scolnick E. M. Nucleotide sequence of two rasH related-genes isolated from the yeast Saccharomyces cerevisiae. Nucleic Acids Res. 1984 Apr 25;12(8):3611–3618. doi: 10.1093/nar/12.8.3611. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Finkel T., Cooper G. M. Detection of a molecular complex between ras proteins and transferrin receptor. Cell. 1984 Apr;36(4):1115–1121. doi: 10.1016/0092-8674(84)90062-x. [DOI] [PubMed] [Google Scholar]
- Furth M. E., Davis L. J., Fleurdelys B., Scolnick E. M. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294–304. doi: 10.1128/jvi.43.1.294-304.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
- Hantke K., Braun V. Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the murein-lipoprotein of the Escherichia coli outer membrane. Eur J Biochem. 1973 Apr;34(2):284–296. doi: 10.1111/j.1432-1033.1973.tb02757.x. [DOI] [PubMed] [Google Scholar]
- Land H., Parada L. F., Weinberg R. A. Cellular oncogenes and multistep carcinogenesis. Science. 1983 Nov 18;222(4625):771–778. doi: 10.1126/science.6356358. [DOI] [PubMed] [Google Scholar]
- McGrath J. P., Capon D. J., Smith D. H., Chen E. Y., Seeburg P. H., Goeddel D. V., Levinson A. D. Structure and organization of the human Ki-ras proto-oncogene and a related processed pseudogene. Nature. 1983 Aug 11;304(5926):501–506. doi: 10.1038/304501a0. [DOI] [PubMed] [Google Scholar]
- Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
- Norris K., Norris F., Christiansen L., Fiil N. Efficient site-directed mutagenesis by simultaneous use of two primers. Nucleic Acids Res. 1983 Aug 11;11(15):5103–5112. doi: 10.1093/nar/11.15.5103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Papageorge A., Lowy D., Scolnick E. M. Comparative biochemical properties of p21 ras molecules coded for by viral and cellular ras genes. J Virol. 1982 Nov;44(2):509–519. doi: 10.1128/jvi.44.2.509-519.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Powers S., Kataoka T., Fasano O., Goldfarb M., Strathern J., Broach J., Wigler M. Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins. Cell. 1984 Mar;36(3):607–612. doi: 10.1016/0092-8674(84)90340-4. [DOI] [PubMed] [Google Scholar]
- Reddy E. P. Nucleotide sequence analysis of the T24 human bladder carcinoma oncogene. Science. 1983 Jun 3;220(4601):1061–1063. doi: 10.1126/science.6844927. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scolnick E. M., Papageorge A. G., Shih T. Y. Guanine nucleotide-binding activity as an assay for src protein of rat-derived murine sarcoma viruses. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5355–5359. doi: 10.1073/pnas.76.10.5355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sefton B. M., Trowbridge I. S., Cooper J. A., Scolnick E. M. The transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus contain tightly bound lipid. Cell. 1982 Dec;31(2 Pt 1):465–474. doi: 10.1016/0092-8674(82)90139-8. [DOI] [PubMed] [Google Scholar]
- Shih T. Y., Stokes P. E., Smythers G. W., Dhar R., Oroszlan S. 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. J Biol Chem. 1982 Oct 10;257(19):11767–11773. [PubMed] [Google Scholar]
- Shih T. Y., Weeks M. O., Gruss P., Dhar R., Oroszlan S., Scolnick E. M. Identification of a precursor in the biosynthesis of the p21 transforming protein of harvey murine sarcoma virus. J Virol. 1982 Apr;42(1):253–261. doi: 10.1128/jvi.42.1.253-261.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shimizu K., Birnbaum D., Ruley M. A., Fasano O., Suard Y., Edlund L., Taparowsky E., Goldfarb M., Wigler M. Structure of the Ki-ras gene of the human lung carcinoma cell line Calu-1. Nature. 1983 Aug 11;304(5926):497–500. doi: 10.1038/304497a0. [DOI] [PubMed] [Google Scholar]
- Taparowsky E., Shimizu K., Goldfarb M., Wigler M. Structure and activation of the human N-ras gene. Cell. 1983 Sep;34(2):581–586. doi: 10.1016/0092-8674(83)90390-2. [DOI] [PubMed] [Google Scholar]
- Tatchell K., Chaleff D. T., DeFeo-Jones D., Scolnick E. M. Requirement of either of a pair of ras-related genes of Saccharomyces cerevisiae for spore viability. Nature. 1984 Jun 7;309(5968):523–527. doi: 10.1038/309523a0. [DOI] [PubMed] [Google Scholar]
- Tsuchida N., Ryder T., Ohtsubo E. Nucleotide sequence of the oncogene encoding the p21 transforming protein of Kirsten murine sarcoma virus. Science. 1982 Sep 3;217(4563):937–939. doi: 10.1126/science.6287573. [DOI] [PubMed] [Google Scholar]
- Willingham M. C., Pastan I., Shih T. Y., Scolnick E. M. Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1980 Apr;19(4):1005–1014. doi: 10.1016/0092-8674(80)90091-4. [DOI] [PubMed] [Google Scholar]
- Willumsen B. M., Christensen A., Hubbert N. L., Papageorge A. G., Lowy D. R. The p21 ras C-terminus is required for transformation and membrane association. Nature. 1984 Aug 16;310(5978):583–586. doi: 10.1038/310583a0. [DOI] [PubMed] [Google Scholar]