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. 1990 Nov;87(22):8960–8964. doi: 10.1073/pnas.87.22.8960

Posttranslationally processed structure of the human platelet protein smg p21B: evidence for geranylgeranylation and carboxyl methylation of the C-terminal cysteine.

M Kawata 1, C C Farnsworth 1, Y Yoshida 1, M H Gelb 1, J A Glomset 1, Y Takai 1
PMCID: PMC55080  PMID: 2123345

Abstract

smg p21A and -B are small GTP-binding proteins that share putative effector and consensus C-terminal sequences with ras p21 proteins. In the present report, we showed that human platelet smg p21B became labeled when intact platelets were incubated with exogenous [3H]mevalonolactone and when a purified preparation of smg p21B was incubated with bovine brain membranes and S-adenosyl-L-[methyl-3H]methionine. In addition, we demonstrated by gas chromatography/mass spectrometry that treatment of smg p21B with Raney nickel released a geranylgeranyl moiety in a molar ratio of about 1:1. In contrast, treatment of smg p21B with NH2OH or KOH yielded no evidence for the presence of a palmitoyl thioester. Extensive digestion of smg p21B with Achromobacter protease I yielded two C-terminal tripeptides that contained serine and cysteine in a molar ratio of 2:1. Both peptides were modified by a thioether-linked geranylgeranyl group. One of the peptides comigrated with a 3H-labeled proteolytic product of methylated smg p21B on reverse-phase HPLC and this peptide appeared at the same retention time as that of the other peptide after being treated with KOH. Since the cDNA-predicted C-terminal sequence of smg p21B contains a unique Ser-Ser-Cys peptide within its C-terminal domain, -Lys-Lys-Ser-Ser-Cys-Gln-Leu-Leu184, these results indicate that smg p21B is posttranslationally modified by geranylgeranylation of Cys-181 and suggest that further modifications cause proteolytic removal of the three predicted C-terminal amino acids followed by partial methylation of the cysteinyl carboxyl group.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderegg R. J., Betz R., Carr S. A., Crabb J. W., Duntze W. Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component. J Biol Chem. 1988 Dec 5;263(34):18236–18240. [PubMed] [Google Scholar]
  2. Baenziger N. L., Majerus P. W. Isolation of human platelets and platelet surface membranes. Methods Enzymol. 1974;31:149–155. doi: 10.1016/0076-6879(74)31015-4. [DOI] [PubMed] [Google Scholar]
  3. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Casey P. J., Solski P. A., Der C. J., Buss J. E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8323–8327. doi: 10.1073/pnas.86.21.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clarke S., Vogel J. P., Deschenes R. J., Stock J. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4643–4647. doi: 10.1073/pnas.85.13.4643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Farnsworth C. C., Wolda S. L., Gelb M. H., Glomset J. A. Human lamin B contains a farnesylated cysteine residue. J Biol Chem. 1989 Dec 5;264(34):20422–20429. [PMC free article] [PubMed] [Google Scholar]
  8. Frech M., John J., Pizon V., Chardin P., Tavitian A., Clark R., McCormick F., Wittinghofer A. Inhibition of GTPase activating protein stimulation of Ras-p21 GTPase by the Krev-1 gene product. Science. 1990 Jul 13;249(4965):169–171. doi: 10.1126/science.2164710. [DOI] [PubMed] [Google Scholar]
  9. Fujiyama A., Tamanoi F. RAS2 protein of Saccharomyces cerevisiae undergoes removal of methionine at N terminus and removal of three amino acids at C terminus. J Biol Chem. 1990 Feb 25;265(6):3362–3368. [PubMed] [Google Scholar]
  10. Fukada Y., Takao T., Ohguro H., Yoshizawa T., Akino T., Shimonishi Y. Farnesylated gamma-subunit of photoreceptor G protein indispensable for GTP-binding. Nature. 1990 Aug 16;346(6285):658–660. doi: 10.1038/346658a0. [DOI] [PubMed] [Google Scholar]
  11. Gautam N., Baetscher M., Aebersold R., Simon M. I. A G protein gamma subunit shares homology with ras proteins. Science. 1989 May 26;244(4907):971–974. doi: 10.1126/science.2499046. [DOI] [PubMed] [Google Scholar]
  12. Glomset J. A., Gelb M. H., Farnsworth C. C. Prenyl proteins in eukaryotic cells: a new type of membrane anchor. Trends Biochem Sci. 1990 Apr;15(4):139–142. doi: 10.1016/0968-0004(90)90213-u. [DOI] [PubMed] [Google Scholar]
  13. Gutierrez L., Magee A. I., Marshall C. J., Hancock J. F. Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J. 1989 Apr;8(4):1093–1098. doi: 10.1002/j.1460-2075.1989.tb03478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  15. Hata Y., Kikuchi A., Sasaki T., Schaber M. D., Gibbs J. B., Takai Y. Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s. J Biol Chem. 1990 May 5;265(13):7104–7107. [PubMed] [Google Scholar]
  16. Hoshijima M., Kikuchi A., Kawata M., Ohmori T., Hashimoto E., Yamamura H., Takai Y. Phosphorylation by cyclic AMP-dependent protein kinase of a human platelet Mr 22,000 GTP-binding protein (smg p21) having the same putative effector domain as the ras gene products. Biochem Biophys Res Commun. 1988 Dec 30;157(3):851–860. doi: 10.1016/s0006-291x(88)80953-7. [DOI] [PubMed] [Google Scholar]
  17. Hurley J. B., Fong H. K., Teplow D. B., Dreyer W. J., Simon M. I. Isolation and characterization of a cDNA clone for the gamma subunit of bovine retinal transducin. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6948–6952. doi: 10.1073/pnas.81.22.6948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jackson J. H., Cochrane C. G., Bourne J. R., Solski P. A., Buss J. E., Der C. J. Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3042–3046. doi: 10.1073/pnas.87.8.3042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kawata M., Kawahara Y., Araki S., Sunako M., Tsuda T., Fukuzaki H., Mizoguchi A., Takai Y. Identification of a major GTP-binding protein in bovine aortic smooth muscle membranes as smg p21, a GTP-binding protein having the same effector domain as ras p21s. Biochem Biophys Res Commun. 1989 Sep 29;163(3):1418–1427. doi: 10.1016/0006-291x(89)91137-6. [DOI] [PubMed] [Google Scholar]
  20. Kawata M., Kikuchi A., Hoshijima M., Yamamoto K., Hashimoto E., Yamamura H., Takai Y. Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets. J Biol Chem. 1989 Sep 15;264(26):15688–15695. [PubMed] [Google Scholar]
  21. Kawata M., Matsui Y., Kondo J., Hishida T., Teranishi Y., Takai Y. A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. Purification, determination of primary structure, and characterization. J Biol Chem. 1988 Dec 15;263(35):18965–18971. [PubMed] [Google Scholar]
  22. Kikuchi A., Sasaki T., Araki S., Hata Y., Takai Y. Purification and characterization from bovine brain cytosol of two GTPase-activating proteins specific for smg p21, a GTP-binding protein having the same effector domain as c-ras p21s. J Biol Chem. 1989 Jun 5;264(16):9133–9136. [PubMed] [Google Scholar]
  23. Kim S., Mizoguchi A., Kikuchi A., Takai Y. Tissue and subcellular distributions of the smg-21/rap1/Krev-1 proteins which are partly distinct from those of c-ras p21s. Mol Cell Biol. 1990 Jun;10(6):2645–2652. doi: 10.1128/mcb.10.6.2645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kitayama H., Sugimoto Y., Matsuzaki T., Ikawa Y., Noda M. A ras-related gene with transformation suppressor activity. Cell. 1989 Jan 13;56(1):77–84. doi: 10.1016/0092-8674(89)90985-9. [DOI] [PubMed] [Google Scholar]
  25. Matsui Y., Kikuchi A., Kawata M., Kondo J., Teranishi Y., Takai Y. Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B. Biochem Biophys Res Commun. 1990 Jan 30;166(2):1010–1016. doi: 10.1016/0006-291x(90)90911-6. [DOI] [PubMed] [Google Scholar]
  26. McCormick F. ras GTPase activating protein: signal transmitter and signal terminator. Cell. 1989 Jan 13;56(1):5–8. doi: 10.1016/0092-8674(89)90976-8. [DOI] [PubMed] [Google Scholar]
  27. Mumby S. M., Casey P. J., Gilman A. G., Gutowski S., Sternweis P. C. G protein gamma subunits contain a 20-carbon isoprenoid. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5873–5877. doi: 10.1073/pnas.87.15.5873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ohmori T., Kikuchi A., Yamamoto K., Kawata M., Kondo J., Takai Y. Identification of a platelet Mr 22,000 GTP-binding protein as the novel smg-21 gene product having the same putative effector domain as the ras gene products. Biochem Biophys Res Commun. 1988 Dec 15;157(2):670–676. doi: 10.1016/s0006-291x(88)80302-4. [DOI] [PubMed] [Google Scholar]
  29. Ohmori T., Kikuchi A., Yamamoto K., Kim S., Takai Y. Small molecular weight GTP-binding proteins in human platelet membranes. Purification and characterization of a novel GTP-binding protein with a molecular weight of 22,000. J Biol Chem. 1989 Jan 25;264(3):1877–1881. [PubMed] [Google Scholar]
  30. Pizon V., Chardin P., Lerosey I., Olofsson B., Tavitian A. Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the 'effector' region. Oncogene. 1988 Aug;3(2):201–204. [PubMed] [Google Scholar]
  31. Pizon V., Lerosey I., Chardin P., Tavitian A. Nucleotide sequence of a human cDNA encoding a ras-related protein (rap1B). Nucleic Acids Res. 1988 Aug 11;16(15):7719–7719. doi: 10.1093/nar/16.15.7719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pollard K. M., Chan E. K., Grant B. J., Sullivan K. F., Tan E. M., Glass C. A. In vitro posttranslational modification of lamin B cloned from a human T-cell line. Mol Cell Biol. 1990 May;10(5):2164–2175. doi: 10.1128/mcb.10.5.2164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Reiss Y., Goldstein J. L., Seabra M. C., Casey P. J., Brown M. S. Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides. Cell. 1990 Jul 13;62(1):81–88. doi: 10.1016/0092-8674(90)90242-7. [DOI] [PubMed] [Google Scholar]
  34. Sakagami Y., Yoshida M., Isogai A., Suzuki A. Peptidal Sex Hormones Inducing Conjugation Tube Formation in Compatible Mating-Type Cells of Tremella mesenterica. Science. 1981 Jun 26;212(4502):1525–1527. doi: 10.1126/science.212.4502.1525. [DOI] [PubMed] [Google Scholar]
  35. Ueda T., Kikuchi A., Ohga N., Yamamoto J., Takai Y. GTPase activating proteins for the smg-21 GTP-binding protein having the same effector domain as the ras proteins in human platelets. Biochem Biophys Res Commun. 1989 Mar 31;159(3):1411–1419. doi: 10.1016/0006-291x(89)92267-5. [DOI] [PubMed] [Google Scholar]
  36. Yamane H. K., Farnsworth C. C., Xie H. Y., Howald W., Fung B. K., Clarke S., Gelb M. H., Glomset J. A. Brain G protein gamma subunits contain an all-trans-geranylgeranylcysteine methyl ester at their carboxyl termini. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5868–5872. doi: 10.1073/pnas.87.15.5868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yamane H. K., Fung B. K. The membrane-binding domain of a 23-kDa G-protein is carboxyl methylated. J Biol Chem. 1989 Nov 25;264(33):20100–20105. [PubMed] [Google Scholar]

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