Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Dec;82(24):8310–8314. doi: 10.1073/pnas.82.24.8310

Effects of phospholipids and ADP-ribosylation on GTP hydrolysis by Escherichia coli-synthesized Ha-ras-encoded p21.

S C Tsai, R Adamik, J Moss, M Vaughan, V Manne, H F Kung
PMCID: PMC390905  PMID: 3001695

Abstract

The Ha-ras protooncogene product p21, which may be involved in control of cellular growth, is a membrane protein that binds guanine nucleotides and hydrolyzes GTP. p21 GTPase activity is stimulated by lysophosphatidylcholine; a delay in activation was observed unless p21 was incubated with the phospholipid prior to assay. Maximal activation by the phospholipid was observed over a narrow concentration range; the presence in the assay mixture of lysophosphatidylcholine at concentrations above this optimum markedly inhibited p21 GTPase. GTP hydrolysis was also stimulated, but to a lesser degree, by phosphatidylcholine. Phosphatidylinositol and phosphatidylserine did not significantly enhance GTPase activity. The stimulatory effect of phospholipid was mimicked, in part, by nonionic detergents. p21 may be related to other GTPases, the regulatory guanine nucleotide-binding G proteins of the hormone-sensitive adenylate cyclase complex and transducin of the retinal light-activated phosphodiesterase system. The G proteins and transducin are heterotrimers; the alpha subunits possess GTPase activity and the beta gamma subunit complex along with agonist-receptor complex or light-activated rhodopsin enhance GTP hydrolysis. p21 GTPase activity was slightly stimulated by rhodopsin, but, in contrast to the GTPase activity of transducin, stimulation was not light-dependent. GTP hydrolysis was enhanced somewhat by beta gamma subunit complex in the absence, but not in the presence, of rhodopsin. Like the G proteins and transducin, activity of p21 was altered by ADP-ribosylation. Modification of p21 catalyzed by an NAD: arginine ADP-ribosyltransferase purified from turkey erythrocytes decreased both GTPase activity and guanine nucleotide binding activity.

Full text

PDF
8310

Images in this article

8313Image
on p.8313
8313Image
on p.8313

Selected References

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

  1. Abood M. E., Hurley J. B., Pappone M. C., Bourne H. R., Stryer L. Functional homology between signal-coupling proteins. Cholera toxin inactivates the GTPase activity of transducin. J Biol Chem. 1982 Sep 25;257(18):10540–10543. [PubMed] [Google Scholar]
  2. Asano T., Katada T., Gilman A. G., Ross E. M. Activation of the inhibitory GTP-binding protein of adenylate cyclase, Gi, by beta-adrenergic receptors in reconstituted phospholipid vesicles. J Biol Chem. 1984 Aug 10;259(15):9351–9354. [PubMed] [Google Scholar]
  3. Brandt D. R., Asano T., Pedersen S. E., Ross E. M. Reconstitution of catecholamine-stimulated guanosinetriphosphatase activity. Biochemistry. 1983 Sep 13;22(19):4357–4362. doi: 10.1021/bi00288a002. [DOI] [PubMed] [Google Scholar]
  4. Brandt D. R., Ross E. M. GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates. J Biol Chem. 1985 Jan 10;260(1):266–272. [PubMed] [Google Scholar]
  5. Burns D. L., Hewlett E. L., Moss J., Vaughan M. Pertussis toxin inhibits enkephalin stimulation of GTPase of NG108-15 cells. J Biol Chem. 1983 Feb 10;258(3):1435–1438. [PubMed] [Google Scholar]
  6. Burns D. L., Moss J., Vaughan M. Release of guanyl nucleotides from the regulatory subunit of adenylate cyclase. J Biol Chem. 1983 Jan 25;258(2):1116–1120. [PubMed] [Google Scholar]
  7. Cassel D., Pfeuffer T. Mechanism of cholera toxin action: covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2669–2673. doi: 10.1073/pnas.75.6.2669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cassel D., Selinger Z. Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes. Biochim Biophys Acta. 1976 Dec 8;452(2):538–551. doi: 10.1016/0005-2744(76)90206-0. [DOI] [PubMed] [Google Scholar]
  9. Cassel D., Selinger Z. Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3307–3311. doi: 10.1073/pnas.74.8.3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cassel D., Selinger Z. Mechanism of adenylate cyclase activation through the beta-adrenergic receptor: catecholamine-induced displacement of bound GDP by GTP. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4155–4159. doi: 10.1073/pnas.75.9.4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cerione R. A., Codina J., Benovic J. L., Lefkowitz R. J., Birnbaumer L., Caron M. G. The mammalian beta 2-adrenergic receptor: reconstitution of functional interactions between pure receptor and pure stimulatory nucleotide binding protein of the adenylate cyclase system. Biochemistry. 1984 Sep 25;23(20):4519–4525. doi: 10.1021/bi00315a003. [DOI] [PubMed] [Google Scholar]
  12. Cerione R. A., Staniszewski C., Benovic J. L., Lefkowitz R. J., Caron M. G., Gierschik P., Somers R., Spiegel A. M., Codina J., Birnbaumer L. Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. J Biol Chem. 1985 Feb 10;260(3):1493–1500. [PubMed] [Google Scholar]
  13. Feramisco J. R., Gross M., Kamata T., Rosenberg M., Sweet R. W. Microinjection of the oncogene form of the human H-ras (T-24) protein results in rapid proliferation of quiescent cells. Cell. 1984 Aug;38(1):109–117. doi: 10.1016/0092-8674(84)90531-2. [DOI] [PubMed] [Google Scholar]
  14. Fung B. K. Characterization of transducin from bovine retinal rod outer segments. I. Separation and reconstitution of the subunits. J Biol Chem. 1983 Sep 10;258(17):10495–10502. [PubMed] [Google Scholar]
  15. Fung B. K., Hurley J. B., Stryer L. Flow of information in the light-triggered cyclic nucleotide cascade of vision. Proc Natl Acad Sci U S A. 1981 Jan;78(1):152–156. doi: 10.1073/pnas.78.1.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gay N. J., Walker J. E. Homology between human bladder carcinoma oncogene product and mitochondrial ATP-synthase. Nature. 1983 Jan 20;301(5897):262–264. doi: 10.1038/301262a0. [DOI] [PubMed] [Google Scholar]
  17. Gibbs J. B., Sigal I. S., Poe M., Scolnick E. M. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5704–5708. doi: 10.1073/pnas.81.18.5704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gill D. M., Meren R. ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3050–3054. doi: 10.1073/pnas.75.7.3050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gilman A. G. G proteins and dual control of adenylate cyclase. Cell. 1984 Mar;36(3):577–579. doi: 10.1016/0092-8674(84)90336-2. [DOI] [PubMed] [Google Scholar]
  20. Gilman A. G. Guanine nucleotide-binding regulatory proteins and dual control of adenylate cyclase. J Clin Invest. 1984 Jan;73(1):1–4. doi: 10.1172/JCI111179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hildebrandt J. D., Codina J., Risinger R., Birnbaumer L. Identification of a gamma subunit associated with the adenylyl cyclase regulatory proteins Ns and Ni. J Biol Chem. 1984 Feb 25;259(4):2039–2042. [PubMed] [Google Scholar]
  22. Hsia J. A., Moss J., Hewlett E. L., Vaughan M. ADP-ribosylation of adenylate cyclase by pertussis toxin. Effects on inhibitory agonist binding. J Biol Chem. 1984 Jan 25;259(2):1086–1090. [PubMed] [Google Scholar]
  23. 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]
  24. Hurley J. B., Simon M. I., Teplow D. B., Robishaw J. D., Gilman A. G. Homologies between signal transducing G proteins and ras gene products. Science. 1984 Nov 16;226(4676):860–862. doi: 10.1126/science.6436980. [DOI] [PubMed] [Google Scholar]
  25. Johnson G. L., Kaslow H. R., Bourne H. R. Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase. J Biol Chem. 1978 Oct 25;253(20):7120–7123. [PubMed] [Google Scholar]
  26. Kanaho Y., Tsai S. C., Adamik R., Hewlett E. L., Moss J., Vaughan M. Rhodopsin-enhanced GTPase activity of the inhibitory GTP-binding protein of adenylate cyclase. J Biol Chem. 1984 Jun 25;259(12):7378–7381. [PubMed] [Google Scholar]
  27. Katada T., Ui M. ADP ribosylation of the specific membrane protein of C6 cells by islet-activating protein associated with modification of adenylate cyclase activity. J Biol Chem. 1982 Jun 25;257(12):7210–7216. [PubMed] [Google Scholar]
  28. Katada T., Ui M. Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. Proc Natl Acad Sci U S A. 1982 May;79(10):3129–3133. doi: 10.1073/pnas.79.10.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kataoka T., Powers S., Cameron S., Fasano O., Goldfarb M., Broach J., Wigler M. Functional homology of mammalian and yeast RAS genes. Cell. 1985 Jan;40(1):19–26. doi: 10.1016/0092-8674(85)90304-6. [DOI] [PubMed] [Google Scholar]
  30. Koski G., Klee W. A. Opiates inhibit adenylate cyclase by stimulating GTP hydrolysis. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4185–4189. doi: 10.1073/pnas.78.7.4185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kurose H., Katada T., Amano T., Ui M. Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha-adrenergic, cholinergic, and opiate receptors in neuroblastoma x glioma hybrid cells. J Biol Chem. 1983 Apr 25;258(8):4870–4875. [PubMed] [Google Scholar]
  32. Kwok-Keung Fung B., Stryer L. Photolyzed rhodopsin catalyzes the exchange of GTP for bound GDP in retinal rod outer segments. Proc Natl Acad Sci U S A. 1980 May;77(5):2500–2504. doi: 10.1073/pnas.77.5.2500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lacal J. C., Santos E., Notario V., Barbacid M., Yamazaki S., Kung H., Seamans C., McAndrew S., Crowl R. Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5305–5309. doi: 10.1073/pnas.81.17.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  35. Leberman R., Egner U. Homologies in the primary structure of GTP-binding proteins: the nucleotide-binding site of EF-Tu and p21. EMBO J. 1984 Feb;3(2):339–341. doi: 10.1002/j.1460-2075.1984.tb01808.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lefkowitz R. J., Caron M. G., Stiles G. L. Mechanisms of membrane-receptor regulation. Biochemical, physiological, and clinical insights derived from studies of the adrenergic receptors. N Engl J Med. 1984 Jun 14;310(24):1570–1579. doi: 10.1056/NEJM198406143102406. [DOI] [PubMed] [Google Scholar]
  37. Manne V., Bekesi E., Kung H. F. Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity. Proc Natl Acad Sci U S A. 1985 Jan;82(2):376–380. doi: 10.1073/pnas.82.2.376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Manne V., Yamazaki S., Kung H. F. Guanosine nucleotide binding by highly purified Ha-ras-encoded p21 protein produced in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6953–6957. doi: 10.1073/pnas.81.22.6953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Manning D. R., Fraser B. A., Kahn R. A., Gilman A. G. ADP-ribosylation of transducin by islet-activation protein. Identification of asparagine as the site of ADP-ribosylation. J Biol Chem. 1984 Jan 25;259(2):749–756. [PubMed] [Google Scholar]
  40. McGrath J. P., Capon D. J., Goeddel D. V., Levinson A. D. Comparative biochemical properties of normal and activated human ras p21 protein. Nature. 1984 Aug 23;310(5979):644–649. doi: 10.1038/310644a0. [DOI] [PubMed] [Google Scholar]
  41. Moss J., Burns D. L., Hsia J. A., Hewlett E. L., Guerrant R. L., Vaughan M. NIH conference. Cyclic nucleotides: mediators of bacterial toxin action in disease. Ann Intern Med. 1984 Nov;101(5):653–666. doi: 10.7326/0003-4819-101-5-653. [DOI] [PubMed] [Google Scholar]
  42. Moss J., Stanley S. J., Watkins P. A. Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes. J Biol Chem. 1980 Jun 25;255(12):5838–5840. [PubMed] [Google Scholar]
  43. Moss J., Vaughan M. Isolation of an avian erythrocyte protein possessing ADP-ribosyltransferase activity and capable of activating adenylate cyclase. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3621–3624. doi: 10.1073/pnas.75.8.3621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Murayama T., Ui M. Loss of the inhibitory function of the guanine nucleotide regulatory component of adenylate cyclase due to its ADP ribosylation by islet-activating protein, pertussis toxin, in adipocyte membranes. J Biol Chem. 1983 Mar 10;258(5):3319–3326. [PubMed] [Google Scholar]
  45. Murayama T., Ui M. [3H]GDP release from rat and hamster adipocyte membranes independently linked to receptors involved in activation or inhibition of adenylate cyclase. Differential susceptibility to two bacterial toxins. J Biol Chem. 1984 Jan 25;259(2):761–769. [PubMed] [Google Scholar]
  46. Navon S. E., Fung B. K. Characterization of transducin from bovine retinal rod outer segments. Mechanism and effects of cholera toxin-catalyzed ADP-ribosylation. J Biol Chem. 1984 May 25;259(10):6686–6693. [PubMed] [Google Scholar]
  47. Newbold R. Cancer: mutant ras proteins and cell transformation. Nature. 1984 Aug 23;310(5979):628–629. doi: 10.1038/310628a0. [DOI] [PubMed] [Google Scholar]
  48. Northup J. K., Sternweis P. C., Smigel M. D., Schleifer L. S., Ross E. M., Gilman A. G. Purification of the regulatory component of adenylate cyclase. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6516–6520. doi: 10.1073/pnas.77.11.6516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sunyer T., Codina J., Birnbaumer L. GTP hydrolysis by pure Ni, the inhibitory regulatory component of adenylyl cyclases. J Biol Chem. 1984 Dec 25;259(24):15447–15451. [PubMed] [Google Scholar]
  50. Sweet R. W., Yokoyama S., Kamata T., Feramisco J. R., Rosenberg M., Gross M. The product of ras is a GTPase and the T24 oncogenic mutant is deficient in this activity. Nature. 1984 Sep 20;311(5983):273–275. doi: 10.1038/311273a0. [DOI] [PubMed] [Google Scholar]
  51. Tamanoi F., Walsh M., Kataoka T., Wigler M. A product of yeast RAS2 gene is a guanine nucleotide binding protein. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6924–6928. doi: 10.1073/pnas.81.22.6924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Toda T., Uno I., Ishikawa T., Powers S., Kataoka T., Broek D., Cameron S., Broach J., Matsumoto K., Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985 Jan;40(1):27–36. doi: 10.1016/0092-8674(85)90305-8. [DOI] [PubMed] [Google Scholar]
  53. Tsai S. C., Adamik R., Kanaho Y., Hewlett E. L., Moss J. Effects of guanyl nucleotides and rhodopsin on ADP-ribosylation of the inhibitory GTP-binding component of adenylate cyclase by pertussis toxin. J Biol Chem. 1984 Dec 25;259(24):15320–15323. [PubMed] [Google Scholar]
  54. Van Dop C., Tsubokawa M., Bourne H. R., Ramachandran J. Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin. J Biol Chem. 1984 Jan 25;259(2):696–698. [PubMed] [Google Scholar]
  55. Van Dop C., Yamanaka G., Steinberg F., Sekura R. D., Manclark C. R., Stryer L., Bourne H. R. ADP-ribosylation of transducin by pertussis toxin blocks the light-stimulated hydrolysis of GTP and cGMP in retinal photoreceptors. J Biol Chem. 1984 Jan 10;259(1):23–26. [PubMed] [Google Scholar]
  56. Watkins P. A., Moss J., Burns D. L., Hewlett E. L., Vaughan M. Inhibition of bovine rod outer segment GTPase by Bordetella pertussis toxin. J Biol Chem. 1984 Feb 10;259(3):1378–1381. [PubMed] [Google Scholar]
  57. Wierenga R. K., Hol W. G. Predicted nucleotide-binding properties of p21 protein and its cancer-associated variant. Nature. 1983 Apr 28;302(5911):842–844. doi: 10.1038/302842a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES