Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Jun;16(6):2689–2699. doi: 10.1128/mcb.16.6.2689

Identification of a novel human Rho protein with unusual properties: GTPase deficiency and in vivo farnesylation.

R Foster 1, K Q Hu 1, Y Lu 1, K M Nolan 1, J Thissen 1, J Settleman 1
PMCID: PMC231259  PMID: 8649376

Abstract

We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.

Full Text

The Full Text of this article is available as a PDF (950.3 KB).

Selected References

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

  1. Adamson P., Marshall C. J., Hall A., Tilbrook P. A. Post-translational modifications of p21rho proteins. J Biol Chem. 1992 Oct 5;267(28):20033–20038. [PubMed] [Google Scholar]
  2. Aepfelbacher M. ADP-ribosylation of Rho enhances adhesion of U937 cells to fibronectin via the alpha 5 beta 1 integrin receptor. FEBS Lett. 1995 Apr 17;363(1-2):78–80. doi: 10.1016/0014-5793(95)00285-h. [DOI] [PubMed] [Google Scholar]
  3. Armstrong S. A., Hannah V. C., Goldstein J. L., Brown M. S. CAAX geranylgeranyl transferase transfers farnesyl as efficiently as geranylgeranyl to RhoB. J Biol Chem. 1995 Apr 7;270(14):7864–7868. doi: 10.1074/jbc.270.14.7864. [DOI] [PubMed] [Google Scholar]
  4. Barfod E. T., Zheng Y., Kuang W. J., Hart M. J., Evans T., Cerione R. A., Ashkenazi A. Cloning and expression of a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain. J Biol Chem. 1993 Dec 15;268(35):26059–26062. [PubMed] [Google Scholar]
  5. Bos J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989 Sep 1;49(17):4682–4689. [PubMed] [Google Scholar]
  6. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  7. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  8. Buss J. E., Quilliam L. A., Kato K., Casey P. J., Solski P. A., Wong G., Clark R., McCormick F., Bokoch G. M., Der C. J. The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein. Mol Cell Biol. 1991 Mar;11(3):1523–1530. doi: 10.1128/mcb.11.3.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Colby W. W., Hayflick J. S., Clark S. G., Levinson A. D. Biochemical characterization of polypeptides encoded by mutated human Ha-ras1 genes. Mol Cell Biol. 1986 Feb;6(2):730–734. doi: 10.1128/mcb.6.2.730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Der C. J., Finkel T., Cooper G. M. Biological and biochemical properties of human rasH genes mutated at codon 61. Cell. 1986 Jan 17;44(1):167–176. doi: 10.1016/0092-8674(86)90495-2. [DOI] [PubMed] [Google Scholar]
  11. Der C. J., Pan B. T., Cooper G. M. rasH mutants deficient in GTP binding. Mol Cell Biol. 1986 Sep;6(9):3291–3294. doi: 10.1128/mcb.6.9.3291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Feig L. A., Cooper G. M. Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins. Mol Cell Biol. 1988 Jun;8(6):2472–2478. doi: 10.1128/mcb.8.6.2472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Foster R., Hu K. Q., Shaywitz D. A., Settleman J. p190 RhoGAP, the major RasGAP-associated protein, binds GTP directly. Mol Cell Biol. 1994 Nov;14(11):7173–7181. doi: 10.1128/mcb.14.11.7173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Garrett M. D., Self A. J., van Oers C., Hall A. Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins. J Biol Chem. 1989 Jan 5;264(1):10–13. [PubMed] [Google Scholar]
  16. Glomset J. A., Farnsworth C. C. Role of protein modification reactions in programming interactions between ras-related GTPases and cell membranes. Annu Rev Cell Biol. 1994;10:181–205. doi: 10.1146/annurev.cb.10.110194.001145. [DOI] [PubMed] [Google Scholar]
  17. Gyuris J., Golemis E., Chertkov H., Brent R. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell. 1993 Nov 19;75(4):791–803. doi: 10.1016/0092-8674(93)90498-f. [DOI] [PubMed] [Google Scholar]
  18. Hall A., Self A. J. The effect of Mg2+ on the guanine nucleotide exchange rate of p21N-ras. J Biol Chem. 1986 Aug 25;261(24):10963–10965. [PubMed] [Google Scholar]
  19. Hall A. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu Rev Cell Biol. 1994;10:31–54. doi: 10.1146/annurev.cb.10.110194.000335. [DOI] [PubMed] [Google Scholar]
  20. Hancock J. F., Paterson H., Marshall C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990 Oct 5;63(1):133–139. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  21. Hart P. A., Marshall C. J. Amino acid 61 is a determinant of sensitivity of rap proteins to the ras GTPase activating protein. Oncogene. 1990 Jul;5(7):1099–1101. [PubMed] [Google Scholar]
  22. John J., Frech M., Wittinghofer A. Biochemical properties of Ha-ras encoded p21 mutants and mechanism of the autophosphorylation reaction. J Biol Chem. 1988 Aug 25;263(24):11792–11799. [PubMed] [Google Scholar]
  23. Jurnak F. Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science. 1985 Oct 4;230(4721):32–36. doi: 10.1126/science.3898365. [DOI] [PubMed] [Google Scholar]
  24. Kaziro Y., Itoh H., Kozasa T., Nakafuku M., Satoh T. Structure and function of signal-transducing GTP-binding proteins. Annu Rev Biochem. 1991;60:349–400. doi: 10.1146/annurev.bi.60.070191.002025. [DOI] [PubMed] [Google Scholar]
  25. Kinsella B. T., Erdman R. A., Maltese W. A. Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA. J Biol Chem. 1991 May 25;266(15):9786–9794. [PubMed] [Google Scholar]
  26. Lamarche N., Hall A. GAPs for rho-related GTPases. Trends Genet. 1994 Dec;10(12):436–440. doi: 10.1016/0168-9525(94)90114-7. [DOI] [PubMed] [Google Scholar]
  27. Lancaster C. A., Taylor-Harris P. M., Self A. J., Brill S., van Erp H. E., Hall A. Characterization of rhoGAP. A GTPase-activating protein for rho-related small GTPases. J Biol Chem. 1994 Jan 14;269(2):1137–1142. [PubMed] [Google Scholar]
  28. Landis C. A., Masters S. B., Spada A., Pace A. M., Bourne H. R., Vallar L. GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature. 1989 Aug 31;340(6236):692–696. doi: 10.1038/340692a0. [DOI] [PubMed] [Google Scholar]
  29. Lyons J., Landis C. A., Harsh G., Vallar L., Grünewald K., Feichtinger H., Duh Q. Y., Clark O. H., Kawasaki E., Bourne H. R. Two G protein oncogenes in human endocrine tumors. Science. 1990 Aug 10;249(4969):655–659. doi: 10.1126/science.2116665. [DOI] [PubMed] [Google Scholar]
  30. Maguire J., Santoro T., Jensen P., Siebenlist U., Yewdell J., Kelly K. Gem: an induced, immediate early protein belonging to the Ras family. Science. 1994 Jul 8;265(5169):241–244. doi: 10.1126/science.7912851. [DOI] [PubMed] [Google Scholar]
  31. McGlade J., Brunkhorst B., Anderson D., Mbamalu G., Settleman J., Dedhar S., Rozakis-Adcock M., Chen L. B., Pawson T. The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion. EMBO J. 1993 Aug;12(8):3073–3081. doi: 10.1002/j.1460-2075.1993.tb05976.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Moores S. L., Schaber M. D., Mosser S. D., Rands E., O'Hara M. B., Garsky V. M., Marshall M. S., Pompliano D. L., Gibbs J. B. Sequence dependence of protein isoprenylation. J Biol Chem. 1991 Aug 5;266(22):14603–14610. [PubMed] [Google Scholar]
  34. Nobes C. D., Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995 Apr 7;81(1):53–62. doi: 10.1016/0092-8674(95)90370-4. [DOI] [PubMed] [Google Scholar]
  35. Nobes C., Hall A. Regulation and function of the Rho subfamily of small GTPases. Curr Opin Genet Dev. 1994 Feb;4(1):77–81. doi: 10.1016/0959-437x(94)90094-9. [DOI] [PubMed] [Google Scholar]
  36. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Randazzo P. A., Kahn R. A. GTP hydrolysis by ADP-ribosylation factor is dependent on both an ADP-ribosylation factor GTPase-activating protein and acid phospholipids. J Biol Chem. 1994 Apr 8;269(14):10758–10763. [PubMed] [Google Scholar]
  38. Reiss Y., Stradley S. J., Gierasch L. M., Brown M. S., Goldstein J. L. Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):732–736. doi: 10.1073/pnas.88.3.732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reynet C., Kahn C. R. Rad: a member of the Ras family overexpressed in muscle of type II diabetic humans. Science. 1993 Nov 26;262(5138):1441–1444. doi: 10.1126/science.8248782. [DOI] [PubMed] [Google Scholar]
  40. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  41. Satoh T., Nakafuku M., Kaziro Y. Function of Ras as a molecular switch in signal transduction. J Biol Chem. 1992 Dec 5;267(34):24149–24152. [PubMed] [Google Scholar]
  42. Seabra M. C., Reiss Y., Casey P. J., Brown M. S., Goldstein J. L. Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit. Cell. 1991 May 3;65(3):429–434. doi: 10.1016/0092-8674(91)90460-g. [DOI] [PubMed] [Google Scholar]
  43. Settleman J., Albright C. F., Foster L. C., Weinberg R. A. Association between GTPase activators for Rho and Ras families. Nature. 1992 Sep 10;359(6391):153–154. doi: 10.1038/359153a0. [DOI] [PubMed] [Google Scholar]
  44. Settleman J., Narasimhan V., Foster L. C., Weinberg R. A. Molecular cloning of cDNAs encoding the GAP-associated protein p190: implications for a signaling pathway from ras to the nucleus. Cell. 1992 May 1;69(3):539–549. doi: 10.1016/0092-8674(92)90454-k. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Takaishi K., Sasaki T., Kameyama T., Tsukita S., Tsukita S., Takai Y. Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows. Oncogene. 1995 Jul 6;11(1):39–48. [PubMed] [Google Scholar]
  47. Trahey M., Wong G., Halenbeck R., Rubinfeld B., Martin G. A., Ladner M., Long C. M., Crosier W. J., Watt K., Koths K. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. doi: 10.1126/science.3201259. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Xu X., Barry D. C., Settleman J., Schwartz M. A., Bokoch G. M. Differing structural requirements for GTPase-activating protein responsiveness and NADPH oxidase activation by Rac. J Biol Chem. 1994 Sep 23;269(38):23569–23574. [PubMed] [Google Scholar]
  50. Yasuda S., Furuichi M., Soeda E. An altered DNA sequence encompassing the ras gene of Harvey murine sarcoma virus. Nucleic Acids Res. 1984 Jul 25;12(14):5583–5588. doi: 10.1093/nar/12.14.5583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zheng Y., Bagrodia S., Cerione R. A. Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. J Biol Chem. 1994 Jul 22;269(29):18727–18730. [PubMed] [Google Scholar]
  52. Zheng Y., Hart M. J., Shinjo K., Evans T., Bender A., Cerione R. A. Biochemical comparisons of the Saccharomyces cerevisiae Bem2 and Bem3 proteins. Delineation of a limit Cdc42 GTPase-activating protein domain. J Biol Chem. 1993 Nov 25;268(33):24629–24634. [PubMed] [Google Scholar]
  53. Zhu J., Reynet C., Caldwell J. S., Kahn C. R. Characterization of Rad, a new member of Ras/GTPase superfamily, and its regulation by a unique GTPase-activating protein (GAP)-like activity. J Biol Chem. 1995 Mar 3;270(9):4805–4812. doi: 10.1074/jbc.270.9.4805. [DOI] [PubMed] [Google Scholar]
  54. Ziman M., Preuss D., Mulholland J., O'Brien J. M., Botstein D., Johnson D. I. Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity. Mol Biol Cell. 1993 Dec;4(12):1307–1316. doi: 10.1091/mbc.4.12.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES