Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jan 15;361(Pt 2):243–254. doi: 10.1042/0264-6021:3610243

Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes.

Marie-Annick Forget 1, Richard R Desrosiers 1, Denis Gingras 1, Richard Béliveau 1
PMCID: PMC1222304  PMID: 11772396

Abstract

The Rho GDP dissociation inhibitor (RhoGDI) regulates the activation-inactivation cycle of Rho small GTPases, such as Cdc42 and RhoA, by extracting them from the membrane. To study the roles of Mg(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), ionic strength and phosphorylation on the interactions of RhoGDI with Cdc42 and RhoA, we developed a new, efficient and reliable method to produce prenylated Rho proteins using the yeast Saccharomyces cerevisiae. It has been previously reported that protein kinase A (PKA)-treatment of isolated membranes increased RhoA extraction from membranes by RhoGDI [Lang, Gesbert, Delespine-Carmagnat, Stancou, Pouchelet and Bertoglio (1996) EMBO J. 16, 510-519]. In the present study, we used an in vitro affinity chromatography system to show that phosphorylation of RhoA and Cdc42 significantly increased their interaction with RhoGDI under physiological conditions of ionic strength. This increase was independent of the nucleotide (GDP or guanosine 5'-[gamma-thio]triphosphate) loaded on to the Rho proteins, as well as of Mg(2+) and PIP(2). Moreover, dephosphorylation of rat brain membranes by alkaline phosphatase significantly decreased the extraction of RhoA and Cdc42 by RhoGDI. Subsequent re-phosphorylation by PKA restored the extraction levels, indicating the reversibility of this process. These results clearly demonstrate that the phosphorylation states of Cdc42 and RhoA regulate their interactions with RhoGDI and, consequently, their extraction from rat brain membranes. We therefore suggest that phosphorylation is a mechanism of regulation of Cdc42 and RhoA activity that is independent of GDP-GTP cycling.

Full Text

The Full Text of this article is available as a PDF (258.9 KB).

Selected References

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

  1. Allal C., Favre G., Couderc B., Salicio S., Sixou S., Hamilton A. D., Sebti S. M., Lajoie-Mazenc I., Pradines A. RhoA prenylation is required for promotion of cell growth and transformation and cytoskeleton organization but not for induction of serum response element transcription. J Biol Chem. 2000 Oct 6;275(40):31001–31008. doi: 10.1074/jbc.M005264200. [DOI] [PubMed] [Google Scholar]
  2. Backlund P. S., Jr Carboxyl methylation of the low molecular weight GTP-binding protein G25K: regulation of carboxyl methylation by rhoGDI. Biochem Biophys Res Commun. 1993 Oct 29;196(2):534–542. doi: 10.1006/bbrc.1993.2283. [DOI] [PubMed] [Google Scholar]
  3. Bailly E., McCaffrey M., Touchot N., Zahraoui A., Goud B., Bornens M. Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2. Nature. 1991 Apr 25;350(6320):715–718. doi: 10.1038/350715a0. [DOI] [PubMed] [Google Scholar]
  4. Bilodeau D., Lamy S., Desrosiers R. R., Gingras D., Béliveau R. Regulation of Rho protein binding to membranes by rhoGDI: inhibition of releasing activity by physiological ionic conditions. Biochem Cell Biol. 1999;77(1):59–69. [PubMed] [Google Scholar]
  5. Bokoch G. M. Regulation of cell function by Rho family GTPases. Immunol Res. 2000;21(2-3):139–148. doi: 10.1385/IR:21:2-3:139. [DOI] [PubMed] [Google Scholar]
  6. Bourmeyster N., Vignais P. V. Phosphorylation of Rho GDI stabilizes the Rho A-Rho GDI complex in neutrophil cytosol. Biochem Biophys Res Commun. 1996 Jan 5;218(1):54–60. doi: 10.1006/bbrc.1996.0011. [DOI] [PubMed] [Google Scholar]
  7. Chardin P. Rnd proteins: a new family of Rho-related proteins that interfere with the assembly of filamentous actin structures and cell adhesion. Prog Mol Subcell Biol. 1999;22:39–50. doi: 10.1007/978-3-642-58591-3_3. [DOI] [PubMed] [Google Scholar]
  8. Chuang T. H., Bohl B. P., Bokoch G. M. Biologically active lipids are regulators of Rac.GDI complexation. J Biol Chem. 1993 Dec 15;268(35):26206–26211. [PubMed] [Google Scholar]
  9. Dong J. M., Leung T., Manser E., Lim L. cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKalpha. J Biol Chem. 1998 Aug 28;273(35):22554–22562. doi: 10.1074/jbc.273.35.22554. [DOI] [PubMed] [Google Scholar]
  10. Fauré J., Vignais P. V., Dagher M. C. Phosphoinositide-dependent activation of Rho A involves partial opening of the RhoA/Rho-GDI complex. Eur J Biochem. 1999 Jun;262(3):879–889. doi: 10.1046/j.1432-1327.1999.00458.x. [DOI] [PubMed] [Google Scholar]
  11. Fritz G., Lang P., Just I. Tissue-specific variations in the expression and regulation of the small GTP-binding protein Rho. Biochim Biophys Acta. 1994 Jul 21;1222(3):331–338. doi: 10.1016/0167-4889(94)90038-8. [DOI] [PubMed] [Google Scholar]
  12. Fukumoto Y., Kaibuchi K., Hori Y., Fujioka H., Araki S., Ueda T., Kikuchi A., Takai Y. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins. Oncogene. 1990 Sep;5(9):1321–1328. [PubMed] [Google Scholar]
  13. Gerner C., Frohwein U., Gotzmann J., Bayer E., Gelbmann D., Bursch W., Schulte-Hermann R. The Fas-induced apoptosis analyzed by high throughput proteome analysis. J Biol Chem. 2000 Dec 15;275(50):39018–39026. doi: 10.1074/jbc.M006495200. [DOI] [PubMed] [Google Scholar]
  14. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gosser Y. Q., Nomanbhoy T. K., Aghazadeh B., Manor D., Combs C., Cerione R. A., Rosen M. K. C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases. Nature. 1997 Jun 19;387(6635):814–819. doi: 10.1038/42961. [DOI] [PubMed] [Google Scholar]
  16. Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998 Jan 23;279(5350):509–514. doi: 10.1126/science.279.5350.509. [DOI] [PubMed] [Google Scholar]
  17. Hancock J. F., Cadwallader K., Marshall C. J. Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras(B). EMBO J. 1991 Mar;10(3):641–646. doi: 10.1002/j.1460-2075.1991.tb07992.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hancock J. F., Cadwallader K., Paterson H., Marshall C. J. A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. EMBO J. 1991 Dec;10(13):4033–4039. doi: 10.1002/j.1460-2075.1991.tb04979.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hata Y., Kaibuchi K., Kawamura S., Hiroyoshi M., Shirataki H., Takai Y. Enhancement of the actions of smg p21 GDP/GTP exchange protein by the protein kinase A-catalyzed phosphorylation of smg p21. J Biol Chem. 1991 Apr 5;266(10):6571–6577. [PubMed] [Google Scholar]
  21. Hori Y., Kikuchi A., Isomura M., Katayama M., Miura Y., Fujioka H., Kaibuchi K., Takai Y. Post-translational modifications of the C-terminal region of the rho protein are important for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins. Oncogene. 1991 Apr;6(4):515–522. [PubMed] [Google Scholar]
  22. Kwak J. Y., Uhlinger D. J. Downregulation of phospholipase D by protein kinase A in a cell-free system of human neutrophils. Biochem Biophys Res Commun. 2000 Jan 7;267(1):305–310. doi: 10.1006/bbrc.1999.1941. [DOI] [PubMed] [Google Scholar]
  23. Kwon T., Kwon D. Y., Chun J., Kim J. H., Kang S. S. Akt protein kinase inhibits Rac1-GTP binding through phosphorylation at serine 71 of Rac1. J Biol Chem. 2000 Jan 7;275(1):423–428. doi: 10.1074/jbc.275.1.423. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Lang P., Gesbert F., Delespine-Carmagnat M., Stancou R., Pouchelet M., Bertoglio J. Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes. EMBO J. 1996 Feb 1;15(3):510–519. [PMC free article] [PubMed] [Google Scholar]
  26. Lapetina E. G., Lacal J. C., Reep B. R., Molina y Vedia L. A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets. Proc Natl Acad Sci U S A. 1989 May;86(9):3131–3134. doi: 10.1073/pnas.86.9.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laudanna C., Campbell J. J., Butcher E. C. Elevation of intracellular cAMP inhibits RhoA activation and integrin-dependent leukocyte adhesion induced by chemoattractants. J Biol Chem. 1997 Sep 26;272(39):24141–24144. doi: 10.1074/jbc.272.39.24141. [DOI] [PubMed] [Google Scholar]
  28. Leonard D. A., Cerione R. A. Solubilization of Cdc42Hs from membranes by Rho-GDP dissociation inhibitor. Methods Enzymol. 1995;256:98–105. doi: 10.1016/0076-6879(95)56014-9. [DOI] [PubMed] [Google Scholar]
  29. Leonard D., Hart M. J., Platko J. V., Eva A., Henzel W., Evans T., Cerione R. A. The identification and characterization of a GDP-dissociation inhibitor (GDI) for the CDC42Hs protein. J Biol Chem. 1992 Nov 15;267(32):22860–22868. [PubMed] [Google Scholar]
  30. Longenecker K., Read P., Derewenda U., Dauter Z., Liu X., Garrard S., Walker L., Somlyo A. V., Nakamoto R. K., Somlyo A. P. How RhoGDI binds Rho. Acta Crystallogr D Biol Crystallogr. 1999 Sep;55(Pt 9):1503–1515. doi: 10.1107/s090744499900801x. [DOI] [PubMed] [Google Scholar]
  31. Martin M. E., Hidalgo J., Rosa J. L., Crottet P., Velasco A. Effect of protein kinase A activity on the association of ADP-ribosylation factor 1 to golgi membranes. J Biol Chem. 2000 Jun 23;275(25):19050–19059. doi: 10.1074/jbc.275.25.19050. [DOI] [PubMed] [Google Scholar]
  32. Michaelson D., Silletti J., Murphy G., D'Eustachio P., Rush M., Philips M. R. Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding. J Cell Biol. 2001 Jan 8;152(1):111–126. doi: 10.1083/jcb.152.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Newcombe A. R., Stockley R. W., Hunter J. L., Webb M. R. The interaction between rac1 and its guanine nucleotide dissociation inhibitor (GDI), monitored by a single fluorescent coumarin attached to GDI. Biochemistry. 1999 May 25;38(21):6879–6886. doi: 10.1021/bi9829837. [DOI] [PubMed] [Google Scholar]
  34. Nomanbhoy T. K., Cerione R. Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy. J Biol Chem. 1996 Apr 26;271(17):10004–10009. doi: 10.1074/jbc.271.17.10004. [DOI] [PubMed] [Google Scholar]
  35. Olofsson B. Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling. Cell Signal. 1999 Aug;11(8):545–554. doi: 10.1016/s0898-6568(98)00063-1. [DOI] [PubMed] [Google Scholar]
  36. Sadra A., Cinek T., Imboden J. B. Multiple probing of an immunoblot membrane using a non-block technique: advantages in speed and sensitivity. Anal Biochem. 2000 Feb 15;278(2):235–237. doi: 10.1006/abio.1999.4453. [DOI] [PubMed] [Google Scholar]
  37. Saikumar P., Ulsh L. S., Clanton D. J., Huang K. P., Shih T. Y. Novel phosphorylation of c-ras p21 by protein kinases. Oncogene Res. 1988;3(3):213–222. [PubMed] [Google Scholar]
  38. Sasaki T., Kato M., Nishiyama T., Takai Y. The nucleotide exchange rates of rho and rac small GTP-binding proteins are enhanced to different extents by their regulatory protein Smg GDS. Biochem Biophys Res Commun. 1993 Aug 16;194(3):1188–1193. doi: 10.1006/bbrc.1993.1948. [DOI] [PubMed] [Google Scholar]
  39. Sasaki T., Kato M., Takai Y. Consequences of weak interaction of rho GDI with the GTP-bound forms of rho p21 and rac p21. J Biol Chem. 1993 Nov 15;268(32):23959–23963. [PubMed] [Google Scholar]
  40. Sasaki T., Takai Y. The Rho small G protein family-Rho GDI system as a temporal and spatial determinant for cytoskeletal control. Biochem Biophys Res Commun. 1998 Apr 28;245(3):641–645. doi: 10.1006/bbrc.1998.8253. [DOI] [PubMed] [Google Scholar]
  41. Sauzeau V., Le Jeune H., Cario-Toumaniantz C., Smolenski A., Lohmann S. M., Bertoglio J., Chardin P., Pacaud P., Loirand G. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem. 2000 Jul 14;275(28):21722–21729. doi: 10.1074/jbc.M000753200. [DOI] [PubMed] [Google Scholar]
  42. Sawada N., Itoh H., Yamashita J., Doi K., Inoue M., Masatsugu K., Fukunaga Y., Sakaguchi S., Sone M., Yamahara K. cGMP-dependent protein kinase phosphorylates and inactivates RhoA. Biochem Biophys Res Commun. 2001 Jan 26;280(3):798–805. doi: 10.1006/bbrc.2000.4194. [DOI] [PubMed] [Google Scholar]
  43. Self A. J., Hall A. Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates. Methods Enzymol. 1995;256:67–76. doi: 10.1016/0076-6879(95)56010-6. [DOI] [PubMed] [Google Scholar]
  44. Self A. J., Hall A. Purification of recombinant Rho/Rac/G25K from Escherichia coli. Methods Enzymol. 1995;256:3–10. doi: 10.1016/0076-6879(95)56003-3. [DOI] [PubMed] [Google Scholar]
  45. Shimizu T., Ihara K., Maesaki R., Kuroda S., Kaibuchi K., Hakoshima T. An open conformation of switch I revealed by the crystal structure of a Mg2+-free form of RHOA complexed with GDP. Implications for the GDP/GTP exchange mechanism. J Biol Chem. 2000 Jun 16;275(24):18311–18317. doi: 10.1074/jbc.M910274199. [DOI] [PubMed] [Google Scholar]
  46. Takahashi K., Sasaki T., Mammoto A., Takaishi K., Kameyama T., Tsukita S., Takai Y. Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J Biol Chem. 1997 Sep 12;272(37):23371–23375. doi: 10.1074/jbc.272.37.23371. [DOI] [PubMed] [Google Scholar]
  47. Takai Y., Kaibuchi K., Kikuchi A., Kawata M. Small GTP-binding proteins. Int Rev Cytol. 1992;133:187–230. doi: 10.1016/s0074-7696(08)61861-6. [DOI] [PubMed] [Google Scholar]
  48. Takai Y., Kaibuchi K., Kikuchi A., Sasaki T. Effects of prenyl modifications on interactions of small G proteins with regulators. Methods Enzymol. 1995;250:122–133. doi: 10.1016/0076-6879(95)50067-7. [DOI] [PubMed] [Google Scholar]
  49. Takai Y., Sasaki T., Tanaka K., Nakanishi H. Rho as a regulator of the cytoskeleton. Trends Biochem Sci. 1995 Jun;20(6):227–231. doi: 10.1016/s0968-0004(00)89022-2. [DOI] [PubMed] [Google Scholar]
  50. Tolias K. F., Couvillon A. D., Cantley L. C., Carpenter C. L. Characterization of a Rac1- and RhoGDI-associated lipid kinase signaling complex. Mol Cell Biol. 1998 Feb;18(2):762–770. doi: 10.1128/mcb.18.2.762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Ueda T., Kikuchi A., Ohga N., Yamamoto J., Takai Y. Purification and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein. J Biol Chem. 1990 Jun 5;265(16):9373–9380. [PubMed] [Google Scholar]
  52. Van Aelst L., D'Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev. 1997 Sep 15;11(18):2295–2322. doi: 10.1101/gad.11.18.2295. [DOI] [PubMed] [Google Scholar]
  53. Wei Y., Zhang Y., Derewenda U., Liu X., Minor W., Nakamoto R. K., Somlyo A. V., Somlyo A. P., Derewenda Z. S. Crystal structure of RhoA-GDP and its functional implications. Nat Struct Biol. 1997 Sep;4(9):699–703. doi: 10.1038/nsb0997-699. [DOI] [PubMed] [Google Scholar]
  54. van der Sluijs P., Hull M., Huber L. A., Mâle P., Goud B., Mellman I. Reversible phosphorylation--dephosphorylation determines the localization of rab4 during the cell cycle. EMBO J. 1992 Dec;11(12):4379–4389. doi: 10.1002/j.1460-2075.1992.tb05538.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES