Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Jun 15;276(Pt 3):833–836. doi: 10.1042/bj2760833

Purification and N-terminal sequence of the p21rho GTPase-activating protein, rho GAP.

M D Garrett 1, G N Major 1, N Totty 1, A Hall 1
PMCID: PMC1151079  PMID: 1905930

Abstract

Eukaryotic cells contain numerous small-molecular-mass GTP-binding proteins, but the processes that they regulate are not known. Different members of this protein family appear to be associated with specific GTPase-activating proteins (GAPs), and we have previously reported the identification of a cytoplasmic GAP (rho GAP) that stimulates the GTPase activity of p21rho but not of other small-molecular-mass GTP-binding proteins. We have now purified rho GAP 2000-fold from human spleen tissue using f.p.l.c. Electrotransfer of this 27.5 kDa protein on to an Immobilon-P transfer membrane followed by reconstitution of its enzymic activity confirmed its identity. Rho GAP was subjected to N-terminal sequence analysis and 15 amino acids were obtained. The sequence showed 53% identity with a region present in IRA1, a protein which stimulates the GTPase activity of RAS proteins in Saccharomyces cerevisiae. These results suggest that there is a family of sequence-related GAP proteins, which to date includes ras GAP and its yeast counterparts IRA1 and IRA2, rho GAP and the Neurofibromatosis gene product NF1.

Full text

PDF
833

Images in this article

835Fig. 2.
on p.835

Selected References

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

  1. Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [PubMed] [Google Scholar]
  2. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  3. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Chardin P., Boquet P., Madaule P., Popoff M. R., Rubin E. J., Gill D. M. The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO J. 1989 Apr;8(4):1087–1092. doi: 10.1002/j.1460-2075.1989.tb03477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Didsbury J., Weber R. F., Bokoch G. M., Evans T., Snyderman R. rac, a novel ras-related family of proteins that are botulinum toxin substrates. J Biol Chem. 1989 Oct 5;264(28):16378–16382. [PubMed] [Google Scholar]
  6. Drivas G. T., Shih A., Coutavas E., Rush M. G., D'Eustachio P. Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line. Mol Cell Biol. 1990 Apr;10(4):1793–1798. doi: 10.1128/mcb.10.4.1793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ellis C., Moran M., McCormick F., Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990 Jan 25;343(6256):377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  10. Hall A. ras and GAP--who's controlling whom? Cell. 1990 Jun 15;61(6):921–923. doi: 10.1016/0092-8674(90)90054-i. [DOI] [PubMed] [Google Scholar]
  11. Isomura M., Kaibuchi K., Yamamoto T., Kawamura S., Katayama M., Takai Y. Partial purification and characterization of GDP dissociation stimulator (GDS) for the rho proteins from bovine brain cytosol. Biochem Biophys Res Commun. 1990 Jun 15;169(2):652–659. doi: 10.1016/0006-291x(90)90380-6. [DOI] [PubMed] [Google Scholar]
  12. Johnson D. I., Pringle J. R. Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity. J Cell Biol. 1990 Jul;111(1):143–152. doi: 10.1083/jcb.111.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaplan D. R., Morrison D. K., Wong G., McCormick F., Williams L. T. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990 Apr 6;61(1):125–133. doi: 10.1016/0092-8674(90)90220-9. [DOI] [PubMed] [Google Scholar]
  14. Kazlauskas A., Ellis C., Pawson T., Cooper J. A. Binding of GAP to activated PDGF receptors. Science. 1990 Mar 30;247(4950):1578–1581. doi: 10.1126/science.2157284. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Madaule P., Axel R., Myers A. M. Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 Feb;84(3):779–783. doi: 10.1073/pnas.84.3.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Major G. N., Gardner E. J., Carne A. F., Lawley P. D. Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine. Nucleic Acids Res. 1990 Mar 25;18(6):1351–1359. doi: 10.1093/nar/18.6.1351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  20. Molloy C. J., Bottaro D. P., Fleming T. P., Marshall M. S., Gibbs J. B., Aaronson S. A. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature. 1989 Dec 7;342(6250):711–714. doi: 10.1038/342711a0. [DOI] [PubMed] [Google Scholar]
  21. Paterson H. F., Self A. J., Garrett M. D., Just I., Aktories K., Hall A. Microinjection of recombinant p21rho induces rapid changes in cell morphology. J Cell Biol. 1990 Sep;111(3):1001–1007. doi: 10.1083/jcb.111.3.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pegg A. E., Wiest L., Foote R. S., Mitra S., Perry W. Purification and properties of O6-methylguanine-DNA transmethylase from rat liver. J Biol Chem. 1983 Feb 25;258(4):2327–2333. [PubMed] [Google Scholar]
  23. Polakis P. G., Snyderman R., Evans T. Characterization of G25K, a GTP-binding protein containing a novel putative nucleotide binding domain. Biochem Biophys Res Commun. 1989 Apr 14;160(1):25–32. doi: 10.1016/0006-291x(89)91615-x. [DOI] [PubMed] [Google Scholar]
  24. Sekine A., Fujiwara M., Narumiya S. Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. J Biol Chem. 1989 May 25;264(15):8602–8605. [PubMed] [Google Scholar]
  25. Szewczyk B., Summers D. F. Preparative elution of proteins blotted to Immobilon membranes. Anal Biochem. 1988 Jan;168(1):48–53. doi: 10.1016/0003-2697(88)90008-5. [DOI] [PubMed] [Google Scholar]
  26. Tanaka K., Matsumoto K., Toh-E A. IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):757–768. doi: 10.1128/mcb.9.2.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tanaka K., Nakafuku M., Tamanoi F., Kaziro Y., Matsumoto K., Toh-e A. IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein. Mol Cell Biol. 1990 Aug;10(8):4303–4313. doi: 10.1128/mcb.10.8.4303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  31. Xu G. F., O'Connell P., Viskochil D., Cawthon R., Robertson M., Culver M., Dunn D., Stevens J., Gesteland R., White R. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell. 1990 Aug 10;62(3):599–608. doi: 10.1016/0092-8674(90)90024-9. [DOI] [PubMed] [Google Scholar]
  32. Yatani A., Okabe K., Polakis P., Halenbeck R., McCormick F., Brown A. M. ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K+ channels. Cell. 1990 Jun 1;61(5):769–776. doi: 10.1016/0092-8674(90)90187-j. [DOI] [PubMed] [Google Scholar]

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

RESOURCES