Home LiteratureArticle Details
PMID: 8198524 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

ADP-ribosylation of Rho proteins by Clostridium botulinum exoenzyme C3 is influenced by phosphorylation of Rho-associated factors.

The Biochemical journal ·Vol. 300 ( Pt 1) ·1994-05-15 ·Pages 133-9

Fritz G, Aktories K

Abstract

Specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study the involvement of phosphorylation in the regulation of the low-molecular-mass GTP-binding protein Rho. Dephosphorylation of CHO cell extracts by alkaline phosphatase treatment resulted in a 80-90% reduction in the C3-catalysed [32P]ADP-ribosylation of Rho proteins in both cytosolic and membrane fractions. Similar results were obtained after dephosphorylation with protein phosphatase type-1 from bovine retina, whereas type-2B and type-2C phosphatases had no effect on the level of subsequent [32P]ADP-ribosylation of Rho by C3. Incubation of CHO cell lysate under phosphorylation conditions increased the subsequent C3-mediated [32P]ADP-ribosylation of Rho proteins. The protein kinase inhibitors H7 and H9 had no effect on [32P]ADP-ribosylation at concentrations which are specific for inhibition of protein kinase A or C. Recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was phosphorylated by protein kinase A; however, the phosphorylation had no stimulatory effect on the ADP-ribosylation of GST-RhoA by C3. An approx. 48 kDa phosphoprotein was identified which bound specifically to recombinant GST-RhoA fusion protein. By gel-permeation chromatography, Rho-containing complexes of approx. 50 kDa and 130-170 kDa were detected. The ADP-ribosylation of Rho in the 130-170 kDa complex was reduced by alkaline phosphatase pretreatment. The data suggest that Rho activity is influenced by phosphorylation of Rho-associated regulatory factors. Phosphorylation/dephosphorylation of these Rho-regulating factors appears to alter the ability of Rho to serve as a substrate for C3-induced [32P]ADP-ribosylation.

MeSH Terms
3T3 Cells ADP Ribose Transferases/metabolism Adenosine Diphosphate Ribose/metabolism Animals Binding Sites Biological Factors/metabolism Botulinum Toxins CHO Cells Cricetinae GTP-Binding Proteins/metabolism Guanine Nucleotide Dissociation Inhibitors Mice Phosphoproteins/metabolism Phosphorylation Recombinant Proteins/metabolism rho-Specific Guanine Nucleotide Dissociation Inhibitors rhoA GTP-Binding Protein
Chemicals
Biological Factors Guanine Nucleotide Dissociation Inhibitors Phosphoproteins Recombinant Proteins rho-Specific Guanine Nucleotide Dissociation Inhibitors Adenosine Diphosphate Ribose ADP Ribose Transferases exoenzyme C3, Clostridium botulinum Botulinum Toxins GTP-Binding Proteins rhoA GTP-Binding Protein
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Fritz G
Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg, Germany.
Aktories K
References (44)
44 references, click to expand
  1. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  2. Cyclic AMP-induced shape changes of astrocytes are accompanied by rapid depolymerization of actin.
    Brain Res. 1990 Oct 1;528(2):189-96 PMID: 1980224
  3. Hormonally induced changes in the cytoskeleton of human thyroid cells in culture.
    J Cell Biol. 1982 Jul;94(1):42-50 PMID: 6288739
  4. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C.
    Biochemistry. 1984 Oct 9;23(21):5036-41 PMID: 6238627
  5. Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin.
    FEBS Lett. 1987 Feb 9;212(1):109-13 PMID: 3100333
  6. A thousand and one protein kinases.
    Cell. 1987 Sep 11;50(6):823-9 PMID: 3113737
  7. Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum.
    Mol Cell Biol. 1988 Jan;8(1):418-26 PMID: 3122025
  8. Molecular cloning and sequencing of the epidermal cell differentiation inhibitor gene from Staphylococcus aureus.
    Biochem Biophys Res Commun. 1991 Jan 31;174(2):459-64 PMID: 1993048
  9. Purification of GTPase-activating protein specific for the rho gene products.
    J Biol Chem. 1991 Apr 25;266(12):7646-50 PMID: 1902224
  10. Purification and N-terminal sequence of the p21rho GTPase-activating protein, rho GAP.
    Biochem J. 1991 Jun 15;276 ( Pt 3):833-6 PMID: 1905930
  11. A stimulatory GDP/GTP exchange protein for smg p21 is active on the post-translationally processed form of c-Ki-ras p21 and rhoA p21.
    Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6442-6 PMID: 1907371
  12. Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3 ADP-ribosyltransferase.
    Eur J Cell Biol. 1991 Apr;54(2):237-45 PMID: 1908779
  13. ADP-ribosylation of a small size GTP-binding protein in bovine neutrophils by the C3 exoenzyme of Clostridium botulinum and effect on the cell motility.
    Biochem Biophys Res Commun. 1991 Oct 31;180(2):615-22 PMID: 1659402
  14. Epidermal cell differentiation inhibitor ADP-ribosylates small GTP-binding proteins and induces hyperplasia of epidermis.
    J Biol Chem. 1992 Feb 5;267(4):2600-4 PMID: 1733958
  15. ADP-ribosylation of small GTP-binding proteins by Bacillus cereus.
    Biochem Biophys Res Commun. 1992 Mar 31;183(3):931-6 PMID: 1567406
  16. Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction.
    J Biol Chem. 1992 May 5;267(13):8719-22 PMID: 1577714
  17. 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):539-49 PMID: 1581965
  18. Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum.
    J Biol Chem. 1992 May 25;267(15):10274-80 PMID: 1587816
  19. ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity.
    J Biol Chem. 1992 Jun 15;267(17):11677-80 PMID: 1601841
  20. Clostridium botulinum C3 ADP-ribosyltransferase.
    Curr Top Microbiol Immunol. 1992;175:115-31 PMID: 1628497
  21. Functional interactions of stimulatory and inhibitory GDP/GTP exchange proteins and their common substrate small GTP-binding protein.
    J Biol Chem. 1992 Jul 25;267(21):14611-5 PMID: 1634508
  22. The regulation of transcription by phosphorylation.
    Cell. 1992 Aug 7;70(3):375-87 PMID: 1643656
  23. 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):389-99 PMID: 1643657
  24. Diversity and versatility of GTPase activating proteins for the p21rho subfamily of ras G proteins detected by a novel overlay assay.
    J Biol Chem. 1992 Aug 15;267(23):16025-8 PMID: 1644791
  25. The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins.
    J Biol Chem. 1992 Sep 5;267(25):17512-9 PMID: 1517204
  26. Bacterial ADP-ribosyltransferase with a substrate specificity of the rho protein disassembles the Golgi apparatus in Vero cells and mimics the action of brefeldin A.
    Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8903-7 PMID: 1409583
  27. Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets.
    J Cell Biol. 1992 Nov;119(4):905-12 PMID: 1385445
  28. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes.
    Cell. 1992 Dec 11;71(6):891-4 PMID: 1458538
  29. Identification and characterization of a cytoskeleton-associated, epidermal growth factor sensitive pp60c-src substrate.
    Oncogene. 1992 Dec;7(12):2429-38 PMID: 1281304
  30. Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP-ribosylation by the C3 exoenzyme of Clostridium botulinum.
    Biochemistry. 1992 Dec 29;31(51):12863-9 PMID: 1334435
  31. ADP-ribosylation of a 21-24 kDa eukaryotic protein(s) by C3, a novel botulinum ADP-ribosyltransferase, is regulated by guanine nucleotide.
    Biochem J. 1987 Oct 15;247(2):363-8 PMID: 3122724
  32. Botulinum ADP-ribosyltransferase C3. Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes.
    Eur J Biochem. 1988 Mar 1;172(2):445-50 PMID: 3127209
  33. Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase triggers acute morphological changes in thyroid epithelial cells.
    FEBS Lett. 1988 May 23;232(2):409-13 PMID: 2837413
  34. ADP-ribosylation of the bovine brain rho protein by botulinum toxin type C1.
    J Biol Chem. 1988 Nov 5;263(31):16303-8 PMID: 3141405
  35. Substrate for botulinum ADP-ribosyltransferase, Gb, has an amino acid sequence homologous to a putative rho gene product.
    J Biol Chem. 1988 Nov 25;263(33):17255-7 PMID: 3141419
  36. Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins.
    J Biol Chem. 1989 Jan 5;264(1):10-3 PMID: 2491843
  37. The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3.
    Biochem Biophys Res Commun. 1989 Jan 16;158(1):209-13 PMID: 2492192
  38. 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):1087-92 PMID: 2501082
  39. Activator protein supporting the botulinum ADP-ribosyltransferase reaction.
    J Biol Chem. 1989 Sep 5;264(25):15000-5 PMID: 2504715
  40. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases.
    Nature. 1990 Jan 25;343(6256):377-81 PMID: 1689011
  41. Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.
    J Cell Biol. 1990 Jul;111(1):103-12 PMID: 2164027
  42. Microinjection of recombinant p21rho induces rapid changes in cell morphology.
    J Cell Biol. 1990 Sep;111(3):1001-7 PMID: 2118140
  43. 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):1321-8 PMID: 2120668
  44. 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;72:248-54 PMID: 942051
Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj3000133
Published
1994-05-15
Pages
133-9
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1138134
Subset
IM
External Links
PubMed source DOI: 10.1042/bj3000133 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2