Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Mar 2;120(6):1529–1537. doi: 10.1083/jcb.120.6.1529

Inhibition of PMA-induced, LFA-1-dependent lymphocyte aggregation by ADP ribosylation of the small molecular weight GTP binding protein, rho

PMCID: PMC2119740  PMID: 7680658

Abstract

Botulinum C3 exoenzyme specifically ADP-ribosylates a group of ras- related small molecular weight GTP-binding proteins, rho, and inhibits their biological activity. Using this enzyme, we examined the function of rho in PMA-induced activation of lymphocyte function-associated antigen-1 (LFA-1) in a B lymphoblastoid cell line, JY. Northern blot analysis revealed that among the three rho genes, rhoA mRNA was predominantly expressed in JY cells. Consistently, only one [32P]ADP- ribosylated band was found when the lysate of the cells was subjected to ADP ribosylation by C3 exoenzyme. When the cells were cultured with C3 exoenzyme, this substrate was ADP-ribosylated in situ in a time- and concentration-dependent manner. Concomitant with this ADP ribosylation, PMA-induced LFA-1/intercellular adhesion molecule (ICAM)-1-dependent aggregation of JY cells was inhibited. This inhibition was blocked by prior treatment of the enzyme with an anti-C3 monoclonal antibody, and overcome by stimulation with higher concentrations of PMA. The C3 exoenzyme-induced inhibition was not affected by shaking of the cell suspension, while inhibition of aggregation by cytochalasin B was abolished by this procedure, suggesting that the inhibitory effect of the C3 exoenzyme treatment was not due to decrease in cell motility. The C3 exoenzyme treatment affected neither protein phosphorylation in JY cells before and after PMA stimulation, nor affected surface expression of LFA-1 and ICAM-1. These results suggest that rhoA protein works downstream of protein kinase C activation linking PMA stimulation to LFA-1 activation and aggregation in JY cells.

Full Text

The Full Text of this article is available as a PDF (2.9 MB).

Selected References

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

  1. Aktories K., Weller U., Chhatwal G. S. Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett. 1987 Feb 9;212(1):109–113. doi: 10.1016/0014-5793(87)81566-1. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Burn P., Kupfer A., Singer S. J. Dynamic membrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes. Proc Natl Acad Sci U S A. 1988 Jan;85(2):497–501. doi: 10.1073/pnas.85.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burridge K., Fath K., Kelly T., Nuckolls G., Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol. 1988;4:487–525. doi: 10.1146/annurev.cb.04.110188.002415. [DOI] [PubMed] [Google Scholar]
  5. Buyon J. P., Slade S. G., Reibman J., Abramson S. B., Philips M. R., Weissmann G., Winchester R. Constitutive and induced phosphorylation of the alpha- and beta-chains of the CD11/CD18 leukocyte integrin family. Relationship to adhesion-dependent functions. J Immunol. 1990 Jan 1;144(1):191–197. [PubMed] [Google Scholar]
  6. Carroll R. C., Butler R. G., Morris P. A., Gerrard J. M. Separable assembly of platelet pseudopodal and contractile cytoskeletons. Cell. 1982 Sep;30(2):385–393. doi: 10.1016/0092-8674(82)90236-7. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chardin P., Madaule P., Tavitian A. Coding sequence of human rho cDNAs clone 6 and clone 9. Nucleic Acids Res. 1988 Mar 25;16(6):2717–2717. doi: 10.1093/nar/16.6.2717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chatila T. A., Geha R. S., Arnaout M. A. Constitutive and stimulus-induced phosphorylation of CD11/CD18 leukocyte adhesion molecules. J Cell Biol. 1989 Dec;109(6 Pt 2):3435–3444. doi: 10.1083/jcb.109.6.3435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Downward J., Graves J. D., Warne P. H., Rayter S., Cantrell D. A. Stimulation of p21ras upon T-cell activation. Nature. 1990 Aug 23;346(6286):719–723. doi: 10.1038/346719a0. [DOI] [PubMed] [Google Scholar]
  11. Dustin M. L., Springer T. A. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature. 1989 Oct 19;341(6243):619–624. doi: 10.1038/341619a0. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Haverstick D. M., Sakai H., Gray L. S. Lymphocyte adhesion can be regulated by cytoskeleton-associated, PMA-induced capping of surface receptors. Am J Physiol. 1992 Apr;262(4 Pt 1):C916–C926. doi: 10.1152/ajpcell.1992.262.4.C916. [DOI] [PubMed] [Google Scholar]
  14. Hibbs M. L., Jakes S., Stacker S. A., Wallace R. W., Springer T. A. The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1 beta subunit: sites required for binding to intercellular adhesion molecule 1 and the phorbol ester-stimulated phosphorylation site. J Exp Med. 1991 Nov 1;174(5):1227–1238. doi: 10.1084/jem.174.5.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hibbs M. L., Xu H., Stacker S. A., Springer T. A. Regulation of adhesion of ICAM-1 by the cytoplasmic domain of LFA-1 integrin beta subunit. Science. 1991 Mar 29;251(5001):1611–1613. doi: 10.1126/science.1672776. [DOI] [PubMed] [Google Scholar]
  16. Hirata K., Kikuchi A., Sasaki T., Kuroda S., Kaibuchi K., Matsuura Y., Seki H., Saida K., Takai Y. Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem. 1992 May 5;267(13):8719–8722. [PubMed] [Google Scholar]
  17. Horwitz A., Duggan K., Buck C., Beckerle M. C., Burridge K. Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage. Nature. 1986 Apr 10;320(6062):531–533. doi: 10.1038/320531a0. [DOI] [PubMed] [Google Scholar]
  18. Jähner D., Hunter T. The ras-related gene rhoB is an immediate-early gene inducible by v-Fps, epidermal growth factor, and platelet-derived growth factor in rat fibroblasts. Mol Cell Biol. 1991 Jul;11(7):3682–3690. doi: 10.1128/mcb.11.7.3682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kikuchi A., Yamamoto K., Fujita T., Takai Y. ADP-ribosylation of the bovine brain rho protein by botulinum toxin type C1. J Biol Chem. 1988 Nov 5;263(31):16303–16308. [PubMed] [Google Scholar]
  20. Kitazawa T., Gaylinn B. D., Denney G. H., Somlyo A. P. G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem. 1991 Jan 25;266(3):1708–1715. [PubMed] [Google Scholar]
  21. Kupfer A., Singer S. J. The specific interaction of helper T cells and antigen-presenting B cells. IV. Membrane and cytoskeletal reorganizations in the bound T cell as a function of antigen dose. J Exp Med. 1989 Nov 1;170(5):1697–1713. doi: 10.1084/jem.170.5.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Lang P., Guizani L., Vitté-Mony I., Stancou R., Dorseuil O., Gacon G., Bertoglio J. ADP-ribosylation of the ras-related, GTP-binding protein RhoA inhibits lymphocyte-mediated cytotoxicity. J Biol Chem. 1992 Jun 15;267(17):11677–11680. [PubMed] [Google Scholar]
  24. Morii N., Kawano K., Sekine A., Yamada T., Narumiya S. Purification of GTPase-activating protein specific for the rho gene products. J Biol Chem. 1991 Apr 25;266(12):7646–7650. [PubMed] [Google Scholar]
  25. Morii N., Ohashi Y., Nemoto Y., Fujiwara M., Ohnishi Y., Nishiki T., Kamata Y., Kozaki S., Narumiya S., Sakaguchi G. Immunochemical identification of the ADP-ribosyltransferase in botulinum C1 neurotoxin as C3 exoenzyme-like molecule. J Biochem. 1990 May;107(5):769–775. doi: 10.1093/oxfordjournals.jbchem.a123123. [DOI] [PubMed] [Google Scholar]
  26. Morii N., Sekine A., Ohashi Y., Nakao K., Imura H., Fujiwara M., Narumiya S. Purification and properties of the cytosolic substrate for botulinum ADP-ribosyltransferase. Identification as an Mr 22,000 guanine nucleotide-binding protein. J Biol Chem. 1988 Sep 5;263(25):12420–12426. [PubMed] [Google Scholar]
  27. Morii N., Teru-uchi T., Tominaga T., Kumagai N., Kozaki S., Ushikubi F., Narumiya S. A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation. J Biol Chem. 1992 Oct 15;267(29):20921–20926. [PubMed] [Google Scholar]
  28. Narumiya S., Sekine A., Fujiwara M. 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):17255–17257. [PubMed] [Google Scholar]
  29. Nemoto Y., Namba T., Kozaki S., Narumiya S. Clostridium botulinum C3 ADP-ribosyltransferase gene. Cloning, sequencing, and expression of a functional protein in Escherichia coli. J Biol Chem. 1991 Oct 15;266(29):19312–19319. [PubMed] [Google Scholar]
  30. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  31. Olofsson B., Chardin P., Touchot N., Zahraoui A., Tavitian A. Expression of the ras-related ralA, rho12 and rab genes in adult mouse tissues. Oncogene. 1988 Aug;3(2):231–234. [PubMed] [Google Scholar]
  32. Otey C. A., Pavalko F. M., Burridge K. An interaction between alpha-actinin and the beta 1 integrin subunit in vitro. J Cell Biol. 1990 Aug;111(2):721–729. doi: 10.1083/jcb.111.2.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Rothlein R., Springer T. A. The requirement for lymphocyte function-associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester. J Exp Med. 1986 May 1;163(5):1132–1149. doi: 10.1084/jem.163.5.1132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rubin E. J., Gill D. M., Boquet P., Popoff M. R. Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum. Mol Cell Biol. 1988 Jan;8(1):418–426. doi: 10.1128/mcb.8.1.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Springer T. A. Adhesion receptors of the immune system. Nature. 1990 Aug 2;346(6283):425–434. doi: 10.1038/346425a0. [DOI] [PubMed] [Google Scholar]
  39. Stasia M. J., Jouan A., Bourmeyster N., Boquet P., Vignais P. V. 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):615–622. doi: 10.1016/s0006-291x(05)81110-6. [DOI] [PubMed] [Google Scholar]
  40. Takaishi K., Kikuchi A., Kuroda S., Kotani K., Sasaki T., Takai Y. Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility. Mol Cell Biol. 1993 Jan;13(1):72–79. doi: 10.1128/mcb.13.1.72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yahara I., Harada F., Sekita S., Yoshihira K., Natori S. Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro. J Cell Biol. 1982 Jan;92(1):69–78. doi: 10.1083/jcb.92.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yeramian P., Chardin P., Madaule P., Tavitian A. Nucleotide sequence of human rho cDNA clone 12. Nucleic Acids Res. 1987 Feb 25;15(4):1869–1869. doi: 10.1093/nar/15.4.1869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. van Kooyk Y., van de Wiel-van Kemenade P., Weder P., Kuijpers T. W., Figdor C. G. Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes. Nature. 1989 Dec 14;342(6251):811–813. doi: 10.1038/342811a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES