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. 2000 Dec 15;352(Pt 3):739–745.

Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor.

G Vitale 1, L Bernardi 1, G Napolitani 1, M Mock 1, C Montecucco 1
PMCID: PMC1221512  PMID: 11104681

Abstract

The lethal factor (LF) produced by toxigenic strains of Bacillus anthracis is a Zn(2+)-endopeptidase that cleaves the mitogen-activated protein kinase kinases (MAPKKs) MEK1, MEK2 and MKK3. Using genetic and biochemical approaches, we have extended the study of LF proteolytic specificity to all known MAPKK family members and found that LF also cleaves MKK4, MKK6 and MKK7, but not MEK5. The peptide bonds hydrolysed by LF within all MAPKKs were identified. Cleavage invariably occurs within the N-terminal proline-rich region preceding the kinase domain, thus disrupting a sequence involved in directing specific protein-protein interactions necessary for the assembly of signalling complexes. Alignment of the sequences flanking the site of cleavage reveals the occurrence of some consensus motifs: position P2 and P1' are occupied by hydrophobic residues and at least one basic residue is present between P4 and P7. The implications of these findings for the biochemical activity and functional specificity of LF are discussed.

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Selected References

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  1. Aepfelbacher M., Zumbihl R., Ruckdeschel K., Jacobi C. A., Barz C., Heesemann J. The tranquilizing injection of Yersinia proteins: a pathogen's strategy to resist host defense. Biol Chem. 1999 Jul-Aug;380(7-8):795–802. doi: 10.1515/BC.1999.099. [DOI] [PubMed] [Google Scholar]
  2. Bernardi L., Vitale G., Montecucco C., Musacchio A. Expression, crystallization and preliminary X-ray diffraction studies of recombinant Bacillus anthracis lethal factor. Acta Crystallogr D Biol Crystallogr. 2000 Nov;56(Pt 11):1449–1451. doi: 10.1107/s0907444900010027. [DOI] [PubMed] [Google Scholar]
  3. Colanzi A., Deerinck T. J., Ellisman M. H., Malhotra V. A specific activation of the mitogen-activated protein kinase kinase 1 (MEK1) is required for Golgi fragmentation during mitosis. J Cell Biol. 2000 Apr 17;149(2):331–339. doi: 10.1083/jcb.149.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dhanasekaran N., Premkumar Reddy E. Signaling by dual specificity kinases. Oncogene. 1998 Sep 17;17(11 REVIEWS):1447–1455. doi: 10.1038/sj.onc.1202251. [DOI] [PubMed] [Google Scholar]
  5. Dixon T. C., Meselson M., Guillemin J., Hanna P. C. Anthrax. N Engl J Med. 1999 Sep 9;341(11):815–826. doi: 10.1056/NEJM199909093411107. [DOI] [PubMed] [Google Scholar]
  6. Duesbery N. S., Webb C. P., Leppla S. H., Gordon V. M., Klimpel K. R., Copeland T. D., Ahn N. G., Oskarsson M. K., Fukasawa K., Paull K. D. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science. 1998 May 1;280(5364):734–737. doi: 10.1126/science.280.5364.734. [DOI] [PubMed] [Google Scholar]
  7. Dérijard B., Raingeaud J., Barrett T., Wu I. H., Han J., Ulevitch R. J., Davis R. J. Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science. 1995 Feb 3;267(5198):682–685. doi: 10.1126/science.7839144. [DOI] [PubMed] [Google Scholar]
  8. Friedlander A. M. Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J Biol Chem. 1986 Jun 5;261(16):7123–7126. [PubMed] [Google Scholar]
  9. Fukuda M., Gotoh I., Gotoh Y., Nishida E. Cytoplasmic localization of mitogen-activated protein kinase kinase directed by its NH2-terminal, leucine-rich short amino acid sequence, which acts as a nuclear export signal. J Biol Chem. 1996 Aug 16;271(33):20024–20028. doi: 10.1074/jbc.271.33.20024. [DOI] [PubMed] [Google Scholar]
  10. Fukuda M., Gotoh Y., Nishida E. Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase. EMBO J. 1997 Apr 15;16(8):1901–1908. doi: 10.1093/emboj/16.8.1901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hammond S. E., Hanna P. C. Lethal factor active-site mutations affect catalytic activity in vitro. Infect Immun. 1998 May;66(5):2374–2378. doi: 10.1128/iai.66.5.2374-2378.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Han J., Lee J. D., Jiang Y., Li Z., Feng L., Ulevitch R. J. Characterization of the structure and function of a novel MAP kinase kinase (MKK6). J Biol Chem. 1996 Feb 9;271(6):2886–2891. doi: 10.1074/jbc.271.6.2886. [DOI] [PubMed] [Google Scholar]
  13. Hanna P. C., Acosta D., Collier R. J. On the role of macrophages in anthrax. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10198–10201. doi: 10.1073/pnas.90.21.10198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanna P. C., Kruskal B. A., Ezekowitz R. A., Bloom B. R., Collier R. J. Role of macrophage oxidative burst in the action of anthrax lethal toxin. Mol Med. 1994 Nov;1(1):7–18. [PMC free article] [PubMed] [Google Scholar]
  15. Klimpel K. R., Arora N., Leppla S. H. Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity. Mol Microbiol. 1994 Sep;13(6):1093–1100. doi: 10.1111/j.1365-2958.1994.tb00500.x. [DOI] [PubMed] [Google Scholar]
  16. Kochi S. K., Schiavo G., Mock M., Montecucco C. Zinc content of the Bacillus anthracis lethal factor. FEMS Microbiol Lett. 1994 Dec 15;124(3):343–348. doi: 10.1111/j.1574-6968.1994.tb07306.x. [DOI] [PubMed] [Google Scholar]
  17. Menard A., Papini E., Mock M., Montecucco C. The cytotoxic activity of Bacillus anthracis lethal factor is inhibited by leukotriene A4 hydrolase and metallopeptidase inhibitors. Biochem J. 1996 Dec 1;320(Pt 2):687–691. doi: 10.1042/bj3200687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miller C. J., Elliott J. L., Collier R. J. Anthrax protective antigen: prepore-to-pore conversion. Biochemistry. 1999 Aug 10;38(32):10432–10441. doi: 10.1021/bi990792d. [DOI] [PubMed] [Google Scholar]
  19. Moriguchi T., Toyoshima F., Gotoh Y., Iwamatsu A., Irie K., Mori E., Kuroyanagi N., Hagiwara M., Matsumoto K., Nishida E. Purification and identification of a major activator for p38 from osmotically shocked cells. Activation of mitogen-activated protein kinase kinase 6 by osmotic shock, tumor necrosis factor-alpha, and H2O2. J Biol Chem. 1996 Oct 25;271(43):26981–26988. doi: 10.1074/jbc.271.43.26981. [DOI] [PubMed] [Google Scholar]
  20. Ménard A., Altendorf K., Breves D., Mock M., Montecucco C. The vacuolar ATPase proton pump is required for the cytotoxicity of Bacillus anthracis lethal toxin. FEBS Lett. 1996 May 20;386(2-3):161–164. doi: 10.1016/0014-5793(96)00422-x. [DOI] [PubMed] [Google Scholar]
  21. Orth K., Palmer L. E., Bao Z. Q., Stewart S., Rudolph A. E., Bliska J. B., Dixon J. E. Inhibition of the mitogen-activated protein kinase kinase superfamily by a Yersinia effector. Science. 1999 Sep 17;285(5435):1920–1923. doi: 10.1126/science.285.5435.1920. [DOI] [PubMed] [Google Scholar]
  22. Paul A., Wilson S., Belham C. M., Robinson C. J., Scott P. H., Gould G. W., Plevin R. Stress-activated protein kinases: activation, regulation and function. Cell Signal. 1997 Sep;9(6):403–410. doi: 10.1016/s0898-6568(97)00042-9. [DOI] [PubMed] [Google Scholar]
  23. Pellizzari R., Guidi-Rontani C., Vitale G., Mock M., Montecucco C. Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNgamma-induced release of NO and TNFalpha. FEBS Lett. 1999 Nov 26;462(1-2):199–204. doi: 10.1016/s0014-5793(99)01502-1. [DOI] [PubMed] [Google Scholar]
  24. Pellizzari R., Rossetto O., Lozzi L., Giovedi' S., Johnson E., Shone C. C., Montecucco C. Structural determinants of the specificity for synaptic vesicle-associated membrane protein/synaptobrevin of tetanus and botulinum type B and G neurotoxins. J Biol Chem. 1996 Aug 23;271(34):20353–20358. doi: 10.1074/jbc.271.34.20353. [DOI] [PubMed] [Google Scholar]
  25. Pezard C., Duflot E., Mock M. Construction of Bacillus anthracis mutant strains producing a single toxin component. J Gen Microbiol. 1993 Oct;139(10):2459–2463. doi: 10.1099/00221287-139-10-2459. [DOI] [PubMed] [Google Scholar]
  26. Raingeaud J., Whitmarsh A. J., Barrett T., Dérijard B., Davis R. J. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol. 1996 Mar;16(3):1247–1255. doi: 10.1128/mcb.16.3.1247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rossetto O., Schiavo G., Montecucco C., Poulain B., Deloye F., Lozzi L., Shone C. C. SNARE motif and neurotoxins. Nature. 1994 Dec 1;372(6505):415–416. doi: 10.1038/372415a0. [DOI] [PubMed] [Google Scholar]
  28. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  29. Schenk P. M., Baumann S., Mattes R., Steinbiss H. H. Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ArgtRNAs. Biotechniques. 1995 Aug;19(2):196-8, 200. [PubMed] [Google Scholar]
  30. Singh Y., Klimpel K. R., Goel S., Swain P. K., Leppla S. H. Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells. Infect Immun. 1999 Apr;67(4):1853–1859. doi: 10.1128/iai.67.4.1853-1859.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Singh Y., Leppla S. H., Bhatnagar R., Friedlander A. M. Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells. J Biol Chem. 1989 Jul 5;264(19):11099–11102. [PubMed] [Google Scholar]
  32. Stenmark H., Vitale G., Ullrich O., Zerial M. Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell. 1995 Nov 3;83(3):423–432. doi: 10.1016/0092-8674(95)90120-5. [DOI] [PubMed] [Google Scholar]
  33. Tanoue T., Adachi M., Moriguchi T., Nishida E. A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat Cell Biol. 2000 Feb;2(2):110–116. doi: 10.1038/35000065. [DOI] [PubMed] [Google Scholar]
  34. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tournier C., Whitmarsh A. J., Cavanagh J., Barrett T., Davis R. J. The MKK7 gene encodes a group of c-Jun NH2-terminal kinase kinases. Mol Cell Biol. 1999 Feb;19(2):1569–1581. doi: 10.1128/mcb.19.2.1569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vitale G., Pellizzari R., Recchi C., Napolitani G., Mock M., Montecucco C. Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. Biochem Biophys Res Commun. 1998 Jul 30;248(3):706–711. doi: 10.1006/bbrc.1998.9040. [DOI] [PubMed] [Google Scholar]
  37. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  38. Widmann C., Gibson S., Jarpe M. B., Johnson G. L. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev. 1999 Jan;79(1):143–180. doi: 10.1152/physrev.1999.79.1.143. [DOI] [PubMed] [Google Scholar]
  39. Windsor L. J., Bodden M. K., Birkedal-Hansen B., Engler J. A., Birkedal-Hansen H. Mutational analysis of residues in and around the active site of human fibroblast-type collagenase. J Biol Chem. 1994 Oct 21;269(42):26201–26207. [PubMed] [Google Scholar]
  40. Xia Y., Wu Z., Su B., Murray B., Karin M. JNKK1 organizes a MAP kinase module through specific and sequential interactions with upstream and downstream components mediated by its amino-terminal extension. Genes Dev. 1998 Nov 1;12(21):3369–3381. doi: 10.1101/gad.12.21.3369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhou G., Bao Z. Q., Dixon J. E. Components of a new human protein kinase signal transduction pathway. J Biol Chem. 1995 May 26;270(21):12665–12669. doi: 10.1074/jbc.270.21.12665. [DOI] [PubMed] [Google Scholar]

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