Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages.
Lethal factor (LF) is the major virulence factor produced by Bacillus anthracis. LF is sufficient to cause death in laboratory animals and cytolysis of peritoneal macrophages and macrophage cell lines. LF contains the characteristic zinc binding motif of metalloproteases and indirect evidence suggest that this hydrolytic activity is essential for its cytotoxicity. To identify the substrate(s) of LF, we have used the yeast two-hybrid system, employing a LF inactive mutant as bait. This approach has led to the identification of the MAP kinase kinases (MAPKKs) Mek1 and Mek2 as proteins capable of specific interaction with LF. LF cleaves Mek1 and Mek2 within their N-terminus in vitro and in vivo, hydrolyzing a Pro8-Ile9 and a Pro10-Arg11 peptide bond in Mek1 and Mek2 respectively. The removal of the amino terminus of MAPKKs eliminates the "docking site" for the MAPKs ERK1 and ERK2, which become phosphorylated in cultured macrophages following toxin challenge. The possible implications of these findings for the cytolysis of macrophage cells induced by LF are discussed. These results open the way to the design and screening of specific inhibitors of LF.
Vitale
G
G
Dipartimento di Scienze Biomediche, Università di Padova, Italy.
Pellizzari
R
R
Recchi
C
C
Napolitani
G
G
Mock
M
M
Montecucco
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000472:pathogenicity; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D017124:Conserved Sequence; D007700:Kinetics; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D014774:Virulence
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D008666:Metalloendopeptidases