Anthrax Lethal Factor Proteolysis and Inactivation of MAPK Kinase

Abstract

Anthrax lethal toxin produced by the bacterium Bacillus anthracis is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), inactivates members of the mitogen-activated protein kinase kinase or MEK family through proteolysis of their NH 2 termini. However, neither the substrate requirements for LF cleavage nor the mechanism by which proteolysis inactivates MEK have been demonstrated. By means of deletion mutant analysis and site-directed mutagenesis, we have identified an LFIR (LF interacting region) in the COOH-terminal kinase domain of MEK1 adjacent to the proline-rich region, which is essential for LF-mediated proteolysis of MEK. Point mutations in this region block proteolysis but do not alter the kinase activity of MEK. Similar mutations in MEK6 also prevent proteolysis, indicating that this region is functionally conserved among MEKs. In addition, NH 2 -terminal proteolysis of MEK1 by LF was found to reduce not only the affinity of MEK1 for its substrate mitogen-activated protein kinase but also its intrinsic kinase activity, indicating that the NH 2 -terminal end of MEK is important not only for substrate interaction but also for catalytic activity.The lethal effects of Bacillus anthracis have been attributed to an exotoxin, which it produces (1). This exotoxin is composed of three proteins: protective antigen (PA), 1 edema factor, and lethal factor (LF) (for recent reviews see Refs. 2 and 3). PA binds to a cell surface receptor (4) and, upon proteolytic activation to a 63-kDa fragment, heptamerizes to form a membrane channel that mediates the entry of three molecules of LF or edema factor into the cell (5-7). Edema factor is an adenylate cyclase and, together with PA, forms a toxin referred to as edema toxin (8). LF is a Zn 2ϩ -metalloprotease, which together with PA forms a toxin referred to as lethal toxin. Lethal toxin is the dominant virulence factor produced by B. anthracis and is the major cause of death in infected animals (9).Although LF has been shown to cleave the NH 2 termini of select members of the mitogen-activated protein kinase kinase or MEK family (10 -12), the substrate requirements that determine LF specificity are unknown. Indirect evidence suggests that epitopes distal to the cleavage site are required for LF-MEK interaction. Yeast two-hybrid assays for binding partners of LF have isolated cDNA for MEK2, which lacks the NH 2 -terminal cleavage site (13). Moreover, although it has been demonstrated that LF-cleaved MEK1 as well as recombinant MEK1, which lacks the seven NH 2 -terminal residues that are removed by LF, has reduced kinase activity (10), it is not clear how the absence of these residues alters MEK activity. Therefore, to identify regions distal to the cleavage site that are required for proteolysis, we have constructed a series of internal and COOH-terminal deletion mutants of MEK1 and have analyzed their cleavability by LF. The results reveal that a functionally conserved COOH-terminal region located adjacent to a proli...

Identification of a Novel Francisella tularensis Factor Required for Intramacrophage Survival and Subversion of Innate Immune Response

“…In addition to NF-B, some bacterial pathogens have evolved mechanisms to suppress MAPKs, another key antimicrobial signaling pathway involved in bacterial clearance. Yersinia, Shigella, and Bacillus anthracis inhibit MAPK signaling to suppress inflammatory response that permits bacterial growth and dissemination (55)(56)(57)(58). It has been reported that F. tularensis LVS suppresses MAPK signaling via intracellular growth locus C and RipA (16,19).…”

Mitochondrial Impairment Mediates Cytolysis in Anthrax Lethal Toxin-Treated Murine Macrophages

“…5 LF cleaves mitogen-activated protein kinase kinases (MAPKKs), thereby disrupting MAPK signaling pathways. [6][7][8][9][10] LT uptake and subsequent MAPKK cleavage are ubiquitous, and also occur in cells that are resistant to LT killing. 11,12 This suggests that either LF targets cell type-specific factors in addition to MAPKKs, or that cells differ in their response to MAPKK cleavage.…”

Anthrax Lethal Toxin Kills Macrophages in a Strain-Specific Manner by Apoptosis or Caspase-1-Mediated Necrosis

“…[5][6][7][8] LF specifically cleaves mitogen-activated protein kinase kinases (MAPKKs), thereby disrupting three MAPK signaling pathways. [9][10][11][12][13] Due to the ubiquitous expression of anthrax toxin receptors (ATRs), [14][15][16] LT uptake and subsequent MAPKK cleavage occur in all mammalian cells tested. 17 Despite the broad entry and activity of LT, it selectively kills only a few cell types.…”