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...

Anthrax lethal factor (LF) is a Zn2؉ -metalloprotease that cleaves and inactivates mitogen-activated protein kinase kinases (MEKs). We have used site-directed mutagenesis to identify a cluster of residues in domain II of LF that lie outside the active site and are required for cellular proteolytic activity toward MEKs. Alanine substituted for Leu 293 514 and Asn 516 resulted in toxicity comparable with N516A alone. The introduction of these mutations reduced LF-mediated cleavage of MEK2 in cell-based assays but altered neither the ability of LF to bind protective antigen nor its ability to translocate across a membrane. Interestingly, direct in vitro measurement of LF activity indicated that decreased toxicity was not always accompanied by reduced proteolytic activity. However, mutations in this region significantly reduced the ability of LF to competitively inhibit B-Raf phosphorylation of MEK. These results provide evidence that elements of domain II are involved in the association of LF into productive complex with MEKs.Anthrax toxin is derived from an exotoxin produced by the Gram-positive bacterium Bacillus anthracis. The toxin is composed of three proteins: protective antigen (PA), 1 edema factor (EF), and lethal factor (LF). PA, by itself, is not toxic; it serves to translocate EF and LF to the cytosol (1-4). Two cell surface receptors for PA (anthrax toxin receptor or ANTXR) have recently been identified (5,6). Following binding to ANTXR, PA is cleaved by cell surface-associated furin, removing a 20-kDa fragment and leaving a 63-kDa fragment (PA 63 ) attached to the ANTXR. This step is necessary to expose a binding site for EF or LF (7) as well as to remove steric hindrances to the subsequent oligomerization of PA into a heptamer (4,8,9). Following EF or LF binding to heptameric PA 63 , the toxin complex internalizes via the endosomal pathway (10 -12). The acidic environment of the endosome triggers a conformational change in the structure of PA, causing it to form a pore through which EF or LF apparently transits to the cytosol (4).EF is an adenylate cyclase (12). EF plus PA (edema toxin) is not lethal but causes edema when injected subcutaneously (13,14). LF is a Zn 2ϩ -metalloprotease that specifically cleaves the NH 2 termini of mitogen-activated protein kinase kinases (MEKs) 1 and 2 (15, 16), 3 (17) and 4, 6, and 7 (18), but not MEK 5 (18), resulting in their inactivation (15,19,20). Combinations of PA plus LF (lethal toxin) do not cause edema, but when injected intravenously rapidly induce hypotensive shock leading to death of the host (13,14).LF is a large 776-amino acid (90.2-kDa) protein (21). The crystal structure of LF has been solved to a resolution of 2.2 Å (22) (see Fig. 1). It is composed of four domains. Domain I comprises the NH 2 -terminal portion, which binds PA. Domain II (residues 263-297 and 385-550) shows structural similarity with the adenosine diphosphate-ribosylating toxin of Bacillus cereus but lacks the residues required for nicotinamide adenine dinucleotide binding and ca...