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Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor
G Vitale1, L Bernardi, G Napolitani
1Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, Università di Padova, Via Trieste 75, 35121 Padova, Italy. gvitale@makek.dstb.uniud.it
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.
Insights
Bacillus anthracis lethal factor (LF) targets multiple mitogen-activated protein kinase kinases (MAPKKs), cleaving them in a specific proline-rich region. This disruption impacts signaling complex assembly and cellular function.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Signaling
Background:
- Bacillus anthracis lethal factor (LF) is a zinc-dependent endopeptidase.
- LF targets specific mitogen-activated protein kinase kinases (MAPKKs), including MEK1, MEK2, and MKK3.
- The precise targets and cleavage mechanisms of LF require further elucidation.
Purpose of the Study:
- To investigate the full proteolytic specificity of LF across the entire MAPKK family.
- To identify the specific peptide bonds hydrolyzed by LF within MAPKKs.
- To understand the structural and functional implications of LF-mediated cleavage.
Main Methods:
- Genetic analysis of LF activity.
- Biochemical assays to determine proteolytic cleavage sites.
- Sequence alignment to identify consensus motifs at cleavage sites.
Main Results:
- LF cleaves MKK4, MKK6, and MKK7 in addition to MEK1, MEK2, and MKK3.
- MEK5 is not cleaved by LF.
- Cleavage occurs in the N-terminal proline-rich region, disrupting protein-protein interaction sites.
Conclusions:
- LF exhibits broad specificity towards MAPKKs, targeting a conserved proline-rich region.
- Cleavage disrupts essential signaling complex assembly, impacting cellular function.
- Identification of consensus motifs provides insight into LF's enzymatic activity and substrate recognition.