scholarly journals Involvement of Domain II in Toxicity of Anthrax Lethal Factor

2004 ◽  
Vol 279 (50) ◽  
pp. 52473-52478 ◽  
Author(s):  
Xudong Liang ◽  
John J. Young ◽  
Sherrie A. Boone ◽  
David S. Waugh ◽  
Nicholas S. Duesbery
Keyword(s):  
Proteolytic Activity ◽  
Active Site ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Anthrax Lethal Factor ◽  
Fold Reduction ◽  
Mitogen Activated Protein

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 Leu293, Lys294, Leu514, Asn516, or Arg491caused a 10–50-fold reduction in LF toxicity. Further, whereas pairwise substitution of alanine for Leu514and either Leu293, Lys294, or Arg491completely abrogated LF toxicity, pairwise mutation of Leu514and Asn516resulted 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, directin vitromeasurement 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.

scholarly journals A new c-Jun N-terminal kinase (JNK)-interacting protein, Sab (SH3BP5), associates with mitochondria

2002 ◽  
Vol 367 (3) ◽  
pp. 577-585 ◽  
Author(s):  
Carolyn WILTSHIRE ◽  
Masato MATSUSHITA ◽  
Satoshi TSUKADA ◽  
David A.F. GILLESPIE ◽  
Gerhard H.W. MAY
Keyword(s):  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Physical Interaction ◽  
Cell Fractionation ◽  
Interacting Protein ◽  
Subcellular Compartment ◽  
Src Homology 3 ◽  
Mitogen Activated Protein ◽  
Src Homology

We have identified a novel c-Jun N-terminal kinase (JNK)-interacting protein, Sab, by yeast two-hybrid screening. Sab binds to and serves as a substrate for JNK in vitro, and was previously found to interact with the Src homology 3 (SH3) domain of Bruton's tyrosine kinase (Btk). Inspection of the sequence of Sab reveals the presence of two putative mitogen-activated protein kinase interaction motifs (KIMs) similar to that found in the JNK docking domain of the c-Jun transcription factor, and four potential serine—proline JNK phosphorylation sites in the C-terminal half of the molecule. Using deletion and site-directed mutagenesis, we demonstrate that the most N-terminal KIM in Sab is essential for JNK binding, and that, as with c-Jun, physical interaction with JNK is necessary for Sab phosphorylation. Interestingly, confocal immunocytochemistry and cell fractionation studies indicate that Sab is associated with mitochondria, where it co-localizes with a fraction of active JNK. These and previously reported properties of Sab suggest a possible role in targeting JNK to this subcellular compartment and/or mediating cross-talk between the Btk and JNK signal transduction pathways.

Manipulation of host signalling pathways by anthrax toxins

2007 ◽  
Vol 402 (3) ◽  
pp. 405-417 ◽  
Author(s):  
Benjamin E. Turk
Keyword(s):  
Protein Kinase ◽  
Pathogenic Bacteria ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Cell Types ◽  
Host Cells ◽  
Mitogen Activated Protein Kinase ◽  
Signalling Pathways ◽  
Common Strategy ◽  
Mitogen Activated Protein

Infectious microbes face an unwelcoming environment in their mammalian hosts, which have evolved elaborate multicelluar systems for recognition and elimination of invading pathogens. A common strategy used by pathogenic bacteria to establish infection is to secrete protein factors that block intracellular signalling pathways essential for host defence. Some of these proteins also act as toxins, directly causing pathology associated with disease. Bacillus anthracis, the bacterium that causes anthrax, secretes two plasmid-encoded enzymes, LF (lethal factor) and EF (oedema factor), that are delivered into host cells by a third bacterial protein, PA (protective antigen). The two toxins act on a variety of cell types, disabling the immune system and inevitably killing the host. LF is an extraordinarily selective metalloproteinase that site-specifically cleaves MKKs (mitogen-activated protein kinase kinases). Cleavage of MKKs by LF prevents them from activating their downstream MAPK (mitogen-activated protein kinase) substrates by disrupting a critical docking interaction. Blockade of MAPK signalling functionally impairs cells of both the innate and adaptive immune systems and induces cell death in macrophages. EF is an adenylate cyclase that is activated by calmodulin through a non-canonical mechanism. EF causes sustained and potent activation of host cAMP-dependent signalling pathways, which disables phagocytes. Here I review recent progress in elucidating the mechanisms by which LF and EF influence host signalling and thereby contribute to disease.

scholarly journals Anthrax toxin lethal factor domain 3 is highly mobile and responsive to ligand binding

2014 ◽  
Vol 70 (11) ◽  
pp. 2813-2822 ◽  
Author(s):  
Kimberly M. Maize ◽  
Elbek K. Kurbanov ◽  
Teresa De La Mora-Rey ◽  
Todd W. Geders ◽  
Dong-Jin Hwang ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Host Immune System ◽  
Peptide Substrates ◽  
Edema Factor ◽  
Mitogen Activated Protein ◽  
Domain 3

The secreted anthrax toxin consists of three components: the protective antigen (PA), edema factor (EF) and lethal factor (LF). LF, a zinc metalloproteinase, compromises the host immune system primarily by targeting mitogen-activated protein kinase kinases in macrophages. Peptide substrates and small-molecule inhibitors bind LF in the space between domains 3 and 4 of the hydrolase. Domain 3 is attached on a hinge to domain 2viaresidues Ile300 and Pro385, and can move through an angular arc of greater than 35° in response to the binding of different ligands. Here, multiple LF structures including five new complexes with co-crystallized inhibitors are compared and three frequently populated LF conformational states termed `bioactive', `open' and `tight' are identified. The bioactive position is observed with large substrate peptides and leaves all peptide-recognition subsites open and accessible. The tight state is seen in unliganded and small-molecule complex structures. In this state, domain 3 is clamped over certain substrate subsites, blocking access. The open position appears to be an intermediate state between these extremes and is observed owing to steric constraints imposed by specific bound ligands. The tight conformation may be the lowest-energy conformation among the reported structures, as it is the position observed with no bound ligand, while the open and bioactive conformations are likely to be ligand-induced.

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

2000 ◽  
Vol 352 (3) ◽  
pp. 739-745 ◽  
Author(s):  
Gaetano VITALE ◽  
Lorenzo BERNARDI ◽  
Giorgio NAPOLITANI ◽  
Michèle MOCK ◽  
Cesare MONTECUCCO
Keyword(s):  
Protein Kinase ◽  
Lethal Factor ◽  
Family Members ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Basic Residue ◽  
Peptide Bonds ◽  
Anthrax Lethal Factor ◽  
Mitogen Activated Protein ◽  
Toxigenic Strains

The lethal factor (LF) produced by toxigenic strains of Bacillus anthracis is a Zn2+-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 proteinŐprotein 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.

scholarly journals Off-target inhibition by active site-targeting SHP2 inhibitors

2018 ◽  
Author(s):  
Ryouhei Tsutsumi ◽  
Hao Ran ◽  
Benjamin G. Neel
Keyword(s):  
Growth Factor ◽  
Active Site ◽  
Tyrosine Phosphatase ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Cellular Context ◽  
Site Targeting

AbstractDue to the involvement of SHP2 (SH2 domain-containing protein tyrosine phosphatase) in human disease, including Noonan syndrome and cancer, several inhibitors targeting SHP2 have been developed. Here, we report that the commonly used SHP2 inhibitor NSC-78788 does not exhibit robust inhibitory effects on growth factor-dependent MAPK (mitogen-activated protein kinase) pathway activation, and that the recently developed active site-targeting SHP2 inhibitors IIB-08, 11a-1, and GS-493 show off-target effects on ligand-evoked activation/trans-phosphorylation of the PDGFRβ (platelet-derived growth factor receptor β). GS-493 also inhibits purified human PDGFRβ and SRC in vitro, whereas PDGFRβ inhibition by IIB-08 and 11a-1 occurs only in the cellular context. Our results argue for extreme caution in inferring specific functions for SHP2 based on studies using these inhibitors.

scholarly journals Anthrax lethal factor-cleavage products of MAPK (mitogen-activated protein kinase) kinases exhibit reduced binding to their cognate MAPKs

2004 ◽  
Vol 378 (2) ◽  
pp. 569-577 ◽  
Author(s):  
A. Jane BARDWELL ◽  
Mahsa ABDOLLAHI ◽  
Lee BARDWELL
Keyword(s):  
Protein Kinase ◽  
Protective Antigen ◽  
Signal Transmission ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Cell Surface Receptor ◽  
Common Mechanism ◽  
Anthrax Lethal Toxin ◽  
Mitogen Activated Protein

Anthrax lethal toxin is the major cause of death in systemic anthrax. Lethal toxin consists of two proteins: protective antigen and LF (lethal factor). Protective antigen binds to a cell-surface receptor and transports LF into the cytosol. LF is a metalloprotease that targets MKKs [MAPK (mitogen-activated protein kinase) kinases]/MEKs [MAPK/ERK (extracellular-signal-regulated kinase) kinases], cleaving them to remove a small N-terminal stretch but leaving the bulk of the protein, including the protein kinase domain, intact. LF-mediated cleavage of MEK1 and MKK6 has been shown to inhibit signalling through their cognate MAPK pathways. However, the precise mechanism by which this proteolytic cleavage inhibits signal transmission has been unclear. Here we show that the C-terminal LF-cleavage products of MEK1, MEK2, MKK3, MKK4, MKK6 and MKK7 are impaired in their ability to bind to their MAPK substrates, suggesting a common mechanism for the LF-induced inhibition of signalling.

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