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 Genome-wide identification of MAPKKK genes and their response to phytoplasma stress in Chinese jujube (Ziziphus jujuba Mill.)

2019 ◽  
Author(s):  
ZhiGuo Liu ◽  
Lixin Wang ◽  
Chaoling Xue ◽  
Yuetong Chu ◽  
Weilin Gao ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Expression Profiles ◽  
Duplication Event ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Chinese Jujube ◽  
Mapk Cascades ◽  
Mitogen Activated Protein ◽  
Phytoplasma Infection ◽  
Gene Structures

Abstract Backgrounds Mitogen activated protein kinase (MAPK) cascades play vital roles in signal transduction in response to various biotic and abiotic stresses. In the previous study we have identified 10 ZjMAPKs and 5 ZjMAPKKs in Chinese jujube genome and found some crucial members of ZjMAPKs and ZjMAPKKs might function importantly in the process of phytoplasma infection. But how these ZjMAPKKs were modulated by ZjMAPKKKs during this process is still elusive and little information is known about the MAPKKKs in Chinese jujube. Results In the current study, 56 ZjMAPKKKs were identified in the jujube genome and all of them contain the key S-TKc (serine/threonine protein kinase) domain which distributed in all 12 chromosomes. Phylogenetic analysis showed that these ZjMAPKKKs could be classified into two subfamilies, of which 41 belonged to Raf, and 15 to MEKK subfamily. In addition, the ZjMAPKKKs in each subfamily share the same conserved motifs and gene structures, one pair of ZjMAPKKKs (15/16) was the only tandem duplication event on Chromosome 5. Furthermore, the expression profiles of these MAPKKKs in response to phytoplasma disease were investigated by qPCR. In the three main infected tissues (witches’ broom leaves, phyllody leaves, apparent normal leaves), the ZjMAPKKK26 and 45 were significantly up regulated and the ZjMAPKKK3, 43 and 50 were down regulated. While the ZjMAPKKK4, 10, 25 and 44 were significant highly induced in the sterile cultivated tissues infected by phytoplasma, and the ZjMAPKKK7, 30, 35, 37, 40, 41, 43 and 46 were significantly down regulated. Conclusions The identification and classification analysis of ZjMAPKKKs was firstly reported and some key individual ZjMAPKKKs genes might play essential roles in response to phytoplasma infection. This could provide initial understanding for the mechanism that how the ZjMAPKKKs were involved in jujube - phytoplasma infection.

scholarly journals Sti1 and Cdc37 Can Stabilize Hsp90 in Chaperone Complexes with a Protein Kinase

2004 ◽  
Vol 15 (4) ◽  
pp. 1785-1792 ◽  
Author(s):  
Paul Lee ◽  
Arsalan Shabbir ◽  
Christopher Cardozo ◽  
Avrom J. Caplan
Keyword(s):  
Protein Kinase ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase Domain ◽  
Relative Contribution ◽  
Expression Studies ◽  
Mitogen Activated Protein ◽  
Gene Expression Studies ◽  
Kinase Pathway

Hsp90 functions in association with several cochaperones for folding of protein kinases and transcription factors, although the relative contribution of each to the overall reaction is unknown. We assayed the role of nine different cochaperones in the activation of Ste11, a Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase. Studies on signaling via this protein kinase pathway was measured by α-factor-stimulated induction of FIG1 or lacZ, and repression of HHF1. Several cochaperone mutants tested had reduced FIG1 induction or HHF1 repression, although to differing extents. The greatest defects were in cpr7Δ, sse1Δ, and ydj1Δ mutants. Assays of Ste11 kinase activity revealed a pattern of defects in the cochaperone mutant strains that were similar to the gene expression studies. Overexpression of CDC37, a chaperone required for protein kinase folding, suppressed defects the sti1Δ mutant back to wild-type levels. CDC37 overexpression also restored stable Hsp90 binding to the Ste11 protein kinase domain in the sti1Δ mutant strain. These data suggest that Cdc37 and Sti1 have functional overlap in stabilizing Hsp90:client complexes. Finally, we show that Cns1 functions in MAP kinase signaling in association with Cpr7.

scholarly journals An ste20 Homologue in Ustilago maydis Plays a Role in Mating and Pathogenicity

2004 ◽  
Vol 3 (1) ◽  
pp. 180-189 ◽  
Author(s):  
David G. Smith ◽  
Maria D. Garcia-Pedrajas ◽  
Wei Hong ◽  
Zhanyang Yu ◽  
Scott E. Gold ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Plant Pathogen ◽  
Genomic Library ◽  
Pathogenic Fungi ◽  
Ustilago Maydis ◽  
Kinase Domain ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Dimorphic Fungi ◽  
Mitogen Activated Protein

ABSTRACT The mitogen-activated protein kinase (MAPK) pathways are conserved from fungi to humans and have been shown to play important roles in mating and filamentous growth for both Saccharomyces cerevisiae and dimorphic fungi and in infectivity for pathogenic fungi. STE20 encodes a protein kinase of the p21-activated protein kinase family that regulates more than one of these cascades in yeasts. We hypothesized that an Ste20p homologue would play a similar role in the dimorphic plant pathogen Ustilago maydis. The full-length copy of the U. maydis gene was obtained from a genomic library; it lacked introns and was predicted to encode a protein of 826 amino acids, whose sequence confirmed its identity as the first Ste20p homologue to be isolated from a plant pathogen. The predicted protein contained both an N-terminal regulatory Cdc42-Rac interactive binding domain and a C-terminal catalytic kinase domain. Disruption of the gene smu1 resulted in a delayed mating response in a mating-type-specific manner and also in a severe reduction in disease production on maize. Unlike the Ustilago bypass of cyclase (ubc) mutations previously identified in genes in the pheromone-responsive MAPK cascade, mutation of smu1 does not by itself act as an extragenic suppressor of the filamentous phenotype of a uac1 mutant. Thus, the direct connection of Smu1p to MAPK cascade function has yet to be established. Even so, Smu1, though not absolutely required for mating, is necessary for wild-type mating and pathogenicity.

scholarly journals A Specific Activation of the Mitogen-Activated Protein Kinase Kinase 1 (Mek1) Is Required for Golgi Fragmentation during Mitosis

2000 ◽  
Vol 149 (2) ◽  
pp. 331-340 ◽  
Author(s):  
Antonino Colanzi ◽  
Thomas J. Deerinck ◽  
Mark H. Ellisman ◽  
Vivek Malhotra
Keyword(s):  
Protein Kinase ◽  
Rat Kidney ◽  
Lethal Factor ◽  
Mitogen Activated Protein Kinase ◽  
Permeabilized Cells ◽  
Golgi Membranes ◽  
Mitogen Activated Protein ◽  
Golgi Fragmentation ◽  
Protein Kinase Kinase

Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal–regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al. 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo.

scholarly journals Mutational Activation of ErbB2 Reveals a New Protein Kinase Autoinhibition Mechanism

2007 ◽  
Vol 283 (3) ◽  
pp. 1588-1596 ◽  
Author(s):  
Ying-Xin Fan ◽  
Lily Wong ◽  
Jinhui Ding ◽  
Nikolay A. Spiridonov ◽  
Richard C. Johnson ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Human Cancer ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase Activity ◽  
Mitogen Activated Protein ◽  
Cancer Mutations ◽  
Mutational Activation ◽  
New Protein

Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the αC helix and β4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to ∼10- and ∼7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the αC-β4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the αC-β4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the αC-β4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of αC-β4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.

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