scholarly journals Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases

1994 ◽  
Vol 303 (1) ◽  
pp. 105-112 ◽  
Author(s):  
P Dent ◽  
Y H Chow ◽  
J Wu ◽  
D K Morrison ◽  
R Jove ◽  
...  
Keyword(s):  
Peptide Mapping ◽  
Mitogen Activated Protein Kinase ◽  
Recombinant Viruses ◽  
Dual Specificity ◽  
Protein Map ◽  
Mitogen Activated Protein ◽  
Michaelis Constants ◽  
High Level ◽  
Specificity Protein

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.

scholarly journals Structure ofMycobacterium smegmatisEis in complex with paromomycin

2014 ◽  
Vol 70 (9) ◽  
pp. 1173-1179 ◽  
Author(s):  
Kyoung Hoon Kim ◽  
Doo Ri An ◽  
Hye Jin Yoon ◽  
Jin Kuk Yang ◽  
Se Won Suh
Keyword(s):  
Crystal Structure ◽  
Aminoglycoside Antibiotic ◽  
Intracellular Survival ◽  
Mitogen Activated Protein Kinase ◽  
Host Immune Responses ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Dual Specificity Protein Phosphatase ◽  
High Level ◽  
Specificity Protein

TheRv2416cgene ofMycobacterium tuberculosis(Mtb) encodes the enhanced intracellular survival (Eis) protein that enhances intracellular survival of the pathogen in host macrophages during infection. TheMtbEis protein is released into the cytoplasm of the phagocyte during intracellular infection and modulates the host immune response. It also contributes to drug resistance by acetylating multiple amine groups of aminoglycosides. Interestingly, the nonpathogenicM. smegmatis(Msm) contains a homologouseisgene (MSMEG_3513). The overall structures ofMtbEis andMsmEis are highly similar to each other, reflecting the high level (58%) of amino-acid sequence identity between them. BothMtbEis andMsmEis are active as aminoglycoside acetyltransferases, while onlyMtbEis functions as anN∊-acetyltransferase to acetylate Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase 7 (MKP-7), leading to the suppression of host immune responses. Here, the crystal structure ofMsmEis in the paromomycin-bound form is reported, revealing detailed interactions between an aminoglycoside antibiotic andMsmEis. The crystal structure ofMsmEis in the paromomycin-bound form has been determined at 3.3 Å resolution. This work provides potentially useful information for structure-guided discovery of Eis inhibitors as a novel antituberculosis drug against drug-resistantMtb.

Expression, purification, and enzymatic characterization of the dual specificity mitogen-activated protein kinase phosphatase, MKP-4

2005 ◽  
Vol 33 (1) ◽  
pp. 34-44 ◽  
Author(s):  
Suk-Bong Hong ◽  
Thomas H. Lubben ◽  
Christine M. Dolliver ◽  
Anthony J. Petrolonis ◽  
Rebecca A. Roy ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Enzymatic Characterization ◽  
Dual Specificity ◽  
Mitogen Activated Protein

Genetic Analysis of rolled, Which Encodes a Drosophila Mitogen-Activated Protein Kinase

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 763-771 ◽  
Author(s):  
Young-Mi Lim ◽  
Kimiko Nishizawa ◽  
Yoshimi Nishi ◽  
Leo Tsuda ◽  
Yoshihiro H Inoue ◽  
...  
Keyword(s):  
Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Female Sterility ◽  
Tor Signaling ◽  
Map Kinase Cascade ◽  
Dominant Female ◽  
Protein Map ◽  
Mitogen Activated Protein ◽  
Kinase Cascade

Abstract Genetic and molecular characterization of the dominant suppressors of D-rafC110 on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rlSu23, was found to bear the same molecular lesion as rlSem, which has been reported to be dominant female sterile. However, rlSu23 and the current stock of rlSem showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed.

scholarly journals Dual-Specificity Phosphatase Regulation in Neurons and Glial Cells

2019 ◽  
Vol 20 (8) ◽  
pp. 1999 ◽  
Author(s):  
Pérez-Sen ◽  
Queipo ◽  
Gil-Redondo ◽  
Ortega ◽  
Gómez-Villafuertes ◽  
...  
Keyword(s):  
Map Kinases ◽  
Protein Phosphatases ◽  
Mapk Signaling ◽  
Protein Stabilization ◽  
Mitogen Activated Protein Kinase ◽  
Brain Diseases ◽  
General Terms ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Specificity Protein

Dual-specificity protein phosphatases comprise a protein phosphatase subfamily with selectivity towards mitogen-activated protein (MAP) kinases, also named MKPs, or mitogen-activated protein kinase (MAPK) phosphatases. As powerful regulators of the intensity and duration of MAPK signaling, a relevant role is envisioned for dual-specificity protein phosphatases (DUSPs) in the regulation of biological processes in the nervous system, such as differentiation, synaptic plasticity, and survival. Important neural mediators include nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) that contribute to DUSP transcriptional induction and post-translational mechanisms of DUSP protein stabilization to maintain neuronal survival and differentiation. Potent DUSP gene inducers also include cannabinoids, which preserve DUSP activity in inflammatory conditions. Additionally, nucleotides activating P2X7 and P2Y13 nucleotide receptors behave as novel players in the regulation of DUSP function. They increase cell survival in stressful conditions, regulating DUSP protein turnover and inducing DUSP gene expression. In general terms, in the context of neural cells exposed to damaging conditions, the recovery of DUSP activity is neuroprotective and counteracts pro-apoptotic over-activation of p38 and JNK. In addition, remarkable changes in DUSP function take place during the onset of neuropathologies. The restoration of proper DUSP levels and recovery of MAPK homeostasis underlie the therapeutic effect, indicating that DUSPs can be relevant targets for brain diseases.

scholarly journals Recurrent Somatic MAP2K1 Mutations in Papillary Thyroid Cancer and Colorectal Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Rong Bu ◽  
Abdul K. Siraj ◽  
Tariq Masoodi ◽  
Sandeep Kumar Parvathareddy ◽  
Kaleem Iqbal ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Thyroid Cancer ◽  
Protein Kinase ◽  
Papillary Thyroid Cancer ◽  
Middle Eastern ◽  
Mitogen Activated Protein Kinase ◽  
Papillary Thyroid ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Specificity Protein

Mitogen-activated protein kinase kinase 1 (MAP2K1) is a dual specificity protein kinase that phosphorylates both threonine and tyrosine residues in ERK. MAP2K1 mutations have been identified in several cancers. However, their role in Middle Eastern papillary thyroid cancer (PTC) and colorectal cancer (CRC) is lacking. In this study, we evaluated the prevalence of MAP2K1 mutations in a large cohort of Middle Eastern PTC and CRC using whole-exome and Sanger sequencing technology. In the discovery cohort of 100 PTC and 100 CRC cases (comprising 50 MAPK mutant and 50 MAPK wildtype cases each), we found one MAP2K1 mutation each in PTC and CRC, both of which were MAPK wildtype. We further analyzed 286 PTC and 289 CRC MAPK wildtype cases and found three MAP2K1 mutant PTC cases and two MAP2K1 mutant CRC cases. Thus, the overall prevalence of MAP2K1 mutation in MAPK wildtype cases was 1.1% (4/336) in PTC and 0.9% (3/339) in CRC. Histopathologically, three of the four MAP2K1 mutant PTC cases were follicular variant and all four tumors were unifocal with absence of extra-thyroidal extension. All the three CRC cases harboring MAP2K1 mutation were of older age (> 50 years) and had moderately differentiated stage II/III tumors located in the left colon. In conclusion, this is the first comprehensive report of MAP2K1 somatic mutations prevalence in PTC and CRC from this ethnicity. The mutually exclusive nature of MAP2K1 and MAPK mutations suggests that each of these mutation may function as an initiating mutation driving tumorigenesis through MAPK signaling pathway.

scholarly journals Characterization of Caenorhabditis elegans Homologs of the Down Syndrome Candidate Gene DYRK1A

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 571-580 ◽  
Author(s):  
William B Raich ◽  
Celine Moorman ◽  
Clay O Lacefield ◽  
Jonah Lehrer ◽  
Dusan Bartsch ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Caenorhabditis Elegans ◽  
Trisomy 21 ◽  
Gene Dosage ◽  
Inducible Promoters ◽  
C Elegans ◽  
Dual Specificity ◽  
Specificity Protein

Abstract The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.

scholarly journals Differential Role of Threonine and Tyrosine Phosphorylation in the Activation and Activity of the Yeast MAPK Slt2

2021 ◽  
Vol 22 (3) ◽  
pp. 1110
Author(s):  
Gema González-Rubio ◽  
Ángela Sellers-Moya ◽  
Humberto Martín ◽  
María Molina
Keyword(s):  
Cell Wall ◽  
Biological Activity ◽  
Biological Significance ◽  
Mitogen Activated Protein Kinase ◽  
Activation Loop ◽  
High Increase ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Full Activation

The Mitogen-Activated Protein Kinase (MAPK) Slt2 is central to signaling through the yeast Cell Wall Integrity (CWI) pathway. MAPKs are regulated by phosphorylation at both the threonine and tyrosine of the conserved TXY motif within the activation loop (T190/Y192 in Slt2). Since phosphorylation at both sites results in the full activation of MAPKs, signaling through MAPK pathways is monitored with antibodies that detect dually phosphorylated forms. However, most of these antibodies also recognize monophosphorylated species, whose relative abundance and functionality are diverse. By using different phosphospecific antibodies and phosphate-affinity (Phos-tag) analysis on distinct Slt2 mutants, we determined that Y192- and T190-monophosphorylated species coexist with biphosphorylated Slt2, although most of the Slt2 pool remains unphosphorylated following stress. Among the monophosphorylated forms, only T190 exhibited biological activity. Upon stimulation, Slt2 is first phosphorylated at Y192, mainly by the MAPKK Mkk1, and this phosphorylation is important for the subsequent T190 phosphorylation. Similarly, dephosphorylation of Slt2 by the Dual Specificity Phosphatase (DSP) Msg5 is ordered, with dephosphorylation of T190 depending on previous Y192 dephosphorylation. Whereas Y192 phosphorylation enhances the Slt2 catalytic activity, T190 is essential for this activity. The conserved T195 residue is also critical for Slt2 functionality. Mutations that abolish the activity of Slt2 result in a high increase in inactive Y192-monophosphorylated Slt2. The coexistence of different Slt2 phosphoforms with diverse biological significance highlights the importance of the precise detection of the Slt2 phosphorylation status.

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