scholarly journals Oxidative stress inhibits MEKK1 by site-specific glutathionylation in the ATP-binding domain

2004 ◽  
Vol 381 (3) ◽  
pp. 675-683 ◽  
Author(s):  
Janet V. CROSS ◽  
Dennis J. TEMPLETON
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase ◽  
Cysteine Residue ◽  
Binding Domain ◽  
Mitogen Activated Protein Kinase ◽  
Atp Binding ◽  
Cysteine Oxidation ◽  
Mitogen Activated Protein ◽  
In Cells

Many intracellular signalling events are accompanied by generation of reactive oxygen species in cells. Oxidation of protein thiol groups is an emerging theme in signal-transduction research. We have found that MEKK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase 1], an upstream activator of the SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, is directly inhibited by cysteine alkylation using NEM (N-ethylmaleimide). The related kinase, ASK1 (apoptosis signal-regulating kinase 1), was not inhibited, but was instead activated by NEM. Inhibition of MEKK1 requires a single unique cysteine residue (Cys1238) in the ATP-binding domain of MEKK1. Oxidative stress induced by menadione (2-methyl-1,4-naphthoquinone) also inhibited MEKK1, but activated ASK1, in cells. MEKK1 inhibition by menadione also required Cys1238. Oxidant-inhibited MEKK1 was re-activated by dithiothreitol and glutathione, supporting reversible cysteine oxidation as a mechanism. Using various chemical probes, we excluded modification by S-nitrosylation or oxidation of cysteine to sulphenic acid. Oxidant-inhibited MEKK1 migrated normally on non-reducing gels, excluding the possibility of intra- or inter-molecular disulphide bond formation. MEKK1 was inhibited by glutathionylation in vitro, and MEKK1 isolated from menadione-treated cells was shown by MS to be modified by glutathione on Cys1238. Our results support a model whereby the redox environment within the cell selectively regulates stress signalling through MEKK1 versus ASK1, and may thereby participate in the induction of apoptosis by oxidative stress.

scholarly journals MSK1 activity is controlled by multiple phosphorylation sites

2005 ◽  
Vol 387 (2) ◽  
pp. 507-517 ◽  
Author(s):  
Claire E. McCOY ◽  
David G. CAMPBELL ◽  
Maria DEAK ◽  
Graham B. BLOOMBERG ◽  
J. Simon C. ARTHUR
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Cascades ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase ◽  
In Cells

MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2.

scholarly journals Nur77 is phosphorylated in cells by RSK in response to mitogenic stimulation

2006 ◽  
Vol 393 (3) ◽  
pp. 715-724 ◽  
Author(s):  
Andrew D. Wingate ◽  
David G. Campbell ◽  
Mark Peggie ◽  
J. Simon C. Arthur
Keyword(s):  
Protein Kinase ◽  
Orphan Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Mitogenic Stimulation ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Ribosomal S6 Kinase ◽  
In Cells

Nur77 is a nuclear orphan receptor that is able to activate transcription independently of exogenous ligand, and has also been shown to promote apoptosis on its localization to mitochondria. Phosphorylation of Nur77 on Ser354 has been suggested to reduce ability of Nur77 to bind DNA; however, the kinase responsible for this phosphorylation in cells has not been clearly established. In the present study, we show that Nur77 is phosphorylated on this site by RSK (ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase), but not by PKB (protein kinase B) or PKA (protein kinase A), in vitro. In cells, phosphorylation of Nur77 in vivo is catalysed by RSK, which is activated downstream of the classical MAPK (mitogen-activated protein kinase) cascade. Phosphorylation of Nur77 by RSK is able to promote the binding of Nur77 to 14-3-3 proteins in vitro, however, no evidence could be seen for this interaction in cells. We have established that two related proteins, Nurr1 and Nor1, are also phosphorylated on the equivalent site by RSK in cells in response to mitogenic stimulation.

scholarly journals Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

2003 ◽  
Vol 23 (20) ◽  
pp. 7177-7188 ◽  
Author(s):  
Hoanh Tran ◽  
Fabienne Maurer ◽  
Yoshikuni Nagamine
Keyword(s):  
Hydrogen Peroxide ◽  
Protein Kinase ◽  
Active Form ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Hydrogen Peroxide Treatment ◽  
Mitogen Activated Protein ◽  
Cytoplasmic Accumulation ◽  
In Cells

ABSTRACT The mRNAs of urokinase plasminogen activator (uPA) and its receptor, uPAR, contain instability-determining AU-rich elements (AREs) in their 3′ untranslated regions. The cellular proteins binding to these RNA sequences (AREuPA/uPAR) are not known. We show here that the mRNA-stabilizing factor HuR functionally interacts with these sequences. HuR stabilized an AREuPA-containing RNA substrate in vitro and stabilized in HeLa Tet-off cells both endogenous uPA and uPAR mRNAs and a β-globin reporter mRNA containing the AREuPA. RNAi-mediated depletion of HuR in BT-549 and MDA-MB-231 cells significantly reduced the steady-state levels of endogenous uPA and uPAR mRNAs. Furthermore, we show that a constitutively active form of mitogen-activated protein kinase-activated protein kinase 2 (MK2), MK2-EE, has an ARE-mRNA-stabilizing effect that correlates with its ability to enhance the cytoplasmic accumulation of endogenous HuR, but not in cells cotransfected with a dominant negative version of MK2, MK2-K76R. These effects were mimicked by hydrogen peroxide treatment (oxidative stress), which resulted in the phosphorylation of endogenous MK2. In addition, hydrogen peroxide treatment enhanced the cytoplasmic binding of HuR to the AREuPA, which was abrogated in cells transfected with MK2-K76R. These results indicate a role for HuR and MK2 in regulating the expression of uPA and uPAR genes at the posttranscriptional level.

Age-Related Changes in Activation of Mitogen-Activated Protein Kinase Cascades by Oxidative Stress

1998 ◽  
Vol 3 (1) ◽  
pp. 23-27 ◽  
Author(s):  
Kathryn Z Guyton ◽  
Myriani Gorospe ◽  
Xiantao Wang ◽  
Yolanda D Mock ◽  
Gertrude C Kokkonen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Age Related ◽  
Mitogen Activated Protein ◽  
Age Related Changes

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

Far1 and Fus3 link the mating pheromone signal transduction pathway to three G1-phase Cdc28 kinase complexes

1993 ◽  
Vol 13 (9) ◽  
pp. 5659-5669 ◽  
Author(s):  
M Tyers ◽  
B Futcher
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Signal Transduction Pathway ◽  
Mitogen Activated Protein Kinase ◽  
G1 Phase ◽  
Transduction Pathway ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Factor ◽  
Mitogen Activated Protein

In the yeast Saccharomyces cerevisiae, the Cdc28 protein kinase controls commitment to cell division at Start, but no biologically relevant G1-phase substrates have been identified. We have studied the kinase complexes formed between Cdc28 and each of the G1 cyclins Cln1, Cln2, and Cln3. Each complex has a specific array of coprecipitated in vitro substrates. We identify one of these as Far1, a protein required for pheromone-induced arrest at Start. Treatment with alpha-factor induces a preferential association and/or phosphorylation of Far1 by the Cln1, Cln2, and Cln3 kinase complexes. This induced interaction depends upon the Fus3 protein kinase, a mitogen-activated protein kinase homolog that functions near the bottom of the alpha-factor signal transduction pathway. Thus, we trace a path through which a mitogen-activated protein kinase regulates a Cdc2 kinase.

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