Activation Of MAPKAP Kinase-2 Research Articles

Oxidative inhibition of MAPKAP kinase 2 (MK2) is predominately mediated through the activation loop cysteine

MAPKAP kinase 2 (MK2) is a member of the mitogen activated protein kinase activated protein kinase family. MK2 is activated by p38 MAP kinase and this signaling pathway is important in cellular responses to inflammatory cytokines and stress (e.g. UV, osmotic shock, oxidative stress). Mammalian MK2 has 7 conserved cysteines, that are not known to participate in disulfide bonds. Among these is a cysteine residue in the activation loop that is conserved in other kinases including protein kinase A (PKA) and protein kinase C (PKC). PKA and PKC are inhibited by oxidative modification at this conserved cysteine residue. A non‐radioactive fluorescent based peptide assay was used to assess MK2 activity after various treatments using either bacterially expressed active‐MK2 or myc tagged MK2 immunoprecipitated from transiently transfected cells. MK2 was inhibited by oxidized glutathione in vitro and in cells by treatment with diamide. Mutation of the activation loop cysteine residue produced an active kinase that was considerably less sensitive to oxidative inhibition. This work was supported by the Research Corporation (CC6942) and a Mentor Protégé grant from Kennesaw State University.

Effect of construct design on MAPKAP kinase-2 activity, thermodynamic stability and ligand-binding affinity

MAPK-activated protein kinase-2 (MAPKAPK2) regulates the synthesis of tumor necrosis factor and other cytokines and is a potential drug target for inflammatory diseases. Five protein constructs were produced in 4–10 mg quantities per liter of culture media using baculovirus-infected insect cells and characterized for kinase activity, thermal stability, and ligand-binding affinity. Compared to construct 1–370, removal of the C-terminal autoinhibitory peptide in 1–338 resulted in a destabilized but partially active nonphosphorylated enzyme; phosphorylation of 1–338 by p38α further increased activity 12-fold. A putative constitutively active mutant, 1–370/T222E/T334E, was 6.3-fold less active than phosphorylated 1–370. ThermoFluor, an equilibrium ligand-binding assay, was used to measure nucleotide analogue affinity for various constructs. Binding of phosphorylated nucleotides was Mg 2+-dependent. Residues 1–40 were required for high-affinity binding of ADP, ATPγS, staurosporine, and K252a. A mutation M138A rendered 1–370 susceptible to p38-inhibitors SB-203580 and SB-202190 with IC50 values of 17.4 and 14.1 μM, respectively. Taken together, these studies provide information on the mechanism of ligand-binding to MAPKAPK2 that can be used in the search for selective small-molecule inhibitors.

Sphingosine-1-phosphate stimulates smooth muscle cell migration through gα i- and pi3-kinase-dependent p38 mapk activation

Background Sphingosine-1-phosphate (S-1-P) is an extracellular mediator released in response to vessel injury. S-1-P binds to G-protein-coupled receptors, which can be Gα i-, Gα q-, or G 12/13-linked. This study examines the role of p38 mitogen-activated protein kinase (p38 MAPK) in vascular smooth muscle cell migration after stimulation with S-1-P, and pathways leading to p38 MAPK activation. S-1-P has previously been shown to stimulate migration of vascular smooth muscle cells (VSMCs) in vitro through ERK1/2 and G i. We hypothesized that S-1-P-induced VSMC migration is also dependent on p38 MAPK activation through a G i-coupled extracellular receptor and phosphoinositide 3-kinase (PI3-K). Methods VSMCs were cultured in vitro. A linear wound assay was performed in the presence of S-1-P and inhibitors of p38 MAPK (SB203580) or epidermal growth factor (EGF) receptor kinase (AG1478). Chemotaxis stimulated by S-1-P was also assayed in a modified Boyden chamber with and without SB203580 pretreatment. Western blotting was performed to examine p38 MAPK activation in response to S-1-P with and without SB203580, AG1478, or inhibitors of G i (pertussis toxin), PI3-K (Wortmannin and LY294002), or MEK1 (PD98059). Western blotting and immunoprecipitation for targets of p38 MAPK (MAPKAP kinase-2) and PI3-K (Akt) were also performed. Results S-1-P stimulated migration of VSMCs in both wound and Boyden transwell assays. This migration was inhibited by SB203580 to the level of control, whereas AG478 had no effect. S-1-P stimulated activation of p38 MAPK that peaked at 10 min, as well as activation of MAPKAP kinase-2. Activation of p38 MAPK was significantly inhibited by SB203580, pertussis toxin, Wortmannin, and LY294002, but not by PD98059 or AG1478; MAPKAP kinase-2 activation was inhibited by SB203580. Akt was activated by S-1-P at 3 to 5 min; this response was inhibited by Wortmannin and LY294002, but not by SB203580 or pertussis toxin. Conclusions S-1-P induced VSMC migration through a G i-linked and a PI3-K coupled, p38 MAPK- dependent process. PI3-K appears to function upstream of p38 MAPK, but was not G i-dependent. S-1-P-stimulated activation of p38 MAPK does not signal via transactivation of the EGF receptor. Understanding signal transduction will allow targeted molecular interventions to treat the response of a vessel to injury.

Chronic exposure of human mesangial cells to high glucose environments activates the p38 MAPK pathway

High glucose (HG) environments activate several protein kinase pathways in mesangial cells, including the mitogen-activated protein kinase (MAPK) pathway, ERK. The p38 MAPK pathway is activated by events that occur in the setting of diabetes, such as protein kinase C (PKC) up-regulation and cellular stresses (osmotic stress and redox changes). Substrates of activated p38 MAPK include transcription factors that are involved in the microvascular complications of diabetes. This current study investigated the mechanisms of HG-mediated activation of p38 MAPK in cultured human mesangial cells (HMCs) and the effects of p38 MAPK activation on the transcription factor activator protein-1 (AP-1). HMCs were cultured in 5 mmol/L D-glucose [normal glucose (NG)] or 30 mmol/L D-glucose (HG) for seven days. Cells were also treated with HG for brief periods of time (0.5 to 4 hours) to assess the acute effects of HG on p38 MAPK. Using Western blotting of HMC lysates, changes in the tyrosine and threonine phosphorylation of p38 MAPK were measured. The kinase activity of immunoprecipitated p38 MAPK was determined by an in vitro assay that measured the phosphorylation and activation of MAPKAP kinase-2, an intermediary signaling protein downstream of p38 MAPK. To investigate the role of osmotic stress in HG activation of p38 MAPK, cells were acutely treated with mannitol (25 to 250 mOsm/L x 5 to 60 min) or were grown seven days in media supplemented with mannitol at concentrations iso-osmotic to HG media. To investigate the role of PKC in HG-mediated p38 MAPK activation, HMCs were treated with the PKC inhibitors GF 109203X, Ro 32-0432, or rottlerin during the last several hours of HG treatment. HG conditioned cells were also treated with the antioxidants L-N-acetylcysteine (L-NAC) or diphenyliodonium (DPI) prior to harvest. To determine a functional significance of HG-mediated p38 MAPK activation, the DNA binding of the transcription factor complex AP-1 was measured by electrophoretic mobility shift assay. The p38 MAPK pathway was not activated by the acute addition of HG to the HMCs. However, activation of p38 MAPK in HMCs grown seven days in HG was demonstrated by increased tyrosine and threonine phosphorylation of p38 MAPK proteins and increased kinase activity of immunoprecipitated p38 MAPK. As assessed by a kinase assay, p38 MAPK activity in cells grown in HG for seven days exceeded that of NG cells by more than 250%. This difference was not due to differences in the amount of p38 MAPK protein between the treatment groups. Acute osmotic activation of p38 MAPK occurred at extremely high mannitol concentrations (250 mOsm/L) that exceeded the osmotic stress of acute HG. Furthermore, in cells grown for seven days in mannitol at concentrations similar to HG, p38 MAPK activity was similar to control values. Phorbol ester (PMA) treatment stimulated a twofold increase in p38 MAPK activity. The addition of GFX or Ro 32-0432 to HG cells, at concentrations that inhibited PMA activation of p38 MAPK, did not inhibit the glucose-mediated p38 MAPK activation. Rottlerin, a PKC delta inhibitor, also failed to reverse the HG-mediated p38 MAPK activation. Treatment of HG cells with L-NAC or DPI inhibited the HG-mediated p38 MAPK phosphorylation. As we have previously shown, DNA binding of the transcription factor complex AP-1 was increased in HG cells. This binding was reversed by treatment of the HG cells with the p38 MAPK inhibitor SB 203580. Chronic exposure of HMC to HG concentrations activates the p38 MAPK pathway. This activation appears to be independent of changes in the amount of total p38 MAPK produced by the cells, independent of chronic osmotic stress and independent of PKC activation. The reversal of p38 MAPK by L-NAC and DPI suggests the glucose-mediated p38 MAPK activation may occur via reactive oxygen species. The activity of AP-1, a transcription factor complex that regulates several genes involved in diabetic nephropathy, is reversed when the p38 MAPK pathway is inhibited. These findings suggest the p38 MAPK pathway may be an important pathway involved in diabetic complications.

Induction of cyclooxygenase-2 expression in human myometrial smooth muscle cells by interleukin-1beta: involvement of p38 mitogen-activated protein kinase.

1. Human myometrial smooth muscle cells (HMSMCs) in culture were exposed to recombinant human interleukin-1beta (IL-1beta, 10 ng ml-1) for 1 to 24 h. Cyclooxygenase-2 (COX-2) mRNA and protein were rapidly induced, with expression sustained at 24 h. 2. Cycloheximide (10 microg ml-1, 6 h) blocked IL-1beta-induced COX-2 protein expression and super-induced COX-2 mRNA expression. Induction of COX-2 mRNA and protein was blocked by dexamethasone (1 microm, 6 h). 3. IL-1beta-induced COX-2 expression was accompanied by a 3-fold increase of prostaglandin E2 release into the culture medium. 4. IL-1beta induced a transient (5-30 min) activation of p42/44 and p38 mitogen-activated protein kinase (MAPK) enzymes in HMSMCs. Activity of p38 MAPK was monitored by in-gel activity of its substrate MAP kinase-activated protein kinase-2 (MAPKAP kinase-2). Induction of MAPKAP kinase-2 activity was prevented by the p38 MAPK inhibitor SB 203580 (10 microm, 5-30 min). 5. COX-2 protein expression detected after 6 h IL-1beta stimulation was blocked by SB 203580 (10 microM). Exposure of HMSMCs to 10 ng ml-1 IL-1beta for only 30 min induced a level of COX-2 protein expression at 6 h culture similar to that detected in cells exposed to the cytokine for 6 h. 6. Exposure of cells to SB 203580 (10 microM during only the first 30 min of IL-1beta stimulation was effective in blocking COX-2 protein expression assayed after 6 h in culture. 7. This study has established that a transient activation of the p38 MAPK cascade is involved in IL-1beta-stimulated COX-2 expression in human myometrial smooth muscle cells. Induction of COX-2 by IL-1beta in HMSMCs provides support for the hypothesis that autocrine prostaglandin signalling in the myometrium, initiated by elevated intrauterine cytokine concentrations, plays a role in regulating myometrial contractility during labour.

An 11-Amino Acid Sequence in the Cytoplasmic Domain of CD40 Is Sufficient for Activation of c-Jun N-Terminal Kinase, Activation of MAPKAP Kinase-2, Phosphorylation of IκBα, and Protection of WEHI-231 Cells from Anti-IgM-Induced Growth Arrest

AbstractWe have previously shown that CD40 causes strong activation of the c-Jun N-terminal kinase (JNK), the p38 mitogen-activated protein kinases (MAPK) and MAPKAP kinase-2, a downstream target of p38 MAPK. To identify signaling motifs in the CD40 cytoplasmic domain that are responsible for activation of these kinases, we have created a set of 11 chimeric receptors consisting of the extracellular and transmembrane domains of CD8 fused to portions of the murine CD40 cytoplasmic domain. These chimeric receptors were expressed in WEHI-231 B lymphoma cells. We found that amino acids 35–45 of the CD40 cytoplasmic domain constitute an independent signaling motif that is sufficient for activation of the JNK and p38 MAPK pathways, as well as for induction of IκBα phosphorylation and degradation. Amino acids 35–45 were also sufficient to protect WEHI-231 cells from anti-IgM-induced growth arrest. This is the same region of CD40 required for binding the TNF receptor-associated factor-2 (TRAF2), TRAF3, and TRAF5 adapter proteins. These data support the idea that one or more of these TRAF proteins couple CD40 to the kinase cascades that activate NF-κB, JNK, and p38 MAPK.

Stress-activated Protein Kinase-2/p38 and a Rapamycin-sensitive Pathway Are Required for C2C12 Myogenesis

The differentiation of C2C12 myoblasts to myotubes was found to be accompanied by a strong activation of p70 S6 kinase and the mitogen-activated protein kinase (MAPK) family member SAPK2/p38, without significant activation of p42 MAPK and only slight activation of SAPK1/JNK and protein kinase Balpha. Consistent with these findings, SB 203580 (a specific inhibitor of SAPK2/p38) or rapamycin (which blocks the activation of p70 S6 kinase) prevented the formation of multinucleated myotubes, as well as the expression of muscle-specific proteins that included SAPK3 (another MAPK family member). PD 098059 (which prevents the activation of p42 MAPK) had no effect on myotube formation. Surprisingly, the slow activation of p70 S6 kinase during differentiation was not only prevented by rapamycin but also by SB 203580, and the activation of MAPKAP kinase-2 (an in vivo substrate of SAPK2/p38) was not only prevented by SB 203580 but also by rapamycin. In contrast, the acute activation of p70 S6 kinase in C2C12 myoblasts induced by phorbol esters was unaffected by SB 203580 and the acute activation of MAPKAP kinase-2 induced by anisomycin was unaffected by rapamycin. These results show for the first time that SAPK2/p38 plays an essential role in C2C12 cell differentiation.

The p38 mitogen-activated protein kinase is activated by ligation of the T or B lymphocyte antigen receptors, Fas or CD40, but suppression of kinase activity does not inhibit apoptosis induced by antigen receptors.

We have investigated the activation of the p38 mitogen-activated protein kinase (MAPK) in normal mouse T and B cells and its role in apoptosis. Cross-linking of the CD3 chains of the TCR complex on proliferating T cells resulted in activation of p38 MAPK and MAPKAP kinase-2. Cross-linking of CD28 failed to activate p38 MAPK or MAPKAP kinase-2, but synergized strongly with low doses of anti-CD3. Cross-linking of Fas on T cells also induced rapid activation of p38 MAPK and MAPKAP kinase-2. The in vivo activation of MAPKAP kinase-2 in response to cross-linking of CD3, Fas, or CD3 and CD28 was shown to be dependent on p38 MAPK activity using a specific inhibitor, SB 203580. SB 203580 did not inhibit activation-induced cell death in T cells when used at concentrations that suppressed activation of MAPKAP kinase-2 in vivo. Cross-linking of the B cell Ag receptor (BCR) or CD40 on freshly isolated or LPS-activated splenic B cells or the immature B lymphoma, WEHI 231, resulted in activation of p38 MAPK and MAPKAP kinase-2. In vivo inhibition of p38 MAPK activity in WEHI 231 cells by SB 203580 had no effect on either BCR-induced apoptosis or anti-CD40-mediated suppression of apoptosis. We conclude that the activation of p38 MAPK and MAPKAP kinase-2 by cross-linking of the TCR, BCR, Fas, or CD40 was not correlated with their roles in regulating lymphocyte survival, and that suppression of kinase activity did not inhibit the induction of apoptosis.

Platelet-derived Growth Factor Activates a Mammalian Ste20 Coupled Mitogen-activated Protein Kinase in Airway Smooth Muscle

We have investigated the mechanisms regulating p38MAPK in airway smooth muscle cells. Incubation of cells with platelet-derived growth factor (PDGF) stimulated MAPKAP kinase-2 activity, a kinase immediately down-stream of P38MAPK. Preincubation of the cells with forskolin (10 μM), which stimulated a 20-fold increase in intracellular cAMP formation, inhibited this response. An antibody raised against subdomain VI of yeast Ste20 Kinase co-immunoprecipitated p38MAPK from cell lysates. The immunoprecipitated kinase(s) was shown to catalyse the phosphorylation of myelin basic protein (MBP) and to activate purified MAPKAP kinase-2. Incubation of cells with PDGF did not increase the amount of p38MAPK isolated in the anti-Ste20 immunoprecipitate. However, the kinase phosphorylated MBP and stimulated purified MAPKAP kinase-2 activity more effectively than kinase from control cells. The preincubation of cells with forskolin (10μM) reduced the amount of p38MAPK in the immunoprecipitate and this correlated with a decrease in kinase activity. We conclude that PDGF induces the activation of p38MAPK, whereas forskolin elicits its dissociation from the complex with Ste20. Therefore, Ste20/p38MAPK may form part of a signal transduction pathway linked to the activation of MAPKAP kinase-2 in ASM cells.

The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.

Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.

SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1

A class of pyridinyl imidazoles inhibit the MAP kinase homologue, termed here reactivating kinase (RK) [Lee et al. (1994) Nature 372, 739–746]. We now show that one of these compounds (SB 203580) inhibits RK in vitro (IC 50 = 0.6 μM), suppresses the activation of MAPKAP kinase-2 and prevents the phosphorylation of heat shock protein (HSP) 27 in response to interleukin-1, cellular stresses and bacterial endotoxin in vivo. These results establish that MAPKAP kinase-2 is a physiological RK substrate, and that HSP27 is phosphorylated by MAPKAP kinase-2 in vivo. The specificity of SB 203580 was indicated by its failure to inhibit 12 other protein kinases in vitro, and by its lack of effect on the activation of RK kinase and other MAP kinase cascades in vivo. We suggest that SB 203580 will be useful for identifying other physiological roles and targets of RK and MAPKAP kinase-2.

Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins

MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser 15 and Ser 40 (hsp25) and Ser 15, Ser 78 and Ser 82 (hsp27). Se 86 of hsp25 and the equivalent residue in hsp27 (Ser 82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co-purifies with MAPKAP kinase-2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase-2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.