Mammalian p38 mitogen-activated protein kinases (MAPKs) are activated by a wide range of cellular stresses as well as in response to inflammatory cytokines. There are four members of the p38MAPK ...family (p38α, p38β, p38γ and p38δ) which are about 60% identical in their amino acid sequence but differ in their expression patterns, substrate specificities and sensitivities to chemical inhibitors such as SB203580. A large body of evidences indicates that p38MAPK activity is critical for normal immune and inflammatory response. The p38MAPK pathway is a key regulator of pro-inflammatory cytokines biosynthesis at the transcriptional and translational levels, which makes different components of this pathway potential targets for the treatment of autoimmune and inflammatory diseases. However, recent studies have shed light on the broad effect of p38MAPK activation in the control of many other aspects of the physiology of the cell, such as control of cell cycle or cytoskeleton remodelling. Here we focus on these emergent roles of p38MAPKs and their implication in different pathologies.
p38 Signalling Pathway Sanz-Ezquerro, Juan José; Cuenda, Ana
International journal of molecular sciences,
01/2021, Volume:
22, Issue:
3
Journal Article
Peer reviewed
Open access
p38 Mitogen activated protein kinases (p38MAPK) are a highly evolutionary conserved group of protein kinases, which are central for cell adaptation to environmental changes as well as for immune ...response, inflammation, tissue regeneration, and tumour formation ....
Fragment‐based drug discovery (FBDD) is a popular method in academia and the pharmaceutical industry for the discovery of early lead candidates. Despite its wide‐spread use, the approach still ...suffers from laborious screening workflows and a limited diversity in the fragments applied. Presented here is the design, synthesis, and biological evaluation of the first fragment library specifically tailored to tackle both these challenges. The 3F library of 115 fluorinated, Fsp3‐rich fragments is shape diverse and natural‐product‐like with desirable physicochemical properties. The library is perfectly suited for rapid and efficient screening by NMR spectroscopy in a two‐stage workflow of 19F NMR and subsequent 1H NMR methods. Hits against four diverse protein targets are widely distributed among the fragment scaffolds in the 3F library and a 67 % validation rate was achieved using secondary assays. This collection is the first synthetic fragment library tailor‐made for 19F NMR screening and the results demonstrate that the approach should find broad application in the FBDD community.
Taking the lead: A large library of fluorinated fragments was designed and synthesized to address the need for better sampling of chemical space and easier screening workflows. The incorporation of fluorine enabled efficient screening by 19F NMR spectroscopy, which was demonstrated for screening against four disease‐relevant targets, providing a series of starting points for drug discovery.
Evidence implicating p38γ and p38δ (p38γ/p38δ) in inflammation are mainly based on experiments using
deficient (p38γ/δKO) mice, which show low levels of TPL2, the kinase upstream of MKK1-ERK1/2 in ...myeloid cells. This could obscure p38γ/p38δ roles, since TPL2 is essential for regulating inflammation. Here we generated a
/
(p38γ/δKIKO) mouse, expressing kinase-inactive p38γ and lacking p38δ. This mouse exhibited normal TPL2 levels, making it an excellent tool to elucidate specific p38γ/p38δ functions. p38γ/δKIKO mice showed a reduced inflammatory response and less susceptibility to LPS-induced septic shock and
infection than wild-type mice. Gene expression analyses in LPS-activated WT and p38γ/δKIKO macrophages revealed that p38γ/p38δ regulated numerous genes implicated in innate immune response. Additionally, phospho-proteomic analyses and
kinase assays showed that the transcription factor myocyte enhancer factor-2D (MEF2D) was phosphorylated at Ser444 via p38γ/p38δ. Mutation of MEF2D Ser444 to the non-phosphorylatable residue Ala increased its transcriptional activity and the expression of
and
mRNA. These results suggest that p38γ/p38δ govern innate immune responses by regulating MEF2D phosphorylation and transcriptional activity.
Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site ...has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response.
The compound BIRB796 inhibits the stress-activated protein kinases p38α and p38β and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also ...inhibits the activity and the activation of SAPK3/p38γ. This occurs at higher concentrations of BIRB796 than those that inhibit p38α and p38β and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations, BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97, further establishing that this is a physiological substrate of SAPK3/p38γ. Our results demonstrate that BIRB796, in combination with SB203580, a compound that inhibits p38α and p38β, but not the other p38 isoforms, can be used to identify physiological substrates of SAPK3/p38γ as well as those of p38α and p38β.
On the basis mainly of pharmacological experiments, the p38α MAP kinase isoform has been established as an important regulator of immune and inflammatory responses. However, the role of the related ...p38γ and p38δ kinases has remained unclear. Here, we show that deletion of p38γ and p38δ impaired the innate immune response to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, by blocking the extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages and dendritic cells. p38γ and p38δ were necessary to maintain steady-state levels of tumor progression locus 2 (TPL2), the MKK kinase that mediates ERK1/2 activation after TLR4 stimulation. TNFα, IL-1β, and IL-10 production were reduced in LPS-stimulated macrophages from p38γ/δ-null mice, whereas IL-12 and IFNβ production increased, in accordance with the known effects of TPL2/ERK1/2 signaling on the induction of these cytokines. Furthermore, p38γ/δ-deficient mice were less sensitive than controls to LPS-induced septic shock, showing lower TNFα and IL-1β levels after challenge. Together, our results establish p38γ and p38δ as key components in innate immune responses.
All four members of the mammalian p38 mitogen-activated protein kinase (MAPK) family (p38α, p38β, p38γ and p38δ) are activated by dual phosphorylation in the TGY motif in the activation loop. This ...phosphorylation is mediated by three kinases, MKK3, MKK6 and MKK4, at least
in vitro. The role of these MKK in the activation of p38α has been demonstrated in studies using fibroblasts that lack MKK3 and/or MKK6. Nonetheless, the physiological upstream activators of the other p38MAPK isoforms have not yet been reported using MKK knockout cells. In this study, we examined p38β, γ and δ activation by MKK3 and MKK6, in cells lacking MKK3, MKK6 or both. We show that MKK3 and MKK6 are both essential for the activation of p38γ and p38β induced by environmental stress, whereas MKK6 is the major p38γ activator in response to TNFα. In contrast, p38δ activation by ultraviolet radiation, hyperosmotic shock, anisomycin or by TNFα is mediated by MKK3. Moreover, in response to osmotic stress, MKK3 and MKK6 are crucial in regulating the phosphorylation of the p38γ substrate hDlg and its activity as scaffold protein. These data indicate that activation of distinct p38MAPK isoforms is regulated by the selective and synchronized action of two kinases, MKK3 and MKK6, in response to cell stress.
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 Bα. 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.
Objective
The role of most p38 MAPK isoforms in inflammatory arthritis is not known. This study was undertaken to evaluate p38γ and p38δ deficiency in the collagen‐induced arthritis (CIA) model.
...Methods
Wild‐type, p38γ−/−, p38δ−/−, and p38γ/δ−/− mice were immunized with chicken type II collagen, and disease activity was evaluated by semiquantitative scoring and histologic assessment. Serum cytokine levels and in vitro T cell cytokine responses were quantified by flow cytometry and multiplex analysis, and serum anticollagen antibody levels by enzyme‐linked immunosorbent assay. Cytokine and p38 MAPK isoform expression in joints were determined by quantitative polymerase chain reaction.
Results
Compound p38γ and p38δ deficiency markedly reduced arthritis severity compared with that in wild‐type mice, whereas lack of either p38γ or p38δ had an intermediate effect. Joint damage was minimal in arthritic p38γ/δ−/− mice compared with wild‐type mice. The p38γ/δ−/− mice had lower levels of pathogenic anticollagen antibodies and interleukin‐1β (IL‐1β) and tumor necrosis factor α than controls. In vitro T cell assays showed reduced proliferation, interferon‐γ (IFNγ) production, and IL‐17 production by lymph node cells from p38γ/δ−/− mice. IL‐17 and IFNγ messenger RNA expression in joints was significantly inhibited in p38γ/δ−/− mice. Wild‐type chimeric mice with p38γ/δ−/− bone marrow did not show decreased CIA.
Conclusion
Reduced disease severity in p38γ/δ−/− mice was associated with lower cytokine production and anticollagen antibody responses than in controls, indicating that p38γ and p38δ are crucial regulators of inflammatory joint destruction in CIA. Our findings indicate that p38γ and p38δ are potential therapeutic targets in complex diseases, such as rheumatoid arthritis, that involve innate and adaptive immune responses.
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