Mitogen-activated Protein Kinase Alpha Research Articles

Early chronic suppression of microglial p38α in a model of Alzheimer's disease does not significantly alter amyloid-associated neuropathology.

The p38 alpha mitogen-activated protein kinase (p38α) is linked to both innate and adaptive immune responses and is under investigation as a target for drug development in the context of Alzheimer's disease (AD) and other conditions with neuroinflammatory dysfunction. While preclinical data has shown that p38α inhibition can protect against AD-associated neuropathology, the underlying mechanisms are not fully elucidated. Inhibitors of p38α may provide benefit via modulation of microglial-associated neuroinflammatory responses that contribute to AD pathology. The present study tests this hypothesis by knocking out microglial p38α and assessing early-stage pathological changes. Conditional knockout of microglial p38α was accomplished in 5-month-old C57BL/6J wild-type and amyloidogenic AD model (APPswe/PS1dE9) mice using a tamoxifen-inducible Cre/loxP system under control of the Cx3cr1 promoter. Beginning at 7.5 months of age, animals underwent behavioral assessment on the open field, followed by a later radial arm water maze test and collection of cortical and hippocampal tissues at 11 months. Additional endpoint measures included quantification of proinflammatory cytokines, assessment of amyloid burden and plaque deposition, and characterization of microglia-plaque dynamics. Loss of microglial p38α did not alter behavioral outcomes, proinflammatory cytokine levels, or overall amyloid plaque burden. However, this manipulation did significantly increase hippocampal levels of soluble Aβ42 and reduce colocalization of Iba1 and 6E10 in a subset of microglia in close proximity to plaques. The data presented here suggest that rather than reducing inflammation per se, the net effect of microglial p38α inhibition in the context of early AD-type amyloid pathology is a subtle alteration of microglia-plaque interactions. Encouragingly from a therapeutic standpoint, these data suggest no detrimental effect of even substantial decreases in microglial p38α in this context. Additionally, these results support future investigations of microglial p38α signaling at different stages of disease, as well as its relationship to phagocytic processes in this particular cell-type.

Synergistic Antiviral Activity of Pamapimod and Pioglitazone against SARS-CoV-2 and Its Variants of Concern.

The SARS-CoV-2 pandemic remains a major public health threat, especially due to newly emerging SARS-CoV-2 Variants of Concern (VoCs), which are more efficiently transmitted, more virulent, and more able to escape naturally acquired and vaccine-induced immunity. Recently, the protease inhibitor Paxlovid® and the polymerase inhibitor molnupiravir, both targeting mutant-prone viral components, were approved for high-risk COVID-19 patients. Nevertheless, effective therapeutics to treat COVID-19 are urgently needed, especially small molecules acting independently of VoCs and targeting genetically stable cellular pathways which are crucial for viral replication. Pamapimod is a selective inhibitor of p38 Mitogen-Activated Protein Kinase alpha (p38 MAPKα) that has been extensively clinically evaluated for the treatment of rheumatoid arthritis. Signaling via p38 has recently been described as a key pathway for the replication of SARS-CoV-2. Here, we reveal that the combination of pamapimod with pioglitazone, an anti-inflammatory and approved drug for the treatment of type 2 diabetes, possesses potent and synergistic activity to inhibit SARS-CoV-2 replication in vitro. Both drugs showed similar antiviral potency across several cultured cell types and similar antiviral activity against SARS-CoV-2 Wuhan type, and the VoCs Alpha, Beta, Gamma, Delta, and Omicron. These data support the combination of pamapimod and pioglitazone as a potential therapy to reduce duration and severity of disease in COVID-19 patients, an assumption currently evaluated in an ongoing phase II clinical study.

Neflamapimod induces vasodilation in resistance mesenteric arteries by inhibiting p38 MAPK\u03b1 and downstream Hsp27 phosphorylation

Neflamapimod, a selective inhibitor of p38 mitogen activated protein kinase alpha (MAPKα), is under clinical investigation for its efficacy in Alzheimer’s disease (AD) and dementia with Lewy Bodies (DLB). Here, we investigated if neflamapimod-mediated acute inhibition of p38 MAPKα could induce vasodilation in resistance-size rat mesenteric arteries. Our pressure myography data demonstrated that neflamapimod produced a dose-dependent vasodilation in mesenteric arteries. Our Western blotting data revealed that acute neflamapimod treatment significantly reduced the phosphorylation of p38 MAPKα and its downstream target heat-shock protein 27 (Hsp27) involved in cytoskeletal reorganization and smooth muscle contraction. Likewise, non-selective inhibition of p38 MAPK by SB203580 attenuated p38 MAPKα and Hsp27 phosphorylation, and induced vasodilation. Endothelium denudation or pharmacological inhibition of endothelium-derived vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) had no effect on such vasodilation. Neflamapimod-evoked vasorelaxation remained unaltered by the inhibition of smooth muscle cell K+ channels. Altogether, our data for the first time demonstrates that in resistance mesenteric arteries, neflamapimod inhibits p38 MAPKα and phosphorylation of its downstream actin-associated protein Hsp27, leading to vasodilation. This novel finding may be clinically significant and is likely to improve systemic blood pressure and cognitive deficits in AD and DLB patients for which neflamapimod is being investigated.

Opposing Oncogenic Functions of p38 Mitogen-activated Protein Kinase Alpha and Beta in Human Pancreatic Cancer Cells.

The p38 family of mitogen-activated protein kinases (MAPK) includes four isoforms: p38α, -β, -γ and -δ. The aim of this study was to elucidate possible functions of p38α and p38β in human pancreatic cancer. Isoform expression was determined in seven human pancreatic cancer cell lines. After shRNA based selective knockdown of p38α and p38β, in vitro growth and migration as well as in vivo tumorigenicity were assessed. All pancreatic cancer cells expressed p38 isoforms. Knockdown of p38α and p38β inhibited in vitro growth. Migration was markedly reduced in p38α shRNA expressing clones, but not altered by p38β knockdown. While in vivo inhibition of p38β decreased tumor formation and growth, the knockdown of p38α significantly enhanced tumorigenicity. p38 MAPKs may exert isoform specific functions in pancreatic cancer. Selective targeting may contribute to individualized treatment of pancreatic cancer in the future.

Chrysophanol, Physcion, Hesperidin and Curcumin Modulate the Gene Expression of Pro-Inflammatory Mediators Induced by LPS in HepG2: In Silico and Molecular Studies.

Hepatitis is an inflammatory condition that can develop hepatocellular carcinoma. Traditional medicine has always been the pillar of medical practice. However, it became less compatible with the current understanding of the diseases and the possible treatment. Therefore, in silico tools could be utilized for building the bridge between the legacy of the past and the current medical approaches allowing access to new therapeutic discoveries. In this work, a Chinese traditional medicine database was screened using structure-based virtual screening to identify molecules that could inhibit p38 alpha mitogen-activated protein kinase (MAPK). Out of the identified compounds, four selected compounds: chrysophanol, physcion, curcumin and hesperidin were isolated from their respective sources and their structures were confirmed by spectroscopic methods. These compounds decreased the gene expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1beta (IL-1β) in lipopolysaccharide (LPS) induced inflammation in a hepatocellular carcinoma cell line (HepG2) in a dose-dependent manner. The molecular docking study revealed the specificity of these compounds towards p38 MAPK rather than other MAPKs. In conclusion, the molecular and in silico studies suggest that the isolated compounds could be a potential treatment for hepatitis by resolving inflammation controlled by MAPKs, thus limiting the development of further complications and lower side effects.

Functions of p38 mitogen-activated protein kinase alpha and beta in human pancreatic cancer cells

Objective To elucidate possible functions of p38α and p38β in human pancreatic cancer. Methods Expression of p38α, p38β, p38γ and p38δ were determined by immunoblot in 7 cultured human pancreatic cancer cell lines. To selectively inhibit p38α or p38β, cell clones were established in a stable manner expression either p38α short hairpin RNA (shRNA) or p38β shRNA. Proliferation, single cell movement, in vitro migration, colony forming ability as well as in vivo tumorigenicity were determined by methyl thiazol tetrazolium (MTT) assay, video-time laps microscopy, boyden-chamber, soft-agar assay, tumor formation and growth in nude mice respectively. Results All pancreatic cancer cells showed p38 isoforms protein expression at various levels. Selective inhibition of p38α or p38β in Mia Paca-2 and Panc-1 cells revealed that both kinases enhance cell proliferation and anchorage-independent growth. Single cell movement and cell invasion were markedly reduced in p38α shRNA expressing clones, but not altered in p38β shRNA expressing clones. Inhibition of p38α enhanced in vivo tumorigenicity profoundly in Mia Paca-2 and Panc-1 cells. In Mia Paca-2 cell, the in vivo tumor formation rate of two p38β shRNA expressing clones is 18.3% and 50.0% respectively, which decreased significantly comparing to the wild type cell and null vector transfection cell. The tumor volume decreased significantly either (P<0.05). In Panc-1 cell, the in vivo tumor formation rate of two p38β shRNA expressing clones is 25.0% and 12.5% respectively. Conclusion p38 mitogen activated protein kinases (p38MAPKs) are expressed in pancreatic cancer cells and the isoforms may exert different functions. Key words: p38 mitogen-activated protein kinase; Pancreatic cancer; p38α; p38β

Insights on the molecular mechanism of anti-inflammatory effect of formula from Islamic traditional medicine: An in-silico study

Background and aimTraditional medicine is an important source for drug discovery. However, many challenges face the scientific community to develop novel drugs from it. To investigate the rationale behind the medical legacy of centuries of precious knowledge from traditional medicine, we aimed at performing virtual screening to identify potential leads from the middle-age textbook, The Canon of Medicine. Experimental procedureA database of chemical constituents of plants mentioned within the book was built and docked against different molecular targets associated with inflammation such as phospholipase A2, p38 alpha mitogen activated protein kinase, cyclooxygenase-2 and leukotriene B4 dehydrogenase, after that literature survey was done to determine the consistency of traditional uses and molecular docking results with the current knowledge obtained from previous studies and reports. Results and conclusionThe in-silico study revealed the ability of several chemical constituents, in the plants under investigation, to bind effectively to different targets associated with inflammation, which was consistent with previous reports, indicating that Islamic traditional medicine can be considered as a reliable promising source for developing new anti-inflammatory agents with low toxicity and minimal side effects.

Isolates from Alpinia officinarum Hance attenuate LPS‐induced inflammation in HepG2: Evidence from in silico and in vitro studies

In an attempt to connect the legacy of centuries of invaluable knowledge from traditional medicine and the current understanding to the molecular mechanism of diseases, we took the advantage of the emergence of in silico screening as a promising tool for identification of potential leads from libraries of natural products. Traditional Chinese Medicine database was subjected to structure based virtual screening for identification of anti-inflammatory compounds using the 3D crystal structure of p38 alpha mitogen activated protein kinase. The molecular docking studies revealed the potential activity of several classes of compounds known to be the constituents of the rhizomes of Alpinia officinarum Hance (Lesser galangal). Five compounds, galangin, kaempferide, isorhamnetin, and two diarylheptanoids, were isolated from the rhizomes of the plant using vacuum liquid chromatography and flash chromatography techniques. The anti-inflammatory activity of these compounds was investigated on HepG2 cells stimulated by lipopolysaccharide. The latter induced the gene expression of proinflammatory cytokines; interleukin-1β, interleukin-6, tumor necrosis factor alpha. Addition of the 5 isolated compounds downregulated this increased gene expression in a dose dependent manner. Thus, these results indicate that the isolated compounds from A.officinarum could be used as a beneficial source for preventing and treating inflammatory diseases.

Neflamapimod: Clinical Phase 2b-Ready Oral Small Molecule Inhibitor of p38α to Reverse Synaptic Dysfunction in Early Alzheimer's Disease.

Neflamapimod (previously code named VX-745) is a clinical phase 2b-ready highly specific inhibitor of the intra-cellular enzyme p38 mitogen activated protein kinase alpha ("p38α") that is being developed as a disease-modifying drug for Alzheimer's disease (AD) that acts via targeting synaptic dysfunction. Neflamapimod was discovered through a proprietary structure-based drug discovery platform at Vertex Pharmaceuticals, and developed previously by Vertex through to phase 2a in rheumatoid arthritis. EIP Pharma licensed the compound in 2014 for development and commercialization as a treatment of central nervous system (CNS) disorders. Neflamapimod is the most advanced in the clinic drug that targets specific molecular mechanisms within neurons that leads to synaptic dysfunction, the pathogenic process that is now considered to be a major driver of the development of memory deficits and disease progression in the early stages of AD. Based on the scientific rationale of targeting synaptic dysfunction and the preclinical data, neflamapimod has the potential to both reverse memory deficits and slow disease progression. Phase 2a clinical data in patients with early-stage AD (MMSE 20-28, biomarker positive) provides evidence that the preclinical science may be translatable to human Alzheimer's, as 6- to 12-weeks of neflamapimod treatment led to significant improvement in episodic memory, the best clinical measure of synaptic dysfunction in AD. A phase 2b six-month placebo-controlled 150-patient clinical study is anticipated to start by end of 2017. This study is designed to definitively demonstrate that neflamapimod reverses memory deficits, and also to provide preliminary evidence that the drug slows disease progression.

P38 Mitogen Activated Protein Kinase alpha (MAPKα) Inhibition to Promote Neurologic Recovery in Rat Stroke Transient Middle Cerebral Artery Occlusion (t-MCAO) Model (P5.228)

P38 Mitogen Activated Protein Kinase alpha (MAPKα) Inhibition to Promote Neurologic Recovery in Rat Stroke Transient Middle Cerebral Artery Occlusion (t-MCAO) Model (P5.228)

TNFα inhibits IGFBP-3 through activation of p38α and casein kinase 2 in human retinal endothelial cells.

We recently reported a reciprocal relationship between tumor necrosis factor alpha (TNFα) and insulin-like receptor growth factor binding protein 3 (IGFBP-3) in whole retina of normal and IGFBP-3 knockout mice. A similar relationship was also observed in cultured retinal endothelial cells (REC). We found that TNFα significantly reduced IGFBP-3 levels and vice-versa, IGFBP-3 can lower TNFα and TNFα receptor expression. Since IGFBP-3 is protective to the diabetic retina and TNFα is causative in the development of diabetic retinopathy, we wanted to better understand the cellular mechanisms by which TNFα can reduce IGFBP-3 levels. For these studies, primary human retinal endothelial cells (REC) were used since these cells undergo TNFα-mediated apoptosis under conditions of high glucose conditions and contribute to diabetic retinopathy. We first cultured REC in normal or high glucose, treated with exogenous TNFα, then measured changes in potential signaling pathways, with a focus on P38 mitogen-activated protein kinase alpha (P38α) and casein kinase 2 (CK2) as these pathways have been linked to both TNFα and IGFBP-3. We found that TNFα significantly increased phosphorylation of P38α and CK2. Furthermore, specific inhibitors of P38α or CK2 blocked TNFα inhibition of IGFBP-3 expression, demonstrating that TNFα reduces IGFBP-3 through activation of P38α and CK2. Since TNFα and IGFBP-3 are key mediators of retinal damage and protection respectively in diabetic retinopathy, increased understanding of the relationship between these two proteins will offer new therapeutic options for treatment.

22 Identifying the Substrates of P38 MAPK alpha in the Heart

<h3></h3> Activation of p38 mitogen activated protein kinase alpha during myocardial ischaemia results in myocardial damage. Identifying and targeting the relevant substrates downstream of p38alpha may potentially provide a therapeutic target to limit myocardial injury. We aim to identify the substrates of p38alpha particularly during myocardial ischaemia. We have adopted the analogue sensitive kinase strategy, as developed by the Shokat lab, of mutating the kinase to allow it to use expanded forms of ATP that wild type kinases are not capable of using. In addition, these ATP analogues are modified with a gamma thiophosphate instead of the gamma phosphate that is transferred during catalysis. This transferred thiophosphate acts as a label and allows for identification and isolation of the substrates of the kinase. We have characterised an analogue sensitive form of p38 and confirmed it to have a similar substrate specificity to wild type p38alpha. We have successfully optimized the conditions for labelling p38alpha substrates in mouse heart homogenates exposed to the analogue sensitive kinase and the expanded form of ATP. We isolated and used MS to identify the substrates and the sites of phosphorylation. 61 proteins with phosphorylation at p38 consensus sequences were identified and we are currently in the process of validating these results. In conclusion we have optimized the conditions for identification and isolation of substrates of p38 alpha in the mouse heart. In the future, we will use the identification of these substrates to elucidate the mechanism through which p38alpha mediates its effect during myocardial ischaemia.

Disruption of TAB1/p38α Interaction Using a Cell-permeable Peptide Limits Myocardial Ischemia/Reperfusion Injury

Targeting the adaptor protein (transforming growth factor-β (TGF-β)-activated protein kinase 1 (TAK1)-binding protein 1) (TAB1)-mediated non-canonical activation of p38α to limit ischemia/reperfusion (I/R) injury after an acute myocardial infarction seems to be attractive since TAB1/p38α interaction occurs specifically in very limited circumstances and possesses unique structural basis. However, so far no TAB1/p38α interaction inhibitor has been reported due to the limited knowledge about the interfaces. In this study, we sought to identify key amino acids essential for the unique mode of interaction with computer-guided molecular simulations and molecular docking. After validation of the predicted three-dimensional (3-D) structure of TAB1/p38α complex, we designed several peptides and evaluated whether they could block TAB1/p38α interaction with selectivity. We found that a cell-permeable peptide worked as a selective TAB1/p38α interaction inhibitor and decreased myocardial I/R injury. To our knowledge, this is the first TAB1/p38α interaction inhibitor.

Functions of p38 mitogen-activated protein kinase alpha and beta in human pancreatic cancer cells

The p38 family of mitogen-activated protein kinases (p38 MAPKs) includes p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13). Among these four isoforms, p38α and p38β are widely expressed and are involved in regulating cell proliferation, cell differentiation and cell apoptosis. Nothing is presently known about the role of the p38 MAPKs in human pancreatic cancers. In this study, we characterized for the first time the effects of p38α and p38β on human pancreatic cancer cells. Expression of p38α and p38β were determined by immunoblot in 7 cultured human pancreatic cancer cell lines. ShRNA inhibition of endogenous p38α and p38β in Miapaca-2 and Panc-1 cells resulted in decreased anchorage-dependent and -independent growth. Single cell movement and cell invasion were markedly reduced in p38α shRNA expressing clones, but not altered in p38β shRNA expressing clones. And more dramatically, in contrast to in vitro invasion, in vivo tumorigenicity was decreased in p38β shRNA expressing clones rather than p38α shRNA expressing clones. We conclude that p38α and p38β may function synergistically in the tumor genesis of pancreatic cancer. Selective targeting may result in individualized treatment depending on the activity pattern in the future.

Mechanism(s) involved in carbon monoxide-releasing molecule-2-mediated cardioprotection during ischaemia-reperfusion injury in isolated rat heart

The purpose of the present study was to determine the mechanism(s) involved in carbon monoxide-releasing molecule-2, carbon monoxide-releasing molecule-2-induced cardioprotection. We used the transition metal carbonyl compound carbon monoxide-releasing molecule-2 that can act as carbon monoxide donor in cardiac ischaemia-reperfusion injury model using isolated rat heart preparation. Langendorff's perfused rat hearts when treated with carbon monoxide-releasing molecule-2 (50 μM) for 10 min before global ischaemia exhibited significant reduction in postischaemic levels of myocardial injury markers, creatine kinase and lactate dehydrogenase in coronary effluent. Similarly, pretreatment with carbon monoxide-releasing molecule-2 showed significantly improved postischaemic recovery of heart rate, coronary flow rate, cardiodynamic parameters and reduced infarct size as compared to vehicle control hearts. Perfusion with p38 mitogen-activated protein kinase inhibitor, SB203580, a specific inhibitor of α and β isoform, before and concomitantly with carbon monoxide-releasing molecule-2 treatment abolished carbon monoxide-releasing molecule-2-induced cardioprotection. However, p38 mitogen-activated protein kinase alpha inhibitor, SCIO-469, was unable to inhibit the cardioprotective effect of carbon monoxide-releasing molecule-2. Furthermore, protective effect of carbon monoxide-releasing molecule-2 was significantly inhibited by the protein kinase C inhibitor, chelerythrine, when added before and concomitantly with carbon monoxide-releasing molecule-2. It was also observed that, perfusion with phosphatidylinositol 3-kinase inhibitor, wortmannin, before and concomitantly with carbon monoxide-releasing molecule-2 was not able to inhibit carbon monoxide-releasing molecule-2-induced cardioprotection. Interestingly, we observed that wortmannin perfusion before ischaemia and continued till reperfusion significantly inhibited carbon monoxide-releasing molecule-2-mediated cardioprotection. Our findings suggest that the carbon monoxide-releasing molecule-2 treatment may activate the p38 mitogen-activated protein kinase β and protein kinase C pathways before ischaemia and phosphatidylinositol 3-kinase pathway during reperfusion which may be responsible for the carbon monoxide-releasing molecule-2-mediated cardioprotective effect.

Functions of p38 mitogen-activated protein kinase alpha and beta in human pancreatic cancer cells

Background and aims: The p38 family of mitogen-activated protein kinases (p38 MAPKs) includes p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13). Among these four isoforms, p38α and p38β are widely expressed and are involved in regulating cell proliferation, cell differentiation and cell apoptosis. However, little is known about the role of the p38 MAPKs in human cancers. The aim of this study was to elucidate possible functions of p38α and p38β in human pancreatic cancer.

Rapid and quantitative detection of p38 kinase pathway in mouse blood monocyte

The p38 alpha mitogen-activated protein kinase (MAPK) is essential in controlling the production of many proinflammatory cytokines, and its specific inhibitor can effectively block their production for treating human diseases. To effectively identify highly specific p38 alpha inhibitors in vivo, we developed an ex vivo mouse blood cell-based assay by flow cytometry to measure the intracellular p38 alpha kinase activation. We first attempted to identify the individual blood cell population in which the p38 alpha kinase pathway is highly expressed and activated. Based on CD11b, combined with Ly-6G cell surface expression, we identified two distinct subsets of non-neutrophilic myeloid cells, CD11b(Med)Ly-6G(-) and CD11b(Lo)Ly-6G(-), and characterized them as monocytes and natural killer (NK) cells, respectively. Then, we demonstrated that only monocytes, not NK cells, expressed a high level of p38 alpha kinase, which was rapidly activated by anisomycin stimulation as evidenced by the phosphorylation of both p38 and its substrate, MAPKAP-K2 (MK2). Finally, the p38 alpha kinase pathway activation in monocytes was fully inhibited by a highly selective p38 alpha kinase inhibitor dose-dependently in vitro and in vivo. In conclusion, we demonstrated an effective method for separating blood monocytes from other cells and for detecting the expression level and activation of the p38 alpha kinase pathway in monocytes, which provided a new approach for the rapid identification of specific p38 alpha inhibitors.

Dissection of the Recognition Properties of p38 MAP Kinase. Determination of the Binding Mode of a New Pyridinyl−Heterocycle Inhibitor Family

The main recognition characteristics of the ATP binding site of p38 mitogen activated protein kinase alpha (p38alpha MAPK) have been explored by a combination of modeling and bioinformatics techniques, making special emphasis in the characteristics of the site that justifies binding specificity with respect to other MAP kinases. Particularly, we have analyzed the binding mode of a new family of p38 MAPK inhibitors based on the pyridinyl-heterocycle core. This family of compounds has a marked pseudosymmetry and can exist in different tautomeric forms, which makes the determination of the binding mode especially challenging. A combination of homology modeling, quantum mechanics, classical docking, and molecular dynamics calculations allowed us to determine the main characteristics defining the binding mode of this new scaffold in the ATP binding site of p38alpha. A set of free energy calculations allowed us to verify the binding mode proposed, giving an overall excellent agreement with the experimental values. Finally, the binding mode of this new family of compounds was compared to that of other members of the pyridinyl and pyrimidinyl heterocycle class.

Kinetic mechanism for p38 MAP kinase alpha. A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate

p38 Mitogen-activated protein kinase alpha (p38 MAPKalpha) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPKalpha, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1-10) that at pH 7.5, 10 mm Mg2+ and 27 degrees C p38 MAPKalpha phosphorylates ATF2Delta115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (beta,gamma-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPKalpha with dissociation constants of KA = 360 microm, KI = 240 microm, and KI > 2000 microm, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPKalpha-ATF2Delta115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd approximately 70 microm. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2Delta115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a beta-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPKalpha inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations > 100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor.

Activation of p38 mitogen-activated protein kinase alpha and beta by insulin and contraction in rat skeletal muscle: potential role in the stimulation of glucose transport.

The stress-activated p38 mitogen-activated protein kinase (MAPK) was recently shown to be activated by insulin in muscle and adipose cells in culture. Here, we explore whether such stimulation is observed in rat skeletal muscle and whether muscle contraction can also affect the enzyme. Insulin injection (2 U over 3.5 min) resulted in increases in p38 MAPK phosphorylation measured in soleus (3.2-fold) and quadriceps (2.2-fold) muscles. Increased phosphorylation (3.5-fold) of an endogenous substrate of p38 MAPK, cAMP response element binder (CREB), was also observed. After in vivo insulin treatment, p38 MAPKalpha and p38 MAPKbeta isoforms were found to be activated (2.1- and 2.4-fold, respectively), using an in vitro kinase assay, in immunoprecipitates from quadriceps muscle extracts. In vitro insulin treatment (1 nmol/l over 4 min) and electrically-induced contraction of isolated extensor digitorum longus (EDL) muscle also doubled the kinase activity of p38 MAPKalpha and p38 MAPKbeta. The activity of both isoforms was inhibited in vitro by 10 micromol/l SB203580 in all muscles. To explore the possible participation of p38 MAPK in the stimulation of glucose uptake, EDL and soleus muscles were exposed to increasing doses of SB203580 before and during stimulation by insulin or contraction. SB203580 caused a significant reduction in the insulin- or contraction-stimulated 2-deoxyglucose uptake. Maximal inhibition (50-60%) occurred with 10 micromol/l SB203580. These results show that p38 MAPKalpha and -beta isoforms are activated by insulin and contraction in skeletal muscle. The data further suggest that activation of p38 MAPK may participate in the stimulation of glucose uptake by both stimuli in rat skeletal muscle.