P38-regulated/activated Protein Kinase Research Articles

The PRAK-NRF2 axis promotes the differentiation of Th17 cells by mediating the redox homeostasis and glycolysis

Oxidative stress is a key feature in both chronic inflammation and cancer. P38 regulated/activated protein kinase (PRAK) deficiency can cause functional disorders in neutrophils and macrophages under high oxidative stress, but the precise mechanisms by which PRAK regulates reactive oxygen species (ROS) elimination and its potential impact on CD4+ T helper subset function are unclear. The present study reveals that the PRAK-NF-E2-related factor 2(NRF2) axis is essential for maintaining the intracellular redox homeostasis of T helper 17(Th17) cells, thereby promoting Th17 cell differentiation and antitumor effects. Through mechanistic analysis, we identify NRF2 as a novel protein substrate of PRAK and find that PRAK enhances the stability of the NRF2 protein through phosphorylation NRF2 Serine(S) 558 independent of protein ubiquitination. High accumulation of cellular ROS caused by loss of PRAK disrupts both glycolysis and PKM2-dependent phosphorylation of STAT3, which subsequently impairs the differentiation of Th17 cells. As a result, Prak knockout (KO) mice display significant resistance to experimental autoimmune encephalomyelitis (EAE) but impaired antitumor immunity in a MC38 tumor model. This work reveals that the PRAK-NRF2-mediated antioxidant pathway is a metabolic checkpoint that controls Th17-cell glycolysis and differentiation. Targeting PRAK is a promising strategy for maintaining an active ROS scavenging system and may lead to potent Th17 cell antitumor immunity.

Ablation of PRAK exacerbates the mitochondria dysfunction in C2C12 myoblasts exposed to palmitate stress

Backgroundp38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating survival and metabolism. However, the role of PRAK in the regulation of palmitate‐induced metabolic stress remains unknown. We examined whether deletion of PRAK would suppress cell survival and mitochondrial function and whether PRAK‐induced detrimental effect was mitigated by irisin in C2C12 myoblasts in response to palmitate (PA) stress.MethodsPRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas‐9 genome editing technology. Both wild type and PRAK‐/‐ C2C12 cells were exposed to palmitate at the concentration of 200 mmol/L for 48 h to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK‐/‐cells was determined to explore its relationship with RPAK in response to palmitate stress. Cell viability, ATP product, glucose uptake, mitochondrial membrane potential and mitochondrial swelling were assessed.ResultsPRAK knockout decreased C2C12 viability in response to PA stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to PA treatment, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential and increasing mitochondrial swelling. However, PRAK knockout induced an impairment of mitochondrial and cell damage in C2C12 cells exposed to PA stress were rescued by treatment of irisin (10ng/ml).ConclusionOur finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function and glucose uptake in C2C12 cells exposed to palmitate induced metabolic stress.

Deletion of PRAK Mitigates the Mitochondria Function and Suppresses Insulin Signaling in C2C12 Myoblasts Exposed to High Glucose.

Background: p38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating cell death and survival. However, the role of PRAK in the regulation of metabolic stress remains unclear. We examined the effects of PRAK on cell survival and mitochondrial function in C2C12 myoblasts in response to high glucose stresses. Methods: PRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas-9 genome editing technology. Both wild type and PRAK−/− C2C12 cells were exposed to high glucose at the concentration of 30 mmol/L to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK−/− cells was determined to explore its relationship with RPAK. Cell viability, ATP product, glucose uptake, mitochondrial damage, and insulin signaling were assessed. Results: PRAK knockout decreased C2C12 viability in response to high glucose stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to high glucose, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential. PRAK knockout induced impairment of mitochondrial and cell damage were rescued by irisin. PRAK knockout caused decrease in phosphorylated PI3 kinase at Tyr 485, IRS-1 and AMPKα and but did not affect non-phosphorylated PI3 kinase, IRS-1 and AMPKα signaling. High glucose caused the further reduction of phosphorylated PI3 kinase, IRS-1 and AMPKα. Irisin treatment preserved phosphorylated PI3 kinase, IRS-1by rescuing PRAK in high glucose treatment. Conclusion: Our finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function, and high glucose stress.

Abstract P312: The Essential Role Of Prak In Preserving Cardiac Function And Insulin Resistance In High Fat Diet-induced Diabetes

Background: p38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating cell death and survival. However, the role of PRAK in mediating cardiac dysfunction and metabolic disorders remains unclear. We examined the effects of deletion of PRAK on modulating cardiac function and insulin resistance in mice exposed to a high fat diet (HFD). Methods: Wild type and PRAK -/- mice at 8 weeks old were exposed to either chow food or HFD for a consecutive 16 weeks. Glucose tolerance test and insulin tolerance test were employed to assess insulin resistance. Echocardiography was employed to assess myocardial function. Western blot was used to determine the molecular signaling involved in phosphorylation of IRS-1, AMPKα, ERK-44/42, and irisin. Real time-PCR was used to assess the hypertrophic genes of the myocardium. Histological analysis was employed to assess the hypertrophic response, interstitial myocardial fibrosis, and apoptosis in the heart. Results: HFD induced metabolic stress is indicated by glucose intolerance and insulin intolerance. PRAK knockout aggravated insulin resistance, as indicated by glucose intolerance and insulin intolerance testing as compared to wild type littermates. As compared to wild type, hyperglycemia and hypercholesterolemia were manifested in PRAK-knockout mice following high fat diet intervention. High fat diet intervention displayed a decline in fractional shortening (FS) and ejection fraction (EF). However, deletion of PRAK exacerbated the decline in EF and FS as compared to wild type mice following HFD treatment. In addition, PRAK knockout mice enhanced the expression of myocardial hypertrophic genes including ANP, BNP, and βMHC in HFD treatment, which was also associated with an increase in cardiomyocyte size and interstitial fibrosis. Western blot indicated that deletion of PRAK induces decreases in phosphorylation of IRS-1, AMPKα, and ERK44/42 as compared to wild type controls. Conclusion: Our finding indicates that deletion of PRAK promoted myocardial dysfunction, cardiac remodeling, and metabolic disorders in response to HFD.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

PRAK Promotes the Pathogen Clearance by Macrophage Through Regulating Autophagy and Inflammasome Activation

The p38 regulated/activated protein kinase (PRAK) is a protein kinase downstream of p38MAPK. The present study investigated its function in the macrophage. Myeloid-specific deletion of Prak resulted in a significant reduction in F4/80+CD11b+ peritoneal macrophages with decreased expression of MHC-II and CD80. Upon infection with Listeria monocytogenes, Prak-deficient mice demonstrated an increased mortality, which was accompanied by a higher bacterial load in multiple tissues and elevated levels of proinflammatory cytokines in the serum. While the Prak-deficient macrophage showed similar potency in phagocytosis assays, its bactericidal activity was severely impaired. Moreover, Prak deficiency was associated with defects in ROS production, inflammasome activation as well as autophagy induction. Therefore, PRAK critically contributes to the clearance of intracellular pathogens by affecting multiple aspects of the macrophage function.

P38-Regulated/activated protein kinase plays a pivotal role in protecting heart against ischemia-reperfusion injury and preserving cardiac performance.

p38-Regulated/activated protein kinase (PRAK) plays a critical role in modulating cellular survival and biological function. However, the function of PRAK in the regulation of myocardial ischemic injury remains unknown. This study is aimed at determining the function of PRAK in modulating myocardial ischemia-reperfusion injury and myocardial remodeling following myocardial infarction. Hearts were isolated from adult male homozygous PRAK-/- and wild-type mice and subjected to global ischemia-reperfusion injury in Langendorff isolated heart perfusion. PRAK-/- mice mitigated postischemic ventricular functional recovery and decreased coronary effluent. Moreover, the infarct size in the perfused heart was significantly increased by deletion of PRAK. Western blot showed that deletion of PRAK decreased the phosphorylation of ERK1/2. Furthermore, the effect of deletion of PRAK on myocardial function and remodeling was also examined on infarcted mice in which the left anterior descending artery was ligated. Echocardiography indicated that PRAK-/- mice had accelerated left ventricular systolic dysfunction, which was associated with increased hypertrophy in the infarcted area. Deletion of PRAK augmented interstitial fibrosis and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL)-positive myocytes. Furthermore, immunostaining analysis shows that CD31-postive vascular density and α-smooth muscle actin capillary staining decreased significantly in PRAK-/- mice. These results indicate that deletion of PRAK enhances susceptibility to myocardial ischemia-reperfusion injury, attenuates cardiac performance and angiogenesis, and increases interstitial fibrosis and apoptosis in the infarcted hearts.

Sodium Butyrate Protects -Against High Fat Diet-Induced Cardiac Dysfunction and Metabolic Disorders in Type II Diabetic Mice.

Histone deacetylases are recently identified to act as key regulators for cardiac pathophysiology and metabolic disorders. However, the function of histone deacetylase (HDAC) in controlling cardiac performance in Type II diabetes and obesity remains unknown. Here, we determine whether HDAC inhibition attenuates high fat diet (HFD)-induced cardiac dysfunction and improves metabolic features. Adult mice were fed with either HFD or standard chow food for 24 weeks. Starting at 12 weeks, mice were divided into four groups randomly, in which sodium butyrate (1%), a potent HDAC inhibitor, was provided to chow and HFD-fed mice in drinking water, respectively. Glucose intolerance, metabolic parameters, cardiac function, and remodeling were assessed. Histological analysis and cellular signaling were examined at 24 weeks following euthanization of mice. HFD-fed mice demonstrated myocardial dysfunction and profound interstitial fibrosis, which were attenuated by HDAC inhibition. HFD-induced metabolic syndrome features insulin resistance, obesity, hyperinsulinemia, hyperglycemia, lipid accumulations, and cardiac hypertrophy, these effects were prevented by HDAC inhibition. Furthermore, HDAC inhibition attenuated myocyte apoptosis, reduced production of reactive oxygen species, and increased angiogenesis in the HFD-fed myocardium. Notably, HFD induced decreases in MKK3, p38, p38 regulated/activated protein kinase (PRAK), and Akt-1, but not p44/42 phosphorylation, which were prevented by HDAC inhibition. These results suggest that HDAC inhibition plays a critical role to preserve cardiac performance and mitigate metabolic disorders in obesity and diabetes, which is associated with MKK3/p38/PRAK pathway. The study holds promise in developing a new therapeutic strategy in the treatment of Type II diabetic-induced heart failure and metabolic disorders. J. Cell. Biochem. 118: 2395-2408, 2017. © 2017 Wiley Periodicals, Inc.

The novel RAGE interactor PRAK is associated with autophagy signaling in Alzheimer's disease pathogenesis.

BackgroundThe receptor for advanced glycation end products (RAGE) has been found to interact with amyloid β (Aβ). Although RAGE does not have any kinase motifs in its cytosolic domain, the interaction between RAGE and Aβ triggers multiple cellular signaling involved in Alzheimer’s disease (AD). However, the mechanism of signal transduction by RAGE remains still unknown. Therefore, identifying binding proteins of RAGE may provide novel therapeutic targets for AD.ResultsIn this study, we identified p38-regulated/activated protein kinase (PRAK) as a novel RAGE interacting molecule. To investigate the effect of Aβ on PRAK mediated RAGE signaling pathway, we treated SH-SY5Y cells with monomeric form of Aβ. We demonstrated that Aβ significantly increased the phosphorylation of PRAK as well as the interaction between PRAK and RAGE. We showed that knockdown of PRAK rescued mTORC1 inactivation induced by Aβ treatment and decreased the formation of Aβ-induced autophagosome.ConclusionsWe provide evidence that PRAK plays a critical role in AD pathology as a key interactor of RAGE. Thus, our data suggest that PRAK might be a potential therapeutic target of AD involved in RAGE-mediated cell signaling induced by Aβ.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0068-5) contains supplementary material, which is available to authorized users.

New insights into the activation, interaction partners and possible functions of MK5/PRAK.

MAP kinase-activated protein kinase 5 (MK5) was first described as a downstream target of the p38 MAP kinase pathway leading to its alternative acronym of p38-regulated/activated protein kinase (PRAK). However, since the discovery that MK5 is a bona fide interaction partner of the atypical MAP kinases ERK3 and ERK4 and that this interaction leads to both the activation and subcellular relocalisation of MK5, there has been considerable debate as to the relative roles of these MAPK pathways in mediating the activation and biological functions of MK5. Here we discuss recent progress in defining novel upstream components of the ERK3/ERK4 signalling pathway, our increased understanding of the mechanism by which MK5 interacts with and is activated by ERK3 and ERK4, and the discovery of novel interaction partners for MK5. Finally, we review recent literature that suggests novel biological functions for MK5 in a range of physiological and pathophysiological conditions including neuronal function and cancer.

Rheb phosphorylation is involved in p38-regulated/activated protein kinase-mediated tumor suppression in liver cancer.

Ras homolog enriched in brain (Rheb) is a key regulator of mammalian target of rapamycin complex 1 (mTORC1). The Rheb-mTORC1 axis is a pivotal pathway that mediates cell growth. It was previously reported that upon energy-stress stimulation, the phosphorylation of Rheb at serine 130 by p38-regulated/activated protein kinase (PRAK) results in the impaired nucleotide binding ability of Rheb and inhibits Rheb-mediated mTORC1 activation. However, the role of Rheb phosphorylation in cancer development remains to be elucidated. The aim of the present study was to determine the effect of Rheb phosphorylation on tumor growth in vitro and in vivo. In addition, tissue samples were obtained from 70 hepatocellular carcinoma (HCC) patients in order to determine any associations between Rheb phosphorylation and the clinicopathological characteristics of patients. In vitro and ex vivo kinase assays were performed to determine the phosphorylation of Rheb by PRAK. A xenograft assay was performed to assess tumorigenicity of MEF cell lines. In addition, western blot and immunohistochemical analyses were performed to detect Rheb protein expression and phosphorylation. The results of the present study revealed that Rheb phosphorylation may be induced through Ras overexpression. In addition, kinase-dead PRAK and dominant-negative PRAK mutation were demonstrated to abolish the Rheb phosphorylation induced by Ras overexpression. Xenograft assays in nude mice revealed that Rheb phosphorylation was involved in PRAK-mediated tumor suppression. Of note, the clinicopathological analysis of 70 HCC samples determined that Rheb phosphorylation was associated with poor proliferation and the progression of HCC. In conclusion, the results of the present study suggested that Rheb phosphorylation may have an important role as an intracellular barrier to cancer development.

MK5 is degraded in response to doxorubicin and negatively regulates doxorubicin-induced apoptosis in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The mechanisms by which hepatoma cells resist apoptosis induced by doxorubicin are largely unknown. MAPKAPK5 (MK5), also named as p38-regulated/activated protein kinase (PRAK), has been identified as a crucial mediator of skin tumorigenesis in mouse and colon cancerogenesis in human. Here, we describe a novel role of MK5 in doxorubicin-induced apoptosis in human hepatoma cells. Expression of MK5 was highly upregulated in hepatoma cell lines. Doxorubicin rather than other chemotherapeutic drugs reduced MK5 protein level in a time- and concentration-dependent manner in hepatoma cells (HepG2 and Hep3B). We further showed that MK5 degradation induced by doxorubicin was via the 26S proteasome. Remarkably, stable overexpression of MK5 led to decreased cleavage of caspase-3 and PARP and attenuated doxorubicin-induced apoptosis, while stable knockdown of endogenous MK5 sensitized hepatoma cells to doxorubicin, which was coupled with increased cleavage of caspase-3 and PARP. Taken together, our results firstly demonstrate that MK5 is degraded in response to doxorubicin and negatively regulates doxorubicin-induced apoptosis, providing novel insights into the molecular mechanism of doxorubicin resistance in hepatoma cells.

Rottlerin-Mediated Inhibition of Chlamydia trachomatis Growth and Uptake of Sphingolipids Is Independent of p38-Regulated/Activated Protein Kinase (PRAK)

We previously found that rottlerin, a plant-derived small molecule compound, profoundly inhibited Chlamydia trachomatis growth and blocked sphingolipid trafficking from host cell Golgi into chlamydial inclusions. Since the p38-regulated/activated protein kinase (PRAK) is a known target of rottlerin and is activated in Chlamydia trachomatis-infected cells, we investigated the potential role of this kinase in rottlerin-mediated anti-chlamydial activity. However, we found that a PRAK-specific inhibitor failed to inhibit chlamydial growth, suggesting that the kinase activity of PRAK may not be required for chlamydial intracellular replication. This conclusion was supported by the observation that chlamydial organisms replicated equally well in mouse embryonic fibroblast cells with or without PRAK. Moreover, neither the PRAK inhibitor nor PRAK deficiency altered host sphingolipid trafficking into chlamydial inclusions. Finally, rottlerin maintained its anti-chlamydial activity in PRAK-deficient cells. Together, these observations have demonstrated that PRAK is not required for either the rottlerin-mediated anti-chlamydial activity or rottlerin inhibition of sphingolipid trafficking, suggesting that rottlerin may achieve its inhibitory role by targeting other host factors.

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases (MAPKs) are a family of proteins that constitute signaling pathways involved in processes that control gene expression, cell division, cell survival, apoptosis, metabolism, differentiation and motility. The MAPK pathways can be divided into conventional and atypical MAPK pathways. The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases, MAPK kinase, and MAPK. Atypical MAPK pathways are not organized into this three-tiered cascade. MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases. The latter are referred to as MAPK-activated protein kinases. This review focuses on one such MAPK-activated protein kinase, MAPK-activated protein kinase 5 (MK5) or p38-regulated/activated protein kinase (PRAK). This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways. Recent findings on the regulation of the activity and subcellular localization, bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

Comment on “Increased MKK4 Abundance with Replicative Senescence Is Linked to the Joint Reduction of Multiple MicroRNAs”

Marasa et al. (Research Article, 27 October 2009, DOI: 10.1126/scisignal.2000442) reported that the human kinase p38-regulated/activated protein kinase (PRAK) was phosphorylated on residue Ser(93) in senescent cells. We have been unable to detect phosphorylation at this site with the antibody that they used, and the commercial supplier of this antibody has discontinued its availability, which casts doubt on whether this residue of PRAK is phosphorylated.

Mechanisms regulating the nuclear translocation of p38 MAP kinase

p38 mitogen-activated protein kinase (MAPK) is of fundamental importance in a cell's response to environmental stresses, cytokines and DNA damage. p38 resides in the cytoplasm of resting cells, and translocates into the nucleus upon activation, yet the exact mechanisms remain largely unclear. We show here that the phosphorylation-dependent nuclear translocation of p38 is a common phenomenon when cells are stimulated with various stresses. On the other hand, the nuclear export of p38 requires its dephosphorylation, and it is exported both in a MK2-dependent and a nuclear export signal (NES)-independent manner. Although different p38-regulated/activated protein kinase (PRAK) mutants all dictate the intracellular localization of p38, results from a PRAK-deficient cell line indicate that it plays no role in this process. Microtubule depolymerizing reagent nocodazole and dynein inhibitor EHNA both block the nuclear translocation of p38, demonstrating roles for microtubules and dynein in p38 transport. Taken together, stress-induced nuclear accumulation of p38 is a phosphorylation-dependent, microtubule- and dynein-associated process.

Effect of PRAK gene knockout on the proliferation of mouse embryonic fibroblasts

p38 regulated/activated protein kinase (PRAK) plays a key role in cell senescence and tumor suppression. The aim of this study was to investigate if PRAK had effect on cell proliferation. The growth of PRAK +/+ and PRAK −/− mouse embryonic fibroblast (MEF) cells was measured by methylthiazoletetrazolium (MTT) colorimetric assay, and the proportion of the cell number in different phases of the cell cycle was analyzed by flow cytometry. The growth curves showed that the growth rate was notably decreased, and cell double time was elongated in PRAK −/− cells; moreover, the number of PRAK −/− cells was decreased by 44.5% compared with that of PRAK +/+ cells cultured for 96 h, suggesting that G2/M transition is inhibited in PRAK −/− cells. Meanwhile, G1/S transition was also inhibited in PRAK −/− cells, observed with flow cytometry analysis. The ratios of G0/G1, G2/M, and S phases of PRAK +/+ cells were 44.9%, 12.2%, and 42.9%, respectively, while those of PRAK −/− cells were 55.3%, 7.3%, and 37.4%, respectively. There were 23.1% increase and 12.7% decrease of the number of PRAK −/− cells in G1 and S phases in comparison with that of PRAK +/+ cells, respectively. Taken together, PRAK gene knockout in MEF cells leads to cell cycle arrest and proliferation inhibition.

Does MK5 reconcile classical and atypical MAP kinases?

MAP kinase-activated protein kinase 5 (MK5) was originally described as a protein kinase activated downstream of the p38 MAP kinase and is also named p38-regulated/activated protein kinase (PRAK). However, while MK5 is most similar in sequence to the two p38 regulated MAPKAP kinases MK2 and MK3, recent data has shown that in contrast to these enzymes MK5 is not activated in response to either cellular stress or pro-inflammatory cytokines. This lack of response to stimuli which cause robust activation of p38 MAP kinase in vivo is supported by data obtained using transgenic mice lacking MK5. Unlike animals lacking MK2 and MK3, MK5 null mice respond normally to endotoxic shock and display an unchanged pattern of cytokine expression in response to LPS. Clues as to the physiological function of MK5 have come from the recent observation that MK5 is uniquely regulated and activated following complex formation with the atypical MAP kinases ERK3 and ERK4. Thus, it is possible that MK5 is unique amongst the MAPKAP kinases in being regulated downstream of signaling pathways other than the classical MAP kinases p38 and ERK1/2.

PRAK Is Essential for ras-Induced Senescence and Tumor Suppression

Like apoptosis, oncogene-induced senescence is a barrier to tumor development. However, relatively little is known about the signaling pathways mediating the senescence response. p38-regulated/activated protein kinase (PRAK) is a p38 MAPK substrate whose physiological functions are poorly understood. Here we describe a role for PRAK in tumor suppression by demonstrating that PRAK mediates senescence upon activation by p38 in response to oncogenic ras. PRAK deficiency in mice enhances DMBA-induced skin carcinogenesis, coinciding with compromised senescence induction. In primary cells, inactivation of PRAK prevents senescence and promotes oncogenic transformation. Furthermore, we show that PRAK activates p53 by direct phosphorylation. We propose that phosphorylation of p53 by PRAK following activation of p38 MAPK by ras plays an important role in ras-induced senescence and tumor suppression.

Oncogene-Induced Senescence Pathways Weave an Intricate Tapestry

The induction of cellular senescence by activated oncogenes acts as a barrier to cell transformation. Now, identify a key component of a senescence pathway that prevents tumorigenesis in a mouse model of skin cancer. They show that the p38-regulated/activated protein kinase (PRAK) induces senescence downstream of oncogenic Ras by directly phosphorylating and activating the tumor-suppressor protein p53.