RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

BACKGROUND: Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication initiation determinant protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING E3 ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. METHODS: The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. RESULTS: RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. CONCLUSION: This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. Video Abstract.

RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

Background Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication origin binding protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. Methods The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. Results RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. Conclusion This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production.

Heat Treatment Enhances the Neuroprotective Effects of Crude Ginseng Saponin by Increasing Minor Ginsenosides.

Ginsenoside is the primary active substance of ginseng and has many pharmacological effects, such as anti-cancer, immune, regulating sugar and lipid metabolism, and antioxidant effects. It also protects the nervous and cardiovascular systems. This study analyzes the effects of thermal processing on the bioactivities of crude ginseng saponin. Heat treatment increased the contents of minor ginsenosides in crude saponins, such as Rg3, and heat-treated crude ginseng saponin (HGS) had better neuroprotective effects than non-treated crude saponin (NGS). HGS reduced glutamate-induced apoptosis and reactive oxygen species generation in pheochromocytoma 12 (PC12) cells, significantly more than NGS. HGS protected PC12 cells against glutamate-induced oxidative stress by upregulating Nrf2-mediated antioxidant signaling and downregulating MAPK-mediated apoptotic signaling. HGS has the potential for the prevention and treatment of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease.

DAXX ameliorates metabolic dysfunction in mice with diet-induced obesity by activating the AMP-activated protein kinase-related kinase MPK38/MELK.

Death domain-associated protein (DAXX) is involved in the activation of adipocyte apoptosis and is downregulated in response to a high-fat diet (HFD), which implies that the inhibition of adipocyte apoptosis may cause obesity. However, the anti-obesity effects of DAXX in diet-induced obesity (DIO) remain to be characterized. Here, we identified DAXX as an interacting partner of murine protein serine-threonine kinase 38 (MPK38). This interaction was mediated by the C-terminal (amino acids 270-643) domain of MPK38 and the N-terminal (amino acids 1-440) domain of DAXX and was increased by diverse signals that activate ASK1/TGF-ß/p53 signaling. MPK38 phosphorylated DAXX at Thr578. Wild-type DAXX, but not a DAXX T578A mutant, stimulated MPK38-dependent ASK1/TGF-ß/p53 signaling by increasing the stability of MPK38 and complex formation between MPK38 and its downstream targets, such as ASK1, Smad3, and p53. This mechanism was also shown in MEF cells that were null (-/-) for DAXX. Furthermore, the adenovirally-mediated reinstatement of DAXX expression activated MPK38 and ameliorated diet-induced defects in glucose and lipid metabolism in mice. These results indicate that DAXX limits obesity-induced metabolic abnormalities in DIO mice by activating MPK38.

Ablation of AMPK-Related Kinase MPK38/MELK Leads to Male-Specific Obesity in Aged Mature Adult Mice.

Murine protein serine-threonine kinase 38 (MPK38)/maternal embryonic leucine zipper kinase (MELK) is implicated in diverse biological processes, including the cell cycle, apoptosis, and tumorigenesis; however, its physiological role is unknown. Using mice lacking MPK38 (MPK38-/-), we found that MPK38-/- male, but not female, mice (7 months of age) became obese while consuming a standard diet, displayed impairments in metabolism and inflammation, became more obese than wild-type mice while consuming a high-fat diet, and exhibited no castration/testosterone replacement-induced metabolic changes. The adenoviral restoration of MPK38 ameliorated the obesity-induced adverse metabolic profile of the obese male, but not female, mice. Seven-month-old MPK38-/- males displayed typical postcastration concentrations of serum testosterone with an accompanying decrease in serum luteinizing hormone (LH) levels, suggesting a role for MPK38 in the age-related changes in serum testosterone in aged mature adult male mice. The stability and activity of MPK38 were increased by dihydrotestosterone but reduced by estradiol (E2). These findings suggest MPK38 as a therapeutic target for obesity-related metabolic disorders in males.

High dietary oleic acid in olive oil-supplemented diet enhanced omega-3 fatty acid in blood plasma of rats.

This study was conducted to investigate the effect of dietary oleic acid in olive oil-supplemented diets on the blood lipid profile and fatty acid composition in blood plasma and adipose tissue of rats. A total of 60 Sprague Dawley rats with mean body weight of 249 g ± 3.04 g were equally divided into three diet groups: control (CON) contained 10% coconut oil, olive50 contained 5% coconut oil and 5% olive oil, and olive100 contained 10% olive oil. Oleic acid (OA) level was highest in olive100 followed by the olive50 and control. The final body weight (BW) of the rats was significantly affected by the intake of OA, in which rats fed olive100 had the lowest final BW, which signified that OA could be associated with weight loss. Olive oil intake significantly increased levels of the high-density lipoprotein cholesterol (HDL-C) and exhibited a potential attenuation effect on the glutamic-oxaloacetic transaminase and the glutamic-pyruvic transaminase, and a potential role in the reduction of triglycerides in the bloodstream of the animals. In terms of fatty acid composition, significantly high OA was observed in the blood plasma and adipose tissues of rats fed olive100. Omega-3 polyunsaturated fatty acids (PUFAs), such as linolenic (C18:3 n-3), eicosapentaenoic (C20:5 n-3), and docosahexaenoic (C22:6 n-3), and n-6 PUFA arachidonic (C20:4 n-6) were also significantly increased in the blood plasma of rats fed olive100. These findings suggest that the intake of dietary high OA may enhance the omega-3 fatty acid levels in the blood plasma of rats and may have a positive effect in reducing risks to cardiovascular disease, as evidenced by weight loss, increased HDL-C levels, and decreased TG levels in the blood plasma of experimental animals.

Thr55 phosphorylation of p21 by MPK38/MELK ameliorates defects in glucose, lipid, and energy metabolism in diet-induced obese mice.

Murine protein serine-threonine kinase 38 (MPK38)/maternal embryonic leucine zipper kinase (MELK), an AMP-activated protein kinase (AMPK)-related kinase, has previously been shown to interact with p53 and to stimulate downstream signaling. p21, a downstream target of p53, is also known to be involved in adipocyte and obesity metabolism. However, little is known about the mechanism by which p21 mediates obesity-associated metabolic adaptation. Here, we identify MPK38 as an interacting partner of p21. p21 and MPK38 interacted through the cyclin-dependent kinase (CDK) binding region of p21 and the C-terminal domain of MPK38. MPK38 potentiated p21-mediated apoptosis and cell cycle arrest in a kinase-dependent manner by inhibiting assembly of CDK2-cyclin E and CDK4-cyclin D complexes via induction of CDK2-p21 and CDK4-p21 complex formation and reductions in complex formation between p21 and its negative regulator mouse double minute 2 (MDM2), leading to p21 stabilization. MPK38 phosphorylated p21 at Thr55, stimulating its nuclear translocation, which resulted in greater association of p21 with peroxisome proliferator-activated receptor γ (PPARγ), preventing the PPARγ transactivation required for adipogenesis. Furthermore, restoration of p21 expression by adenoviral delivery in diet-induced obese mice ameliorated obesity-induced metabolic abnormalities in a MPK38 phosphorylation-dependent manner. These results suggest that MPK38 functions as a positive regulator of p21, regulating apoptosis, cell cycle arrest, and metabolism during obesity.

Thioredoxin-interacting protein-derived peptide (TN13) inhibits LPS-induced inflammation by inhibiting p38 MAPK signaling.

Inflammation comprises an innate immune response, and is mainly induced by macrophages to protect the host from pathogens and mechanical injuries. The p38 mitogen-activated protein kinase (MAPK) pathway is a key regulator of inflammatory responses in macrophages. Here, we investigated the anti-inflammatory effects of thioredoxin-interacting protein-derived peptide (TN13) in macrophages in vitro and in vivo. Human immunodeficiency virus (HIV) trans-activator protein (TAT)-conjugated TN13 (TAT-TN13) was found to penetrate RAW 264.7 cells and decrease p38 MAPK activation in a dose-dependent manner. We also showed that TAT-TN13 could significantly inhibit lipopolysaccharide (LPS)-induced expression of macrophage activation-related receptors including CD80, CD86, and MHC II, as well as the transcriptional activation of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) in RAW 264.7 cells and primary mouse splenic macrophages. Furthermore, TAT-TN13 decreased the LPS-induced production of proinflammatory cytokines and mediators such as tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), nitric oxide (NO), inducible NO synthase (iNOS), and cyclooxygenase 2 (COX-2) in RAW 264.7 cells and mice. These results indicate that TAT-TN13 can inhibit macrophage-derived inflammation by inhibiting p38 MAPK activity and might represent a potential novel drug for the treatment of inflammation-related diseases.

Dual Roles of Serine-Threonine Kinase Receptor-Associated Protein (STRAP) in Redox-Sensitive Signaling Pathways Related to Cancer Development.

Serine-threonine kinase receptor-associated protein (STRAP) is a transforming growth factor ß (TGF-ß) receptor-interacting protein that has been implicated in both cell proliferation and cell death in response to various stresses. However, the precise roles of STRAP in these cellular processes are still unclear. The mechanisms by which STRAP controls both cell proliferation and cell death are now beginning to be unraveled. In addition to its biological roles, this review also focuses on the dual functions of STRAP in cancers displaying redox dysregulation, where it can behave as a tumor suppressor or an oncogene (i.e., it can either inhibit or promote tumor formation), depending on the cellular context. Further studies are needed to define the functions of STRAP and the redox-sensitive intracellular signaling pathways that enhance either cell proliferation or cell death in human cancer tissues, which may help in the development of effective treatments for cancer.

Smad proteins differentially regulate obesity-induced glucose and lipid abnormalities and inflammation via class-specific control of AMPK-related kinase MPK38/MELK activity.

Smad proteins have been implicated in metabolic processes, but little is known about how they regulate metabolism. Because Smad 2, 3, 4, and 7 have previously been shown to interact with murine protein serine-threonine kinase 38 (MPK38), an AMP-activated protein kinase (AMPK)-related kinase that has been implicated in obesity-associated metabolic defects, we investigated whether Smad proteins regulate metabolic processes via MPK38. Smads2/3/4 increased, but Smad7 decreased, MPK38-mediated apoptosis signal-regulating kinase-1 (ASK1)/transforming growth factor-ß (TGF-ß)/p53 signaling. However, MPK38-mediated phosphorylation-defective Smad mutants (Smad2 S245A, Smad3 S204A, Smad4 S343A, and Smad7 T96A) had no such effect. In addition, Smads2/3/4 increased, but Smad7 decreased, the stability of MPK38. Consistent with this, Smads2/3/4 attenuated complex formation between MPK38 and its negative regulator thioredoxin (Trx), whereas Smad7 increased this complex formation. However, an opposite effect was observed on complex formation between MPK38 and its positive regulator zinc-finger-like protein 9 (ZPR9). When Smads were overexpressed in high-fat diet (HFD)-fed obese mice using an adenoviral delivery system, Smads2/3/4 improved, but Smad7 worsened, obesity-associated metabolic parameters and inflammation in a MPK38 phosphorylation-dependent manner. These findings suggest that Smad proteins have class-specific impacts on obesity-associated metabolism by differentially regulating MPK38 activity in diet-induced obese mice.

Characterization of the changes in eicosanoid profiles of activated macrophages treated with 20(S)-ginsenoside Rg3.

In this study, we used ultra-performance liquid chromatography coupled with tandem mass spectrometry to assess the levels of eicosanoids from RAW264.7 macrophages treated with lipopolysaccharides (LPS) and 20(S)-ginsenoside Rg3 (Rg3). The production of nitric oxide (NO) and the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were increased in inflammatory macrophages treated with LPS. Rg3 treatment, however, decreased the levels of NO, TNF-α, and IL-6 in activated macrophages. Eicosanoids, known as major metabolites correlated with inflammation, have pro- or anti-inflammatory activities. For a detailed characterization of the eicosanoids altered by treatment with LPS and Rg3, the eicosanoids were profiled by multiple reaction monitoring. A total of 69 macrophage eicosanoids were analyzed and the profiling dataset was statistically analyzed. Principal component and hierarchical cluster analyses differentiated control cells from cells treated with LPS, Rg3, or LPS+Rg3 for 12 or 24h. Furthermore, 18 differentially regulated eicosanoids were found between macrophages treated with LPS for 24h and those treated with LPS+Rg3 for 24h (fold change>2, p value<0.05). These results indicate that Rg3 alters eicosanoid metabolism in activated macrophages treated with LPS. Furthermore, we also identified several eicosanoids correlated with the anti-inflammatory activity of Rg3.

Zinc finger protein ZPR9 functions as an activator of AMPK-related serine/threonine kinase MPK38/MELK involved in ASK1/TGF-ß/p53 signaling pathways.

Murine protein serine-threonine kinase 38 (MPK38), an AMP-activated protein kinase (AMPK)-related kinase, has been implicated in the induction of apoptosis signal-regulating kinase 1 (ASK1)-, transforming growth factor-ß (TGF-ß)-, and p53-mediated activity involved in metabolic homeostasis. Here, zinc finger protein ZPR9 was found to be an activator of MPK38. The association of MPK38 and ZPR9 was mediated by cysteine residues present in each of these two proteins, Cys269 and Cys286 of MPK38 and Cys305 and Cys308 of ZPR9. MPK38 phosphorylated ZPR9 at Thr252. Wild-type ZPR9, but not the ZPR9 mutant T252A, enhanced ASK1, TGF-ß, and p53 function by stabilizing MPK38. The requirement of ZPR9 Thr252 phosphorylation was validated using CRISPR/Cas9-mediated ZPR9 (T252A) knockin cell lines. The knockdown of endogenous ZPR9 showed an opposite trend, resulting in the inhibition of MPK38-dependent ASK1, TGF-ß, and p53 function. This effect was also demonstrated in mouse embryonic fibroblast (MEF) cells that were haploinsufficient (+/-) for ZPR9, NIH 3T3 cells with inducible knockdown of ZPR9, and CRISPR/Cas9-mediated ZPR9 knockout cells. Furthermore, high-fat diet (HFD)-fed mice displayed reduced MPK38 kinase activity and ZPR9 expression compared to that in mice on control chow, suggesting that ZPR9 acts as a physiological activator of MPK38 that may participate in obesity.

Coordinate Activation of Redox-Dependent ASK1/TGF-ß Signaling by a Multiprotein Complex (MPK38, ASK1, SMADs, ZPR9, and TRX) Improves Glucose and Lipid Metabolism in Mice.

AIMS: To explore the molecular connections between redox-dependent apoptosis signal-regulating kinase 1 (ASK1) and transforming growth factor-ß (TGF-ß) signaling pathways and to examine the physiological processes in which coordinated regulation of these two signaling pathways plays a critical role. RESULTS: We provide evidence that the ASK1 and TGF-ß signaling pathways are interconnected by a multiprotein complex harboring murine protein serine-threonine kinase 38 (MPK38), ASK1, Sma- and Mad-related proteins (SMADs), zinc-finger-like protein 9 (ZPR9), and thioredoxin (TRX) and demonstrate that the activation of either ASK1 or TGF-ß activity is sufficient to activate both the redox-dependent ASK1 and TGF-ß signaling pathways. Physiologically, the restoration of the downregulated activation levels of ASK1 and TGF-ß signaling in genetically and diet-induced obese mice by adenoviral delivery of SMAD3 or ZPR9 results in the amelioration of adiposity, hyperglycemia, hyperlipidemia, and impaired ketogenesis. INNOVATION AND CONCLUSION: Our data suggest that the multiprotein complex linking ASK1 and TGF-ß signaling pathways may be a potential target for redox-mediated metabolic complications.

A crucial role for the phosphorylation of STRAP at Ser(188) by MPK38 in STRAP-dependent cell death through ASK1, TGF-ß, p53, and PI3K/PDK1 signaling pathways.

Serine-threonine kinase receptor-associated protein (STRAP) is a TGF-ß receptor-interacting protein that participates in the regulation of cell proliferation and cell death in response to various stresses. Here, we demonstrate that STRAP phosphorylation plays an important role in determining the pro- or anti-apoptotic function of STRAP. Murine protein serine/threonine kinase 38 (MPK38) phosphorylates STRAP at Ser(188) via direct interaction. Complex formation between STRAP and MPK38 is mediated by Cys(152) and Cys(270) of STRAP and Cys(339) and Cys(377) of MPK38, suggesting the redox dependency of this interaction. MPK38-mediated STRAP Ser(188) phosphorylation contributes to the pro-apoptotic function of STRAP by modulating key steps in STRAP-dependent ASK1, TGF-ß, p53, and PI3K/PDK1 signaling pathways. Moreover, knockdown of endogenous MPK38 using an inducible MPK38 shRNA system and in vivo activation of MPK38 by treatment of HEK293 and STRAP-null MEF cells with 1-chloro-2,4-dinitrobenzene (DNCB), a specific inhibitor of Trx reductase, provide evidence that STRAP Ser(188) phosphorylation plays a key role in STRAP-dependent cell death. Adenoviral delivery of MPK38 in mice also demonstrates that STRAP Ser(188) phosphorylation in the liver is tightly associated with cell death and proliferation through ASK1, TGF-ß, p53, and PI3K/PDK1 pathways, resulting in apoptotic cell death.

Thioredoxin inhibits MPK38-induced ASK1, TGF-ß, and p53 function in a phosphorylation-dependent manner.

Murine protein serine-threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family. The factors that regulate MPK38 activity and function are not yet elucidated. Here, thioredoxin (Trx) was shown to be a negative regulator of MPK38. The redox-dependent association of MPK38 and Trx was mediated through the C-terminal domain of MPK38. Single and double amino acid substitution mutagenesis of MPK38 (C286S, C339S, C377S, and C339S/C377S) and Trx (C32S, C35S, and C32S/C35S) demonstrated that Cys(339) and Cys(377) of MPK38 and Cys(32) and Cys(35) of Trx are required for MPK38-Trx complex formation. MPK38 directly interacted with and phosphorylated Trx at Thr(76). Expression of wild-type Trx, but not the Trx mutants C32S/C35S and T76A, inhibited MPK38-induced ASK1, TGF-ß, and p53 function by destabilizing MPK38. The E3 ubiquitin-protein ligase Mdm2 played a critical role in the regulation of MPK38 stability by Trx. Treatment of cells with 1-chloro-2,4-dinitrobenzene, a specific inhibitor of Trx reductase, decreased MPK38-Trx complex formation and subsequently increased MPK38 stability and activity, indicating that Trx negatively regulates MPK38 activity in vivo. Finally, we used ASK1-, Smad3-, and p53-null mouse embryonic fibroblasts to demonstrate that ASK1, Smad3, and p53 play important roles in the activity and function of MPK38, suggesting a functional link between MPK38 and ASK1, TGF-ß, and p53 signaling pathways. These results indicate that Trx functions as a physiological inhibitor of MPK38, which plays an important role in inducing ASK1-, TGF-ß-, and p53-mediated activity.

PDK1 protein phosphorylation at Thr354 by murine protein serine-threonine kinase 38 contributes to negative regulation of PDK1 protein activity.

Murine protein serine-threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family, which acts as cellular energy sensors. In this study, MPK38-induced PDK1 phosphorylation was examined to elucidate the biochemical mechanisms underlying phosphorylation-dependent regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) activity. The results showed that MPK38 interacted with and inhibited PDK1 activity via Thr(354) phosphorylation. MPK38-PDK1 complex formation was mediated by the amino-terminal catalytic kinase domain of MPK38 and the pleckstrin homology domain of PDK1. This activity was dependent on insulin, a PI3K/PDK1 stimulator, as well as various apoptotic stimuli, including TNF-α, H(2)O(2), thapsigargin, and ionomycin. MPK38 inhibited PDK1 activity in a kinase-dependent manner and alleviated PDK1-mediated suppression of TGF-ß (or ASK1) signaling, probably via the phosphorylation of PDK1 at Thr(354). In addition, MPK38-mediated inhibition of PDK1 activity was accompanied by the modulation of PDK1 binding to its positive and negative regulators, serine/threonine kinase receptor-associated protein and 14-3-3, respectively. Together, these findings suggest an important role for MPK38-mediated phosphorylation of PDK1 in the negative regulation of PDK1 activity.

Murine protein serine-threonine kinase 38 activates p53 function through Ser15 phosphorylation.

Murine protein serine-threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family. In this study, we show that MPK38 physically associates with p53 via the carboxyl-terminal domain of MPK38 and the central DNA-binding domain of p53. This interaction is increased by 5-fluorouracil or doxorubicin treatment and is responsible for Ser(15) phosphorylation of p53. Ectopic expression of wild-type Mpk38, but not kinase-dead Mpk38, stimulates p53-mediated transcription in a dose-dependent manner and up-regulates p53 targets, including p53, p21, MDM2, and BAX. Consistently, knockdown of MPK38 shows an opposite trend, inhibiting p53-mediated transcription. MPK38 functionally enhances p53-mediated apoptosis and cell cycle arrest in a kinase-dependent manner by stimulating p53 nuclear translocation. We also demonstrate that MPK38-mediated p53 activation is induced by removing MDM2, a negative regulator of p53, from the p53-MDM2 complex as well as by stabilization of interaction between p53 and its positive regulators, including NM23-H1, serine/threonine kinase receptor-associated protein, and 14-3-3. This leads to the enhancement of p53 stability. Together, these results suggest that MPK38 may act as a novel regulator for promoting p53 activity through direct phosphorylation of p53 at Ser(15).

Positive regulation of apoptosis signal-regulating kinase 1 signaling by ZPR9 protein, a zinc finger protein.

A zinc finger protein, ZPR9, has been identified as a physiological substrate of murine protein serine/threonine kinase 38 (MPK38), which is involved in various cellular responses, including the cell cycle, apoptosis, embryonic development, and oncogenesis. Here, ZPR9 was found to physically interact with apoptosis signal-regulating kinase 1 (ASK1) through a disulfide linkage involving Cys(1351) and Cys(1360) of ASK1 and Cys(305) and Cys(308) of ZPR9. ASK1 directly phosphorylated ZPR9 at Ser(314) and Thr(318), suggesting that ZPR9 can act as an ASK1 substrate. Ectopic expression of wild-type ZPR9, but not an S314A/T318A mutant, stimulated ASK1 kinase activity and positively regulated ASK1-mediated signaling to both JNK and p38 kinases by destabilizing complex formation between ASK1 and its negative regulators, Trx and 14-3-3, or by increasing complex formation between ASK1 and its substrate MKK3. ZPR9 functionally stimulated ASK1-induced AP-1 transcriptional activity as well as H(2)O(2)-mediated apoptosis in a phosphorylation-dependent manner. ASK1-mediated phosphorylation of ZPR9 at Ser(314) and Thr(318) was also responsible for ZPR9-induced apoptosis. Moreover, ZPR9 inhibited PDK1-mediated signaling through ASK1 activation. These results suggest that ZPR9 functions as a novel positive regulator of ASK1.

B-MYB positively regulates serine-threonine kinase receptor-associated protein (STRAP) activity through direct interaction.

Serine-threonine kinase receptor-associated protein (STRAP) functions as a regulator of both TGF-ß and p53 signaling. However, the regulatory mechanism of STRAP activity is not understood. In this study, we report that B-MYB is a new STRAP-interacting protein, and that an amino-terminal DNA-binding domain and an area (amino acids 373-468) between the acidic and conserved regions of B-MYB mediate the B-MYB·STRAP interaction. Functionally, B-MYB enhances STRAP-mediated inhibition of TGF-ß signaling pathways, such as apoptosis and growth inhibition, by modulating complex formation between the TGF-ß receptor and SMAD3 or SMAD7. Furthermore, coexpression of B-MYB results in a dose-dependent increase in STRAP-mediated stimulation of p53-induced apoptosis and cell cycle arrest via direct interaction. Confocal microscopy showed that B-MYB prevents the normal translocation of SMAD3 in response to TGF-ß1 and stimulates p53 nuclear translocation. These results suggest that B-MYB acts as a positive regulator of STRAP.

Murine protein serine/threonine kinase 38 stimulates TGF-beta signaling in a kinase-dependent manner via direct phosphorylation of Smad proteins.

The present study demonstrated that murine protein serine/threonine kinase 38 (MPK38) coimmunoprecipitates with Smad proteins (Smad2, -3, -4, and -7) and that this association is mediated by the catalytic kinase domain of MPK38. The association between MPK38 and Smad2, -3, and -4 was significantly increased by TGF-ß or ASK1 signals, whereas these signals decreased association of MPK38 with Smad7. MPK38 stimulated TGF-ß-induced transcription required for TGF-ß-mediated biological functions, such as apoptosis and cell growth arrest, in a kinase-dependent manner. Knockdown of endogenous MPK38 showed an opposite effect, inhibiting TGF-ß signaling. MPK38-mediated phosphorylation of Smad proteins (Ser(245) of Smad2, Ser(204) of Smad3, Ser(343) of Smad4, and Thr(96) of Smad7) was also found to be crucial to the positive regulation of TGF-ß signaling induced by MPK38. In addition, MPK38 enhanced nuclear translocation of Smad3, as well as redistribution of Smad7 from the nucleus to the cytoplasm, in response to TGF-ß. Together, these results indicate that MPK38 functions as a stimulator of TGF-ß signaling through direct interaction with and phosphorylation of Smad proteins.