Murine Protein Serine-threonine Kinase 38 Research Articles

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-β/p53 signaling. MPK38 phosphorylated DAXX at Thr578. Wild-type DAXX, but not a DAXX T578A mutant, stimulated MPK38-dependent ASK1/TGF-β/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.

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.

Zinc finger protein ZPR9 functions as an activator of AMPK-related serine/threonine kinase MPK38/MELK involved in ASK1/TGF-\u03b2/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-β (TGF‐β)-, 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‐β, 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‐β, 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-β Signaling by a Multiprotein Complex (MPK38, ASK1, SMADs, ZPR9, and TRX) Improves Glucose and Lipid Metabolism in Mice.

To explore the molecular connections between redox-dependent apoptosis signal-regulating kinase 1 (ASK1) and transforming growth factor-β (TGF-β) signaling pathways and to examine the physiological processes in which coordinated regulation of these two signaling pathways plays a critical role. We provide evidence that the ASK1 and TGF-β 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-β activity is sufficient to activate both the redox-dependent ASK1 and TGF-β signaling pathways. Physiologically, the restoration of the downregulated activation levels of ASK1 and TGF-β 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. Our data suggest that the multiprotein complex linking ASK1 and TGF-β signaling pathways may be a potential target for redox-mediated metabolic complications.

Coordinate regulation of ASK1/TGF‐β signaling contributes to improved glucose and lipid metabolism in mice

We explore the molecular connections between ASK1 and TGF‐β signaling pathways and examine the physiological processes in which coordinate regulation of these two signaling pathways plays a critical role. ASK1 and TGF‐β signaling pathways are interconnected by complex containing Smad proteins (Smad2, 3, 4, and 7) and ZPR9, a zinc finger protein, under the control of apoptosis signal‐regulating kinase 1 (ASK1) and its upstream kinase, murine protein serine‐threonine kinase 38 (MPK38), and a signal for either ASK1 or TGF‐β activity is sufficient to activate both ASK1 and TGF‐β signaling pathways via phosphorylation of Smad(s) and ZPR9 by ASK1 and MPK38. The Smad(s)‐ZPR9 complex is required for the coordinate regulation of ASK1 and TGF‐β signaling in response to ASK1 or TGF‐β signals. We also defined Smad(s) as differential regulators for ASK1 and its downstream targets (MKK3, p38, and JNK), as well as ZPR9 as a regulator of Smad(s) stability. In the physiological setting, restoration of down‐regulated ASK1 and TGF‐β signaling in a genetic mouse model of obesity by adenoviral delivery of Smad3 or ZPR9 resulted in the amelioration of hyperglycemia and hyperlipidemia.

The crystal structure of MPK38 in complex with OTSSP167, an orally administrative MELK selective inhibitor

Murine protein serine/threonine kinase 38 (MPK38), also known as maternal embryonic leucine zipper kinase (MELK), has been associated with various human cancers and plays an important role in the formation of cancer stem cells. OTSSP167, a MELK selective inhibitor, exhibits a strong in vitro activity, conferring an IC50 of 0.41nM and in vivo effect on various human cancer xenograft models. Here, we report the crystal structure of MPK38 (T167E), an active mutant, in complex with OTSSP167 and describe its detailed protein-inhibitor interactions. Comparison with the previous determined structure of MELK bound to the nanomolar inhibitors shows that OTSSP167 effectively fits into the active site, thus offering an opportunity for structure-based development and optimization of MELK inhibitors.

The structures of the kinase domain and UBA domain of MPK38 suggest the activation mechanism for kinase activity.

Murine protein serine/threonine kinase 38 (MPK38) is the murine orthologue of human maternal embryonic leucine-zipper kinase (MELK), which belongs to the SNF1/AMPK family. MELK is considered to be a promising drug target for anticancer therapy because overexpression and hyperactivation of MELK is correlated with several human cancers. Activation of MPK38 requires the extended sequence (ExS) containing the ubiquitin-associated (UBA) linker and UBA domain and phosphorylation of the activation loop. However, the activation mechanism of MPK38 is unknown. This paper reports the crystal structure of MPK38 (T167E), which mimics a phosphorylated state of the activation loop, in complex with AMP-PNP. In the MPK38 structure, the UBA linker forces an inward movement of the αC helix. Phosphorylation of the activation loop then induces movement of the activation loop towards the C-lobe and results in interlobar cleft closure. These processes generate a fully active state of MPK38. This structure suggests that MPK38 has a similar molecular mechanism regulating activation as in other kinases of the SNF1/AMPK family.

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).

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-β (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.

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.

Murine Protein Serine/Threonine Kinase 38 Stimulates TGF-β 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-β or ASK1 signals, whereas these signals decreased association of MPK38 with Smad7. MPK38 stimulated TGF-β-induced transcription required for TGF-β-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-β 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-β 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-β. Together, these results indicate that MPK38 functions as a stimulator of TGF-β signaling through direct interaction with and phosphorylation of Smad proteins.

Reciprocal Negative Regulation of PDK1 and ASK1 Signaling by Direct Interaction and Phosphorylation

Cell survival and death-inducing signals are tightly associated with each other, and the decision as to whether a cell survives or dies is determined by controlling the relationship between these signals. However, the mechanism underlying the reciprocal regulation of such signals remains unclear. In this study, we reveal a functional association between PDK1 (3-phosphoinositide-dependent protein kinase 1), a critical mediator of cell survival, and ASK1 (apoptosis signal-regulating kinase 1), an apoptotic stress-activated MAPKKK. The physical association between PDK1 and ASK1 is mediated through the pleckstrin homology domain of PDK1 and the C-terminal regulatory domain of ASK1 and is decreased by ASK1-activating stimuli, such as H(2)O(2), tumor necrosis factor alpha, thapsigargin, and ionomycin, as well as insulin, a PDK1 stimulator. Wild-type PDK1, but not kinase-dead PDK1, negatively regulates ASK1 activity by phosphorylating Ser(967), a binding site for 14-3-3 protein, on ASK1. PDK1 functionally suppresses ASK1-mediated AP-1 transactivation and H(2)O(2)-mediated apoptosis in a kinase-dependent manner. On the other hand, ASK1 has been shown to inhibit PDK1 functions, including PDK1-mediated regulation of apoptosis and cell growth, by phosphorylating PDK1 at Ser(394) and Ser(398), indicating that these putative phosphorylation sites are involved in the negative regulation of PDK1 activity. These results provide evidence that PDK1 and ASK1 directly interact and phosphorylate each other and act as negative regulators of their respective kinases in resting cells.

Murine Protein Serine/Threonine Kinase 38 Activates Apoptosis Signal-regulating Kinase 1 via Thr838 Phosphorylation

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H(2)O(2) or tumor necrosis factor alpha treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr(838) phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H(2)O(2)-mediated apoptosis by enhancing the ASK1 activity through Thr(838) phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.

Phosphorylation of a novel zinc-finger-like protein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38)

We have identified previously a new murine protein serine/threonine kinase, MPK38, closely related to the sucrose-non-fermenting protein kinase family [Gil, Yang, Lee, Choi and Ha (1997) Gene 195, 295–301]. Using the C-terminal half of the putative human counterpart of MPK38, HPK38, as a bait in a yeast two-hybrid screen of a human HeLa cDNA library, it was discovered that the zinc-finger-motif-containing protein, termed zinc-finger-like protein 9 (ZPR9), bound both HPK38 and MPK38. In a co-expression assay, ZPR9 associated with MPK38 in vivo, and we showed that the ZPR9 is also phosphorylated by MPK38. In addition, ZPR9 physically interacts with itself in mammalian cells. The ZPR9 cDNA hybridized with a mRNA species of approx. 1.7kb in Northern-blot analysis. The ZPR9 transcript was detected in all tissues examined, including lung, kidney, spleen, liver and brain. Co-expression of ZPR9 with MPK38 caused the accumulation of ZPR9 in the nucleus. These findings suggest a potentially important role for ZPR9 in MPK38-mediated signal transduction, and that ZPR9 is a physiological substrate of MPK38 in vivo.

Phosphorylation of a novel zinc-finger-like protein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38).

We have identified previously a new murine protein serine/threonine kinase, MPK38, closely related to the sucrose-non-fermenting protein kinase family [Gil, Yang, Lee, Choi and Ha (1997) Gene 195, 295-301]. Using the C-terminal half of the putative human counterpart of MPK38, HPK38, as a bait in a yeast two-hybrid screen of a human HeLa cDNA library, it was discovered that the zinc-finger-motif-containing protein, termed zinc-finger-like protein 9 (ZPR9), bound both HPK38 and MPK38. In a co-expression assay, ZPR9 associated with MPK38 in vivo, and we showed that the ZPR9 is also phosphorylated by MPK38. In addition, ZPR9 physically interacts with itself in mammalian cells. The ZPR9 cDNA hybridized with a mRNA species of approx. 1.7 kb in Northern-blot analysis. The ZPR9 transcript was detected in all tissues examined, including lung, kidney, spleen,liver and brain. Co-expression of ZPR9 with MPK38 caused the accumulation of ZPR9 in the nucleus. These findings suggest a potentially important role for ZPR9 in MPK38-mediated signal transduction, and that ZPR9 is a physiological substrate of MPK38 in vivo.