Mammalian Kinase Research Articles

P-049 Verteporfin suppresses cell proliferation, survival and migration of TCam-2 Human Seminoma Cells via inhibits the YAP-TEAD complex

Abstract Study question Does verteporfin administration effect on proliferation, survival and migration of TCam-2 human seminoma cells? Summary answer Verteporfin may be a promising anticancer agent for TCam-2 human seminoma cells via inhibition of cell viability, proliferation, migration and stimulation of apoptosis. What is known already Seminoma is germ cell tumor of the testis and TCam-2 cells is the first seminoma-derived cell line that retains many of the characteristic traits of seminoma. The Hippo signalling is a evolutionarily highly conserved pathway that controls organ size, tissue homeostasis, and cancer development. In mammals, the Hippo pathway consists of the serine/threonine kinases Mammalian Sterile 20-protein Kinase 1 and 2 (MST1/2), Large Tumor Suppressor Homologues 1 and 2 (LATS1/2), Yes-Associated Protein (YAP), and its transcriptional cofactor TAZ. Verteporfin inhibits YAP–TEAD complex, blocking cell proliferation and survival. Study design, size, duration TCam-2 cells were treated with different concentrations of verteporfin (1, 5, 10, 20 and 40 µM) for 24, 48 and 72 hours (h). Participants/materials, setting, methods TCam-2 cells were treated with verteporfin at different doses and incubation times. Afterwards, protein expression of Hippo pathway proteins was determined by western blot, mRNA levels were analyzed by qRT-PCR and protein localizations were evaluated by immunoflourescence staining. Cell migration was determinated with wound-healing scratch assay; cell viability, early-late apoptosis and necrosis rates were evaluated by flow cytometry. Main results and the role of chance We detected that 40 μM verteporfin decreased LATS1/2 and p-LATS1/2 protein expression after 48h and 72h treatment (p < 0.05, **p<0.01). MST1/2 and p-MST1/2 was significantly decreased in groups treated with 20 and 40 µM verteporfin after 48 and 72 h of treatment (p < 0.05, ****p<0.0001). YAP expression decreased at 5, 20 and 40 μM verteporfin treatment after 48h and all verteporfin concentrations after 72h (**p<0.01, ***p<0.001, ****p<0.0001). p-YAP expression significantly decreased at all verteporfin concentrations after 48h and only at 40 μM concentrations after 72h (**p<0.01). TEAD4 expression decreased at all verteporfin concentrations after 48 and 72h (****p<0.0001). After 48 and 72h of verteporfin treatment, no significant difference was observed in the expression of the MST1, MST2, LATS1, LATS2 and YAP1 mRNA levels in TCam-2 cells, while TEAD4 mRNA expression level was significantly decreased at 20 and 40 µM compared to control (**p<0.01, ***p<0.001). We also showed that Hippo signaling pathway proteins localized in the cell nucleus especially YAP1 and TEAD4, were translocated to cytoplasm depending on the increasing dose of verteporfin in TCam-2 cells. Verteporfin decreased cell proliferation (****p<0.0001), migration and cell viability (****p<0.0001), as well as increased the early-late apoptosis (**p<0.01, ***p<0.001, ****p<0.0001) in TCam-2 human seminoma cells dose-dependent manner. Limitations, reasons for caution The TCam-2 cell line was gifted by Prof. Dr. Hubert Schorle (Department of Developmental Pathology and Institute of Molecular Diagnostic Pathology, Faculty of Medicine, Germany, Bonn) to our laboratory. There is no limitation in the scope of the study. Wider implications of the findings We determined for the first time the effect of verteporfin treatment on the protein expression and mRNA levels of Hippo signaling pathway in TCam-2 human seminoma cells. We suggested that Hippo pathway and verteporfin relationship may be a therapeutic target for seminoma by affecting cell proliferation, survival, migration and apoptosis. Trial registration number not applicable

Crystal structure of the kinase domain of a receptor tyrosine kinase from a choanoflagellate, Monosiga brevicollis.

Genomic analysis of the unicellular choanoflagellate, Monosiga brevicollis (MB), revealed the remarkable presence of cell signaling and adhesion protein domains that are characteristically associated with metazoans. Strikingly, receptor tyrosine kinases, one of the most critical elements of signal transduction and communication in metazoans, are present in choanoflagellates. We determined the crystal structure at 1.95 Å resolution of the kinase domain of the M. brevicollis receptor tyrosine kinase C8 (RTKC8, a member of the choanoflagellate receptor tyrosine kinase C family) bound to the kinase inhibitor staurospaurine. The chonanoflagellate kinase domain is closely related in sequence to mammalian tyrosine kinases (~ 40% sequence identity to the human Ephrin kinase domain EphA3) and, as expected, has the canonical protein kinase fold. The kinase is structurally most similar to human Ephrin (EphA5), even though the extracellular sensor domain is completely different from that of Ephrin. The RTKC8 kinase domain is in an active conformation, with two staurosporine molecules bound to the kinase, one at the active site and another at the peptide-substrate binding site. To our knowledge this is the first example of staurospaurine binding in the Aurora A activation segment (AAS). We also show that the RTKC8 kinase domain can phosphorylate tyrosine residues in peptides from its C-terminal tail segment which is presumably the mechanism by which it transmits the extracellular stimuli to alter cellular function.

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

The C-terminus of WNK1 kinase is evolutionarily conserved and mediates unique phase behavior

With-no-lysine 1 (WNK1) is a serine-threonine kinase with two isoforms, long (L)-WNK1 and kidney-specific (KS)-WNK1. L-WNK1 is an ancient kinase that is activated by hypertonic stress. Alternatively, KS-WNK1 emerged more recently, it is only expressed in the kidney, it is regulated by K+, and it lacks a functional kinase domain. Both WNK1 isoforms contain identical large C-term domains (CTD) with intrinsically disordered regions (IDRs) that drive the protein into discrete puncta. Our research has identified these puncta as membraneless biomolecular condensates that form via phase separation. In cells these condensates are visible at physiologic levels of hypertonic stress and at native levels of protein expression. In the kidney these condensates appear during K+ stress and are termed WNK bodies. We hypothesized that the large CTD of WNK1 mediates its phase behavior and that this function is evolutionarily conserved. To determine whether the CTD is required for condensate formation we performed live cell imaging studies of expressed mRuby2-tagged WNK1 constructs in cells. Full-length L-WNK1 was compared to two truncated constructs (1-494 or 1-1242), revealing that the CTD is required for condensate formation. To screen for the specific regions of the CTD that undergo phase separation, we used an optogenetic approach that mapped two areas within the WNK1 CTD that facilitate phase separation: (480-1242) and (1770-2126). Both regions were intrinsically disordered, exhibited low sequence complexity, and contained coiled-coil and prion-like domains- features typical of proteins that exhibit strong phase behavior. A bioinformatic analysis of human, C. elegan, and Drosophila WNK kinases revealed that these kinases shared nearly identical disorder tendency plots. A deeper analysis of CTD sequence complexity across 27 species ranging from humans to protists revealed that mammalian WNK kinases are enriched in prolines and serines, whereas invertebrates favor glutamine. Thus, the WNK CTD underwent a permissive Q-to-P/S amino acid shift during evolution, likely to preserve phase behavior. This conserved CTD has physiologic implications in the kidney as KS-WNK1 has an identical CTD and undergoes phase separation into WNK bodies during potassium deficiency. Mice that lack KS-WNK1 (KS-WNK1 KO) do not form WNK bodies during potassium deficiency and have a Gitelman-like phenotype with a 33% decrease in phospo-NCC [P = 0.01]. This decrease in pNCC correlated with decreased thiazide sensitivity—KO mice treated with HCTZ (25mg/kg IP) had no significant reduction in blood pressure, whereas WT littermates had a 4.5mmHg reduction in mean arterial pressure [P ≤ 0.05]. Based on these findings we conclude that during evolution KS-WNK1 conserved the intrinsically disordered WNK CTD to assemble WNK bodies to serve as protein scaffolds that enhance NCC activation during K+ stress. R01 DK098145, R01 DK119252, K08 DK118211 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A point mutation in the kinase domain of CRK10 leads to xylem vessel collapse and activation of defence responses in Arabidopsis.

Cysteine-rich receptor-like kinases (CRKs) are a large family of plasma membrane-bound receptors ubiquitous in higher plants. However, despite their prominence, their biological roles have remained largely elusive so far. In this study we report the characterization of an Arabidopsis mutant named crk10-A397T in which alanine 397 has been replaced by a threonine in the αC helix of the kinase domain of CRK10, known to be a crucial regulatory module in mammalian kinases. The crk10-A397T mutant is a dwarf that displays collapsed xylem vessels in the root and hypocotyl, whereas the vasculature of the inflorescence develops normally. In situ phosphorylation assays with His-tagged wild type and crk10-A397T versions of the CRK10 kinase domain revealed that both alleles are active kinases capable of autophosphorylation, with the newly introduced threonine acting as an additional phosphorylation site in crk10-A397T. Transcriptomic analysis of wild type and crk10-A397T mutant hypocotyls revealed that biotic and abiotic stress-responsive genes are constitutively up-regulated in the mutant, and a root-infection assay with the vascular pathogen Fusarium oxysporum demonstrated that the mutant has enhanced resistance to this pathogen compared with wild type plants. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10, the first such mutant to have been identified for a CRK in Arabidopsis.

Methionine restriction and cancer treatment: a systems biology study of yeast to investigate the possible key players.

Dietary restriction, mainly carbon and/or methionine restriction are among the upcoming supporting interventions along with chemotherapy in various cancers. Although dietary restriction has been proven to be beneficial, the main cellular machineries affected by its administration lacks deeper information considerably, a notable pitfall in its use as a personalized nutritional approach. In this study, cellular effects of methionine restriction on a yeast model are explored via systems biology approaches. The methionine biosynthesis network, constructed by integrating interaction data with gene ontology terms, was analysed topologically, and proved to be informative about the intertwined relationship of methionine biosynthesis and cancer. Experimentally, effects of methionine restriction on the yeast model were explored in vivo, with transcriptome analyses. The integrative analysis of the transcriptional data together with the reconstructed network gave insight into cellular machineries such as TOR, MAPK, and sphingolipid-mediated signaling cascades as the mostly responsive cellular pathways in the methionine-restricted cases with Sch9p (functional orthologue of mammalian S6 kinase) being placed at the intersection of these signaling routes.

Vitamin B3 inhibits apoptosis and promotes autophagy of islet β cells under high glucose stress

Background: Hyperglycemia is a typical symptom of diabetes. High glucose induces apoptosis of islet β cells. While autophagy functions in cytoprotection and autophagic cell death. The interaction between autophagy and apoptosis is important in the modulation of the function of islet β cells. Vitamin B3 can induce autophagy and inhibit islet β apoptosis.Method: The mechanism of vitamin B3-mediated protective effect on the function of islet β cells was explored by the method of western blot, immunofluorescence and flow cytometry.Results: In the present study, high glucose stress increased the apoptosis rate, while vitamin B3 reduced the apoptosis rate. The effect of vitamin B3 on autophagy flux under normal and high glucose stress was also investigated. Vitamin B3 increased the number of autophagosomes and increased the light chain (LC)3-II/LC3-I ratio. In contrast, vitamin B3 decreased sequestosome 1 (SQSTM1)/p62 protein expression and inhibited the phosphorylation of mammalian ribosomal protein S6 kinase β-1 (p70S6K/S6K1), which was a substrate of mammalian target of rapamycin (mTOR) under normal and high glucose stress. To further verify the protective effect of vitamin B3 on apoptosis, we treated islet β cell RIN-m5F with autophagy inhibitor 3-methyladenine (3-MA). Vitamin B3 decreased the apoptosis rate under high glucose stress, while the inhibition of apoptosis by vitamin B3 was blocked after adding 3-MA.Conclusion: Our data suggested that vitamin B3 reduced the apoptosis rate of β cells, possibly through inducing autophagy under high glucose stress.

The Prevalence of CHEK1 and CHEK2 Mutations in Prostate Cancer: a Retrospective Cohort Study.

Prostate cancer (PCa) is one of the most common types of cancer among men. Mutations and accumulation of chromosomal deviations are correlated with the development and aggressiveness of PCa. Cell cycle checkpoint pathways and DNA repair mechanisms are reported to deviate in cancers. Mammalian checkpoint kinase 1/2 (CHEK1/CHEK2) genes act as key signal transducers inside the genomic integrity checkpoints. CHEK1 and CHEK2 gene mutations were reported in a few different types of cancers. In PCa, CHEK2 mutations were studied, but CHEK1 gene variations were not well investigated. This study aimed to investigate the occurrence of variations in the CHEK1 and CHEK2 genes in PCa in the Jordanian population. Formalin-fixed paraffin-embedded PCa specimens of radical prostatectomy surgical procedures from 74 Jordanian patients were subjected to DNA extraction, polymerase chain reactions and Sanger sequencing to screen the mutations in selected exons of CHEK1 and CHEK2 tumor suppressor genes. The presence of F281L (T/C) (1.4%) homologous missense point mutation in the kinase domain of the CHEK2 gene and P188P (1.4%) silent point mutation in the kinase domain of the CHEK1 gene. In addition, the 1100delC mutation was not detected in the studied PCa specimens. In line with previous reports, the presence of CHEK2 mutation with a frequency of 1.4% supported the possible role of genetic variants of this gene in the development of PCa. No 1100delC mutation was detected in this study. No association was found in this study between CHEK1 mutations and the development of PCa. Further studies are needed with larger cohorts along with a screening of more exons in order to shed more light on the frequency of CHEK2 gene mutations and their role in the development of PCa in Jordan.

Genetic complementation screening and molecular docking give new insight on phosphorylation-dependent Mastl kinase activation

Mastl is a mitotic kinase that is essential for error-free chromosome segregation. It is an atypical member of AGC kinase family, possessing a unique non-conserved middle region. The mechanism of Mastl activation has been studied extensively in vitro. Phosphorylation of several residues were identified to be crucial for activation. These sites correspond to T193 and T206 in the activation loop and S861 in the C-terminal tail of mouse Mastl. To date, the significance of these phosphosites was not confirmed in intact mammalian cells. Here, we utilize a genetic complementation approach to determine the essentials of mammalian Mastl kinase activation. We used tamoxifen-inducible conditional knockout mouse embryonic fibroblasts to delete endogenous Mastl and screened various mutants for their ability to complement its loss. S861A mutant was able to complement endogenous Mastl loss. In parallel, we performed computational molecular docking studies to evaluate the significance of this residue for kinase activation. Our in-depth sequence and structure analysis revealed that Mastl pS861 does not belong to a conformational state, where the phosphoresidue contributes to C-tail docking. C-tail of Mastl is relatively short and it lacks a hydrophobic (HF) motif that would otherwise help its anchoring over N-lobe, required for the final steps of kinase activation. Our results show that phosphorylation of Mastl C-tail turn motif (S861) is dispensable for kinase function in cellulo. Communicated by Ramaswamy H. Sarma

Targeting Echinococcus multilocularis PIM kinase for improving anti-parasitic chemotherapy.

The potentially lethal zoonosis alveolar echinococcosis (AE) is caused by the metacestode larval stage of the tapeworm Echinococcus multilocularis. Current AE treatment options are limited and rely on surgery as well as on chemotherapy involving benzimidazoles (BZ). BZ treatment, however, is mostly parasitostatic only, must be given for prolonged time periods, and is associated with adverse side effects. Novel treatment options are thus urgently needed. By applying a broad range of kinase inhibitors to E. multilocularis stem cell cultures we identified the proto-oncogene PIM kinase as a promising target for anti-AE chemotherapy. The gene encoding the respective E. multilocularis ortholog, EmPim, was characterized and in situ hybridization assays indicated its expression in parasite stem cells. By yeast two-hybrid assays we demonstrate interaction of EmPim with E. multilocularis CDC25, indicating an involvement of EmPim in parasite cell cycle regulation. Small molecule compounds SGI-1776 and CX-6258, originally found to effectively inhibit human PIM kinases, exhibited detrimental effects on in vitro cultured parasite metacestode vesicles and prevented the formation of mature vesicles from parasite stem cell cultures. To improve compound specificity for EmPim, we applied a high throughput in silico modelling approach, leading to the identification of compound Z196138710. When applied to in vitro cultured metacestode vesicles and parasite cell cultures, Z196138710 proved equally detrimental as SGI-1776 and CX-6258 but displayed significantly reduced toxicity towards human HEK293T and HepG2 cells. Repurposing of kinase inhibitors initially designed to affect mammalian kinases for helminth disease treatment is often hampered by adverse side effects of respective compounds on human cells. Here we demonstrate the utility of high throughput in silico approaches to design small molecule compounds of higher specificity for parasite cells. We propose EmPim as a promising target for respective approaches towards AE treatment.

ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response

The unfolded protein response (UPR) is an elaborate signaling network that evolved to maintain proteostasis in the endoplasmic reticulum (ER) and mitochondria (mt). These organelles are functionally and physically associated, and consequently, their stress responses are often intertwined. It is unclear how these two adaptive stress responses are coordinated during ER stress. The inositol-requiring enzyme-1 (IRE1), a central ER stress sensor and proximal regulator of the UPRER, harbors dual kinase and endoribonuclease (RNase) activities. IRE1 RNase activity initiates the transcriptional layer of the UPRER, but IRE1’s kinase substrate(s) and their functions are largely unknown. Here, we discovered that sphingosine 1-phosphate (S1P) lyase (SPL), the enzyme that degrades S1P, is a substrate for the mammalian IRE1 kinase. Our data show that IRE1-dependent SPL phosphorylation inhibits SPL’s enzymatic activity, resulting in increased intracellular S1P levels. S1P has previously been shown to induce the activation of mitochondrial UPR (UPRmt) in nematodes. We determined that IRE1 kinase-dependent S1P induction during ER stress potentiates UPRmt signaling in mammalian cells. Phosphorylation of eukaryotic translation initiation factor 2α (eif2α) is recognized as a critical molecular event for UPRmt activation in mammalian cells. Our data further demonstrate that inhibition of the IRE1-SPL axis abrogates the activation of two eif2α kinases, namely double-stranded RNA-activated protein kinase (PKR) and PKR–like ER kinase upon ER stress. These findings show that the IRE1-SPL axis plays a central role in coordinating the adaptive responses of ER and mitochondria to ER stress in mammalian cells.

Isc10, an inhibitor of the Smk1 MAPK, prevents activation loop autophosphorylation and substrate phosphorylation through separate mechanisms

Many eukaryotic protein kinases are activated by the intramolecular autophosphorylation of activation loop residues. Smk1 is a meiosis-specific mitogen-activated protein kinase (MAPK) in yeast that autophosphorylates its activation loop tyrosine and thereby upregulates catalytic output. This reaction is controlled by an inhibitor, Isc10, that binds the MAPK during meiosis I and an activator, Ssp2, that binds Smk1/Isc10 during meiosis II. Upon completion of the meiotic divisions, Isc10 is degraded, and Smk1 undergoes autophosphorylation to generate the high activity form of the MAPK that controls spore formation. How Isc10 inhibits Smk1 is not clear. Here, we use a bacterial coexpression/reconstitution system to define a domain in the carboxy-terminal half of Isc10 that specifically inhibits Smk1 autophosphorylation. Nevertheless, Smk1 bound by this domain is able to phosphorylate other substrates, and it phosphorylates the amino-terminal half of Isc10 on serine 97. In turn, the phosphorylated motif in Isc10 inhibits the Smk1 active site. These data show that Isc10 inhibits autophosphorylation and the phosphorylation of substrates by separate mechanisms. Furthermore, we demonstrate Isc10 can inhibit the autophosphorylation of the mammalian intestinal cell kinase ICK1 (also known as CILK1), suggesting a conserved mechanism of action. These findings define a novel class of developmentally regulated molecules that prevent the self-activation of MAPKs and MAPK-like enzymes.

Abstract PO029: Targeting non-canonical Hippo pathway in NRF2-mutant liver cancer

Abstract NRF2 is an “undruggable” oncogenic transcription factor recurrently mutated in solid tumors such as liver and lung cancers. Our recent study demonstrates that NRF2 acts by coordinating the redox and metabolic stress responses as well as drug resistance programs in liver and other cancers. Developing an NRF2 inhibitor is a major goal of cancer drug discovery. Here, we identify the non-canonical Hippo pathway protein serine/threonine kinase 38 (STK38/NDR1) as a new NRF2 kinase that is a requirement for its stability and function. STK38 directly phosphorylates NRF2 at two sites (S33 and T559), and both sites contribute to full NRF2 activation. Loss of STK38 disables the redox response in NRF2 driven cancers and leads to tumor regression in vivo. Conversely, STK38 can also activate NRF2 and promote de novo liver cancer development in mice. This oncogenic effect is reflected in low frequency (3%) genomic amplifications in human liver cancers. Importantly, inhibition of the upstream STK38-activating mammalian Hippo kinases STK3/4 (MST2/1) by XMU-MP-1 inactivates STK38-NRF2 regulatory axis in vitroand in vivo. More importantly, XMU-MP-1 produces single agent activity against NRF2-driven liver and lung cancers in vivo in primary and patient derived xenograft (PDX)-based mouse models. In addition, we performed an in-silicoscreen and identified TAE-684 as an STK38 inhibitor that was subsequently confirmed to block STK38 activity in an in vitro kinase assay. TAE-684 treatment resulted in significant growth impairment of NRF2-mutant PDXs in vivo but no activity was observed in NRF2 wildtype counterparts. Together, these results uncover a surprising role of Hippo-related kinase STK38 in NRF2 activation and point to a paradoxical vulnerability in NRF2-driven cancers. Citation Format: Viraj Sanghvi, Aleksander Rust, Josef Leibold, Scott Lowe, Agnes Viale, John Chodera, Ronald C. Hendrickson, Elisa de Stanchina, Hans-Guido Wendel. Targeting non-canonical Hippo pathway in NRF2-mutant liver cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO029.

Regulating the regulator: a survey of mechanisms from transcription to translation controlling expression of mammalian cell cycle kinase Aurora A.

Aurora Kinase A (AURKA) is a positive regulator of mitosis with a strict cell cycle-dependent expression pattern. Recently, novel oncogenic roles of AURKA have been uncovered that are independent of the kinase activity and act within multiple signalling pathways, including cell proliferation, survival and cancer stem cell phenotypes. For this, cellular abundance of AURKA protein is per se crucial and must be tightly fine-tuned. Indeed, AURKA is found overexpressed in different cancers, typically as a result of gene amplification or enhanced transcription. It has however become clear that impaired processing, decay and translation of AURKA mRNA can also offer the basis for altered AURKA levels. Accordingly, the involvement of gene expression mechanisms controlling AURKA expression in human diseases is increasingly recognized and calls for much more research. Here, we explore and create an integrated view of the molecular processes regulating AURKA expression at the level of transcription, post-transcription and translation, intercalating discussion on how impaired regulation underlies disease. Given that targeting AURKA levels might affect more functions compared to inhibiting the kinase activity, deeper understanding of its gene expression may aid the design of alternative and therapeutically more successful ways of suppressing the AURKA oncogene.

The mitochondrial NAD kinase functions as a major metabolic regulator upon increased energy demand

ObjectiveThe mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (MNADK) mediates de novo mitochondrial NADP biosynthesis by catalyzing the phosphorylation of NAD to yield NADP. In this study, we investigated the function and mechanistic basis by which MNADK regulates metabolic homeostasis. MethodsGeneralized gene set analysis by aggregating human patient genomic databases, metabolic studies with genetically engineered animal models, mitochondrial bioenergetic analysis, as well as gain- and loss- of-function studies were performed to address the functions and mechanistic basis by which MNADK regulates energy metabolism and redox state associated with metabolic disease. ResultsHuman MNADK common gene variants or decreased expression of the gene are significantly associated with the occurrence of type-2 diabetes, non-alcoholic fatty liver disease (NAFLD), or hepatocellular carcinoma (HCC). Ablation of the MNADK gene in mice led to decreased fat oxidation, coincident with increased respiratory exchange ratio (RER) and decreased energy expenditure upon energy demand triggered by endurance exercise or fasting. On an atherogenic high-fat diet (HFD), MNADK-null mice exhibited hepatic insulin resistance and glucose intolerance, indicating a type-2 diabetes-like phenotype in the absence of MNADK. MNADK deficiency led to a decrease in mitochondrial NADP(H) but an increase in cellular reactive oxygen species (ROS) in mouse livers. Consistently, protein levels of the major metabolic regulators or enzymes were decreased, while their acetylation modifications were increased in the livers of MNADK-null mice. Feeding mice with a HFD caused S-nitrosylation (SNO) modification, a posttranslational modification that represses protein activities, on MNADK protein in the liver. Reconstitution of an SNO-resistant MNADK variant, MNADK-S193, into MNADK-null mice mitigated hepatic steatosis induced by HFD. ConclusionMNADK, the only known mammalian mitochondrial NAD kinase, plays important roles in preserving energy homeostasis to mitigate the risk of metabolic disorders.

Thymidine Kinase+/- Mammalian Cell Mutagenicity Assays for Assessment of Nanomaterials.

The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM characterization, sample preparation and dose selection, relevant to all genotoxicity assays, are found in an accompanying paper. The present paper describes methods for evaluation of mutagenicity in the mammalian (mouse) thymidine kinase (Tk) gene occurring in L5178Y mouse lymphoma (ML) cells and in the designated TK gene in human lymphoblastoid TK6 cells. Mutations change the functional genotype from TK+/- to TK-/-, detectable as cells surviving on media selective for the lack of thymidine kinase (TK) function. Unlike cells with TK enzyme function, the TK-/- cells are unable to integrate the toxic selection agent, allowing these cells to survive as rare mutant colonies. The ML assay has been shown to detect a broad spectrum of genetic damage, including both small scale (point) mutations and chromosomal alterations. This assay is a widely used mammalian cell gene mutation assay for regulatory purposes and is included in the core battery of genotoxicity tests for regulatory decision-making. The TK6 assay is an assay using a human cell line derived similarly via mutagenic manipulations and optimal selection. Details are provided on the materials required, cell culture methods, selection of test chemical concentrations, cytotoxicity, treatment time, mutation expression, cloning, and data calculation and interpretation. The methods describe the microwell plate version of the assays without metabolic activation.

Ceramide Kinase Inhibition Drives Ferroptosis and Sensitivity to Cisplatin in Mutant KRAS Lung Cancer by Dysregulating VDAC-Mediated Mitochondria Function.

Our findings depict how CERK inhibition may serve as a new key point in combination therapeutic strategy for NSCLC, specifically precision therapeutics targeting NSCLC possessing a KRAS mutation.

Knockout of circRNA single stranded interacting protein 1 (circRBMS1) played a protective role in myocardial ischemia-reperfusion injury though inhibition of miR-2355-3p/Mammalian Sterile20-like kinase 1 (MST1) axis

ABSTRACT Evidence suggests circRBMS1 regulates mRNA to mediate cell apoptosis, inflammation, and oxidative stress in different diseases. MST1 is reported to be the target and activator of apoptosis-related molecules and signaling pathways. Hence, the present study aims to investigate the role of circ-RBMS1/miR-2355-3p/MST1 in the development of I/R injury. In vitro experiments showed increased circ-RBMS1 and decreased miR-2355-3p in H/R-induced HCMs. CircRBMS1 served as a sponge for miR-2355-3p and miR-2355-3p targeted MST1. Furthermore, knockout of circRBMS1 attenuated cell apoptosis, oxidized stress, and inflammation in H/R-induced HCMs. In vivo experiments indicated circRBMS1 knockdown attenuated cardiac function damage, cell apoptosis, oxidative stress injury and inflammatory response through miR-2355-3p/MST1 axis in mice. In summary, these results demonstrated circRBMS1 played a protective role in myocardial I/R injury though inhibition of miR-2355-3p/MST1 axis. It might provide a new therapeutic target for cardiac I/R injury.

Isc10 is a Dual Mechanism Inhibitor of CMGC Kinases

Autophosphorylation of the activation loop is a common step that converts many protein kinases from an inactive to an active state. Despite the physiological importance of these reactions, the mechanisms that control activation loop autophosphorylation are poorly understood. Smk1 is a meiosis‐specific MAPK in yeast that autophosphorylates its activation loop on tyrosine (Y), thereby upregulating its own catalytic activity. Previously, we identified Isc10 as an inhibitor of Smk1. Here, we show that Isc10 inhibits Smk1 by two mechanisms. First, the C‐terminal segment of Isc10 binds to Smk1 and specifically inhibits the cis‐autophosphorylation of Y209 in its activation‐loop. Second, prior to Smk1 autophosphorylation, the low activity state Smk1 that is bound to Isc10 phosphorylates it on serine 97 (S97). The phosphorylated S97, in turn, becomes trapped in the Smk1 active site. This prevents phosphorylation of downstream targets before activating signals promote Isc10 degradation and the release of active Smk1. Further, Isc10 inhibits autophosphorylation of the mammalian intestinal cell kinase ICK1 (also known as CILK1), suggesting a conserved mechanism of action. Taken together, these findings define a novel class of developmentally‐regulated molecules that inhibit CMGC‐group kinases.

Malarial PI4K inhibitor induced diaphragmatic hernias in rat: Potential link with mammalian kinase inhibition.

BackgroundMMV390048 is an aminopyridine plasmodial PI4K inhibitor, selected as a Plasmodium blood‐stage schizonticide for a next generation of malaria treatments to overcome resistance to current therapies. MMV390048 showed an acceptable preclinical safety profile and progressed up to Phase 2a clinical trials. However, embryofetal studies revealed adverse developmental toxicity signals, including diaphragmatic hernias and cardiovascular malformations in rats but not rabbits.MethodsIn vivo exposures of free plasma concentrations of compound in rats were assessed in relation to in vitro human kinase inhibition by MMV390048, using the ADP‐Glo™ Kinase Assay.ResultsWe demonstrate a potential link between the malformations seen in the embryofetal developmental (EFD) studies and inhibition of the mammalian PI4Kβ paralogue, as well as inhibition of the off‐target kinases MAP4K4 and MINK1. PI3Kγ may also play a role in the embryofetal toxicity as its in vitro inhibition is covered by in vivo exposure. The exposures in the rabbit embryofetal development studies did not reach concentrations likely to cause PI4K inhibition. Overall, we hypothesize that the in vivo malformations observed could be due to inhibition of the PI4K target in combination with the off‐targets, MAP4K4 and MINK1. However, these relationships are by association and not mechanistically proven.ConclusionsDeciphering if the EFD effects are dependent on PI4K inhibition, and/or via inhibition of other off‐target kinases will require the generation of novel, more potent, and more specific PI4K inhibitors.