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A conditional knock-in mouse was generated in which the TAK1 catalytic subunit was largely replaced by the kinase-inactive TAK1[D175A] mutant in immune cells. The activation of p38α MAP kinase, c-Jun N-terminal kinases 1 and 2 (JNK1/2) and the canonical IKK complex induced by stimulation with several TLR-activating ligands was reduced in bone marrow-derived macrophages (BMDM) from TAK1[D175A] mice. TLR signalling in TAK1[D175A] BMDM was catalysed by the residual wild-type TAK1 in these cells because it was abolished by either of two structurally unrelated TAK1 inhibitors (NG25 and 5Z-7-oxozeaenol) whose off-target effects do not overlap. The secretion of inflammatory mediators and production of the mRNAs encoding these cytokines induced by TLR ligation was greatly reduced in peritoneal neutrophils or BMDM from TAK1[D175A] mice. The Pam3CSK4- or LPS-stimulated activation of MAP kinases and the canonical IKK complex, as well as cytokine secretion, was also abolished in TAK1 knock-out human THP1 monocytes or macrophages. The results establish that TAK1 protein kinase activity is required for TLR-dependent signalling and cytokine secretion in myeloid cells from mice. We discuss possible reasons why other investigators, studying myeloid mice with a conditional knock-out of TAK1 or a different conditional kinase-inactive knock-in of TAK1, reported TAK1 to be a negative regulator of LPS-signalling and cytokine production in mouse macrophages and neutrophils.

The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, Escherichia coli, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or Escherichia coli. The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.

Corneal endothelial cell (CEC) dysfunction causes corneal edema and severe visual impairment that require transplantation to restore vision. To address the unmet need of organ shortage, descemetorhexis without endothelial keratoplasty has been specifically employed to treat early stage Fuchs endothelial corneal dystrophy, which is pathophysiologically related to oxidative stress and exhibits centrally located corneal guttae. After stripping off central Descemet's membrane, rho-associated protein kinase (ROCK) inhibitor has been found to facilitate CEC migration, an energy-demanding task, thereby achieving wound closure. However, the correlation between ROCK inhibition and the change in bioenergetic status of CECs remained to be elucidated. Through transcriptomic profiling, we found that the inhibition of ROCK activity by the selective inhibitor, ripasudil or Y27632, promoted enrichment of oxidative phosphorylation (OXPHOS) gene set in bovine CECs (BCECs). Functional analysis revealed that ripasudil, a clinically approved anti-glaucoma agent, enhanced mitochondrial respiration, increased spare respiratory capacity, and induced overexpression of electron transport chain components through upregulation of AMP-activated protein kinase (AMPK) pathway. Accelerated BCEC migration and in vitro wound healing by ripasudil were diminished by OXPHOS and AMPK inhibition, but not by glycolysis inhibition. Correspondingly, lamellipodial protrusion and actin assembly that were augmented by ripasudil became reduced with additional OXPHOS or AMPK inhibition. These results indicate that ROCK inhibition induces metabolic reprogramming toward OXPHOS to support migration of CECs.

Nuclear receptor subfamily 6 group A member 1 (NR6A1) is involved in promoting the apoptotic process of vascular smooth muscle cells (VSMCs) which is a critical process involved in atherosclerosis, but the action mechanism remains to be determined. Therefore, we studied the underlying mechanisms by which NR6A1 accelerated VSMC apoptosis in atherosclerosis. An atherosclerosis model has been established in apolipoprotein E-deficient rats with a high-fat diet for 12 weeks, which was characterized by pathological aortic plaques, increased lipid deposition and collagen content in aortic tissues, and high cholesterol and triglycerides levels in the serum. NR6A1 was experimentally shown to increase at protein level rather than messenger RNA level in atherosclerotic rats. Immunofluorescence exhibited the main location of NR6A1 in the cell nucleus of rat aortic tissues. By performing ectopic expression experiments, NR6A1 was demonstrated to suppress the viability and expedite the apoptosis of VSMCs, corresponding to augmented caspase-3, caspase-8, and caspase-9 activities. It was further unraveled that NR6A1 could activate receptor-interacting serine/threonine-protein kinase 3 (RIPK3) by inducing its phosphorylation. Conversely, RIPK3 inhibitor GSK872 undermined the proapoptotic effect of NR6A1 on VSMCs. The co-immunoprecipitation assay identified that linear ubiquitin chain assembly complex (LUBAC) can be pulled down by NR6A1. Furthermore. LUBAC inhibited the expression of NR6A1 by promoting its linear ubiquitination, thereby dephosphorylating RIPK3 and consequently inhibiting the VSMC apoptosis. Overall, LUBAC-induced linear ubiquitination of NR6A1 can potentially arrest the apoptosis of VSMCs in atherosclerosis by downregulating RIPK3 and attenuating caspase activity. This finding suggests promising athero-protective targets by limiting VSMC apoptosis.

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JAK2 is cytokine-activated non-receptor tyrosine kinase. Although JAK2 is mainly localized at the plasma membrane, it is also present on the centrosome. In this study, we demonstrated that JAK2 localization to the centrosome depends on the SH2 domain and intact kinase activity. We created JAK2 mutants deficient in centrosomal localization ΔSH2, K882E and (ΔSH2, K882E). We showed that JAK2 WT clone strongly enhances cell proliferation as compared to control cells while JAK2 clones ΔSH2, K882E and (ΔSH2, K882E) proliferate slower than JAK2 WT cells. These mutant clones also progress much slower through the cell cycle as compared to JAK2 WT clone and the enhanced proliferation of JAK2 WT cells is accompanied by increased S −> G2 progression. Both the SH2 domain and the kinase activity of JAK2 play a role in prolactin-dependent activation of JAK2 substrate STAT5. We showed that JAK2 is an important regulator of centrosome function as the SH2 domain of JAK2 regulates centrosome amplification. The cells overexpressing ΔSH2 and (ΔSH2, K-E) JAK2 have almost three-fold the amplified centrosomes of WT cells. In contrast, the kinase activity of JAK2 is dispensable for centrosome amplification. Our observations provide novel insight into the role of SH2 domain and kinase activity of JAK2 in centrosome localization of JAK2 and in the regulation of cell growth and centrosome biogenesis.

Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

In vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson’s disease (PD). However, difficulty in detecting endogenous PINK1 protein in rodent brains and cell lines has prevented the rigorous investigation of the in vivo role of PINK1. Here we report that PINK1 kinase form is selectively expressed in the human and monkey brains. CRISPR/Cas9-mediated deficiency of PINK1 causes similar neurodegeneration in the brains of fetal and adult monkeys as well as cultured monkey neurons without affecting mitochondrial protein expression and morphology. Importantly, PINK1 mutations in the primate brain and human cells reduce protein phosphorylation that is important for neuronal function and survival. Our findings suggest that PINK1 kinase activity rather than its mitochondrial function is essential for the neuronal survival in the primate brains and that its kinase dysfunction could be involved in the pathogenesis of PD.

During salinity stress, osmoregulatory processes in euryhaline fish need to modify for their survival, and glucose is the preferred mode of extra energy during such conditions. These organisms must have a proper mechanism to maintain glucose homeostasis during such modified osmoregulatory process across different body fluids. Hence, we studied high salinity effect on regulation of glucose homeostasis in Mozambique tilapia. The fish were induced to 15‰ salinity for 21 days. Glucose, glycogen, ion concentrations, Na+-K+-ATPase, pyruvate kinase, γ-amylase activities and GLUT mRNA expressions were investigated in liver, intestine, gill and white muscle tissues. At the end of experiment, Na+ ion concentrations, glucose content and activity of Na+-K+-ATPase especially in the gill and intestine were increased, while decrease in liver and gill glycogen content was seen. Lower concentration of glycogen decrease was observed in the intestine and white muscle of the treated group. High pyruvate kinase activity was noticed in liver and gill tissues that correlates with high Na+-K+-ATPase activity. Elevated γ-amylase activity was observed in the liver and intestine suggesting breakdown of glycogen; however, gill and white muscle did not show any increased activity. Increase in GLUT1 and GLUT4 mRNA expressions was observed especially in the gill and intestine, while increase in GLUT2 mRNA expressions was observed in the liver. Upregulations of GLUTs suggest higher influx of glucose into the cell for catabolism to provide energy and further to drive the enhanced osmoregulatory process. These findings suggest glucose homeostasis being regulated in Mozambique tilapia during salinity acclimation.

Innate immunity is the first line of host defense against pathogens. This process is modulated by multiple antiviral protein modifications, such as phosphorylation and ubiquitination. Here, we showed that cellular S-nitrosoglutathione reductase (GSNOR) is actively involved in innate immunity activation. GSNOR deficiency in mouse embryo fibroblasts (MEFs) and RAW264.7 macrophages reduced the antiviral innate immune response and facilitated herpes simplex virus-1 (HSV-1) and vesicular stomatitis virus (VSV) replication. Concordantly, HSV-1 infection in Gsnor−/- mice and wild-type mice with GSNOR being inhibited by N6022 resulted in higher mortality relative to the respective controls, together with severe infiltration of immune cells in the lungs. Mechanistically, GSNOR deficiency enhanced cellular TANK-binding kinase 1 (TBK1) protein S-nitrosation at the Cys423 site and inhibited TBK1 kinase activity, resulting in reduced interferon production for antiviral responses. Our study indicated that GSNOR is a critical regulator of antiviral responses and S-nitrosation is actively involved in innate immunity., Highlights • GSNOR deficiency impairs antiviral innate response. • GSNOR deficiency enhances S-nitrosation of TBK1 on Cys423 inhibits TBK1 activity. • Deficiency of Gsnor promotes viral infection in vivo. • Mice treated with GSNOR inhibitor N6022 are permissive to HSV-1 infection.

The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase family. Using various stimulated rodent cells and kinase activation techniques, we identified a 46-kDa ERK. The kinetics of activation of this ERK isoform was similar to that of ERK1 and ERK2 under most but not all circumstances. We purified this isoform from rat cells followed by its cloning. The sequence of this isoform revealed that it is an alternatively spliced version of the 44-kDa ERK1 and therefore we termed it ERK1b. Interestingly, this isoform had a 26-amino acid insertion between residues 340 and 341 of ERK1, which results from Intron 7 insertion to the sequence. Examining the expression pattern, we found that ERK1b is detected mainly in rat and particularly in Ras-transformed Rat1 cells. In this cell line, ERK1b was more sensitive to extracellular stimulation than ERK1 and ERK2. Moreover, unlike ERK1 and ERK2, ERK1b had a very low binding affinity to MEK1. This low interaction led to nuclear localization of this isoform when expressed together with MEK1 under conditions in which ERK1 and ERK2 are retained in the cytoplasm. In addition, ERK1b was not coimmunoprecipitated with MEK1. We identified a new, 46-kDa ERK alternatively spliced isoform. Our results indicate that this isoform is the major one to respond to exogenous stimulation in Ras-transformed cells, probably due to its differential regulation by MAPK/ERK kinase and by phosphatases.

Tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR-TKIs) are currently used therapy for non-small cell lung cancer (NSCLC) patients; however, drug resistance during cancer treatment is a critical problem. Survivin is an anti-apoptosis protein, which promotes cell proliferation and tumor growth that highly expressed in various human cancers. Here, we show a novel synthetic compound derived from gefitinib, do-decyl-4-(4-(3-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)propyl) piper-azin-1-yl)-4-oxobutanoate, which is named as SP101 that inhibits survivin expression and tumor growth in both the EGFR-wild type and -T790M of NSCLC. SP101 blocked EGFR kinase activity and induced apoptosis in the A549 (EGFR-wild type) and H1975 (EGFR-T790M) lung cancer cells. SP101 reduced survivin proteins and increased active caspase 3 for inducing apoptosis. Ectopic expression of survivin by a survivin-expressed vector attenuated the SP101-induced cell death in lung cancer cells. Moreover, SP101 inhibited the gefitinib-resistant tumor growth in the xenograft human H1975 lung tumors of nude mice. SP101 substantially reduced survivin proteins but conversely elicited active caspase 3 proteins in tumor tissues. Besides, SP101 exerted anticancer abilities in the gefitinib resistant cancer cells separated from pleural effusion of a clinical lung cancer patient. Consistently, SP101 decreased the survivin proteins and the patient-derived xenografted lung tumor growth in nude mice. Anti-tumor ability of SP101 was also confirmed in the murine lung cancer model harboring EGFR T790M-L858R. Together, SP101 is a new EGFR inhibitor with inhibiting survivin that can be developed for treating EGFR wild-type and EGFR-mutational gefitinib-resistance in human lung cancers.

Autophagy is a major cellular quality control system responsible for the degradation of proteins and organelles in response to stress and damage to maintain homeostasis. Ubiquitination of autophagy-related proteins or regulatory components is important for the precise control of autophagy pathways. Here, we show that the deubiquitinase ubiquitin-specific protease 11 (USP11) restricts autophagy and that KO of USP11 in mammalian cells results in elevated autophagic flux. We also demonstrate that depletion of the USP11 homolog H34C03.2 in Caenorhabditis elegans triggers hyperactivation of autophagy and protects the animals against human amyloid-β peptide 42 aggregation-induced paralysis. USP11 coprecipitated with autophagy-specific class III phosphatidylinositol 3-kinase complex I and limited its interaction with nuclear receptor-binding factor 2, thus decreasing lipid kinase activity of class III phosphatidylinositol 3-kinase complex I and subsequent recruitment of effectors such as WD-repeat domain phosphoinositide-interacting proteins to the autophagosomal membrane. Accordingly, more WD-repeat domain phosphoinositide-interacting protein 2 puncta accumulated in USP11 KO cells. In addition, USP11 interacts with and stabilizes the serine/threonine kinase mechanistic target of rapamycin, thereby further contributing to the regulation of autophagy induction. Taken together, our data suggested that USP11 impinges on the autophagy pathway at multiple sites and that inhibiting USP11 alleviates symptoms of proteotoxicity, which is a major hallmark of neurodegenerative diseases.

The Pim (proviral integration site for Moloney murine leukemia virus) proteins compose a serine threonine kinase family whose members regulate cell proliferation, migration and cell survival. However, whether Pim kinases participate in innate immune responses is unclear. Here, we show for the first time that Pim1 plays an essential role in the production of interferon (IFN)-β by macrophages after their Toll-like receptor (TLR) pathway is activated by pathogen-associated molecular patterns (PAMPs). Specifically, Pim1 was quickly upregulated in an NF-κB-dependent manner after TLR stimulation with PAMPs. Pim1 deficiency reduced TLR3- or TLR4-stimulated IFN-β and IFN-stimulated gene (ISG) expression but not proinflammatory cytokine expression in macrophages. Mechanistically, Pim1 specifically upregulates IRF3 phosphorylation and nuclear translocation. However, this role is not dependent on Pim1 kinase activity. Rather, Pim1 appears to promote IRF3 phosphorylation by enhancing the formation of IFN-β signaling complexes composed of TRIF, TRAF3, TBK1, and IRF3. Poly (I:C)-treated Pim1−/− mice produced less serum IFN-β and were less likely to survive than wild-type mice. These findings show for the first time that Pim1 participates in TLR-mediated IFN-β production, thus revealing a novel target for controlling antiviral innate immune responses.

Cocaine as a highly addictive psychostimulant can cause changes in the body at the cellular and molecular levels over a long period of time. It reminds us that cocaine may have a potential role in post-transcriptional regulation, but the alteration of insula-expression profile in adolescent cocaine use disorder (CUD) has not been reported. To reveal the mechanisms underlying the post-transcriptional regulation of cocaine, we investigate the transcriptome in the insula of cocaine-induced mice based on high-throughput strand-specific RNA sequencing. We analyzed the alterations of messenger RNA (mRNA) expression profile in the insula of cocaine-induced condition place preference (CPP) mice and then correlated it with microRNAs to reveal their involvement in the formation of cocaine-induced CPP. In this study, a total of 27786 genes were identified, 5750 new genes (novel expressed transcripts of unannotated in the reference genome) were discovered, among which 1,205 were annotated functionally. A total of 198 differentially expressed genes (DEG) that functioned in synaptic transmission, cholinergic, developmental process, neurotransmitter metabolic process, drug catabolism, cellular response to drug, MAP kinase activity, ceramidase activity, and drug resistance were significantly enriched. Further analysis showed that 26045 mRNAs formed 45,208 network-relationship pairs with 1770 microRNAs. In the current study, our work was the first to reveal that alterations of RNAs in the insula, as a core brain region of the neural circuits of interoception, were involved in the process of cocaine-induced CPP of adolescent mice. These findings enrich the biology and expand the molecular regulatory network related to adolescence CUD. They provided the possibility that some DEGs may be used as novel biomarkers for the diagnosis or evaluation of substance use disorder, and also provided clues for elucidating the neurobiological mechanism of substance use disorder.

The S-nitrosoglutathione reductase (GSNOR) is a key denitrosating enzyme that regulates protein S-nitrosation, a process which has been found to be involved in the pathogenesis of Parkinson's disease (PD). However, the physiological function of GSNOR in PD remains unknown. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model, we found that GSNOR expression was significantly increased and accompanied by autophagy mediated by MPTP-induced cyclin dependent kinase 5 (CDK5), behavioral dyskinesias and dopaminergic neuron loss. Whereas, knockout of GSNOR, or treatment with the GSNOR inhibitor N6022, alleviated MPTP-induced PD-like pathology and neurotoxicity. Mechanistically, deficiency of GSNOR inhibited MPTP-induced CDK5 kinase activity and CDK5-mediated autophagy by increasing S-nitrosation of CDK5 at Cys83. Our study indicated that GSNOR is a key regulator of CDK5 S-nitrosation and is actively involved in CDK5-mediated autophagy induced by MPTP.

The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-κB signaling to promote their survival, but the effectors of type I strains remain unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser-468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-κB activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-κB, culminating in the enhanced production of IL-6, IL-12, and TNF-α in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-κB pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent.

To gain insight into the potential protective mechanisms of low phenylalanine diet (LPD) in phenylketonuria (PKU), gene expression profiles were studied in the cerebral cortex and hippocampus of a PKU mouse model (BTBR-Pahenu2). PKU mice were fed with low Phe diet (LPD-PKU group) and normal diet (PKU group). Wild-type mice were treated with normal diet (WT group) as control. After 12 weeks, we detected gene expression in the cerebral cortex and hippocampus of the three groups by RNA-sequencing, and then screened the differentially-expressed genes (DEGs) among the groups by bioinformatics analyses. We found that the transcriptional profiles of both cerebral cortex and hippocampus changed markedly between PKU and WT mice. Furthermore, LPD changed the transcriptional profiles of the cerebral cortex and the hippocampus of PKU mice significantly, especially in the cerebral cortex, with overlaps of genes that changed with the disease and altered by LPD treatment. In the cerebral cortex, hundreds of DEGs enriched in a wide spectrum of biological processes, molecular function, and cellular component, including nervous system development, axon development and guidance, calcium ion binding, modulation of chemical synaptic transmission, and regulation of protein kinase activity. In the hippocampus, the overlapping genes were enriched in positive regulation of long term synaptic, negative regulation of excitatory postsynaptic potential, positive regulation of synapse assembly. Our results showed that genes impaired in PKU and then rescued by LPD might indicate the potential protective capability of LPD in the PKU brain.

BackgroundA major goal in the discovery of cellular signaling networks is to identify regulated phosphorylation sites (“phosphosites”) and map them to the responsible protein kinases. The V2 vasopressin receptor is a G-protein coupled receptor (GPCR) that is responsible for regulation of renal water excretion through control of aquaporin-2-mediated osmotic water transport in kidney collecting duct cells. Genome editing experiments have demonstrated that virtually all vasopressin-triggered phosphorylation changes are dependent on protein kinase A (PKA), but events downstream from PKA are still obscure.MethodsHere, we used: 1) Tandem mass tag-based quantitative phosphoproteomics to experimentally track phosphorylation changes over time in native collecting ducts isolated from rat kidneys; 2) a clustering algorithm to classify time course data based on abundance changes and the amino acid sequences surrounding the phosphosites; and 3) Bayes’ Theorem to integrate the dynamic phosphorylation data with multiple prior “omic” data sets covering expression, subcellular location, known kinase activity, and characteristic surrounding sequences to identify a set of protein kinases that are regulated secondary to PKA activation.ResultsPhosphoproteomic studies revealed 185 phosphosites regulated by vasopressin over 15 min. The resulting groups from the cluster algorithm were integrated with Bayes’ Theorem to produce corresponding ranked lists of kinases likely responsible for each group. The top kinases establish three PKA-dependent protein kinase modules whose regulation mediate the physiological effects of vasopressin at a cellular level. The three modules are 1) a pathway involving several Rho/Rac/Cdc42-dependent protein kinases that control actin cytoskeleton dynamics; 2) mitogen-activated protein kinase and cyclin-dependent kinase pathways that control cell proliferation; and 3) calcium/calmodulin-dependent signaling.ConclusionsOur findings identify a novel set of downstream small GTPase effectors and calcium/calmodulin-dependent kinases with potential roles in the regulation of water permeability through actin cytoskeleton rearrangement and aquaporin-2 trafficking. The proposed signaling network provides a stronger hypothesis for the kinases mediating V2 vasopressin receptor responses, encouraging future targeted examination via reductionist approaches. Furthermore, the Bayesian analysis described here provides a template for investigating signaling via other biological systems and GPCRs.

Topoisomerase II Binding Protein 1 (TOPBP1) is an important activator of the DNA damage response kinase Ataxia Telangiectasia and Rad3-related (ATR), although the mechanism by which this activation occurs is not yet known. TOPBP1 contains nine copies of the BRCA1 C-terminal repeat (BRCT) motif, which allows protein-protein and protein-DNA interactions. TOPBP1 also contains an ATR activation domain (AAD), which physically interacts with ATR and its partner ATR-interacting protein (ATRIP) in a manner that stimulates ATR kinase activity. It is unclear which of TOPBP1's nine BRCT domains participate in the reaction, as well as the individual roles played by these relevant BRCT domains. To address this knowledge gap, here, we delineated a minimal TOPBP1 that can activate ATR at DNA double-strand breaks in a regulated manner. We named this minimal TOPBP1 "Junior" and we show that Junior is composed of just three regions: BRCT0-2, the AAD, and BRCT78. We further defined the individual functions of these three regions by showing that BRCT0-2 is required for recruitment to DNA double-strand breaks and is dispensable thereafter, and that BRCT78 is dispensable for recruitment but essential to allow the AAD to multimerize and activate ATR. The delineation of TOPBP1 Junior creates a leaner, simplified, and better understood TOPBP1 and provides insight into the mechanism of ATR activation.

The posttranslational regulation of the neuronal proteome is critical for brain homeostasis but becomes dysregulated in the aged or diseased brain, in which abnormal posttranslational modifications (PTMs) are frequently observed. While the full extent of modified substrates that comprise the "PTM-ome" are slowly emerging, how the upstream enzymes catalyzing these processes are regulated themselves is not well understood, particularly in the context of neurodegeneration. Here, we describe the reciprocal regulation of a kinase, the microtubule affinity-regulating kinase 2 (MARK2), and an acetyltransferase, CREB-binding protein (CBP), two enzymes known to extensively modify tau proteins in the progression of Alzheimer's disease. We found that MARK2 negatively regulates CBP and, conversely, CBP directly acetylates and inhibits MARK2 kinase activity. These findings highlight a reciprocal negative feedback loop between a kinase and an acetyltransferase, which has implications for how PTM interplay is coordinated on substrates including tau. Our study suggests that PTM profiles occur through the posttranslational control of the master PTM remodeling enzymes themselves.

Akt is a Ser/Thr protein kinase that regulates cell growth and metabolism and is considered a therapeutic target for cancer. Regulation of Akt by membrane recruitment and post-translational modifications (PTMs) has been extensively studied. The most well-established mechanism for cellular Akt activation involves phosphorylation on its activation loop on Thr308 by PDK1 and on its C-terminal tail on Ser473 by mTORC2. In addition, dual phosphorylation on Ser477 and Thr479 has been shown to activate Akt. Other C-terminal tail PTMs have been identified, but their functional impacts have not been well-characterized. Here, we investigate the regulatory effects of phosphorylation of Tyr474 and O-GlcNAcylation of Ser473 on Akt. We use expressed protein ligation as a tool to produce semisynthetic Akt proteins containing phosphoTyr474 and O-GlcNAcSer473 to dissect the enzymatic functions of these PTMs. We find that O-GlcNAcylation at Ser473 and phosphorylation at Tyr474 can also partially increase Akt's kinase activity toward both peptide and protein substrates. Additionally, we performed kinase assays employing human protein microarrays to investigate global substrate specificity of Akt, comparing phosphorylated versus O-GlcNAcylated Ser473 forms. We observed a high similarity in the protein substrates phosphorylated by phosphoSer473 Akt and O-GlcNAcSer473 Akt. Two Akt substrates identified using microarrays, PPM1H, a protein phosphatase, and NEDD4L, an E3 ubiquitin ligase, were validated in solution-phase assays and cell transfection experiments.

Myosins belong to a large superfamily of actin-dependent molecular motors. Nonmuscle myosin II (NM II) is involved in the morphology and function of neurons, but little is known about how NM II activity is regulated. Brain-derived neurotrophic factor (BDNF) is a prevalent neurotrophic factor in the brain that encourages growth and differentiation of neurons and synapses. In this study, we report that BDNF upregulates the phosphorylation of myosin regulatory light chain (MLC2), to increases the activity of NM II. The role of BDNF on modulating the phosphorylation of MLC2 was validated by using Western blotting in primary cultured hippocampal neurons. This result was confirmed by injecting BDNF into the dorsal hippocampus of mice and detecting the phosphorylation level of MLC2 by Western blotting. We further perform coimmunoprecipitation assay to confirm that this process depends on the activation of the LYN kinase through binding with tyrosine kinase receptor B, the receptor of BDNF, in a kinase activity-dependent manner. LYN kinase subsequently phosphorylates MLCK, further promoting the phosphorylation of MLC2. Taken together, our results suggest a new molecular mechanism by which BDNF regulates MLC2 activity, which provides a new perspective for further understanding the functional regulation of NM II in the nervous system.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19), known to be more common in the elderly, who also show more severe symptoms and are at higher risk of hospitalization and death. Here, we show that the expression of the angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 cell receptor, increases during aging in mouse and human lungs. ACE2 expression increases upon telomere shortening or dysfunction in both cultured mammalian cells and invivo in mice. This increase is controlled at the transcriptional level, and Ace2 promoter activity is DNA damage response (DDR)-dependent. Both pharmacological global DDR inhibition of ATM kinase activity and selective telomeric DDR inhibition by the use of antisense oligonucleotides prevent Ace2 upregulation following telomere damage in cultured cells and in mice. We propose that during aging telomere dysfunction due to telomeric shortening or damage triggers DDR activation and this causes the upregulation of ACE2, the SARS-CoV-2 cell receptor, thus contributing to make the elderly more susceptible to the infection.

Rationale Cell-based therapeutics have been extensively used for cardiac repair yet underperform due to inability of the donated cells to survive in near anoxia after cardiac injury. Cellular metabolism is linked to maintenance of cardiac stem cell (CSC) renewal, proliferation and survival. Ex vivo expansion alters (CSC) metabolism increasing reliance on oxygen dependent respiration. Whether promoting ‘metabolic flexibility’ in CSCs augments their ability to survive in near anoxia and repair the heart after injury remains untested. Objective Determine the effect of LIN28a induced metabolic flexibility on cardiac tissue derived stem like cell (CTSC) survival and repair after cardiac injury. Methods and results LIN28a expression coincides during heart development but is lost in adult CTSCs. Reintroduction of LIN28a in adult CTSC (CTSC-LIN) increased proliferation, survival, expression of pluripotency genes and reduced senescence compared to control (CTSC-GFP). Metabolomic analysis show glycolytic intermediates upregulated in CTSC-LIN together with increased lactate production, pyruvate kinase activity, glucose uptake, ECAR and expression of glycolytic enzymes compared to CTSC-GFP. Additionally, CTSC-LIN showed significantly reduced ROS generation and increase antioxidant markers. In response to H2O2 induced oxidative stress, CTSC-LIN showed increased survival and expression of glycolytic genes. LIN28a salutary effects on CTSCs were linked to PDK1/let-7 signaling pathway with loss of PDK1 or alteration of let-7 abrogating LIN28a effects. Following transplantation in the heart after myocardial infarction (MI), CTSC-LIN showed 6% survival rate at day 7 after injection compared to control cells together with increased proliferation and significant increase in cardiac structure and function 8 weeks after MI. Finally, CSTC-LIN showed enhanced ability to secrete paracrine factors under hypoxic conditions and ability to promote cardiomyocyte proliferation following ischemic cardiac injury. Conclusions LIN28a modification promotes metabolic flexibility in CTSCs enhancing proliferation and survival post transplantation including ability to repair the heart after myocardial injury., Graphical abstract Image 1, Highlights • Cell therapy is hampered by stem cell loss after transplantation in the heart. • Genetic modification of stem cells with LIN28 promotes cardiac repair after injury. • Metabolic flexibility of LIN28a stem cells increased survival in the ischemic heart. • LIN28a salutary effects are linked to modulation of let-7/PDK1 axis.

In this research, we identified genes associated with ammonia nitrogen (TAN) stress response and resistance in juveniles of the Zebra II strain and a wild population of the Manila clam Ruditapes philippinarum. Both groups were subjected to a 96 h acute toxicity test using TAN concentrations of 17.617 ± 0.634 and 16.670 ± 0.7 mg/l, respectively. We then collected samples, conducted transcriptome sequencing and screened the sequences for differentially expressed genes (DEGs) related to TAN stress response. We identified 2908 and 2861 DEGs in the Zebra II and wild clam groups, respectively, and the two groups had 626 DEGs in common. The verified DEGs had less of a detoxification effect in the wild population than that in the Zebra II group. Gene Ontology database analysis showed that Zebra II juveniles were mainly enriched in protein phosphorylation, purine nucleoside binding, and kinase activity, whereas the wild population juveniles were primarily enriched in oxidases activity, organic acid metabolic processes, and extracellular regions. Kyoto Encyclopedia of Genes and Genomes pathway analysis mainly highlighted aminoacyl tRNA biosynthesis in Zebra II juveniles and sphingolipid metabolism, FOXO signaling, biosynthesis of aminoacyl tRNA, and other pathways in the wild population. These results show that the toxic effect of TAN on the Manila clam is related to a variety of pathways, which are mainly related to immune response, inflammatory response, metabolic pathways, and nerve conduction. This study provides basic data and theoretical reference for revealing the molecular regulation mechanism of the improved TAN tolerance of Zebra II strain as compared with the wild population of Ruditapes philippinarum.

The ubiquitously expressed ABL1 and ABL2 protein kinases play many important roles in cell function. Although they have been implicated in neuron development, maintenance and signaling, there are no good tool compounds to evaluate the effects of ABL kinase inhibition in the brain. Asciminib is a recently approved drug that specifically and potently inhibits the tyrosine kinase activity of ABL1, ABL2 and that of the chimeric BCR-ABL1 oncoprotein which causes chronic myeloid leukemia. Herein we show that asciminib does not penetrate the intact blood-brain barrier (BBB) following administration to rats, which curtails its utility for assessing the in vivo effects of ABL kinase inhibition in the brain. However, we describe another specific ABL kinase inhibitor, possessing physicochemical characteristics suitable for BBB penetration, and which after administration (either i.v., i.p. or p.o.) to mice achieves substantial, pharmacologically relevant brain concentrations. This bipyridine compound (4) therefore has potential for elucidating the role of ABL kinases in the brain in non-clinical studies.

Despite major advances, not all patients achieve rheumatoid arthritis (RA) remission, thus highlighting a pressing need for new therapeutic treatments. Given this scenario, this study sought to evaluate Se-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl] 4-chlorobenzoselenolate (Se-DMC) potential on a complete Freund's adjuvant (CFA)-induced unilateral arthritis model. The effects of Se-DMC (5 mg/kg; oral dose) and meloxicam (5 mg/kg; oral dose), both administered to animals daily for 14 days, on paw edema, mechanical sensitivity, neurobehavioral deficits (anxiogenic- and depressive-like behaviors), Na+/K+-ATPase activity, oxidative stress, and inflammation were evaluated in male Swiss mice exposed to CFA (intraplantar injection of 0.1 mL; 10 mg/mL). Se-DMC reduced the paw withdrawal threshold and CFA-induced paw edema. Histopathological results revealed the antiedematogenic potential of the compound, which was evidenced by lower quantities of dilated lymphatic vessels compared with the CFA group. Se-DMC reduced mRNA relative expression levels of tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) in the hippocampus and paw of CFA mice. The CFA-induced anxiogenic- and depressive-like behaviors were reversed by Se-DMC to the control levels in the elevated plus-maze and tail suspension tests. Se-DMC reduced the paw reactive species levels and restored the superoxide dismutase (hippocampus and paw) and Na+/K+-ATPase (hippocampus) activities previously increased by CFA. Moreover, CFA administration inhibited serum creatinine kinase activity, albeit the Se-DMC effects did not appear to involve the modulation of this enzyme and were equal to or greater than meloxicam. Se-DMC attenuates CFA-induced inflammatory response, nociception, and neurobehavioral deficits in mice.

Towards a better understanding of the formation mechanism of salt on intramuscular triglyceride (TG) hydrolysis occurring in biceps femoris (BF) muscles during dry-salting process, the changes of TG hydrolysis, TG hydrolysis activity and phosphorylation of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) as well as their regulatory proteins (Perilipin1, ABHD5, G0S2) with different salt content (0%, 1%, 3%, 5%) and salting time (the first and third day) were analyzed. The results showed that dry-salting significantly increased the TG hydrolase activity and hydrolysis extent with salting process proceed (P 0.05), especially upon the treatment with 3% amount of salt. The SDS-PAGE and Western-blot results further demonstrated that the promotion of salt on TG hydrolysis in intramuscular adipocytes was mainly attributed to the activation of protein kinase activity and protein phosphorylation process. Accordingly, the ATGL and HSL were activated, and meanwhile, the TG hydrolysis pivotal switch perilipin1 was also turned on by phosphorylation modification.

The intestinal barrier function relies on the presence of a single layer of epithelial cells. Barrier dysfunction is associated with the inflammatory bowel diseases (IBD). Understanding the mechanisms involved in intestinal wound healing in order to sustain the barrier function has a great therapeutic potential. Activation of protease-activated receptor-2 (PAR2) induces COX-2 expression in intestinal epithelial cells via EGFR transactivation. COX-2 is well known for its protective effects in the gastrointestinal tract. Therefore, we hypothesized that PAR-2 activation induces a wound healing response in intestinal epithelial cells through COX-2-derived lipid mediators and EGFR transactivation. Immunofluorescence and calcium assay were used to characterize CMT-93 mouse colonic epithelial cell line for PAR2 expression and its activity, respectively. Treatment with PAR2 activating peptide 2-furoyl-LIGRLO-NH(2) (2fLI), but not by its inactive reverse-sequence peptide (2fO) enhanced wound closure in scratch wounded monolayers. The EGFR tyrosine kinase inhibitor (PD153035), broad-spectrum matrix metalloproteinase inhibitor (GM6001) and Src tyrosine kinase inhibitor (PP2) inhibited PAR2-induced wound healing. However, PAR2 activation did not induce COX-2 expression in CMT-93 cells and inhibition of COX-2 by COX-2 selective inhibitor (NS-398) did not alter PAR2-induced wound healing. In conclusion, PAR2 activation drives wound healing in CMT-93 cells via EGFR transactivation. Matrix metalloproteinases and Src tyrosine kinase activity may involve in EGFR transactivation and PAR2-induced wound healing is independent of COX-2 activity. These findings provide a mechanism whereby PAR2 can participate in the resolution of intestinal wounds in gastrointestinal inflammatory diseases.

Body weight (BW) is a critical economic trait for meat production in sheep, and it is a complex trait affected by numerous elements. The aim of this study was to investigate the genetic mechanisms of sheep BW by a label-free proteomics approach. The result showed, a total of 27, 14, 61, and 65 differentially abundant proteins (DAPs) were identified in the Hu_HBW vs. Hu_LBW, DP_HBW vs. DP_LBW, Hu_HBW vs. DP_HBW, and Hu_LBW vs. DP_LBW comparisons, respectively. Five proteins (including ILK, AHCYL2, MLIP, CYB5A, and SMTNL1) related to fat synthesis and muscle development were detected in the Hu sheep group. In the Dorper sheep group, the screened DAPs strictly related to muscle development and fat synthesis were significantly enriched in MAP kinase activity (MAPK12), Arachidonic acid metabolism, and Steroid hormone biosynthesis (PGFS, LOC101107119) pathways. Several DAPs related to immune responses (SERPINA1, FGG, SERPINC1, and LOC101108131), fat deposition (APOH, GC, AHSG, SKP1, ACSL1, ACAT1, and ACADS), and muscle development (LMOD3 and LRRC39) were detected in the Hu vs. Dorper sheep comparison. These analyses indicated that the BW of sheep is regulated via a variety of pathways, and these DAPs can be further investigated as candidate markers for predicting the BW of sheep. SIGNIFICANCE: Body weight is one of the key traits in sheep and involves multiple coordinated regulatory mechanisms, but the genetic mechanism of BW is still unclear in sheep. In the current study, the label-free method was used to identify the proteins and pathways related to BW using LT muscle of Hu sheep and Dorper sheep with different BW. These findings will provide new candidate proteins and vital pathways into the molecular mechanisms involved growth traits in sheep.

Disruption of fetal growth results in severe consequences to human health, including increased fetal and neonatal morbidity and mortality, as well as potential lifelong health problems. Molecular mechanisms promoting fetal growth represent potential therapeutic strategies to treat and/or prevent fetal growth restriction (FGR). Here, we identify a previously unknown role for the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) in promoting fetal and placental growth. We demonstrate that inactivation of MAP3K4 kinase activity causes FGR due in part to placental insufficiency. Significantly, MAP3K4 kinase-inactive mice display highly penetrant lethality prior to weaning and persistent growth reduction of surviving adults. Additionally, we elucidate molecular mechanisms by which MAP3K4 promotes growth through control of the insulin-like growth factor 1 receptor (IGF1R), insulin receptor (IR), and Akt signaling pathway. Specifically, MAP3K4 kinase inactivation in trophoblast stem (TS) cells results in reduced IGF1R and IR expression and decreased Akt activation. We observe these changes in TS cells also occur in differentiated trophoblasts created through in vitro differentiation of cultured TS cells and in vivo in placental tissues formed by TS cells. Furthermore, we show that MAP3K4 controls this pathway by promoting Igf1r transcript expression in TS cells through activation of CREB-binding protein (CBP). In the MAP3K4 kinase-inactive TS cells, Igf1r transcripts are repressed because of reduced CBP activity and increased histone deacetylase 6 expression and activity. Together, these data demonstrate a critical role for MAP3K4 in promoting fetal and placental growth by controlling the activity of the IGF1R/IR and Akt signaling pathway.

Uncontrolled gluconeogenesis results in elevated hepatic glucose production in type 2 diabetes (T2D). The small ubiquitin-related modifier (SUMO)-specific protease 2 (SENP2) is known to catalyze deSUMOylation of target proteins, with broad effects on cell growth, signal transduction, and developmental processes. However, the role of SENP2 in hepatic gluconeogenesis and the occurrence of T2D remain unknown. Herein, we established SENP2 hepatic knockout mice and found that SENP2 deficiency could protect against high-fat diet-induced hyperglycemia. Pyruvate- or glucagon-induced elevation in blood glucose was attenuated by disruption of SENP2 expression, whereas overexpression of SENP2 in the liver facilitated high-fat diet-induced hyperglycemia. Using an in vitro assay, we showed that SENP2 regulated hepatic glucose production. Mechanistically, the effects of SENP2 on gluconeogenesis were found to be mediated by the cellular fuel sensor kinase, 5'-AMP-activated protein kinase alpha (AMPKα), which is a negative regulator of gluconeogenesis. SENP2 interacted with and deSUMOylated AMPKα, thereby promoting its ubiquitination and reducing its protein stability. Inhibition of AMPKα kinase activity dramatically reversed impaired hepatic gluconeogenesis and reduced blood glucose levels in SENP2-deficient mice. Our study highlights the novel role of hepatic SENP2 in regulating gluconeogenesis and furthers our understanding of the pathogenesis of T2D.