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Neuronal communication crucially relies on exocytosis of neurotransmitters from synaptic vesicles (SVs) which are clustered at synapses. To ensure reliable neurotransmitter release, synapses need to maintain an adequate pool of SVs at all times. Decades of research have established that SVs are clustered by synapsin 1, an abundant SV-associated phosphoprotein. The classical view postulates that SVs are crosslinked in a scaffold of protein-protein interactions between synapsins and their binding partners. Recent studies have shown that synapsins cluster SVs via liquid-liquid phase separation (LLPS), thus providing a new framework for the organization of the synapse. We discuss the evidence for phase separation of SVs, emphasizing emerging questions related to its regulation, specificity, and reversibility.

The ATP binding cassette (ABC) transporter molecule ABCA1 participates in the cholesterol transport within and through cell membranes. We recently demonstrated that in dog spermatozoa, capacitation could be decreased with probucol (PRO), an ABCA1 specific antagonist. In this study, a dose-effect relationship of PRO on dog sperm capacitation, tyrosine phosphorylation and cholesterol efflux from the sperm plasma membrane was investigated. A total of 16 ejaculates from dogs of different breeds, aged 2-4 years were used. Sperm motility and membrane integrity in the main fraction was determined by CASA. Samples were stained with a boron dipyrromethene difluoride (BODIPY) fluorophore (P9672, Sigma- Aldrich, A) diluted in DMSO at a final concentration of 0.4 μM. All samples were divided into 5 aliquots, with 0, 100, 250, 500 and 1000 μM of PRO. After incubation at 37 °C for 2 h, PI was added and flow cytometry performed. All aliquots were examined for capacitation and acrosome reaction by using the CTC assay and tyrosine phosphorylation (TP). Membrane integrity was measured in all aliquots to investigate the effect of PRO on cell membranes. Membrane integrity did not differ between controls (0 μM), and 100, 250 and 500 μM PRO, but decreased with 1000 μM PRO (p 0.05). Increasing PRO concentration decreased the percentage alive cells with cholesterol efflux per PRO group (0 μM: 77.8 ± 10.6%, 100 μM: 63.7 ± 11.7%, 250 μM: 52.1 ± 12.9%, 500 μM: 37.7 ± 11.6%, 1000 μM: 33.1 ± 14.4%; p 0.05), decreased head and entire tail phosphorylated cells (0 μM: 34.6%, 1000 μM: 5.1% p 0.05); and decreased the percentage capacitated cells (maximum with PRO 500 μM: capacitated vs. control: 54.2 ± 17% vs 25 ± 7.7%, p 0.05). Conclusion: PRO decreased the cholesterol efflux, and decreased tyrosine phosphorylation and capacitation in a dose-dependent manner. This suggests a strong involvement of the ABCA1 transporter in different functional aspects of sperm capacitation in dogs.

In mammals, nemo-like kinase 2 (NLK2) is a conservative protein kinase involved in Wnt/β-catenin signaling pathway and immune response. However, the role of NLK2 in immune response in teleost remain unclear. In this study, we identified an ortholog of mammalian NLK from grass carp (Ctenopharyngodon idellus) named CiNLK2. CiNLK2 shares a high level of homology with the counterparts, especially with that of Cyprinus carpio. CiNLK2 was ubiquitously expressed in all tested tissues (liver, brain, spleen, gill, kidney and eye) and its expression was up-regulated under the treatment with poly I:C or GCRV. Overexpression of CiNLK2 suppressed the production of IFN I in CIK cells whether or not treated with poly I:C. However, knockdown of CiNLK2 increased the expression level of IFN I. The analysis of subcellular localization showed that CiNLK2 protein was scattered throughout the cytoplasm and nucleus. In terms of mechanism, CiNLK2 can directly interact with MAVS and inhibit MAVS-induced IFN I response. Moreover, CiNLK2 increased the phosphorylation level of MAVS, which led to the degradation of MAVS protein. On the other hand, CiNLK2 suppressed the phosphorylation and nuclear translocation of IRF3. In general, CiNLK2 served as an inhibitor for IFN I response by targeting MAVS-IRF3 signal axis.

Protein-protein interaction networks - interactomes - are charted with the hope to understand how phenotypes emerge and how they are altered in disease states. Early efforts to map interactomes have focused on the assembly of context agnostic, reference networks. However, recent studies have mapped interactomes across different cell lines and tissues, finding highly variable interactomes due to the rewiring of protein-protein interactions in different contexts. Increasing evidence points to significant links between protein structure and interactome diversity seen across cell types and tissues. We discuss how recent findings support the key role of alternative splicing and phosphorylation, two well-established regulators of protein structural and functional diversity, in defining cell type- and tissue-specific interactomes. Moreover, we show that intrinsically disordered protein regions are most favorably equipped to support interactome rewiring by acting as hubs of protein structure and function regulation.

Bruton's tyrosine kinase (BTK) is a critical enzyme which is involved in multiple signaling pathways that regulate cellular survival, activation, and proliferation, making it a major cancer therapeutic target. We applied the novel integrated structure-based pharmacophore modeling, machine learning, and other in silico studies to screen the Korean chemical database (KCB) to identify the potential BTK inhibitors (BTKi). Further evaluation of these inhibitors on three different human cancer cell lines showed significant cell growth inhibitory activity. Among the 13 compounds shortlisted, four demonstrated consistent cell inhibition activity among breast, gastric, and lung cancer cells (IC50 below 3 μM). The selected compounds also showed significant kinase inhibition activity (IC50 below 5 μM). The current study suggests the potential of these inhibitors for targeting BTK malignant tumors.

Although several studies have focused on cancer diagnosis and therapy, prostate cancer (PC) remains an intractable disease. Androgen deprivation therapy (ADT), which is used to treat early stage PC can lead to the development of castration-resistant prostate cancer (CRPC), which is highly associated with androgen receptor (AR) mutations. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) is a chaperone that shuttles between the nucleus and the cytoplasm. Studies suggest that NOLC1 regulates PC progression; however, the underlying mechanisms remain unclear. Herein, we showed that NOLC1 knockdown suppresses PC cell proliferation by altering the signaling pathways and the expression of various proteins involved in DNA replication, amino acid metabolism, and RNA processing. Mechanistically, NOLC1 knockdown suppressed cell cycle progression by inhibiting AKT phosphorylation and β-catenin accumulation. Finally, we showed that NOLC1 expression is higher in human PC than in human hyperplastic prostate tissues. Altogether, we demonstrated that NOLC1 knockdown suppresses the progression of both AR-positive and AR-negative PC cells by inducing changes in the expression of several genes leading to cell cycle arrest. Thus, NOLC1 might be a novel and promising therapeutic target for PC.

In plants, transitory starch is synthetized during the day and degraded at night to provide the continuous carbon needed for growth and development. Starch metabolism is highly coordinated, as the starch degradation rate must be coupled to the amount of starch synthetized during the day. Maltose is one of the chloroplastic products obtained from starch degradation, and maltose is exported to the cytosol where disproportionating enzyme-2 (DPE2) is responsible for its metabolism. The amount of DPE2 remained unchanged throughout the day, but its activity notably increased at the end of the day (7 p.m.), suggesting that posttranslational modification drives the mechanism underlying the regulatory activity of this enzyme. Sucrose nonfermenting-related kinase-1 (SnRK1), a protein kinase that controls the activity of several metabolic enzymes, was able to interact and phosphorylate DPE2 at three different residues localized in the α-glucanotransferase domain. This phosphorylation acts as a positive regulator of DPE2, increasing its activity. Complementation of dpe2-deficient mutants with the wild-type (WT) and S786A forms of DPE2 showed that the nonphosphorylated form of DPE2 only partially restored starch degradation, suggesting that phosphorylation at S786 is involved in enzyme regulation.

Atherosclerosis (AS) is the pathological basis of many cardiovascular and cerebrovascular diseases, in which macrophage-derived foam cells are the critical step and a typical pathological feature of early atherosclerosis. We previously confirmed that low-molecular-weight fucoidan (LMWF) had a good anti-AS effect, but the mechanism is still unclear. Here with aim to investigate the inhibitory effect of LMWF on foam cells and its molecular mechanism. Oil red O staining showed that LMWF effectively alleviated lipid accumulation and the formation of foam cells. Flow cytometry detection showed that LMWF promoted foam cells apoptosis. In addition, immunofluorescence showed that LMWF inhibited macrophage scavenger receptor A1 (SR-A1)-mediated lipid uptake and promoted ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol outflow. Western blot showed that LMWF downregulated SR-A1 protein expression and upregulated ABCA1 protein expression by inhibiting p38 mitogen activated protein kinase (p38MAPK) phosphorylation. Moreover, the mRNA transcriptions of Stat1, Elk-1, and Myc were downregulated when treated with LMWF. It concluded that, LMWF achieved bidirectional regulation of SR-A1 and ABCA1, then prevented the formation of foam cells, finally ameliorated the development of AS.

The adaptor protein GAREM has two subtypes. Each is involved in Erk activation signaling downstream of the cell growth factor receptor in cultured cells. Regarding their role in individual animals, we have previously reported that mice deficient in GAREM2, which is highly expressed in the brain, exhibit emotional changes. In this paper, we report an amino acid substitution mutation (K291R) in GAREM1, in a patient with idiopathic short stature, which indicates that the mutant exhibits dominant-negative properties. The GAREM K291R mutant did not promote Erk activation in EGF-stimulated cultured cells. Similar features were also observed in cells in which GAREM1 expression was suppressed by genome editing; along with Erk, phosphorylation of S6 kinase and 4EBP1, whose activation is necessary for cell proliferation and biological growth, were inhibited Furthermore, we generated mice deficient in GAREM1 and showed that the mutant mice are lighter in weight. Overall, the results of this paper suggest that GAREM1 is required for normal growth and for maintaing average body size in humans and mice.

To evaluate the phosphorylation of estrogen receptor α at serine-118 (phospho-ERα S118) in the endometrium, ovarian endometrioma, and deep infiltrating endometriosis (DIE).Experimental study.University-affiliated hospital and academic research laboratory.Twenty-five patients underwent a hysterectomy, 18 patients underwent surgical removal of ovarian endometrioma, and 6 patients underwent DIE.Tissue samples were obtained from patients who underwent surgical procedures.Immunostaining for phospho-ERα S118, ERα, or phosphorylated p44/42 mitogen-activated protein kinase (phospho-p44/42 MAPK) was performed to evaluate the endometrium with or without endometriosis, ovarian endometrioma, and DIE. For in vitro analysis, endometrial epithelial cells (Ishikawa cells) were stimulated with estradiol (E2) or tumor necrosis factor alpha (TNFα), and the expression levels of phospho-ERα S118 and phospho-p44/42 MAPK were evaluated via Western blotting.First, phospho-ERα S118 level was significantly higher in the glands and stroma of ovarian endometriosis samples than in those of endometrial and DIE samples. Second, colocalization of phospho-p44/42 MAPK and phospho-ERα S118 was observed in the glands of ovarian endometrioma. The proportions of cells strongly expressing phospho-p44/42 and phospho-ERα were 87% in phosphor-p44/42 MAPK-positive cells and 79% in phosphor-ERα-positive cells. Third, E2 stimulation significantly enhanced phospho-ERα S118 after 15 and 30 minutes in in vitro analysis using endometrial epithelial cells. Fourth, TNFα stimulation modestly but significantly enhanced phospho-ERα S118 after 15 and 30 minutes. Fifth, in Ishikawa cells, treatment with a p44/42 inhibitor (PD98059) significantly reduced phospho-ERα S118 by TNFα but not by E2.ERα-S118 phosphorylation was increased in ovarian endometriosis. Our findings may provide a new perspective for understanding the mechanism of increased ERα action in the pathophysiology of endometriosis.

We previously showed that the adaptive response of BRAFV600-mutated melanoma cells to BRAF inhibition emerges from a subpopulation of cells expressing an intermittent lower level of the mRNA-binding protein HuR. In this study, following initial overexpression experiments in which we confirm our previous results, we use wild-type and mutants HuR full-length mRNA constructs and in vivo-interacting assays and demonstrate that a highly conserved interferon-γ-activated inhibitor of translation (GAIT)-like motif located upstream of the GU-rich elements of HuR major polyadenylation site (PAS2), interacts with constituents of the GAIT complex and affects HuR post-transcriptional expression regulation. Knockdown of the ribosomal protein L13a or the inhibition of the DAPK1-ZIPK axis involved in L13a phosphorylation, reduces the proportion of HuR

Cellulose is an interesting biopolymer offering numerous functionalization possibilities for various applications. Yet, cellulose functionalization usually involves expensive chemicals and complex processes. Here, we aim to utilize inexpensive fertilizer-grade phosphate for cellulose functionalization. Cellulose microfibers (CMF) were isolated from Giant Reed (GR) and were then phosphorylated using either a reagent-grade or a fertilizer-grade diammonium hydrogen phosphate (DAP) in the presence of urea following a water-based protocol. The effect of DAP on the phosphorylation reaction was mainly studied by conductometric titration, ICP-OES and FTIR, while further characterization was performed by SEM/EDX, TGA and XRD to investigate the morphology, composition, charge content, structure, and thermal degradation of the phosphorylated materials. It was found that cellulose phosphorylation using DAP fertilizer gave materials with the same charge content as that registered when using the reagent-grade DAP. Optimizing the reaction conditions with respect to the amount of fertilizer-grade DAP used for the phosphorylation gave high charge content (7000 mmol·g

Triacylglycerols (TGs) serve as reservoirs for diacylglycerols and fatty acids, which play important roles in synthesizing energy and membrane lipids that are required for cell cycle progression. In the yeast, Saccharomyces cerevisiae, Tgl4, the functional ortholog of murine adipose triacylglycerol lipase (ATGL), is activated by Cdk1/Cdc28-mediated phosphorylation and facilitates the G1/S transition. However, little is known about how Tgl4 is inactivated during the cell cycle. To monitor the phosphorylation status and the stability of endogenous Tgl4, we raised a specific antibody against Tgl4. We found that in contrast to the previous suggestion, Tgl4 was a stable protein throughout the cell cycle. We also showed that Tgl4 was dephosphorylated upon entry into G1 phase. These results suggest that Tgl4 is a stable protein and is inactivated during G1 phase by dephosphorylation.

Benzodiazepines like midazolam were generally considered one of the possible causes affecting postoperative cognitive recovery. As a new kind of rapid-effect benzodiazepine, remimazolam tosylate was widely used in clinical anesthesia for its pharmacological advantage, but few studies reported its effects on cognitive function in the elderly. Here, we aimed to research the effects of remimazolam tosylate on cognitive function in aged mice and its underlying biological mechanisms. We measured the memory function of aged mice immediately and one month after intraperitoneal injection of remimazolam tosylate compared to the saline control. The brain metabolism level was detected by Positron Emission Tomography/Computed tomography (PET/CT). Compared with the control, we observed a decrease in memory ability， as well as an increase in tau phosphorylation level and a decrease in phosphatase level in the short term; however, one month later, contrary to the previous results, we observed better memory and brain metabolism and lower tau phosphorylation levels in the experimental group compared to the control. Therefore, we concluded that remimazolam tosylate did not cause long-term damage to the cognitive function of aged mice and even delayed the decline of memory function in the aging process to some extent.

Salt stress adversely affects plant growth, development, and crop yield. Rice (Oryza sativa L.) is one of the most salt-sensitive cereal crops, especially at the early seedling stage. Mitogen-activated protein kinase (MAPK/MPK) cascades have been shown to play critical roles in salt response in Arabidopsis. However, the roles of the MPK cascade signaling in rice salt response and substrates of OsMPK remain largely unknown. Here, we report that the salt-induced OsMPK4-Ideal Plant Architecture 1 (IPA1) signaling pathway regulates the salt tolerance in rice. Under salt stress, OsMPK4 could interact with IPA1 and phosphorylate IPA1 at Thr180, leading to degradation of IPA1. Genetic evidence shows that IPA1 is a negative regulator of salt tolerance in rice, whereas OsMPK4 promotes salt response in an IPA1-dependent manner. Taken together, our results uncover an OsMPK4-IPA1 signal cascade that modulates the salt stress response in rice and sheds new light on the breeding of salt-tolerant rice varieties.

Our previous study confirmed that polydatin (PD) can alleviate sepsis-induced multiorgan dysfunction (in the vascular endothelium, kidney, and small intestine) by activating Sirt1 and that PD protects against traumatic brain injury in rats via increased Sirt1 and inhibition of the p38-mediated mitogen-activated protein kinase (MAPK) pathway. We aim to investigate whether PD may also attenuate sepsis-associated encephalopathy (SAE).In this study, we constructed an SAE mouse model by cecal ligation and puncture (CLP) and measured Sirt1 protein activity, p38 phosphorylation, brain tissue pathological damage, pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), mitochondrial function (mitochondrial membrane potential, ATP content, and reactive oxygen species), neurological function, and animal survival time. Sirt1 selective inhibitor Ex527 and p38 inhibitor SB203580 were used to explore the possible mechanism of PD in SAE.We confirmed that PD inhibits neuroinflammation evidenced by reduced proinflammatory cytokines. In addition, PD protects mitochondria as demonstrated by restored mitochondrial membrane potential and adenosine triphosphate (ATP) content, and decreased reactive oxygen species (ROS) level. As we expected, p38 inhibition reduces neuroinflammation and mitochondrial damage. In contrast, Sirt1 inhibition aggravates cerebral cortex mitochondrial damage and neuroinflammation and promotes phosphorylation of p38. Mechanistically, PD treatment suppressed p38 phosphorylation and consequently reduced the neuroinflammatory response, and these effects were blocked by the Sirt selective inhibitor Ex527.This study, to the best of our knowledge, is the first to demonstrate that PD alleviates SAE, at least partially, by upregulating Sir1-mediated neuroinflammation inhibition and mitochondrial function protection.

This study reports that hairy and enhancer of split homolog-1 (HES1), known to repress gene transcription in progenitor cells of several cell lineages, was strongly expressed in cells and tissues of T-cell lymphoma expressing the oncogenic chimeric tyrosine kinase nucleophosmin (NPM)-anaplastic lymphoma kinase [ALK; ALK

Plants under pathogen attack produce high levels of the gaseous phytohormone ethylene to induce plant defense responses via the ethylene signaling pathway. The 1-aminocyclopropane-1-carboxylate synthase (ACS) is a critical rate-limiting enzyme of ethylene biosynthesis. Transcriptional and post-translational upregulation of ACS2 and ACS6 by the mitogen-activated protein kinases MPK3 and MPK6 are previously shown to be crucial for pathogen-induced ethylene biosynthesis in Arabidopsis. Here, we report that the fungal pathogen Botrytis cinerea-induced ethylene biosynthesis in Arabidopsis is under the negative feedback regulation by ethylene signaling pathway. The ethylene response factor ERF1A is further found to act downstream of ethylene signaling to negatively regulate the B. cinerea-induced ethylene biosynthesis via indirectly suppressing the expression of ACS2 and ACS6. Interestingly, ERF1A is shown to also upregulate defensin genes directly and therefore promote Arabidopsis resistance to B. cinerea. Furthermore, ERF1A is identified to be a substrate of MPK3 and MPK6, which phosphoactivate ERF1A to enhance its functions in suppressing ethylene biosynthesis and inducing defensin gene expression. Taken together, our data reveal that ERF1A and its phosphorylation by MPK3/MPK6 not only mediate the negative-feedback regulation of the B. cinerea-induced ethylene biosynthesis, but also upregulate defensin gene expression to increase Arabidopsis resistance to B. cinerea.

Neuropathic and inflammatory pain are major clinical challenges due to their ambiguous mechanisms and limited treatment approaches. N-methyl-D-aspartate receptor (NMDAR) and calcium-calmodulin-dependent protein kinase II (CaMKII) are responsible for nerve system sensation and are required for the induction and maintenance of pain. However, the roles of NMDAR and CaMKII in regulating orofacial pain are still less well known. Here, we established a neuropathic pain model by transecting a mouse inferior alveolar nerve (IAN) and an inflammatory pain model by injecting complete Freund

Nuclear receptor Pregnane X Receptor (PXR; NR1I2) has transcriptional regulation functions for energy homeostasis in the liver. Mouse PXR has a conserved phosphorylation motif at serine 347 (serine 350 in humans) within the ligand-binding domain. PXR phosphorylated at this motif is expressed in mouse livers in response to fasting. Mice with a PXR∗Ser347Ala knockin mutation (PXR KI) were generated to block phosphorylation, and utilized to investigate the role of Ser347 phosphorylation in vivo. PXR KI mice had decreased body weight at 8-weeks of age and had much greater weight loss after fasting compared with PXR WT mice. The cDNA microarray analysis of hepatic mRNAs showed that cell death or apoptotic signaling was induced in fasting PXR KI mice. Moreover, increasing hepatic lipids, triglycerides and the development of hypertriglyceridemia were observed in fasting PXR KI mice. These findings are indicative that blocking phosphorylation prevents mice from maintaining hepatic energy homeostasis. Thus, phosphorylated PXR may be an essential factor to prevent the liver from developing damage caused by fasting.

Ribosomal protein S6 (rpS6) phosphorylation mediates the hypertrophic growth of kidney proximal tubule cells. However, the role of rpS6 phosphorylation in podocyte hypertrophy and podocyte loss during the pathogenesis of focal segmental glomerulosclerosis (FSGS) remains undefined. Here, we examined rpS6 phosphorylation levels in kidney biopsy specimens from patients with FSGS and in podocytes from mouse kidneys with Adriamycin-induced FSGS. Using genetic and pharmacologic approaches in the mouse model of FSGS, we investigated the role of rpS6 phosphorylation in podocyte hypertrophy and loss during development and progression of FSGS. Phosphorylated rpS6 was found to be markedly increased in the podocytes of patients with FSGS and Adriamycin-induced FSGS mice. Genetic deletion of the Tuberous sclerosis 1 gene in kidney glomerular podocytes activated mammalian target of rapamycin complex 1 signaling to rpS6 phosphorylation, resulting in podocyte hypertrophy and pathologic features similar to those of patients with FSGS including podocyte loss, leading to segmental glomerulosclerosis. Since protein phosphatase 1 is known to negatively regulate rpS6 phosphorylation, treatment with an inhibitor increased phospho-rpS6 levels, promoted podocyte hypertrophy and exacerbated formation of FSGS lesions. Importantly, blocking rpS6 phosphorylation (either by generating congenic rpS6 knock-in mice expressing non-phosphorylatable rpS6 or by inhibiting ribosomal protein S6 kinase 1-mediated rpS6 phosphorylation with an inhibitor) significantly blunted podocyte hypertrophy, inhibited podocyte loss, and attenuated formation of FSGS lesions. Thus, our study provides genetic and pharmacologic evidence indicating that specifically targeting rpS6 phosphorylation can attenuate the development of FSGS lesions by inhibiting podocyte hypertrophy and associated podocyte depletion.

Transcranial ultrasound stimulation is an emerging technique for the development of a non-invasive neuromodulation device for the treatment of various types of neurodegenerations and brain damages. However, there are very few studies that have quantified the optimal ultrasound dosage and the long-term associated effects of transcranial ultrasound treatments of brain diseases. In this study, we used a simple ex vivo hippocampal tissues stimulated by different dosages of ultrasound in combination with different chemical treatments to quantify the required energy for a measurable effect. After determining the most desirable ex vivo stimulation conditions, it was then replicated for the in vivo mouse brains. It was discovered that transcranial ultrasound promoted the increase of Tbr2-expressing neural progenitors in an ASIC1a-dependent manner. Furthermore, such effect was observable at least a week after the initial ultrasound treatments and was not abolished by auditory toxicity.

Rapamycin has been recommended to treat Kaposiform hemangioendothelioma (KHE) with Kasabach-Merritt phenomenon (KMP), but the underlying mechanism of the clinical effect has not been established. Therefore, we determined rapamycin cytotoxicity on KHE cells in vitro and the underlying mechanism.KHE primary cells were derived from a tumor specimen and treated with rapamycin. Immunofluorescence was applied to identify the cells. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay. Cell cycle and apoptosis were assessed using flow cytometry (FCM). Western blots (WB) were performed to determine phosphorylation of mammalian target of rapamycin (mTOR), p70 S6 kinase (S6K1), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), as well light chain 3 (LC3) expression.Rapamycin inhibited the growth of KHE primary cells in a dose- and time-dependent manner. Cell cycle progression was arrested in the G0/G1 phase and apoptosis was induced. WB results showed that LC3-II/I expression was significantly elevated in KHE primary cells treated with rapamycin, while the level of p-mTOR, p-S6K1, and p-4E-BP1 expression was reduced. LC3 fluorescent spots were increased in the rapamycin treatment group.Rapamycin inhibited KHE primary cell proliferation, induced apoptosis and autophagy, and blocked the mTOR signaling pathway.

It has been nearly three decades since the discovery of the first bacterial serine/threonine protein kinase (STPK). Since then, a blend of technological advances has led to the characterization of a multitude of STPKs and phosphorylation substrates in several bacterial species that finely regulate intricate signaling cascades. Years of intense research from several laboratories have demonstrated unexpected roles for serine/threonine phosphorylation, regulating not only bacterial growth and cell division but also antibiotic persistence, virulence and infection, metabolism, chromosomal biology, and cellular differentiation. This review aims to provide an account of the most recent and significant developments in this up and growing field in microbiology.

Nuclear factor κB (NF-κB) is a ubiquitous regulator of the signalome and is indispensable for various biological cell functions. NF-κB consists of five transcription factors that execute both cytoplasmic and nuclear signaling processes in cells. NF-κB is the only signaling molecule that governs both pro- and anti-apoptotic, and pro- and anti-inflammatory responses. This is due to the canonical and non-canonical components of the NF-κB signaling pathway. Together, these pathways orchestrate cancer-related inflammation, hyperplasia, neoplasia, and metastasis. Non-canonical NF-κB pathways are particularly involved in the chemoresistance of cancer cells. In view of its pivotal role in cancer progression, NF-κB represents a potentially significant therapeutic target for modifying tumor cell behavior. Several phytochemicals are known to modulate NF-κB pathways through the stabilization of its inhibitor, IκB by inhibiting the phosphorylation and ubiquitination thereof. Several natural pharmacophores are known to inhibit the nuclear translocation of NF-κB and associated pro-inflammatory responses and cell survival pathways. In view of this, and the high degree of specificity exhibited by various phytochemicals for the NF-κB component, we herein present an in-depth overview of these phytochemicals and discuss their mode of interaction with the NF-κB signaling pathways for controlling fate of tumor cells for cancer-directed interventions.

Protein kinase C (PKC) isozymes are maintained in a 'ready-to-go' but 'safe' autoinhibited conformation until second messenger binding unleashes an autoinhibitory pseudosubstrate to allow substrate phosphorylation. However, to gain this 'ready-to-go' conformation, PKC must be processed by a series of complex priming phosphorylations, the mechanism of which was enigmatic until now. Recent findings snapped the pieces of the phosphorylation puzzle into place to unveil a process that involves a newly described motif (TOR interaction motif, TIM), a well-described kinase [mechanistic target of rapamycin complex 2 (mTORC2)], and an often-used mechanism (autophosphorylation) to prime PKC to signal. This review highlights new insights into how phosphorylation controls PKC and discusses them in the context of common mechanisms for AGC kinase regulation by phosphorylation and autophosphorylation.

Clostridium acetobutylicum has been extensively exploited to produce biofuels and solvents and its biofilm could dramatically improve the productivities. However, genetic control of C. acetobutylicum biofilm has not been dissected so far. Here, to identify potential genes controlling C. acetobutylicum biofilm formation, over 40 gene candidates associated with extracellular matrix, cell surface, cell signaling or gene transcription, were tried to be disrupted to examine their individual impact. A total of 25 disruptants were finally obtained over years of attempts, for which biofilm and relevant phenotypes were characterized. Most of these disruptants formed robust biofilm still, or suffered both growth and biofilm defect. Only a strain with a disrupted histidine kinase gene (CA_C2730, designated bfcK in this study) abolished biofilm formation without impairing cell growth or solvent production. Further analysis revealed that bfcK could control flagellar biogenesis and cell motility at protein levels. The bfcK also appeared to repress the phosphorylation of a serine/threonine protein kinase (encoded by CA_C0404) that might negatively regulate biofilm formation. Based on these findings, possible bfcK-mediated mechanisms for biofilm formation were proposed. This is a big step toward understanding the biofilm formation in C. acetobutylicum and will help further engineering of its biofilm-based industrial processes.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen belongs to betaherpesvirus subfamily. RNA2.7 is a highly conserved long non-coding RNA accounting for more than 20% of total viral transcripts. In our study, functions of HCMV RNA2.7 were investigated by comparison of host cellular transcriptomes between cells infected with HCMV clinical strain and RNA2.7 deleted mutant. It was demonstrated that RNA polymerase II (Pol II)-dependent host gene transcriptions were significantly activated when RNA2.7 was removed during infection. A 145 ​nt-in-length motif within RNA2.7 was identified to inhibit the phosphorylation of Pol II Serine-2 (Pol II S2) by reducing the interaction between Pol II and phosphorylated cyclin-dependent kinase 9 (pCDK9). Due to the loss of Pol II S2 phosphorylation, cellular DNA pre-replication complex (pre-RC) factors, including Cdt1 and Cdc6, were significantly decreased, which prevented more cells from entering into S phase and facilitated viral DNA replication. Our results provide new insights of HCMV RNA2.7 functions in regulation of host cellular transcription.

The target of rapamycin (TOR), discovered 30 years ago, is a highly conserved serine/threonine protein kinase that plays a central role in regulating cell growth and metabolism. It is activated by nutrients, growth factors, and cellular energy. TOR forms two structurally and functionally distinct complexes, TORC1 and TORC2. TOR signaling activates cell growth, defined as an increase in biomass, by stimulating anabolic metabolism while inhibiting catabolic processes. With emphasis on mammalian TOR (mTOR), we comprehensively reviewed the literature and identified all reported direct substrates. In the context of recent structural information, we discuss how mTORC1 and mTORC2, despite having a common catalytic subunit, phosphorylate distinct substrates. We conclude that the two complexes recruit different substrates to phosphorylate a common, minimal motif.

In Alzheimer's disease (AD), hyper-phosphorylation and aggregation of tau correlate with clinical progression and represent a valid therapeutic target. A recent 20-year prospective study revealed an association between moderate to high frequency of Finnish sauna bathing and a lower incidence of dementia and AD, but the molecular mechanisms underlying these benefits remain uncertain. Here, we tested the hypothesis that sauna-like conditions could lower tau phosphorylation by increasing body temperature. We observed a decrease in tau phosphorylation in wild-type and hTau mice as well as in neuron-like cells when exposed to higher temperatures. These effects were correlated with specific changes in phosphatase and kinase activities, but not with inflammatory or heat shock responses. We also used a drug strategy to promote thermogenesis: topical application of menthol, which led to a sustained increase in body temperature in hTau mice, concomitant with a significant decrease in tau phosphorylation. Our results suggest that sauna-like conditions or menthol treatment could lower tau pathology through mild hyperthermia, and may provide promising therapeutic strategies for AD and other tauopathies.

Memory storage is a conserved survivability feature, present in virtually any complex species . During the last few decades, much effort has been devoted to understanding how memories are formed and which molecular switches define whether a memory should be stored for a short or a long period of time. Among these, de novo protein synthesis is known to be required for the conversion of short- to long-term memory. There are a number translational control pathways involved in synaptic plasticity and memory consolidation, including the phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α), which has emerged as a critical molecular switch for long-term memory consolidation. In this review, we discuss findings pertaining to the requirement of de novo protein synthesis to memory formation, how local dendritic and axonal translation is regulated in neurons, and how these can influence memory consolidation. We also highlight the importance of eIF2α-dependent translation initiation to synaptic plasticity and memory formation. Finally, we contextualize how aberrant phosphorylation of eIF2α contributes to Alzheimer’s disease (AD) pathology and how preventing disruption of eIF2-dependent translation may be a therapeutic avenue for preventing and/or restoring memory loss in AD.

Background Gintonin is a ginseng-derived exogenous G-protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin exerts its neuronal and non-neuronal in vitro and in vivo effects through LPA receptor subtypes. However, it is unknown whether gintonin can bind to the plasma membrane of cells and can transactivate the epidermal growth factor (EGF) receptor. In the present study, we examined whether gintonin-biotin conjugates directly bound to LPA receptors and transactivated the EGF receptor. Methods We designed gintonin-biotin conjugates through gintonin biotinylation and examined whether gintonin-biotin conjugate binding sites co-localized with the LPA receptor subtype binding sites. We further examined whether gintonin-biotin transactivated the EGF receptor via LPA receptor regulation via phosphor-EGF and cell migration assays. Results Gintonin-biotin conjugates elicit [Ca2+]i transient similar to that observed with unbiotinylated gintonin in cultured PC3 cells, suggesting that biotinylation does not affect physiological activity of gintonin. We proved that gintonin-biotin conjugate binding sites co-localized with the LPA1/6 receptor binding sites. Gintonin-biotin binding to the LPA1 receptor transactivates the epidermal growth factor (EGF) receptor through phosphorylation, while the LPA1/3 receptor antagonist, Ki16425, blocked phosphorylation of the EGF receptor. Additionally, an EGF receptor inhibitor AG1478 blocked gintonin-biotin conjugate-mediated cell migration. Conclusions We observed the binding between ginseng-derived gintonin and the plasma membrane target proteins corresponding to the LPA1/6 receptor subtypes. Moreover, gintonin transactivated EGF receptors via LPA receptor regulation. Our results suggest that gintonin directly binds to the LPA receptor subtypes and transactivates the EGF receptor. It may explain the molecular basis of ginseng physiology/pharmacology in biological systems.

Endothelial nitric oxide synthase (eNOS) is a critical regulatory enzyme that controls vascular tone via the production of nitric oxide. Although thrombin also modulates vascular tone predominantly via the activation of protease-activated receptors (PARs), the time course and mechanisms involved in how thrombin controls eNOS enzymatic activity are unknown. eNOS enzymatic activity is enhanced by the phosphorylation of eNOS-Ser1177 and reduced by the phosphorylation of eNOS-Thr495. In this study, we hypothesized that thrombin regulates vascular tone through the differential phosphorylation of eNOS. Using rat descending aorta, we show that thrombin modulates vascular tone in an eNOS-dependent manner via activated PAR-1. We also show that thrombin causes a temporal biphasic response. Protein kinase C (PKC) is associated with second phase of thrombin-induced response. Western blot analysis demonstrated thrombin phosphorylated eNOS-Ser1177 and eNOS-Thr495 in human umbilical vein endothelial cells. A PKC inhibitor suppressed the thrombin-induced phosphorylation of eNOS-Thr495, but not that of eNOS-Ser1177. Our results suggest that thrombin induces a temporal biphasic vascular response through the differential phosphorylation of eNOS via activated PAR-1. Thrombin causes transient vasorelaxation by the phosphorylation of eNOS-Ser1177, and subsequent attenuation of vasorelaxation by the phosphorylation of eNOS-Thr495 via PKC, leading to the modulation of vascular tone.

The epigenetic phenomenon is known to derive the phenotypic variation of an organism through an interconnected cellular network of histone modifications, DNA methylation and RNA regulatory network. Transcription for protein coding genes is a highly regulated process and carried out by a large multi-complex RNA Polymerase II. The carboxy terminal domain (CTD) of the largest subunit of RNA Polymerase II consists of a conserved and highly repetitive heptad sequence Tyr

p38δ is a member of p38 mitogen-activated protein kinases (MAPKs) family that displays cell- and tissue-specific expression patterns. Recent studies demonstrate that p38δ is centrally involved in several pathologic events, such as diabetes, neurodegeneration diseases, inflammatory diseases, and cancer, and suggest that it may be a potential target for diagnosis and therapy of specific diseases. A nanobody is a new type of antibody that exhibits high antigen-binding activity, solubility, stability, and easy production. This study utilized phage display to isolate nanobodies specifically against p38δ from a fully synthetic nanobody library. Two of them, nanobodies Nb13-6 and Nb13-1, display high binding activity to p38δ, less cross-reactivity with other p38 MAPKs, and high thermal and pH stabilities. Modeling and docking analysis indicated that Nb13-6 is mostly linked to the activation loop of p38δ. Furthermore, detailed studies revealed that Nb13-6 inhibited the protein kinase activity of p38δ and the growth of cancer cells. Therefore, this study provides p38δ-specific nanobodies that are promisingly exploited for diagnosing and treating p38δ-associated diseases.

Nutrient sensing and signaling are essential for adjusting growth and development to available resources. Deprivation of the essential mineral phosphorus (P) inhibits root growth.1 The molecular processes that sense P limitation to trigger early root growth inhibition are not known yet. Target of rapamycin (TOR) kinase is a central regulatory hub in eukaryotes to adapt growth to internal and external nutritional cues.2,3 How nutritional signals are transduced to TOR to control plant growth remains unclear. Here, we identify Arabidopsis-root-specific kinase 1 (ARSK1), which attenuates initial root growth inhibition in response to P limitation. We demonstrate that ARSK1 phosphorylates and stabilizes the regulatory-associated protein of TOR 1B (RAPTOR1B), a component of the TOR complex 1, to adjust root growth to P availability. These findings uncover signaling components acting upstream of TOR to balance growth to P availability.

Manganese (Mn) is an essential micronutrient for all living organisms. However, excess supply of Mn as occurring in acid or waterlogged soils has toxic effects on plant physiology and development. Although a variety of Mn transporter families has been characterized, our understanding of how these transporters are regulated to uphold and adjust Mn homeostasis in plants remains rudimentary. Here, we identify two calcineurin-B-like proteins, CBL2/3, and their interacting kinases, CIPK3/9/26, as key regulators for plant Mn homeostasis. We found that Arabidopsis mutants lacking CBL2 and 3 or their interacting protein kinases CIPK3/9/26 exhibit remarkable high-Mn tolerance. Intriguingly, CIPK3/9/26 interact with and phosphorylate the tonoplast-localized Mn and iron (Fe) transporter MTP8 primarily at Ser35, which is conserved among MTP8 proteins from various species. Mn transport complementation assays in yeast combined with multiple physiological assays indicate that CBL-CIPK-mediated phosphorylation of MTP8 negatively regulates its transport activity from the cytoplasm to the vacuole. Moreover, we report that sequential phosphorylation of MTP8 initially at Ser31/32 by the calcium-dependent protein kinases CPK5 and subsequently by CIPK26 at Ser35 provides an activation/deactivation fine-tuning mechanism for differential Mn transport regulation. Collectively, our studies define a two-tiered calcium-controlled mechanism for dynamically orchestrating Mn homeostasis under conditions of fluctuating Mn supply.

The transcription factor nuclear factor of kappa-light-chain-enhancer of activated B cells (NF-κB) is expressed in brown adipocytes, but its role remains largely unknown in the cells. This issue was addressed in current study by examining NF-κB in brown adipocytes in vitro and in vivo. NF-κB activity was increased by differentiation of brown adipocytes through elevation of p65 (RelA) expression. The transcriptional activity of NF-κB was induced by the cold stimulation with an elevation in S276 phosphorylation of p65 protein. Inactivation of NF-κB in brown adipocytes made the knockout mice [uncoupling protein 1 (Ucp1)–CreER–p65f/f, U-p65-KO] intolerant to the cold environment. The brown adipocytes exhibited an increase in apoptosis, a decrease in cristae density and uncoupling activity in the interscapular brown adipose tissue (iBAT) of p65-KO mice. The alterations became severer after cold exposure of the KO mice. The brown adipocytes of mice with NF-κB activation (p65 overexpression, p65-OE) exhibited a set of opposite alterations with a reduction in apoptosis, an increase in crista density and uncoupling activity. In mechanism, NF-κB inhibited expression of the adenine nucleotide translocase 2 (ANT2) in the control of apoptosis. Data suggest that NF-κB activity is increased in brown adipocytes by differentiation and cold stimulation to protect the cells from apoptosis through down-regulation of ANT2 expression.

Cellular mechanosensing is pivotal for virtually all biological processes, and many molecular mechano-sensors and their way of function are being uncovered. In this work, we suggest that c-Src kinase acts as a direct mechano-sensor. c-Src is responsible for, among others, cell proliferation, and shows increased activity in stretched cells. In its native state, c-Src has little basal activity, because its kinase domain binds to an SH2 and SH3 domain. However, it is known that c-Src can bind to p130Cas, through which force can be transmitted to the membrane. Using molecular dynamics simulations, we show that force acting between the membrane-bound N-terminus of the SH3 domain and p130Cas induces partial SH3 unfolding, thereby impeding rebinding of the kinase domain onto SH2/SH3 and effectively enhancing kinase activity. Forces involved in this process are slightly lower or similar to the forces required to pull out c-Src from the membrane through the myristoyl linker, and key interactions involved in this anchoring are salt bridges between negative lipids and nearby basic residues in c-Src. Thus, c-Src appears to be a candidate for an intriguing mechanosensing mechanism of impaired kinase inhibition, which can be potentially tuned by membrane composition and other environmental factors.

Cyanobacteria possesses the simplest circadian clock, composed of three proteins that act as a phosphorylation oscillator: KaiA, KaiB, and KaiC. The timing of this oscillator is determined by the fold-switch of KaiB, a structural rearrangement of its C-terminal half that is accompanied by a change in the oligomerization state. During the day, KaiB forms a stable tetramer (gsKaiB), whereas it adopts a monomeric thioredoxin-like fold during the night (fsKaiB). Although the structures and functions of both native states are well studied, little is known about the sequence and structure determinants that control their structural interconversion. Here, we used confinement molecular dynamics (CCR-MD) and folding simulations using structure-based models to show that the dissociation of the gsKaiB dimer is a key energetic event for the fold-switch. Hydrogen-deuterium exchange mass spectrometry (HDXMS) recapitulates the local stability of protein regions reported by CCR-MD, with both approaches consistently indicating that the energy and backbone flexibility changes are solely associated with the region that fold-switches between gsKaiB and fsKaiB and that the localized regions that differentially stabilize gsKaiB also involve regions outside the dimer interface. Moreover, two mutants (R23C and R75C) previously reported to be relevant for altering the rhythmicity of the Kai clock were also studied by HDXMS. Particularly, R75C populates dimeric and monomeric states with a deuterium incorporation profile comparable to the one observed for fsKaiB, emphasizing the importance of the oligomerization state of KaiB for the fold-switch. These findings suggest that the information necessary to control the rhythmicity of the cyanobacterial biological clock is, to a great extent, encoded within the KaiB sequence.

Tyrosine kinases are enzymes that can transfer a phosphate group from ATP to a specific protein tyrosine, serine or threonine residue within a cell, operating as a switch that can turn 'on' and 'off' causing different physiological alterations in the body. Mutated kinases have been shown to display an equilibrium shift toward the activated state. Types I-III have been studied intensively leading to drugs like imatinib (type II), cobimetinib (type III), among others. It is the same scenario for types V-VII; however, there is a lacuna in information regarding type IV inhibitors, although recently some advances have surfaced. This review aims to accumulate the knowledge gained so far about type IV inhibitors.

The etiology of idiopathic Parkinson's disease (iPD) is multifactorial, and both genetics and environmental exposures are risk factors. While mutations in leucine-rich repeat kinase-2 (LRRK2) that are associated with increased kinase activity are the most common cause of autosomal dominant PD, the role of LRRK2 in iPD, independent of mutations, remains uncertain. In this review, we discuss how the architecture of LRRK2 influences kinase activation and how enhanced LRRK2 substrate phosphorylation might contribute to pathogenesis. We describe how oxidative stress and endolysosomal dysfunction, both of which occur in iPD, can activate non-mutated LRRK2 to a similar degree as pathogenic mutations. Similarly, environmental toxicants that are linked epidemiologically to iPD risk can also activate LRRK2. In aggregate, current evidence suggests an important role for LRRK2 in iPD.

The cytoskeleton and cell-matrix adhesions constitute a dynamic network that controls cellular behavior during development and cancer. The Focal Adhesion Kinase (FAK) is a central actor of these cell dynamics, promoting cell-matrix adhesion turnover and active membrane fluctuations. However, the initial steps leading to FAK activation and subsequent promotion of cell dynamics remain elusive. Here, we report that the serine/threonine kinase PKCθ participates in the initial steps of FAK activation. PKCθ, which is strongly expressed in aggressive human breast cancers, controls the dynamics of cell-matrix adhesions and active protrusions through direct FAK activation, thereby promoting cell invasion and lung metastases. Using various tools for in vitro and live cell studies, we precisely decipher the molecular mechanisms of FAK activation. PKCθ directly interacts with the FAK FERM domain to open FAK conformation through PKCθ’s specific V3 domain, while phosphorylating FAK at newly identified serine/threonine residues within nascent adhesions, inducing cell dynamics and aggressive behavior. This study thus places PKCθ-directed FAK opening and phosphorylations as an original mechanism controlling dynamic, migratory, and invasive abilities of aggressive breast cancer cells, further strengthening the emerging oncogenic function of PKCθ.

Icariin (ICA), a bioactive flavonoid compound derived from Epimedium, have been demonstrated possessing anti-oxidative stress, anti-inflammation in the cardiovascular disease. But its effects on cardiomyocyte hypertrophy and the underlying mechanisms remains unclear. Here we found that ICA alleviated ISO-induced H9c2 or NRCM myocytes hypertrophy, assessed by surface area and the expression of ANP, BNP and β-MHC. Furthemore, ICA reversed cardiomcytes enlargment by suppresing apoptotic injury and increasing autophagic flux. By contrast, 3-MA, an autophagy inhibitor, could abolished the antihypertrophic and pro-autophagic flux effects of ICA. Mechanistically, ICA increased the phosphorylation levels of AMPK and autophagy-related markers beclin-1, Atg5 and the LC3II/I ratio, and decreased phosphorylated mTOR. But the effects of ICA on ISO-induced cardiomyocytes hypertrophy were attenuated by selective AMPK inhibitor Compound C. In conclusion, these findings indicated that ICA attenuated cardiomyocyte hypertrophy induced by ISO and prevented cell injury, and the specific mechanism was mediated by AMPK/mTOR pathway to enhance autophagy and reduce autophagy-related cardiomyocyte apoptosis.

Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) is one of important kinases in inflammation to phosphorylate inhibitor of nuclear factor kappa-B (IκBα) and then activate nuclear factor kappa-B (NF-κB). Inhibition of IKKβ has been a therapeutic strategy for inflammatory and autoimmune diseases. Here we report that IKKβ is constitutively activated in healthy donors and healthy IkkβC46A (cysteine 46 mutated to alanine) knock-in mice although they possess intensive IKKβ–IκBα–NF-κB signaling activation. These indicate that IKKβ activation probably plays homeostatic role instead of causing inflammation. Compared to IkkβWT littermates, lipopolysaccharides (LPS) could induce high mortality rate in IkkβC46A mice which is correlated to breaking the homeostasis by intensively activating p-IκBα–NF-κB signaling and inhibiting phosphorylation of 5ʹ adenosine monophosphate-activated protein kinase (p-AMPK) expression. We then demonstrated that IKKβ kinase domain (KD) phosphorylates AMPKα1 via interacting with residues Thr183, Ser184, and Thr388, while IKKβ helix–loop–helix motifs is essential to phosphorylate IκBα according to the previous reports. Kinase assay further demonstrated that IKKβ simultaneously catalyzes phosphorylation of AMPK and IκBα to mediate homeostasis. Accordingly, activation of AMPK rather than inhibition of IKKβ could substantially rescue LPS-induced mortality in IkkβC46A mice by rebuilding the homeostasis. We conclude that IKKβ activates AMPK to restrict inflammation and IKKβ mediates homeostatic function in inflammation via competitively phosphorylating AMPK and IκBα.

Hepatocellular carcinoma (HCC), a heterogeneous cancer with high mortality, is resistant to single targeted therapy; thus, combination therapy based on synthetic lethality is a promising therapeutic strategy for HCC. Poly (adenosine diphosphate [ADP]-ribose) polymerase 1 (PARP1) is the most recognized target for synthetic lethality; however, the therapeutic effect of PARP1 inhibition on HCC is disappointing. Therefore, exploring new synthetic lethal partners for the efficient manipulation of HCC is urgently required. In this study, we identified Src and PARP1 as novel synthetic lethal partners, and the combination therapy produced significant anti-tumor effects without causing obvious side effects. Mechanistically, Src interacted with PARP1 and phosphorylated PARP1 at the Y992 residue, which further mediated resistance to PARP1 inhibition. Overall, this study revealed that Src-mediated PARP1 phosphorylation induced HCC resistance to PARP1 inhibitors and indicated a therapeutic window of the Y992 phosphorylation of PARP1 for HCC patients. Moreover, synthetic lethal therapy by co-targeting PARP1 and Src have the potential to broaden the strategies for HCC and might benefit HCC patients with high Src activation and resistance to PARP1 inhibitors alone.

Impairment of the astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2) is associated with neurological disorders such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and manganism, a neurological disorder caused by overexposure to manganese (Mn) which shares the features of sporadic PD. Mechanisms of Mn-induced neurotoxicity include dysregulation of EAAT2 following activation of the transcription factor Yin Yang 1 (YY1) by transcriptional upregulation, but the posttranslational mechanisms by which YY1 is activated to repress EAAT2 remain to be elucidated. In the present study, we tested if Mn activates YY1 through posttranslational phosphorylation in cultured H4 human astrocytes, leading to EAAT2 repression. The results demonstrate that Mn exposure induced phosphorylation of YY1 at serine residues via kinases Aurora B kinase (AurkB) and Casein kinase II (CK2), leading to YY1 nuclear translocation, YY1/HDAC interactions, binding to the EAAT2 promoter, and consequent decreases in EAAT2 promoter activity and mRNA/protein levels. Although further studies are warranted to fully elucidate the mechanisms of Mn-induced YY1 phosphorylation and resultant EAAT2 impairment, our findings indicate that serine phosphorylation of YY1 via AurkB and CK2 is critical, at least in part, to its activation and transcriptional repression of EAAT2.

S6K1 serves as an important signaling regulator of cell proliferation and growth in the mTOR signaling pathway. Excessive activation of the mTOR/S6K1 signaling pathway promotes abnormal cell growth and survival, thereby resulting in tumorigenesis. The roles of S6K1 in protein synthesis and metabolism are well known, but an additional role of S6K1 as a gene transcription regulator has not been much understood. Here, we demonstrated that S6K1 is dynamically distributed in the cytoplasm and nuclei of human cervical cancer cells. S6K1 nuclear localization was serum dependent and serum deprivation or rapamycin treatment inhibited S6K1 Thr389 phosphorylation and, thereby, S6K1 was retained in the cytoplasm. Furthermore, we found that endogenous S6K1 interacted with CREB in the cervical cancer cells. Additionally, S6K1 upregulated the CRE-driven promoter luciferase activity. The proto-oncogene c-JUN, which has several CREs, was attenuated in the S6K1 knockdown cervical cancer cells. The binding of CREB/S6K1 to the c-JUN promoter, altered by serum restimulation, was associated with active epigenetic markers. In HeLa cell, 891 promoter regions, to which S6K1 directly binds, were detected. Our findings suggested that active S6K1, which is dynamically translocated into the nucleus, directly binds to chromatin and could play a role in epigenetic mechanisms or transcription factor recruitment.

Skeletal muscle atrophy caused by various conditions including aging, nerve damage, and steroid administration, is a serious health problem worldwide. We recently reported that neuron-derived neurotrophic factor (NDNF) functions as a muscle-derived secreted factor, also known as myokine, which exerts protective actions on endothelial cell and cardiomyocyte function. Here, we investigated whether NDNF regulates skeletal muscle atrophy induced by steroid administration and sciatic denervation. NDNF-knockout (KO) mice and age-matched wild-type (WT) mice were subjected to continuous dexamethasone (DEX) treatment or sciatic denervation. NDNF-KO mice exhibited decreased gastrocnemius muscle weight and reduced cross sectional area of myocyte fiber after DEX treatment or sciatic denervation compared with WT mice. Administration of an adenoviral vector expressing NDNF (Ad-NDNF) or recombinant NDNF protein to gastrocnemius muscle of WT mice increased gastrocnemius muscle weight after DEX treatment. NDNF-KO mice showed increased expression of ubiquitin E3-ligases, including atrogin-1 and MuRF-1, in gastrocnemius muscle after DEX treatment, whereas Ad-NDNF reduced expression of atrogin-1 and MuRF-1 in gastrocnemius muscle of WT mice after DEX treatment. Pretreatment of cultured C2C12 myocytes with NDNF protein reversed reduced myotube diameter and increased expression of atrogin-1 and MuRF-1 after DEX stimulation. Treatment of C2C12 myocytes increased Akt phosphorylation. Pretreatment of C2C12 myotubes with the PI3-kinase/Akt inhibitor reversed NDNF-induced increase in myotube fiber diameter after DEX treatment. In conclusion, our findings indicated that NDNF prevents skeletal muscle atrophy in vivo and in vitro through reduction of ubiquitin E3-ligases expression, suggesting that NDNF could be a novel therapeutic target of muscle atrophy.