Phosphorylation Of Ribosomal Protein S6 Kinase Research Articles

One Gene, Many Facets: Multiple Immune Pathway Dysregulation in SOCS1 Haploinsufficiency.

BackgroundInborn errors of immunity (IEI) present with a large phenotypic spectrum of disease, which can pose diagnostic and therapeutic challenges. Suppressor of cytokine signaling 1 (SOCS1) is a key negative regulator of cytokine signaling, and has recently been associated with a novel IEI. Of patients described to date, it is apparent that SOCS1 haploinsufficiency has a pleiotropic effect in humans.ObjectiveWe sought to investigate whether dysregulation of immune pathways, in addition to STAT1, play a role in the broad clinical manifestations of SOCS1 haploinsufficiency.MethodsWe assessed impacts of reduced SOCS1 expression across multiple immune cell pathways utilizing patient cells and CRISPR/Cas9 edited primary human T cells.ResultsSOCS1 haploinsufficiency phenotypes straddled across the International Union of Immunological Societies classifications of IEI. We found that reduced SOCS1 expression led to dysregulation of multiple intracellular pathways in immune cells. STAT1 phosphorylation is enhanced, comparably with STAT1 gain-of-function mutations, and STAT3 phosphorylation is similarly reduced with concurrent reduction of Th17 cells. Furthermore, reduced SOCS1 E3 ligase function was associated with increased FAK1 in immune cells, and increased AKT and p70 ribosomal protein S6 kinase phosphorylation. We also found Toll-like receptor responses are increased in SOCS1 haploinsufficiency patients.ConclusionsSOCS1 haploinsufficiency is a pleiotropic monogenic IEI. Dysregulation of multiple immune cell pathways may explain the variable clinical phenotype associated with this new condition. Knowledge of these additional dysregulated immune pathways is important when considering the optimum management for SOCS1 haploinsufficient patients.

Triterpenoids Extracted From Antrodia cinnamomea Mycelia Attenuate Acute Alcohol-Induced Liver Injury in C57BL/6 Mice via Suppression Inflammatory Response.

Excessive alcohol consumption causes liver injury–induced mortality. Here we systematically analyzed the structure of triterpenoids extracted from Antrodia cinnamomea mycelia (ACT) and investigated their protective effects against acute alcohol-induced liver injury in mice. Liquid chromatography–mass spectrometry and liquid chromatography with tandem mass spectrometry were performed to determine the structures of ACT constituents. Alcohol-induced liver injury was generated in C57BL/6 mice by oral gavage of 13 g/kg white spirit (a wine at 56% ABV). Mice were treated with either silibinin or ACT for 2 weeks. Liver injury markers and pathological signaling were then quantified with enzyme-linked immunosorbent assays, antibody array assays, and Western blots, and pathological examinations were performed using hematoxylin-eosin staining and periodic acid–Schiff staining. Triterpenoids extracted from A. cinnamomea mycelia contain 25 types of triterpenoid compounds. A 2-weeks alcohol consumption treatment caused significant weight loss, liver dyslipidemia, and elevation of alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, and alkaline phosphatase activities in the serum and/or liver. These effects were markedly reversed after 2-weeks ACT administration. Triterpenoids extracted from A. cinnamomea mycelia alleviated the organ structural changes and inflammatory infiltration of alcohol-damaged tissues. Triterpenoids extracted from A. cinnamomea mycelia inhibited proinflammatory cytokine levels and enhanced anti-inflammatory cytokine levels. Acute alcohol treatment promoted inflammation with significant correlations to hypoxia-inducible factor 1α (HIF-1α), which was reduced by ACT and was partially related to modulation of the protein kinase B (Akt)/70-kDa ribosomal protein S6 kinase phosphorylation (p70S6K) and Wnt/β-catenin signaling pathways. In conclusion, ACT protected against acute alcohol-induced liver damage in mice mainly through its suppression of the inflammatory response, which may be related to HIF-1α signaling.

Protective effects of glucagon-like peptide-1 on cardiac remodeling by inhibiting oxidative stress through mammalian target of rapamycin complex1/p70 ribosomal proteinS6 kinase pathway in diabetes mellitus.

Aims/IntroductionAlthough increased reactive oxygen species (ROS) generation is a major mechanism leading to cardiac remodeling in diabetes mellitus, research into the effects of anti‐oxidation on diabetic cardiac remodeling remains scarce and controversial. Glucagon‐like peptide‐1 (GLP‐1) shows potential anti‐oxidative effects besides lowering blood glucose. The objective of this research was to investigate the effects of GLP‐1 on cardiac remodeling and the molecular mechanism involved in diabetes mellitus.Materials and MethodsStreptozotocin‐induced diabetic rats received exenatide treatment for 3 months. Cardiac function, cardiac weight index and myocardial interstitial fibrosis were measured. Cardiomyocytes were cultured in high‐glucose medium with GLP‐1 treatment. The ROS production, apoptosis and the levels of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase protein expression in cardiomyocytes were analyzed.ResultsExperimental diabetes mellitus showed impaired cardiac diastolic function, increased brain natriuretic peptide expression and increased interstitial collagen deposition in the myocardium, which were ameliorated by exenatide treatment. Exenatide reduced myocardial ROS production and apoptosis in diabetes mellitus. Also, high glucose‐induced ROS generation and apoptosis in cardiomyocytes were inhibited by GLP‐1, as well as the levels of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase phosphorylation. Furthermore, GLP‐1 treatment upregulated adenosine monophosphate‐activated protein kinase activity in high‐glucose‐induced cardiomyocyte.ConclusionsGlucagon‐like peptide‐1 protects the cardiomyocytes from oxidative stress and apoptosis in diabetes mellitus, which might contribute to the improvement of cardiac remodeling. The cardiac protection of GLP‐1 might be dependent on inhibition of mammalian target of rapamycin complex 1/p70 ribosomal protein S6 kinase, through an adenosine monophosphate‐activated protein kinase‐mediated pathway.

Intermittent bolus feeding promotes greater lean growth than continuous feeding in a neonatal piglet model

Orogastric tube feeding is indicated in neonates with an impaired ability to ingest food normally and can be administered with an intermittent bolus or continuous feeding schedule. The objectives were to 1) compare the long-term effect of continuous with intermittent feeding on growth using the newborn pig as a model, 2) determine whether feeding frequency alters lean tissue and fat mass gain, and 3) identify the signaling mechanisms by which protein deposition is controlled in skeletal muscle in response to feeding frequency. Neonatal pigs were fed the same amount of a balanced formula by orogastric tube either as an intermittent bolus meal every 4 h (INT) or as a continuous infusion (CON). Body composition was assessed at the start and end of the study by dual-energy X-ray absorptiometry, and hormone and substrate profiles, muscle mass, protein synthesis, and indexes of nutrient and insulin signaling were measured after 21 d. Body weight, lean mass, spine length, and skeletal muscle mass were greater in the INT group than in the CON group. Skeletal muscle fractional protein synthesis rates were greater in the INT group after a meal than in the CON group and were associated with higher circulating branched-chain amino acid and insulin concentrations. Skeletal muscle protein kinase B (PKB) and ribosomal protein S6 kinase phosphorylation and eukaryotic initiation factor (eIF) 4E-eIF4G complex formation were higher, whereas eIF2α phosphorylation was lower in the INT group than in the CON group, indicating enhanced activation of insulin and amino acid signaling to translation initiation. These results suggest that when neonates are fed the same amounts of nutrients as intermittent meals rather than continuously there is greater lean growth. This response can be ascribed, in part, to the pulsatile pattern of amino acids, insulin, or both induced by INT, which enables the responsiveness of anabolic pathways to feeding to be sustained chronically in skeletal muscle.

Dihydrocelastrol exerts potent antitumor activity in mantle cell lymphoma cells via dual inhibition of mTORC1 and mTORC2.

Mantle cell lymphoma (MCL) is a distinct and highly aggressive subtype of B-cell non-Hodgkin lymphoma. Dihydrocelastrol (DHCE) is a dihydro-analog of celastrol, which is isolated from the traditional Chinese medicinal plant Tripterygium wilfordii. The present study aimed to investigate the effects of DHCE treatment on MCL cells, and to determine the mechanism underlying its potent antitumor activity invitro and invivo using the Cell Counting kit-8 assay, clonogenic assay, apoptosis assay, cell cycle analysis, immunofluorescence staining, western blotting and tumor xenograft models. The results demonstrated that DHCE treatment exerted minimal cytotoxic effects on normal cells, but markedly suppressed MCL cell proliferation by inducing G0/G1 phase cell cycle arrest, and inhibited MCL cell viability by stimulating apoptosis via extrinsic and intrinsic pathways. In addition, the results revealed that DHCE suppressed cell growth and proliferation by inhibiting mammalian target of rapamycin complex (mTORC)1-mediated phosphorylation of ribosomal proteinS6 kinase and eukaryotic initiation factor4E binding protein. Simultaneously, DHCE induced apoptosis and inhibited cell survival by suppressing mTORC2-mediated phosphorylation of protein kinaseB and nuclear factor-κB activity. In addition to invitro findings, DHCE treatment reduced the MCL tumor burden in a xenograft mouse model, without indications of toxicity. Furthermore, combined treatment with DHCE and bortezomib, a proteasome inhibitor, induced a synergistic cytotoxic effect on MCL cells. These findings indicated that DHCE may have the potential to serve as a novel therapeutic agent for the treatment of MCL through dually inhibiting mTORC1 and mTORC2.

Effects of long-term exposures to low iron and branched-chain amino acid containing diets on aging skeletal muscle of Fisher 344 × Brown Norway rats.

Aging skeletal muscle displays an altered iron status that may promote oxidative stress and sarcopenia. A diet containing low iron (LI) could reduce muscle iron status and attenuate age-related muscle atrophy. Supplemental branched-chain amino acids (BCAA) may also alleviate sarcopenia by promoting muscle protein synthesis and iron status improvement. This study examined individual and combined effects of LI and BCAA diets on anabolic signaling and iron status in skeletal muscle of aging rats. Twenty-nine-month-old male Fisher 344 × Brown Norway rats consumed the following control-base diets: control + regular iron (35 mg iron/kg) (CR; n = 11); control + LI (∼6 mg iron/kg) (CL; n = 11); 2×BCAA + regular iron (BR; n = 10); and 2×BCAA + LI (BL; n = 12) for 12 weeks. Although LI and/or 2×BCAA did not affect plantaris muscle mass, 2×BCAA groups showed lower muscle iron content than did CR and CL groups (P < 0.05). p70 ribosomal protein S6 kinase phosphorylation was greater in 2×BCAA and LI animals compared with CR animals (P < 0.05). Interactions between IRON and BCAA were observed for proteins indicative of mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1 alpha) and oxidative capacity (cytochrome c oxidase subunit 2 and citrate synthase) (P < 0.05) wherein the combined diet (BL) negated potential benefits of individual diets. Antioxidant capacity, superoxide dismutase activity, and oxidative injury (3-nitrotyrosine, protein carbonyls, and 4-hydroxynonenal) were similar between groups. In conclusion, 12 weeks of LI and 2×BCAA diets showed significant impacts on increasing anabolic signaling as well as ameliorating iron status; however, these interventions did not affect muscle mass.

Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient

Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine.

Tributylhexadecylphosphonium Bromide, a Novel Nuclear Factor of Activated T Cells Signaling Inhibitor, Blocks Interleukin-2 Induction Associated with Inhibition of p70 Ribosomal Protein S6 Kinase Phosphorylation

Transcriptional factors of the nuclear factor of activated T cells (NFAT) family are involved in T cell signaling. Many NFAT signaling inhibitors, such as cyclosporin A (CsA) and tacrolimus, abrogate dephosphorylation of NFAT proteins by inhibiting calcineurin activity. In pursuit of a novel type of NFAT signaling inhibitor, we screened our chemical library using the NFAT-dependent reporter assay and identified tributylhexadecylphosphonium bromide (THPB) as a selective NFAT signaling inhibitor. THPB inhibited NFAT-dependent reporter activity, and the induction of interleukin-2 (IL-2) at both mRNA and protein levels by calcium stimulation. Moreover, THPB had an additive effect on the inhibition of IL-2 induction with CsA. Unlike CsA, THPB did not affect dephosphorylation of NFAT1, but suppressed phosphorylation of p70 ribosomal protein S6 kinase (p70S6K). These results suggest that THPB may be a novel type of NFAT signaling inhibitor that acts in association with inhibition of p70S6K phosphorylation.

Ribosomal Protein S6 Kinase (RSK)-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein

BackgroundEpithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP) has been implicated in cellular EMT program; however, the major signaling determinant(s) responsible for MSP-induced EMT is unknown.ResultsThe study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration.ConclusionsMSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

Decreased Phosphorylation of RSK May Predict Response for Neoadjuvant Chemotherapy.

Abstract Neo-adjuvant chemotherapy is an essential part of the management of locally advanced breast cancer. The resistance in breast cancer towards chemotherapeutic agents makes chemotherapy an incommodious problem. Current clinical factors cannot predict therapy response sufficiently in the treated patients. Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. The resistance mechanisms responsible for doxorubicin-resistance of breast cancer have been the subject of numerous investigations. Identification of proteins that are associated with doxorubicin resistance is a first step toward better response prediction and tailored treatment of patients.In this study, we compared the expression of core proteins and their phosphorylated forms of several canonical pathways (MAPK signaling, Apoptosis, PI3K/Akt Signaling, Cell Cycle/Checkpoint and TGF-β/Smad signaling) in breast cancer tissues which were divided into two classes based on the type of response to doxorubicin therapy by immunoblotting. As a result, we found that the phosphorylated form of ribosomal protein S6 kinase (RSK) was low in doxorubicin-resistant breast cancer tissues compared with doxorubicin-sensitive breast cancer tissues.We also investigated RSK's phosphorylation rate and doxorubicin sensitivity of various breast cancer cell lines including MCF-7, MCF-7s (sphere form), MDA-MB-231, MDA-MB-231s, MDA-MB-436, MDA-MB-453, MDA-MB-468, ZR75-1, BT474, Hs578T, T47D and MCF-10A to determine the phosphorylation rate of RSK in relation to the drug sensitivity of the breast cancer cell lines. Expectedly, the phosphorylation of RSK was low in doxorubicin resistant breast cancer cell line such as MDA-MB-453, ZR75-1 and Hs578T. Additionally, MCF-7s and MDA-MB-231s showed decreased phosphorylation rates compared with MCF-7 and MDA-MB-231.In conclusion, decreased phosphorylation of RSK may predict response and further clinical studies should evaluate for molecular function of phosphorylated RSK during doxorubicin therapy. Furthermore, immunohistochemistry for the detection of RSK's phosphorylation rate in breast cancer tissues is being developed for the analysis of increased and independent group of patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2146.

Amino acids inhibit Agrp gene expression via an mTOR-dependent mechanism

Metabolic fuels act on hypothalamic neurons to regulate feeding behavior and energy homeostasis, but the signaling mechanisms mediating these effects are not fully clear. Rats placed on a low-protein diet (10% of calories) exhibited increased food intake (P < 0.05) and hypothalamic Agouti-related protein (Agrp) gene expression (P = 0.002). Direct intracerebroventricular injection of either an amino acid mixture (RPMI 1640) or leucine alone (1 mug) suppressed 24-h food intake (P < 0.05), indicating that increasing amino acid concentrations within the brain is sufficient to suppress food intake. To define a cellular mechanism for these direct effects, GT1-7 hypothalamic cells were exposed to low amino acids for 16 h. Decreasing amino acid availability increased Agrp mRNA levels in GT1-7 cells (P < 0.01), and this effect was attenuated by replacement of the amino acid leucine (P < 0.05). Acute exposure to elevated amino acid concentrations increased ribosomal protein S6 kinase phosphorylation via a rapamycin-sensitive mechanism, suggesting that amino acids directly stimulated mammalian target of rapamycin (mTOR) signaling. To test whether mTOR signaling contributes to amino acid inhibition of Agrp gene expression, GT1-7 cells cultured in either low or high amino acids for 16 h and were also treated with rapamcyin (50 nM). Rapamycin treatment increased Agrp mRNA levels in cells exposed to high amino acids (P = 0.01). Taken together, these observations indicate that amino acids can act within the brain to inhibit food intake and that a direct, mTOR-dependent inhibition of Agrp gene expression may contribute to this effect.

Steady-state Kinetic Mechanism of PDK1

PDK1 catalyzes phosphorylation of Thr in the conserved activation loop region of a number of its downstream AGC kinase family members. In addition to the consensus sequence at the site of phosphorylation, a number of PDK1 substrates contain a PIF sequence (PDK1-interacting fragment), which binds and activates the kinase domain of PDK1 (PDK1(deltaPH)). To gain further insight to PIF-dependent catalysis, steady-state kinetic and inhibition studies were performed for His6-PDK1(deltaPH)-catalyzed phosphorylation of PDK1-Tide (Tide), which contains an extended "PIF" sequence C-terminal to the consensus sequence for PDK1 phosphorylation. In two-substrate kinetics, a large degree of negative binding synergism was observed to occur on formation of the active ternary complex (alphaKd(ATP) = 40 microM and alphaKd(Tide) = 80 microM) from individual transitory binary complexes (Kd(ATP) = 0.6 microM and Kd(Tide) = 1 microM). On varying ATP concentrations, the ADP product and the (T/E)-PDK1-Tide product analog (p'Tide) behaved as competitive and noncompetitive inhibitors, respectively; on varying Tide concentrations, ADP and p'Tide behaved as noncompetitive and competitive inhibitors, respectively. Also, negative binding synergism was associated with formation of dead-end inhibited ternary complexes. Time progress curves in pre-steady-state studies under "saturating" or kcat conditions showed (i) no burst or lag phenomena, (ii) no change in reaction velocity when adenosine 5'-O-(thiotriphosphate) was used as a phosphate donor, and (iii) no change in reaction velocity on increasing relative microviscosity (0 < or = eta/eta0 < or = 3). Taken together, PDK1-catalyzed trans-phosphorylation of PDK1-Tide approximates a Rapid Equilibrium Random Bi Bi system, where motions in the central ternary complex are largely rate-determining.

Differential activation of mTOR signaling by contractile activity in skeletal muscle.

The cellular mechanisms by which contractile activity stimulates skeletal muscle hypertrophy are beginning to be elucidated and appear to include activation of the phosphatidylinositol 3-kinase signaling substrate mammalian target of rapamycin (mTOR). We examined the time course and location of mTOR phosphorylation in response to an acute bout of contractile activity. Rat hindlimb muscle contractile activity was elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Plantaris (Pla), tibialis anterior (TA), and soleus (Sol) muscles from stimulated and control limbs were collected immediately or 6 h after stimulation. HFES resulted in mTOR phosphorylation immediately after (3.4 +/- 0.9-fold, P < 0.01) contractile activity in Pla, whereas TA was unchanged compared with controls. mTOR phosphorylation remained elevated in Pla (3.6 +/- 0.6-fold) and increased in TA (4.6 +/- 0.9-fold, P < 0.05) 6 h after HFES. Interestingly, mTOR activation occurred predominantly in fibers expressing type IIa but not type I myosin heavy chain isoform. Furthermore, HFES induced modest ribosomal protein S6 kinase phosphorylation immediately after exercise in Pla (0.4 +/- 0.1-fold, P < 0.05) but not TA and more markedly 6 h after in both Pla and TA (1.4 +/- 0.4-fold vs. 2.4 +/- 0.3-fold, respectively, P < 0.01). Akt/PKB phosphorylation was similar to controls at both time points. These results suggest that mTOR signaling is increased after a single bout of muscle contractile activity. Despite reports that mTOR is activated downstream of Akt/PKB, in this study, HFES induced mTOR signaling independent of Akt/PKB phosphorylation. Fiber type-dependent mTOR phosphorylation may be a molecular basis by which some fiber types are more susceptible to contraction-induced hypertrophy.