Phosphorylation State Of Protein Kinase Research Articles

Analysis of protein kinases by Phos-tag SDS-PAGE

Protein kinases regulate almost all biological processes including cell proliferation, differentiation, apoptosis, and gene expression. Dysregulation of protein phosphorylation caused by abnormal activity and expression of protein kinases results in the onset of various diseases such as cancer and metabolic syndromes. The activities of a large number of protein kinases are regulated by phosphorylation. Therefore, analysis of the phosphorylation status of protein kinases is important for elucidation of biological phenomena and the pathogenesis of diseases. To investigate protein phosphorylation, phosphate-binding tag molecule “Phos-tag” was developed. In addition, various techniques and tools using Phos-tag such as Phos-tag SDS-PAGE, have been developed for analysis and profiling of protein phosphorylation. Here, we describe the methods and analytical techniques that use Phos-tag for investigation of protein kinase phosphorylation and the applications of phosphorylation analysis. SignificanceProtein kinases play pivotal roles in regulating many biological processes and pathogenesis of diseases. Determination of phosphorylation status of protein kinases can provide the essential information for their activation. This review provides analytical techniques for analysis of phosphorylation status of protein kinases by Phos-tag SDS-PAGE. We believe that this review would help readers to study in kinomics research.

Ruxolitinib in Combination with Vorinostat Suppresses Tumor Growth and Alters the Metabolic Phenotype in Haematological Diseases

Background and Objectives Jak-2 dysregulation has a significative role as an oncogenic driver and as a prominent therapeutic target in hematological malignancies. Ruxolitinib is pyrrolo[2.3-d]pyrimidine derivative with inhibitory activity against JAK1 and JAK2, moderate activity against TYK2 and minor activity against JAK3. Vorinostat is an HDAC inhibitor able to reduce Jak-2 expression affecting Jak-2 mRNA expression and increasing Jak-2 proteasomal deterioration. In this study we hypothesized that Ruxolitinib and Vorinostat combination could have a synergistic effects on haematological disease. Methods. We tested Ruxolitinib and Vorinostat alone and in combination for 24 and 48 hrs in 12 lymphoma and myeloma cell lines. The synergism was assessed by Chou-Talalay method. The cell cycle, the apoptosis, the caspase activation, the autophagy, the interleukin expression, the ROS generation, the ATP levels, the lactate, the phosphorylation status of protein kinases were evaluated in all 12 cell lines after 24 hrs. Co-coltures with bone marrow stromal cells were also performed after 24 to 120 hrs of treatments. Results. Treatment of 12 cell lines with low doses of Ruxolitinib and Vorinostat as single agents for 24h was rather ineffective at blocking cell growth if compared with the efficacy of the combination of the two drugs. The combination was additive in two lines of mantle cell lymphoma, in one line of Hodgkin lymphoma, in one line of myeloma, in one line of T cell lymphoma and in on line of B cell lymphoma. In the remaining cell lines the combination of the two drugs was highly synergistic. This synergistic cytotoxic action was maintained over time up to 120 hrs also in the presence of stromal cells. Ruxolitinib and Vorinostat combination influenced the cell cycle distribution increasing G2-M phase arrest in all 12 cell lines after 24 hrs. The levels of p21 and p27 resulted up-regulated while the levels of Cyclin D and Aurora A were downregulated in all 12 cell line after combined treatment. The apoptotic AnnexinV-positive cells after 24 hrs of treatment with Ruxolitinib or Vorinostat alone was less than 10%. However, the percentage of apoptotic cells grew up to 50 % after treatment of the cells with Ruxolitinib in combination with Vorinostat. After 24 of combined treatment, we detected a cleavage of caspase 8 and caspase 3 in all 12 cell line, suggesting activation of the extrinsic apoptotic pathway. Quantification of caspase 9 in all 12 cell lines has resulted in observation of an increase in cleaved form only in the six most sensitive cell lines, suggesting a caspase activation mitochondrial events regulating the intrinsic apoptosis. Bax, Bid and BAD levels increased in all 12 cell lines treated with combined drugs, but apoptotic inhibitors BLC-2 and MCL-1 were down regulated only in the 6 more sensitive cell lines. Our experiment showed that Ruxolitinib in combination with Vorinostat reduced IL-10 and IL-6 secretion in all 12 cell lines in a comparable way. The expression of IL-17A after combined treatment was significantly decreased in only the 6 most sensitive cell lines. Ruxolitinib and Vorinostat alone and in combination induced a decrease of the amount of autophagy-related protein p62 in cancer cells, indicating the activation of autophagic system. Autophagy and apoptosis increased the oxidative stress leading to ROS production and in this work, we showed that Ruxolitinib and Vorinostat synergistically induce apoptosis in 6 more sensitive cell lines, associated with a marked increase in ROS generation. Interestingly, our data have shown that combined therapy alters tumor cell metabolism by reducing the glycolic state by causing greater sensitivity of the six cell lines to the combined treatment. Conclusions. In conclusions, the combination of Ruxolitinib and Vorinostat is able to decrease glucose metabolism and this partial reversion of Warburg effect is associated to ROS production, apoptosis and cell growth inhibition. The drugs tested under different cell culture conditions provide informations on drug efficacy and could help predict pharmacological effects that may be difficult to translate from in vitro and ex vivo conditions to the clinic. This study gives evidence of synergistic interaction between Ruxolitinib and Vorinostat in lymphoid cells and provides the rationale for clinical studies with the combination of both agents in patients. DisclosuresNo relevant conflicts of interest to declare.

Intestinal Glucose Absorption Was Reduced by Vertical Sleeve Gastrectomy via Decreased Gastric Leptin Secretion.

The unique effects of gastric resection after vertical sleeve gastrectomy (VSG) on type 2 diabetes mellitus remain unclear. This work aimed to investigate the effects of VSG on gastric leptin expression and intestinal glucose absorption in high-fat diet-induced obesity. Male C57BL/6J mice were fed a high-fat diet (HFD) to induce obesity. HFD mice were randomized into VSG and sham-operation groups, and the relevant parameters were measured at 8weeks postoperation. Higher gastric leptin expression and increased intestinal glucose transport were observed in the HFD mice. Furthermore, VSG reduced gastric leptin expression and the intestinal absorption of alimentary glucose. Both exogenous leptin replenishment during the oral glucose tolerance test (OGTT) and the addition of leptin into the everted isolated jejunum loops in vitro restored the glucose transport capacity in VSG-operated mice, and this effect was abolished when the glucose transporter GLUT2 was blocked with phloretin. Moreover, phloretin almost completely suppressed glucose transport in the HFD mice. Intestinal immunohistochemistry in the obese mice showed increased GLUT2 and diminished sodium glucose co-transporter 1 (SGLT-1) in the apical membrane of enterocytes. Decreased GLUT2 and enhanced SGLT1 were observed following VSG. VSG also reduced the phosphorylation status of protein kinase C isoenzyme β II (PKCβ II) in the jejunum, which was stimulated by the combination of leptin and glucose. Our data demonstrated that the decreased secretion of gastric leptin in VSG results in a decrease in intestinal glucose absorption via modulation of GLUT2 translocation.

Muscle Protein Anabolic Resistance to Essential Amino Acids Does Not Occur in Healthy Older Adults Before or After Resistance Exercise Training

The muscle protein anabolic response to contraction and feeding may be blunted in older adults. Acute bouts of exercise can improve the ability of amino acids to stimulate muscle protein synthesis (MPS) by activating mechanistic target of rapamycin complex 1 (mTORC1) signaling, but it is not known whether exercise training may improve muscle sensitivity to amino acid availability. The aim of this study was to determine if muscle protein anabolism is resistant to essential amino acids (EAAs) and whether resistance exercise training (RET) improves muscle sensitivity to EAA in healthy older adults. In a longitudinal study, 19 healthy older adults [mean ± SD age: 71±4 y body mass index (kg/m2): 28±3] were trained for 12 wk with a whole-body program of progressive RET (60-75% 1-repetition maximum). Body composition, strength, and metabolic health were measured pre- and posttraining. We also performed stable isotope infusion experiments with muscle biopsies pre- and posttraining to measure MPS and markers of amino acid sensing in the basal state and in response to 6.8 g of EAA ingestion. RET increased muscle strength by 16%, lean mass by 2%, and muscle cross-sectional area by 27% in healthy older adults (P<0.05). MPS and mTORC1 signaling (i.e., phosphorylation status of protein kinase B, 4E binding protein 1, 70-kDa S6 protein kinase, and ribosomal protein S6) increased after EAA ingestion (P<0.05) pre- and posttraining. RET increased basal MPS by 36% (P<0.05); however, RET did not affect the response of MPS and mTORC1 signaling to EAA ingestion. RET increases strength and basal MPS, promoting hypertrophy in healthy older adults. In these subjects, a small dose of EAAs stimulates muscle mTORC1 signaling and MPS, and this response to EAAs does not improve after RET. Our data indicate that anabolic resistance to amino acids may not be a problem in healthy older adults. This trial was registered at www.clinicaltrials.gov as NCT02999802.

A method for profiling the phosphorylation state of tyrosine protein kinases

Protein kinases are known to be implicated in various biological phenomena and diseases through their involvement in protein phosphorylation. Therefore, analysis of the activity of protein kinases by examination of their phosphorylation state is important to elucidate their mechanisms. However, a method for analyzing the phosphorylation state of entire protein kinases in cells is not established. In the present study, we developed a new profiling method to analyze the expression and phosphorylation state of protein kinases using a Multi-PK antibody and Phos-tag 2D-PAGE. When HL-60 cells were differentiated into macrophage-like cells induced by 12-O-tetradecanoylphorbol-13-acetate, we observed significant changes in the expression and phosphorylation state of immunoreactive spots by this method. These results show that tyrosine kinase expression levels and phosphorylation state are changed by differentiation. Taken together, the developed method will be a useful tool for analysis of intracellular tyrosine protein kinases.

Neurobiological Correlates of Pain Avoidance Behavior in Opioid vs. Alcohol Dependence

In contrast to their analgesic properties, excessive use of either alcohol or opioids produces a paradoxical emergence of heightened pain sensitivity to noxious stimuli, termed hyperalgesia, which may promote further drinking or use of opioids to manage worsening pain symptoms. Hyperalgesia has traditionally been measured in rodents via reflex‐based assays, including the von Frey method. To better model the cognitive/motivational dimension of pain in a state of alcohol/opioid dependence and withdrawal, we employed a recently developed non‐reflex‐based method for measuring pain avoidance‐like behavior in animals (Mechanical Conflict Avoidance test). Adult male Wistar rats were administered an escalating dose regimen of morphine (opioid‐dependent group) or repeated saline (control group). Morphine‐dependent rats exhibited significantly greater avoidance of noxious stimuli during withdrawal. Such pain avoidance‐like behavior exhibited a modest correlation with mechanical paw withdrawal thresholds as measured via the von Frey method (r=−0.4413, p=0.0452), indicating that the two behavioral assays measure overlapping but not entirely similar pain‐like conditions. We next investigated individual relationships between pain avoidance‐like behavior and alterations in protein phosphorylation in central motivation‐related brain areas. We discovered that pain avoidance‐like behavior was significantly correlated with alterations in phosphorylation status of protein kinases (ERK, CaMKII), transcription factors (CREB), presynaptic markers of neurotransmitter release (Synapsin), and the rate‐limiting enzyme for dopamine synthesis (TH) across specific brain regions. Separate groups of male Wistar rats were made dependent on alcohol via exposure to chronic, intermittent ethanol vapor and compared to air‐exposed (non‐dependent) controls. Here, alcohol‐dependent animals exhibited enhanced pain avoidance‐like behavior of shorter probe heights that were innocuous (i.e., not avoided) in non‐dependent animals, indicative of a state of allodynia. We are currently examining the neural correlates of pain avoidance in these groups to compare to our results in opioid‐dependent animals. Our findings suggest that alterations in phosphorylation events in specific brain centers may support cognitive/motivational responses to avoid pain, and that qualitatively distinct dimensions of pain avoidance‐like behavior (hyperalgesia vs. allodynia) may exist in opioid‐ vs. alcohol‐dependent animals.Support or Funding InformationNational Institute on Alcohol Abuse and Alcoholism (T32AA007577, ARP; R00AA020839, SE), LSUHSC School of Medicine start‐up funds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neurobiological Correlates of Pain Avoidance-Like Behavior in Morphine-Dependent and Non-Dependent Rats

Repeated use of opioids can lead to the development of analgesic tolerance and dependence. Additionally, chronic opioid exposure can cause a paradoxical emergence of heightened pain sensitivity to noxious stimuli, termed hyperalgesia, which may drive continued or escalated use of opioids to manage worsening pain symptoms. Opioid-induced hyperalgesia has traditionally been measured in rodents via reflex-based assays, including the von Frey method. To better model the cognitive/motivational dimension of pain in a state of opioid dependence and withdrawal, we employed a recently developed non-reflex-based method for measuring pain avoidance-like behavior in animals (mechanical conflict avoidance test). Adult male Wistar rats were administered an escalating dose regimen of morphine (opioid-dependent group) or repeated saline (control group). Morphine-dependent rats exhibited significantly greater avoidance of noxious stimuli during withdrawal. We next investigated individual relationships between pain avoidance-like behavior and alterations in protein phosphorylation in central motivation-related brain areas. We discovered that pain avoidance-like behavior was significantly correlated with alterations in phosphorylation status of protein kinases (ERK, CaMKII), transcription factors (CREB), presynaptic markers of neurotransmitter release (Synapsin), and the rate-limiting enzyme for dopamine synthesis (TH) across specific brain regions. Our findings suggest that alterations in phosphorylation events in specific brain centers may support cognitive/motivational responses to avoid pain.

Co-Targeting Jak Signaling Pathway and Histone Deacetylases Produces Synergistic Activity in Haematologic Malignancies Cell Lines

Background and Objectives. Jak pathway is abnormally activated in multiple hematologic malignancies. Ruxolitinib, a JAK1/2 inhibitor, is currently prescribed in the clinical practice for the treatment of myelofibrosis; it is orally available and has a very manageable toxicity profile. Aberrant expression and activity of HDACs are also implicated in several types of tumors and HDAC inhibitors are under investigation in vitro and in clinical trials for the treatment of cancer. Vorinostat is a pan HDAC inhibitor with recognized efficacy in hematological disease, and ongoing studies are evaluating the best combination. Based on these considerations we screened a series of hematologic malignancies cell lines, in order to explore whether low doses of Ruxolitinib and Vorinostat might identify a new possible drug combination, with low toxicity profile. Methods. We tested Ruxolitinib and Vorinostat alone and in combination in 12 cell lines: Hodgkin Lymphoma (L1236, L540), B cell lymphomas (RPMI, WSU-NHL, Karpas422, RL) mantle cell lymphoma (Granta519, Jeko1), multiple myeloma (U266, RPMI8266), chronic limphocytic leukemia (MEK1), anaplastic lymphoma (Karpas299) and cutaneous T cell lymphoma (HUT78). The synergism was assessed by Chou-Talalay method. Effect of the treatment on cell cycle, apoptosis, caspase activation and ROS generation was evaluated by flow cytometry. Jak expression, apoptosis-regulating proteins and the phosphorylation status of protein kinases were studied by western blot analysis. Co-coltures with bone marrow stromal cells were also performed. Results. At 24h of treatment with ruxolitinib all the cell lines tested showed an homogeneous pattern of response, with IC50 around 20 nM; at 48h IC50 ranged from 0.1 to 2nM, with 4 cell lines more sensitive to the treatment (WSU-NHL, Karpas422, RL, HUT78) compared with the rest. The treatment with vorinostat induced an homogeneous response, both at 24h (IC50=40 uM) and 48h (IC50=5-10 uM). The combination treatment with a ratio R:V=0.1 nM:1 uM and 0.5nM:5uM showed a various range of results: a clear synergistic interaction of Ruxolitinib and Vorinostat was observed at 24h using low concentrations of two compounds in L1236, RL, RPMI, MEK1 and Karpas 299. There was no synergistic effects in mantle cell lymphoma cell lines, and a minor synergism in Karpas 422, a cell line that is known for carrying both t:(14;18) and t:(4;11) chromosomal translocation. The effect of the combination was additive on the remaining cell lines. The results were similar at 48 hours of treatment. The combination induced a marked increase in G2-M arrest and enhanced cell apoptosis in all cell lines. Accordingly the expression of apoptosis-regulating proteins, in particular BIM and Bad (SET-2), were up-regulated with down-regulation of Bcl-2 and Mcl-1 (HEL). Particularly evident was the effect of the combination on autophagy with decreased expression of p62 and a dramatic reactive oxygen species (ROS) accumulation in the cell lines in which the combination of the two drugs resulted highly synergistic. In addition, cell lines more sensitive to the combination of the two drugs showed a greater effect of the treatment on phosphorylation status of Jak, STAT3, STAT5, Akt and mTOR proteins. By co-culturing cells lines and primary BMSCs, we observed that Ruxolitinib and Vorinostat alone did not exert their anti-tumour activity in some cell lines. Conclusions. This study showed that the combination of Ruxolitinib and Vorinostat provokes significant changes in cell viability, apoptosis, cell cycle arrest, autophagy and ROS generation in several cell lines of hematologic malignancies alone and co-cultured. Because the bone microenvironment plays such an important role in the resistance to conventional therapies, the ability of Ruxolitinib combined with Vorinostat to overcome these factors is encouraging. Collectively, these findings create a compelling rationale to determine the in vivo activity of Ruxolitinib/Vorinostat combination. DisclosuresNo relevant conflicts of interest to declare.

Acetylshikonin from Zicao Prevents Obesity in Rats on a High-Fat Diet by Inhibiting Lipid Accumulation and Inducing Lipolysis.

Various drugs have been developed to treat obesity, but these have undesirable secondary effects, and an efficient but non-toxic anti-obesity drug from natural sources is desired. This study investigated the anti-obesity effects and mechanisms of action of acetylshikonin (AS)—which is used in traditional Chinese medicine—in rats on a high-fat diet (HFD). Rats were fed a normal diet or an HFD; the latter group was received no treatment or were treated with 100, 300, or 900 mg/kg AS extract by intragastric administration for 6 weeks. In addition, 3T3-L1 adipocytes were treated with AS and the effects on adipogenesis and lipolysis were evaluated by western blot analysis of adipogenic transcription factors and lipid-metabolizing enzyme levels and the phosphorylation status of protein kinase (PK) A and hormone-sensitive lipase (HSL). AS prevented HFD-induced obesity including reduction in body weight, white adipose tissue content, liver mass, and serum triglyceride and free fatty acid levels in rats. It also suppressed the expression of adipogenic differentiation transcription factors and decreased the expression of the adipocyte-specific proteins HSL and adipose triglyceride lipase (ATGL). Furthermore, AS treatment induced lipolysis, leading to the release of glycerol and increased in PKA and HSL phosphorylation. These findings demonstrate that AS has anti-obesity effects in a rat model and may be a safe treatment for obesity in humans.

Inhibitory Effect of Dried Pomegranate Concentration Powder on Melanogenesis in B16F10 Melanoma Cells; Involvement of p38 and PKA Signaling Pathways.

Plants rich in antioxidant substances may be useful for preventing skin aging. Pomegranates, containing flavonoids and other polyphenolic compounds, are widely consumed due to their beneficial properties. We examined the underlying mechanisms of dried pomegranate concentrate powder (PCP) on melanin synthesis in B16F10 melanoma cells. The antioxidant effects of PCP were determined by measuring free radical scavenging capacity and transcript levels of antioxidant enzymes. To explore the inhibitory effects of PCP on melanin synthesis, we measured tyrosinase activity and melanin content in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. In addition, the levels of tyrosinase-related protein-1 (TRP-1), TRP-2, tyrosinase, and microphthalmia-associated transcription factor (MITF) expression were determined by Western blotting. Changes in the phosphorylation status of protein kinase A (PKA), cAMP response element-binding protein (CREB), mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase Akt, and glycogen kinase 3β (GSK3β) were also examined. The free radical scavenging activity of PCP increased in a dose-dependent manner. In PCP-treated B16F10 cells, transcript levels of glutathione peroxidase-1 (GPx-1) were increased compared with α-MSH-stimulated cells. In addition, PCP led to the down-regulation of phospho-p38, phospho-PKA, phospho-CREB, phospho-GSK3β, MITF, and TRP-1 compared with α-MSH-stimulated B16F10 cells. We believe this effect may be associated with PCP activity, which leads to the inhibition of melanin production and tyrosinase activity. These results suggest that PCP decreases tyrosinase activity and melanin production via inactivation of the p38 and PKA signaling pathways, and subsequently decreases phosphorylation of CREB, MITF, and melanogenic enzymes. These observations provided new insights on the molecular mechanisms of the skin-whitening property of PCP.

Changes in insulin receptor signaling underlie neoadjuvant metformin administration in breast cancer: a prospective window of opportunity neoadjuvant study.

IntroductionThe antidiabetic drug metformin exhibits potential anticancer properties that are believed to involve both direct (insulin-independent) and indirect (insulin-dependent) actions. Direct effects are linked to activation of AMP-activated protein kinase (AMPK) and an inhibition of mammalian target of rapamycin mTOR signaling, and indirect effects are mediated by reductions in circulating insulin, leading to reduced insulin receptor (IR)-mediated signaling. However, the in vivo impact of metformin on cancer cell signaling and the factors governing sensitivity in patients remain unknown.MethodsWe conducted a neoadjuvant, single-arm, “window of opportunity” trial to examine the clinical and biological effects of metformin on patients with breast cancer. Women with untreated breast cancer who did not have diabetes were given 500 mg of metformin three times daily for ≥2 weeks after diagnostic biopsy until surgery. Fasting blood and tumor samples were collected at diagnosis and surgery. Blood glucose and insulin were assayed to assess the physiologic effects of metformin, and immunohistochemical analysis of tumors was used to characterize cellular markers before and after treatment.ResultsLevels of IR expression decreased significantly in tumors (P = 0.04), as did the phosphorylation status of protein kinase B (PKB)/Akt (S473), extracellular signal-regulated kinase 1/2 (ERK1/2, T202/Y204), AMPK (T172) and acetyl coenzyme A carboxylase (S79) (P = 0.0001, P < 0.0001, P < 0.005 and P = 0.02, respectively). All tumors expressed organic cation transporter 1, with 90% (35 of 39) exhibiting an Allred score of 5 or higher.ConclusionsReduced PKB/Akt and ERK1/2 phosphorylation, coupled with decreased insulin and IR levels, suggest insulin-dependent effects are important in the clinical setting. These results are consistent with beneficial anticancer effects of metformin and highlight key factors involved in sensitivity, which could be used to identify patients with breast cancer who may be responsive to metformin-based therapies.Trial registrationClinicalTrials.gov identifier: NCT00897884. Registered 8 May 2009.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0540-0) contains supplementary material, which is available to authorized users.

New Targets and Potential Strategy to Enhance the Anticancer Efficacy BKM-120 and BEZ235 in Lymphoma Cell Lines

Abstract Background. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays an important role in many physiological functions, including cell cycle progression, differentiation, survival, motility, apoptosis, protein synthesis and metabolism modification. The PI3K/AKT/mTOR signaling pathway also inhibits cell autophagy, a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. BKM120 and BEZ235 are synthetic small molecules belonging to the class of imidazo-quinolones that show preclinical activity against a range of solid and hematological malignancies. BKM-120 inhibits the catalytic subunit of class I PI3K by competitive binding to its ATP binding site, while BEZ235 is a dual class I PI3K/mTOR inhibitor. In this study we investigated the effects of BKM120 and BEZ235 on survival rate, apoptosis, signaling pathways expression, autophagy, metabolism modification, cell cycle arrest and tubulin polymerization kinetic in lymphoma cell lines. Method. Lymphoma cell lines (WSU-NHL, Jeko-1 and Karpas-299) were treated with different concentrations of BKM120 and BEZ235 (Novartis) for 24 and 48h and the IC50 values were evaluated using MTT assay. To assess for apoptosis, we used annexin V/PI staining kit/ flow cytometer analysis and Western Blot to evaluate caspase 3, 8, 9 and PARP expression. The cell cycle was performed applying PI incorporation and flow cytometer analysis. A flow cytometry based technique for the analysis of tubulin polymerization using a-tubulin staining was done to test whether the two drugs could cause a mitotic block. Western blot was utilized for phosphorylation status of protein kinases and for monitoring autophagy and metabolism. Results and conclusions. BKM120 and BEZ235 induced significant increase of apoptosis evidenced by annexin IV/PI staining and confirmed by the cleavage of caspases -3, -9 -8 and PARP. BKM120 and BEZ235 induced an up regulation of pro apoptotic protein Bim, Bax and Bad. Treatment for 24h with BKM120 and BEZ235 resulted in different effects on cell cycle. BKM120 induced an increase of G2-phase with down regulation of Cyclin D and E, and an up-regulation of Cyclin A, p21 and p27. The increase in G2-M caused by BKM120 treatment occurred in a dose dependent manner. BEZ235 induced an increase of G0/G1-phase with up regulation of Cyclin A, D, E and p21 and p27. Quantitative analysis of a-tubulins polymerization of the cell lines revealed that treatment with BKM120 induced an accumulation of mitotic cells. BKM120 and BEZ235 are inhibitors of intracellular pathways in targeting p-Akt, p- mTOR, pS6K, 4EBP1, MYC and STAT. BKM120 and BEZ235 increased the levels of type II LC3 and p62, hallmarks of autophagy, in addition to increasing caspase 3 cleavage and annexin positive cells, suggesting that the two drugs induced both apoptosis and autophagy. The combination of BKM120 and BEZ235 with chloroquine which are in clinical use, with the lysosomotropic autophagy inhibitor chloroquine demonstrating marked cooperates with inhibition of autophagy to elicit apoptosis through the intrinsic mitochondrial pathway (BAX, BIM and BAD). Treatment with BKM120 and BEZ235 induced an increase of GLUT1 and HIF-1 protein expression. GLUT1 plays a role in regulation of ROS levels in particular after BEZ235 treatment. We concluded that BKM120 and BEZ235 inhibit PI3K/AKT and mTOR signaling, induce autophagy-apoptosis, affect lymphoid cell metabolism and promote ER stress via ROS increase. Moreover, we observed that BKM120 can act as a microtubule destabilizer inducing cell cycle arrest. In these study, we highlight new targets of BKM120 and BEZ235 in addition to the known PI3K/AKT signaling pathway; these results can help to identify new potential strategies to enhance the anticancer efficacy BKM-120 and BEZ235 in lymphoma cell lines. Disclosures No relevant conflicts of interest to declare.

From cyclic AMP pathway to research the ketamine-related learning and memory

Background Studies have shown that cAMP response element binding protein (CREB) plays an important role in learning and memorization by activating relevant gene transcription directly or indirectly.In addition,CREB which is downstream protein regulated by cyclic AMP (cAMP) involves in the learning and memory deficits caused by ketamine in minor rats.Objective To discuss the effect of ketamine,cAMP pathway and relevant regulate protein extracellular signal regulating kinase (ERK) on learning and memory. Content It has been shown that cAMP alters the phosphorylation state of protein kinase A (PKA) and activation of CREB,the latter is an important nuclear protein,its regulating promoter involves in genetic transcription of cAMP response element (CRE),which has many biological functions including regulation of learning and memory. Trend To explore a new method for treatment of learning and memory deficits induced by ketamine from cAMP and ERK signaling pathway. Key words: Ketamine; cAMP; cAMP response element binding protein; Extracellular signal regulating kinase; Phosphodiesterase 4; Learning and memory

Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.

Hypoxia Decreases Insulin Signaling Pathways in Adipocytes

OBJECTIVE—Obesity is characterized by an overgrowth of adipose tissue that leads to the formation of hypoxic areas within this tissue. We investigated whether this phenomenon could be responsible for insulin resistance by studying the effect of hypoxia on the insulin signaling pathway in adipocytes.RESEARCH DESIGN AND METHODS—The hypoxic signaling pathway was modulated in adipocytes from human and murine origins through incubation under hypoxic conditions (1% O2) or modulation of hypoxia-inducible factor (HIF) expression. Insulin signaling was monitored through the phosphorylation state of several key partners of the pathway and glucose transport.RESULTS—In both human and murine adipocytes, hypoxia inhibits insulin signaling as revealed by a decrease in the phosphorylation of insulin receptor. In 3T3-L1 adipocytes, this inhibition of insulin receptor phosphorylation is followed by a decrease in the phosphorylation state of protein kinase B and AS160, as well as an inhibition of glucose transport in response to insulin. These processes were reversible under normoxic conditions. The mechanism of inhibition seems independent of protein tyrosine phosphatase activities. Overexpression of HIF-1α or -2α or activation of HIF transcription factor with CoCl2 mimicked the effect of hypoxia on insulin signaling, whereas downregulation of HIF-1α and -2α by small interfering RNA inhibited it.CONCLUSIONS—We have demonstrated that hypoxia creates a state of insulin resistance in adipocytes that is dependent upon HIF transcription factor expression. Hypoxia could be envisioned as a new mechanism that participates in insulin resistance in adipose tissue of obese patients.

FTY720 Induces Apoptosis in Hepatocellular Carcinoma Cells through Activation of Protein Kinase C δ Signaling

This study was aimed at elucidating the mechanism by which FTY720, a synthetic sphingosine immunosuppressant, mediated antitumor effects in hepatocellular carcinoma (HCC) cells. The three HCC cell lines examined, Hep3B, Huh7, and PLC5, exhibited differential susceptibility to FTY720-mediated suppression of cell viability, with IC(50) values of 4.5, 6.3, and 11 mumol/L, respectively. Although FTY720 altered the phosphorylation state of protein kinase B and p38, our data refuted the role of these two signaling kinases in FTY720-mediated apoptosis. Evidence indicates that the antitumor effect of FTY720 was attributable to its ability to stimulate reactive oxygen species (ROS) production, which culminated in protein kinase C (PKC)delta activation and subsequent caspase-3-dependent apoptosis. We showed that FTY720 activated PKC delta through two distinct mechanisms: phosphorylation and caspase-3-dependent cleavage. Cotreatment with the caspase-3 inhibitor Z-VAD-FMK abrogated the effect of FTY720 on facilitating PKC delta proteolysis. Equally important, pharmacologic inhibition or shRNA-mediated knockdown of PKC delta protected FTY720-treated Huh7 cells from caspase-3 activation. Moreover, FTY720 induced ROS production to different extents among the three cell lines, in the order of Hep3B > Huh7 >> PLC5, which inversely correlated with the respective glutathione S-transferase pi expression levels. The low level of ROS generation might underlie the resistant phenotype of PLC5 cells to the apoptotic effects of FTY720. Blockade of ROS production by an NADPH oxidase inhibitor protected Huh7 cells from FTY720-induced PKC delta activation and caspase-3-dependent apoptosis. Together, this study provides a rationale to use FTY720 as a scaffold to develop potent PKC delta-activating agents for HCC therapy.

Effects of resistance exercise with and without creatine supplementation on gene expression and cell signaling in human skeletal muscle

To test the hypothesis that creatine supplementation would enhance the anabolic responses of muscle cell signaling and gene expression to exercise, we studied nine subjects who received either creatine or a placebo (maltodextrin) for 5 days in a double-blind fashion before undergoing muscle biopsies: at rest, immediately after exercise (10 x 10 repetitions of one-leg extension at 80% 1 repetition maximum), and 24 and 72 h later (all in the morning after fasting overnight). Creatine supplementation decreased the phosphorylation state of protein kinase B (PKB) on Thr308 at rest by 60% (P < 0.05) and that of eukaryotic initiation factor 4E-binding protein on Thr37/46 (4E-BP1) by 30% 24 h postexercise (P < 0.05). Creatine increased mRNA for collagen 1 (alpha(1)), glucose transporter-4 (GLUT-4), and myosin heavy chain I at rest by 250%, 45%, and 80%, respectively, and myosin heavy chain IIA (MHCIIA) mRNA immediately after exercise by 70% (all P < 0.05). Immediately after exercise, and independent of creatine, mRNA for muscle atrophy F-box (MAFbx), MHCIIA, peroxisome proliferator-activated receptor gamma coactivator-1alpha, and interleukin-6 were upregulated (60-350%; P < 0.05); the phosphorylation state of p38 both in the sarcoplasm and nucleus were increased (12- and 25-fold, respectively; both P < 0.05). Concurrently, the phosphorylation states of PKB (Thr308) and 4E-BP1 (Thr37/46) were decreased by 50% and 75%, respectively (P < 0.05). Twenty-four hours postexercise, MAFbx, myostatin, and GLUT-4 mRNA expression decreased below preexercise values (-35 to -50%; P < 0.05); calpain 1 mRNA increased 70% 72 h postexercise (P < 0.05) and at no other time. In conclusion, 5 days of creatine supplementation do not enhance anabolic signaling but increase the expression of certain targeted genes.

Pten (phosphatase and tensin homologue gene) haploinsufficiency promotes insulin hypersensitivity.

Aims/hypothesisInsulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake.Materials and methodsInsulin sensitivity in Pten heterozygous (Pten+/−) mice was investigated in i.p. insulin challenge and glucose tolerance tests. Glucose uptake was monitored in vitro in primary cultures of myocytes from Pten+/− mice, and in vivo by positron emission tomography. The phosphorylation status of protein kinase B (PKB/Akt), a downstream signalling protein in the PI3K pathway, and glycogen synthase kinase 3β (GSK3β), a substrate of PKB/Akt, was determined by western immunoblotting.ResultsFollowing i.p. insulin challenge, blood glucose levels in Pten+/− mice remained depressed for up to 120 min, whereas glucose levels in wild-type mice began to recover after approximately 30 min. After glucose challenge, blood glucose returned to normal about twice as rapidly in Pten+/− mice. Enhanced glucose uptake was observed both in Pten+/− myocytes and in skeletal muscle of Pten+/− mice by PET. PKB and GSK3β phosphorylation was enhanced and prolonged in Pten+/− myocytes.Conclusions/interpretationPten is a key negative regulator of insulin-stimulated glucose uptake in vitro and in vivo. The partial reduction of Pten due to Pten haploinsufficiency is enough to elicit enhanced insulin sensitivity and glucose tolerance in Pten+/− mice.

Short-term activation of peroxysome proliferator-activated receptor β/δ increases fatty acid oxidation but does not restore insulin action in muscle cells from type 2 diabetic patients

Defective fatty acid oxidation in skeletal muscle is one of the possible causes of insulin resistance. Peroxisome proliferator-activated receptor beta activators are strong inducers of fatty acid oxidation. The aim of this study was to verify whether activation of fatty acid oxidation by PPARbeta agonists in human skeletal muscle cells prepared from type 2 diabetic patients could improve the reduced responses to insulin that characterized this cell model. GW0742 (10 nM) significantly increased fatty acid oxidation and oxidative gene expression in myotubes prepared from both healthy subjects and type 2 diabetic patients. In cells from control subjects, incubation with the agonist for 48 h affected neither insulin-induced rate of glycogen synthesis nor the phosphorylation state of protein kinase B (PKB serine 473). Myotubes from type 2 diabetic patients displayed marked reduction in the effects of insulin on glycogen synthesis and on PKB phosphorylation. However, treatment with PPARbeta agonists did not restore these defects. Therefore, these results indicate that induction of fatty acid oxidation with PPARbeta activators during short-term exposition is not sufficient to correct for insulin resistance in muscle cells from type 2 diabetic patients. This suggests that additional studies are needed to better characterize the link between fatty acid oxidation and insulin sensitivity in humans.

Reactivation of the Epstein–Barr virus from viral latency by an S-adenosylhomocysteine hydrolase/14-3-3 ζ/PLA2-dependent pathway

The S-adenosylhomocysteine hydrolase (SAH) and 14-3-3 zeta/phospholipase A2 (PLA2) are transcriptionally activated in parallel to the induction of the Epstein-Barr virus (EBV) lytic cycle by the ganglioside IV(3)NeuAc-nLcOse(4)Cer. For analysis of the initiation of the viral reactivation, SAH and 14-3-3 zeta/PLA2 were overexpressed. Expression of EA-D, BZLF1, and BHRF1 was increased in response to both, SAH- and 14-3-3 zeta/PLA2 overexpression indicating the initiation of the EBV lytic cycle. Expression of 14-3-3 zeta/PLA2 was shown to be increased in SAH overexpressing cells. Additionally, SAH-triggered initiation of viral reactivation could be inhibited by PLA2-specific inhibitors. The phosphorylation status of protein kinase C (PKC) was shown to be increased in SAH-overexpressing cells. PKC-specific inhibitors arrested SAH-triggered initiation of viral reactivation. Surprisingly, 14-3-3 zeta/PLA2-induced initiation of viral reactivation did not correlate with PKC activation. PKC-specific inhibitors were of no influence. SAH initiated EBV reactivation via the BZLF1-Zp and the BZLF1-Rp promoter, whereas 14-3-3 zeta/PLA2 was connected to the promoter Rp only. Our results suggest two routes of viral reactivation involving SAH, one associated with PKC and BZLF1-Zp, the other associated with 14-3-3 zeta/PLA2 and BZLF1-Rp.