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Sample records for akt ser473 phosphorylation

  1. PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

    PubMed Central

    Tsuchiya, A; Kanno, T; Nishizaki, T

    2014-01-01

    Insulin stimulated translocation of the glucose transporter GLUT4 from the cytosol to the plasma membrane in a concentration (1 nM–1 μM)-dependent manner and increased glucose uptake in 3T3-L1 adipocytes. Insulin-induced GLUT4 translocation to the cell surface was prevented by the phosphoinositide 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor BX912 or the Akt1/2 inhibitor MK2206, and by knocking-down PI3K, PDK1 or Akt1/2. Insulin increased phosphorylation of Akt1/2 at Thr308/309 and Ser473/474, to activate Akt1/2, in the adipocytes. Insulin-induced phosphorylation of Akt1/2 was suppressed by wortmannin and knocking-down PI3K, while no significant inhibition of the phosphorylation was obtained with BX912 or knocking-down PDK1. In the cell-free Akt assay, PI3K phosphorylated Akt1 both at Thr308 and Ser473 and Akt2 at Ser474 alone. In contrast, PDK1 phosphorylates Akt1 at Thr308 and Akt2 at Thr309. The results of this study indicate that PI3K activates Akt1, independently of PDK1, and Akt2 by cooperating with PDK1 in the insulin signal transduction pathway linked to GLUT4 translocation. PMID:24169049

  2. Apigenin Attenuates Atherogenesis through Inducing Macrophage Apoptosis via Inhibition of AKT Ser473 Phosphorylation and Downregulation of Plasminogen Activator Inhibitor-2

    PubMed Central

    Zeng, Ping; Liu, Bin; Wang, Qun; Fan, Qin; Diao, Jian-Xin; Tang, Jing; Fu, Xiu-Qiong; Sun, Xue-Gang

    2015-01-01

    Macrophage survival is believed to be a contributing factor in the development of early atherosclerotic lesions. Dysregulated apoptosis of macrophages is involved in the inflammatory process of atherogenesis. Apigenin is a flavonoid that possesses various clinically relevant properties such as anti-inflammatory, antiplatelet, and antitumor activities. Here we showed that apigenin attenuated atherogenesis in apoE−/− mice in an in vivo test. In vitro experiments suggested that apigenin induced apoptosis of oxidized low density lipoprotein- (OxLDL-) loaded murine peritoneal macrophages (MPMs). Proteomic analysis showed that apigenin reduced the expression of plasminogen activator inhibitor 2 (PAI-2). PAI-2 has antiapoptotic effects in OxLDL-loaded MPMs. Enhancing PAI-2 expression significantly reduced the proapoptosis effects of apigenin. Molecular docking assay with AutoDock software predicted that residue Ser473 of Akt1 is a potential binding site for apigenin. Lentiviral-mediated overexpression of Akt1 wild type weakened the proapoptosis effect of apigenin in OxLDL-loaded MPMs. Collectively, apigenin executes its anti-atherogenic effects through inducing OxLDL-loaded MPMs apoptosis. The proapoptotic effects of apigenin were at least partly attributed to downregulation of PAI-2 through suppressing phosphorylation of AKT at Ser473. PMID:25960827

  3. The loss of telomerase activity in highly differentiated CD8+CD28-CD27- T cells is associated with decreased Akt (Ser473) phosphorylation.

    PubMed

    Plunkett, Fiona J; Franzese, Ornella; Finney, Helene M; Fletcher, Jean M; Belaramani, Lavina L; Salmon, Mike; Dokal, Inderjeet; Webster, David; Lawson, Alastair D G; Akbar, Arne N

    2007-06-15

    The enzyme telomerase is essential for maintaining the replicative capacity of memory T cells. Although CD28 costimulatory signals can up-regulate telomerase activity, human CD8(+) T cells lose CD28 expression after repeated activation. Nevertheless, telomerase is still inducible in CD8(+)CD28(-) T cells. To identify alternative costimulatory pathways that may be involved, we introduced chimeric receptors containing the signaling domains of CD28, CD27, CD137, CD134, and ICOS in series with the CD3 zeta (zeta) chain into primary human CD8(+) T cells. Although CD3 zeta-chain signals alone were ineffective, triggering of all the other constructs induced proliferation and telomerase activity. However, not all CD8(+)CD28(-) T cells could up-regulate this enzyme. The further fractionation of CD8(+)CD28(-) T cells into CD8(+)CD28(-) CD27(+) and CD8(+)CD28(-)CD27(-) subsets showed that the latter had significantly shorter telomeres and extremely poor telomerase activity. The restoration of CD28 signaling in CD8(+)CD28(-)CD27(-) T cells could not reverse the low telomerase activity that was not due to decreased expression of human telomerase reverse transcriptase, the enzyme catalytic subunit. Instead, the defect was associated with decreased phosphorylation of the kinase Akt, that phosphorylates human telomerase reverse transcriptase to induce telomerase activity. Furthermore, the defective Akt phosphorylation in these cells was specific for the Ser(473) but not the Thr(308) phosphorylation site of this molecule. Telomerase down-regulation in highly differentiated CD8(+)CD28(-)CD27(-) T cells marks their inexorable progress toward a replicative end stage after activation. This limits the ability of memory CD8(+) T cells to be maintained by continuous proliferation in vivo. PMID:17548608

  4. Neuronal Ablation of p-Akt at Ser473 Leads to Altered 5-HT1A/2A Receptor Function

    PubMed Central

    Saunders, Christine; Siuta, Michael; Robertson, Sabrina D.; Davis, Adeola R.; Sauer, Jennifer; Matthies, Heinrich J.G.; Gresch, Paul J.; Airey, David; Lindsley, Craig W.; Schetz, John A.; Niswender, Kevin D.

    2014-01-01

    The serotonergic system regulates a wide range of behavior, including mood and impulsivity, and its dysregulation has been associated with mood disorders, autism spectrum disorder, and addiction. Diabetes is a risk factor for these conditions. Insulin resistance in the brain is specifically associated with susceptibility to psychostimulant abuse. Here, we examined whether phosphorylation of Akt, a key regulator of the insulin signaling pathway, controls serotonin (5-HT) signaling. To explore how impairment in Akt function regulates 5-HT homeostasis, we used a brain-specific rictor knockout (KO) mouse model of impaired neuronal phosphorylation of Akt at Ser473. Cortical 5-HT1A and 5-HT2A receptor binding was significantly elevated in rictor KO mice. Concomitant with this elevated receptor expression, the 5-HT1A receptor agonist 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) led to an increased hypothermic response in rictor KO mice. The increased cortical 5-HT1A receptor density was associated with higher 5-HT1A receptor levels on the cortical cell surface. In contrast, rictor KO mice displayed significantly reduced head-twitch response (HTR) to the 5-HT2A/C agonist 2,5-dimethoxy-4-iodoamphetamine (DOI), with evidence of impaired 5-HT2A/C receptor signaling. In vitro, pharmacological inhibition of Akt significantly increased 5-HT1A receptor expression and attenuated DOI-induced 5-HT2A receptor signaling, thereby lending credence to the observed in vivo cross-talk between neuronal Akt signaling and 5-HT receptor regulation. These data reveal that defective central Akt function alters 5-HT signaling as well as 5-HT-associated behaviors, demonstrating a novel role for Akt in maintaining neuronal 5-HT receptor function. PMID:24090638

  5. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue.

    PubMed

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J

    2016-05-01

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1-3 kinases are specifically activated by two phosphorylation events on residues Thr(308) and Ser(473) upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser(473) and Thr(308) phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser(473) and Thr(308) phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser(473)-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons. PMID:26945062

  6. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue*

    PubMed Central

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J.

    2016-01-01

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1–3 kinases are specifically activated by two phosphorylation events on residues Thr308 and Ser473 upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser473 and Thr308 phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser473 and Thr308 phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser473-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons. PMID:26945062

  7. Phosphorylation of AKT and abdominal aortic aneurysm formation.

    PubMed

    Ghosh, Abhijit; Lu, Guanyi; Su, Gang; McEvoy, Brendan; Sadiq, Omar; DiMusto, Paul D; Laser, Adriana; Futchko, John S; Henke, Peter K; Eliason, Jonathan L; Upchurch, Gilbert R

    2014-01-01

    It is hypothesized that differential AKT phosphorylation between sexes is important in abdominal aortic aneurysm (AAA) formation. Male C57BL/6 mice undergoing elastase treatment showed a typical AAA phenotype (80% over baseline, P < 0.001) and significantly increased phosphorylated AKT-308 (p308) and total-AKT (T-AKT) at day 14 compared with female mice. Elastase-treated Raw cells produced increased p308 and significant amounts of matrix metalloproteinase 9 (MMP-9), and these effects were suppressed by LY294002 treatment, a known AKT inhibitor. Male and female rat aortic smooth muscle cells treated with elastase for 1, 6, or 24 hours demonstrated that the p308/T-AKT and AKT-Ser-473/T-AKT ratios peaked at 6 hours and were significantly higher in the elastase-treated cells compared with controls. Similarly, male cells had higher phosphorylated AKT/T-AKT levels than female cells. LY294002 also inhibited elastase-induced p308 formation more in female smooth muscle cells than in males, and the corresponding cell media had less pro-MMP-9. AKT siRNA significantly decreased secretion of pro-MMP-9, as well as pro-MMP-2 and active MMP-2 from elastase-treated male rat aortic smooth muscle cells. IHC of male mice AAA aortas showed increased p308, AKT-Ser-473, and T-AKT compared with female mice. Aortas from male AAA patients had a significantly higher p308/T-AKT ratio than female AAA tissues. These data suggest that AKT phosphorylation is important in the upstream regulation of MMP activity, and that differential phosphorylation may be important in sex differences in AAA. PMID:24332015

  8. Akt-Ser473 Phosphorylation as a Marker for Predicting Taxane Chemotherapy Outcome | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Institute of Health, National Cancer Institute is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize a marker for predicting taxane chemotherapy outcome.

  9. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation.

    PubMed

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-07-18

    Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li2CO3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li2CO3 did not affect PI3K-mediated PI(3,4,5)P3 production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li2CO3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li2CO3 significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li2CO3 significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity. PMID:24950409

  10. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    SciTech Connect

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-04-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3{beta} (GSK-3{beta}) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-{alpha} (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.

  11. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

    SciTech Connect

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-07-18

    Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li{sub 2}CO{sub 3} significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li{sub 2}CO{sub 3} did not affect PI3K-mediated PI(3,4,5)P{sub 3} production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li{sub 2}CO{sub 3} on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li{sub 2}CO{sub 3} significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li{sub 2}CO{sub 3} significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity.

  12. Asymmetric Dimethylarginine Stimulates Akt1 Phosphorylation via Heat Shock Protein 70-Facilitated Carboxyl-Terminal Modulator Protein Degradation in Pulmonary Arterial Endothelial Cells.

    PubMed

    Sun, Xutong; Kellner, Manuela; Desai, Ankit A; Wang, Ting; Lu, Qing; Kangath, Archana; Qu, Ning; Klinger, Christina; Fratz, Sohrab; Yuan, Jason X-J; Jacobson, Jeffrey R; Garcia, Joe G N; Rafikov, Ruslan; Fineman, Jeffrey R; Black, Stephen M

    2016-08-01

    Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial nitric oxide synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at threonine (T) 308 and serine (S) 473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells to ADMA enhanced Akt1 phosphorylation at both threonine 308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog activity, or membrane Akt1 levels. Heat shock protein (Hsp) 90 plays a pivotal role in maintaining Akt1 activity, and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but, surprisingly, overexpression of a dominant-negative Hsp90 mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of dominant-negative Hsp90 increased Hsp70 levels, and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, carboxyl-terminal modulator protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMP protein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo, and that increasing CTMP levels may be an avenue to treat pulmonary hypertension. PMID:26959555

  13. Hypoxia activates Akt and induces phosphorylation of GSK-3 in PC12 cells.

    PubMed

    Beitner-Johnson, D; Rust, R T; Hsieh, T C; Millhorn, D E

    2001-01-01

    Akt is a serine/threonine kinase that has been shown to play a central role in promoting cell survival and opposing apoptosis. We evaluated the effect of hypoxia on Akt in rat pheochromocytoma (PC12) cells. PC12 cells were exposed to varying levels of hypoxia, including 21%, 15%, 10%, 5%, and 1% O(2). Hypoxia dramatically increased phosphorylation of Akt (Ser(473)). This effect peaked after 6 h exposure to hypoxia, but persisted strongly for up to 24 h. Phosphorylation of Akt was paralleled with a progressive increase in phosphorylation of glycogen synthase kinase-3 (GSK-3), one of its downstream substrates. The effect of hypoxia on phosphorylation of Akt was completely blocked by pretreatment of the cells with wortmannin (100 nM), indicating that this effect is mediated by phosphatidylinositol 3-kinase (P13K). In contrast, whereas hypoxia also strongly induced phosphorylation of the transcription factors CREB and EPAS1, these effects persisted in the presence of wortmannin. Thus, hypoxia regulates both P13K-dependent and P13K-independent signaling pathways. Furthermore, activation of the P13K and Akt signaling pathways may be one mechanism by which cells adapt and survive under conditions of hypoxia. PMID:11257444

  14. Antiplatelet activity of loureirin A by attenuating Akt phosphorylation: In vitro studies.

    PubMed

    Hao, Hong-Zhen; He, Ao-Di; Wang, Dao-Chun; Yin, Zhao; Zhou, Ya-Jun; Liu, Gang; Liang, Ming-Lu; Da, Xing-Wen; Yao, Guang-Qiang; Xie, Wen; Xiang, Ji-Zhou; Ming, Zhang-Yin

    2015-01-01

    Loureirin A is a flavonoid extracted from Dragon׳s Blood that has been used to promote blood circulation and remove stasis in Chinese traditional medicine. However, the mechanisms of these effects are not fully understood. We explored the anti-platelet activity and underlying mechanism of loureirin A in vitro. Our results indicated that loureirin A negatively affected agonist-induced platelet aggregation such as collagen, collagen-related peptide (CRP), ADP and thrombin. Loureirin A inhibited collagen-induced platelet ATP secretion and thrombin-stimulated P-selectin expression in a dose-dependent manner. Platelet spreading on immobilized fibrinogen was significantly impaired in the presence of loureirin A. Immunoblotting analysis indicated that 100μM of loureirin A almost completely eliminated collagen-induced Akt phosphorylation at Ser473. Interestingly, a submaximal dose (50μM) of loureirin A had an additive inhibitory effect with the phosphoinositide 3-kinase (PI3K) inhibitor Ly294002 on collage-induced Akt phosphorylation in platelets. Taken together, loureirin A had an inhibitory effect on platelet activation, perhaps through an impairment of PI3K/Akt signaling. PMID:25445049

  15. Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2 + 2a) phosphorylation

    PubMed Central

    Birk, Jesper B.; Richter, Erik A.; Ribel-Madsen, Rasmus; Pehmøller, Christian; Hansen, Bo Falck; Beck-Nielsen, Henning; Hirshman, Michael F.; Goodyear, Laurie J.; Vaag, Allan; Poulsen, Pernille; Wojtaszewski, Jørgen F. P.

    2013-01-01

    Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. Previously, we demonstrated that phosphorylation of Thr308 on Akt (p-Akt-Thr308), Akt2 activity, and GS activity in muscle were positively associated with insulin sensitivity. Here, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. One-hundred eighty-one nondiabetic twins were examined with the euglycemic hyperinsulinemic clamp combined with excision of muscle biopsies. Insulin signaling was evaluated at the levels of the insulin receptor, IRS-1-associated PI3K (IRS-1-PI3K), Akt, and GS employing activity assays and phosphospecific Western blotting. The insulin-stimulated GS activity was positively associated with p-Akt-Thr308 (P = 0.01) and Akt2 activity (P = 0.04) but not p-Akt-Ser473 or IRS-1-PI3K activity. Furthermore, p-Akt-Thr308 and Akt2 activity were negatively associated with NH2-terminal GS phosphorylation (P = 0.001 for both), which in turn was negatively associated with insulin-stimulated GS activity (P < 0.001). We found no association between COOH-terminal GS phosphorylation and Akt or GS activity. Employing whole body Akt2-knockout mice, we validated the necessity for Akt2 in insulin-mediated GS activation. However, since insulin did not affect NH2-terminal phosphorylation in mice, we could not use this model to validate the observed association between GS NH2-terminal phosphorylation and Akt activity in humans. In conclusion, our study suggests that although COOH-terminal dephosphorylation is likely necessary for GS activation, Akt2-dependent NH2-terminal dephosphorylation may be the site for “fine-tuning” insulin-mediated GS activation in humans. PMID:23321478

  16. Apigenin up-regulates transgelin and inhibits invasion and migration of colorectal cancer through decreased phosphorylation of AKT.

    PubMed

    Chunhua, Li; Donglan, Lin; Xiuqiong, Fu; Lihua, Zhang; Qin, Fan; Yawei, Liu; Liang, Zhao; Ge, Wen; Linlin, Jing; Ping, Zeng; Kun, Li; Xuegang, Sun

    2013-10-01

    Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. Apigenin is a flavonoid that possesses various clinically relevant properties such as anti-tumour, anti-platelet and anti-inflammatory activities. Our results showed that apigenin has anti-proliferation, anti-invasion and anti-migration effects in three kinds of colorectal adenocarcinoma cell lines, namely SW480, DLD-1 and LS174T. Proteomic analysis with SW480 indicated that apigenin up-regulated the expression of transgelin (TAGLN) in mitochondria to exert its anti-tumour growth and anti-metastasis effects. Real-time quantitative polymerase chain reaction (RQ-PCR) and western blot confirm the up-regulation in all the three colorectal adenocarcinoma cells. An inverse correlation was observed between TAGLN expression and CRC metastasis in tissue microarray staining. TAGLN siRNA increased the viability of SW480. Apigenin decreased the expression of MMP-9 in a dose-dependent manner. Transfection of three truncated forms of TAGLN and wild type has identified TAGLN as a repressor of MMP-9 expression. A synergetic effect was observed in overexpression of TAGLN wild type and apigenin treatment which manifested as lowered phosphorylation of AKT Ser473 and ATK Thr308. In an orthotopic CRC model, apigenin inhibited tumour growth and metastasis to liver and lung. In conclusion, our research provided direct evidence that apigenin inhibited tumour growth and metastasis both in vitro and in vivo. Apigenin up-regulated TAGLN and hence down-regulated MMP-9 expression through decreasing phosphorylation of Akt at Ser473 and in particular Thr308 to prevent cell proliferation and migration. PMID:23773626

  17. Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation

    PubMed Central

    Xie, Chanlu; Hua, Sheng; Li, Jianfang; Wang, Tingfeng; Yao, Mu; Vignarajan, Soma; Teng, Ying; Hejazi, Leila; Liu, Bingya; Dong, Qihan

    2014-01-01

    A constitutive activation of protein kinase B (AKT) in a hyper-phosphorylated status at Ser473 is one of the hallmarks of anti-EGFR therapy-resistant colorectal cancer (CRC). The aim of this study was to examine the role of cytosolic phospholipase A2α (cPLA2α) on AKT phosphorylation at Ser473 and cell proliferation in CRC cells with mutation in phosphoinositide 3-kinase (PI3K). AKT phosphorylation at Ser473 was resistant to EGF stimulation in CRC cell lines of DLD-1 (PIK3CAE545K mutation) and HT-29 (PIK3CAP499T mutation). Over-expression of cPLA2α by stable transfection increased basal and EGF-stimulated AKT phosphorylation and proliferation in DLD-1 cells. In contrast, silencing of cPLA2α with siRNA or inhibition with Efipladib decreased basal and EGF-stimulated AKT phosphorylation and proliferation in HT-29. Treating animals transplanted with DLD-1 with Efipladib (10 mg/kg, i.p. daily) over 14 days reduced xenograft growth by >90% with a concomitant decrease in AKT phosphorylation. In human CRC tissue, cPLA2α expression and phosphorylation were increased in 63% (77/120) compared with adjacent normal mucosa determined by immunohistochemistry. We conclude that cPLA2α is required for sustaining AKT phosphorylation at Ser473 and cell proliferation in CRC cells with PI3K mutation, and may serve as a potential therapeutic target for treatment of CRC resistant to anti-EGFR therapy. PMID:25365190

  18. AKT-p53 axis protect cancer cells from autophagic cell death during nutrition deprivation.

    PubMed

    Sudhagar, S; Sathya, S; Gokulapriya, G; Lakshmi, B S

    2016-03-18

    An altered metabolism supports growth of tumor. AKT, a major signal integrator plays a key role in cell metabolism. We have shown that nutritional deprivation activates AKT as observed by increased phosphorylation of both Thr308 and Ser473. Pharmacological inhibition or silencing of AKT by siRNA affects cell viability during starvation. The tumor suppressor, p53 is also observed to be elevated during nutritional deprivation due to AKT. Silencing of AKT and p53 enhanced autophagy as evidenced by increased acidic vesicular organelles and LC3B II levels, suggesting AKT-p53 to play a significant role in cell survival through regulating autophagy during nutritional deprivation. PMID:26903300

  19. Lycium barbarum polysaccharide attenuates the cytotoxicity of mutant huntingtin and increases the activity of AKT.

    PubMed

    Fang, Fang; Peng, Ting; Yang, Shiming; Wang, Weixi; Zhang, Yinong; Li, He

    2016-08-01

    Huntington's disease (HD) is an inherited neurodegenerative disease that is caused by the abnormal expansion of CAG repeats in the gene encoding huntingtin (Htt). Reduced AKT phosphorylation and inhibited AKT activity have been shown to be involved in mutant Htt (mHtt)-induced cell death. Lycium barbarum polysaccharide (LBP), the main bioactive component of Lycium barbarum, reportedly has neuroprotective roles in neural injuries, including neurodegenerative diseases. Here, we report that treatment with LBP can increased the viability of HEK293 cells that stably expressed mHtt containing 160 glutamine repeats and significantly improved motor behavior and life span in HD-transgenic mice. Furthermore, we found that in LBP-treated HEK293 cells expressing mHtt, mHtt levels were reduced and the phosphorylation of AKT at Ser473 (p-AKT-Ser473) was significantly increased. We also found that treatment with LBP increased p-AKT-Ser473 and decreased mHtt in the cortex, hippocampus and striatum in HD-transgenic mice. The level of phosphorylation of p-GSK3β-Ser9 remained unchanged in both cultured cells and HD-transgenic mice. Our findings suggest that LBP alleviates the cytotoxicity of mHtt by activating AKT and reducing mHtt levels, indicating that LBP may be potentially useful for treating HD. PMID:27196502

  20. Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177

    PubMed Central

    Walther, Stefanie; Pluteanu, Florentina; Renz, Susanne; Nikonova, Yulia; Maxwell, Joshua T.; Yang, Li-Zhen; Schmidt, Kurt; Edwards, Joshua N.; Wakula, Paulina; Groschner, Klaus; Maier, Lars S.; Spiess, Joachim; Blatter, Lothar A.; Pieske, Burkert

    2014-01-01

    Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca2+-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling. PMID:25015964

  1. Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177.

    PubMed

    Walther, Stefanie; Pluteanu, Florentina; Renz, Susanne; Nikonova, Yulia; Maxwell, Joshua T; Yang, Li-Zhen; Schmidt, Kurt; Edwards, Joshua N; Wakula, Paulina; Groschner, Klaus; Maier, Lars S; Spiess, Joachim; Blatter, Lothar A; Pieske, Burkert; Kockskämper, Jens

    2014-09-01

    Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling. PMID:25015964

  2. Age-related decrease in constructive activation of Akt/PKB in SAMP10 hippocampus.

    PubMed

    Nie, Kun; Yu, Jian-Chun; Fu, Yu; Cheng, Hai-Yan; Chen, Fu-Yan; Qu, You; Han, Jing-Xian

    2009-01-01

    Aging is the greatest risk factor for neurodegenerative diseases such as Alzheimer's disease (AD). Age-dependent alterations of cell signaling play an important role in the onset of AD. The serine/threonine kinase Akt is a critical cell signaling to neuronal survival. Using the senescence-accelerated mouse SAMP10, we investigated the effect of aging on AKT signaling in hippocampus tissue. During aging, the expression of Akt mRNA and protein remained stable. However, the constructive phosphorylation of Akt(Ser473) displayed a continuous decrease after 6 months in SAMP10. When compared with the control SAMR1, aged SAMP10 mice showed significant reduced phosphorylation of Akt(Ser473). SAMP10 at the age of 6 months showed obvious deterioration in performance of learning and memory tasks. Thus, the data reported here suggested a potential link between the age-related alteration of Akt(Ser473) and the deterioration in performance of learning and memory tasks in SAMP10 mouse. PMID:19013131

  3. Site Specific Activation of AKT Protects Cells from Death Induced by Glucose Deprivation

    PubMed Central

    Gao, Meng; Liang, Jiyong; Lu, Yiling; Guo, Huifang; German, Peter; Bai, Shanshan; Jonasch, Eric; Yang, Xingsheng; Mills, Gordon B.; Ding, Zhiyong

    2013-01-01

    The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKTThr308 phosphorylation and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose deprivation-induced AKTThr308 phosphorylation is dependent on PDK1 and PI3K but not EGFR or IGF1R. Prolonged glucose deprivation induces the formation of a complex of AKT, PDK1, and the GRP78 chaperone protein, directing phosphorylation of AKTThr308 but AKTSer473. Our results reveal a novel mechanism of AKT activation under prolonged glucose deprivation that protects cells from metabolic stress. The selective activation of AKTThr308 phosphorylation that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling. PMID:23396361

  4. Thr308 determines Akt1 nuclear localization in insulin-stimulated keratinocytes

    SciTech Connect

    Goren, Itamar; Mueller, Elke; Pfeilschifter, Josef

    2008-07-18

    Here, we determined the localization and activation of protein kinase B (Akt) in acute cutaneous wound tissue in mice. Akt1 represented the major Akt isoform that was expressed and activated in wound margin keratinocytes and also in the cultured human keratinocyte line HaCaT. Mutation of Akt1 protein, exchanging the activation-essential Ser473 and Thr308 residues for inactive Ala or phosphorylation-mimicking Asp and Glu residues, revealed that phosphorylation of Ser473 represented an essential prerequisite for auto-phosphorylation of Thr308 within the Akt1 protein in keratinocytes. Moreover, cell culture experiments and transfection studies using Thr308 mutated Akt1 proteins demonstrated that phosphorylation of Akt1 at Thr308 appeared to selectively exclude the active kinase from the nucleus and direct the kinase to the cytoplasmic compartment in keratinocytes upon insulin stimulation. In summary, our data show that phosphorylation of Thr308 during insulin-mediated Akt1 activation is an essential prerequisite to exclude Akt1 from the nuclear compartment.

  5. PI3K/Akt promotes feedforward mTORC2 activation through IKKα

    PubMed Central

    Dan, Han C.; Antonia, Ricardo J.; Baldwin, Albert S.

    2016-01-01

    The ser-thr Akt plays a critical role in the regulation of cell survival, cell growth and proliferation, as well as energy metabolism and is dysregulated in many cancers. The regulation of Akt activity depends on the phosphorylation at two sites: (i) Thr308 in the activation loop by phosphoinositide-dependent kinase-1 (PDK1) and (ii) Ser473 hydrophobic motif at the carboxyl terminus by a second activity termed PDK2, which is the mTORC2 complex composed of mTOR, rictor, and Sin1. Previously we demonstrated that IKKα, a component of the IKK complex that controls NF-κB activation, participates in the Akt-dependent regulation of mTORC1. Here we have explored a potential involvement of IKKα in controlling Akt activity and whether this may involve mTORC2. The experiments show that IKKα associates with mTORC2 in several cancer cells in a manner dependent on PI3K/Akt activity and that IKKα positively promotes Akt phosphorylation at Ser473 and at Thr308. Moreover, IKKα enhances mTORC2 kinase activity directed to Akt on Ser473 and Akt-mediated phosphorylation of FOXO3a and GSK3β, but not other Akt-associated targets such as TSC2 and PRAS40, indicating the existence of multiple mechanisms of Akt activation in cells. In addition, loss of IKKα suppresses growth factor-induced Akt activation associated with mTORC1 inhibition. These results indicate that IKKα serves as a feedforward regulator of mTORC2 and that IKKα could serve as a key therapeutic target to block mTORC2 and Akt activation in some cancers. PMID:27027448

  6. Immunohistochemical analysis of the Akt/mTOR/4E-BP1 signalling pathway in canine haemangiomas and haemangiosarcomas.

    PubMed

    Murai, A; Abou Asa, S; Kodama, A; Sakai, H; Hirata, A; Yanai, T

    2012-11-01

    The specific signalling pathways that are deregulated in canine endothelial tumours have not yet fully elucidated. Therefore, the aim of the present study was to examine activation of the Akt/mammalian target of rapamycin (mTOR)/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) signalling pathway in spontaneously arising canine haemangiomas (HAs) and haemangiosarcomas (HSAs) in order to identify novel molecular targets for treatment. Surgically-resected samples of HA (n = 27), HSA (n = 37), granulation tissue (n = 4) and normal skin (n = 4) were investigated by immunohistochemistry. Approximately 80% of the HSA samples had moderate to intense expression of phosphorylated Akt at Ser473 (p-Akt Ser473), p-Akt Thr308, p-4E-BP1 Thr37/46 and eukaryotic initiation factor 4E, which was significantly higher than in the HAs and was similar to the expression in activated endothelial cells (ECs). Although p-mTOR complex1 (p-mTORC1) Ser2448 was expressed by most of the activated ECs, only 35% of the HSA samples had weak to moderate expression. Because mTORC2 and phosphorylates Akt Ser473 was activated in HSA samples, the present findings suggest that the mTORC2/Akt/4E-BP1 pathway, regulated independently of mTORC1, may be important for targeting therapy in canine HSAs. PMID:22789858

  7. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    NASA Astrophysics Data System (ADS)

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-01

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  8. Modulation in Activation and Expression of PTEN, Akt1, and PDK1: Further Evidence Demonstrating Altered Phosphoinositide 3-kinase Signaling in Postmortem Brain of Suicide Subjects

    PubMed Central

    Dwivedi, Yogesh; Rizavi, Hooriyah S.; Zhang, Hui; Roberts, Rosalinda C.; Conley, Robert R.; Pandey, Ghanshyam N.

    2010-01-01

    Background Phosphoinositide 3-kinase (PI 3-K) signaling plays a crucial role in neuronal growth and plasticity. Recently, we demonstrated that suicide brain is associated with decreased activation and expression of selective catalytic and regulatory subunits of PI 3-K. The present investigation examined the regulation and functional significance of compromised PI 3-K in suicide brain at the level of upstream phosphatase and tensin homolog on chromosome ten (PTEN) and downstream substrates 3-phosphoinositide-dependent kinase 1 (PDK1) and Akt. Method mRNA expression of Akt1, Akt3, PTEN, and PDK1 by competitive RT-PCR; protein expression of Akt1, Akt3, PTEN, PDK1, phosphorylated-Akt1 (Ser473), phosphorylated-Akt1(Thr308), phosphorylated-PDK1, and phosphorylated-PTEN by Western blot; and catalytic activities of Akt1, Akt3, and PDK1 by enzymatic assays were determined in prefrontal cortex (PFC) and hippocampus obtained from suicide subjects and nonpsychiatric controls. Results No significant changes in the expression of Akt1 or Akt3 were observed; however, catalytic activity of Akt1, but not of Akt3, was decreased in PFC and hippocampus of suicide subjects, which was associated with decreased phosphorylation of Akt1 at Ser473 and Thr308. The catalytic activity of PDK1 and the level of phosphorylated-PDK1 were also decreased in both brain areas without any change in expression levels of PDK1. On the other hand, mRNA and protein expression of PTEN was increased, whereas the level of phosphorylated-PTEN was decreased. Conclusion Our study demonstrates abnormalities in PI 3-K signaling at several levels in brain of suicide subjects and suggests the possible involvement of aberrant PI 3-K/Akt signaling in the pathogenic mechanisms of suicide. PMID:20163786

  9. Carboxy-terminal modulator protein attenuated extracellular matrix deposit by inhibiting phospho-Akt, TGF-β1 and α-SMA in kidneys of diabetic mice.

    PubMed

    Chen, Ning; Hao, Jun; Li, Lisha; Li, Fan; Liu, Shuxia; Duan, Huijun

    2016-06-10

    Glomerulosclerosis and tubular interstitial extracellular matrix deposit and fibrosis are the main features of diabetic nephropathy, which are mediated by activation of PI3K/Akt signal pathway. Carboxy-terminal modulator protein (CTMP) is known as a negative regulator of PI3K/Akt pathway. Whether CTMP regulates renal extracellular matrix metabolism of diabetic nephropathy is still not known. Here, renal decreased CTMP, enhanced phospho-Akt (Ser 473), TGF-β1, α-SMA and extracellular matrix deposit are found in diabetic mice. Furthermore, high glucose decreases CTMP expression accompanied by enhanced phospho-Akt (Ser 473), TGF-β1 and α-SMA in cultured human renal proximal tubular epithelial cells (HKC), which are effectively prevented by transfection of pYr-ads-4-musCTMP vector. Moreover, delivery of pYr-ads-4-musCTMP vector into kidneys via tail vein of diabetic mice increases CTMP expression by 8.84 times followed by 60.00%, 76.50% and 24.37% decreases of phospho-Akt (Ser 473), TGF-β1 and α-SMA compared with diabetic mice receiving pYr-adshuttle-4 vector. Again, increased renal extracellular matrix accumulation of diabetic mice is also inhibited with delivery of pYr-ads-4-musCTMP vector. Our results indicate that CTMP attenuates renal extracellular matrix deposit by regulating the phosphorylation of Akt, TGF-β1 and α-SMA expression in diabetic mice. PMID:27166156

  10. Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

    SciTech Connect

    Truong Le Xuan Nguyen; Choi, Joung Woo; Lee, Sang Bae; Ye, Keqiang; Woo, Soo-Dong; Lee, Kyung-Hoon; Ahn, Jee-Yin . E-mail: jyahn@med.skku.ac.kr

    2006-10-20

    Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.

  11. Constitutive phosphorylation of the mTORC2/Akt/4E-BP1 pathway in newly derived canine hemangiosarcoma cell lines

    PubMed Central

    2012-01-01

    Background Canine hemangiosarcoma (HSA) is a malignant tumor with poor long-term prognosis due to development of metastasis despite aggressive treatment. The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is involved in its endothelial pathologies; however, it remains unknown how this pathway plays a role in canine HSA. Here, we characterized new canine HSA cell lines derived from nude mice-xenografted canine HSAs and investigated the deregulation of the signaling pathways in these cell lines. Results Seven canine HSA cell lines were established from 3 xenograft canine HSAs and showed characteristics of endothelial cells (ECs), that is, uptake of acetylated low-density lipoprotein and expression of canine-specific CD31 mRNA. They showed varied morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, PDGF-B, and their receptors. Cell proliferation was stimulated by these growth factors and fetal bovine serum (FBS) in 1 cell line and by FBS alone in 3 cell lines. However, cell proliferation was not stimulated by growth factors and FBS in the remaining 3 cell lines. Phosphorylated p44/42 Erk1/2 was increased by FBS stimulation in 4 cell lines. In contrast, phosphorylation of Akt at Ser473, mTOR complex 1 (mTORC1) at Ser2448, and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) at Ser65 was high in serum-starved condition and not altered by FBS stimulation in 6 cell lines, despite increased phosphorylation of these residues in normal canine ECs. This suggested that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in these 6 canine HSA cell lines. After cell inoculation into nude mice, canine HSA tumors were formed from 4 cell lines and showed Akt and 4E-BP1 phosphorylation identical to the parental cell lines. Conclusions Our findings suggest that the present cell lines may be useful tools for investigating the role of the mTORC2/Akt/4E-BP1 pathway in canine HSA formation both

  12. The Masticatory Contractile Load Induced Expression and Activation of Akt1/PKBα in Muscle Fibers at the Myotendinous Junction within Muscle-Tendon-Bone Unit

    PubMed Central

    Korkmaz, Yüksel; Klinz, Franz J.; Moghbeli, Mehrnoush; Addicks, Klaus; Raab, Wolfgang H. -M.; Bloch, Wilhelm

    2010-01-01

    The cell specific detection of enzyme activation in response to the physiological contractile load within muscle-tendon-bone unit is essential for understanding of the mechanical forces transmission from muscle cells via tendon to the bone. The hypothesis that the physiological mechanical loading regulates activation of Akt1/PKBα at Thr308 and at Ser473 in muscle fibers within muscle-tendon-bone unit was tested using quantitative immunohistochemistry, confocal double fluorescence analysis, and immunoblot analysis. In comparison to the staining intensities in peripheral regions of the muscle fibers, Akt1/PKBα was detected with a higher staining intensity in muscle fibers at the myotendinous junction (MTJ) areas. In muscle fibers at the MTJ areas, Akt1/PKBα is dually phosphorylated at Thr308 and Ser473. The immunohistochemical results were confirmed by immunoblot analysis. We conclude that contractile load generated by masticatory muscles induces local domain-dependent expression of Akt1/PKBα as well as activation by dually phosphorylation at Thr308 and Ser473 in muscle fibers at the MTJ areas within muscle-tendon-bone unit. PMID:20454577

  13. Hydrogen Peroxide-Induced Akt Phosphorylation Regulates Bax Activation

    PubMed Central

    Sadidi, Mahdieh; Lentz, Stephen I.; Feldman, Eva L.

    2009-01-01

    Reactive oxygen species such as hydrogen peroxide (H2O2) are involved in many cellular processes that positively and negatively regulate cell fate. H2O2, acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H2O2 was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H2O2-induced activation of PI3K/Akt influences posttranslational modification of Bax and inactivate a key component of the cell death machinery. PMID:19278624

  14. Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

    PubMed Central

    Chan, Tung O.; Zhang, Jin; Tiegs, Brian C.; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M.; Armen, Roger S.; Rodeck, Ulrich; Penn, Raymond B.

    2015-01-01

    The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr308 in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr308 dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser473) increased phosphatase resistance of the phosphorylated activation loop (pThr308) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr308 phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. PMID:26201515

  15. PKC{eta} is a negative regulator of AKT inhibiting the IGF-I induced proliferation

    SciTech Connect

    Shahaf, Galit; Rotem-Dai, Noa; Koifman, Gabriela; Raveh-Amit, Hadas; Frost, Sigal A.; Livneh, Etta

    2012-04-15

    The PI3K-AKT pathway is frequently activated in human cancers, including breast cancer, and its activation appears to be critical for tumor maintenance. Some malignant cells are dependent on activated AKT for their survival; tumors exhibiting elevated AKT activity show sensitivity to its inhibition, providing an Achilles heel for their treatment. Here we show that the PKC{eta} isoform is a negative regulator of the AKT signaling pathway. The IGF-I induced phosphorylation on Ser473 of AKT was inhibited by the PKC{eta}-induced expression in MCF-7 breast adenocarcinoma cancer cells. This was further confirmed in shRNA PKC{eta}-knocked-down MCF-7 cells, demonstrating elevated phosphorylation on AKT Ser473. While PKC{eta} exhibited negative regulation on AKT phosphorylation it did not alter the IGF-I induced ERK phosphorylation. However, it enhanced ERK phosphorylation when stimulated by PDGF. Moreover, its effects on IGF-I/AKT and PDGF/ERK pathways were in correlation with cell proliferation. We further show that both PKC{eta} and IGF-I confer protection against UV-induced apoptosis and cell death having additive effects. Although the protective effect of IGF-I involved activation of AKT, it was not affected by PKC{eta} expression, suggesting that PKC{eta} acts through a different route to increase cell survival. Hence, our studies show that PKC{eta} provides negative control on AKT pathway leading to reduced cell proliferation, and further suggest that its presence/absence in breast cancer cells will affect cell death, which could be of therapeutic value.

  16. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  17. A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation

    PubMed Central

    2014-01-01

    Background Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC. Methods Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A. Results Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr308 and AKT-Ser473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC

  18. DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

    SciTech Connect

    Xu, Yong; Fang, Shi-ji; Zhu, Li-juan; Zhu, Lun-qing; Zhou, Xiao-zhong

    2014-10-24

    Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

  19. Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigenesis via FOXO inactivation

    PubMed Central

    Salazar, M; Lorente, M; García-Taboada, E; Pérez Gómez, E; Dávila, D; Zúñiga-García, P; María Flores, J; Rodríguez, A; Hegedus, Z; Mosén-Ansorena, D; Aransay, A M; Hernández-Tiedra, S; López-Valero, I; Quintanilla, M; Sánchez, C; Iovanna, J L; Dusetti, N; Guzmán, M; Francis, S E; Carracedo, A; Kiss-Toth, E; Velasco, G

    2015-01-01

    Tribbles pseudokinase-3 (TRIB3) has been proposed to act as an inhibitor of AKT although the precise molecular basis of this activity and whether the loss of TRIB3 contributes to cancer initiation and progression remain to be clarified. In this study, by using a wide array of in vitro and in vivo approaches, including a Trib3 knockout mouse, we demonstrate that TRIB3 has a tumor-suppressing role. We also find that the mechanism by which TRIB3 loss enhances tumorigenesis relies on the dysregulation of the phosphorylation of AKT by the mTORC2 complex, which leads to an enhanced phosphorylation of AKT on Ser473 and the subsequent hyperphosphorylation and inactivation of the transcription factor FOXO3. These observations support the notion that loss of TRIB3 is associated with a more aggressive phenotype in various types of tumors by enhancing the activity of the mTORC2/AKT/FOXO axis. PMID:25168244

  20. Tamoxifen-induced cytotoxicity in breast cancer cells is mediated by glucose-regulated protein 78 (GRP78) via AKT (Thr308) regulation.

    PubMed

    Pujari, Radha; Jose, Jemy; Bhavnani, Varsha; Kumar, Natesh; Shastry, Padma; Pal, Jayanta K

    2016-08-01

    Glucose regulated protein 78 (GRP78) has recently been suggested to be associated with drug resistance in breast cancer patients. However, the precise role of GRP78 in drug resistance and the involved signaling pathways are not clearly understood. In the present study, we show that among a panel of drugs, namely Paclitaxel (TAX), Doxorubicin (DOX), 5-fluorouracil (5-FU), UCN-01 and Tamoxifen (TAM) used, TAM alone up-regulated the expression of GRP78 significantly and induced apoptosis in MCF-7 and MDA-MB-231 cells. Interestingly, inhibition of GRP78 by a specific pharmacological inhibitor, VER-155008 augmented TAM-induced apoptosis, and overexpression of GRP78 rendered the cells resistant to TAM-induced cell death suggesting a role for GRP78 in TAM-induced cytotoxicity. Mechanistically, the expression of phosphorylated AKT as determined by Western blot analyses revealed that TAM selectively upregulated phosphorylation of AKT at Thr308 but not at Ser473, and siRNA silencing of GRP78 resulted in inhibition of AKT phosphorylation at Thr308 but not at Ser473. Further, a GRP78 inhibitor, VER155008 inhibited TAM-induced phosphorylation of GSK3β, a downstream substrate of AKT. These results, thus suggests a role for GRP78 in TAM-induced AKT activation. Additionally, co-localization studies by immunofluorescence, and immunoprecipitation experiments demonstrated a complex formation of AKT and GRP78. Furthermore, in glucose-free medium, the cells were sensitized to TAM-induced cell death that was associated with reduced AKT phosphorylation at Thr308, thus strengthening the association of AKT regulation with drug response. Collectively, our findings identify a role of GRP78 in AKT regulation in response to TAM in breast cancer cells. PMID:27262235

  1. Wall stretch and thromboxane A2 activate NO synthase (eNOS) in pulmonary arterial smooth muscle cells via H2O2 and Akt-dependent phosphorylation.

    PubMed

    Kim, Hae Jin; Yoo, Hae Young; Jang, Ji Hyun; Lin, Hai Yue; Seo, Eun Yeong; Zhang, Yin Hua; Kim, Sung Joon

    2016-04-01

    Pulmonary arteries (PAs) have high compliance, buffering the wide ranges of blood flow. Here, we addressed a hypothesis that PA smooth muscle cells (PASMCs) express nitric oxide synthases (NOS) that might be activated by mechanical stress and vasoactive agonists. In the myograph study of endothelium-denuded rat PAs, NOS inhibition (L-NAME) induced strong contraction (96 % of 80 mM KCl-induced contraction (80K)) in the presence of 5 nM U46619 (thromboxane A2 (TXA2) analogue) with relatively high basal stretch (2.94 mN, S(+)). With lower basal stretch (0.98 mN, S(-)), however, L-NAME application following U46619 (TXA2/L-NAME) induced weak contraction (27 % of 80K). Inhibitors of nNOS and iNOS had no such effect in S(+) PAs. In endothelium-denuded S(+) mesenteric and renal arteries, TXA2/L-NAME-induced contraction was only 18 and 21 % of 80K, respectively. Expression of endothelial-type NOS (eNOS) in rat PASMCs was confirmed by RT-PCR and immunohistochemistry. Even in S(-) PAs, pretreatment with H2O2 (0.1-10 μM) effectively increased the sensitivity to TXA2/L-NAME (105 % of 80K). Vice versa, NADPH oxidase inhibitors, reactive oxygen species scavengers, or an Akt inhibitor (SC-66) suppressed TXA2/L-NAME-induced contraction in S(+) PAs. In a human PASMC line, immunoblot analysis showed the following: (1) eNOS expression, (2) Ser(1177) phosphorylation by U46619 and H2O2, and (3) Akt activation (Ser(473) phosphorylation) by U46619. In the cell-attached patch clamp study, H2O2 facilitated membrane stretch-activated cation channels in rat PASMCs. Taken together, the muscular eNOS in PAs can be activated by TXA2 and mechanical stress via H2O2 and Akt-mediated signaling, which may counterbalance the contractile signals from TXA2 and mechanical stimuli. PMID:26729266

  2. Cell-cycle-regulated activation of Akt kinase by phosphorylation at its carboxyl terminus

    PubMed Central

    Liu, Pengda; Begley, Michael; Michowski, Wojciech; Inuzuka, Hiroyuki; Ginzberg, Miriam; Gao, Daming; Tsou, Peiling; Gan, Wenjian; Papa, Antonella; Kim, Byeong Mo; Wan, Lixin; Singh, Amrik; Zhai, Bo; Yuan, Min; Wang, Zhiwei; Gygi, Steven P.; Lee, Tae Ho; Lu, Kun-Ping; Toker, Alex; Pandolfi, Pier Paolo; Asara, John M.; Kirschner, Marc W.; Sicinski, Piotr; Cantley, Lewis; Wei, Wenyi

    2014-01-01

    Akt, also known as protein kinase B, plays key roles in cell proliferation, survival and metabolism. Akt hyperactivation contributes to many pathophysiological conditions, including human cancers1–3, and is closely associated with poor prognosis and chemo- or radio-therapeutic resistance4. Phosphorylation of Akt at S473 (ref. 5) and T308 (ref. 6) activates Akt. However, it remains unclear whether further mechanisms account for full Akt activation, and whether Akt hyperactivation is linked to misregulated cell cycle progression, another cancer hallmark7. Here we report that Akt activity fluctuates across the cell cycle, mirroring cyclin A expression. Mechanistically, phosphorylation of S477 and T479 at the Akt extreme carboxy terminus by cyclin-dependent kinase 2 (Cdk2)/cyclin A or mTORC2, under distinct physiological conditions, promotes Akt activation through facilitating, or functionally compensating for, S473 phosphorylation. Furthermore, deletion of the cyclin A2 allele in the mouse olfactory bulb leads to reduced S477/T479 phosphorylation and elevated cellular apoptosis. Notably, cyclin A2-deletion-induced cellular apoptosis in mouse embryonic stem cells is partly rescued by S477D/T479E-Akt1, supporting a physiological role for cyclin A2 in governing Akt activation. Together, the results of our study show Akt S477/T479 phosphorylation to be an essential layer of the Akt activation mechanism to regulate its physiological functions, thereby providing a new mechanistic link between aberrant cell cycle progression and Akt hyperactivation in cancer. PMID:24670654

  3. Similar requirement for clathrin in EGF- and HGF- stimulated Akt phosphorylation.

    PubMed

    Lucarelli, Stefanie; Pandey, Rohan; Judge, Gurjeet; Antonescu, Costin N

    2016-01-01

    Receptor tyrosine kinases, such as the epidermal growth factor (EGF) receptor (EGFR) and Met lead to activation of intracellular signals including Akt, a critical regulator of cell survival, metabolism and proliferation. Upon binding their respective ligands, each of these receptors is recruited into clathrin coated pits (CCPs) eventually leading to endocytosis. We have recently shown that phosphorylation of Gab1 and Akt following EGFR activation requires clathrin, but does not require receptor endocytosis. We examined whether clathrin regulates Akt signaling downstream of Met, as it does for EGFR signaling. Stimulation with the Met ligand Hepatocyte Growth Factor (HGF) leads to enrichment of phosphorylated Gab1 (pGab1) within CCPs in ARPE-19 cells. Perturbation of clathrin using the inhibitor pitstop2 decreases HGF-stimulated Akt phosphorylation. These results indicate that clathrin may regulate Met signaling leading to Akt phosphorylation similarly as it does for EGFR signaling. PMID:27489582

  4. Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia.

    PubMed

    Suizu, Futoshi; Hirata, Noriyuki; Kimura, Kohki; Edamura, Tatsuma; Tanaka, Tsutomu; Ishigaki, Satoko; Donia, Thoria; Noguchi, Hiroko; Iwanaga, Toshihiko; Noguchi, Masayuki

    2016-06-15

    A primary cilium is a microtubule-based sensory organelle that plays an important role in human development and disease. However, regulation of Akt in cilia and its role in ciliary development has not been demonstrated. Using yeast two-hybrid screening, we demonstrate that Inversin (INVS) interacts with Akt. Mutation in the INVS gene causes nephronophthisis type II (NPHP2), an autosomal recessive chronic tubulointerstitial nephropathy. Co-immunoprecipitation assays show that Akt interacts with INVS via the C-terminus. In vitro kinase assays demonstrate that Akt phosphorylates INVS at amino acids 864-866 that are required not only for Akt interaction, but also for INVS dimerization. Co-localization of INVS and phosphorylated form of Akt at the basal body is augmented by PDGF-AA Akt-null MEF cells as well as siRNA-mediated inhibition of Akt attenuated ciliary growth, which was reversed by Akt reintroduction. Mutant phosphodead- or NPHP2-related truncated INVS, which lack Akt phosphorylation sites, suppress cell growth and exhibit distorted lumen formation and misalignment of spindle axis during cell division. Further studies will be required for elucidating functional interactions of Akt-INVS at the primary cilia for identifying the molecular mechanisms underlying NPHP2. PMID:27220846

  5. Neuregulin-1β promotes glucose uptake via PI3K/Akt in neonatal rat cardiomyocytes.

    PubMed

    Pentassuglia, Laura; Heim, Philippe; Lebboukh, Sonia; Morandi, Christian; Xu, Lifen; Brink, Marijke

    2016-05-01

    Nrg1β is critically involved in cardiac development and also maintains function of the adult heart. Studies conducted in animal models showed that it improves cardiac performance under a range of pathological conditions, which led to its introduction in clinical trials to treat heart failure. Recent work also implicated Nrg1β in the regenerative potential of neonatal and adult hearts. The molecular mechanisms whereby Nrg1β acts in cardiac cells are still poorly understood. In the present study, we analyzed the effects of Nrg1β on glucose uptake in neonatal rat ventricular myocytes and investigated to what extent mTOR/Akt signaling pathways are implicated. We show that Nrg1β enhances glucose uptake in cardiomyocytes as efficiently as IGF-I and insulin. Nrg1β causes phosphorylation of ErbB2 and ErbB4 and rapidly induces the phosphorylation of FAK (Tyr(861)), Akt (Thr(308) and Ser(473)), and its effector AS160 (Thr(642)). Knockdown of ErbB2 or ErbB4 reduces Akt phosphorylation and blocks the glucose uptake. The Akt inhibitor VIII and the PI3K inhibitors LY-294002 and Byl-719 abolish Nrg1β-induced phosphorylation and glucose uptake. Finally, specific mTORC2 inactivation after knockdown of rictor blocks the Nrg1β-induced increases in Akt-p-Ser(473) but does not modify AS160-p-Thr(642) or the glucose uptake responses to Nrg1β. In conclusion, our study demonstrates that Nrg1β enhances glucose uptake in cardiomyocytes via ErbB2/ErbB4 heterodimers, PI3Kα, and Akt. Furthermore, although Nrg1β activates mTORC2, the resulting Akt-Ser(473) phosphorylation is not essential for glucose uptake induction. These new insights into pathways whereby Nrg1β regulates glucose uptake in cardiomyocytes may contribute to the understanding of its regenerative capacity and protective function in heart failure. PMID:26979522

  6. Diaminothiazoles inhibit angiogenesis efficiently by suppressing Akt phosphorylation.

    PubMed

    Thomas, Sannu A; Thamkachy, Reshma; Ashokan, Bindu; Komalam, Reena J; Sreerekha, Keerthi V; Bharathan, Asha; Santhoshkumar, Thankayyan R; Rajasekharan, Kallikat N; Sengupta, Suparna

    2012-06-01

    The prevention of neovessel formation or angiogenesis is a recent popular strategy for limiting and curing cancer. Diaminothiazoles are a class of compounds that have been reported to show promise in the treatment of cancer by inhibiting cancer cell proliferation and inducing apoptosis, because of their effects on microtubules and as inhibitors of cyclin-dependent kinases. Many microtubule-targeting agents are being studied for their antiangiogenic activity, and a few have shown promising activity in the treatment of cancer. Here, we report that diaminothiazoles can be highly effective as antiangiogenic agents, as observed in the chick membrane assay. The lead compound, 4-amino-5-benzoyl-2-(4-methoxyphenylamino)thiazole (DAT1), inhibits endothelial cell processes such as invasion, migration, and tubule formation, which require a functional cytoskeleton. DAT1 also decreases the expression of cell adhesion markers. The antiangiogenic activities of DAT1 occur at concentrations that are not cytotoxic to the normal endothelium. Analysis of intracellular signaling pathways shows that DAT1 inhibits Akt phosphorylation, which is actively involved in the angiogenic process. The antiangiogenic properties of diaminothiazoles, in addition to their promising antimitotic and cytotoxic properties in cancer cell lines, give them an extra advantage in the treatment of cancer. PMID:22414853

  7. Interdomain conformational changes in Akt activation revealed by chemical cross-linking and tandem mass spectrometry.

    PubMed

    Huang, Bill X; Kim, Hee-Yong

    2006-06-01

    Akt, a serine/threonine kinase, plays a critical role in cell survival. Upon growth factor receptor stimulation, cytosolic Akt is recruited to the plasma membrane by phospholipid binding and activated through phosphorylation at Thr(308) and Ser(473). Although crystal structures for the parts of Akt have been reported, neither the three-dimensional structure of the whole molecule nor sequential conformational changes during activation have been demonstrated. In this study, we demonstrated that Akt undergoes dramatic interdomain conformational changes during activation processes by probing the three-dimensional structure of full-length Akt in solution using chemical cross-linking and tandem mass spectrometry. The cross-linking results not only provided new structural information but also revealed distinctive spatial arrangements of individual domains in the Akt molecule in resting, membrane-interacted, phosphorylated, and substrate-bound states. Our data allowed a new model for stepwise interdomain conformational changes in Akt activation sequence, setting a stage for the further investigation on Akt-membrane, Akt-protein, and/or Akt-drug interactions in solution to understand molecular mechanisms involved in physiological and pathophysiological processes of cell survival. PMID:16531397

  8. Anticancer effect of celastrol on human triple negative breast cancer: possible involvement of oxidative stress, mitochondrial dysfunction, apoptosis and PI3K/Akt pathways.

    PubMed

    Shrivastava, Shweta; Jeengar, Manish Kumar; Reddy, V Sudhakar; Reddy, G Bhanuprakash; Naidu, V G M

    2015-06-01

    Signaling via the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) is crucial for divergent physiological processes including transcription, translation, cell-cycle progression and apoptosis. The aim of work was to elucidate the anti-cancer effect of celastrol and the signal transduction pathways involved. Cytotoxic effect of celastrol was assessed by MTT assay on human triple negative breast cancer cells (TNBCs) and compared with that of MCF-7. Apoptosis induction was determined by AO/EtBr staining, mitochondrial membrane potential by JC-1, Annexin binding assays and modulation of apoptotic proteins and its effect on PI3K/Akt/mTOR pathway by western blotting. Celastrol induced apoptosis in TNBC cells, were supported by DNA fragmentation, caspase-3 activation and PARP cleavage. Meanwhile, celastrol triggered reactive oxygen species production with collapse of mitochondrial membrane potential, down-regulation of Bcl-2 and up-regulation of Bax expression. Celastrol effectively decreased PI3K 110α/85α enzyme activity, phosphorylation of Akt(ser473) and p70S6K1 and 4E-BP1. Although insulin treatment increased the phosphorylation of Akt(ser473), p70S6K1, 4E-BP1, celastrol abolished the insulin mediated phosphorylation. It clearly indicates that celastrol acts through PI3k/Akt/mTOR axis. We also found that celastrol inhibited the Akt/GSK3β and Akt/NFkB survival pathway. PI3K/Akt/mTOR inhibitor, PF-04691502 and mTOR inhibitor rapamycin enhanced the apoptosis-inducing effect of celastrol. These data demonstrated that celastrol induces apoptosis in TNBC cells and indicated that apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway. PMID:25818165

  9. Akt mediated phosphorylation of LARP6; critical step in biosynthesis of type I collagen

    PubMed Central

    Zhang, Yujie; Stefanovic, Branko

    2016-01-01

    La ribonucleoprotein domain family, member 6 (LARP6) is the RNA binding protein, which regulates translation of collagen mRNAs and synthesis of type I collagen. Posttranslational modifications of LARP6 and how they affect type I collagen synthesis have not been studied. We show that in lung fibroblasts LARP6 is phosphorylated at 8 serines, 6 of which are located within C-terminal domain. Phosphorylation of LARP6 follows a hierarchical order; S451 phosphorylation being a prerequisite for phosphorylations of other serines. Inhibition of PI3K/Akt pathway reduced the phosphorylation of LARP6, but had no effect on the S451A mutant, suggesting that PI3K/Akt pathway targets S451 and we have identified Akt as the responsible kinase. Overexpression of S451A mutant had dominant negative effect on collagen biosynthesis; drastically reduced secretion of collagen and induced hyper-modifications of collagen α2 (I) polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for regulating translation and folding of collagen polypeptides. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production by human lung fibroblasts with EC50 of 150 nM. This effect can be explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of various forms of fibrosis. PMID:26932461

  10. The Novel Small Molecule Inhibitor, OSU-T315, Suppresses Vestibular Schwannoma and Meningioma Growth by Inhibiting PDK2 Function in the AKT Pathway Activation

    PubMed Central

    Mercado-Pimentel, ME; Igarashi, S; Dunn, AM; Behbahani, M; Miller, C; Read, CM; Jacob, A

    2016-01-01

    Activation of PKB/AKT signaling, which requires PDK1 and PDK2 function, drives Vestibular Schwannoma (VS) and meningioma growth. PDK2 function is defined as a molecule that phosphorylates AKT-Ser473. Integrin-Linked Kinase (ILK) functions as PDK2 in PKB/AKT activation in many cancers; therefore, we hypothesized that OSU-T315, a small molecule ILK inhibitor, will inhibit the ILK-PDK2 function in PKB/AKT signaling activation in VS and meningioma cell growth. OSU-T315 decreased cell viability at IC50 < 2μM in VS (HEI193) and meningioma (Ben-Men-1) cell lines, in primary cells at < 3.5μM, while in normal primary Schwann cells at 7.1μM. OSU-T315 inhibits AKT signaling by decreasing phosphorylation at AKT-Ser473, AKT-Thr308, ILK-Ser246 and ILK-Thr173. In addition, OSU-T315 affected the phosphorylation or expression levels of AKT downstream proliferation effectors as well as autophagy markers. Flow cytometry shows that OSU-T315 increased the percentage of cells arrested at G2/M for both, HEI193 (39.99%) and Ben-Men-1 (26.96%) cells, compared to controls (21.54%, 8.47%). Two hours of OSU-T315 treatment increased cell death in both cell lines (34.3%, 9.1%) versus untreated (12.1%, 8.1%). Though longer exposure increased cell death in Ben-Men-1, TUNEL assays showed that OSU-T315 does not induce apoptosis. OSU-T315 was primarily cytotoxic for HEI193 and Ben-Men-1 inducing a dysregulated autophagy. Our studies suggest that OSU-T315 has translational potential as a chemotherapeutic agent against VS and meningioma.

  11. Expression of phosphorylated Akt/mTOR and clinical significance in human ameloblastoma

    PubMed Central

    Li, Ning; Sui, Jianfu; Liu, Hao; Zhong, Ming; Zhang, Min; Wang, Yan; Hao, Fengyu

    2015-01-01

    This study aimed to evaluate the expression of AKT and phosphorylated AKT (p-Akt) in human ameloblastoma (AB). Immunohistochemistry showed human AB was positive for Akt and Akt expression was mainly found in the cytoplasm of epithelial cells. The Akt expression in AB was significantly higher than that in normal oral mucosa (NOM), but still lower than that in oral squamous cell carcinoma (OSCC). NOM was negative for p-Akt, but AB was positive for p-Akt. In some AB tissues, p-Akt expression was found in both cytoplasm and nucleus. Akt expression in AB was significantly different from that in NOM and OSCC. The p-Akt in AB was markedly higher than that in NOM, but lower than that in OSCC. mTOR expressed in cytoplasm in AB, but not in NOM. P-mTOR expressed on cell membrane in NOM, while in cytoplasm and nucleus in Ab. Results of western blot assay showed that Akt expression was found in all the AB tissues, and increased in tissues with malignant transformation. In addition, the p-Akt expression also markedly increased in AB, but was still lower than that in OSCC tissues. Compared to NOM, mTOR and p-mTOR expression significantly increased in AB. BandScan 5.0 software was used to detect the optical density of protein bands. Results showed p-Akt, mTOR and p-mTOR expression in AB was markedly different from that in control group. PMID:26131097

  12. RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3.

    PubMed

    Cailliau, Katia; Lescuyer, Arlette; Burnol, Anne-Françoise; Cuesta-Marbán, Álvaro; Widmann, Christian; Browaeys-Poly, Edith

    2015-08-01

    Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling. PMID:26109071

  13. Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1.

    PubMed

    He, Chang-Liang; Bian, Yang-Yang; Xue, Yu; Liu, Ze-Xian; Zhou, Kai-Qiang; Yao, Cui-Fang; Lin, Yan; Zou, Han-Fa; Luo, Fang-Xiu; Qu, Yuan-Yuan; Zhao, Jian-Yuan; Ye, Ming-Liang; Zhao, Shi-Min; Xu, Wei

    2016-01-01

    In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells. PMID:26876154

  14. Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1

    PubMed Central

    He, Chang-Liang; Bian, Yang-Yang; Xue, Yu; Liu, Ze-Xian; Zhou, Kai-Qiang; Yao, Cui-Fang; Lin, Yan; Zou, Han-Fa; Luo, Fang-Xiu; Qu, Yuan-Yuan; Zhao, Jian-Yuan; Ye, Ming-Liang; Zhao, Shi-Min; Xu, Wei

    2016-01-01

    In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells. PMID:26876154

  15. Early Endosomal Antigen 1 (EEA1) Is an Obligate Scaffold for Angiotensin II-induced, PKC-α-dependent Akt Activation in Endosomes*

    PubMed Central

    Nazarewicz, Rafal Robert; Salazar, Gloria; Patrushev, Nikolay; Martin, Alejandra San; Hilenski, Lula; Xiong, Shiqin; Alexander, R. Wayne

    2011-01-01

    Akt/protein kinase B (PKB) activation/phosphorylation by angiotensin II (Ang II) is a critical signaling event in hypertrophy of vascular smooth muscle cells (VSMCs). Conventional wisdom asserts that Akt activation occurs mainly in plasma membrane domains. Recent evidence that Akt activation may take place within intracellular compartments challenges this dogma. The spatial identity and mechanistic features of these putative signaling domains have not been defined. Using cell fractionation and fluorescence methods, we demonstrate that the early endosomal antigen-1 (EEA1)-positive endosomes are a major site of Ang II-induced Akt activation. Akt moves to and is activated in EEA1 endosomes. The expression of EEA1 is required for phosphorylation of Akt at both Thr-308 and Ser-473 as well as for phosphorylation of its downstream targets mTOR and S6 kinase, but not for Erk1/2 activation. Both Akt and phosphorylated Akt (p-Akt) interact with EEA1. We also found that PKC-α is required for organizing Ang II-induced, EEA1-dependent Akt phosphorylation in VSMC early endosomes. EEA1 expression enables PKC-α phosphorylation, which in turn regulates Akt upstream signaling kinases, PDK1 and p38 MAPK. Our results indicate that PKC-α is a necessary regulator of EEA1-dependent Akt signaling in early endosomes. Finally, EEA1 down-regulation or expression of a dominant negative mutant of PKC-α blunts Ang II-induced leucine incorporation in VSMCs. Thus, EEA1 serves a novel function as an obligate scaffold for Ang II-induced Akt activation in early endosomes. PMID:21097843

  16. Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3.

    PubMed

    Yoshizaki, Yuki; Mori, Yutaro; Tsuzaki, Yoshihito; Mori, Takayasu; Nomura, Naohiro; Wakabayashi, Mai; Takahashi, Daiei; Zeniya, Moko; Kikuchi, Eriko; Araki, Yuya; Ando, Fumiaki; Isobe, Kiyoshi; Nishida, Hidenori; Ohta, Akihito; Susa, Koichiro; Inoue, Yuichi; Chiga, Motoko; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi; Sohara, Eisei

    2015-11-13

    Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal. PMID:26435498

  17. Low Phosphorylated AKT Expression in Laryngeal Cancer: Indications for a Higher Metastatic Risk

    SciTech Connect

    Nijkamp, Monique M.; Span, Paul N.; Stegeman, Hanneke; Grénman, Reidar; Kaanders, Johannes H.A.M.; Bussink, Johan

    2013-10-01

    Purpose: To validate the association of phosphorylated (p)AKT with lymph node metastasis in an independent, homogeneous cohort of patients with larynx cancer. Methods and Materials: Seventy-eight patients with laryngeal cancer were included. Epidermal growth factor receptor, pAKT, vimentin, E-cadherin, hypoxia, and blood vessels were visualized in biopsy material using immunohistochemistry. Positive tumor areas and spatial relationships between markers were assessed by automated image analysis. In 6 laryngeal cancer cell lines, E-cadherin and vimentin messenger RNA was quantified by real-time polymerase chain reaction and by immunohistochemistry before and after treatment with the pAKT inhibitor MK-2206. Results: A significant correlation was found between low pAKT in the primary tumor and positive lymph node status (P=.0005). Tumors with lymph node metastases had an approximately 10-fold lower median pAKT value compared with tumors without lymph node metastases, albeit with large intertumor variations, validating our previous results. After inhibition of pAKT in laryngeal cancer cells with MK-2206, up-regulation of vimentin and a downregulation of E-cadherin occurred, consistent with epithelial–mesenchymal transition. Conclusion: Low pAKT expression in larynx tumors is associated with lymph node metastases. Further, inhibition of pAKT in laryngeal cancer induces epithelial–mesenchymal transition, predisposing for an increased metastatic risk.

  18. Fangchinoline suppresses the growth and invasion of human glioblastoma cells by inhibiting the kinase activity of Akt and Akt-mediated signaling cascades.

    PubMed

    Guo, Bingyu; Xie, Peng; Su, Jingyuan; Zhang, Tingting; Li, Xiaoming; Liang, Guobiao

    2016-02-01

    Glioblastoma multiforme (GBM) is one of the most palindromic and malignant central nervous system neoplasms, and the current treatment is not effectual for GBM. Research of specific medicine for GBM is significant. Fangchinoline possesses a wide range of pharmacological activities and attracts more attentions due to its anti-tumor effects. In this study, two WHO grade IV human GBM cell lines (U87 MG and U118 MG) were exposed to fangchinoline, and we found that fangchinoline specifically inhibits the kinase activity of Akt and markedly suppresses the phosphorylation of Thr308 and Ser473 of Akt in human GBM cells. We also observed that fangchinoline inhibits tumor cell proliferation and invasiveness and induces apoptosis through suppressing the Akt-mediated signaling cascades, including Akt/p21, Akt/Bad, and Akt/matrix metalloproteinases (MMPs). These data demonstrated that fangchinoline exerts its anti-tumor effects in human glioblastoma cells, at least partly by inhibiting the kinase activity of Akt and suppressing Akt-mediated signaling cascades. PMID:26408176

  19. Phospholipid transfer protein (PLTP) reduces phosphorylation of tau in human neuronal cells (HCN2)

    PubMed Central

    Dong, Weijiang; Albers, John J.; Vuletic, Simona

    2009-01-01

    Tau function is regulated by phosphorylation, and abnormal tau phosphorylation in neurons is one of the key processes associated with development of Alzheimer’s disease and other tauopathies. In this study we provide evidence that phospholipid transfer protein (PLTP), one of the main lipid transfer proteins in the brain, significantly reduces levels of phosphorylated tau, and increases levels of the inactive form of glycogen synthase kinase-3β (GSK3β) in HCN2 cells. Furthermore, inhibition of the phosphatidylinositol-3 kinase (PI3K) reversed the PLTP-induced increase in levels of GSK3β phosphorylated at serine 9 (pGSK3βSer9) and partially reversed the PLTP-induced reduction in tau phosphorylation. We provide evidence that the PLTP-induced changes are not due to activation of Disabled-1 (Dab1), since PLTP reduced levels of total and phosphorylated Dab1 in HCN2 cells. We have also shown that inhibition of tyrosine kinase activity of insulin receptor (IR) and/or insulin-like growth factor 1 (IGF1) receptor (IGFR) reverses PLTP-induced increase in levels of phosphorylated Akt (pAktThr308 and pAktSer473), suggesting that PLTP-mediated activation of the PI3K/Akt pathway is dependent on IR/IGFR receptor tyrosine kinase activity. Our study suggests that PLTP may be an important modulator of signal transduction pathways in human neurons. PMID:19472218

  20. Inhibition of Rb Phosphorylation Leads to mTORC2-Mediated Activation of Akt.

    PubMed

    Zhang, Jinfang; Xu, Kai; Liu, Pengda; Geng, Yan; Wang, Bin; Gan, Wenjian; Guo, Jianping; Wu, Fei; Chin, Y Rebecca; Berrios, Christian; Lien, Evan C; Toker, Alex; DeCaprio, James A; Sicinski, Piotr; Wei, Wenyi

    2016-06-16

    The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell-cycle progression. However, it remains largely elusive whether the hyper-phosphorylated, non-E2F1-interacting form of Rb has any physiological role. Here we report that hyper-phosphorylated Rb directly binds to and suppresses the function of mTORC2 but not mTORC1. Mechanistically, Rb, but not p107 or p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D or using CDK4/6 inhibitors releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer. PMID:27237051

  1. 14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells.

    PubMed

    Gómez-Suárez, M; Gutiérrez-Martínez, I Z; Hernández-Trejo, J A; Hernández-Ruiz, M; Suárez-Pérez, D; Candelario, A; Kamekura, R; Medina-Contreras, O; Schnoor, M; Ortiz-Navarrete, V; Villegas-Sepúlveda, N; Parkos, C; Nusrat, A; Nava, P

    2016-06-01

    Akt activation has been associated with proliferation, differentiation, survival and death of epithelial cells. Phosphorylation of Thr308 of Akt by phosphoinositide-dependent kinase 1 (PDK1) is critical for optimal stimulation of its kinase activity. However, the mechanism(s) regulating this process remain elusive. Here, we report that 14-3-3 proteins control Akt Thr308 phosphorylation during intestinal inflammation. Mechanistically, we found that IFNγ and TNFα treatment induce degradation of the PDK1 inhibitor, 14-3-3η, in intestinal epithelial cells. This mechanism requires association of 14-3-3ζ with raptor in a process that triggers autophagy and leads to 14-3-3η degradation. Notably, inhibition of 14-3-3 function by the chemical inhibitor BV02 induces uncontrolled Akt activation, nuclear Akt accumulation and ultimately intestinal epithelial cell death. Our results suggest that 14-3-3 proteins control Akt activation and regulate its biological functions, thereby providing a new mechanistic link between cell survival and apoptosis of intestinal epithelial cells during inflammation. PMID:26846144

  2. Aloe-emodin suppresses esophageal cancer cell TE1 proliferation by inhibiting AKT and ERK phosphorylation

    PubMed Central

    Chang, Xiaobin; Zhao, Jimin; Tian, Fang; Jiang, Yanan; Lu, Jing; Ma, Junfen; Zhang, Xiaoyan; Jin, Guoguo; Huang, Youtian; Dong, Zigang; Liu, Kangdong; Dong, Ziming

    2016-01-01

    Aberrant AKT and extracellular signal-regulated kinase (ERK) activation is often observed in various human cancers. Both AKT and ERK are important in the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase kinase/ERK signaling pathways, which play vital roles in cell proliferation, differentiation and survival. Compounds that are able to block these pathways have therefore a promising use in cancer treatment and prevention. The present study revealed that AKT and ERK are activated in esophageal cancer TE1 cells. Aloe-emodin, an anthraquinone present in aloe latex, can suppress TE1 cell proliferation and anchor-independent cell growth. Aloe-emodin can also reduce the number of TE1 cells in S phase. Protein analysis indicated that aloe-emodin inhibits the phosphorylation of AKT and ERK in a dose-dependent manner. Overall, the present data indicate that aloe-emodin can suppress TE1 cell growth by inhibiting AKT and ERK phosphorylation, and suggest its clinical use for cancer therapy. PMID:27602169

  3. AKT inhibitor suppresses hyperthermia-induced Ndrg2 phosphorylation in gastric cancer cells

    PubMed Central

    Tao, Yurong; Guo, Yan; Liu, Wenchao; Zhang, Jian; Li, Xia; Shen, Lan; Ru, Yi; Xue, Yan; Zheng, Jin; Liu, Xinping; Zhang, Jing; Yao, Libo

    2013-01-01

    Hyperthermia is one of the most effective adjuvant treatments for various cancers with few side effects. However, the underlying molecular mechanisms still are not known. N-myc downstream-regulated gene 2 (NDRG2), a tumor suppressor, has been shown to be involved in diverse cellular stresses including hypoxia, lipotoxicity, etc. In addition, Ndrg2 has been reported to be related to progression of gastric cancer. In the current study, our data showed that the apoptosis rate of MKN28 cells increased relatively rapidly to 13.4% by 24 h after treatment with hyperthermia (42°C for 1 h) compared to 5.1% in control cells (P < 0.05). Nevertheless, there was no obvious change in the expression level of total Ndrg2 during this process. Further investigation demonstrated that the relative phosphorylation levels of Ndrg2 at Ser332, Thr348 increased up to 3.2- and 1.9-fold (hyperthermia group vs control group) at 3 h in MKN28 cells, respectively (P < 0.05). We also found that heat treatment significantly increased AKT phosphorylation. AKT inhibitor VIII (10 µM) decreased the phosphorylation level of Ndrg2 induced by hyperthermia. Accordingly, the apoptosis rate rose significantly in MKN28 cells (16.4%) treated with a combination of AKT inhibitor VIII and hyperthermia compared to that (6.8%) of cells treated with hyperthermia alone (P < 0.05). Taken together, these data demonstrated that Ndrg2 phosphorylation could be induced by hyperthermia in an AKT-dependent manner in gastric cancer cells. Furthermore, AKT inhibitor VIII suppressed Ndrg2 phosphorylation and rendered gastric cancer cells susceptible to apoptosis induced by hyperthermia. PMID:23558861

  4. PARP1 inhibitors attenuate AKT phosphorylation via the upregulation of PHLPP1

    SciTech Connect

    Wang, Shuai; Wang, Huibo; Davis, Ben C.; Liang, Jiyong; Cui, Rutao; Chen, Sai-Juan; Xu, Zhi-Xiang

    2011-08-26

    Highlights: {yields} PARP1 inhibitors cause a cytotoxic effect independent of DNA repair impairment. {yields} PARP1 inhibitors attenuated AKT-FOXO3A signaling by activating PHLPP1. {yields} PHLPP1 regulates the sensitivity of cancer cells to PARP1 inhibitors. -- Abstract: Poly(ADP-ribose) polymerase-1 (PARP1) inhibitors are emerging as an important class of drugs for treating BRCA-deficient cancers. Recent discoveries have shown that PARP1 inhibitors may treat other cancer patients in addition to the relatively small proportion of patients carrying BRCA mutations. However, the additional targets by which PARP1 inhibitor-mediated tumor suppression remain poorly understood. In this study, we show that two PARP1 inhibitors, PJ-34 and 3-AB, attenuate AKT phosphorylation at serine 473 (S473) independent of DNA repair impairment. These inhibitors decrease the AKT-associated phosphorylation of FOXO3A, enhance the nuclear retention of FOXO3A, and activate its transcriptional activity. We further demonstrate that treatment with PJ-34 or 3-AB dramatically increases the level of PHLPP1. Overexpression of PHLPP1 enhances the PARP1 inhibitor-induced downregulation of AKT phosphorylation and increases tumor cell death. In contrast, knockdown of PHLPP1 abrogates the PARP1 inhibitor-mediated AKT inhibition and desensitizes cells to its treatment. Therefore, our findings not only show the robust role of PARP1 inhibitors in AKT inhibition but also develop a novel strategy to increase the effectiveness of cancer treatment via PARP1 inhibitor-induced PHLPP1 upregulation.

  5. O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling

    PubMed Central

    Shi, Jianhua; Gu, Jin-hua; Dai, Chun-ling; Gu, Jianlan; Jin, Xiaoxia; Sun, Jianming; Iqbal, Khalid; Liu, Fei; Gong, Cheng-Xin

    2015-01-01

    Apoptosis plays an important role in neural development and neurological disorders. In this study, we found that O-GlcNAcylation, a unique protein posttranslational modification with O-linked β-N-acetylglucosamine (GlcNAc), promoted apoptosis through attenuating phosphorylation/activation of AKT and Bad. By using co-immunoprecipitation and mutagenesis techniques, we identified O-GlcNAc modification at both Thr308 and Ser473 of AKT. O-GlcNAcylation-induced apoptosis was attenuated by over-expression of AKT. We also found a dynamic elevation of protein O-GlcNAcylation during the first four hours of cerebral ischemia, followed by continuous decline after middle cerebral artery occlusion (MCAO) in the mouse brain. The elevation of O-GlcNAcylation coincided with activation of cell apoptosis. Finally, we found a negative correlation between AKT phosphorylation and O-GlcNAcylation in ischemic brain tissue. These results indicate that cerebral ischemia induces a rapid increase of O-GlcNAcylation that promotes apoptosis through down-regulation of AKT activity. These findings provide a novel mechanism through which O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling. PMID:26412745

  6. Wnt5a promotes migration of human osteosarcoma cells by triggering a phosphatidylinositol-3 kinase/Akt signals

    PubMed Central

    2014-01-01

    Wnt5a is classified as a non-transforming Wnt family member and plays complicated roles in oncogenesis and cancer metastasis. However, Wnt5a signaling in osteosarcoma progression remains poorly defined. In this study, we found that Wnt5a stimulated the migration of human osteosarcoma cells (MG-63), with the maximal effect at 100 ng/ml, via enhancing phosphorylation of phosphatidylinositol-3 kinase (PI3K)/Akt. PI3K and Akt showed visible signs of basal phosphorylation and elevated phosphorylation at 15 min after stimulation with Wnt5a. Pharmaceutical inhibition of PI3K with LY294002 significantly blocked the Wnt5a-induced activation of Akt (p-Ser473) and decreased Wnt5a-induced cell migration. Akt siRNA remarkably inhibited Wnt5a-induced cell migration. Additionally, Wnt5a does not alter the total expression and phosphorylation of β-catenin in MG-63 cells. Taken together, we demonstrated for the first time that Wnt5a promoted osteosarcoma cell migration via the PI3K/Akt signaling pathway. These findings could provide a rationale for designing new therapy targeting osteosarcoma metastasis. PMID:24524196

  7. Marine lipopeptide Iturin A inhibits Akt mediated GSK3β and FoxO3a signaling and triggers apoptosis in breast cancer.

    PubMed

    Dey, Goutam; Bharti, Rashmi; Dhanarajan, Gunaseelan; Das, Subhasis; Dey, Kaushik Kumar; Kumar, B N Prashanth; Sen, Ramkrishna; Mandal, Mahitosh

    2015-01-01

    Akt kinase is a critical component of the PI3K/Akt signaling pathway, which is frequently over expressed in human cancers including breast. Therapeutic regimens for inhibiting breast cancer with aberrant Akt activity are essential. Here, we evaluated antitumor effect of a marine bacteria derived lipopeptide 'Iturin A' on human breast cancer in vitro and in vivo through disrupting Akt pathway. Proliferation of MDA-MB-231 and MCF-7 breast cancer cells were significantly inhibited by Iturin A and it induced apoptosis as confirmed by increased Sub G1 populations, DNA fragmentation, morphological changes and western blot analysis. Furthermore, Iturin A inhibited EGF induced Akt phosphorylation (Ser473 and Thr308) and its downstream targets GSK3β and FoxO3a. Iturin A inactivated MAPK as well as Akt kinase leading to the translocation of FoxO3a to the nucleus. Gene silencing of Akt in MDA-MB-231 and MCF-7 cells reduced the sensitivity of cancer cells to Iturin A. Interestingly, overexpression of Akt with Akt plasmid in cancer cells caused highly susceptible to induce apoptosis by Iturin A treatment. In a xenograft model, Iturin A inhibited tumor growth with reduced expressions of Ki-67, CD-31, P-Akt, P-GSK3β, P-FoxO3a and P-MAPK. Collectively, these findings imply that Iturin A has potential anticancer effect on breast cancer. PMID:25974307

  8. Marine lipopeptide Iturin A inhibits Akt mediated GSK3β and FoxO3a signaling and triggers apoptosis in breast cancer

    PubMed Central

    Dey, Goutam; Bharti, Rashmi; Dhanarajan, Gunaseelan; Das, Subhasis; Dey, Kaushik Kumar; Kumar, B N Prashanth; Sen, Ramkrishna; Mandal, Mahitosh

    2015-01-01

    Akt kinase is a critical component of the PI3K/Akt signaling pathway, which is frequently over expressed in human cancers including breast. Therapeutic regimens for inhibiting breast cancer with aberrant Akt activity are essential. Here, we evaluated antitumor effect of a marine bacteria derived lipopeptide ‘Iturin A’ on human breast cancer in vitro and in vivo through disrupting Akt pathway. Proliferation of MDA-MB-231 and MCF-7 breast cancer cells were significantly inhibited by Iturin A and it induced apoptosis as confirmed by increased Sub G1 populations, DNA fragmentation, morphological changes and western blot analysis. Furthermore, Iturin A inhibited EGF induced Akt phosphorylation (Ser473 and Thr308) and its downstream targets GSK3β and FoxO3a. Iturin A inactivated MAPK as well as Akt kinase leading to the translocation of FoxO3a to the nucleus. Gene silencing of Akt in MDA-MB-231 and MCF-7 cells reduced the sensitivity of cancer cells to Iturin A. Interestingly, overexpression of Akt with Akt plasmid in cancer cells caused highly susceptible to induce apoptosis by Iturin A treatment. In a xenograft model, Iturin A inhibited tumor growth with reduced expressions of Ki-67, CD-31, P-Akt, P-GSK3β, P-FoxO3a and P-MAPK. Collectively, these findings imply that Iturin A has potential anticancer effect on breast cancer. PMID:25974307

  9. Inhibition of protein kinase CK2 by CX-5011 counteracts imatinib-resistance preventing rpS6 phosphorylation in chronic myeloid leukaemia cells: new combined therapeutic strategies

    PubMed Central

    Salizzato, Valentina; Borgo, Christian; Cesaro, Luca; Pinna, Lorenzo A.; Donella-Deana, Arianna

    2016-01-01

    Chronic myeloid leukaemia (CML) is a myeloproliferative disorder promoted by the constitutive tyrosine kinase activity of Bcr-Abl oncoprotein. Although treatment with the Bcr-Abl-inhibitor imatinib represents the first-line therapy against CML, almost 20-30% of patients develop chemotherapeutic resistance and require alternative therapy. Here we show that a strong hyper-phosphorylation/activation of ERK1/2, Akt Ser473, and 40S ribosomal protein S6 (rpS6) is detectable in imatinib-resistant KCL22 and K562 CML cells as compared to the -sensitive cell variants. In imatinib-resistant CML cells, high concentration of imatinib is required to strongly inhibit Bcr-Abl, ERK1/2 and Akt Ser473 phosphorylation, but under these conditions the phosphorylation of rpS6, a common downstream effector of MEK/ERK1/2 and PI3K/Akt/mTOR pathways is only slightly reduced. By contrast, down-regulation of the protein kinase CK2 by the inhibitor CX-5011 or by silencing the CK2 subunits does not affect the activation state of MEK/ERK1/2 or PI3K/Akt/mTOR signalling, but causes a drop in rpS6 phosphorylation in parallel with reduced protein synthesis. CK2-inhibition by CX-5011 induces cell death by apoptosis and acts synergistically with imatinib or the MEK-inhibitor U0126 in reducing the viability of imatinib-resistant CML cells. The ternary mixture containing CX-5011, imatinib and U0126 represents the most effective synergistic combination to counteract CML cell viability. These results disclose a novel CK2-mediated mechanism of acquired imatinib-resistance resulting in hyper-phosphorylation of rpS6. We suggest that co-targeting CK2 and MEK protein kinases is a promising strategy to restore responsiveness of resistant CML cells to imatinib. PMID:26919095

  10. Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of purine synthesis.

    PubMed

    Saha, Arindam; Connelly, Stephen; Jiang, Jingjing; Zhuang, Shunhui; Amador, Deron T; Phan, Tony; Pilz, Renate B; Boss, Gerry R

    2014-07-17

    The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mammalian target of rapamycin 2 and IκB kinase regulate Akt activity and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the nonoxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the nonoxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for 2 days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis. PMID:24981175

  11. Deoxycholyltaurine rescues human colon cancer cells from apoptosis by activating EGFR-dependent PI3K/Akt signaling.

    PubMed

    Raufman, Jean-Pierre; Shant, Jasleen; Guo, Chang Yue; Roy, Sanjit; Cheng, Kunrong

    2008-05-01

    Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF-alpha-stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid-induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT-29 and H508 human colon cancer cells. DCT caused dose- and time-dependent Akt (Ser(473)) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT-induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK-3-paramyosin substrate. Transfection of HT-29 cells with kinase-dead EGFR (K721M) reduced DCT-induced Akt phosphorylation. In HT-29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT-induced cell proliferation. DCT-induced PI3K/Akt activation resulted in downstream phosphorylation of GSK-3 (Ser(21/9)) and BAD (Ser(136)), and nuclear translocation (activation) of NF-kappaB, thereby confirming that DCT-induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT-induced activation of post-EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation. PMID:18064605

  12. Smad3 Sensitizes Hepatocelluar Carcinoma Cells to Cisplatin by Repressing Phosphorylation of AKT

    PubMed Central

    Zhou, Hong-Hao; Chen, Lin; Liang, Hui-Fang; Li, Guang-Zhen; Zhang, Bi-Xiang; Chen, Xiao-Ping

    2016-01-01

    Background: Heptocelluar carcinoma (HCC) is insensitive to chemotherapy due to limited bioavailability and acquired drug resistance. Smad3 plays dual roles by inhibiting cell growth initially and promoting the progression of advanced tumors in HCC. However, the role of smad3 in chemosensitivity of HCC remains elusive. Methods: The role of smad3 in chemosensitivity of HCC was measured by cell viability, apoptosis, plate colony formation assays and xenograft tumor models. Non-smad signaling was detected by Western blotting to search for the underlying mechanisms. Results: Smad3 enhanced the chemosensitivity of HCC cells to cisplatin. Smad3 upregulated p21Waf1/Cip1 and downregulated c-myc and bcl2 with the treatment of cisplatin. Moreover, overexpression of smad3 repressed the phosphorylation of AKT, and vice versa. Inhibition of PI3K/AKT pathway by LY294002 restored chemosensitivity of smad3-deficiency cells to cisplatin in HCC. Conclusion: Smad3 sensitizes HCC cells to the effects of cisplatin by repressing phosphorylation of AKT and combination of inhibitor of AKT pathway and conventional chemotherapy may be a potential way to solve drug resistance in HCC. PMID:27110775

  13. Akt-mediated Phosphorylation of XLF Impairs Non-homologous End Joining DNA Repair

    PubMed Central

    Liu, Pengda; Gan, Wenjian; Guo, Chunguang; Xie, Anyong; Gao, Daming; Guo, Jianping; Zhang, Jinfang; Willis, Nicholas; Su, Arthur; Asara, John M.; Scully, Ralph; Wei, Wenyi

    2015-01-01

    SUMMARY Deficiency in repair of damaged DNA leads to genomic instability and is closely associated with tumorigenesis. Most DNA double-strand-breaks (DSBs) are repaired by two major mechanisms, homologous-recombination (HR) and non-homologous-end-joining (NHEJ). Although Akt has been reported to suppress HR, its role in NHEJ remains elusive. Here, we report that Akt phosphorylates XLF at Thr181 to trigger its dissociation from the DNA ligase IV/XRCC4 complex, and promotes its interaction with 14-3-3β leading to XLF cytoplasmic retention, where cytosolic XLF is subsequently degraded by SCFβ-TRCP in a CKI-dependent manner. Physiologically, upon DNA damage, XLF-T181E expressing cells display impaired NHEJ and elevated cell death. Whereas a cancer-patient-derived XLF-R178Q mutant, deficient in XLF-T181 phosphorylation, exhibits an elevated tolerance of DNA damage. Together, our results reveal a pivotal role for Akt in suppressing NHEJ and highlight the tight connection between aberrant Akt hyper-activation and deficiency in timely DSB repair, leading to genomic instability and tumorigenesis. PMID:25661488

  14. CD133 promotes gallbladder carcinoma cell migration through activating Akt phosphorylation

    PubMed Central

    Zhen, Jiaojiao; Ai, Zhilong

    2016-01-01

    Gallbladder carcinoma (GBC) is the fifth most common malignancy of gastrointestinal tract. The prognosis of gallbladder carcinoma is extremely terrible partially due to metastasis. However, the mechanisms underlying gallbladder carcinoma metastasis remain largely unknown. CD133 is a widely used cancer stem cell marker including in gallbladder carcinoma. Here, we found that CD133 was highly expressed in gallbladder carcinoma as compared to normal tissues. CD133 was located in the invasive areas in gallbladder carcinoma. Down-regulation expression of CD133 inhibited migration and invasion of gallbladder carcinoma cell without obviously reducing cell proliferation. Mechanism analysis revealed that down-regulation expression of CD133 inhibited Akt phosphorylation and increased PTEN protein level. The inhibitory effect of CD133 down-regulation on gallbladder carcinoma cell migration could be rescued by Akt activation. Consistent with this, addition of Akt inhibitor Wortmannin markedly inhibited the migration ability of CD133-overexpressing cells. Thus, down-regulation of CD133 inhibits migration of gallbladder carcinoma cells through reducing Akt phosphorylation. These findings explore the fundamental biological aspect of CD133 in gallbladder carcinoma progression, providing insights into gallbladder carcinoma cell migration. PMID:26910892

  15. ERK and AKT phosphorylation status in lung cancer and emphysema using nanocapillary isoelectric focusing

    PubMed Central

    Crosbie, Philip A J; Crosbie, Emma J; Aspinall-O'Dea, Mark; Walker, Michael; Harrison, Rebecca; Pernemalm, Maria; Shah, Rajesh; Joseph, Leena; Booton, Richard; Pierce, Andrew; Whetton, Anthony D

    2016-01-01

    Background Emphysema is an independent risk factor for the development of lung cancer in smokers. Activation of oncogenic signalling proteins AKT and ERK by phosphorylation has an established role in the development of lung cancer and has also been implicated in the pathogenesis of emphysema. The aim of this study was to compare the protein level and phosphorylation status of AKT and ERK in paired lung cancer and emphysema tissue using a highly sensitive phosphoprotein analysis approach. Methods An antibody-based, nanocapillary isoelectric focusing (cIEF) assay was used to determine the relative quantities and phosphorylation status of AKT and ERK in tumour and matched lung tissue from patients, with or without evidence of emphysema, undergoing curative resection for non-small cell lung cancer. Results 20 patients with adenocarcinoma (n=9) or squamous cell carcinoma (n=11) of the lung were included (mean age 67.3 years (SD 7.5, range 47–80 years)), 12 were men and all were current (n=10) or former smokers (n=10). Paired macroscopically normal lung tissue was either histologically normal (n=7) or showed emphysema (n=13). Total and phosphorylated AKT levels were fourfold (p=0.0001) and fivefold (p=0.001) higher in tumour compared with matched lung, respectively. There was no correlation with tumour histology, stage or differentiation; however, total AKT signal in tumour was significantly correlated with fluorodeoxyglucose avidity on positron emission tomography-CT scan (r=0.53, p=0.035). Total ERK was not differentially expressed, but doubly phosphorylated (activated) ERK was threefold higher in emphysema (23.5%, SD 9.2) than either matched tumour (8.8%, SD 8.6) or normal lung tissue (8.3%, SD 9.0) and correlated with the histological severity of emphysema (p=0.005). Conclusions cIEF offers opportunities for quantifying subtle shifts in the phosphorylation status of oncoproteins in nanogram amounts of lung tissue. ERK activation is a feature of emphysema. PMID

  16. Novel RNA chaperone domain of RNA-binding protein La is regulated by AKT phosphorylation

    PubMed Central

    Kuehnert, Julia; Sommer, Gunhild; Zierk, Avery W.; Fedarovich, Alena; Brock, Alexander; Fedarovich, Dzmitry; Heise, Tilman

    2015-01-01

    The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La's RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation. PMID:25520193

  17. Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation

    PubMed Central

    Chen, Long; Li, Zhiguo; Ahmad, Nihal; Liu, Xiaoqi

    2016-01-01

    Insulin receptor substrate (IRS) proteins play important roles by acting as a platform in transducing signals from transmembrane receptors upon growth factor stimulation. Although tyrosine phosphorylation on IRS proteins plays critical roles in signal transduction, phosphorylation of IRS proteins on serine/threonine residues are believed to play various regulatory roles on IRS protein function. However, studies on serine/threonine phosphorylation of IRS proteins are very limited, especially for insulin receptor substrate 2 (IRS2), one member of the IRS protein family. In this study, we identify Polo-like kinase 1 (Plk1) as the responsible kinase for phosphorylation of IRS2 on two serine residues, Ser 556 and Ser 1098. Phosphorylation of IRS2 on these two serine residues by Plk1 prevents the activation of the PI3K pathway upon growth factor stimulation by inhibiting the binding between IRS2 and the PI3K pathway components and increasing IRS2 protein degradation. Of significance, we show that IRS2 phosphorylation is cell cycle regulated and that Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation. PMID:25830382

  18. Regulation of Akt during torpor in the hibernating ground squirrel, Ictidomys tridecemlineatus

    PubMed Central

    McMullen, David C.

    2010-01-01

    The 13-lined ground squirrel (Ictidomys tridecemlineatus) is capable of entering into extended periods of torpor during winter hibernation. The state of torpor represents a hypometabolic shift wherein the rate of oxygen consuming processes are strongly repressed in an effort to maintain cellular homeostasis as the availability of food energy becomes limited. We are interested in studying hibernation/torpor because of the robust state of tolerance to constrained oxygen delivery, oligemia, and hypothermia achieved by the tissues of hibernating mammals. The role of the serine/threonine kinase Akt (also known as PKB) has been examined in torpor in previous studies. However, this is the first study that examines the level of Akt phosphorylation in the liver during the two transition phases of the hibernation cycle: entrance into torpor, and the subsequent arousal from torpor. Our results indicate that Akt is activated in the squirrel liver by phosphorylation of two key residues (Thr308 and Ser473) during entrance into torpor and arousal from torpor. Moreover, we observed increased phosphorylation of key substrates of Akt during the two transition stages of torpor. Finally, this study reports the novel finding that PRAS40, a component of the TORC1 multi-protein complex and a potentially important modulator of metabolism, is regulated during torpor. PMID:20352231

  19. Rictor Phosphorylation on the THR-1135 Site Does Not Require Mammalian Target of Rapamycin Complex 2

    PubMed Central

    Boulbes, Delphine; Chen, Chien-Hung; Shaikenov, Tattym; Agarwal, Nitin K.; Peterson, Timothy R.; Addona, Terri A.; Keshishian, Hasmik; Carr, Steven A.; Magnuson, Mark A.; Sabatini, David M.; Sarbassov, Dos D.

    2010-01-01

    In animal cells growth factors coordinate cell proliferation and survival by regulating the PI3K/Akt signaling pathway. Deregulation of this signaling pathway is common in a variety of human cancers. The PI3K dependent signaling kinase complex defined as mTORC2 functions as a regulatory Ser-473 kinase of Akt. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component rictor on Thr-1135. The phosphorylation of this site is induced by the growth factor stimulation and expression of the oncogenic forms of ras or PI3K. Rictor phosphorylation is sensitive to inhibition of PI3K, mTOR, or expression of ILK. The substitution of wild-type rictor with its specific phospho-mutants in rictor null mouse embryonic fibroblasts did not alter the growth factor-dependent phosphorylation of Akt indicating that the rictor Thr-1135 phosphorylation is not critical in regulation of the mTORC2 kinase activity. We found that this rictor phosphorylation takes place in the mTORC2-deficient cells suggesting that this modification might play a role in regulation not only mTORC2 but also the mTORC2-independent function of rictor. PMID:20501647

  20. Pyrrolidinium fullerene induces apoptosis by activation of procaspase-9 via suppression of Akt in primary effusion lymphoma

    SciTech Connect

    Watanabe, Tadashi; Nakamura, Shigeo; Ono, Toshiya; Ui, Sadaharu; Yagi, Syota; Kagawa, Hiroki; Watanabe, Hisami; Ohe, Tomoyuki; Mashino, Tadahiko; Fujimuro, Masahiro

    2014-08-15

    Highlights: • Seven fullerenes were evaluated in terms of their cytotoxic effects on B-lymphomas. • Pyrrolidinium fullerene induced apoptosis of KSHV-infected B-lymphoma PEL cells. • The activation of Akt is essential for PEL cell survival. • Pyrrolidinium fullerene activated caspase-9 by inactivating Akt in PEL cells. • Pyrrolidinium fullerene have potential as novel drugs for the treatment of PEL. - Abstract: Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin’s B-cell lymphoma and is an aggressive neoplasm caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. In general, PEL cells are derived from post-germinal center B-cells and are infected with KSHV. To evaluate potential novel anti-tumor compounds against KSHV-associated PEL, seven water-soluble fullerene derivatives were evaluated as potential drug candidates for the treatment of PEL. Herein, we discovered a pyrrolidinium fullerene derivative, 1,1,1′,1′-tetramethyl [60]fullerenodipyrrolidinium diiodide, which induced apoptosis of PEL cells via a novel mechanism, the caspase-9 activation by suppressing the caspase-9 phosphorylation, causing caspase-9 inactivation. Pyrrolidinium fullerene treatment reduced significantly the viability of PEL cells compared with KSHV-uninfected lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9 via procaspase-9 cleavage. Pyrrolidinium fullerene additionally reduced the Ser473 phosphorylation of Akt and Ser196 of procaspase-9. Ser473-phosphorylated Akt (i.e., activated Akt) phosphorylates Ser196 in procaspase-9, causing inactivation of procaspase-9. We also demonstrated that Akt inhibitors suppressed the proliferation of PEL cells compared with KSHV-uninfected cells. Our data therefore suggest that Akt activation is essential for cell survival in PEL and a pyrrolidinium fullerene derivative induced apoptosis by activating caspase-9 via suppression of Akt in PEL cells. In addition, we evaluated

  1. Akt is activated in chronic lymphocytic leukemia cells and delivers a pro-survival signal: the therapeutic potential of Akt inhibition

    PubMed Central

    Zhuang, Jianguo; Hawkins, Stephen F.; Glenn, Mark A.; Lin, Ke; Johnson, Gillian G.; Carter, Anthony; Cawley, John C.; Pettitt, Andrew R.

    2010-01-01

    Background The aims of the present study were to ascertain the activation status of Akt in the primary cells of chronic lymphocytic leukemia and to investigate the effects of specific Akt inhibition on chronic lymphocytic leukemia-cell survival. Design and Methods Anti-phospho-Akt (Ser473 or Thr308) antibodies and western blotting were used to establish the activation status of Akt. The effects of two different, specific small-molecule inhibitors (A-443654 or Akti-1/2) or small interfering RNA on cell survival and downstream targets of Akt were assessed. Apoptosis was determined by fluorescence-activated cell sorting analysis of phosphatidylserine exposure and by measurement of PARP cleavage. The phosphorylation status of GSK-3 and MDM2, two immediate downstream substrates of Akt, levels of the anti-apoptotic proteins BCL2 and MCL1, and expression of p53 and p21 were all measured by western blotting. Results Fully activated Akt was demonstrable in all chronic lymphocytic leukemia clones examined (n=26). These results were validated with extensive controls and it was shown that a harsh method of cell extraction is needed for detection of the active enzyme. Specific inhibition of Akt induced extensive apoptosis of chronic lymphocytic leukemia cells, which was associated with both a rapid loss of MCL1 through proteasomal degradation and increased expression of p53. Moreover, the Akt inhibitors, at concentrations that induced extensive apoptosis in chronic lymphocytic leukemia cells, had little or no effect on normal peripheral blood mononuclear cells. Conclusions Chronic lymphocytic leukemia clones consistently contain activated Akt which plays a pivotal role in maintaining cell survival. Inhibition of the Akt pathway may be of potential value as a novel therapeutic strategy in chronic lymphocytic leukemia. PMID:19713228

  2. Protein kinase Cδ regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

    PubMed Central

    Sud, Neetu; Wedgwood, Stephen; Black, Stephen M.

    2008-01-01

    In this study, we explore the roles of the delta isoform of PKC (PKCδ) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKCδ with either rottlerin or with the peptide, δV1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKCδ inhibition using either rottlerin or the overexpression of a dominant negative PKCδ mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKCδ inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKCδ is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKCδ-mediated Akt activation and NO generation in maintaining eNOS expression. PMID:18192589

  3. EGFR phosphorylation of DCBLD2 recruits TRAF6 and stimulates AKT-promoted tumorigenesis

    PubMed Central

    Feng, Haizhong; Lopez, Giselle Y.; Kim, Chung Kwon; Alvarez, Angel; Duncan, Christopher G.; Nishikawa, Ryo; Nagane, Motoo; Su, An-Jey A.; Auron, Philip E.; Hedberg, Matthew L.; Wang, Lin; Raizer, Jeffery J.; Kessler, John A.; Parsa, Andrew T.; Gao, Wei-Qiang; Kim, Sung-Hak; Minata, Mutsuko; Nakano, Ichiro; Grandis, Jennifer R.; McLendon, Roger E.; Bigner, Darell D.; Lin, Hui-Kuan; Furnari, Frank B.; Cavenee, Webster K.; Hu, Bo; Yan, Hai; Cheng, Shi-Yuan

    2014-01-01

    Aberrant activation of EGFR in human cancers promotes tumorigenesis through stimulation of AKT signaling. Here, we determined that the discoidina neuropilin-like membrane protein DCBLD2 is upregulated in clinical specimens of glioblastomas and head and neck cancers (HNCs) and is required for EGFR-stimulated tumorigenesis. In multiple cancer cell lines, EGFR activated phosphorylation of tyrosine 750 (Y750) of DCBLD2, which is located within a recently identified binding motif for TNF receptor-associated factor 6 (TRAF6). Consequently, phosphorylation of DCBLD2 Y750 recruited TRAF6, leading to increased TRAF6 E3 ubiquitin ligase activity and subsequent activation of AKT, thereby enhancing EGFR-driven tumorigenesis. Moreover, evaluation of patient samples of gliomas and HNCs revealed an association among EGFR activation, DCBLD2 phosphorylation, and poor prognoses. Together, our findings uncover a pathway in which DCBLD2 functions as a signal relay for oncogenic EGFR signaling to promote tumorigenesis and suggest DCBLD2 and TRAF6 as potential therapeutic targets for human cancers that are associated with EGFR activation. PMID:25061874

  4. Casein kinase 2 dependent phosphorylation of neprilysin regulates receptor tyrosine kinase signaling to Akt.

    PubMed

    Siepmann, Martin; Kumar, Sathish; Mayer, Günter; Walter, Jochen

    2010-01-01

    Neprilysin (NEP) is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp) abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1) stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling. PMID:20957047

  5. mTORC2 Phosphorylation of Akt1: A Possible Mechanism for Hydrogen Sulfide-Induced Cardioprotection

    PubMed Central

    Zhou, Yue; Wang, Daying; Gao, Xiufang; Lew, Karsheng; Richards, Arthur Mark; Wang, Peipei

    2014-01-01

    Hydrogen sulfide (H2S) is known to have cardiac protective effects through Akt activation. Akt acts as a ‘central sensor’ for myocyte survival or death; its activity is regulated by multiple kinases including PI3K, mTORC2, PDK1 and phosphatases including PTEN, PP2A and PHLPPL. Based on the previous finding that PI3K inhibitor LY294002 abolishes H2S-induced Akt phosphorylation and cardioprotection, it is accepted that PI3K is the mediator of H2S-induced Akt phosphorylation. However, LY294002 inhibits both PI3K and mTOR, and PI3K only recruits Akt to the membrane where Akt is phosphorylated by Akt kinases. We undertook a series of experiments to further evaluate the role of mTORC2, PDK1, PTEN, PP2A and PHLPPL in H2S-induced Akt phosphorylation and cardioprotection, which, we believe, has not been investigated before. Hearts from adult Sprague-Dawley rats were isolated and subjected to (i) normoxia, (ii) global ischemia and (iii) ischemia/reperfusion in the presence or absence of 50 µM of H2S donor NaHS. Cardiac mechanical function and lactate dehydrogenase (LDH) release were assessed. All hearts also were Western analyzed at the end of perfusion for Akt and a panel of appropriate Akt regulators and targets. Hearts pretreated with 50 µM NaHS had improved function at the end of reperfusion (Rate pressure product; 19±4×103 vs. 10±3×103 mmHg/min, p<0.05) and reduced cell injury (LDH release 19±10 vs. 170±87 mU/ml p<0.05) compared to untreated hearts. NaHS significantly increased phospho-Akt, phospho-mTOR, phospho-Bim and Bcl-2 in reperfused hearts (P<0.05). Furthermore using H9c2 cells we demonstrate that NaHS pretreatment reduces apoptosis following hypoxia/re-oxygenation. Importantly, PP242, a specific mTOR inhibitor, abolished both cardioprotection and protein phosphorylation in isolated heart and reduced apoptotic effects in H9c2 cells. Treating hearts with NaHS only during reperfusion produced less cardioprotection through a similar mechanism. These data

  6. Tert-butylhydroquinone lowers blood pressure in AngII-induced hypertension in mice via proteasome-PTEN-Akt-eNOS pathway.

    PubMed

    Xu, Bing-Can; Long, Hui-Bao; Luo, Ke-Qin

    2016-01-01

    Tert-butylhydroquinone (tBHQ), as an antioxidant, has been widely used for many years to prevent oxidization of food products. The aim of this study was to investigate whether tBHQ activates endothelial nitric oxide synthase (eNOS) to prevent endothelial dysfunction and lower blood pressure. The role of Akt in tBHQ-induced eNOS phosphorylation was examined in human umbilical vein endothelial cells (HUVEC) or in mice. tBHQ treatment of HUVEC increased both Akt-Ser473 phosphorylation, accompanied with increased eNOS-Ser1177 phosphorylation and NO release. Mechanically, pharmacologic or genetic inhibition of Akt abolished tBHQ-enhanced NO release and eNOS phosphorylation in HUVEC. Gain-function of PTEN or inhibition of 26S proteasome abolished tBHQ-enhanced Akt phosphorylation in HUVEC. Ex vivo analysis indicated that tBHQ improved Ach-induced endothelium-dependent relaxation in LPC-treated mice aortic arteries, which were abolished by inhibition of Akt or eNOS. In animal study, administration of tBHQ significantly increased eNOS-Ser1177 phosphorylation and acetylcholine-induced vasorelaxation, and lowered AngII-induced hypertension in wildtype mice, but not in mice deficient of Akt or eNOS. In conclusion, tBHQ via proteasome-dependent degradation of PTEN increases Akt phosphorylation, resulting in upregulation of eNOS-derived NO production and consequent improvement of endothelial function in vivo. In this way, tBHQ lowers blood pressure in hypertensive mice. PMID:27435826

  7. Tert-butylhydroquinone lowers blood pressure in AngII-induced hypertension in mice via proteasome-PTEN-Akt-eNOS pathway

    PubMed Central

    Xu, Bing-Can; Long, Hui-Bao; Luo, Ke-Qin

    2016-01-01

    Tert-butylhydroquinone (tBHQ), as an antioxidant, has been widely used for many years to prevent oxidization of food products. The aim of this study was to investigate whether tBHQ activates endothelial nitric oxide synthase (eNOS) to prevent endothelial dysfunction and lower blood pressure. The role of Akt in tBHQ-induced eNOS phosphorylation was examined in human umbilical vein endothelial cells (HUVEC) or in mice. tBHQ treatment of HUVEC increased both Akt-Ser473 phosphorylation, accompanied with increased eNOS-Ser1177 phosphorylation and NO release. Mechanically, pharmacologic or genetic inhibition of Akt abolished tBHQ-enhanced NO release and eNOS phosphorylation in HUVEC. Gain-function of PTEN or inhibition of 26S proteasome abolished tBHQ-enhanced Akt phosphorylation in HUVEC. Ex vivo analysis indicated that tBHQ improved Ach-induced endothelium-dependent relaxation in LPC-treated mice aortic arteries, which were abolished by inhibition of Akt or eNOS. In animal study, administration of tBHQ significantly increased eNOS-Ser1177 phosphorylation and acetylcholine-induced vasorelaxation, and lowered AngII-induced hypertension in wildtype mice, but not in mice deficient of Akt or eNOS. In conclusion, tBHQ via proteasome-dependent degradation of PTEN increases Akt phosphorylation, resulting in upregulation of eNOS-derived NO production and consequent improvement of endothelial function in vivo. In this way, tBHQ lowers blood pressure in hypertensive mice. PMID:27435826

  8. PTEN-inhibition by zinc ions augments interleukin-2-mediated Akt phosphorylation.

    PubMed

    Plum, Laura Marie; Brieger, Anne; Engelhardt, Gabriela; Hebel, Silke; Nessel, Andreas; Arlt, Marcus; Kaltenberg, Jennifer; Schwaneberg, Ulrich; Huber, Michael; Rink, Lothar; Haase, Hajo

    2014-07-01

    Free zinc ions (Zn(2+)) participate in several signaling pathways. The aim of the present study was to investigate a potential involvement of Zn(2+) in the PI3K/Akt pathway of interleukin (IL)-2 signaling in T-cells. The IL-2 receptor triggers three major pathways, ERK1/2, JAK/STAT5, and PI3K/Akt. We have previously shown that an IL-2-mediated release of lysosomal Zn(2+) into the cytoplasm activates ERK1/2, but not STAT5. In the present study, Akt phosphorylation in response to IL-2 was abrogated by the Zn(2+) chelator N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, and was induced by treatment with Zn(2+) and the ionophore pyrithione. The latter were ineffective in cells that were treated with siRNA against the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatase that degrades the lipid second messenger PI(3,4,5)P3, which is produced by PI3K and leads to activation of Akt. Inhibition of recombinant PTEN by Zn(2+)in vitro yielded an IC50 of 0.59 nM. Considering a resting free cytoplasmic Zn(2+) level of 0.2 nM in the T-cell line CTLL-2, this seems ideally suited for dynamic regulation by cellular Zn(2+). Oxidation with H2O2 and supplementation with Zn(2+) led to similar changes in the CD spectrum of PTEN. Moreover, Zn(2+) partially prevented the oxidation of cysteines 71 and 124. Hence, we hypothesize that zinc signals affect the IL-2-dependent PI3K/Akt pathway by inhibiting the negative regulator PTEN through binding with a sub-nanomolar affinity to cysteine residues that are essential for its catalytic activity. PMID:24759986

  9. Isoflurane Is More Deleterious to Developing Brain Than Desflurane: The Role of the Akt/GSK3β Signaling Pathway

    PubMed Central

    Tao, Guorong; Xue, Qingsheng; Luo, Yan; Li, Guohui; Xia, Yimeng; Yu, Buwei

    2016-01-01

    Demand is increasing for safer inhalational anesthetics for use in pediatric anesthesia. In this regard, researchers have debated whether isoflurane is more toxic to the developing brain than desflurane. In the present study, we compared the effects of postnatal exposure to isoflurane with those of desflurane on long-term cognitive performance and investigated the role of the Akt/GSK3β signaling pathway. Postnatal day 6 (P6) mice were exposed to either isoflurane or desflurane, after which the phosphorylation levels of Akt/GSK3β and learning and memory were assessed at P8 or P31. The phosphorylation levels of Akt/GSK3β and learning and memory were examined after intervention with lithium. We found that isoflurane, but not desflurane, impaired spatial learning and memory at P31. Accompanied by behavioral change, only isoflurane decreased p-Akt (ser473) and p-GSK3β (ser9) expressions, which led to GSK3β overactivation. Lithium prevented GSK3β overactivation and alleviated isoflurane-induced cognitive deficits. These results suggest that isoflurane is more likely to induce developmental neurotoxicity than desflurane in context of multiple exposures and that the Akt/GSK3β signaling pathway partly participates in this process. GSK3β inhibition might be an effective way to protect against developmental neurotoxicity. PMID:27057548

  10. AKT1 Inhibits Epithelial-to-Mesenchymal Transition in Breast Cancer through Phosphorylation-Dependent Twist1 Degradation.

    PubMed

    Li, Chia-Wei; Xia, Weiya; Lim, Seung-Oe; Hsu, Jennifer L; Huo, Longfei; Wu, Yun; Li, Long-Yuan; Lai, Chien-Chen; Chang, Shih-Shin; Hsu, Yi-Hsin; Sun, Hui-Lung; Kim, Jongchan; Yamaguchi, Hirohito; Lee, Dung-Fang; Wang, Hongmei; Wang, Yan; Chou, Chao-Kai; Hsu, Jung-Mao; Lai, Yun-Ju; LaBaff, Adam M; Ding, Qingqing; Ko, How-Wen; Tsai, Fuu-Jen; Tsai, Chang-Hai; Hortobagyi, Gabriel N; Hung, Mien-Chie

    2016-03-15

    Epithelial-to-mesenchymal transition (EMT) is an essential physiologic process that promotes cancer cell migration, invasion, and metastasis. Several lines of evidence from both cellular and genetic studies suggest that AKT1/PKBα, but not AKT2 or AKT3, serves as a negative regulator of EMT and breast cancer metastasis. However, the underlying mechanism by which AKT1 suppresses EMT remains poorly defined. Here, we demonstrate that phosphorylation of Twist1 by AKT1 is required for β-TrCP-mediated Twist1 ubiquitination and degradation. The clinically used AKT inhibitor MK-2206, which possesses higher specificity toward AKT1, stabilized Twist1 and enhanced EMT in breast cancer cells. However, we discovered that resveratrol, a naturally occurring compound, induced β-TrCP-mediated Twist1 degradation to attenuate MK-2206-induced EMT in breast cancer cells. Taken together, our findings demonstrate that resveratrol counteracts the unexpected metastatic potential induced by anti-AKT therapy and therefore suggest that the addition of resveratrol to an anti-AKT therapeutic regimen may provide extra support for limiting EMT. PMID:26759241

  11. A novel AKT inhibitor, AZD5363, inhibits phosphorylation of AKT downstream molecules, and activates phosphorylation of mTOR and SMG-1 dependent on the liver cancer cell type

    PubMed Central

    ZHANG, YUNCHENG; ZHENG, YUANWEN; FAHEEM, ALI; SUN, TIANTONG; LI, CHUNYOU; LI, ZHE; ZHAO, DIANTANG; WU, CHAO; LIU, JUN

    2016-01-01

    Due to frequent phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway dysregulation, AKT is typically accepted as a promising anticancer therapeutic target. mTOR, in particular, represents a suitable therapeutic target for hepatocellular carcinoma, whilst suppressor with morphogenetic effect on genitalia family member-1 (SMG-1) is believed to serve a potential tumor suppressor role in human cancer. Despite SMG-1 and mTOR belonging to the same PI3K-related kinase family, the interactions between them are not yet fully understood. In the present study, a novel pyrrolopyrimidine-derived compound, AZD5363, was observed to suppress proliferation in liver cancer Hep-G2 and Huh-7 cells by inhibiting the phosphorylation of downstream molecules in the AKT signal pathway, in a dose- and time-dependent manner. AZD5363 activated the phosphorylation of mTOR, dependent on the liver cancer cell type, as it may have differing effects in various liver cancer cell lines. Additionally, AZD5363 also activated SMG-1 within the same liver cancer cells types, which subsequently activated the phosphorylation of mTOR. In conclusion, the present study indicates that AZD5363 inhibited phosphorylation of AKT downstream molecules, and activated phosphorylation of mTOR and SMG-1, dependent on the liver cancer type. PMID:26998062

  12. Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

    SciTech Connect

    Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra; Galadari, Sehamuddin

    2010-04-09

    Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

  13. KIF14 promotes AKT phosphorylation and contributes to chemoresistance in triple-negative breast cancer.

    PubMed

    Singel, Stina M; Cornelius, Crystal; Zaganjor, Elma; Batten, Kimberly; Sarode, Venetia R; Buckley, Dennis L; Peng, Yan; John, George B; Li, Hsiao C; Sadeghi, Navid; Wright, Woodring E; Lum, Lawrence; Corson, Timothy W; Shay, Jerry W

    2014-03-01

    Despite evidence that kinesin family member 14 (KIF14) can serve as a prognostic biomarker in various solid tumors, how it contributes to tumorigenesis remains unclear. We observed that experimental decrease in KIF14 expression increases docetaxel chemosensitivity in estrogen receptor-negative/progesterone receptor-negative/human epidermal growth factor receptor 2-negative, "triple-negative" breast cancers (TNBC). To investigate the oncogenic role of KIF14, we used noncancerous human mammary epithelial cells and ectopically expressed KIF14 and found increased proliferative capacity, increased anchorage-independent grown in vitro, and increased resistance to docetaxel but not to doxorubicin, carboplatin, or gemcitabine. Seventeen benign breast biopsies of BRCA1 or BRCA2 mutation carriers showed increased KIF14 mRNA expression by fluorescence in situ hybridization compared to controls with no known mutations in BRCA1 or BRCA2, suggesting increased KIF14 expression as a biomarker of high-risk breast tissue. Evaluation of 34 cases of locally advanced TNBC showed that KIF14 expression significantly correlates with chemotherapy-resistant breast cancer. KIF14 knockdown also correlates with decreased AKT phosphorylation and activity. Live-cell imaging confirmed an insulin-induced temporal colocalization of KIF14 and AKT at the plasma membrane, suggesting a potential role of KIF14 in promoting activation of AKT. An experimental small-molecule inhibitor of KIF14 was then used to evaluate the potential anticancer benefits of downregulating KIF14 activity. Inhibition of KIF14 shows a chemosensitizing effect and correlates with decreasing activation of AKT. Together, these findings show an early and critical role for KIF14 in the tumorigenic potential of TNBC, and therapeutic targeting of KIF14 is feasible and effective for TNBC. PMID:24784001

  14. O-GlcNAcylation enhances the invasion of thyroid anaplastic cancer cells partially by PI3K/Akt1 pathway

    PubMed Central

    Zhang, Peng; Wang, Chunli; Ma, Tao; You, Shengyi

    2015-01-01

    Background The PI3K family participates in multiple signaling pathways to regulate cellular functions. PI3K/Akt signaling pathway plays an important role in tumorigenesis and development. O-GlcNAcylation, a posttranslational modification, is thought to modulate a wide range of biological processes, such as transcription, cell growth, signal transduction, and cell motility. O-GlcNAcylation is catalyzed by the nucleocytoplasmic enzymes, OGT and OGA, which adds or removes O-GlcNAc moieties, respectively. Abnormal O-GlcNAcylation has been implicated in a variety of human diseases. However, the role of O-GlcNAcylation in tumorigenesis and progression of cancer is still under-investigated. Understanding the O-GlcNAc-associated molecular mechanism might be significant for diagnosis and therapy of cancer. Methods Human thyroid anaplastic cancer 8305C cells were used to evaluate the role of O-GlcNAcylation in tumorigenesis and progression of cancer. The global O-GlcNAc level of intracellular proteins was up-regulated by OGA inhibitor Thiamet-G treatment or OGT over-expression. Cell proliferation was assessed by MTT assay. Invasion in vitro was determined by Transwell assay, and phosphorylation of Akt1 at Ser473 was assessed by Western blot for activity of Akt1. PI3K-specific inhibitor LY294002 and RNA interference of Akt1 were used to investigate the impact of PI3K/Akt signaling on the regulation of O-GlcNAcylation during tumor progression. Results Cell models with remarkably up-regulated O-GlcNAcylation were constructed, and then cell proliferation and invasion were determined. The results indicated that the proliferation was not affected by OGA inhibition or OGT overexpression, while the invasion of 8305C cells with OGA inhibition or OGT overexpression was obviously increased. Akt1 activity was stimulated by elevated O-GlcNAcylation by mediating phosphorylation at Ser473. The enhanced invasion of thyroid cancer cells by Thiamet-G treatment or OGT overexpression was

  15. Essential role of PH domain and leucine-rich repeat protein phosphatase 2 in Nrf2 suppression via modulation of Akt/GSK3β/Fyn kinase axis during oxidative hepatocellular toxicity.

    PubMed

    Rizvi, F; Shukla, S; Kakkar, P

    2014-01-01

    Instances of sustained oxidative activity have been shown to involve dysregulation of Nrf2-mediated transcriptional induction; however, mechanisms warranting Nrf2-repression remain unclear. In this study, using primary rat hepatocytes, we have attempted to identify factors that may negatively influence Nrf2 survival pathway. Though studies indicate a conspicuous association between Akt and Nrf2, a confirmatory link between the two is unaddressed. On inhibiting PI3K/Akt pathway, we observed compromised activities of antioxidant and detoxification enzymes culminating in oxidative cytotoxicity. This was accompanied by reduced nuclear retention of Nrf2 and its ARE binding affinity, increased Nrf2 ubiquitination and concurrent decline in its downstream targets. Moreover, Akt inhibition enhanced nuclear translocation as well as phosphorylation of Fyn kinase, an enzyme linked to Nrf2 degradation, by relieving GSK3β from phosphorylation-mediated repression. The involvement of Akt and Fyn kinase in influencing Nrf2 signaling was further confirmed in oxidatively stressed hepatocytes by using tert-butyl hydroperoxide (tBHP). tBHP-induced decrease in Nrf2 levels was associated with enhanced Fyn kinase phosphorylation, Fyn kinase nuclear translocation and decreased levels of phosphorylated GSK3β(Ser9) in a time-dependent manner. Interestingly, tBHP induced site-specific deactivation of Akt as only Akt(Ser473) phosphorylation was observed to be affected. Further, protein expression as well as nuclear localization of PHLPP2, a phosphatase specific for Akt(Ser473), was found to be significantly enhanced in tBHP-stressed hepatocytes. Silencing of PHLPP2 not only resulted in considerable restoration of Nrf2 signaling, enhanced Nrf2-ARE binding and reduced Nrf2 ubiquitination but also significantly suppressed tBHP-induced ROS generation and alterations in mitochondrial permeability. We infer that cellular PHLPP2 levels may aggravate oxidative toxicity by suppressing Nrf2/ARE

  16. Akt/Protein Kinase B-Dependent Phosphorylation and Inactivation of WEE1Hu Promote Cell Cycle Progression at G2/M Transition

    PubMed Central

    Katayama, Kazuhiro; Fujita, Naoya; Tsuruo, Takashi

    2005-01-01

    The serine/threonine kinase Akt is known to promote cell growth by regulating the cell cycle in G1 phase through activation of cyclin/Cdk kinases and inactivation of Cdk inhibitors. However, how the G2/M phase is regulated by Akt remains unclear. Here, we show that Akt counteracts the function of WEE1Hu. Inactivation of Akt by chemotherapeutic drugs or the phosphatidylinositide-3-OH kinase inhibitor LY294002 induced G2/M arrest together with the inhibitory phosphorylation of Cdc2. Because the increased Cdc2 phosphorylation was completely suppressed by wee1hu gene silencing, WEE1Hu was associated with G2/M arrest induced by Akt inactivation. Further analyses revealed that Akt directly bound to and phosphorylated WEE1Hu during the S to G2 phase. Serine-642 was identified as an Akt-dependent phosphorylation site. WEE1Hu kinase activity was not affected by serine-642 phosphorylation. We revealed that serine-642 phosphorylation promoted cytoplasmic localization of WEE1Hu. The nuclear-to-cytoplasmic translocation was mediated by phosphorylation-dependent WEE1Hu binding to 14-3-3θ but not 14-3-3β or -σ. These results indicate that Akt promotes G2/M cell cycle progression by inducing phosphorylation-dependent 14-3-3θ binding and cytoplasmic localization of WEE1Hu. PMID:15964826

  17. Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

    PubMed Central

    Xu, Daichao; Shan, Bing; Lee, Byung-Hoon; Zhu, Kezhou; Zhang, Tao; Sun, Huawang; Liu, Min; Shi, Linyu; Liang, Wei; Qian, Lihui; Xiao, Juan; Wang, Lili; Pan, Lifeng; Finley, Daniel; Yuan, Junying

    2015-01-01

    Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells. DOI: http://dx.doi.org/10.7554/eLife.10510.001 PMID:26523394

  18. Pistacia chinensis Methanolic Extract Attenuated MAPK and Akt Phosphorylations in ADP Stimulated Rat Platelets In Vitro.

    PubMed

    Park, Ji Young; Hong, Mei; Jia, Qi; Lee, Young-Chul; Yayeh, Taddesse; Hyun, Eujin; Kwak, Dong-Mi; Cho, Jae Youl; Rhee, Man Hee

    2012-01-01

    Pistacia chinensis (Chinese pistache) is a widely grown plant in southern China where the galls extract is a common practice in folk medicine. However, extracts from this plant have never been attempted for their cardiovascular protective effects in experimental setting. Here therefore we aimed to investigate the antiplatelet activity of Pistacia chinensis methanolic extract (PCME) in ADP stimulated rat platelets in vitro. PCME (2.5-20 μg/mL) inhibited ADP-induced platelet aggregation. While PCME diminished [Ca(2+)]i, ATP, and TXA2 release in ADP-activated platelets, it enhanced cAMP production in resting platelets. Likewise, PCME inhibited fibrinogen binding to αIIbβ3 and downregulated JNK, ERK, and Akt phosphorylations. Thus, PCME contains potential antiplatelet compounds that could be deployed for their therapeutic values in cardiovascular pathology. PMID:22899962

  19. Pistacia chinensis Methanolic Extract Attenuated MAPK and Akt Phosphorylations in ADP Stimulated Rat Platelets In Vitro

    PubMed Central

    Park, Ji Young; Hong, Mei; Jia, Qi; Lee, Young-Chul; Yayeh, Taddesse; Hyun, Eujin; Kwak, Dong-Mi; Cho, Jae Youl; Rhee, Man Hee

    2012-01-01

    Pistacia chinensis (Chinese pistache) is a widely grown plant in southern China where the galls extract is a common practice in folk medicine. However, extracts from this plant have never been attempted for their cardiovascular protective effects in experimental setting. Here therefore we aimed to investigate the antiplatelet activity of Pistacia chinensis methanolic extract (PCME) in ADP stimulated rat platelets in vitro. PCME (2.5–20 μg/mL) inhibited ADP-induced platelet aggregation. While PCME diminished [Ca2+]i, ATP, and TXA2 release in ADP-activated platelets, it enhanced cAMP production in resting platelets. Likewise, PCME inhibited fibrinogen binding to αIIbβ3 and downregulated JNK, ERK, and Akt phosphorylations. Thus, PCME contains potential antiplatelet compounds that could be deployed for their therapeutic values in cardiovascular pathology. PMID:22899962

  20. Atorvastatin attenuates cognitive deficits through Akt1/caspase-3 signaling pathway in ischemic stroke.

    PubMed

    Yang, Jie; Pan, Ying; Li, Xuejing; Wang, Xianying

    2015-12-10

    Neuronal damage in the hippocampal formation is more sensitive to ischemic stimulation and easily injured, causing severe learning and memory impairment. Therefore, protection of hippocampal neuronal damage is the main contributor for learning and memory impairment during cerebral ischemia. Atorvastatin has been reported to ameliorate ischemic brain damage after ischemia reperfusion (I/R). However, its molecular mechanism has not been elucidated clearly. In this study, we established four-vessel occlusion model in rats with cerebral ischemia. Here, we demonstrated that atorvastatin significantly improves the behavior of I/R-rat in open field tasks. We also found that atorvastatin significantly shortens the distance and time of loading onto the hidden platform in the positioning navigation process, decreases the latency in the space exploration process when cognitive testing with Morris water maze was performed during ischemic stroke in rats. Furthermore, the survival rate of neurons in the CA1 area of the hippocampus and the phosphorylation of Akt (Ser473) in the neurons are increased, whereas the expression of caspase-3 are inhibited by atorvastatin. However, after an intracerebroventricular injection of LY294002 (an inhibitor of Akt1), the above neuroprotective effects of atorvastatin are attenuated. In summary, our results imply atorvastatin may improve the survival rate of hippocampal neurons and reduce the impairment of learning and memory by downregulating the activation of the caspase-3 via increasing the phosphorylation of Akt1 during ischemia/reperfusion. PMID:26597376

  1. Endothelium-Independent Hypoxic Contraction Is Prevented Specifically by Nitroglycerin via Inhibition of Akt Kinase in Porcine Coronary Artery

    PubMed Central

    Liu, Huixia; Li, Yanjing; An, Yuanming; He, Peixin; Wu, Liling; Gao, Yuansheng; Dou, Dou

    2016-01-01

    Objective. Hypoxia-induced sustained contraction of porcine coronary artery is endothelium-independent and mediated by PI3K/Akt/Rho kinase. Nitroglycerin (NTG) is a vasodilator used to treat angina pectoris and acute heart failure. The present study was to determine the role of NTG in hypoxia-induced endothelium-independent contraction and the underlying mechanism. Methods and Results. Organ chamber technique was used to measure the isometric vessel tension of isolated porcine coronary arteries. Protein levels of phosphorylated and total Akt were determined by western blot. A sustained contraction of porcine coronary arteries induced by hypoxia was significantly reduced by NTG but not by isoproterenol. This contraction was also inhibited by DETA NONOate, 8-Br-cGMP, which can be reversed by ODQ, and Rp-8-Br-PET-cGMPS. The restored contraction was blocked by LY294002. The reduction of Akt-p at Ser-473 by NTG, DETA NONOate, and 8-Br-cGMP was significantly inhibited by ODQ, PKG-I. The decrease in Akt-p level by NTG and 8-Br-cGMP was prevented by calyculin A but not by okadaic acid. Conclusions. These results demonstrated that the endothelium-independent sustained hypoxic vasoconstriction can be prevented by NTG and that the inhibition of PI3K/Akt signaling pathway may be involved. PMID:26839558

  2. Cross regulation between cGMP-dependent protein kinase and Akt in vasodilatation of porcine pulmonary artery.

    PubMed

    Liu, Juan; Liu, Huixia; Li, Yanjing; Xu, Xiaojian; Chen, Zhengju; Liu, Limei; Yu, Xiaoxing; Gao, Yuansheng; Dou, Dou

    2014-11-01

    cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation. PMID:24977346

  3. Human recombinant H2 relaxin induces AKT and GSK3β phosphorylation and HTR-8/SVneo cell proliferation.

    PubMed

    Astuti, Yoni; Nakabayashi, Koji; Deguchi, Masashi; Ebina, Yasuhiko; Yamada, Hideto

    2015-01-01

    Relaxin is essential for trophoblast development during pregnancy. Evidence shows that relaxin increases trophoblast cell migration capacity. Here, we show the effect of relaxin on protein kinase B (AKT) activation and glycogen synthase kinase 3-beta (GSK3β) inactivation as well as on the proliferation of HTR-8/SVneo cells, a model of human extravillous trophoblast (EVT). HTR-8/SVneo cells were treated with different doses of human recombinant (rH2) relaxin in serum-deprived conditions and treated for increasing time with 1 ng/mL of rH2 relaxin. Western blot analysis was performed to detect pAKT, AKT, pGSK3β, GSK3β, and actin expression. Proliferation of HTR-8/SVneo cells was analyzed by MTS assay. rH2 relaxin treatment increased the ratio of pAKT/AKT, pGSK3β/GSK3β, and proliferation in HTR-8/SVneo cells. Furthermore, AKT and GSK3β activation by rH2 relaxin was inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor. This study suggests that rH2 relaxin induces AKT and GSK3β phosphorylation as well as proliferation in HTR-8/SVneo cells. PMID:25868609

  4. Factor XII stimulates ERK1/2 and Akt through uPAR, integrins, and the EGFR to initiate angiogenesis

    PubMed Central

    LaRusch, Gretchen A.; Mahdi, Fakhri; Shariat-Madar, Zia; Adams, Gregory; Sitrin, Robert G.; Zhang, Wan Ming; McCrae, Keith R.

    2010-01-01

    Factor XII (FXII) and high molecular weight kininogen (HK) mutually block each other's binding to the urokinase plasminogen activator receptor (uPAR). We investigated if FXII stimulates cells by interacting with uPAR. FXII (3-62nM) with 0.05mM Zn2+ induces extracellular signal-related kinase 1/2 (ERK1/2; mitogen-activated protein kinase 44 [MAPK44] andMAPK42) and Akt (Ser473) phosphorylation in endothelial cells. FXII-induced phosphorylation of ERK1/2 or Akt is a zymogen activity, not an enzymatic event. ERK1/2 or Akt phosphorylation is blocked upstream by PD98059 or Wortmannin or LY294002, respectively. An uPAR signaling region for FXII is on domain 2 adjacent to uPAR's integrin binding site. Cleaved HK or peptides from HK's domain 5 blocks FXII-induced ERK1/2 and Akt phosphorylation. A β1 integrin peptide that binds uPAR, antibody 6S6 to β1 integrin, or the epidermal growth factor receptor (EGFR) inhibitor AG1478 blocks FXII-induced phosphorylation of ERK1/2 and Akt. FXII induces endothelial cell proliferation and 5-bromo-2′deoxy-uridine incorporation. FXII stimulates aortic sprouting in normal but not uPAR-deficient mouse aorta. FXII produces angiogenesis in matrigel plugs in normal but not uPAR-deficient mice. FXII knockout mice have reduced constitutive and wound-induced blood vessel number. In sum, FXII initiates signaling mediated by uPAR, β1 integrin, and the EGFR to induce human umbilical vein endothelial cell proliferation, growth, and angiogenesis. PMID:20228268

  5. Activation of PI3-kinase stimulates endocytosis of ROMK via Akt1/SGK1-dependent phosphorylation of WNK1.

    PubMed

    Cheng, Chih-Jen; Huang, Chou-Long

    2011-03-01

    WNK kinases stimulate endocytosis of ROMK channels to regulate renal K+ handling. Phosphatidylinositol 3-kinase (PI3K)-activating hormones, such as insulin and IGF 1, phosphorylate WNK1, but how this affects the regulation of ROMK abundance is unknown. Here, serum starvation of ROMK-transfected HEK cells led to an increase of ROMK current density; subsequent addition of insulin or IGF1 inhibited ROMK currents in a PI3K-dependent manner. Serum and insulin also increased phosphorylation of the downstream kinases Akt1 and SGK1 as well as WNK1. A biotinylation assay suggested that insulin and IGF1 inhibit ROMK by enhancing its endocytosis, a process that WNK1 may mediate. Knockdown of WNK1 with siRNA or expression of a phospho-deficient WNK1 mutant (T58A) both prevented insulin-induced inhibition of ROMK currents, suggesting that phosphorylation at Threonine-58 of WNK1 is important to mediate the inhibition of ROMK by PI3K-activating hormones or growth factors. In vitro and in vivo kinase assays supported the notion that Akt1 and SGK1 can phosphorylate WNK1 at this site, and we established that Akt1 and SGK1 synergistically inhibit ROMK through WNK1. We used dominant-negative intersectin and dynamin constructs to show that SGK1-mediated phosphorylation of WNK1 inhibits ROMK by promoting its endocytosis. Taken together, these results suggest that PI3K-activating hormones inhibit ROMK by enhancing its endocytosis via a mechanism that involves phosphorylation of WNK1 by Akt1 and SGK1. PMID:21355052

  6. AB044. AGE/RAGE/Akt pathway contributes to prostate cancer cell proliferation by promoting Rb phosphorylation and degradation

    PubMed Central

    Bao, Jiming; Bao, Yawei; Zhao, Shanchao; He, Minyi; Luo, Haihua; Ren, Zhonglu; Lv, Yongjie; Hong, Yingqia

    2016-01-01

    Objective Metabolomic research has revealed that metabolites play an important role in prostate cancer development and progression. Previous studies have suggested that prostate cancer cell proliferation is induced by advanced glycation end products (AGEs) exposure, but the mechanism of this induction remains unknown. This study aim to investigate the molecular mechanisms underlying the proliferative response of prostate cancer cell to the interaction of AGEs and the receptor for advanced glycation end products (RAGE). Methods To investigate this mechanism, we used Western blotting to evaluate the responses of the retinoblastoma (Rb), p-Rb and PI3K/Akt pathway to AGEs stimulation. We also examined the effect of knocking down Rb and blocking the PI3K/Akt pathway on AGEs induced PC-3 cell proliferation. Results Our results indicated that AGE-RAGE interaction enhanced Rb phosphorylation and subsequently decreased total Rb levels. Bioinformatics analysis further indicated a negative correlation between RAGE and RB1 expression in prostate cancer tissue. Furthermore, we observed that AGEs stimulation activated the PI3K/Akt signaling pathway and that blocking PI3K/Akt signaling abrogated AGEs-induced cell proliferation. Conclusions We report, for the first time, that AGE-RAGE interaction enhances prostate cancer cell proliferation by phosphorylation of Rb via the PI3K/Akt signaling pathway.

  7. Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

    PubMed Central

    Garay, Camilo; Judge, Gurjeet; Lucarelli, Stefanie; Bautista, Stephen; Pandey, Rohan; Singh, Tanveer; Antonescu, Costin N.

    2015-01-01

    Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation. PMID:26246598

  8. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Sorescu, George; Boyd, Nolan; Shiojima, Ichiro; Walsh, Kenneth; Du, Jie; Jo, Hanjoong

    2002-01-01

    Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction

  9. BRAF, KIT and NRAS mutations and expression of c-KIT, phosphorylated extracellular signal-regulated kinase and phosphorylated AKT in Japanese melanoma patients.

    PubMed

    Oyama, Satomi; Funasaka, Yoko; Watanabe, Atsushi; Takizawa, Toshihiro; Kawana, Seiji; Saeki, Hidehisa

    2015-05-01

    To clarify the status of gene mutation and activation of growth signal in melanoma of Japanese patients in vivo, we analyzed the mutation of BRAF exon 15, NRAS exon 2, and KIT exons 9, 11, 13, 17 and 18 in melanoma cells obtained by laser capture microdissection, and performed direct sequencing in 20 cases of acral lentiginous melanoma (ALM) and 17 cases of superficial spreading melanoma (SSM). In the study of the mutation of BRAF, pyrosequencing was also done. To examine the cell proliferation signaling, immunohistochemistry for phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT (phosphorylated AKT) and c-KIT was done. The mutation of BRAF p.V600E was detected in 13 cases of ALM (65.0%) and 12 cases of SSM (70.6%). No NRAS mutation was found in all cases. The mutation in exons 9, 11, and 18 of KIT was detected in nine cases. The mutation of BRAF and KIT showed no correlation with clinical stage, lymph node metastasis, tumor thickness, ulceration and histology. pERK and pAKT was observed in small population of melanoma cells and there was no correlation with gene mutation. Our results indicate that the mutations of BRAF and KIT exist in Japanese melanoma patients, however, the cell growth signaling may be regulated by not only these mutated genes, but by other unknown regulatory factors, which may affect the prognosis of melanoma. PMID:25766129

  10. Hydrophobic motif site-phosphorylated protein kinase CβII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.

    PubMed

    Das, Falguni; Ghosh-Choudhury, Nandini; Mariappan, Meenalakshmi M; Kasinath, Balakuntalam S; Choudhury, Goutam Ghosh

    2016-04-01

    PKCβII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCβII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCβII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCβII, dominant negative PKCβII, and PKCβII hydrophobic motif phosphorylation-deficient mutant, we found that PKCβII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCβII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCβII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCβII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCβII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCβII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy. PMID:26739493

  11. Cadmium Activates Multiple Signaling Pathways That Coordinately Stimulate Akt Activity to Enhance c-Myc mRNA Stability

    PubMed Central

    Tsai, Jia-Shiuan; Chao, Cheng-Han; Lin, Lih-Yuan

    2016-01-01

    Cadmium is a known environmental carcinogen. Exposure of Cd leads to the activation of several proto-oncogenes in cells. We investigated here the mechanism of c-Myc expression in hepatic cells under Cd treatment. The c-Myc protein and mRNA levels increased in dose- and time-dependent manners in HepG2 cells with Cd treatment. This increase was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated by Cd. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. We conclude that Cd-induced accumulation of c-Myc requires the activation of several signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins. PMID:26751215

  12. PI-103 and Quercetin Attenuate PI3K-AKT Signaling Pathway in T- Cell Lymphoma Exposed to Hydrogen Peroxide

    PubMed Central

    Maurya, Akhilendra Kumar; Vinayak, Manjula

    2016-01-01

    Phosphatidylinositol 3 kinase—protein kinase B (PI3K-AKT) pathway has been considered as major drug target site due to its frequent activation in cancer. AKT regulates the activity of various targets to promote tumorigenesis and metastasis. Accumulation of reactive oxygen species (ROS) has been linked to oxidative stress and regulation of signaling pathways for metabolic adaptation of tumor microenvironment. Hydrogen peroxide (H2O2) in this context is used as ROS source for oxidative stress preconditioning. Antioxidants are commonly considered to be beneficial to reduce detrimental effects of ROS and are recommended as dietary supplements. Quercetin, a ubiquitous bioactive flavonoid is a dietary component which has attracted much of interest due to its potential health-promoting effects. Present study is aimed to analyze PI3K-AKT signaling pathway in H2O2 exposed Dalton’s lymphoma ascite (DLA) cells. Further, regulation of PI3K-AKT pathway by quercetin as well as PI-103, an inhibitor of PI3K was analyzed. Exposure of H2O2 (1mM H2O2 for 30min) to DLA cells caused ROS accumulation and resulted in increased phosphorylation of PI3K and downstream proteins PDK1 and AKT (Ser-473 and Thr-308), cell survival factors BAD and ERK1/2, as well as TNFR1. However, level of tumor suppressor PTEN was declined. Both PI-103 & quercetin suppressed the enhanced level of ROS and significantly down-regulated phosphorylation of AKT, PDK1, BAD and level of TNFR1 as well as increased the level of PTEN in H2O2 induced lymphoma cells. The overall result suggests that quercetin and PI3K inhibitor PI-103 attenuate PI3K-AKT pathway in a similar mechanism. PMID:27494022

  13. Hyaluronic acid regulates a key redox control factor Nrf2 via phosphorylation of Akt in bovine articular chondrocytes

    PubMed Central

    Onodera, Yuta; Teramura, Takeshi; Takehara, Toshiyuki; Fukuda, Kanji

    2015-01-01

    One important pharmacological function of hyaluronic acid (HA) in chondrocytes is reduction of cellular superoxide generation and accumulation. Here we demonstrated a relationship between HA supplementation and accumulation of Nuclear factor-erythroid-2-related factor 2 (Nrf2), which is a master transcription factor in cellular redox reactions, in cultured chondrocytes derived from bovine joint cartilage. In HA-treated chondrocytes, expression of Nrf2 and its downstream genes was upregulated. In HA-treated chondrocytes, Akt was phosphorylated, and inhibition of Akt activity or suppression of HA receptors CD44 and/or RHAMM with siRNAs prevented HA-mediated Nrf2 accumulation. Furthermore, Nrf2 siRNA inhibited the HA effect on antioxidant enzymes. These results show that HA might contribute to ROS reduction through Nrf2 regulation by activating Akt. Our study suggests a new mechanism for extracellular matrix (ECM)-mediated redox systems in chondrocytes. PMID:26106522

  14. Suppressing cyclooxygenase-2 prevents nonalcoholic and inhibits apoptosis of hepatocytes that are involved in the Akt/p53 signal pathway.

    PubMed

    Wu, Jialing; Chen, Chong; Hu, Xi; Cai, Xianbin; Guan, Yinghong; Hu, Hui; Wang, Qinjia; Chen, Xiaofeng; Cai, Bozhi; Jing, Xubin

    2016-01-22

    Cyclooxygenase-2 (COX-2) can exert pro-inflammatory effects in nonalcoholic steatohepatitis (NASH). The aim of this study was to determine if the inhibition of COX-2 attenuates hepatocyte apoptosis in steatohepatitis and to examine the underlying molecular mechanism. Male wild type C57BL6/J mice and COX-2 knock out (COX-2-/-) mice were fed a methionine choline deficient (MCD) diet for 3 weeks. The wild type mice were also treated with celecoxib or a combination of celecoxib and a Akt specific inhibitor, miltefosine (MTF). After that, liver histology, serum alanine aminotransferase (ALT) levels, hepatic triglyceride (TG) levels, hepatocyte apoptosis, phosphorylated Akt (Ser473, pAkt) and p53 protein levels in mice livers were assessed. Celecoxib attenuated the severity of liver steatohepatitis and reduced the number of apoptotic cells, accompanied by increasing the activity of Akt and decreasing expression of p53. On the contrary, MTF can abrogate the effects of celecoxib on anti-apoptosis and anti-steatohepatitis. Moreover, the effects on the COX-2-/- mice that were fed the MCD diet were similar to that for celecoxib. The findings suggested that suppressing COX-2 can improve steatohepatitis by inhibiting hepatocyte apoptosis in mice via regulating the Akt/p53 pathway. Celecoxib treatment may be a favorable treatment option for NASH. PMID:26723251

  15. Osthole relaxes pulmonary arteries through endothelial phosphatidylinositol 3-kinase/Akt-eNOS-NO signaling pathway in rats.

    PubMed

    Yao, Li; Lu, Ping; Li, Yumei; Yang, Lijing; Feng, Hongxuan; Huang, Yong; Zhang, Dandan; Chen, Jianguo; Zhu, Daling

    2013-01-15

    Pulmonary arterial hypertension is a life-threatening disease lacking effective therapies. Osthole is a natural coumarin compound isolated from Angelica pubescens Maxim., which possesses hypotensive effect. Although its effects on isolated thoracic aorta (systemic circulating system) are clarified, it remains unclear whether Osthole relaxes isolated pulmonary arteries (PAs) (pulmonary circulating system). The aim of this study was to investigate the effects of Osthole on isolated PAs and the underlying mechanisms. We examined PA relaxation induced by Osthole in isolated human and rat PA rings with force-electricity transducers, the expression and activity of endothelial nitric oxide synthase (eNOS) and protein kinase B (Akt) with western blot, and nitric oxide (NO) production using DAF-FM DA fluorescent indicator. The results showed that Osthole elicited a dose-dependent vasorelaxation activity with phenylephrine-precontracted human and rat PA rings, which can be diminished by endothelium denudation and inhibition of eNOS, while having no effect on rat mesenteric arteries. Osthole increased NO release as well as activation of Akt and eNOS, indicated with increased phosphorylations of Akt at Ser-473 and eNOS at Ser-1177 in endothelial cells. PI3K inhibitor LY294002 also blocked Osthole induced vasodilation. In summary, dilative effect of Osthole was dependent on endothelial integrity and NO production, and was mediated by endothelial PI3K/Akt-eNOS-NO pathway. These may provide a new pulmonary vasodilator for the therapy of pulmonary arterial hypertension. PMID:23220709

  16. Protection against MPP(+)-induced neurotoxicity in SH-SY5Y cells by tormentic acid via the activation of PI3-K/Akt/GSK3β pathway.

    PubMed

    Zhao, Qing; Ye, Junli; Wei, Na; Fong, Chichun; Dong, Xiaoli

    2016-07-01

    The cause of Parkinson's disease (PD) could be ascribed to the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta, and thus molecules with neuroprotective ability may have therapeutic value against PD. In the current study, the neuroprotective effects and underlying mechanisms of tormentic acid (TA), a naturally occurring triterpene extracted from medicinal plants such as Rosa rugosa and Potentilla chinensis, were evaluated in a widely used cellular PD model in which neurotoxicity was induced by MPP(+) in cultured SH-SY5Y cells. We found that TA at 1-30 μM substantially protected against MPP(+)-induced neurotoxicity, as evidenced by the increase in cell viability, decrease in lactate dehydrogenase release and the reduction in apoptotic nuclei. Moreover, TA effectively inhibited the elevated intracellular accumulation of reactive oxygen species as well as Bax/Bcl-2 ratio caused by MPP(+). Most importantly, TA markedly reversed the inhibition of protein expression of phosphorylated Akt (Ser 473) and phosphorylated GSK3β (Ser 9) caused by MPP(+). LY294002, the specific inhibitor of PI3-K, significantly abrogated the up-regulated phosphorylated Akt and phosphorylated GSK3β offered by TA, suggesting that the neuroprotection of TA was mainly dependent on the activation of PI3-K/Akt/GSK3β signaling pathway. The results taken together indicate that TA may be a potential candidate for further preclinical study aimed at the prevention and treatment of PD. PMID:26994872

  17. Diallyl trisulfide, a constituent of processed garlic, inactivates Akt to trigger mitochondrial translocation of BAD and caspase-mediated apoptosis in human prostate cancer cells.

    PubMed

    Xiao, Dong; Singh, Shivendra V

    2006-03-01

    We have shown previously that apoptosis induction by diallyl trisulfide (DATS), a constituent of processed garlic, in PC-3 and DU145 human prostate cancer cells is associated with c-Jun N-terminal kinase and extracellular signal-regulated kinase-mediated phosphorylation of Bcl-2. However, pharmacological inhibition of these kinases offers only partial protection against the cell death caused by DATS. Here, we demonstrate that DATS inactivates Akt to trigger apoptosis in prostate cancer cells. Treatment of PC-3/DU145 cells with apoptosis inducing concentration of DATS (40 microM) resulted in a rapid decrease in Ser(473) and Thr(308) phosphorylation of Akt leading to inhibition of its kinase activity. The DATS-mediated inactivation of Akt was associated with downregulation of insulin-like growth factor receptor 1 protein level and inhibition of its autophosphorylation. DATS treatment (40 microM) also caused a decrease in Ser(155) and Ser(136) phosphorylation of BAD (a proapoptotic protein), which is a downstream target of Akt. Phosphorylation sequesters BAD in the cytoplasm owing to increased binding with 14-3-3 proteins. The interaction between BAD and 14-3-3beta was reduced markedly upon a 4 h treatment with 40 microM DATS in both cell lines. Consistent with these results, DATS treatment (40 microM, 4 h) promoted mitochondrial translocation of BAD as revealed by immunocytochemistry. Ectopic expression of constitutively active Akt conferred statistically significant protection against DATS-induced apoptosis. The DATS-induced apoptosis in both cell lines was significantly attenuated in the presence of pan caspase inhibitor zVAD-fmk and caspase 9 specific inhibitor zLEHD-fmk. In conclusion, the present study demonstrates that DATS-induced apoptosis in human prostate cancer cells is mediated, at least in part, by inactivation of Akt signaling axis. PMID:16169930

  18. AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion

    PubMed Central

    Hong, Seung-Keun; Jeong, Joseph H.; Chan, Andrew M.; Park, Jong-In

    2013-01-01

    Upregulated ERK1/2 activity is often correlated with AKT activation during prostate cancer (PCa) progression, yet their functional relation needs elucidation. Using androgen-deprived LNCaP cells, in which ERK1/2 activation occurs in strong correlation with AKT activation, we found that AKT-mediated B-Raf regulation is necessary for ERK1/2 activation. Specifically, in response to androgen deprivation, AKT upregulated B-Raf phosphorylation at Ser445 without affecting A-Raf or C-Raf-1. This effect of AKT was abolished by Arg25 to Ala mutation or truncating (Δ4-129) the pleckstrin homology domain of AKT, indicating that the canonical AKT regulation is important for this signaling. Intriguingly, although a constitutively active AKT containing N-terminal myristoylation signal could sufficiently upregulate B-Raf phosphorylation at Ser445 in LNCaP cells, subsequent MEK/ERK activation still required hormone deprivation. In contrast, AKT activity was sufficient to induce not only B-Raf phosphorylation but also MEK/ERK activation in the hormone refractory LNCaP variant, C4-2. These data indicate that androgen depletion may induce MEK/ERK activation through a synergy between AKT-dependent and -independent mechanisms and that the latter may become deregulated in association with castration resistance. In support, consistent AKT-mediated BRaf regulation was also detected in a panel of PCa lines derived from the cPten-/- L mice before and after castration. Our results also demonstrate that AKT regulates androgen receptor levels partly via the Raf/MEK/ERK pathway. This study reveals a novel crosstalk between ERK1/2 and AKT in PCa cells. PMID:23701950

  19. The PI3K/Akt Pathway Regulates Oxygen Metabolism via Pyruvate Dehydrogenase (PDH)-E1α Phosphorylation.

    PubMed

    Cerniglia, George J; Dey, Souvik; Gallagher-Colombo, Shannon M; Daurio, Natalie A; Tuttle, Stephen; Busch, Theresa M; Lin, Alexander; Sun, Ramon; Esipova, Tatiana V; Vinogradov, Sergei A; Denko, Nicholas; Koumenis, Constantinos; Maity, Amit

    2015-08-01

    Inhibition of the PI3K/Akt pathway decreases hypoxia within SQ20B human head and neck cancer xenografts. We set out to understand the molecular mechanism underlying this observation. We measured oxygen consumption using both a Clark electrode and an extracellular flux analyzer. We made these measurements after various pharmacologic and genetic manipulations. Pharmacologic inhibition of the PI3K/mTOR pathway or genetic inhibition of Akt/PI3K decreased the oxygen consumption rate (OCR) in vitro in SQ20B and other cell lines by 30% to 40%. Pharmacologic inhibition of this pathway increased phosphorylation of the E1α subunit of the pyruvate dehydrogenase (PDH) complex on Ser293, which inhibits activity of this critical gatekeeper of mitochondrial respiration. Expressing wild-type PTEN in a doxycycline-inducible manner in a cell line with mutant PTEN led to an increase in PDH-E1α phosphorylation and a decrease in OCR. Pretreatment of SQ20B cells with dichloroacetate (DCA), which inhibits PDH-E1α phosphorylation by inhibiting dehydrogenase kinases (PDK), reversed the decrease in OCR in response to PI3K/Akt/mTOR inhibition. Likewise, introduction of exogenous PDH-E1α that contains serine to alanine mutations, which can no longer be regulated by phosphorylation, also blunted the decrease in OCR seen with PI3K/mTOR inhibition. Our findings highlight an association between the PI3K/mTOR pathway and tumor cell oxygen consumption that is regulated in part by PDH phosphorylation. These results have important implications for understanding the effects of PI3K pathway activation in tumor metabolism and also in designing cancer therapy trials that use inhibitors of this pathway. PMID:25995437

  20. Insulin Resistance Prevents AMPK-induced Tau Dephosphorylation through Akt-mediated Increase in AMPKSer-485 Phosphorylation*

    PubMed Central

    Kim, Bhumsoo; Figueroa-Romero, Claudia; Pacut, Crystal; Backus, Carey; Feldman, Eva L.

    2015-01-01

    Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including obesity, diabetes, and dyslipidemia, and insulin resistance (IR) is the central feature of MetS. Recent studies suggest that MetS is a risk factor for Alzheimer disease (AD). AMP-activated kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme and a key player in regulating energy metabolism. In this report, we examined the role of IR on the regulation of AMPK phosphorylation and AMPK-mediated Tau phosphorylation. We found that AMPKSer-485, but not AMPKThr-172, phosphorylation is increased in the cortex of db/db and high fat diet-fed obese mice, two mouse models of IR. In vitro, treatment of human cortical stem cell line (HK-5320) and primary mouse embryonic cortical neurons with the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR), induced AMPK phosphorylation at both Thr-172 and Ser-485. AMPK activation also triggered Tau dephosphorylation. When IR was mimicked in vitro by chronically treating the cells with insulin, AICAR specifically induced AMPKSer-485, but not AMPKThr-172, hyperphosphorylation whereas AICAR-induced Tau dephosphorylation was inhibited. IR also resulted in the overactivation of Akt by AICAR treatment; however, preventing Akt overactivation during IR prevented AMPKSer-485 hyperphosphorylation and restored AMPK-mediated Tau dephosphorylation. Transfection of AMPKS485A mutant caused similar results. Therefore, our results suggest the following mechanism for the adverse effect of IR on AD pathology: IR → chronic overactivation of Akt → AMPKSer-485 hyperphosphorylation → inhibition of AMPK-mediated Tau dephosphorylation. Together, our results show for the first time a possible contribution of IR-induced AMPKSer-485 phosphorylation to the increased risk of AD in obesity and diabetes. PMID:26100639

  1. Insulin Resistance Prevents AMPK-induced Tau Dephosphorylation through Akt-mediated Increase in AMPKSer-485 Phosphorylation.

    PubMed

    Kim, Bhumsoo; Figueroa-Romero, Claudia; Pacut, Crystal; Backus, Carey; Feldman, Eva L

    2015-07-31

    Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including obesity, diabetes, and dyslipidemia, and insulin resistance (IR) is the central feature of MetS. Recent studies suggest that MetS is a risk factor for Alzheimer disease (AD). AMP-activated kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme and a key player in regulating energy metabolism. In this report, we examined the role of IR on the regulation of AMPK phosphorylation and AMPK-mediated Tau phosphorylation. We found that AMPK(Ser-485), but not AMPK(Thr-172), phosphorylation is increased in the cortex of db/db and high fat diet-fed obese mice, two mouse models of IR. In vitro, treatment of human cortical stem cell line (HK-5320) and primary mouse embryonic cortical neurons with the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), induced AMPK phosphorylation at both Thr-172 and Ser-485. AMPK activation also triggered Tau dephosphorylation. When IR was mimicked in vitro by chronically treating the cells with insulin, AICAR specifically induced AMPK(Ser-485), but not AMPK(Thr-172), hyperphosphorylation whereas AICAR-induced Tau dephosphorylation was inhibited. IR also resulted in the overactivation of Akt by AICAR treatment; however, preventing Akt overactivation during IR prevented AMPK(Ser-485) hyperphosphorylation and restored AMPK-mediated Tau dephosphorylation. Transfection of AMPK(S485A) mutant caused similar results. Therefore, our results suggest the following mechanism for the adverse effect of IR on AD pathology: IR → chronic overactivation of Akt → AMPK(Ser-485) hyperphosphorylation → inhibition of AMPK-mediated Tau dephosphorylation. Together, our results show for the first time a possible contribution of IR-induced AMPK(Ser-485) phosphorylation to the increased risk of AD in obesity and diabetes. PMID:26100639

  2. Distinct Time Course of the Decrease in Hepatic AMP-Activated Protein Kinase and Akt Phosphorylation in Mice Fed a High Fat Diet

    PubMed Central

    Shiwa, Mami; Yoneda, Masayasu; Okubo, Hirofumi; Ohno, Haruya; Kobuke, Kazuhiro; Monzen, Yuko; Kishimoto, Rui; Nakatsu, Yusuke; Asano, Tomoichiro; Kohno, Nobuoki

    2015-01-01

    AMP-activated protein kinase (AMPK) plays an important role in insulin resistance, which is characterized by the impairment of the insulin-Akt signaling pathway. However, the time course of the decrease in AMPK and Akt phosphorylation in the liver during the development of obesity and insulin resistance caused by feeding a high fat diet (HFD) remains controversial. Moreover, it is unclear whether the impairment of AMPK and Akt signaling pathways is reversible when changing from a HFD to a standard diet (SD). Male ddY mice were fed the SD or HFD for 3 to 28 days, or fed the HFD for 14 days, followed by the SD for 14 days. We examined the time course of the expression and phosphorylation levels of AMPK and Akt in the liver by immunoblotting. After 3 days of feeding on the HFD, mice gained body weight, resulting in an increased oil red O staining, indicative of hepatic lipid accumulation, and significantly decreased AMPK phosphorylation, in comparison with mice fed the SD. After 14 days on the HFD, systemic insulin resistance occurred and Akt phosphorylation significantly decreased. Subsequently, a change from the HFD to SD for 3 days, after 14 days on the HFD, ameliorated the impairment of AMPK and Akt phosphorylation and systemic insulin resistance. Our findings indicate that AMPK phosphorylation decreases early upon feeding a HFD and emphasizes the importance of prompt lifestyle modification for decreasing the risk of developing diabetes. PMID:26266809

  3. Halofuginone inhibits Smad3 phosphorylation via the PI3K/Akt and MAPK/ERK pathways in muscle cells: Effect on myotube fusion

    SciTech Connect

    Roffe, Suzy; Hagai, Yosey; Pines, Mark; Halevy, Orna

    2010-04-01

    Halofuginone, a novel inhibitor of Smad3 phosphorylation, has been shown to inhibit muscle fibrosis and to improve cardiac and skeletal muscle functions in the mdx mouse model of Duchenne muscular dystrophy. Here, we demonstrate that halofuginone promotes the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) family members in a C2 muscle cell line and in primary myoblasts derived from wild-type and mdx mice diaphragms. Halofuginone enhanced the association of phosphorylated Akt and MAPK/extracellular signal-regulated protein kinase (ERK) with the non-phosphorylated form of Smad3, accompanied by a reduction in Smad3 phosphorylation levels. This reduction was reversed by inhibitors of the phosphoinositide 3'-kinase/Akt (PI3K/Akt) and MAPK/ERK pathways, suggesting their specific role in mediating halofuginone's inhibitory effect on Smad3 phosphorylation. Halofuginone enhanced Akt, MAPK/ERK and p38 MAPK phosphorylation and inhibited Smad3 phosphorylation in myotubes, all of which are crucial for myotube fusion. In addition, halofuginone increased the association Akt and MAPK/ERK with Smad3. As a consequence, halofuginone promoted myotube fusion, as reflected by an increased percentage of C2 and mdx myotubes containing high numbers of nuclei, and this was reversed by specific inhibitors of the PI3K and MAPK/ERK pathways. Together, the data suggest a role, either direct or via inhibition of Smad3 phosphorylation, for Akt or MAPK/ERK in halofuginone-enhanced myotube fusion, a feature which is crucial to improving muscle function in muscular dystrophies.

  4. JNK and STAT3 signaling pathways converge on Akt-mediated phosphorylation of EZH2 in bronchial epithelial cells induced by arsenic

    PubMed Central

    Chen, Bailing; Liu, Jia; Chang, Qingshan; Beezhold, Kevin; Lu, Yongju; Chen, Fei

    2013-01-01

    The molecular mechanisms by which arsenic (As3+) causes human cancers remain to be fully elucidated. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb-repressive complexes 2 (PRC2) that promotes trimethylation of lysine 27 of histone H3, leading to altered expression of tumor suppressors or oncogenes. In the present study, we determined the effect of As3+ on EZH2 phosphorylation and the signaling pathways important for As3+-induced EZH2 phosphorylation in human bronchial epithelial cell line BEAS-2B. The involvement of kinases in As3+-induced EZH2 phosphorylation was validated by siRNA-based gene silencing. The data showed that As3+ can induce phosphorylation of EZH2 at serine 21 in human bronchial epithelial cells and that the phosphorylation of EZH2 requires an As3+-activated signaling cascade from JNK and STAT3 to Akt. Transfection of the cells with siRNA specific for JNK1 revealed that JNK silencing reduced serine727 phosphorylation of STAT3, Akt activation and EZH2 phosphorylation, suggesting that JNK is the upstream kinase involved in As3+-induced EZH2 phosphorylation. Because As3+ is capable of inducing miRNA-21 (miR-21), a STAT3-regulated miRNA that represses protein translation of PTEN or Spry2, we also tested the role of STAT3 and miR-21 in As3+-induced EZH2 phosphorylation. Ectopic overexpression of miR-21 promoted Akt activation and phosphorylation of EZH2, whereas inhibiting miR-21 by transfecting the cells with anti-miR-21 inhibited Akt activation and EZH2 phosphorylation. Taken together, these results demonstrate a contribution of the JNK, STAT3 and Akt signaling axis to As3+-induced EZH2 phosphorylation. Importantly, these findings may reveal new molecular mechanisms underlying As3+-induced carcinogenesis. PMID:23255093

  5. AKT hyper-phosphorylation associated with PI3K mutations in lymphatic endothelial cells from a patient with lymphatic malformation

    PubMed Central

    Boscolo, Elisa; Coma, Silvia; Luks, Valerie L.; Greene, Arin; Klagsbrun, Michael; Warman, Matthew L.; Bischoff, Joyce

    2014-01-01

    Lymphatic malformations (LM) are characterized by abnormal formation of lymphatic vessels and tissue overgrowth. The lymphatic vessels present in LM lesions may become blocked and enlarged as lymphatic fluid collects, forming a mass or cyst. Lesions are typically diagnosed during childhood, and are often disfiguring and life threatening. Available treatments consist of sclerotherapy, surgical removal and therapies to diminish complications. We isolated lymphatic endothelial cells (LM-LEC) from a surgically removed microcystic LM lesion. LM-LEC and normal human dermal-LEC (HD-LEC) expressed endothelial (CD31, VE-Cadherin) as well as lymphatic endothelial (Podoplanin, PROX1, LYVE1)-specific markers. Targeted gene sequencing analysis in patient-derived LM-LEC revealed the presence of two mutations in class I phosphoinositide 3-kinases (PI3K) genes. One is an inherited, premature stop codon in the PI3K regulatory subunit PIK3R3. The second is a somatic missense mutation in the PI3K catalytic subunit PIK3CA; this mutation has been found in association with overgrowth syndromes and cancer growth. LM-LEC exhibited angiogenic properties: both cellular proliferation and sprouting in collagen were significantly increased compared to HD-LEC. AKT-Thr308 was constitutively hyper-phosphorylated in LM-LEC. Treatment of LM-LEC with PI3-Kinase inhibitors Wortmannin and LY294 decreased cellular proliferation and prevented the phosphorylation of AKT-Thr308 in both HD-LEC and LM-LEC. Treatment with the mTOR inhibitor rapamycin also diminished cellular proliferation, sprouting and AKT phosphorylation, but only in LM-LEC. Our results implicate disrupted PI3K-AKT signaling in LEC isolated from a human lymphatic malformation lesion. PMID:25424831

  6. Akt attenuates apoptotic death through phosphorylation of H2A under hydrogen peroxide-induced oxidative stress in PC12 cells and hippocampal neurons

    PubMed Central

    Park, Ji Hye; Kim, Chung Kwon; Lee, Sang Bae; Lee, Kyung-Hoon; Cho, Sung-Woo; Ahn, Jee-Yin

    2016-01-01

    Although the essential role of protein kinase B (PKB)/Akt in cell survival signaling has been clearly established, the mechanism by which Akt mediates the cellular response to hydrogen peroxide (H2O2)-induced oxidative stress remains unclear. We demonstrated that Akt attenuated neuronal apoptosis through direct association with histone 2A (H2A) and phosphorylation of H2A at threonine 17. At early time points during H2O2 exposure of PC12 cells and primary hippocampal neurons, when the cells can tolerate the level of DNA damage, Akt was activated and phosphorylated H2A, leading to inhibition of apoptotic death. At later time points, Akt delivered the NAD+-dependent protein deacetylase Sirtuin 2 (Sirt 2) to the vicinity of phosphorylated H2A in response to irreversible DNA damage, thereby inducing H2A deacetylation and subsequently leading to apoptotic death. Ectopically expressed T17A-substituted H2A minimally interacted with Akt and failed to prevent apoptosis under oxidative stress. Thus Akt-mediated H2A phosphorylation has an anti-apoptotic function in conditions of H2O2-induced oxidative stress in neurons and PC12 cells. PMID:26899247

  7. Decreased Phosphorylated Protein Kinase B (Akt) in Individuals with Autism Associated with High Epidermal Growth Factor Receptor (EGFR) and Low Gamma-Aminobutyric Acid (GABA)

    PubMed Central

    Russo, Anthony J

    2015-01-01

    Dysregulation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway could contribute to the pathogenesis of autism spectrum disorders. In this study, phosphorylated Akt concentration was measured in 37 autistic children and 12, gender and age similar neurotypical, controls using an enzyme-linked immunosorbent assay. Akt levels were compared to biomarkers known to be associated with epidermal growth factor receptor (EGFR) and c-Met (hepatocyte growth factor (HGF) receptor) pathways and severity levels of 19 autism-related symptoms. We found phosphorylated Akt levels significantly lower in autistic children and low Akt levels correlated with high EGFR and HGF and low gamma-aminobutyric acid, but not other biomarkers. Low Akt levels also correlated significantly with increased severity of receptive language, conversational language, hypotonia, rocking and pacing, and stimming, These results suggest a relationship between decreased phosphorylated Akt and selected symptom severity in autistic children and support the suggestion that the AKT pathways may be associated with the etiology of autism. PMID:26508828

  8. Activated α2-macroglobulin binding to cell surface GRP78 induces T-loop phosphorylation of Akt1 by PDK1 in association with Raptor.

    PubMed

    Misra, Uma Kant; Pizzo, Salvatore Vincent

    2014-01-01

    PDK1 phosphorylates multiple substrates including Akt by PIP3-dependent mechanisms. In this report we provide evidence that in prostate cancer cells stimulated with activated α2-macroglobulin (α2M*) PDK1 phosphorylates Akt in the T-loop at Thr(308) by using Raptor in the mTORC1 complex as a scaffold protein. First we demonstrate that PDK1, Raptor, and mTOR co-immunoprecipitate. Silencing the expression, not only of PDK1, but also Raptor by RNAi nearly abolished Akt phosphorylation at Akt(Thr308) in Raptor-immunoprecipitates of α2M*-stimulated prostate cancer cells. Immunodepleting Raptor or PDK from cell lysates of cells treated with α2M* drastically reduced Akt phosphorylation at Thr(308), which was recovered by adding the supernatant of Raptor- or PDK1-depleted cell lysates, respectively. Studies of insulin binding to its receptor on prostate cancer cells yielded similar results. We thus demonstrate that phosphorylating the T-loop Akt residue Thr(308) by PDK1 requires Raptor of the mTORC1 complex as a platform or scaffold protein. PMID:24516643

  9. Multikinase inhibitor sorafenib exerts cytocidal efficacy against Non-Hodgkin lymphomas associated with inhibition of MAPK14 and AKT phosphorylation.

    PubMed

    Chapuy, Bjoern; Schuelper, Nikolai; Panse, Melanie; Dohm, Andrea; Hand, Elisabeth; Schroers, Roland; Truemper, Lorenz; Wulf, Gerald G

    2011-02-01

    Intracellular signal transduction by kinase-mediated phosphorylation is essential for the survival and growth of lymphoma cells. This study analysed the multikinase inhibitor sorafenib for its cytotoxic activity against lymphoma cells. We found that sorafenib reduced cell viability at low micromolar concentrations in a time-dependent manner in cell lines and primary cell suspensions representing major types of aggressive B- and T-cell lymphomas. In cells surviving short term exposure, proliferative arrest occurred leading to complete loss of in vitro clonogenicity. Previously described sorafenib targets within the RAF kinase family were found to be expressed and phosphorylated in all cell lines, and sorafenib perturbed the activation of classical RAF/MEK/ERK pathway targets. However, using a global phoshoprotein array, the most consistent downstream effect of sorafenib in NHL cells was the inhibition of mitogen-activated protein kinase 14 (MAPK14) and panAKT phosphorylation. In conclusion, sorafenib has significant in vitro efficacy against aggressive B- and T-cell lymphoma cells, associated with inhibition of MAPK14 and panAKT. PMID:21689083

  10. Gentiopicroside and sweroside from Veratrilla baillonii Franch. induce phosphorylation of Akt and suppress Pck1 expression in hepatoma cells.

    PubMed

    Huang, Xian-Ju; Li, Jun; Mei, Zhi-Yi; Chen, Guoxun

    2016-06-01

    The use of phytochemicals and herbal medicines has accompanied human history. Advances in modern biomedical sciences have allowed us to investigate the functional mechanisms of herbal medicines and phytochemicals. Veratrilla baillonii Franch. has long been used as a medicinal herb in southwestern China. Here, we analyzed the effects of an ethanol extract from V. baillonii (VBFE) on the expression levels of the cytosolic form of the phosphoenolpyruvate carboxykinase gene (Pck1) mRNA and components of the insulin signalling cascade in HL1C hepatoma cells. Compared with the insulin control, VBFE treatment inhibited the expression of Pck1 mRNA in a dose-dependent manner. This was associated with the phosphorylation of Akt and Erk1/2 in a time-dependent manner. Further analysis of the purified components of VBFE indicated that gentiopicroside and sweroside from VBFE, alone and in combination, suppressed Pck1 expression and induced Akt and Erk1/2 phosphorylation. In conclusion, gentiopicroside and sweroside suppress Pck1 expression and induce phosphorylation of components in the insulin signalling cascade. This is the first study to demonstrate that gentiopicroside and sweroside show insulin-mimicking effects on the regulation of Pck1 expression. Further studies are warranted to explore the potential of gentiopicroside and sweroside in the control of blood glucose in animals. PMID:27248905

  11. IL-6 cytoprotection in hyperoxic acute lung injury occurs via PI3K/Akt-mediated Bax phosphorylation

    PubMed Central

    Kolliputi, Narasaiah; Waxman, Aaron B.

    2009-01-01

    IL-6 overexpression protects mice from hyperoxic acute lung injury in vivo, and treatment with IL-6 protects cells from oxidant-mediated death in vitro. The mechanisms of protection, however, are not clear. We characterized the expression, localization, and regulation of Bax, a proapoptotic member of the Bcl-2 family, in wild-type (WT) and IL-6 lung-specific transgenic (Tg+) mice exposed to 100% O2 and in human umbilical vein endothelial cells (HUVEC) treated with H2O2 and IL-6. In control HUVEC treated with H2O2 or in WT mice exposed to 100% O2, a marked induction of Bax translocation and dimerization was associated with increased JNK and p38 kinase activity. In contrast, specific JNK or p38 kinase inhibitors or treatment with IL-6 inhibited Bax mitochondrial translocation and apoptosis of HUVEC. IL-6 Tg+ mice exposed to 100% O2 exhibited enhanced phosphatidylinositol 3-kinase (PI3K)/Akt kinase and increased serine phosphorylation of Bax at Ser184 compared with WT mice. The PI3K-specific inhibitor LY-2940002 blocked this IL-6-induced Bax phosphorylation and promoted cell death. Furthermore, IL-6 potently blocked hyperoxia- or oxidant-induced Bax insertion into mitochondrial membranes. Thus IL-6 functions in a cytoprotective manner, in part, by suppressing Bax translocation and dimerization through PI3K/Akt-mediated Bax phosphorylation. PMID:19376889

  12. Akt1-mediated Gata3 phosphorylation controls the repression of IFNγ in memory-type Th2 cells

    PubMed Central

    Hosokawa, Hiroyuki; Tanaka, Tomoaki; Endo, Yusuke; Kato, Miki; Shinoda, Kenta; Suzuki, Akane; Motohashi, Shinichiro; Matsumoto, Masaki; Nakayama, Keiichi I.; Nakayama, Toshinori

    2016-01-01

    Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells. PMID:27053161

  13. Akt1-mediated Gata3 phosphorylation controls the repression of IFNγ in memory-type Th2 cells.

    PubMed

    Hosokawa, Hiroyuki; Tanaka, Tomoaki; Endo, Yusuke; Kato, Miki; Shinoda, Kenta; Suzuki, Akane; Motohashi, Shinichiro; Matsumoto, Masaki; Nakayama, Keiichi I; Nakayama, Toshinori

    2016-01-01

    Th2 cells produce Th2 cytokines such as IL-4, IL-5 and IL-13, but repress Th1 cytokine IFNγ. Recent studies have revealed various distinct memory-type Th2 cell subsets, one of which produces a substantial amount of IFNγ in addition to Th2 cytokines, however it remains unclear precisely how these Th2 cells produce IFNγ. We herein show that phosphorylation of Gata3 at Ser308, Thr315 and Ser316 induces dissociation of a histone deacetylase Hdac2 from the Gata3/Chd4 repressive complex in Th2 cells. We also identify Akt1 as a Gata3-phosphorylating kinase, and the activation of Akt1 induces derepression of Tbx21 and Ifng expression in Th2 cells. Moreover, T-bet-dependent IFNγ expression in IFNγ-producing memory Th2 cells appears to be controlled by the phosphorylation status of Gata3 in human and murine systems. Thus, this study highlights the molecular basis for posttranslational modifications of Gata3 that control the regulation of IFNγ expression in memory Th2 cells. PMID:27053161

  14. Long chain n-3 polyunsaturated fatty acids increase the efficacy of docetaxel in mammary cancer cells by downregulating Akt and PKCε/δ-induced ERK pathways.

    PubMed

    Chauvin, Lucie; Goupille, Caroline; Blanc, Charly; Pinault, Michelle; Domingo, Isabelle; Guimaraes, Cyrille; Bougnoux, Philippe; Chevalier, Stephan; Mahéo, Karine

    2016-04-01

    Taxanes can induce drug resistance by increasing signaling pathways such as PI3K/Akt and ERK, which promote survival and cell growth in human cancer cells. We have previously shown that long chain n-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6n-3) decrease resistance of experimental mammary tumors to anticancer drugs. Our objective was to determine whether DHA could increase tumor sensitivity to docetaxel by down-regulating these survival pathways. In docetaxel-treated MDA-MB-231 cells, phosphorylated-ERK1/2 levels were increased by 60% in membrane and nuclear compartments, compared to untreated cells. Our data showed that ERK1/2 activation depended on PKC activation since: i) enzastaurin (a pan-PKC inhibitor) blocked docetaxel-induced ERK1/2 phosphorylation ii) docetaxel increased PKC activity by 30% and phosphatidic acid level by 1.6-fold iii) inhibition of PKCε and PKCδ by siRNA resulted in reduced phosphorylated ERK1/2 levels. In DHA-supplemented cells, docetaxel was unable to increase PKCε and δ levels in membrane and nuclear fractions, resulting in diminished ERK1/2 phosphorylation and increased docetaxel efficacy. Reduced membrane level of PKCε and PKCδ was associated with significant incorporation of DHA in all phospholipids, including phosphatidylcholine which is a major source of phosphatidic acid. Additionally, examination of the Akt pathway showed that DHA could repress docetaxel-induced Ser473Akt phosphorylation. In rat mammary tumors, dietary DHA supplementation during docetaxel chemotherapy repressed ERK and Akt survival pathways and in turn strongly improved taxane efficacy. The P-ERK level was negatively correlated with tumor regression. These findings are of potential clinical importance in treating chemotherapy-refractory cancer. PMID:26821209

  15. Piperlongumine, an alkaloid causes inhibition of PI3 K/Akt/mTOR signaling axis to induce caspase-dependent apoptosis in human triple-negative breast cancer cells.

    PubMed

    Shrivastava, Shweta; Kulkarni, Prasad; Thummuri, Dinesh; Jeengar, Manish Kumar; Naidu, V G M; Alvala, Mallika; Redddy, G Bhanuprakash; Ramakrishna, Sistla

    2014-07-01

    The phosphatidylinositol 3-kinase (PI3 K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth and survival under physiological conditions. However, aberrant PI3 K/Akt/mTOR signaling has been implicated in many human cancers, including human triple negative breast cancer. Therefore, dual inhibitors of PI3 K/Akt and mTOR signaling could be valuable agents for treating breast cancer. The objective of this study was to investigate the effect of piperlongumine (PPLGM), a natural alkaloid on PI3 K/Akt/mTOR signaling, Akt mediated regulation of NF-kB and apoptosis evasion in human breast cancer cells. Using molecular docking studies, we found that PPLGM physically interacts with the conserved domain of PI3 K and mTOR kinases and the results were comparable with standard dual inhibitor PF04691502. Our results demonstrated that treatment of different human triple-negative breast cancer cells with PPLGM resulted in concentration- and time-dependent growth inhibition. The inhibition of cancer cell growth was associated with G1-phase cell cycle arrest and down-regulation of the NF-kB pathway leads to activation of the mitochondrial apoptotic pathway. It was also found that PPLGM significantly decreased the expression of p-Akt, p70S6K1, 4E-BP1, cyclin D1, Bcl-2, p53 and increased expression of Bax, cytochrome c in human triple-negative breast cancer cells. Although insulin treatment increased the phosphorylation of Akt (Ser473), p70S6K1, 4E-BP1, PPLGM abolished the insulin mediated phosphorylation, it clearly indicates that PPLGM acts through PI3 k/Akt/mTOR axis. Our results suggest that PPLGM may be an effective therapeutic agent for the treatment of human triple negative breast cancer. PMID:24729100

  16. Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation, but Not for Hypertrophy, Aneuploidy and Hyperfunction of Pancreatic β-Cells

    PubMed Central

    Wittenberg, Avigail Dreazen; Azar, Shahar; Klochendler, Agnes; Stolovich-Rain, Miri; Avraham, Shlomit; Birnbaum, Lea; Binder Gallimidi, Adi; Katz, Maximiliano; Dor, Yuval; Meyuhas, Oded

    2016-01-01

    Constitutive expression of active Akt (Akttg) drives hyperplasia and hypertrophy of pancreatic β-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in β-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-). rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in β-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in β-cells. In vitro experiments with Akttg and rpS6P-/-;Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation. PMID:26919188

  17. Phosphorylated Ribosomal Protein S6 Is Required for Akt-Driven Hyperplasia and Malignant Transformation, but Not for Hypertrophy, Aneuploidy and Hyperfunction of Pancreatic β-Cells.

    PubMed

    Wittenberg, Avigail Dreazen; Azar, Shahar; Klochendler, Agnes; Stolovich-Rain, Miri; Avraham, Shlomit; Birnbaum, Lea; Binder Gallimidi, Adi; Katz, Maximiliano; Dor, Yuval; Meyuhas, Oded

    2016-01-01

    Constitutive expression of active Akt (Akttg) drives hyperplasia and hypertrophy of pancreatic β-cells, concomitantly with increased insulin secretion and improved glucose tolerance, and at a later stage the development of insulinoma. To determine which functions of Akt are mediated by ribosomal protein S6 (rpS6), an Akt effector, we generated mice that express constitutive Akt in β-cells in the background of unphosphorylatable ribosomal protein S6 (rpS6P-/-). rpS6 phosphorylation deficiency failed to block Akttg-induced hypertrophy and aneuploidy in β-cells, as well as the improved glucose homeostasis, indicating that Akt carries out these functions independently of rpS6 phosphorylation. In contrast, rpS6 phosphorylation deficiency efficiently restrained the reduction in nuclear localization of the cell cycle inhibitor p27, as well as the development of Akttg-driven hyperplasia and tumor formation in β-cells. In vitro experiments with Akttg and rpS6P-/-;Akttg fibroblasts demonstrated that rpS6 phosphorylation deficiency leads to reduced translation fidelity, which might underlie its anti-tumorigenic effect in the pancreas. However, the role of translation infidelity in tumor suppression cannot simply be inferred from this heterologous experimental model, as rpS6 phosphorylation deficiency unexpectedly elevated the resistance of Akttg fibroblasts to proteotoxic, genotoxic as well as autophagic stresses. In contrast, rpS6P-/- fibroblasts exhibited a higher sensitivity to these stresses upon constitutive expression of oncogenic Kras. The latter result provides a possible mechanistic explanation for the ability of rpS6 phosphorylation deficiency to enhance DNA damage and protect mice from Kras-induced neoplastic transformation in the exocrine pancreas. We propose that Akt1 and Kras exert their oncogenic properties through distinct mechanisms, even though both show addiction to rpS6 phosphorylation. PMID:26919188

  18. miR-302 Attenuates Amyloid-β-Induced Neurotoxicity through Activation of Akt Signaling.

    PubMed

    Li, Hsin-Hua; Lin, Shi-Lung; Huang, Chien-Ning; Lu, Fung-Jou; Chiu, Pai-Yi; Huang, Wen-Nung; Lai, Te-Jen; Lin, Chih-Li

    2016-01-01

    Deficiency of insulin signaling has been linked to diabetes and ageing-related neurodegenerative diseases such as Alzheimer's disease (AD). In this regard, brains exhibit defective insulin receptor substrate-1 (IRS-1) and hence result in alteration of insulin signaling in progression of AD, the most common cause of dementia. Consequently, dysregulation of insulin signaling plays an important role in amyloid-β (Aβ)-induced neurotoxicity. As the derivation of induced pluripotent stem cells (iPSC) involves cell reprogramming, it may provide a means for regaining the control of ageing-associated dysfunction and neurodegeneration via affecting insulin-related signaling. To this, we found that an embryonic stem cell (ESC)-specific microRNA, miR-302, silences phosphatase and tensin homolog (PTEN) to activate Akt signaling, which subsequently stimulates nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) elevation and hence inhibits Aβ-induced neurotoxicity. miR-302 is predominantly expressed in iPSCs and is known to regulate several important biological processes of anti-oxidative stress, anti-apoptosis, and anti-aging through activating Akt signaling. In addition, we also found that miR-302-mediated Akt signaling further stimulates Nanog expression to suppress Aβ-induced p-Ser307 IRS-1 expression and thus enhances tyrosine phosphorylation and p-Ser 473-Akt/p-Ser 9-GSK3β formation. Furthermore, our in vivo studies revealed that the mRNA expression levels of both Nanog and miR-302-encoding LARP7 genes were significantly reduced in AD patients' blood cells, providing a novel diagnosis marker for AD. Taken together, our findings demonstrated that miR-302 is able to inhibit Aβ-induced cytotoxicity via activating Akt signaling to upregulate Nrf2 and Nanog expressions, leading to a marked restoration of insulin signaling in AD neurons. PMID:26890744

  19. Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation*

    PubMed Central

    Dudakovic, Amel; Evans, Jared M.; Li, Ying; Middha, Sumit; McGee-Lawrence, Meghan E.; van Wijnen, Andre J.; Westendorf, Jennifer J.

    2013-01-01

    Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro. PMID:23940046

  20. Targeted deletion of Kif18a protects from colitis-associated colorectal (CAC) tumors in mice through impairing Akt phosphorylation

    SciTech Connect

    Zhu, Houbao; Xu, Wangyang; Zhang, Hongxin; Liu, Jianbing; Xu, Haimin; Lu, Shunyuan; Dang, Suying; Kuang, Ying; Jin, Xiaolong; Wang, Zhugang

    2013-08-16

    Highlights: •Kif18A is up-regulated in CAC of mouse model. •Kif18a{sup −/−} mice are protected from CAC. •Tumor cells from Kif18a{sup −/−} mice undergo more apoptosis. •Kif18A deficiency induces poor Atk phosphorylation. -- Abstract: Kinesins are a superfamily of molecular motors involved in cell division or intracellular transport. They are becoming important targets for chemotherapeutic intervention of cancer due to their crucial role in mitosis. Here, we demonstrate that the kinesin-8 Kif18a is overexpressed in murine CAC and is a crucial promoter during early CAC carcinogenesis. Kif18a-deficient mice are evidently protected from AOM–DSS-induced colon carcinogenesis. Kif18A is responsible for proliferation of colonic tumor cells, while Kif18a ablation in mice promotes cell apoptosis. Mechanistically, Kif18a is responsible for induction of Akt phosphorylation, which is known to be associated with cell survival regulation. In conclusion, Kif18a is critical for colorectal carcinogenesis in the setting of inflammation by mechanisms of increased PI3K-AKT signaling. Inhibition of Kif18A activity may be useful in the prevention or chemotherapeutic intervention of CAC.

  1. Akt phosphorylates myc-associated zinc finger protein (MAZ), releases P-MAZ from the p53 promoter, and activates p53 transcription.

    PubMed

    Lee, Wei-Ping; Lan, Keng-Hsin; Li, Chung-Pin; Chao, Yee; Lin, Han-Chieh; Lee, Shou-Dong

    2016-05-28

    The p53 protein is a cell cycle regulator. When the cell cycle progresses, p53 plays an important role in putting a brake on the G1 phase to prevent unwanted errors during cell division. Akt is a downstream kinase of receptor tyrosine kinase. Upon activation, Akt phorphorylates IKK that then phosphorylates IκB and releases NF-κB, leading to transcriptional activation of Dmp1. Dmp1 is a transcriptional activator of Arf. It has been known that oncogene activation stabilizes p53 through transcriptional activation of Arf, which then binds and inhibits Mdm2. In the current study, we show that myc-associated zinc finger protein (MAZ) is a transcriptional repressor of the p53 promoter. Akt phosphorylates MAZ at Thr385, and the phosphorylated MAZ is released from the p53 promoter, leading to transcriptional activation of p53, a new mechanism that contributes to increased p53 protein pool during oncogene activation. PMID:26902421

  2. SRPK1 and Akt Protein Kinases Phosphorylate the RS Domain of Lamin B Receptor with Distinct Specificity: A Combined Biochemical and In Silico Approach

    PubMed Central

    Nikolakaki, Eleni; Vlassi, Metaxia; Giannakouros, Thomas

    2016-01-01

    Activated Akt has been previously implicated in acting on RS domain-containing proteins. However, it has been questioned whether its action is direct or it is mediated by co-existing SR kinase activity. To address this issue we studied in detail the phosphorylation of Lamin B Receptor (LBR) by Akt. Using synthetic peptides and a set of recombinant proteins expressing mutants of the LBR RS domain we now demonstrate that while all serines of the RS domain represent more or less equal phosphoacceptor sites for SRPK1, Ser80 and Ser82 are mainly targeted by Akt. 3D-modeling combined with molecular dynamics (MD) simulations show that amongst short, overlapping LBR RS-containing peptides complying with the minimum Akt recognition consensus sequence, only those bearing phosphosites either at Ser80 or Ser82 are able to fit into the active site of Akt, at least as effectively as its known substrate, GSK3-β. Combined our results provide evidence that Akt kinases directly phosphorylate an RS domain-containing protein and that both the residues N-terminal the phosphosite and at position +1 are essential for Akt specificity, with the latter substrate position being compatible with the arginine residue of RS-repeats. PMID:27105349

  3. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

    SciTech Connect

    Luo, Chunxia; Yi, Bin; Bai, Li; Xia, Yongzhi; Wang, Guansong; Qian, Guisheng; Feng, Hua

    2010-07-02

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  4. Serine 1179 phosphorylation of endothelial nitric oxide synthase caused by 2,4,6-trinitrotoluene through PI3K/Akt signaling in endothelial cells

    SciTech Connect

    Sun Yang; Sumi, Daigo; Kumagai, Yoshito . E-mail: yk-em-tu@md.tsukuba.ac.jp

    2006-07-01

    Although 2,4,6-trinitrotoluene (TNT) has been found to uncouple nitric oxide synthase (NOS), thereby leading to reactive oxygen species (ROS), cellular response against TNT still remains unclear. Exposure of bovine aortic endothelial cells (BAECs) to TNT (100 {mu}M) resulted in serine 1179 phosphorylation of endothelial NOS (eNOS). With specific inhibitors (wortmannin and LY294002), we found that PI3K/Akt signaling participated in the eNOS phosphorylation caused by TNT, whereas the ERK pathway did not. ROS were generated following exposure of BAECs to TNT. However, TNT-mediated phosphorylation of either eNOS or Akt was drastically blocked by NAC and PEG-CAT. Interestingly, pretreatment with apocynin, a specific inhibitor for NADPH oxidase, diminished the phosphorylation of eNOS and Akt. These results suggest that TNT affects NADPH oxidase, thereby generating hydrogen peroxide, which is capable of activating PI3K/Akt signaling associated with eNOS Ser 1179 phosphorylation.

  5. Guizhi Fuling Wan, a Traditional Chinese Herbal Formula, Sensitizes Cisplatin-Resistant Human Ovarian Cancer Cells through Inactivation of the PI3K/AKT/mTOR Pathway

    PubMed Central

    Guo, Xiaojuan; Bian, Hua; Yang, Lei; Chen, Zhong; Zang, Wenhua; Yang, Jingke

    2016-01-01

    The aim of the study was to explore the possible mechanisms that Guizhi Fuling Wan (GFW) enhances the sensitivity of the SKOV3/DDP ovarian cancer cells and the resistant xenograft tumours to cisplatin. Rat medicated sera containing GFW were prepared by administering GFW to rats, and the primary bioactive constituents of the sera were gallic acid, paeonol, and paeoniflorin analysed by HPLC/QqQ MS. Cell counting kit-8 analysis was shown that coincubation of the sera with cisplatin/paclitaxel enhanced significantly the cytotoxic effect of cisplatin or paclitaxel in SKOV3/DDP cells. The presence of the rat medicated sera containing GFW resulted in an increase in rhodamine 123 accumulation by flow cytometric assays and a decrease in the protein levels of P-gp, phosphorylation of AKT at Ser473, and mTOR in a dose-dependent manner in SKOV3/DDP cells by western blot analysis, but the sera had no effect on the protein levels of PI3K p110α and total AKT. The low dose of GFW enhanced the anticancer efficacy of cisplatin and paclitaxel treatment in resistant SKOV3/DDP xenograft tumours. GFW could sensitize cisplatin-resistant SKOV3/DDP cells by inhibiting the protein level and function of P-gp, which may be medicated through inactivation of the PI3K/AKT/mTOR pathway. PMID:27293459

  6. Blood glucose fluctuation accelerates renal injury involved to inhibit the AKT signaling pathway in diabetic rats.

    PubMed

    Ying, Changjiang; Zhou, Xiaoyan; Chang, Zhenzhen; Ling, Hongwei; Cheng, Xingbo; Li, Wei

    2016-07-01

    Blood glucose fluctuation is associated with diabetic nephropathy. However, the mechanism by which blood glucose fluctuation accelerates renal injury is not fully understood. The aim of the present study was to assess the effects of blood glucose fluctuation on diabetic nephropathy in rats and investigate its underlying mechanism. Diabetes in the rats was induced by a high sugar, high-fat diet, and a single dose of STZ (35 mg/kg)-injected intraperitoneally. Unstable blood sugar models were induced by subcutaneous insulin injection and intravenous glucose injection alternately. Body weight, glycosylated hemoglobin A1c (HbAlc), blood urea nitrogen (BUN), serum creatinine (Scr), and Creatinine clearance (Ccr) were assessed. T-SOD activity and MDA level were measured by assay kit. Change in renal tissue ultrastructure was observed by light microscopy and electron microscopy. Phosphorylated ser/thr protein kinase (p-AKT) (phosphor-Ser473), phosphorylated glycogen synthase kinase-3 beta (p-GSK-3β) (phosphor-Ser9), Bcl-2-associated X protein (BAX), B cell lymphoma/leukemia 2 (BCL-2), and cleaved-cysteinyl aspartate-specific proteinase-3 (caspase-3) levels were detected by immunohistochemistry and Western blot. We observed that BUN and Scr were increased in diabetic rats, and Ccr was decreased. Furthermore, blood glucose fluctuations could exacerbate the Ccr changes. Renal tissue ultrastructure was also seriously injured by glucose variability in diabetic rats. In addition, glucose fluctuation increased the oxidative stress of renal tissue. Moreover, fluctuating blood glucose decreased p-AKT level and BCL-2, and increased p-GSK-3β, BAX, cleaved-caspase-3 levels, and ratio of BAX/BCL-2 in the kidneys of diabetic rats. In conclusion, these results suggest that blood glucose fluctuation accelerated renal injury is due, at least in part to its oxidative stress promoting and inhibiting the AKT signaling pathway in diabetic rats. PMID:26860515

  7. TNFα Mediated IL-6 Secretion Is Regulated by JAK/STAT Pathway but Not by MEK Phosphorylation and AKT Phosphorylation in U266 Multiple Myeloma Cells

    PubMed Central

    Lee, Chansu; Oh, Jeong-In; Park, Juwon; Choi, Jee-Hye; Bae, Eun-Kyung; Lee, Hyun Jung; Jung, Woo June; Lee, Dong Soon; Ahn, Kwang-Sung; Yoon, Sung-Soo

    2013-01-01

    IL-6 and TNFα were significantly increased in the bone marrow aspirate samples of patients with active multiple myeloma (MM) compared to those of normal controls. Furthermore, MM patients with advanced aggressive disease had significantly higher levels of IL-6 and TNFα than those with MM in plateau phase. TNFα increased interleukin-6 (IL-6) production from MM cells. However, the detailed mechanisms involved in signaling pathways by which TNFα promotes IL-6 secretion from MM cells are largely unknown. In our study, we found that TNFα treatments induce MEK and AKT phosphorylation. TNFα-stimulated IL-6 production was abolished by inhibition of JAK2 and IKKβ or by small interfering RNA (siRNA) targeting TNF receptors (TNFR) but not by MEK, p38, and PI3K inhibitors. Also, TNFα increased phosphorylation of STAT3 (ser727) including c-Myc and cyclin D1. Three different types of JAK inhibitors decreased the activation of the previously mentioned pathways. In conclusion, blockage of JAK/STAT-mediated NF-κB activation was highly effective in controlling the growth of MM cells and, consequently, an inhibitor of TNFα-mediated IL-6 secretion would be a potential new therapeutic agent for patients with multiple myeloma. PMID:24151609

  8. Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients

    PubMed Central

    Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Matsushima-Nishiwaki, Rie; Akamatsu, Shigeru; Enomoto, Yukiko; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

    2016-01-01

    It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9–25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25–50 µm), large aggregates (50–70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients. PMID:27187380

  9. Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients.

    PubMed

    Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Matsushima-Nishiwaki, Rie; Akamatsu, Shigeru; Enomoto, Yukiko; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

    2016-01-01

    It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9-25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25-50 µm), large aggregates (50-70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients. PMID:27187380

  10. Sustained Oxidative Stress Causes Late Acute Renal Failure via Duplex Regulation on p38 MAPK and Akt Phosphorylation in Severely Burned Rats

    PubMed Central

    Cai, Xiaoqing; Wang, Dexin; Wu, Kaimin; Chen, Hongli; Li, Jia; Lei, Wei

    2013-01-01

    Background Clinical evidence indicates that late acute renal failure (ARF) predicts high mortality in severely burned patients but the pathophysiology of late ARF remains undefined. This study was designed to test the hypothesis that sustained reactive oxygen species (ROS) induced late ARF in a severely burned rat model and to investigate the signaling mechanisms involved. Materials and Methods Rats were exposed to 100°C bath for 15 s to induce severe burn injury (40% of total body surface area). Renal function, ROS generation, tubular necrosis and apoptosis, and phosphorylation of MAPK and Akt were measured during 72 hours after burn. Results Renal function as assessed by serum creatinine and blood urea nitrogen deteriorated significantly at 3 h after burn, alleviated at 6 h but worsened at 48 h and 72 h, indicating a late ARF was induced. Apoptotic cells and cleavage caspase-3 in the kidney went up slowly and turned into significant at 48 h and 72 h. Tubular cell ROS production shot up at 6 h and continuously rose during the 72-h experiment. Scavenging ROS with tempol markedly attenuated tubular apoptosis and renal dysfunction at 72 h after burn. Interestingly, renal p38 MAPK phosphorylation elevated in a time dependent manner whereas Akt phosphorylation increased during the first 24 h but decreased at 48 h after burn. The p38 MAPK specific inhibitor SB203580 alleviated whereas Akt inhibitor exacerbated burn-induced tubular apoptosis and renal dysfunction. Furthermore, tempol treatment exerted a duplex regulation through inhibiting p38 MAPK phosphorylation but further increasing Akt phosphorylation at 72 h postburn. Conclusions These results demonstrate that sustained renal ROS overproduction induces continuous tubular cell apoptosis and thus a late ARF at 72 h after burn in severely burned rats, which may result from ROS-mediated activation of p38 MAPK but a late inhibition of Akt phosphorylation. PMID:23349934

  11. A polysaccharide from Grifola frondosa relieves insulin resistance of HepG2 cell by Akt-GSK-3 pathway.

    PubMed

    Ma, Xiaolei; Zhou, Fuchuan; Chen, Yuanyuan; Zhang, Yuanyuan; Hou, Lihua; Cao, Xiaohong; Wang, Chunling

    2014-07-01

    Grifola frondosa is an important fungal research resource. However, there was little report about hyperglycemic activity of Grifola frondosa polysaccharide on insulin resistance in vitro. In this study, the hypoglycemic activity of a polysaccharide obtained from Grifola frondosa (GFP) on HepG2 cell and hpyerglycemic mechanism were investigated. The purity of the isolated polysaccharides was examined by HPLC. In this research, it was found that GFP enhanced the absorption of glucose of HepG2 cells in a dose dependent manner at 24 h of 30 ugmL⁻¹. GC-MS and FT-IR spectroscopy analysis results showed that glucose and galactose were the dominant monosaccharides in GFP and the major component of GFP was β-pyranoside. Western-blotting results showed that the HepG2 cell model treated with GFP activated the insulin receptor protein (IRS) in the cell membrane and increased phosphorylated-AktSer473 expression, which had an inhibition of glycogen synthase kinase (GSK-3). The down-regulation of GSK-3 stimulated synthesis of intracellular glycogen. The results above suggested that the GFP increased the metabolism of glucose and stimulated synthesis of intracellular glycogen through the Akt/GSK-3 pathway. PMID:24908430

  12. Sestrin 3 Protein Enhances Hepatic Insulin Sensitivity by Direct Activation of the mTORC2-Akt Signaling

    PubMed Central

    Tao, Rongya; Xiong, Xiwen; Liangpunsakul, Suthat

    2015-01-01

    Sestrin proteins have been implicated in multiple biological processes including resistance to oxidative and genotoxic stresses, protection against aging-related pathologies, and promotion of metabolic homeostasis; however, the underlying mechanisms are incompletely understood. Some evidence suggests that sestrins may inhibit mTORC1 (mechanistic target of rapamycin complex 1) through inhibition of RagA/B GTPases or activation of AMPK; however, whether sestrins are also involved in mTORC2 regulation and function is unclear. To investigate the functions and mechanisms of Sestrin 3 (Sesn3), we generated Sesn3 liver-specific transgenic and knockout mice. Our data show that Sesn3 liver-specific knockout mice exhibit insulin resistance and glucose intolerance, and Sesn3 transgenic mice were protected against insulin resistance induced by a high-fat diet. Using AMPK liver-specific knockout mice, we demonstrate that the Sesn3 insulin-sensitizing effect is largely independent of AMPK. Biochemical analysis reveals that Sesn3 interacts with and activates mTORC2 and subsequently stimulates Akt phosphorylation at Ser473. These findings suggest that Sesn3 can activate Akt via mTORC2 to regulate hepatic insulin sensitivity and glucose metabolism. PMID:25377878

  13. Vitamin B₂ Sensitizes Cancer Cells to Vitamin-C-Induced Cell Death via Modulation of Akt and Bad Phosphorylation.

    PubMed

    Chen, Ni; Yin, Shutao; Song, Xinhua; Fan, Lihong; Hu, Hongbo

    2015-08-01

    Vitamin C is an essential dietary nutrient that has a variety of biological functions. Recent studies have provided promising evidence for its additional health benefits, including anticancer activity. Vitamin B2, another essential dietary nutrient, often coexists with vitamin C in some fruits, vegetables, or dietary supplements. The objective of the present study is to determine whether the combination of vitamin C and B2 can achieve a synergistic anticancer activity. MDA-MB-231, MCF-7, and A549 cells were employed to evaluate the combinatory effects of vitamin C and B2. We found that the combination of vitamin C and B2 resulted in a synergistic cell death induction in all cell lines tested. Further mechanistic investigations revealed that vitamin B2 sensitized cancer cells to vitamin C through inhibition of Akt and Bad phosphorylation. Our findings identified vitamin B2 as a promising sensitizer for improving the efficacy of vitamin-C-based cancer chemoprevention and chemotherapy. PMID:26165392

  14. Lack of SIRPα phosphorylation and concomitantly reduced SHP-2-PI3K-Akt2 signaling decrease osteoblast differentiation.

    PubMed

    Holm, Cecilia Koskinen; Engman, Sara; Sulniute, Rima; Matozaki, Takashi; Oldenborg, Per-Arne; Lundberg, Pernilla

    2016-09-01

    Normal differentiation of bone forming osteoblasts is a prerequisite for maintenance of skeletal health and is dependent on intricate cellular signaling pathways, including the essential transcription factor Runx2. The cell surface glycoprotein CD47 and its receptor signal regulatory protein alpha (SIRPα) have both been suggested to regulate bone cell differentiation. Here we investigated osteoblastic differentiation of bone marrow stromal cells from SIRPα mutant mice lacking the cytoplasmic signaling domain of SIRPα. An impaired osteoblastogenesis in SIRPα-mutant cell cultures was demonstrated by lower alkaline phosphatase activity and less mineral formation compared to wild-type cultures. This reduced osteoblastic differentiation potential in SIRPα-mutant stromal cells was associated with a significantly reduced expression of Runx2, osterix, osteocalcin, and alkaline phosphatase mRNA, as well as a reduced phosphorylation of SHP-2 and Akt2, as compared with that in wild-type stromal cells. Addition of a PI3K-inhibitor to wild-type stromal cells could mimic the impaired osteoblastogenesis seen in SIRPα-mutant cells. In conclusion, our data suggest that SIRPα signaling through SHP-2-PI3K-Akt2 strongly influences osteoblast differentiation from bone marrow stromal cells. PMID:27422603

  15. Galactose-1 phosphate uridylyltransferase (GalT) gene: A novel positive regulator of the PI3K/Akt signaling pathway in mouse fibroblasts.

    PubMed

    Balakrishnan, Bijina; Chen, Wyman; Tang, Manshu; Huang, Xiaoping; Cakici, Didem Demirbas; Siddiqi, Anwer; Berry, Gerard; Lai, Kent

    2016-01-29

    The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan-Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3β (-74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT(2) Profiler PCR Custom Arrays, the mRNA abundance of Akt1, Pdpk1, Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 min, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels. PMID:26773505

  16. GGA3 mediates TrkA endocytic recycling to promote sustained Akt phosphorylation and cell survival

    PubMed Central

    Li, Xuezhi; Lavigne, Pierre; Lavoie, Christine

    2015-01-01

    Although TrkA postendocytic sorting significantly influences neuronal cell survival and differentiation, the molecular mechanism underlying TrkA receptor sorting in the recycling or degradation pathways remains poorly understood. Here we demonstrate that Golgi-localized, γ adaptin-ear–containing ADP ribosylation factor-binding protein 3 (GGA3) interacts directly with the TrkA cytoplasmic tail through an internal DXXLL motif and mediates the functional recycling of TrkA to the plasma membrane. We find that GGA3 depletion by siRNA delays TrkA recycling, accelerates TrkA degradation, attenuates sustained NGF-induced Akt activation, and reduces cell survival. We also show that GGA3’s effect on TrkA recycling is dependent on the activation of Arf6. This work identifies GGA3 as a key player in a novel DXXLL-mediated endosomal sorting machinery that targets TrkA to the plasma membrane, where it prolongs the activation of Akt signaling and survival responses. PMID:26446845

  17. Equol inhibits proliferation of human gastric carcinoma cells via modulating Akt pathway

    PubMed Central

    Yang, Zhi-Ping; Zhao, Yan; Huang, Fang; Chen, Jie; Yao, Ya-Hong; Li, Jun; Wu, Xiao-Nan

    2015-01-01

    AIM: To investigate the anti-tumor effects of equol in gastric cancer cells and the underlying molecular mechanisms. METHODS: MGC-803 cells were employed for in vitro experiments in this study. Cells were treated with control (vehicle, 0.1% DMSO) or equol under specified dose titration or time courses. Cell viability was examined by MTS assay, and the levels of Ki67 were determined by qPCR and immunofluorescent assay. Changes in cell cycle distribution and apoptosis rate were detected by flow cytometry. The mRNA expression of cyclin E1 and P21WAF1 was determined by qPCR. The protein levels of cell cycle regulators, PARP and Caspase-3 cleavage, and the phosphorylation of Akt were examined by Western blot. In addition, to characterize the role of elevated Akt activation in the anti-tumor effect exerted by equol, Ly294002, a PI3K/AKT pathway inhibitor, was used to pretreat MGC-803 cells. RESULTS: Equol (5, 10, 20, 40, or 80 μmol/L) inhibited viability of MGC-803 cells in a dose- and time-dependent manner after treatment for 24, 36, or 48 h (P < 0.05 for all). Equol also decreased the mRNA (P < 0.05 for 12 and 24 h treatment) and protein levels of Ki67. Equol treatment significantly induced G0/G1 cell cycle arrest (P < 0.05), with the percentages of G0/G1 cells of 32.23% ± 3.62%, 36.31% ± 0.24%, 45.58% ± 2.29%, and 65.10% ± 2.04% for equol (0, 10, 20, or 30 μmol/L) treatment, respectively, accompanied by a significant decrease of CDK2/4 (P < 0.05 for 24 and 48 h treatment) and Cyclin D1/Cyclin E1 (P < 0.05), and an increased level of P21WAF1 (P < 0.05). A marked increase of apoptosis was observed, with the percentages of apoptotic cells of 5.01% ± 0.91%, 14.57% ± 0.99%, 37.40% ± 0.58%, and 38.46% ± 2.01% for equol (0, 5, 10, or 20 μmol/L) treatment, respectively, accompanied by increased levels of cleaved PARP and caspase-3. In addition, we found that equol treatment increased P-Akt (Ser473 and Thr308) at 12 and 24 h compared to vehicle-treated control

  18. Phosphorylation of the translation initiation factor eIF2α at serine 51 determines the cell fate decisions of Akt in response to oxidative stress.

    PubMed

    Rajesh, K; Krishnamoorthy, J; Kazimierczak, U; Tenkerian, C; Papadakis, A I; Wang, S; Huang, S; Koromilas, A E

    2015-01-01

    Phosphorylation of the α subunit of the translation initiation factor eIF2 at serine 51 (eIF2αP) is a master regulator of cell adaptation to various forms of stress with implications in antitumor treatments with chemotherapeutic drugs. Herein, we demonstrate that genetic loss of the eIF2α kinases PERK and GCN2 or impaired eIF2αP by genetic means renders immortalized mouse fibroblasts as well as human tumor cells increasingly susceptible to death by oxidative stress. We also show that eIF2αP facilitates Akt activation in cells subjected to oxidative insults. However, whereas Akt activation has a pro-survival role in eIF2αP-proficient cells, the lesser amount of activated Akt in eIF2αP-deficient cells promotes death. At the molecular level, we demonstrate that eIF2αP acts through an ATF4-independent mechanism to control Akt activity via the regulation of mTORC1. Specifically, eIF2αP downregulates mTORC1 activity, which in turn relieves the feedback inhibition of PI3K resulting in the upregulation of the mTORC2-Akt arm. Inhibition of mTORC1 by rapamycin restores Akt activity in eIF2αP-deficient cells but renders them highly susceptible to Akt-mediated death by oxidative stress. Our data demonstrate that eIF2αP acts as a molecular switch that dictates either cell survival or death by activated Akt in response to oxidative stress. Hence, we propose that inactivation of eIF2αP may be a suitable approach to unleash the killing power of Akt in tumor cells treated with pro-oxidant drugs. PMID:25590801

  19. Matrix-Dependent Regulation of AKT in Hepsin-Overexpressing PC3 Prostate Cancer Cells12

    PubMed Central

    Wittig-Blaich, Stephanie M; Kacprzyk, Lukasz A; Eismann, Thorsten; Bewerunge-Hudler, Melanie; Kruse, Petra; Winkler, Eva; Strauss, Wolfgang S L; Hibst, Raimund; Steiner, Rudolf; Schrader, Mark; Mertens, Daniel; Sültmann, Holger; Wittig, Rainer

    2011-01-01

    The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox)-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser473, which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases. PMID:21750652

  20. Tripeptide SQL Inhibits Platelet Aggregation and Thrombus Formation by Affecting PI3K/Akt Signaling.

    PubMed

    Su, Xing-li; Su, Wen; He, Zhi-long; Ming, Xin; Kong, Yi

    2015-09-01

    Centipede has been prescribed for the treatment of cardiovascular diseases in Asian countries for several hundred years. Previously, a new antiplatelet tripeptide SQL (H-Ser-Gln-Leu-OH) was isolated and characterized from centipede. In this study, we investigated its antithrombotic activities in vivo and underlying mechanism. It was found that SQL inhibited platelet aggregation induced by adenosine diphosphate, thrombin, epinephrine, and collagen and attenuated thrombus formation in both the ferric chloride-induced arterial thrombosis model and arteriovenous shunt thrombosis model in rats. It did not prolong the bleeding time in mice even at the dose of 10 mg/kg that showed potent antithrombosis effects. Molecular docking revealed that SQL binds PI3Kβ with the binding free energy of -24.341 kcal/mol, which is close to that of cocrystallized ligand (-24.220 kcal/mol). Additionally, SQL displayed inhibition on the late (180 seconds) but did not influence the early (60 seconds) Akt Ser473 phosphorylation in the immunoblot assay. These results suggest that SQL inhibits thrombus formation in vivo and that SQL inhibits PI3K-mediated signaling or even the PI3K itself in platelets. This study may help elucidate the mechanism for centipede treating cardiovascular diseases. PMID:25923322

  1. Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin.

    PubMed

    Tee, Andrew R; Anjum, Rana; Blenis, John

    2003-09-26

    The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo. PMID:12867426

  2. Platycodin-D Induced Autophagy in Non-Small Cell Lung Cancer Cells via PI3K/Akt/mTOR and MAPK Signaling Pathways

    PubMed Central

    Zhao, Ruolin; Chen, Meijuan; Jiang, Zequn; Zhao, Fengming; Xi, Beili; Zhang, Xu; Fu, Haian; Zhou, Kunfu

    2015-01-01

    Platycodin-D (PD) is an effective triterpene saponin extracted from the root of Platycodon grandiflorum which has been used clinically to treat pulmonary diseases in traditional Chinese medicine. Recently, it has been reported that PD has anti-tumor effects in various cancer models through the induction of apoptosis. However, whether PD induces autophagy in both cell lines and its molecular mechanisms have not been elucidated. Here, our present study confirmed that PD induced autophagy in both NCI-H460 and A549 cells via up-regulating the expression levels of Atg-3, Atg-7 and Beclin-1. Meanwhile, PD contributed to the up-regulation of LC3-II at both protein and mRNA levels. Further detection of the PI3K/Akt/mTOR signaling pathway compared to LY294002 (PI3K kinase inhibitor), RAP (mTOR kinase inhibitor) and insulin (an activator of PI3K/Akt/mTOR signaling pathway) showed that PD induced autophagy through inhibiting the pathway at p-Akt (Ser473), p-p70S6K (Thr389) and p-4EBP1 (Thr37/46) in both cell lines. Moreover, the examination of MAPK signaling pathway showed that PD treatment increased the phosphorylation of JNK and p38 MAPK, while decreased the phosphorylation of Erk1/2 in both cell lines. Additionally, the effects assessed with a panel of pharmacologic inhibitors, including U0126 (Erk1/2 kinase inhibitor), SP600125 (JNK kinase inhibitor) and SB203580 (p38 MAPK kinase inhibitor) suggested that the activation of JNK and p38 MAPK participated in PD-induced autophagy. Taken together, these findings suggested that PD induced autophagy in NCI-H460 and A549 cells through inhibiting PI3K/Akt/mTOR signaling pathway and activating JNK and p38 MAPK signaling pathways. Therefore, PD may be an alternative compound for NSCLC therapy. PMID:26078792

  3. Platycodin-D Induced Autophagy in Non-Small Cell Lung Cancer Cells via PI3K/Akt/mTOR and MAPK Signaling Pathways.

    PubMed

    Zhao, Ruolin; Chen, Meijuan; Jiang, Zequn; Zhao, Fengming; Xi, Beili; Zhang, Xu; Fu, Haian; Zhou, Kunfu

    2015-01-01

    Platycodin-D (PD) is an effective triterpene saponin extracted from the root of Platycodon grandiflorum which has been used clinically to treat pulmonary diseases in traditional Chinese medicine. Recently, it has been reported that PD has anti-tumor effects in various cancer models through the induction of apoptosis. However, whether PD induces autophagy in both cell lines and its molecular mechanisms have not been elucidated. Here, our present study confirmed that PD induced autophagy in both NCI-H460 and A549 cells via up-regulating the expression levels of Atg-3, Atg-7 and Beclin-1. Meanwhile, PD contributed to the up-regulation of LC3-II at both protein and mRNA levels. Further detection of the PI3K/Akt/mTOR signaling pathway compared to LY294002 (PI3K kinase inhibitor), RAP (mTOR kinase inhibitor) and insulin (an activator of PI3K/Akt/mTOR signaling pathway) showed that PD induced autophagy through inhibiting the pathway at p-Akt (Ser473), p-p70S6K (Thr389) and p-4EBP1 (Thr37/46) in both cell lines. Moreover, the examination of MAPK signaling pathway showed that PD treatment increased the phosphorylation of JNK and p38 MAPK, while decreased the phosphorylation of Erk1/2 in both cell lines. Additionally, the effects assessed with a panel of pharmacologic inhibitors, including U0126 (Erk1/2 kinase inhibitor), SP600125 (JNK kinase inhibitor) and SB203580 (p38 MAPK kinase inhibitor) suggested that the activation of JNK and p38 MAPK participated in PD-induced autophagy. Taken together, these findings suggested that PD induced autophagy in NCI-H460 and A549 cells through inhibiting PI3K/Akt/mTOR signaling pathway and activating JNK and p38 MAPK signaling pathways. Therefore, PD may be an alternative compound for NSCLC therapy. PMID:26078792

  4. The acute effects of strength, endurance and concurrent exercises on the Akt/mTOR/p70(S6K1) and AMPK signaling pathway responses in rat skeletal muscle.

    PubMed

    de Souza, E O; Tricoli, V; Bueno Junior, C; Pereira, M G; Brum, P C; Oliveira, E M; Roschel, H; Aoki, M S; Urginowitsch, C

    2013-04-01

    The activation of competing intracellular pathways has been proposed to explain the reduced training adaptations after concurrent strength and endurance exercises (CE). The present study investigated the acute effects of CE, strength exercises (SE), and endurance exercises (EE) on phosphorylated/total ratios of selected AMPK and Akt/mTOR/p70(S6K1) pathway proteins in rats. Six animals per exercise group were killed immediately (0 h) and 2 h after each exercise mode. In addition, 6 animals in a non-exercised condition (NE) were killed on the same day and under the same conditions. The levels of AMPK, phospho-Thr(172)AMPK (p-AMPK), Akt, phospho-Ser(473)Akt (p-Akt), p70(S6K1), phospho-Thr(389)-p70(S6K1) (p-p70(S6K1)), mTOR, phospho-Ser(2448)mTOR (p-mTOR), and phospho-Thr(1462)-TSC2 (p-TSC2) expression were evaluated by immunoblotting in total plantaris muscle extracts. The only significant difference detected was an increase (i.e., 87%) in Akt phosphorylated/total ratio in the CE group 2 h after exercise compared to the NE group (P = 0.002). There were no changes in AMPK, TSC2, mTOR, or p70(S6K1) ratios when the exercise modes were compared to the NE condition (P ≥ 0.05). In conclusion, our data suggest that low-intensity and low-volume CE might not blunt the training-induced adaptations, since it did not activate competing intracellular pathways in an acute bout of strength and endurance exercises in rat skeletal muscle. PMID:23598645

  5. The acute effects of strength, endurance and concurrent exercises on the Akt/mTOR/p70S6K1 and AMPK signaling pathway responses in rat skeletal muscle

    PubMed Central

    Souza, E.O.de; Tricoli, V.; Bueno, C.; Pereira, M.G.; Brum, P.C.; Oliveira, E.M.; Roschel, H.; Aoki, M.S.; Urginowitsch, C.

    2013-01-01

    The activation of competing intracellular pathways has been proposed to explain the reduced training adaptations after concurrent strength and endurance exercises (CE). The present study investigated the acute effects of CE, strength exercises (SE), and endurance exercises (EE) on phosphorylated/total ratios of selected AMPK and Akt/mTOR/p70S6K1 pathway proteins in rats. Six animals per exercise group were killed immediately (0 h) and 2 h after each exercise mode. In addition, 6 animals in a non-exercised condition (NE) were killed on the same day and under the same conditions. The levels of AMPK, phospho-Thr172AMPK (p-AMPK), Akt, phospho-Ser473Akt (p-Akt), p70S6K1, phospho-Thr389-p70S6K1 (p-p70S6K1), mTOR, phospho-Ser2448mTOR (p-mTOR), and phospho-Thr1462-TSC2 (p-TSC2) expression were evaluated by immunoblotting in total plantaris muscle extracts. The only significant difference detected was an increase (i.e., 87%) in Akt phosphorylated/total ratio in the CE group 2 h after exercise compared to the NE group (P = 0.002). There were no changes in AMPK, TSC2, mTOR, or p70S6K1 ratios when the exercise modes were compared to the NE condition (P ≥ 0.05). In conclusion, our data suggest that low-intensity and low-volume CE might not blunt the training-induced adaptations, since it did not activate competing intracellular pathways in an acute bout of strength and endurance exercises in rat skeletal muscle. PMID:23598645

  6. Garlic Oil Suppressed Nitrosodiethylamine-Induced Hepatocarcinoma in Rats by Inhibiting PI3K-AKT-NF-κB Pathway.

    PubMed

    Zhang, Cui-Li; Zeng, Tao; Zhao, Xiu-Lan; Xie, Ke-Qin

    2015-01-01

    To explore the underlying mechanisms for the protective effects of garlic oil (GO) against nitrosodiethylamine (NDEA)-induced hepatocarcinoma, 60 male Wistar rats were randomized into 4 groups (n=15): control group, NDEA group, and two GO plus NDEA groups. The rats in GO plus NDEA groups were pretreated with GO (20 or 40 mg/kg) for 7 days. Then, all rats except those in control group were gavaged with NDEA for 20 weeks, and the rats in GO plus NDEA groups were continuously administered with GO. The results showed that GO co-treatment significantly suppressed the NDEA-induced increases of alpha fetal protein (AFP) level in serum, nuclear atypia in H&E staining, sirius red-positive areas and proliferating cell nuclear antigen (PCNA) expression. The molecular mechanisms exploration revealed that the protein levels of phosphatidylinositol 3 kinase (PI3K)-p85, PI3K-p110, total AKT, p-AKT (Ser473) and p-AKT (Thr308) in the liver of NDEA group rats were higher than those in control group rats. In addition, NDEA treatment induced IκB degradation and NF-κB p65 phosphorylation, and up-regulated the protein levels of downstream pro-inflammatory mediators. GO co-treatment significantly reversed all the above adverse effects induced by NDEA. These results suggested that the protective effects of GO against NDEA-induced hepatocarcinoma might be associated with the suppression of PI3K- AKT-NF-κB pathway. PMID:25999787

  7. Insulin-Dependent Regulation of mTORC2-Akt-FoxO Suppresses TLR4 Signaling in Human Leukocytes: Relevance to Type 2 Diabetes.

    PubMed

    Zhang, Zhiyong; Amorosa, Louis F; Coyle, Susette M; Macor, Marie A; Birnbaum, Morris J; Lee, Leonard Y; Haimovich, Beatrice

    2016-08-01

    Leukocyte signaling in patients with systemic insulin resistance is largely unexplored. We recently discovered the presence of multiple Toll-like receptor 4 (TLR4) signaling intermediates in leukocytes from patients with type 2 diabetes or acute insulin resistance associated with cardiopulmonary bypass surgery. We extend this work to show that in addition to matrix metalloproteinase 9, hypoxia-inducible factor 1α, and cleaved AMPKα, patient leukocytes also express IRS-1 phosphorylated on Ser(312), Akt phosphorylated on Thr(308), and elevated TLR4 expression. Similar signaling intermediates were detected in leukocytes and neutrophils treated with lipopolysaccharide (LPS), a ligand of TLR4, in vitro. In contrast, insulin, but not LPS, induced mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt on Ser(473) and FoxO1/O3a on Thr(24/32) in leukocytes and neutrophils. Insulin suppressed LPS-induced responses in a dose- and time-dependent manner. AS1842856, a FoxO1 inhibitor, also suppressed TLR4 signaling. We propose that insulin is a homeostatic regulator of leukocyte responses to LPS/TLR4 and that the signaling intermediates expressed in leukocytes of patients with type 2 diabetes indicate TLR4 signaling dominance and deficient insulin signaling. The data suggest that insulin suppresses LPS/TLR4 signals in leukocytes through the mTORC2-Akt-FoxO signaling axis. Better understanding of leukocyte signaling in patients with type 2 diabetes may shed new light on disease causation and progression. PMID:27207509

  8. Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19)

    PubMed Central

    Sangphech, Naunpun; Osborne, Barbara A.; Palaga, Tanapat

    2014-01-01

    Macrophages play critical roles in innate immune defense by sensing microbes using pattern-recognition receptors. Lipopolysaccharide (LPS) stimulates macrophages via TLR, which leads to activation of downstream signaling cascades. In this study, we investigated the roles of a conserved signaling pathway, Notch signaling, in regulating the downstream signaling cascades of the LPS/TLR4 pathways in macrophages. Using a phosphoproteomic approach and a gamma-secretase inhibitor (GSI) to suppress the processing and activation of Notch signaling, we identified regulator of G protein signaling 19 (RGS19) as a target protein whose phosphorylation was affected by GSI treatment. RGS19 is a guanosine triphosphatase (GTPase)-activating protein that functions to negatively regulate G protein-coupled receptors via Gαi/Gαq-linked signaling. Stimulation of RAW264.7 cells with LPS increased the level of the phosphorylated form of RGS19, while LPS stimulation in the presence of GSI decreased its level. GSI treatment did not alter the mRNA level of rgs19. Treatment with GSI or silencing of rgs19 in macrophages impaired the phosphorylation of Akt Thr308 upon LPS stimulation. Furthermore, targeted deletion of a DNA-binding protein and binding partner of the Notch receptor, RBP-Jκ/CSL, in macrophages resulted in delayed and decreased Akt phosphorylation. Because the PI3K/Akt pathway regulates cell survival in various cell types, the cell cycle and cell death were assayed upon GSI treatment, phosphatidylinositol 3 kinase (PI3K) inhibitor treatment or silencing of rgs19. GSI treatment resulted in decreased cell populations in the G1 and S phases, while it increased the cell population of cell death. Similarly, silencing of rgs19 resulted in a decreased cell population in the G1 phase and an increased cell population in the subG1 phase. Inhibition of Akt phosphorylation by PI3K inhibitor in LPS-stimulated macrophages increased cell population in G1 phase, suggesting a possible cell cycle

  9. Inactivation of the phosphatidylinositol 3-kinase/Akt pathway is involved in BMP9-mediated tumor-suppressive effects in gastric cancer cells.

    PubMed

    Duan, Liang; Ye, Liwei; Wu, Rui; Wang, Haiyan; Li, Xueru; Li, Huan; Yuan, Shimei; Zha, He; Sun, Hui; Zhang, Yunyuan; Chen, Xian; Zhang, Yan; Zhou, Lan

    2015-06-01

    Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily signaling factors. Expression of several BMPs (BMP2, BMP4, and BMP7) is correlated to poor prognosis in gastric cancer patients. The function of BMP9, the latest discovered and most powerful osteogenetic factor, in gastric cancer is relatively unclear. In this report, we investigated the expression, function and underlying molecular mechanisms of BMP9 in gastric cancer. The results show that BMP9 expression was markedly decreased in gastric cancer tissues and cell lines. Enforced BMP9 expression in the gastric cancer cell lines SGC-7901 and MNK-45 increased apoptosis and reduced viability and migration. The in vivo function of BMP9 was evaluated in a xenograft mouse model. Tumors derived from SGC-7901 cells with enforced BMP9 expression (SGC-7901/BMP9) showed significantly reduced size and weight compared to that from control cells. Enforced BMP9 expression resulted in decreased Akt activity shown as lower levels of phosphorylation at Ser473 and Thr308 in Akt. The PI3K/Akt inhibitor LY294002 potentiated BMP9's viability and migration suppression, and apoptosis induction, which was associated with reduced expression of snail and VEGF and increased expression of E-cadherin. In addition, tumors derived from SGC-7901/BMP9 showed reduced Akt activity and VEGF expression, and increased E-cadherin expression. Therefore, our studies reveal for the first time that inhibition of the PI3K-Akt pathway is involved in the tumor suppressor effects of BMP9 in gastric cancer. PMID:25640278

  10. Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation*

    PubMed Central

    Secondo, Agnese; Esposito, Alba; Sirabella, Rossana; Boscia, Francesca; Pannaccione, Anna; Molinaro, Pasquale; Cantile, Maria; Ciccone, Roselia; Sisalli, Maria Josè; Scorziello, Antonella; Di Renzo, Gianfranco; Annunziato, Lucio

    2015-01-01

    NGF induces neuronal differentiation by modulating [Ca2+]i. However, the role of the three isoforms of the main Ca2+-extruding system, the Na+/Ca2+ exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca2+]i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca2+ content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na+ currents and 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na+]i significantly increased in cells overexpressing NCX1.4 as well as ER Ca2+ content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca2+]i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation

  11. Protective effect of rutin on LPS-induced acute lung injury via down-regulation of MIP-2 expression and MMP-9 activation through inhibition of Akt phosphorylation.

    PubMed

    Chen, Wen-Ying; Huang, Yi-Chun; Yang, Ming-Ling; Lee, Chien-Ying; Chen, Chun-Jung; Yeh, Chung-Hsin; Pan, Pin-Ho; Horng, Chi-Ting; Kuo, Wu-Hsien; Kuan, Yu-Hsiang

    2014-10-01

    Lipopolysaccharide (LPS), also called endotoxin, is the important pathogen of acute lung injury (ALI), which is a clinical syndrome that still lacks effective therapeutic medicine. Rutin belongs to vitamin P and possesses various beneficial effects. In this study, we investigate the potential protective effects and the mechanisms of rutin on LPS-induced ALI. Pre-administration with rutin inhibited LPS-induced arterial blood gas exchange and neutrophils infiltration in the lungs. LPS-induced expression of macrophage inflammatory protein (MIP)-2 and activation of matrix metalloproteinase (MMP)-9 were suppressed by rutin. In addition, the inhibitory concentration of rutin on phosphorylation of Akt was similar as MIP-2 expression and MMP-9 activation. In conclusion, rutin is a potential protective agent for ALI via suppressing the blood gas exchange and neutrophil infiltration. The mechanism of rutin is down-regulation of MIP-2 expression and MMP-9 activation through inhibition of Akt phosphorylation. PMID:25091621

  12. Cardamonin inhibits angiotensin II-induced vascular smooth muscle cell proliferation and migration by downregulating p38 MAPK, Akt, and ERK phosphorylation.

    PubMed

    Shen, Yan-Jing; Zhu, Xue-Xin; Yang, Xian; Jin, Bo; Lu, Jin-Jian; Ding, Bin; Ding, Zhi-Shan; Chen, Su-Hong

    2014-07-01

    Cardamonin is a chalconoid isolated from various herbs, such as Alpinia katsumadai and Carya cathayensis Sarg. This study examined the effect of cardamonin on angiotensin II (Ang II)-induced proliferation and migration in rat vascular smooth muscle cells (VSMCs) as well as its underlying mechanisms. The results showed that cardamonin significantly inhibited Ang II-induced proliferation and migration in rat VSMCs in a concentration-dependent manner. Moreover, cardamonin suppressed Ang II-induced phosphorylation of p38 MAPK, Akt, and extracellular regulated protein kinase (ERK). These findings indicate that the downregulation of p38 MAPK, Akt, and ERK phosphorylation might be, at least in part, involved in cardamonin-suppressed proliferation and migration induced by Ang II in rat VSMCs. As proliferation and migration of VSMCs play critical roles in the pathogenesis of atherosclerosis, cardamonin might be a potential candidate for atherosclerosis treatment. PMID:24595849

  13. Microcystin-LR promotes proliferation by activating Akt/S6K1 pathway and disordering apoptosis and cell cycle associated proteins phosphorylation in HL7702 cells.

    PubMed

    Liu, Jinghui; Wang, Hao; Wang, Beilei; Chen, Tao; Wang, Xiaofeng; Huang, Pu; Xu, Lihong; Guo, Zonglou

    2016-01-01

    Our previous studies had shown that MC-LR inhibited PP2A activity and hyperphosphorylated PP2A substrates at 24 h exposure in HL7702 cells. Although the cytoskeleton was rearranged, the cellular effects were not observed. The purpose of the present study with HL7702 cell exposed to MC-LR for 1-72 h was to further uncover the adverse effects of MC-LR comprehensively. The results showed that there were no obvious difference in apoptosis rate and cell-cycle distribution but the cell proliferation was changed since 36 h exposure while the uptake of MC-LR and its binding to PP2A/C kept unchanged since 1h exposure. PP2A activity had not manifested continued decline compare to 24h exposure and PP2A regulator α4 was found to release its associated PP2A/C since 1h exposure. The increasing of p-Akt-T308, p-Akt-S473, p-S6K1, p-S6, and p-4E-BP1 since 1h MC-LR exposure indicated that Akt/S6K1 cascade had been activated as early as 1h MC-LR treatment. And, PI3K/Akt inhibitor (LY294002) blocked MC-LR-induced Akt/S6K1 activation and proliferation. Besides, MC-LR also led to hyperphosphorylation of c-Myc, c-Jun, Bcl-2 and Bad and activation of Cdk1. Our study indicated that MC-LR exposure promoted HL7702 cell proliferation and the main mechanism was the activation of Akt/S6K1 cascade. Meanwhile, hyperphosphorylation of Bcl-2, Bad, c-Myc and c-Jun might also be involved. And, the inhibition of PP2A was the major reason for these molecular changes. PMID:26506538

  14. Amoxicillin and amoxicillin/clavulanate reduce ethanol intake and increase GLT-1 expression as well as AKT phosphorylation in mesocorticolimbic regions.

    PubMed

    Goodwani, Sunil; Rao, P S S; Bell, Richard L; Sari, Youssef

    2015-10-01

    Studies have shown that administration of the β-lactam antibiotic ceftriaxone (CEF) attenuates ethanol consumption and cocaine seeking behavior as well as prevents ethanol-induced downregulation of glutamate transporter 1 (GLT-1) expression in central reward brain regions. However, it is not known if these effects are compound-specific. Therefore, the present study examined the effects of two other β-lactam antibiotics, amoxicillin (AMOX) and amoxicillin/clavulanate (Augmentin, AUG), on ethanol drinking, as well as GLT-1 and phosphorylated-AKT (pAKT) levels in the nucleus accumbens (Acb) and medial prefrontal cortex (mPFC) of alcohol-preferring (P) rats. P rats were exposed to free-choice of ethanol (15% and 30%) for five weeks and were given five consecutive daily i.p. injections of saline vehicle, 100 mg/kg AMOX or 100mg/kg AUG. Both compounds significantly decreased ethanol intake and significantly increased GLT-1 expression in the Acb. AUG also increased GLT-1 expression in the mPFC. Results for changes in pAKT levels matched those for GLT-1, indicating that β-lactam antibiotic-induced reductions in ethanol intake are negatively associated with increases in GLT-1 and pAKT levels within two critical brains regions mediating drug reward and reinforcement. These findings add to a growing literature that pharmacological increases in GLT-1 expression are associated with decreases in ethanol intake and suggest that one mechanism mediating this effect may be increased phosphorylation of AKT. Thus, GLT-1 and pAKT may serve as molecular targets for the treatment of alcohol and drug abuse/dependence. PMID:26168897

  15. Phosphorylation of Akt Mediates Anti-Inflammatory Activity of 1-p-Coumaroyl β-D-Glucoside Against Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells

    PubMed Central

    Vo, Van Anh; Lee, Jae-Won; Kim, Ji-Young; Park, Jun-Ho; Lee, Hee Jae; Kim, Sung-Soo; Kwon, Yong-Soo

    2014-01-01

    Hydroxycinnamic acids have been reported to possess numerous pharmacological activities such as antioxidant, anti-inflammatory, and anti-tumor properties. However, the biological activity of 1-p-coumaroyl β-D-glucoside (CG), a glucose ester derivative of p-coumaric acid, has not been clearly examined. The objective of this study is to elucidate the anti-inflammatory action of CG in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. In the present study, CG significantly suppressed LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2 and the protein expression of iNOS and COX-2. CG also inhibited LPS-induced secretion of pro-inflammatory cytokines, IL-1β and TNF-α. In addition, CG significantly suppressed LPS-induced degradation of IκB. To elucidate the underlying mechanism by which CG exerts its anti-inflammatory action, involvement of various signaling pathways were examined. CG exhibited significantly increased Akt phosphorylation in a concentration-dependent manner, although MAPKs such as Erk, JNK, and p38 appeared not to be involved. Furthermore, inhibition of Akt/PI3K signaling pathway with wortmannin significantly, albeit not completely, abolished CG-induced Akt phosphorylation and anti-inflammatory actions. Taken together, the present study demonstrates that Akt signaling pathway might play a major role in CG-mediated anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells. PMID:24634601

  16. PFKL/miR-128 axis regulates glycolysis by inhibiting AKT phosphorylation and predicts poor survival in lung cancer.

    PubMed

    Yang, Jie; Li, Jingqiu; Le, Yanping; Zhou, Chengwei; Zhang, Shun; Gong, Zhaohui

    2016-01-01

    MicroRNAs (miRNAs) affect cancer cell glucose metabolism by targeting mRNAs of diverse enzymes that have been implicated in oxidative phosphorylation (OXPHOS) and glycolytic pathways. However, the mechanisms that underlie miRNA-mediated regulation of phosphofructokinase (PFK), a key rate-limiting enzyme in glycolysis, remain largely unknown. Here, we show that miR-128 directly targets PFK liver type (PFKL) in lung cancer cells and regulates endogenous expression of PFKL at both the mRNA and protein levels. In line with this, overexpression of miR-128 decreased glucose uptake and lactate production, as well as increased cellular ATP content. Interestingly, knockdown of miR-128 was shown to promote lung cancer cell growth and colony formation. Moreover, we observed that miR-128 expression inversely correlated with PFKL mRNA levels in clinic lung cancer samples and that increased PFKL expression predicted poor overall survival in lung cancer patients. Mechanistically, we showed that miR-128 regulates PFKL via a feedback loop that involves inhibition of the AKT signaling pathway. Together, our results suggest that miR-128 acts as a metabolic regulator in lung cancer cells that may be therapeutically exploited. PMID:27186417

  17. PFKL/miR-128 axis regulates glycolysis by inhibiting AKT phosphorylation and predicts poor survival in lung cancer

    PubMed Central

    Yang, Jie; Li, Jingqiu; Le, Yanping; Zhou, Chengwei; Zhang, Shun; Gong, Zhaohui

    2016-01-01

    MicroRNAs (miRNAs) affect cancer cell glucose metabolism by targeting mRNAs of diverse enzymes that have been implicated in oxidative phosphorylation (OXPHOS) and glycolytic pathways. However, the mechanisms that underlie miRNA-mediated regulation of phosphofructokinase (PFK), a key rate-limiting enzyme in glycolysis, remain largely unknown. Here, we show that miR-128 directly targets PFK liver type (PFKL) in lung cancer cells and regulates endogenous expression of PFKL at both the mRNA and protein levels. In line with this, overexpression of miR-128 decreased glucose uptake and lactate production, as well as increased cellular ATP content. Interestingly, knockdown of miR-128 was shown to promote lung cancer cell growth and colony formation. Moreover, we observed that miR-128 expression inversely correlated with PFKL mRNA levels in clinic lung cancer samples and that increased PFKL expression predicted poor overall survival in lung cancer patients. Mechanistically, we showed that miR-128 regulates PFKL via a feedback loop that involves inhibition of the AKT signaling pathway. Together, our results suggest that miR-128 acts as a metabolic regulator in lung cancer cells that may be therapeutically exploited. PMID:27186417

  18. Inhibition of akt phosphorylation diminishes mitochondrial biogenesis regulators, tricarboxylic acid cycle activity and exacerbates recognition memory deficit in rat model of Alzheimer's disease.

    PubMed

    Shaerzadeh, Fatemeh; Motamedi, Fereshteh; Khodagholi, Fariba

    2014-11-01

    3-Methyladenine (3-MA), as a PI3K inhibitor, is widely used for inhibition of autophagy. Inhibition of PI3K class I leads to inhibition of Akt phosphorylation, a central molecule involved in diverse arrays of intracellular cascades in nervous system. Accordingly, in the present study, we aimed to determine the alterations of specific mitochondrial biogenesis markers and mitochondrial function in 3-MA-injected rats following amyloid beta (Aβ) insult. Our data revealed that inhibition of Akt phosphorylation downregulates master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Our data also showed that decrease in PGC-1α level presumably is due to decrease in the phosphorylation of cAMP-response element binding and AMP-activated kinase, two upstream activators of PGC-1α. As a consequence, the level of some mitochondrial biogenesis factors including nuclear respiratory factor-1, mitochondrial transcription factor A, and Cytochrome c decreased significantly. Also, activities of tricarboxylic acid cycle (TCA) enzymes such as Aconitase, a-ketoglutarate dehydrogenase, and malate dehydrogenase reduced in the presence of 3-MA with or without Aβ insult. Decrease in mitochondrial biogenesis factors and TCA enzyme activity in the rats receiving 3-MA and Aβ were more compared to the rats that received either alone; indicating the additive destructive effects of these two agents. In agreement with our molecular results, data obtained from behavioral test (using novel objective recognition test) indicated that inhibition of Akt phosphorylation with or without Aβ injection impaired novel recognition (non-spatial) memory. Our results suggest that 3-MA amplified deleterious effects of Aβ by targeting central molecule Akt. PMID:25135709

  19. PARP-inhibitor treatment prevents hypertension induced cardiac remodeling by favorable modulation of heat shock proteins, Akt-1/GSK-3β and several PKC isoforms.

    PubMed

    Deres, Laszlo; Bartha, Eva; Palfi, Anita; Eros, Krisztian; Riba, Adam; Lantos, Janos; Kalai, Tamas; Hideg, Kalman; Sumegi, Balazs; Gallyas, Ferenc; Toth, Kalman; Halmosi, Robert

    2014-01-01

    Spontaneously hypertensive rat (SHR) is a suitable model for studies of the complications of hypertension. It is known that activation of poly(ADP-ribose) polymerase enzyme (PARP) plays an important role in the development of postinfarction as well as long-term hypertension induced heart failure. In this study, we examined whether PARP-inhibitor (L-2286) treatment could prevent the development of hypertensive cardiopathy in SHRs. 6-week-old SHR animals were treated with L-2286 (SHR-L group) or placebo (SHR-C group) for 24 weeks. Wistar-Kyoto rats were used as aged-matched, normotensive controls (WKY group). Echocardiography was performed, brain-derived natriuretic peptide (BNP) activity and blood pressure were determined at the end of the study. We detected the extent of fibrotic areas. The amount of heat-shock proteins (Hsps) and the phosphorylation state of Akt-1(Ser473), glycogen synthase kinase (GSK)-3β(Ser9), forkhead transcription factor (FKHR)(Ser256), mitogen activated protein kinases (MAPKs), and protein kinase C (PKC) isoenzymes were monitored. The elevated blood pressure in SHRs was not influenced by PARP-inhibitor treatment. Systolic left ventricular function and BNP activity did not differ among the three groups. L-2286 treatment decreased the marked left ventricular (LV) hypertrophy which was developed in SHRs. Interstitial collagen deposition was also decreased by L-2286 treatment. The phosphorylation of extracellular signal-regulated kinase (ERK)1/2(Thr183-Tyr185), Akt-1(Ser473), GSK-3β(Ser9), FKHR(Ser256), and PKC ε(Ser729) and the level of Hsp90 were increased, while the activity of PKC α/βII(Thr638/641), ζ/λ(410/403) were mitigated by L-2286 administration. We could detect signs of LV hypertrophy without congestive heart failure in SHR groups. This alteration was prevented by PARP inhibition. Our results suggest that PARP-inhibitor treatment has protective effect already in the early stage of hypertensive myocardial remodeling. PMID

  20. Mouse hippocampal phosphorylation footprint induced by generalized seizures: Focus on ERK, mTORC1 and Akt/GSK-3 pathways.

    PubMed

    Gangarossa, Giuseppe; Sakkaki, Sophie; Lory, Philippe; Valjent, Emmanuel

    2015-12-17

    Exacerbated hippocampal activity has been associated to critical modifications of the intracellular signaling pathways. We have investigated rapid hippocampal adaptive responses induced by maximal electroshock seizure (MES). Here, we demonstrate that abnormal and exacerbated hippocampal activity induced by MES triggers specific and temporally distinct patterns of phosphorylation of extracellular signal-related kinase (ERK), mammalian target of rapamycin complex (mTORC) and Akt/glycogen synthase kinase-3 (Akt/GSK-3) pathways in the mouse hippocampus. While the ERK pathway is transiently activated, the mTORC1 cascade follows a rapid inhibition followed by a transient activation. This rebound of mTORC1 activity leads to the selective phosphorylation of p70S6K, which is accompanied by an enhanced phosphorylation of the ribosomal subunit S6. In contrast, the Akt/GSK-3 pathway is weakly altered. Finally, MES triggers a rapid upregulation of several plasticity-associated genes as a consequence exacerbated hippocampal activity. The results reported in the present study are reminiscent of the one observed in other models of generalized seizures, thus defining a common molecular footprint induced by intense and aberrant hippocampal activities. PMID:26545981

  1. N-Hydroxycinnamide derivatives of osthole inhibit cell migration and invasion by suppressing Smad2 and Akt pathways in human colorectal adenocarcinoma cells.

    PubMed

    Liu, Ling-Yu; Huang, Wei-Jan; Ho, Feng-Ming; Lin, Ren-Jye; Lin, Shyr-Yi; Suk, Fat-Moon; Liang, Yu-Chih

    2014-06-25

    WJ1376-1 and WJ1398-1 are new synthetic compounds developed based on the structure of the Chinese herbal medicine osthole. Previously, we reported that WJ1376-1 and WJ1398-1 can induce cell-cycle arrest by activating ATR kinase (ataxia telangiectasia and rad3 related kinase) and inhibiting the phosphorylation of Aurora A kinase. In this study, we determined that WJ1376-1 and WJ1398-1 strongly inhibited the migration and invasion in human colorectal cancer cells at concentrations as low as 1μM. In the transforming growth factor (TGF)-β-induced epithelial-mesenchymal transition model, WJ1376-1 and WJ1398-1 potently downregulated the transcription factor Snail1, the mesenchymal protein vimentin, and matrix metalloprotease-9, but upregulated the epithelial protein E-cadherin. WJ1376-1 and WJ1398-1 also inhibited the TGF-β-induced phosphorylation of Smad2 and of Akt at Ser 473, and the nuclear translocation of Smad2 was substantially lower in WJ1376-1- and WJ1398-1-treated cells than it was in control cells. In transient transfection experiments, we observed that WJ1376-1 and WJ1398-1 strongly inhibited TGF-β-stimulated activity of a Smad reporter. Finally, WJ1376-1 and WJ1398-1 blocked TGF-β-induced phosphorylation of the TGF-β Type I receptor (TGF-βRI). These results suggest that WJ1376-1 and WJ1398-1 inhibit cell migration and invasion by suppressing TGF-βRI phosphorylation and subsequently hindering both Smad2 and phosphatidylinositol 3-kinase/Akt signaling pathways. PMID:24727557

  2. Ciliary Neurotrophic Factor Promotes the Migration of Corneal Epithelial Stem/progenitor Cells by Up-regulation of MMPs through the Phosphorylation of Akt.

    PubMed

    Chen, Jialin; Chen, Peng; Backman, Ludvig J; Zhou, Qingjun; Danielson, Patrik

    2016-01-01

    The migration of limbal epithelial stem cells is important for the homeostasis and regeneration of corneal epithelium. Ciliary neurotrophic factor (CNTF) has been found to promote corneal epithelial wound healing by activating corneal epithelial stem/progenitor cells. However, the possible effect of CNTF on the migration of corneal epithelial stem/progenitor cells is not clear. This study found the expression of CNTF in mouse corneal epithelial stem/progenitor cells (TKE2) to be up-regulated after injury, on both gene and protein level. CNTF promoted migration of TKE2 in a dose-dependent manner and the peak was seen at 10 ng/ml. The phosphorylation level of Akt (p-Akt), and the expression of MMP3 and MMP14, were up-regulated after CNTF treatment both in vitro and in vivo. Akt and MMP3 inhibitor treatment delayed the migration effect by CNTF. Finally, a decreased expression of MMP3 and MMP14 was observed when Akt inhibitor was applied both in vitro and in vivo. This study provides new insights into the role of CNTF on the migration of corneal epithelial stem/progenitor cells and its inherent mechanism of Up-regulation of matrix metalloproteinases through the Akt signalling pathway. PMID:27174608

  3. Ciliary Neurotrophic Factor Promotes the Migration of Corneal Epithelial Stem/progenitor Cells by Up-regulation of MMPs through the Phosphorylation of Akt

    PubMed Central

    Chen, Jialin; Chen, Peng; Backman, Ludvig J.; Zhou, Qingjun; Danielson, Patrik

    2016-01-01

    The migration of limbal epithelial stem cells is important for the homeostasis and regeneration of corneal epithelium. Ciliary neurotrophic factor (CNTF) has been found to promote corneal epithelial wound healing by activating corneal epithelial stem/progenitor cells. However, the possible effect of CNTF on the migration of corneal epithelial stem/progenitor cells is not clear. This study found the expression of CNTF in mouse corneal epithelial stem/progenitor cells (TKE2) to be up-regulated after injury, on both gene and protein level. CNTF promoted migration of TKE2 in a dose-dependent manner and the peak was seen at 10 ng/ml. The phosphorylation level of Akt (p-Akt), and the expression of MMP3 and MMP14, were up-regulated after CNTF treatment both in vitro and in vivo. Akt and MMP3 inhibitor treatment delayed the migration effect by CNTF. Finally, a decreased expression of MMP3 and MMP14 was observed when Akt inhibitor was applied both in vitro and in vivo. This study provides new insights into the role of CNTF on the migration of corneal epithelial stem/progenitor cells and its inherent mechanism of Up-regulation of matrix metalloproteinases through the Akt signalling pathway. PMID:27174608

  4. AML sensitivity to YM155 is modulated through AKT and Mcl-1.

    PubMed

    de Necochea-Campion, Rosalia; Diaz Osterman, Carlos J; Hsu, Heng-Wei; Fan, Junjie; Mirshahidi, Saied; Wall, Nathan R; Chen, Chien-Shing

    2015-09-28

    HL60 and U937 (acute myeloid leukemia (AML) cell lines) were assessed for sensitivity to YM155, and found to have distinct sensitive and resistant phenotypes, respectively. In HL60 cells, YM155 inhibition of growth proliferation was due to apoptosis which was measured by annexin V/PI staining. YM155 induced apoptosis through activation of intrinsic and extrinsic pathways that also culminated in caspase-3 activity and PARP cleavage. YM155 sensitivity was partially associated with this compound's ability to down-regulate survivin transcription since this was more pronounced in the HL60 cell line. However, marked differences were also observed in XIAP, Bcl-2, and Mcl-1L, and Mcl-1s. Furthermore, YM155 treatment completely inhibited production of total Akt protein in HL60, but not U937 cells. Importantly, Akt activity (pAkt-Ser473) levels were maintained in YM155 treated U937 cells which may help stabilize other anti-apoptotic proteins. Combination treatments with an Akt inhibitor, MK-2206, reduced levels of pAkt-Ser473 in U937 cells and synergistically sensitized them to YM155 cytotoxicity. Collectively our results indicate that Akt signaling may be an important factor mediating YM155 response in AML, and combinatorial therapies with Akt inhibitors could improve treatment efficacy in YM155-resistant cells. PMID:26118775

  5. ROS-mediated EB1 phosphorylation through Akt/GSK3β pathway: implication in cancer cell response to microtubule-targeting agents

    PubMed Central

    Grand, Marion Le; Rovini, Amandine; Bourgarel-Rey, Veronique; Honore, Stephane; Bastonero, Sonia; Braguer, Diane; Carre, Manon

    2014-01-01

    Microtubule-targeting agents (MTAs) are largely administered in adults and children cancers. Better deciphering their mechanism of action is of prime importance to develop more convenient therapy strategies. Here, we addressed the question of how reactive oxygen species (ROS) generation by mitochondria can be necessary for MTA efficacy. We showed for the first time that EB1 associates with microtubules in a phosphorylation-dependent manner, under control of ROS. By using phospho-defective mutants, we further characterized the Serine 155 residue as critical for EB1 accumulation at microtubule plus-ends, and both cancer cell migration and proliferation. Phosphorylation of EB1 on the Threonine 166 residue triggered opposite effects, and was identified as a requisite molecular switch in MTA activities. We then showed that GSK3β activation was responsible for MTA-triggered EB1 phosphorylation, resulting from ROS-mediated inhibition of upstream Akt. We thus disclosed here a novel pathway by which generation of mitochondrial ROS modulates microtubule dynamics through phosphorylation of EB1, improving our fundamental knowledge about this oncogenic protein, and pointing out the need to re-examine the current dogma of microtubule targeting by MTAs. The present work also provides a strong mechanistic rational to the promising therapeutic strategies that currently combine MTAs with anti-Akt targeted therapies. PMID:24930764

  6. IL-8 up-regulates proliferative angiogenesis in ischemic myocardium in rabbits through phosphorylation of Akt/GSK-3βser9 dependent pathways

    PubMed Central

    Xie, Qiying; Sun, Zelin; Chen, Meifang; Zhong, Qiaoqing; Yang, Tianlun; Yi, Jun

    2015-01-01

    Background: Therapeutic myocardial angiogenesis is an important compensatory mechanism in severely coronary stenosis. Previous studies demonstrated that interleukin-8 (IL-8) not only plays an important role in inflammation, but also a potent angiogenic factor through p38 mitogen-activated protein kinase (p38MAPK), nuclear factor-kappaB (NK-κB)-dependent pathway in carcinoma. Our study sought to investigate the effects of IL-8 on the angiogenesis and the underlying mechanism in the ischemic myocardium. Methods: Acute myocardial infarction animal model was established with male rabbits by directly suturing the left anterior descending branch, then lentivirus-mediated IL-8 was quarterly injected into the borderline of infarction area immediately. We employed CoCl2 induced hypoxic HUVECs for in vitro ischemia study. Left ventricular end-diastolic diameter (LVEDd) and ejection fraction (EF) were measured by echocardiography in pre-operation and at 6th week after operation. CD34 was detected with immunohistochemisty to analyse angiogenesis. Western blot was performed with regard to IL-8, protein kinase B (PKB/Akt) and Glycogen synthase kinase-3βser9 (GSK-3βser9). For the HUVECs’ proliferation and apoptosis, multiscan spectrum reader at A570 nm and annexin V-FITC/PI staining method were used respectively. Results: The levels of IL-8, phosphorylated Akt and GSK-3βser9 in focal myocardium significantly increased, and the over expression of IL-8 led to an increasing in angiogenesis in rabbits. Hypoxia inhibited cell proliferation and promoted apoptosis. IL-8 induced cell proliferation, phosphorylation of Akt and GSK-3βser9, inhibited apoptosis and Caspase3 expression in HUVECs, which were attenuated by anti-IL-8 or the Akt inhibitor LY294002. Conclusions: The present results indicate that IL-8 can increase angiogenesis in myocardial infarction, which maybe through enhancing Akt and GSK-3βser9 expression, and inhibiting myocardial apoptosis. PMID:26550160

  7. Enoxaparin sensitizes human non-small-cell lung carcinomas to gefitinib by inhibiting DOCK1 expression, vimentin phosphorylation, and Akt activation.

    PubMed

    Pan, Yan; Li, Xin; Duan, Jianhui; Yuan, Lan; Fan, Shengjun; Fan, Jingpu; Xiaokaiti, Yilixiati; Yang, Haopeng; Wang, Yefan; Li, Xuejun

    2015-01-01

    Gefitinib is widely used for the treatment of lung cancer in patients with sensitizing epidermal growth factor receptor mutations, but patients tend to develop resistance after an average of 10 months. Low molecular weight heparins, such as enoxaparin, potently inhibit experimental metastasis. This study aimed to determine the potential of combined enoxaparin and gefitinib (enoxaparin + gefitinib) treatment to inhibit tumor resistance to gefitinib both in vitro and in vivo. A549 and H1975 cell migration was analyzed in wound closure and Transwell assays. Akt and extracellular signal-related kinase 1/2 signaling pathways were identified, and a proteomics analysis was conducted using SDS-PAGE/liquid chromatography-tandem mass spectrometry analysis. Molecular interaction networks were visualized using the Cytoscape bioinformatics platform. Protein expression of dedicator of cytokinesis 1 (DOCK1) and cytoskeleton intermediate filament vimentin were identified using an enzyme-linked immunosorbent assay, Western blot, and small interfering RNA transfection of A549 cells. In xenograft A549-luc-C8 tumors in nude mice, enoxaparin + gefitinib inhibited tumor growth and reduced lung colony formation compared with gefitinib alone. Furthermore, the combination had stronger inhibitory effects on cell migration than either agent used individually. Additional enoxaparin administration resulted in better effective inhibition of Akt activity compared with gefitinib alone. Proteomics and network analysis implicated DOCK1 as the key node molecule. Western blot verified the effective inhibition of the expression of DOCK1 and vimentin phosphorylation by enoxaparin + gefitinib compared with gefitinib alone. DOCK1 knockdown confirmed its role in cell migration, Akt expression, and vimentin phosphorylation. Our data indicate that enoxaparin sensitizes gefitinib antitumor and antimigration activity in lung cancer by suppressing DOCK1 expression, Akt activity, and vimentin phosphorylation

  8. The modulation of vascular ATP-sensitive K+ channel function via the phosphatidylinositol 3-kinase-Akt pathway activated by phenylephrine.

    PubMed

    Haba, Masanori; Hatakeyama, Noboru; Kinoshita, Hiroyuki; Teramae, Hiroki; Azma, Toshiharu; Hatano, Yoshio; Matsuda, Naoyuki

    2010-08-01

    The present study examined the modulator role of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway activated by the alpha-1 adrenoceptor agonist phenylephrine in ATP-sensitive K(+) channel function in intact vascular smooth muscle. We evaluated the ATP-sensitive K(+) channel function and the activity of the PI3K-Akt pathway in the rat thoracic aorta without endothelium. The PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) (10(-5) M) augmented relaxation in response to the ATP-sensitive K(+) channel opener levcromakalim (10(-8) to 3 x 10(-6) M) in aortic rings contracted with phenylephrine (3 x 10(-7) M) but not with 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F(2alpha) (U46619; 3 x 10(-8) M), although those agents induced similar contraction. ATP-sensitive K(+) channel currents induced by levcromakalim (10(-6) M) in the presence of phenylephrine (3 x 10(-7) M) were enhanced by the nonselective alpha-adrenoceptor antagonist phentolamine (10(-7) M) and LY294002 (10(-5) M). Levels of the regulatory subunits of PI3K p85-alpha and p55-gamma increased in the membrane fraction from aortas without endothelium treated with phenylephrine (3 x 10(-7) M) but not with U46619 (3 x 10(-8) M). Phenylephrine simultaneously augmented Akt phosphorylation at Ser473 and Thr308. Therefore, activation of the PI3K-Akt pathway seems to play a role in the impairment of ATP-sensitive K(+) channel function in vascular smooth muscle exposed to alpha-1 adrenergic stimuli. PMID:20519555

  9. Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells

    PubMed Central

    Ming, Xiu-Fen; Viswambharan, Hema; Barandier, Christine; Ruffieux, Jean; Kaibuchi, Kozo; Rusconi, Sandro; Yang, Zhihong

    2002-01-01

    Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB. PMID:12446767

  10. Impaired exercise training-induced muscle fiber hypertrophy and Akt/mTOR pathway activation in hypoxemic patients with COPD.

    PubMed

    Costes, Frédéric; Gosker, Harry; Feasson, Léonard; Desgeorges, Marine; Kelders, Marco; Castells, Josiane; Schols, Annemie; Freyssenet, Damien

    2015-04-15

    Exercise training (ExTr) is largely used to improve functional capacity in patients with chronic obstructive pulmonary disease (COPD). However, ExTr only partially restores muscle function in patients with COPD, suggesting that confounding factors may limit the efficiency of ExTr. In the present study, we hypothesized that skeletal muscle adaptations triggered by ExTr could be compromised in hypoxemic patients with COPD. Vastus lateralis muscle biopsies were obtained from patients with COPD who were either normoxemic (n = 15, resting arterial Po2 = 68.5 ± 1.5 mmHg) or hypoxemic (n = 8, resting arterial Po2 = 57.0 ± 1.0 mmHg) before and after a 2-mo ExTr program. ExTr induced a significant increase in exercise capacity both in normoxemic and hypoxemic patients with COPD. However, ExTr increased citrate synthase and lactate dehydrogenase enzyme activities only in skeletal muscle of normoxemic patients. Similarly, muscle fiber cross-sectional area and capillary-to-fiber ratio were increased only in patients who were normoxemic. Expression of atrogenes (MuRF1, MAFbx/Atrogin-1) and autophagy-related genes (Beclin, LC3, Bnip, Gabarapl) remained unchanged in both groups. Phosphorylation of Akt (Ser473), GSK-3β (Ser9), and p70S6k (Thr389) was nonsignificantly increased in normoxemic patients in response to ExTr, but it was significantly decreased in hypoxemic patients. We further showed on C2C12 myotubes that hypoxia completely prevented insulin-like growth factor-1-induced phosphorylation of Akt, GSK-3β, and p70S6K. Together, our observations suggest a role for hypoxemia in the adaptive response of skeletal muscle of patients with COPD in an ExTr program. PMID:25701004

  11. Alternative splicing of Caspase 9 is modulated by the PI3K/Akt pathway via phosphorylation of SRp30a

    PubMed Central

    Shultz, Jacqueline C.; Goehe, Rachel W.; Wijesinghe, D. Shanaka; Murudkar, Charuta; Hawkins, Amy J.; Shay, Jerry W.; Minna, John D.; Chalfant, Charles E.

    2010-01-01

    Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non-small cell lung cancers (NSCLC) regardless of their pathological classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K-RasV12 expression, EGFR overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage-independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of PI3K or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227 and 234. Taken together, our findings demonstrate that oncogenic factors activating the PI3Kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a. PMID:21045158

  12. Carboxyl-Terminal Modulator Protein Positively Acts as an Oncogenic Driver in Head and Neck Squamous Cell Carcinoma via Regulating Akt phosphorylation.

    PubMed

    Chang, Jae Won; Jung, Seung-Nam; Kim, Ju-Hee; Shim, Geun-Ae; Park, Hee Sung; Liu, Lihua; Kim, Jin Man; Park, Jongsun; Koo, Bon Seok

    2016-01-01

    The exact regulatory mechanisms of carboxyl-terminal modulator protein (CTMP) and its downstream pathways in cancer have been controversial and are not completely understood. Here, we report a new mechanism of regulation of Akt serine/threonine kinase, one of the most important dysregulated signals in head and neck squamous cell carcinoma (HNSCC) by the CTMP pathway and its clinical implications. We find that HNSCC tumor tissues and cell lines had relatively high levels of CTMP expression. Clinical data indicate that CTMP expression was significantly associated with positive lymph node metastasis (OR = 3.8, P = 0.033) and correlated with poor prognosis in patients with HNSCC. CTMP was also positively correlated with Akt/GSK-3β phosphorylation, Snail up-regulation and E-cadherin down-regulation, which lead to increased proliferation and epithelial-to-mesenchymal transition, suggesting that CTMP expression results in enhanced tumorigenic and metastatic properties of HNSCC cells. Moreover, CTMP suppression restores sensitivity to cisplatin chemotherapy. Intriguingly, all the molecular responses to CTMP regulation are identical regardless of p53 status in HNSCC cells. We conclude that CTMP promotes Akt phosphorylation and functions as an oncogenic driver and prognostic marker in HNSCC irrespective of p53. PMID:27328758

  13. Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer

    PubMed Central

    Nacerddine, Karim; Beaudry, Jean-Bernard; Ginjala, Vasudeva; Westerman, Bart; Mattiroli, Francesca; Song, Ji-Ying; van der Poel, Henk; Ponz, Olga Balagué; Pritchard, Colin; Cornelissen-Steijger, Paulien; Zevenhoven, John; Tanger, Ellen; Sixma, Titia K.; Ganesan, Shridar; van Lohuizen, Maarten

    2012-01-01

    Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis. PMID:22505453

  14. Carboxyl-Terminal Modulator Protein Positively Acts as an Oncogenic Driver in Head and Neck Squamous Cell Carcinoma via Regulating Akt phosphorylation

    PubMed Central

    Chang, Jae Won; Jung, Seung-Nam; Kim, Ju-Hee; Shim, Geun-Ae; Park, Hee Sung; Liu, Lihua; Kim, Jin Man; Park, Jongsun; Koo, Bon Seok

    2016-01-01

    The exact regulatory mechanisms of carboxyl-terminal modulator protein (CTMP) and its downstream pathways in cancer have been controversial and are not completely understood. Here, we report a new mechanism of regulation of Akt serine/threonine kinase, one of the most important dysregulated signals in head and neck squamous cell carcinoma (HNSCC) by the CTMP pathway and its clinical implications. We find that HNSCC tumor tissues and cell lines had relatively high levels of CTMP expression. Clinical data indicate that CTMP expression was significantly associated with positive lymph node metastasis (OR = 3.8, P = 0.033) and correlated with poor prognosis in patients with HNSCC. CTMP was also positively correlated with Akt/GSK-3β phosphorylation, Snail up-regulation and E-cadherin down-regulation, which lead to increased proliferation and epithelial-to-mesenchymal transition, suggesting that CTMP expression results in enhanced tumorigenic and metastatic properties of HNSCC cells. Moreover, CTMP suppression restores sensitivity to cisplatin chemotherapy. Intriguingly, all the molecular responses to CTMP regulation are identical regardless of p53 status in HNSCC cells. We conclude that CTMP promotes Akt phosphorylation and functions as an oncogenic driver and prognostic marker in HNSCC irrespective of p53. PMID:27328758

  15. C-terminal fragment of tetanus toxin heavy chain activates Akt and MEK/ERK signalling pathways in a Trk receptor-dependent manner in cultured cortical neurons.

    PubMed Central

    Gil, Carles; Chaib-Oukadour, Imane; Aguilera, José

    2003-01-01

    Previous publications from our group [Gil, Chaib, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177-182; Gil, Chaib, Blasi and Aguilera (2001) Biochem. J. 356, 97-103] have reported the activation, in rat brain synaptosomes, of several phosphoproteins, such as neurotrophin tyrosine kinase (Trk) A receptor, phospholipase Cgamma-1, protein kinase C (PKC) isoforms and extracellular-signal-regulated kinases 1 and 2 (ERK-1/2). In the present study, we examined, by means of phospho-specific antibodies, the activation of the signalling cascades involving neurotrophin Trk receptor, Akt kinase and ERK pathway, in cultured cortical neurons from foetal rat brain, by tetanus toxin (TeTx) as well as by the C-terminal part of its heavy chain (H(C)-TeTx). TeTx and H(C)-TeTx induce fast and transient phosphorylation of Trk receptor at Tyr(674) and Tyr(675), but not at Tyr(490), although the potency of TeTx in this action was higher when compared with H(C)-TeTx action. Moreover, H(C)-TeTx and TeTx also induced phosphorylation of Akt (at Ser(473) and Thr(308)) and of ERK-1/2 (Thr(202)/Tyr(204)), in a time- and concentration-dependent manner. The detection of TeTx- and H(C)-TeTx-induced phosphorylation at Ser(9) of glycogen synthase kinase 3beta confirms Akt activation. In the extended analysis of the ERK pathway, phosphorylation of the Raf, mitogen-activated protein kinase kinase (MEK)-1/2 and p90Rsk kinases and phosphorylation of the transcription factor cAMP-response-element-binding protein were detected. The use of tyrphostin AG879, an inhibitor of Trk receptors, demonstrates their necessary participation in the H(C)-TeTx-induced activation of Akt and ERK pathways, as well as in the phosphorylation of phospholipase Cgamma-1. Furthermore, both pathways are totally dependent on phosphatidylinositol 3-kinase action, and they are independent of PKC action, as assessed using wortmannin and Ro-31-8220 as inhibitors. The activation of PKC isoforms was determined by their translocation

  16. The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) protects cells against cold-shock-induced apoptosis by maintaining phosphorylation of protein kinase B (AKT).

    PubMed

    Carpenter, Dale; Hsiang, Chinhui; Jiang, Xianzhi; Osorio, Nelson; BenMohamed, Lbachir; Jones, Clinton; Wechsler, Steven L

    2015-10-01

    The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) blocks apoptosis and inhibits caspase-3 activation. We previously showed that serum starvation (removal of serum from tissue culture media), which takes several days to induce apoptosis, results in decreased levels of both AKT (protein kinase B) and phosphorylated AKT (pAKT) in cells not expressing LAT. In contrast in mouse neuroblastoma cells expressing LAT, AKT, and pAKT levels remained high. AKT is a serine/threonine protein kinase that promotes cell survival. To examine the effect of LAT on AKT-pAKT using a different and more rapid method of inducing apoptosis, a stable cell line expressing LAT was compared to non-LAT expressing cells as soon as 15 min following recovery from cold-shock-induced apoptosis. Expression of LAT appeared to inhibit dephosphorylation of pAKT. This protection correlated with blocking numerous pro-apoptotic events that are inhibited by pAKT. These results support the hypothesis that inhibiting dephosphorylation of pAKT may be one of the pathways by which LAT protects cells against apoptosis. PMID:26071090

  17. Glycogen Repletion in Brown Adipose Tissue upon Refeeding Is Primarily Driven by Phosphorylation-Independent Mechanisms

    PubMed Central

    Carmean, Christopher M.; Huang, Y. Hanna; Brady, Matthew J.

    2016-01-01

    Glycogen storage in brown adipose tissue (BAT) is generally thought to take place through passive, substrate-driven activation of glycogenesis rather than programmatic shifts favoring or opposing the storage and/or retention of glycogen. This perception exists despite a growing body of evidence suggesting that BAT glycogen storage is actively regulated by covalent modification of key glycogen-metabolic enzymes, protein turnover, and endocrine hormone signaling. Members of one such class of covalent-modification regulators, glycogen-binding Phosphoprotein Phosphatase-1 (PP1)-regulatory subunits (PPP1Rs), targeting PP1 to glycogen-metabolic enzymes, were dynamically regulated in response to 24 hr of starvation and/or 24 hr of starvation followed by ad libitum refeeding. Over-expression of the PPP1R Protein Targeting to Glycogen (PTG), under the control of the aP2 promoter in mice, inactivated glycogen phosphorylase (GP) and enhanced basal- and starvation-state glycogen storage. Total interscapular BAT glycogen synthase and the constitutive activity of GS were conditionally affected. During starvation, glucose-6-phosphate (G-6-P) levels and the relative phosphorylation of Akt (p-Ser-473-Akt) were both increased in PTG-overexpressing (Tg) mice, suggesting that elevated glycogen storage during starvation modifies broader cellular metabolic pathways. During refeeding, Tg and WT mice reaccumulated glycogen similarly despite altered GS and GP activities. All observations during refeeding suggest that the phosphorylation states of GS and GP are not physiologically rate-controlling, despite there being a clear balance of endogenous kinase- and phosphatase activities. The studies presented here reveal IBAT glycogen storage to be a tightly-regulated process at all levels, with potential effects on nutrient sensing in vivo. PMID:27213961

  18. Macrophage migration inhibitory factor induces phosphorylation of Mdm2 mediated by phosphatidylinositol 3-kinase/Akt kinase: Role of this pathway in decidual cell survival.

    PubMed

    Costa, Adriana Fraga; Gomes, Sara Zago; Lorenzon-Ojea, Aline R; Martucci, Mariane; Faria, Miriam Rubio; Pinto, Décio Dos Santos; Oliveira, Sergio F; Ietta, Francesca; Paulesu, Luana; Bevilacqua, Estela

    2016-05-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has an anti-apoptotic effect through several downstream targets, which includes activation of the transformed mouse 3T3 cell double-minute 2 (Mdm2) protein, its translocation to the nucleus and degradation of the tumor suppressor p53. We show that Mif, the Macrophage Migration Inhibitory Factor, an important cytokine at the maternal fetal interface in several species, triggers phosphorylation of Mdm2 protein in a PI3K/Akt-dependent manner, thereby preventing apoptosis in cultured mouse decidual cells. Inhibition of Akt and PI3K suppresses the pathway. Mif treatment also changes the nuclear translocation of p53 and interferes with the apoptotic fate of these cells when challenged with reactive oxygen species. In conclusion, an important mechanism has been found underlying decidual cell survival through Akt signaling pathway activated by Mif, suggesting a role for this cytokine in decidual homeostasis and in the integrity of the maternal-fetal barrier that is essential for successful gestation. PMID:27208405

  19. Leptin Effect on Acetylation and Phosphorylation of Pgc1α in Muscle Cells Associated With Ampk and Akt Activation in High-Glucose Medium.

    PubMed

    García-Carrizo, Francisco; Nozhenko, Yuriy; Palou, Andreu; Rodríguez, Ana M

    2016-03-01

    Leptin is crucial in energy metabolism, including muscle regulation. Peroxisome proliferator activated receptor gamma co-activator 1α (PGC1α) orchestrates energy metabolism and is tightly controlled by post-translational covalent modifications such as phosphorylation and acetylation. We aimed to further the knowledge of PGC1α control by leptin (at physiological levels) in muscle cells by time-sequentially analysing the activation of AMP activated protein kinase (AMPK), P38 mitogen-activated protein kinase (P38 MAPK) and Akt (Protein kinase B)--all known to phosphorylate PGC1α and to be involved in the regulation of its acetylation status--in C2C12 myotubes placed in a high-glucose serum-free medium. We also studied the protein levels of PGC1α, Sirtuin 1, adiponectin, COX IV, mitofusin 2 (Mfn2), and pyruvate dehydrogenase kinase 4 (PDK4). Our main findings suggest an important role of leptin regulating AMPK and Akt phosphorylation, Mfn2 induction and PGC1α acetylation status, with the novelty that the latter in transitorily increased in response to leptin, an effect dependent, at least in part, on AMPK regulation. These post-translational reversible changes in PGC1α in response to leptin, especially the increase in acetylation status, may be related to the physiological role of the hormone in modulating muscle cell response to the physiological/nutritional status. PMID:26218179

  20. MARCKSL1 exhibits anti-angiogenic effects through suppression of VEGFR-2-dependent Akt/PDK-1/mTOR phosphorylation.

    PubMed

    Kim, Boh-Ram; Lee, Seung-Hoon; Park, Mi-Sun; Seo, Seung-Hee; Park, Young-Min; Kwon, Young-Joo; Rho, Seung-Bae

    2016-02-01

    Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1) plays a pivotal role in the regulation of apoptosis and has been shown to maintain antitumor and metastasis-suppressive properties. In the present study, we examined the effects of MARCKSL1 as a novel anti-angiogenic agent on the inhibition of angiogenesis-mediated cell migration. MARCKSL1 also reduced vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) proliferation, as well as capillary-like tubular structure formation in vitro. MARCKSL1 disrupted phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) in ovarian tumorigenesis. In addition, MARCKSL1 showed potent anti-angiogenic activity and reduced the levels of VEGF and hypoxia-inducible factor 1α (HIF-1α) expression, an essential regulator of angiogenesis. Consistently, MARCKSL1 decreased VEGF‑induced phosphorylation of the PI3K/Akt signaling pathway components, including phosphoinositide-dependent protein kinase 1 (PDK-1), mammalian target of rapamycin (mTOR), tuberous sclerosis complex 2 (TSC-2), p70 ribosomal protein S6 kinase (p70S6K), and glycogen synthase kinase 3β (GSK-3β) protein. Collectively, our results provide evidence for the physiological/biological function of an endothelial cell system involved in angiogenesis through suppression of Akt/PDK-1/mTOR phosphorylation by interaction with VEGFR-2. PMID:26555156

  1. Hepatitis C virus NS5A promotes insulin resistance through IRS-1 serine phosphorylation and increased gluconeogenesis

    PubMed Central

    Parvaiz, Fahed; Manzoor, Sobia; Iqbal, Jawed; Sarkar-Dutta, Mehuli; Imran, Muhammad; Waris, Gulam

    2015-01-01

    AIM: To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus (HCV) nonstructural protein 5A (NS5A). METHODS: The human hepatoma cell lines, Huh7 and Huh7.5, were infected with HCV or transiently-transfected with a vector expressing HCV NS5A. The effect of HCV NS5A on the status of the critical players involved in insulin signaling was analyzed using real-time quantitative polymerase chain reaction and Western blot assays. Data were analyzed using Graph Pad Prism version 5.0. RESULTS: To investigate the effect of insulin treatment on the players involved in insulin signaling pathway, we analyzed the status of insulin receptor substrate-1 (IRS-1) phosphorylation in HCV infected cells or Huh7.5 cells transfected with an HCV NS5A expression vector. Our results indicated that there was an increased phosphorylation of IRS-1 (Ser307) in HCV infected or NS5A transfected Huh7.5 cells compared to their respective controls. Furthermore, an increased phosphorylation of Akt (Ser473) was observed in HCV infected and NS5A transfected cells compared to their mock infected cells. In contrast, we observed decreased phosphorylation of Akt Thr308 phosphorylation in HCV NS5A transfected cells. These results suggest that Huh7.5 cells either infected with HCV or ectopically expressing HCV NS5A alone have the potential to induce insulin resistance by the phosphorylation of IRS-1 at serine residue (Ser307) followed by decreased phosphorylation of Akt Thr308, Fox01 Ser256 and GSK3β Ser9, the downstream players of the insulin signaling pathway. Furthermore, increased expression of PECK and glucose-6-phosphatase, the molecules involved in gluconeogenesis, in HCV NS5A transfected cells was observed. CONCLUSION: Taken together, our results suggest the role of HCV NS5A in the induction of insulin resistance by modulating various cellular targets involved in the insulin signaling pathway. PMID:26604643

  2. AKT/mTOR substrate P70S6K is frequently phosphorylated in gallbladder cancer tissue and cell lines

    PubMed Central

    Leal, Pamela; Garcia, Patricia; Sandoval, Alejandra; Buchegger, Kurt; Weber, Helga; Tapia, Oscar; Roa, Juan C

    2013-01-01

    Background Gallbladder carcinoma is a highly malignant tumor and a public health problem in some parts of the world. It is characterized by a poor prognosis and its resistance to radio and chemotherapy. There is an urgent need to develop novel therapeutic alternatives for the treatment of gallbladder carcinoma. The mammalian target of the rapamycin (mTOR) signaling pathway is activated in about 50% of human malignancies, and its role in gallbladder carcinoma has previously been suggested. In the present study, we investigated the phosphorylation status of the mTOR substrate p70S6K in preneoplastic and neoplastic gallbladder tissues and evaluated the effect of three mTOR inhibitors on cell growth and migration in gallbladder carcinoma cell lines. Methods Immunohistochemical staining of phospho-p70S6K was analyzed in 181 gallbladder carcinoma cases, classified according to lesion type as dysplasia, early carcinoma, or advanced carcinoma. Protein expression of AKT/mTOR members was also evaluated in eight gallbladder carcinoma cell lines by Western blot analysis. We selected two gallbladder carcinoma cell lines (G415 and TGBC-2TKB) to evaluate the effect of rapamycin, RAD001, and AZD8055 on cell viability, cell migration, and protein expression. Results Our results showed that phospho-p70S6K is highly expressed in dysplasia (66.7%, 12/18), early cancer (84.6%, 22/26), and advanced cancer (88.3%, 121/137). No statistical correlation was observed between phospho-p70S6K status and any clinical or pathological features, including age, gender, ethnicity, wall infiltration level, or histological differentiation (P < 0.05). In vitro treatment with rapamycin, RAD001, and AZD8055 reduced cell growth, cell migration, and phospho-p70S6K expression significantly in G-415 and TGBC-2TKB cancer cells (P < 0.001). Conclusion Our findings confirm the upregulation of this signaling pathway in gallbladder carcinoma and provide a rationale for the potential use of mTOR inhibitors as a

  3. Phosphorylation and changes in the distribution of nucleolin promote tumor metastasis via the PI3K/Akt pathway in colorectal carcinoma.

    PubMed

    Wu, Dong-ming; Zhang, Peng; Liu, Ru-yan; Sang, Ya-xiong; Zhou, Cong; Xu, Guang-chao; Yang, Jin-liang; Tong, Ai-ping; Wang, Chun-ting

    2014-05-21

    Here, we investigated the molecular mechanism underlying the changes in the distribution of nucleolin. Our study identified PI3K/Akt signaling as an essential pathway regulating the distribution of nucleolin. Furthermore, nucleolin can interact with phospho-PI3K-p55, and changes in the distribution of nucleolin were related to its phosphorylation. Subsequently, we analyzed the correlation of VEGF and nucleolin, and found that distribution of nucleolin related to metastatic potential. Finally, blocking cell surface nucleolin influences the process of epithelial-mesenchymal transitions. This indicates that nucleolin may be a novel cancer therapy target and a predictive marker for tumor migration in colorectal carcinoma. PMID:24713430

  4. Grape seed extract enhances eNOS expression and NO production through regulating calcium-mediated AKT phosphorylation in H2O2-treated endothelium.

    PubMed

    Feng, Zhe; Wei, Ri-Bao; Hong, Quan; Cui, Shao-Yuan; Chen, Xiang-Mei

    2010-10-01

    GSE (grape seed extract) has been shown to exhibit protective effects against cardiovascular events and atherosclerosis, although the underlying molecular mechanisms of action are unknown. Herein, we assessed the ability of GSE to enhance eNOS (endothelial nitric oxide synthase) expression and NO (nitric oxide) production in H2O2 (hydrogen peroxide)-treated HUVECs (human umbilical vein endothelial cells). GSE enhanced eNOS expression and NO release in H2O2-treated cells in a dose-dependent manner. GSE inhibited intracellular ROS (reactive oxygen species) and reduced intracellular calcium in a dose-dependent manner in H2O2-treated cells, as shown by confocal microscopy. ROS was inhibited in cells pretreated with 5.0 microM GSE, 2.0 microM TG (thapsigargin) and 20.0 microM 2-APB (2-aminoethoxydiphenyl borate) instead of 0.25 microM extracellular calcium. In addition, GSE enhanced eNOS expression and reduced ROS production via increasing p-AKT (AKT phosphorylation) with high extracellular calcium (13 mM). In conclusion, GSE protected against endothelial injury by up-regulation of eNOS and NO expression via inhibiting InsP3Rs (inositol 1,4,5-trisphosphate receptors)-mediated intracellular excessive calcium release and by activating p-AKT in endothelial cells. PMID:20513234

  5. The Nuclear Zinc Finger Protein Zfat Maintains FoxO1 Protein Levels in Peripheral T Cells by Regulating the Activities of Autophagy and the Akt Signaling Pathway.

    PubMed

    Ishikura, Shuhei; Iwaihara, Yuri; Tanaka, Yoko; Luo, Hao; Nishi, Kensuke; Doi, Keiko; Koyanagi, Midori; Okamura, Tadashi; Tsunoda, Toshiyuki; Shirasawa, Senji

    2016-07-15

    Forkhead box O1 (FoxO1) is a key molecule for the development and functions of peripheral T cells. However, the precise mechanisms regulating FoxO1 expression in peripheral T cells remain elusive. We previously reported that Zfat(f/f)-CD4Cre mice showed a marked decline in FoxO1 protein levels in peripheral T cells, partially through proteasomal degradation. Here we have identified the precise mechanisms, apart from proteasome-mediated degradation, of the decreased FoxO1 levels in Zfat-deficient T cells. First, we confirmed that tamoxifen-inducible deletion of Zfat in Zfat(f/f)-CreERT2 mice coincidently decreases FoxO1 protein levels in peripheral T cells, indicating that Zfat is essential for maintaining FoxO1 levels in these cells. Although the proteasome-specific inhibitors lactacystin and epoxomicin only moderately increase FoxO1 protein levels, the inhibitors of lysosomal proteolysis bafilomycin A1 and chloroquine restore the decreased FoxO1 levels in Zfat-deficient T cells to levels comparable with those in control cells. Furthermore, Zfat-deficient T cells show increased numbers of autophagosomes and decreased levels of p62 protein, together indicating that Zfat deficiency promotes lysosomal FoxO1 degradation through autophagy. In addition, Zfat deficiency increases the phosphorylation levels of Thr-308 and Ser-473 of Akt and the relative amounts of cytoplasmic to nuclear FoxO1 protein levels, indicating that Zfat deficiency causes Akt activation, leading to nuclear exclusion of FoxO1. Our findings have demonstrated a novel role of Zfat in maintaining FoxO1 protein levels in peripheral T cells by regulating the activities of autophagy and the Akt signaling pathway. PMID:27226588

  6. Absence of the GPR37/PAEL receptor impairs striatal Akt and ERK2 phosphorylation, DeltaFosB expression, and conditioned place preference to amphetamine and cocaine.

    PubMed

    Marazziti, Daniela; Di Pietro, Chiara; Mandillo, Silvia; Golini, Elisabetta; Matteoni, Rafaele; Tocchini-Valentini, Glauco P

    2011-06-01

    The orphan G-protein-coupled receptor 37 (GPR37) colocalizes with the dopamine (DA) transporter (DAT) in mouse nigrostriatal presynaptic membranes, and its genetic ablation in homozygous null-mutant (GPR37-KO) mice provokes the marked increase of plasma membrane expression of DAT, alteration of psychostimulant-induced locomotor activity, and reduction of catalepsy induced by DA-receptor antagonists. We report that extracts from GPR37-KO mice displayed biochemical alterations of the nigrostriatal signaling pathways mediated by D1 and D2 dopaminergic receptors. Null-mutant mice showed an increase of the basal phosphorylation level of the D2-regulated Akt kinase. The basal phosphorylation of the D1-activated ERK2 kinase was not altered, but acute treatments with amphetamine or cocaine failed to produce its specific increase, as detected in samples from wild-type littermates. Furthermore, the chronic administration of cocaine to GPR37-KO mice did not increase the expression of the ΔFosB transcription factor isoforms. Consistently, behavioral analysis showed that null-mutant animals did not respond to the incentive properties of amphetamine or cocaine, in conditioned place preference tests. Thus, the lack of GPR37 affects both ERK2- and Akt-mediated striatal signaling pathways, impairing the biochemical and behavioral responses typically induced by acute and chronic administration of psychostimulant drugs. PMID:21372109

  7. The miR-130 family promotes cell migration and invasion in bladder cancer through FAK and Akt phosphorylation by regulating PTEN

    PubMed Central

    Egawa, Hiroshi; Jingushi, Kentaro; Hirono, Takayuki; Ueda, Yuko; Kitae, Kaori; Nakata, Wataru; Fujita, Kazutoshi; Uemura, Motohide; Nonomura, Norio; Tsujikawa, Kazutake

    2016-01-01

    Bladder cancer causes an estimated 150,000 deaths per year worldwide. Although 15% of the recurrent bladder cancer becomes an invasive type, currently used targeted therapy for malignant bladder cancer is still not efficient. We focused on the miR-130 family (miR-130b, miR-301a, and miR-301b) that was significantly upregulated in bladder cancer specimens than that of the normal urothelial specimens. We analyzed the functional significance of miR-130 family using a 5637 bladder cancer cell line and revealed that miR-130 family of inhibitors suppressed cell migration and invasion by downregulating focal adhesion kinase (FAK) and Akt phosphorylation. Mechanistic analyses indicate that the miR-130 family directly targets phosphatase and tensin homolog deleted from chromosome 10 (PTEN), resulting in the upregulation of FAK and Akt phosphorylation. In clinical bladder cancer specimens, downregulation of PTEN was found to be closely correlated with miR-130 family expression levels. Overall, the miR-130 family has a crucial role in malignant progression of bladder cancer and thus the miR-130 family could be a promising therapeutic target for invasive bladder cancer. PMID:26837847

  8. MK-2206 co-treatment with 5-fluorouracil or doxorubicin enhances chemosensitivity and apoptosis in gastric cancer by attenuation of Akt phosphorylation

    PubMed Central

    Jin, Piaopiao; Wong, Chi Chun; Mei, Sibin; He, Xingkang; Qian, Yun; Sun, Leimin

    2016-01-01

    The anticancer effect of MK-2206, an Akt inhibitor, has been explored in some types of cancers, but its effect on gastric cancer is unclear. In this study, we aimed to investigate its anticancer effect in gastric cancer cells. Cell viability and colony formation assays showed that MK-2206 effectively inhibited the proliferation of SGC-7901 and MKN45 cells. The 50% inhibitory concentration values after 24, 48, and 72 hours’ treatment were 22.92, 13.68, and 8.55 μM in SGC-7901 cells and 19.21, 13.10, and 9.11 μM in MKN45 cells, respectively. Treatment with MK-2206 induced apoptosis in SGC-7901 cells as indicated by flow cytometry assay. The combination indexes of MK-2206 and doxorubicin were 0.59 in SGC-7901 cells and 0.57 in MKN45 cells, whereas for 5-fluorouracil (5-FU) the indexes were 0.17 in SGC-7901 cells and 0.73 in MKN45 cells, indicating that MK-2206 could work synergistically with doxorubicin or 5-FU to inhibit cell growth. Furthermore, a small dose (1 μM) of MK-2206 co-treatment with doxorubicin or 5-FU was sufficient for complete inhibition of chemotherapeutic alteration of phosphorylated Akt expression and significant enhancement of pro-apoptosis effect through the activation of caspase pathway. Therefore, MK-2206 effectively inhibits gastric cancer cell growth by attenuation of Akt phosphorylation and synergistically enhances the antitumor effect of doxorubicin and 5-FU via caspase-dependent apoptosis. PMID:27499633

  9. Beneficial effect of Sparassis crispa on stroke through activation of Akt/eNOS pathway in brain of SHRSP

    PubMed Central

    Yoshitomi, Hisae; Iwaoka, Emiko; Kubo, Masahide; Shibata, Masaru

    2010-01-01

    Sparassis crispa (S. crispa) is a mushroom used as a natural medicine that recently became cultivatable in Japan. In this study, we investigated not only the preventive effects of S. crispa against stroke and hypertension in stroke-prone spontaneously hypertensive rats (SHRSP) but also the mechanism involved by using studies of the cerebral cortex at a young age. Six-week-old male SHRSP were divided into 2 groups, a control group and an S. crispa group administered 1.5% S. crispa in feed, and we then observed their survival. In addition, rats of the same age were treated with 1.5% S. crispa for 4 weeks and we measured body weight, blood pressure, blood flow from the tail, NOx production, and the levels of expression of several proteins in the cerebral cortex by western blot analysis. Our results showed that the S. crispa group had a delayed incidence of stroke and death and significantly decreased blood pressure and increased blood flow after the administration. Moreover, the quantity of urinary excretion and the nitrate/nitrite concentration in cerebral tissue were higher than those of control SHRSP rats. In the cerebral cortex, phosphor-eNOS (Ser1177) and phosphor-Akt (Ser473) in S. crispa-treated SHRSP were increased compared with those of control SHRSP rats. In conclusion, S. crispa could ameliorate cerebrovascular endothelial dysfunction by promoting recovery of Akt-dependent eNOS phosphorylation and increasing NO production in the cerebral cortex. S. crispa may be useful for preventing stroke and hypertension. PMID:21076883

  10. Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine

    PubMed Central

    Zhang, Feng-Ying; Hu, Yi; Que, Zhong-You; Wang, Ping; Liu, Yun-Hui; Wang, Zhen-Hua; Xue, Yi-Xue

    2015-01-01

    Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated β-catenin (p-β-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1). PMID:26473829

  11. Activation of protein kinase B (PKB/Akt) and risk of lung cancer among rural women in India who cook with biomass fuel

    SciTech Connect

    Roychoudhury, Sanghita; Mondal, Nandan Kumar; Mukherjee, Sayali; Dutta, Anindita; Siddique, Shabana; Ray, Manas Ranjan

    2012-02-15

    The impact of indoor air pollution (IAP) from biomass fuel burning on the risk of carcinogenesis in the airways has been investigated in 187 pre-menopausal women (median age 34 years) from eastern India who cooked exclusively with biomass and 155 age-matched control women from same locality who cooked with cleaner fuel liquefied petroleum gas. Compared with control, Papanicolau-stained sputum samples showed 3-times higher prevalence of metaplasia and 7-times higher prevalence of dysplasia in airway epithelial cell (AEC) of biomass users. Immunocytochemistry showed up-regulation of phosphorylated Akt (p-Akt{sup ser473} and p-Akt{sup thr308}) proteins in AEC of biomass users, especially in metaplastic and dysplastic cells. Compared with LPG users, biomass-using women showed marked rise in reactive oxygen species (ROS) generation and depletion of antioxidant enzyme, superoxide dismutase (SOD) indicating oxidative stress. There were 2–5 times more particulate pollutants (PM{sub 10} and PM{sub 2.5}), 72% more nitrogen dioxide and 4-times more particulate-laden benzo(a)pyrene, but no change in sulfur dioxide in indoor air of biomass-using households, and high performance liquid chromatography estimated 6-fold rise in the concentration of benzene metabolite trans,trans-muconic acid (t,t-MA) in urine of biomass users. Metaplasia and dysplasia, p-Akt expression and ROS generation were positively associated with PM and t,t-MA levels. It appears that cumulative exposure to biomass smoke increases the risk of lung carcinogenesis via oxidative stress-mediated activation of Akt signal transduction pathway. -- Highlights: ► Carcinogenesis in airway cells was examined in biomass and LPG using women. ► Metaplasia and dysplasia of epithelial cells were more prevalent in biomass users. ► Change in airway cytology was associated with oxidative stress and Akt activation. ► Biomass users had greater exposure to respirable PM, B(a)P and benzene. ► Cooking with biomass

  12. Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

    PubMed Central

    Chen, Szu-Fu; Tsai, Hsin-Ju; Hung, Tai-Ho; Chen, Chien-Cheng; Lee, Chao Yu; Wu, Chun-Hu; Wang, Pei-Yi; Liao, Nien-Chieh

    2012-01-01

    Background Traumatic brain injury (TBI) induces a complex sequence of apopototic cascades that contribute to secondary tissue damage. The aim of this study was to investigate the effects of salidroside, a phenolic glycoside with potent anti-apoptotic properties, on behavioral and histological outcomes, brain edema, and apoptosis following experimental TBI and the possible involvement of the phosphoinositide 3-kinase/protein kinase B (PI3K)/Akt signaling pathway. Methodology/Principal Findings Mice subjected to controlled cortical impact injury received intraperitoneal salidroside (20, or 50 mg/kg) or vehicle injection 10 min after injury. Behavioral studies, histology analysis and brain water content assessment were performed. Levels of PI3K/Akt signaling-related molecules, apoptosis-related proteins, cytochrome C (CytoC), and Smac/DIABLO were also analyzed. LY294002, a PI3K inhibitor, was administered to examine the mechanism of protection. The protective effect of salidroside was also investigated in primary cultured neurons subjected to stretch injury. Treatment with 20 mg/kg salidroside_significantly improved functional recovery and reduced brain tissue damage up to post-injury day 28. Salidroside_also significantly reduced neuronal death, apoptosis, and brain edema at day 1. These changes were associated with significant decreases in cleaved caspase-3, CytoC, and Smac/DIABLO at days 1 and 3. Salidroside increased phosphorylation of Akt on Ser473 and the mitochondrial Bcl-2/Bax ratio at day 1, and enhanced phosphorylation of Akt on Thr308 at day 3. This beneficial effect was abolished by pre-injection of LY294002. Moreover, delayed administration of salidroside at 3 or 6 h post-injury reduced neuronal damage at day 1. Salidroside treatment also decreased neuronal vulnerability to stretch-induced injury in vitro. Conclusions/Significance Post-injury salidroside improved long-term behavioral and histological outcomes and reduced brain edema and apoptosis

  13. Claudin-18 inhibits cell proliferation and motility mediated by inhibition of phosphorylation of PDK1 and Akt in human lung adenocarcinoma A549 cells.

    PubMed

    Shimobaba, Shun; Taga, Saeko; Akizuki, Risa; Hichino, Asami; Endo, Satoshi; Matsunaga, Toshiyuki; Watanabe, Ryo; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Sugatani, Junko; Ikari, Akira

    2016-06-01

    Abnormal expression of claudin subtypes has been reported in various cancers. However, the pathological role of each claudin has not been clarified in detail. Claudin-18 was absent in human non-small cell and small cell lung cancers, although it is expressed in normal lung tissues. Here, we examined the effect of claudin-18 expression on the expression of junctional proteins, cell proliferation, and cell motility using human lung adenocarcinoma A549 cells. Real-time PCR and western blotting showed that exogenous expression of claudin-18 had no effect on the expression of junctional proteins including claudin-1, zonula occludens-1 (ZO-1), occludin, and E-cadherin. Claudin-18 was mainly distributed in cell-cell contact areas concomitant with ZO-1. Cell proliferation was significantly decreased at 48 and 72h after seeding of claudin 18-expressing cells. Claudin-18 suppressed cell motility, whereas it increased cell death in anoikis. Claudin-18 decreased phosphorylated (p)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and p-Akt levels without affecting p-epidermal growth factor receptor and p-phosphatidylinositol-3 kinase (PI3K) levels. Furthermore, claudin-18 was bound with PDK1 and suppressed the nuclear localization of PDK1. We suggest that claudin-18 suppresses the abnormal proliferation and motility of lung epithelial cells mediated by inhibition of the PI3K/PDK1/Akt signaling pathway. PMID:26919807

  14. Stimulation of EphB2 attenuates tau phosphorylation through PI3K/Akt-mediated inactivation of glycogen synthase kinase-3β

    PubMed Central

    Jiang, Jun; Wang, Zhi-Hao; Qu, Min; Gao, Di; Liu, Xiu-Ping; Zhu, Ling-Qiang; Wang, Jian-Zhi

    2015-01-01

    Abnormal tau hyperphosphorylation is an early pathological marker of Alzheimer’s disease (AD), however, the upstream factors that regulate tau phosphorylation are not illustrated and there is no efficient strategy to arrest tau hyperphosphorylation. Here, we find that activation of endogenous EphB2 receptor by ligand stimulation (ephrinB1/Fc) or by ectopic expression of EphB2 plus the ligand stimulation induces a remarkable tau dephosphorylation at multiple AD-associated sites in SK-N-SH cells and human embryonic kidney cells that stably express human tau (HEK293-tau). In cultured hippocampal neurons and the hippocampus of human tau transgenic mice, dephosphorylation of tau proteins was also detected by stimulation of EphB2 receptor. EphB2 activation inhibits glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase, and activates phosphatidylinositol-3-kinase (PI3K)/Akt both in vitro and in vivo, whereas simultaneous inhibition of PI3K or upregulation of GSK-3β abolishes the EphB2 stimulation-induced tau dephosphorylation. Finally, we confirm that ephrinB1/Fc treatment induces tyrosine phosphorylation (activation) of EphB2, while deletion of the tyrosine kinase domain (VM) of EphB2 eliminates the receptor stimulation-induced GSK-3β inhibition and tau dephosphorylation. We conclude that activation of EphB2 receptor kinase arrests tau hyperphosphorylation through PI3K-/Akt-mediated GSK-3β inhibition. Our data provide a novel membranous target to antagonize AD-like tau pathology. PMID:26119563

  15. Salidroside improves endothelial function and alleviates atherosclerosis by activating a mitochondria-related AMPK/PI3K/Akt/eNOS pathway.

    PubMed

    Xing, Sha-Sha; Yang, Xiao-Yan; Zheng, Tao; Li, Wen-Jing; Wu, Dan; Chi, Jiang-Yang; Bian, Fang; Bai, Xiang-Li; Wu, Guang-Jie; Zhang, You-Zhi; Zhang, Cun-Tai; Zhang, Yong-Hui; Li, Yong-Sheng; Jin, Si

    2015-09-01

    Salidroside (SAL) is a phenylpropanoid glycoside isolated from the medicinal plant Rhodiola rosea. A recent study has reported that SAL can efficiently decrease atherosclerotic plaque formation in low-density lipoprotein receptor-deficient mice. This study was to investigate the molecular mechanism of antiatherogenic effects of SAL. Given the importance of endothelial nitric oxide synthase (eNOS) in atherosclerosis, we sought to elucidate whether SAL could stimulate eNOS activation and also to explore its upstream signaling pathway. Six-week old apoE(-/-) male mice were fed a high-fat diet for 8weeks and then were administered with SAL for another 8weeks. SAL significantly improved endothelial function associated with increasing eNOS activation, thus reduced the atherosclerotic lesion area. SAL increased eNOS-Ser1177 phosphorylation and decreased eNOS-Thr495 phosphorylation, indicative of eNOS activation in endothelium. The aortic sinus lesions in SAL treated mice displayed reduced inflammation. SAL significantly activated AMP-activated protein kinase (AMPK). Both AMPK inhibitor and AMPK small interfering RNA (siRNA) abolished SAL-induced Akt-Ser473 and eNOS-Ser1177 phosphorylation. In contrast, LY294002, the PI3k/Akt pathway inhibitor, abolished SAL-induced phosphorylation and expression of eNOS. High performance liquid chromatography (HPLC) analysis revealed that SAL decreased cellular ATP content and increased the cellular AMP/ATP ratio, which was associated with the activation of AMPK. SAL was found to decrease the mitochondrial membrane potential (ΔΨm), which is a likely consequence of reduced ATP production. The action of SAL to reduce atherosclerotic lesion formation may at least be attributed to its effect on improving endothelial function by promoting nitric oxide (NO) production, which was associated with mitochondrial depolarization and subsequent activation of the AMPK/PI3K/Akt/eNOS pathway. Taken together, our data described the effects of SAL on

  16. Modulating Roles of Amiloride in Irradiation-Induced Antiproliferative Effects in Glioblastoma Multiforme Cells Involving Akt Phosphorylation and the Alternative Splicing of Apoptotic Genes

    PubMed Central

    Tang, Jen-Yang

    2013-01-01

    Apoptosis is a key mechanism for enhanced cellular radiosensitivity in radiation therapy. Studies suggest that Akt signaling may play a role in apoptosis and radioresistance. This study evaluates the possible modulating role of amiloride, an antihypertensive agent with a modulating effect to alternative splicing for regulating apoptosis, in the antiproliferative effects induced by ionizing radiation (IR) in glioblastoma multiforme (GBM) 8401 cells. Analysis of cell viability showed that amiloride treatment significantly inhibited cell proliferation in irradiated GBM8401 cells (p<0.05) in a time-dependent manner, especially in cells treated with amiloride with IR post-treatment. In comparison with GBM8401 cells treated with amiloride alone, with GBM8401 cells treated with IR alone, and with human embryonic lung fibroblast control cells (HEL 299), GBM8401 cells treated with IR combined with amiloride showed increased overexpression of phosphorylated Akt, regardless of whether IR treatment was performed before or after amiloride administration. The alternative splicing pattern of apoptotic protease-activating factor-1 (APAF1) in cells treated with amiloride alone, IR alone, and combined amiloride-IR treatments showed more consistent cell proliferation compared to that in other apoptosis-related genes such as baculoviral IAP repeat containing 5 (BIRC5), Bcl-X, and homeodomain interacting protein kinase-3 (HIPK3). In GBM8401 cells treated with amiloride with IR post-treatment, the ratio of prosurvival (-XL,-LC) to proapoptotic (-LN,-S) splice variants of APAF1 was lower than that seen in cells treated with amiloride with IR pretreatment, suggesting that proapoptotic splice variants of APAF1 (APAF1-LN,-S) were higher in the glioblastoma cells treated with amiloride with IR post-treatment, as compared to glioblastoma cells and fibroblast control cells that had received other treatments. Together, these results suggest that amiloride modulates cell radiosensitivity

  17. FV-429 induces apoptosis and inhibits glycolysis by inhibiting Akt-mediated phosphorylation of hexokinase II in MDA-MB-231 cells.

    PubMed

    Zhou, Yuxin; Lu, Na; Qiao, Chen; Ni, Ting; Li, Zhiyu; Yu, Boyang; Guo, Qinglong; Wei, Libin

    2016-09-01

    In this study, the anticancer effect of a newly synthesized flavonoid FV-429, against human breast cancer MDA-MB-231 cells, and the underlying mechanisms were investigated. FV-429 triggered the apoptosis and simultaneously inhibited the glycolysis of MDA-MB-231 cells. Both the HK II activity and its level in mitochondria were significantly down regulated by FV-429. Moreover, FV-429 weakened the interaction between HKII and VDAC, stimulated the detachment of HK II from the mitochondria, and resulted in the opening of the mitochondrial permeability transition pores. Thus FV-429 induced the mitochondrial-mediated apoptosis, showing increased Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (MMP) and activation of caspase-3 and -9, cytochrome c (Cyt c) release, and apoptosis inducing factor (AIF) transposition. Further research revealed that the phosphorylation of mitochondrial HKII via Akt was responsible for the dissociation of HKII and the decreased HKII activity induced by FV-429. Taken together, FV-429 inhibited the phosphorylation of HKII, down-regulated its activity, and stimulated the release of HKII from the mitochondria, resulting the inhibited glycolysis and mitochondrial-mediated apoptosis. The studies provide a molecular basis for the development of flavonoid compounds as novel anticancer agents for breast cancer. © 2015 Wiley Periodicals, Inc. PMID:26258875

  18. Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

    SciTech Connect

    Ilowski, Maren; Putz, Christine; Weiss, Thomas S.; Brand, Stephan; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang Erwin

    2010-04-16

    Background/Aim: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells. Methods: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [{sup 3}H]-thymidine assay. Results: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1). Conclusion: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.

  19. Subcutaneous Adipocytes Promote Melanoma Cell Growth by Activating the Akt Signaling Pathway

    PubMed Central

    Kwan, Hiu Yee; Fu, Xiuqiong; Liu, Bin; Chao, Xiaojuan; Chan, Chi Leung; Cao, Huihui; Su, Tao; Tse, Anfernee Kai Wing; Fong, Wang Fun; Yu, Zhi-Ling

    2014-01-01

    Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt. PMID:25228694

  20. Novel antibodies to phosphorylated α-synuclein serine 129 and NFL serine 473 demonstrate the close molecular homology of these epitopes.

    PubMed

    Rutherford, Nicola J; Brooks, Mieu; Giasson, Benoit I

    2016-01-01

    Pathological inclusions containing aggregated, highly phosphorylated (at serine129) α-synuclein (αS pSer129) are characteristic of a group of neurodegenerative diseases termed synucleinopathies. Antibodies to the pSer129 epitope can be highly sensitive in detecting αS inclusions in human tissue and experimental models of synucleinopathies. However, the generation of extensively specific pSer129 antibodies has been problematic, in some cases leading to the misinterpretation of αS inclusion pathology. One common issue is cross-reactivity to the low molecular mass neurofilament subunit (NFL) phosphorylated at Ser473. Here, we generated a series of monoclonal antibodies to the pSer129 αS and pSer473 NFL epitopes. We determined the relative abilities of the known αS kinases, polo-like kinases (PLK) 1, 2 and 3 and casein kinase (CK) II in phosphorylating NFL and αS, while using this information to characterize the specificity of the new antibodies. NFL can be phosphorylated by PLK1, 2 and 3 at Ser473; however CKII shows the highest phosphorylation efficiency and specificity for this site. Conversely, PLK3 is the most efficient kinase at phosphorylating αS at Ser129, but there is overlay in the ability of these kinases to phosphorylate both epitopes. Antibody 4F8, generated to the pSer473 NFL epitope, was relatively specific for phosphorylated NFL, however it could uniquely cross-react with pSer129 αS when highly phosphorylated, further showing the structural similarity between these phospho-epitopes. All of the new pSer129 antibodies detected pathological αS inclusions in human brains and mouse and cultured cell experimental models of induced synucleinopathies. Several of these pSer129 αS antibodies reacted with the pSer473 NFL epitope, but 2 clones (LS3-2C2 and LS4-2G12) did not. However, LS3-2C2 demonstrated cross-reactivity with other proteins. Our findings further demonstrate the difficulties in generating specific pSer129 αS antibodies, but highlights

  1. Effect of dehydroepiandrosterone (DHEA) on Akt and protein kinase C zeta (PKCζ) phosphorylation in different tissues of C57BL6, insulin receptor substrate (IRS)1(-/-), and IRS2(-/-) male mice fed a high-fat diet.

    PubMed

    Aoki, Kazutaka; Tajima, Kazuki; Taguri, Masataka; Terauchi, Yasuo

    2016-05-01

    We have previously reported that dehydroepiandrosterone (DHEA) suppresses the activity and mRNA expression of the hepatic gluconeogenic enzyme glucose-6-phosphatase (G6Pase), and hepatic glucose production in db/db mice. Tyrosine phosphorylation levels of Insulin receptor substrate (IRS)1 and IRS2 reportedly differ between the liver and muscle tissue and the effect of DHEA on insulin signaling has not been elucidated. Therefore, we examined DHEA's effect on the liver and muscle tissue of IRS1(-/-) and IRS2(-/-) mice. Eight-week-old male C57BL6, IRS1(-/-), and IRS2(-/-) mice were fed a high-fat diet (HFD), or an HFD containing 0.2% DHEA for 4 weeks. In a separate experiment, 8-week-old male C57BL6 mice were fed an HFD or an HFD containing 0.2% androstenedione for 4 weeks. In an insulin tolerance test, DHEA administration decreased the initial plasma glucose levels in the C57BL6, IRS1(-/-), and IRS2(-/-) mice but did not decrease the ratios to the basal blood glucose level. Although DHEA administration increased Akt phosphorylation in the liver of the C57BL6, IRS1(-/-), and IRS2(-/-) mice, androstenedione administration did not increase Akt phosphorylation in the liver of C57BL6 mice. DHEA administration did not increase Akt and PKCζ phosphorylation in the muscle tissue of C57BL6, IRS1(-/-), or IRS2(-/-) mice. However, androstenedione administration increased Akt and PKCζ phosphorylation in the muscle tissue of C57BL6 mice. These findings suggest that the effect of DHEA on insulin action in the liver is self-mediated by DHEA or DHEA sulfate (DHEA-S) in the presence of IRS1, IRS2, or both. PMID:26976654

  2. A heteroglycan from the cyanobacterium Nostoc commune modulates LPS-induced inflammatory cytokine secretion by THP-1 monocytes through phosphorylation of ERK1/2 and Akt.

    PubMed

    Olafsdottir, Astridur; Thorlacius, Gudny Ella; Omarsdottir, Sesselja; Olafsdottir, Elin Soffia; Vikingsson, Arnor; Freysdottir, Jona; Hardardottir, Ingibjorg

    2014-09-25

    Cyanobacteria (blue-green algae) have been consumed as food and used in folk medicine since ancient times to alleviate a variety of diseases. Cyanobacteria of the genus Nostoc have been shown to produce complex exopolysaccharides with antioxidant and antiviral activity. Furthermore, Nostoc sp. are common in cyanolichen symbiosis and lichen polysaccharides are known to have immunomodulating effects. Nc-5-s is a heteroglycan isolated from free-living colonies of Nostoc commune and its structure has been characterized in detail. The aim of this study was to determine the effects of Nc-5-s on the inflammatory response of lipopolysaccharide (LPS)-stimulated human THP-1 monocytes and how the effects are mediated. THP-1 monocytes primed with interferon-γ and stimulated with LPS in the presence of Nc-5-s secreted less of the pro-inflammatory cytokine interleukin (IL)-6 and more of the anti-inflammatory cytokine IL-10 than THP-1 monocytes stimulated without Nc-5-s. In contrast, Nc-5-s increased LPS-induced secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-8. Nc-5-s decreased LPS-induced phosphorylation of the extracellular regulated kinase (ERK)1/2 and Akt kinase, but did not affect phosphorylation of the p38 kinase, activation of the nuclear factor kappa B pathway, nor DNA binding of c-fos. These results show that Nc-5-s has anti-inflammatory effects on IL-6 and IL-10 secretion by THP-1 monocytes, but its effects are pro-inflammatory when it comes to TNF-α and IL-8. Furthermore, they show that the effects of Nc-5-s may be mediated through the ERK1/2 pathway and/or the Akt/phosphoinositide 3-kinase pathway and their downstream effectors. The ability of Nc-5-s to decrease IL-6 secretion, increase IL-10 secretion and moderate ERK1/2 activation indicates a potential for its development as an anti-inflammatory agent. PMID:24877713

  3. Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: Involvement of the adaptive antioxidant response

    SciTech Connect

    Xue, Peng; Hou, Yongyong; Zhang, Qiang; Woods, Courtney G.; Yarborough, Kathy; Liu, Huiyu; Sun, Guifan; Andersen, Melvin E.; Pi, Jingbo

    2011-04-08

    Highlights: {yields} In 3T3-L1 adipocytes iAs{sup 3+} decreases insulin-stimulated glucose uptake. {yields} iAs{sup 3+} attenuates insulin-induced phosphorylation of AKT S473. {yields} iAs{sup 3+} activates the cellular adaptive oxidative stress response. {yields} iAs{sup 3+} impairs insulin-stimulated ROS signaling. {yields} iAs{sup 3+} decreases expression of adipogenic genes and GLUT4. -- Abstract: There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 {mu}M) inorganic arsenite (iAs{sup 3+}) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs{sup 3+} exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs{sup 3+} exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4

  4. Prostaglandin D(2) induces apoptosis of human osteoclasts through ERK1/2 and Akt signaling pathways.

    PubMed

    Yue, Li; Haroun, Sonia; Parent, Jean-Luc; de Brum-Fernandes, Artur J

    2014-03-01

    In a recent study we have shown that prostaglandin D2 (PGD2) induces human osteoclast (OC) apoptosis through the activation of the chemoattractant receptor homologous molecule expressed on T-helper type 2 cell (CRTH2) receptor and the intrinsic apoptotic pathway. However, the molecular mechanisms underlying this response remain elusive. The objective of this study is to investigate the intracellular signaling pathways mediating PGD2-induced OC apoptosis. OCs were generated by in vitro differentiation of human peripheral blood mononuclear cells (PBMCs), and then treated with or without the selective inhibitors of mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase, (MEK)-1/2, phosphatidylinositol3-kinase (PI3K) and NF-κB/IκB kinase-2 (IKK2) prior to the treatments of PGD2 as well as its agonists and antagonists. Fluorogenic substrate assay and immunoblotting were performed to determine the caspase-3 activity and key proteins involved in Akt, ERK1/2 and NF-κB signaling pathways. Treatments with both PGD2 and a CRTH2 agonist decreased ERK1/2 (Thr202/Tyr204) and Akt (Ser473) phosphorylation, whereas both treatments increased β-arrestin-1 phosphorylation (Ser412) in the presence of naproxen, which was used to eliminate endogenous prostaglandin production. In the absence of naproxen, treatment with a CRTH2 antagonist increased both ERK1/2 and Akt phosphorylations, and reduced the phosphorylation of β-arrestin-1. Treatment of OCs with a selective MEK-1/2 inhibitor increased caspase-3 activity and OC apoptosis induced by both PGD2 and a CRTH2 agonist. Moreover, a CRTH2 antagonist diminished the selective MEK-1/2 inhibitor-induced increase in caspase-3 activity in the presence of endogenous prostaglandins. In addition, treatment of OCs with a selective PI3K inhibitor decreased ERK1/2 (Thr202/Tyr204) phosphorylation caused by PGD2, whereas increased ERK1/2 (Thr202/Tyr204) phosphorylation by a CRTH2 antagonist was attenuated with a PI3K

  5. AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

    SciTech Connect

    Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar; Lang, Florian

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protects against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.

  6. Expression of a phosphorylated p130Cas substrate domain attenuates the phosphatidylinositol 3-kinase/Akt survival pathway in tamoxifen resistant breast cancer cells

    PubMed Central

    Soni, Shefali; Lin, Bor-Tyh; August, Avery; Nicholson, Robert I.; Kirsch, Kathrin H.

    2009-01-01

    Elevated expression of p130Cas/BCAR1 (breast cancer anti estrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. Specifically, p130Cas signaling has been associated with antiestrogen resistance, for which the mechanism is currently unknown. TAM-R cells, which were established by long-term exposure of estrogen (E2)-dependent MCF-7 cells to tamoxifen, displayed elevated levels of total and activated p130Cas. Here we have investigated the effects of p130Cas inhibition on growth factor signaling in tamoxifen resistance. To inhibit p130Cas, a phosphorylated substrate domain of p130Cas, that acts as a dominant-negative (DN) p130Cas molecule by blocking signal transduction downstream of the p130Cas substrate domain, as well as knockdown by siRNA was employed. Interference with p130Cas signaling/expression induced morphological changes, which were consistent with a more epithelial-like phenotype. The phenotypic reversion was accompanied by reduced migration, attenuation of the ERK and phosphatidylinositol 3-kinase/Akt pathways, and induction of apoptosis. Apoptosis was accompanied by downregulation of the expression of the anti-apoptotic protein Bcl-2. Importantly, these changes re-sensitized TAM-R cells to tamoxifen treatment by inducing cell death. Therefore, our findings suggest that targeting the product of the BCAR1 gene by a peptide which mimics the phosphorylated substrate domain may provide a new molecular avenue for treatment of antiestrogen resistant breast cancers. PMID:19330798

  7. Cicletanine stimulates eNOS phosphorylation and NO production via Akt and MAP kinase/Erk signaling in sinusoidal endothelial cells.

    PubMed

    Liu, Songling; Rockey, Don C

    2013-07-15

    The function of the endothelial isoform of nitric oxide synthase (eNOS) and production of nitric oxide (NO) is altered in a number of disease states. Pharmacological approaches to enhancing NO synthesis and thus perhaps endothelial function could have substantial benefits in patients. We analyzed the effect of cicletanine, a synthetic pyridine with potent vasodilatory characteristics, on eNOS function and NO production in normal (liver) and injured rat sinusoidal endothelial cells, and we studied the effect of cicletanine-induced NO on stellate cell contraction and portal pressure in an in vivo model of liver injury. Sinusoidal endothelial cells were isolated from normal and injured rat livers. After exposure to cicletanine, eNOS phosphorylation, NO synthesis, and the signaling pathway regulating eNOS activation were measured. Cicletanine led to an increase in eNOS (Ser¹¹⁷⁷) phosphorylation, cytochrome c reductase activity, L-arginine conversion to L-citrulline, as well as NO production. The mechanism of the effect of cicletanine appeared to be via the protein kinase B (Akt) and MAP kinase/Erk signaling pathways. Additionally, cicletanine improved NO synthesis in injured sinusoidal endothelial cells. NO production induced by cicletanine in sinusoidal endothelial cells increased protein kinase G (PKG) activity as well as relaxation of stellate cells. Finally, administration of cicletanine to mice with portal hypertension induced by bile duct ligation led to reduction of portal pressure. The data indicate that cicletanine might improve eNOS activity in injured sinusoidal endothelial cells and likely activates hepatic stellate cell NO/PKG signaling. It raises the possibility that cicletanine could improve intrahepatic vascular function in portal hypertensive patients. PMID:23639812

  8. Kaempferol Suppresses Transforming Growth Factor-β1-Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-179.

    PubMed

    Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

    2015-07-01

    Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non-small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1-induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1-mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1-mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1-induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1-mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1-induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1-induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. PMID:26297431

  9. Kaempferol Suppresses Transforming Growth Factor-β1–Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-1791

    PubMed Central

    Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

    2015-01-01

    Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non–small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1–induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1–mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1–mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1–induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1–mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1–induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1–induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. PMID:26297431

  10. Antiplatelet Activity of Morus alba Leaves Extract, Mediated via Inhibiting Granule Secretion and Blocking the Phosphorylation of Extracellular-Signal-Regulated Kinase and Akt

    PubMed Central

    Rhee, Man Hee; Sung, Yoon-Young; Yang, Won-Kyung; Kim, Seung Hyung; Kim, Ho-Kyoung

    2014-01-01

    Ethnopharmacological Relevance. Morus alba L. leaves (MAE) have been used in fork medicine for the treatment of beriberi, edema, diabetes, hypertension, and atherosclerosis. However, underlying mechanism of MAE on cardiovascular protection remains to be elucidated. Therefore, we investigated whether MAE affect platelet aggregation and thrombosis. Materials and Methods. The anti-platelet activity of MAE was studied using rat platelets. The extent of anti-platelet activity of MAE was assayed in collagen-induced platelet aggregation. ATP and serotonin release was carried out. The activation of integrin αIIbβ3 and phosphorylation of signaling molecules, including MAPK and Akt, were investigated with cytofluorometer and immunoblotting, respectively. The thrombus formation in vivo was also evaluated in arteriovenous shunt model of rats. Results. HPLC chromatographic analysis revealed that MAE contained rutin and isoquercetin. MAE dose-dependently inhibited collagen-induced platelet aggregation. MAE also attenuated serotonin secretion and thromboxane A2 formation. In addition, the extract in vivo activity showed that MAE at 100, 200, and 400 mg/kg significantly and dose-dependently attenuated thrombus formation in rat arterio-venous shunt model by 52.3% (P < 0.001), 28.3% (P < 0.01), and 19.1% (P < 0.05), respectively. Conclusions. MAE inhibit platelet activation, TXB2 formation, serotonin secretion, aggregation, and thrombus formation. The plant extract could be considered as a candidate to anti-platelet and antithrombotic agent. PMID:24701244

  11. Antiplatelet Activity of Morus alba Leaves Extract, Mediated via Inhibiting Granule Secretion and Blocking the Phosphorylation of Extracellular-Signal-Regulated Kinase and Akt.

    PubMed

    Kim, Dong-Seon; Ji, Hyun Dong; Rhee, Man Hee; Sung, Yoon-Young; Yang, Won-Kyung; Kim, Seung Hyung; Kim, Ho-Kyoung

    2014-01-01

    Ethnopharmacological Relevance. Morus alba L. leaves (MAE) have been used in fork medicine for the treatment of beriberi, edema, diabetes, hypertension, and atherosclerosis. However, underlying mechanism of MAE on cardiovascular protection remains to be elucidated. Therefore, we investigated whether MAE affect platelet aggregation and thrombosis. Materials and Methods. The anti-platelet activity of MAE was studied using rat platelets. The extent of anti-platelet activity of MAE was assayed in collagen-induced platelet aggregation. ATP and serotonin release was carried out. The activation of integrin α IIb β 3 and phosphorylation of signaling molecules, including MAPK and Akt, were investigated with cytofluorometer and immunoblotting, respectively. The thrombus formation in vivo was also evaluated in arteriovenous shunt model of rats. Results. HPLC chromatographic analysis revealed that MAE contained rutin and isoquercetin. MAE dose-dependently inhibited collagen-induced platelet aggregation. MAE also attenuated serotonin secretion and thromboxane A2 formation. In addition, the extract in vivo activity showed that MAE at 100, 200, and 400 mg/kg significantly and dose-dependently attenuated thrombus formation in rat arterio-venous shunt model by 52.3% (P < 0.001), 28.3% (P < 0.01), and 19.1% (P < 0.05), respectively. Conclusions. MAE inhibit platelet activation, TXB2 formation, serotonin secretion, aggregation, and thrombus formation. The plant extract could be considered as a candidate to anti-platelet and antithrombotic agent. PMID:24701244

  12. Cytoplasmic localization of wild-type survivin is associated with constitutive activation of the PI3K/Akt signaling pathway and represents a favorable prognostic factor in patients with acute myeloid leukemia

    PubMed Central

    Serrano-López, Juana; Serrano, Josefina; Figueroa, Vianihuini; Torres-Gomez, Antonio; Tabares, Salvador; Casaño, Javier; Fernandez-Escalada, Noemi; Sánchez-Garcia, Joaquín

    2013-01-01

    Survivin is over-expressed in most hematologic malignancies but the prognostic significance of the subcompartmental distribution of wild-type or splicing variants in acute myeloid leukemia has not been addressed yet. Using western blotting, we assessed the expression of wild-type survivin and survivin splice variants 2B and Delta-Ex3 in nuclear and cytoplasmic protein extracts in samples taken from 105 patients at the time of their diagnosis of acute myeloid leukemia. Given that survivin is a downstream effector of the PI3K/Akt signaling pathway, survivin expression was also correlated with pSer473-Akt. Wild-type survivin and the 2B splice variant were positive in 76.3% and 78.0% of samples in the nucleus, cytoplasm or both, whereas the Delta-Ex3 isoform was only positive in the nucleus in 37.7% of samples. Cytoplasmic localization of wild-type survivin was significantly associated with the presence of high levels of pSer473-Akt (P<0.001). Inhibition of the PI3K/Akt pathway with wortmannin and Ly294002 caused a significant reduction in the expression of cytoplasmic wild-type survivin. The presence of cytoplasmic wild-type survivin and pSer473-Akt was associated with a lower fraction of quiescent leukemia stem cells (P=0.02). The presence of cytoplasmic wild-type survivin and pSer473-Akt were favorable independent prognostic factors. Moreover, the activation of the PI3K/Akt pathway with expression of cytoplasmic wild-type survivin identified a subgroup of acute myeloid leukemia patients with an excellent outcome (overall survival rate of 60.0±21.9% and relapse-free survival of 63.0±13.5%). Our findings suggest that cytoplasmic wild-type survivin is a critical downstream effector of the PI3K/Akt pathway leading to more chemosensitive cells and a more favorable outcome in acute myeloid leukemia. PMID:23812937

  13. Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb{sup 2+}-induced neuronal death in cultured hippocampal neurons

    SciTech Connect

    Li Chenchen Xing Tairan Tang Mingliang Yong Wu Yan Dan Deng Hongmin Wang Huili Wang Ming Chen Jutao Ruan Diyun

    2008-06-15

    Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb{sup 2+} causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb{sup 2+}. Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb{sup 2+}-induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb{sup 2+} treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 {mu}M) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb{sup 2+}. And that Pb{sup 2+}-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb{sup 2+} and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.

  14. Up-regulation of the Ang II/AT1 receptor may compensate for the loss of gastric antrum ICC via the PI3k/Akt signaling pathway in STZ-induced diabetic mice.

    PubMed

    Zhang, C M; Huang, X; Lu, H L; Meng, X M; Liu, D H; Kim, Young-Chul; Xu, W X

    2016-03-01

    The classic renin-angiotensin system (RAS) is a complex system in which angiotensin II (Ang II) has been identified as an important endogenous regulator that influences both smooth muscle contraction and cell growth. Although a local RAS is known to exist in the gastrointestinal tract, it is unclear whether Ang II is involved in the loss of gastric interstitial cells of Cajal (ICC) in diabetic mice. The present study was designed to investigate the effect of Ang II on ICC survival in streptozotocin (STZ)-induced diabetic mice. Western blot, immunofluorescence, isometric muscle recording, enzyme-linked immunosorbent assay (ELISA) and a cell counting kit-8 were used in this research. Our results demonstrate that the c-Kit and membrane-bound stem cell factor (mSCF) protein expression levels in gastric smooth muscle were decreased in STZ-induced diabetic mice. However, the angiotensin receptor type 1 (AT1R) expression levels in gastric smooth muscle and angiotensin-converting enzyme (ACE) expression levels in gastric mucosa were increased. The effect of Ang II on the tonic contraction of gastric smooth muscle was potentiated in diabetic mice, and the plasma Ang II level was enhanced. Ang II increased mSCF expression, cell proliferation, and Akt-Ser473 phosphorylation in cultured gastric smooth muscle cells (GSMCs). These effects were reduced by specific inhibitors ZD7155 (an AT1R antagonist) and LY294002 (a PI3-kinase inhibitor). Our results suggest that Ang II increases mSCF expression and cell proliferation in cultured GSMCs in a PI3K/Akt signaling-dependent manner. ACE and AT1R up-regulation in the stomach may help compensate for ICC loss in STZ-induced diabetic mice. PMID:26773730

  15. Harmine induces cell cycle arrest and mitochondrial pathway-mediated cellular apoptosis in SW620 cells via inhibition of the Akt and ERK signaling pathways.

    PubMed

    Liu, Jiming; Li, Qiang; Liu, Zhilong; Lin, Liuming; Zhang, Xiangqiang; Cao, Mingrong; Jiang, Jianwei

    2016-06-01

    Harmine, a β-carboline alkaloid isolated from the seeds of Peganum harmala, possesses both antitumor and anti‑nociceptive effects and inhibits human DNA topoisomerase. However, no detailed data are available concerning the mechanisms of harmine in human colorectal carcinoma SW620 cells. In the present study, we demonstrated that harmine inhibited the proliferation of SW620 cells in a dose-dependent manner using MTT and clone formation assays, and the IC50 value of harmine on the growth inhibition of SW620 cells for 48 h was 5.13 µg/ml. PI staining showed that harmine altered the cell cycle distribution by decreasing the proportion of cells in the G0-G1 phase and increasing the proportion in the S and G2-M phase. The expression level of cyclin D1 was decreased, while the expression of cyclin A, E2 and B1, CDK1/cdc2, Myt-1 and p-cdc2 (Tyr15) were increased, which was in accordance with the S and G2/M phase arrest. Hoechst 33258 staining revealed nuclear fragmentation, chromosomal condensation and cell shrinkage in the SW620 cells treated with harmine. Flow cytometry revealed that the percentage of apoptotic sub-G1 cells increased from 7.19 to 26.58%, while in the control group, sub-G1 cells only increased from 1.53 to 1.60%. Furthermore, early and late apoptotic cells were increased from 11.96 to 26.38% when incubated with the indicated concentration of harmine for 48 h, while in the control group, <8% of cells underwent apoptosis. JC-1 staining revealed that harmine decreased mitochondrial transmembrane potential (ΔΨm). The apoptosis of SW620 cells was also detected by western blot analysis, showing caspase-3 and -9, and PARP activation; the downregulation of Bcl-2, Mcl-1, Bcl-xL; and the upregulation of Bax. The expression of p-ERK, p-Akt (Ser473) and p-Akt (Thr308) was inhibited, and phosphorylation of downstream targets of Akt, such as p-FoxO3a and p-GSK‑3β were also attenuated. In conclusion, harmine induces cell cycle arrest and

  16. Curcumol Suppresses Breast Cancer Cell Metastasis by Inhibiting MMP-9 Via JNK1/2 and Akt-Dependent NF-κB Signaling Pathways.

    PubMed

    Ning, Ling; Ma, Hui; Jiang, Zhuyun; Chen, Lu; Li, Li; Chen, Qianfeng; Qi, Hongyi

    2016-06-01

    Curcumolhas been reported to possess antitumor activity. However, its effect and mechanisms against tumor metastasis are still unclear. This study is to investigate the inhibitory effect of curcumol on breast cancer cell metastasis and elucidate the underlying molecular mechanisms. Our results showed that noncytotoxicity was caused by curcumol within 10 to 40 µg/mL in MDA-MB-231 and 4T1 cells for 24 hours, whereas sustained treatment with curcumol for 14 days significantly suppressed the clonogenic activity of cells. Importantly, curcumol at noncytotoxic concentrations suppressed the migration ability of both MDA-MB-231 and 4T1 cells. Moreover, curcumol suppressed the migration and invasion of MDA-MB-231 cells in the Boyden chamber migration and invasion assay and inhibited the adhesion of MDA-MB-231 cells onto the matrigel. Further investigations revealed that curcumol decreased the enzyme activity and protein expression of matrix metalloproteinase (MMP-9) in MDA-MB-231 cells. Moreover, curcumol inhibited the activation of c-Jun N-terminal kinase (JNK) 1/2 and Akt (Ser473). Meanwhile, it also inhibited the nuclear translocation and transcriptional activity of nuclear factor κB (NF-κB). Furthermore, JNK inhibitor SP600125 and Akt (Ser473) inhibitor LY294002 enhanced the inhibition of curcumol on NF-κB p65 nuclear translocation. Finally, supplementation with SP600125, LY294002, or NF-κB inhibitor Ammonium pyrrolidinedithiocarbamate (PDTC) significantly enhanced the inhibitory effect of curcumol on MMP-9 expression and cell migration, invasion, and adhesion in MDA-MB-231 cells. Our findings provide evidence for the suppression of breast cancer cell metastasis by curcumol and suggest that the inhibition of MMP-9 via JNK1/2 and Akt (Ser473)-dependent NF-κB signaling pathways may be the underlying mechanisms. PMID:27125675

  17. 1,25-Dihydroxyvitamin D{sub 3} induces biphasic NF-{kappa}B responses during HL-60 leukemia cells differentiation through protein induction and PI3K/Akt-dependent phosphorylation/degradation of I{kappa}B

    SciTech Connect

    Tse, A.K.-W.; Wan, C.-K.; Shen, X.-L.; Zhu, G.-Y.; Cheung, H.-Y.; Yang, M.; Fong, W.-F. . E-mail: wffong@hkbu.edu.hk

    2007-05-01

    1,25-Dihydroxyvitamin D{sub 3} (VD{sub 3}) induces differentiation in a number of leukemia cell lines and under various conditions is able to either stimulate or inhibit nuclear factor kappa B (NF-{kappa}B) activity. Here we report a time-dependent biphasic regulation of NF-{kappa}B in VD{sub 3}-treated HL-60 leukemia cells. After VD{sub 3} treatment there was an early {approx} 4 h suppression and a late 8-72 h prolonged reactivation of NF-{kappa}B. The reactivation of NF-{kappa}B was concomitant with increased IKK activities, IKK-mediated I{kappa}B{alpha} phosphorylation, p65 phosphorylation at residues S276 and S536, p65 nuclear translocation and p65 recruitment to the NF-{kappa}B/vitamin D responsive element promoters. In parallel with NF-{kappa}B stimulation, there was an up-regulation of NF-{kappa}B controlled inflammatory and anti-apoptotic genes such as TNF{alpha}, IL-1{beta} and Bcl-xL. VD{sub 3}-triggered reactivation of NF-{kappa}B was associated with PI3K/Akt phosphorylation. PI3K/Akt antagonists suppressed VD{sub 3}-stimulated I{kappa}B{alpha} phosphorylation as well as NF-{kappa}B-controlled gene expression. The early {approx} 4 h VD{sub 3}-mediated NF-{kappa}B suppression coincided with a prolonged increase of I{kappa}B{alpha} protein which require de novo protein synthesis, lasted for as least 72 h and was insensitive to MAPK, IKK or PI3K/Akt inhibitors. Our data suggest a novel biphasic regulation of NF-{kappa}B in VD{sub 3}-treated leukemia cells and our results may have provided the first molecular explanation for the contradictory observations reported on VD{sub 3}-mediated immune-regulation.

  18. MAPKs (ERK1/2, p38) and AKT can be phosphorylated by shear stress independently of platelet endothelial cell adhesion molecule-1 (CD31) in vascular endothelial cells.

    PubMed

    Sumpio, Bauer E; Yun, Sangseob; Cordova, Alfredo C; Haga, Masae; Zhang, Jin; Koh, Yongbok; Madri, Joseph A

    2005-03-25

    PECAM-1 (CD31) is a member of the Ig superfamily of cell adhesion molecules and is expressed on endothelial cells (EC) as several circulating blood elements including platelets, polymorphonuclear leukocytes, monocytes, and lymphocytes. PECAM-1 tyrosine phosphorylation has been observed following mechanical stimulation of EC but its role in mechanosensing is still incompletely understood. The aim of this study was to investigate the involvement of PECAM-1 in signaling cascades in response to fluid shear stress (SS) in vascular ECs. PECAM-1-deficient (KO) and PECAM-reconstituted murine microvascular ECs, 50 and 100% confluent bovine aortic EC (BAEC), and human umbilical vein EC (HUVEC) transfected with antisense PECAM-1 oligonucleotides were exposed to oscillatory SS (14 dynes/cm2) for 0, 5, 10, 30 or 60 min. The tyrosine phosphorylation level of PECAM-1 immunoprecipitated from SS-stimulated PECAM-reconstituted, but not PECAM-1-KO, murine ECs increased. Although PECAM-1 was phosphorylated in 100% confluent BAEC and HUVEC, its phosphorylation level in 50% confluent BAECs or HUVEC was not detected by SS. Likewise PECAM-1 phosphorylation was robust in the wild type and scrambled-transfected HUVEC but not in the PECAM-1 antisense-HUVEC. ERK(1/2), p38 MAPK, and AKT were activated by SS in all cell types tested, including the PECAM-1-KO murine ECs, 50% confluent BAECs, and HUVEC transfected with antisense PECAM-1. This suggests that PECAM-1 may not function as a major mechanoreceptor for activation of MAPK and AKT in ECs and that there are likely to be other mechanoreceptors in ECs functioning to detect shear stress and trigger intercellular signals. PMID:15668248

  19. CXCR1 promotes malignant behavior of gastric cancer cells in vitro and in vivo in AKT and ERK1/2 phosphorylation.

    PubMed

    Wang, Junpu; Hu, Wanming; Wu, Xiaoying; Wang, Kuansong; Yu, Jun; Luo, Baihua; Luo, Gengqiu; Wang, Weiyuan; Wang, Huiling; Li, Jinghe; Wen, Jifang

    2016-05-01

    CXCR1 is a member of the chemokine receptor family, which was reported to play an important role in several cancers. The present study investigated the influence of CXCR1 stable knockdown or overexpression on the malignant behavior of gastric cancer cells in vitro and in vivo and the potential mechanisms. MKN45 and BGC823 cells were stably transfected with plasmid pYr-1.1-CXCR1-shRNA (knockdown) and pIRES2-ZsGreen1-CXCR1 (overexpression), respectively. Malignant behavior was evaluated in vitro for changes in proliferation by MTT and colony forming assays; cell cycle and apoptosis by flow cytometry; and migration and invasion using transwell and wound-healing assays. Proliferation, cell cycle, apoptosis, migration and invasion-related signaling molecule expression were measured by real-time RT-PCR and western blot analysis. CXCR1 knockdown and overexpressing xenografts were monitored for in vivo tumor growth. Stable knockdown of CXCR1 inhibited MKN45 cell proliferation, migration and invasion, but were reversed in BGC823 cells stably overexpressing CXCR1. In addition, MKN45 cells stably transfected with CXCR1 shRNA inhibited AKT and ERK1/2 phosphorylation, protein expression of cyclin D1, EGFR, VEGF, MMP-9, MMP-2 and Bcl-2, and increased protein expression of Bax and E-cadherin (all P<0.05). In vivo CXCR1-shRNA-MKN45 cells transplanted into nude mice formed smaller tumors than non-transfected or scrambled-shRNA cells (both P<0.05). In contrast BGC823 cells overexpressing CXCR1 formed larger tumors in mice than cells carrying an empty expression plasmid or non-transfected cells (both P<0.05). CXCR1 promoted gastric cancer cell proliferation, migration and invasion. The present study provides preclinical data to support CXCR1 as a novel therapeutic target for gastric cancer. PMID:26983663

  20. α-Solanine induces ROS-mediated autophagy through activation of endoplasmic reticulum stress and inhibition of Akt/mTOR pathway.

    PubMed

    Hasanain, M; Bhattacharjee, A; Pandey, P; Ashraf, R; Singh, N; Sharma, S; Vishwakarma, A L; Datta, D; Mitra, K; Sarkar, J

    2015-01-01

    α-Solanine is a glycoalkaloid found in species of the nightshade family including potato. It was primarily reported to have toxic effects in humans. However, there is a growing body of literature demonstrating in vitro and in vivo anticancer activity of α-solanine. Most of these studies have shown activation of apoptosis as the underlying mechanism in antitumor activity of α-solanine. In this study, we report α-solanine as a potential inducer of autophagy, which may act synergistically or in parallel with apoptosis to exert its cytotoxic effect. Induction of autophagy was demonstrated by several assays including electron microscopy, immunoblotting of autophagy markers and immunofluorescence for LC3 (microtubule-associated protein 1 (MAP1) light chain-3) puncta. α-Solanine-induced autophagic flux was demonstrated by additionally enhanced--turnover of LC3-II and--accumulation of LC3-specific puncta after co-incubation of cells with either of the autophagolysosome inhibitors--chloroquine and--bafilomycin A1. We also demonstrated α-solanine-induced oxidative damage in regulating autophagy where pre-incubation of cells with reactive oxygen species (ROS) scavenger resulted in suppression of CM-H2DCFDA (5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester) fluorescence as well as decrease in LC3-II turnover. α-Solanine treatment caused an increase in the expression of endoplasmic reticulum (ER) stress proteins (BiP, activating transcription factor 6 (ATF6), X-box-binding protein 1, PERK, inositol-requiring transmembrane kinase/endonuclease 1, ATF4 and CCAAT-enhancer-binding protein (C/EBP)-homologous protein) suggesting activation of unfolded protein response pathway. Moreover, we found downregulation of phosphorylated Akt (Thr308 and Ser473), mammalian target of rapamycin (mTOR; Ser2448 and Ser2481) and 4E-BP1 (Thr37/46) by α-solanine implying suppression of the Akt/mTOR pathway. Collectively, our results signify that α-solanine induces

  1. α-Solanine induces ROS-mediated autophagy through activation of endoplasmic reticulum stress and inhibition of Akt/mTOR pathway

    PubMed Central

    Hasanain, M; Bhattacharjee, A; Pandey, P; Ashraf, R; Singh, N; Sharma, S; Vishwakarma, A L; Datta, D; Mitra, K; Sarkar, J

    2015-01-01

    α-Solanine is a glycoalkaloid found in species of the nightshade family including potato. It was primarily reported to have toxic effects in humans. However, there is a growing body of literature demonstrating in vitro and in vivo anticancer activity of α-solanine. Most of these studies have shown activation of apoptosis as the underlying mechanism in antitumor activity of α-solanine. In this study, we report α-solanine as a potential inducer of autophagy, which may act synergistically or in parallel with apoptosis to exert its cytotoxic effect. Induction of autophagy was demonstrated by several assays including electron microscopy, immunoblotting of autophagy markers and immunofluorescence for LC3 (microtubule-associated protein 1 (MAP1) light chain-3) puncta. α-Solanine-induced autophagic flux was demonstrated by additionally enhanced – turnover of LC3-II and – accumulation of LC3-specific puncta after co-incubation of cells with either of the autophagolysosome inhibitors – chloroquine and – bafilomycin A1. We also demonstrated α-solanine-induced oxidative damage in regulating autophagy where pre-incubation of cells with reactive oxygen species (ROS) scavenger resulted in suppression of CM-H2DCFDA (5 (and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester) fluorescence as well as decrease in LC3-II turnover. α-Solanine treatment caused an increase in the expression of endoplasmic reticulum (ER) stress proteins (BiP, activating transcription factor 6 (ATF6), X-box-binding protein 1, PERK, inositol-requiring transmembrane kinase/endonuclease 1, ATF4 and CCAAT-enhancer-binding protein (C/EBP)-homologous protein) suggesting activation of unfolded protein response pathway. Moreover, we found downregulation of phosphorylated Akt (Thr308 and Ser473), mammalian target of rapamycin (mTOR; Ser2448 and Ser2481) and 4E-BP1 (Thr37/46) by α-solanine implying suppression of the Akt/mTOR pathway. Collectively, our results signify that

  2. Expression of FLNa in human melanoma cells regulates the function of integrin α1β1 and phosphorylation and localisation of PKB/AKT/ERK1/2 kinases.

    PubMed

    Krebs, Kristi; Ruusmann, Anu; Simonlatser, Grethel; Velling, Teet

    2015-12-01

    FLNa is a ubiquitous cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and functions as a signalling intermediate. We investigated FLNa's role in the function of integrin-type collagen receptors, EGF-EGFR signalling and regulation of PKB/Akt and ERK1/2. Using FLNa-deficient M2 human melanoma cells, and same cells expressing EGFP-FLNa (M2F) or its Ig-like repeats 1-8+24, 8-15+24 and 16-24, we found that in M2F and M2 8-15+24 cells, EGF induced the increased phosphorylation of PKB/Akt and ERK1/2. In M2F cells EGF induced the localisation of these kinases to cell nucleus and lamellipodia, respectively, and the ERK1/2 phosphorylation-dependent co-immunoprecipitation of FLNa with ERK1/2. Only M2F and M2 8-15+24 cells adhered to and spread on type I collagen whereas on fibronectin all cells behaved similarly. α1β1 and α2β1 were the integrin-type collagen receptors expressed on these cells with primarily α1β1 localising to focal contacts and affecting cell adhesion and migration in a manner dependent on FLNa or its Ig-like repeats 8-15. Our results suggest a role for FLNa repeats 8-15 in the α1-subunit-dependent regulation of integrin α1β1 function, EGF-EGFR signalling to PKB/Akt and ERK1/2, identify ERK1/2 in EGF-induced FLNa-associated protein complexes, and show that the function of different integrins is subjected to differential regulation by FLNa. PMID:26572583

  3. Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

    SciTech Connect

    Teramura, Takeshi; Takehara, Toshiyuki; Onodera, Yuta; Nakagawa, Koichi; Hamanishi, Chiaki; Fukuda, Kanji

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Mechanical stimulation is an important factor for regulation of stem cell fate. Black-Right-Pointing-Pointer Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. Black-Right-Pointing-Pointer Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. Black-Right-Pointing-Pointer This reaction could be reproduced only by transfection of dominant active Rho. Black-Right-Pointing-Pointer Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

  4. Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway

    SciTech Connect

    Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Hui-Young; Hong, Suntaek; Kim, Seong-Jin; Kim, Byung-Chul

    2013-06-14

    Highlights: •H{sub 2}O{sub 2} inhibits TGF-β1-induced cell cycle arrest. •H{sub 2}O{sub 2} induces Smad3 linker phosphorylation through Akt-ERK1/2 pathway. •H{sub 2}O{sub 2}-mediated suppression of TGF-β signal requires Smad3 linker phosphorylation. •This is a first report about interplay between H{sub 2}O{sub 2} and growth inhibition pathway. -- Abstract: Hydrogen peroxide (H{sub 2}O{sub 2}) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H{sub 2}O{sub 2} are less understood. Here we report an important mechanism for antagonistic effects of H{sub 2}O{sub 2} on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H{sub 2}O{sub 2} (0.05–0.2 mM) completely blocked TGF-β1-mediated induction of p15{sup INK4B} expression and increase of its promoter activity. Interestingly, H{sub 2}O{sub 2} selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H{sub 2}O{sub 2} increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H{sub 2}O{sub 2} on TGF-β1-induced increase of p15{sup INK4B}-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H{sub 2}O{sub 2} as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing

  5. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

    2016-04-01

    Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. PMID:26921637

  6. Metformin inhibits growth of eutopic stromal cells from adenomyotic endometrium via AMPK activation and subsequent inhibition of AKT phosphorylation: a possible role in the treatment of adenomyosis.

    PubMed

    Xue, Jing; Zhang, Hui; Liu, Wei; Liu, Ming; Shi, Min; Wen, Zeqing; Li, Changzhong

    2013-10-01

    Adenomyosis is a finding that is associated with dysmenorrhea and heavy menstrual bleeding, associated with PI3K/AKT signaling overactivity. To investigate the effect of metformin on the growth of eutopic endometrial stromal cells (ESCs) from patients with adenomyosis and to explore the involvement of AMP-activated protein kinase (AMPK) and PI3K/AKT pathways. Primary cultures of human ESCs were derived from normal endometrium (normal endometrial stromal cells (N-ESCs)) and adenomyotic eutopic endometrium (adenomyotic endometrial stroma cells (A-ESCs)). Expression of AMPK was determined using immunocytochemistry and western blot analysis. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays were used to determine the effects of metformin and compound C on ESCs and also to detect growth and proliferation of ESCs. AMPK and PI3K/AKT signaling was determined by western blotting. A-ECSs exhibited greater AMPK expression than N-ESCs. Metformin inhibited proliferation of ESCs in a concentration-dependent manner. The IC50 was 2.45 mmol/l for A-ESCs and 7.87 mmol/l for N-ESCs. Metformin increased AMPK activation levels (p-AMPK/AMPK) by 2.0±0.3-fold in A-ESCs, 2.3-fold in A-ESCs from the secretory phase, and 1.6-fold in the proliferation phase. The average reduction ratio of 17β-estradiol on A-ESCs was 2.1±0.8-fold in proliferative phase and 2.5±0.5-fold in secretory phase relative to the equivalent groups not treated with 17β-estradiol. The inhibitory effects of metformin on AKT activation (p-AKT/AKT) were more pronounced in A-ESCs from the secretory phase (3.2-fold inhibition vs control) than in those from the proliferation phase (2.3-fold inhibition vs control). Compound C, a selective AMPK inhibitor, abolished the effects of metformin on cell growth and PI3K/AKT signaling. Metformin inhibits cell growth via AMPK activation and subsequent inhibition of PI3K/AKT signaling in A-ESCs, particularly during the secretory phase, suggesting a greater

  7. Phosphorylated AKT inhibits the apoptosis induced by DRAM-mediated mitophagy in hepatocellular carcinoma by preventing the translocation of DRAM to mitochondria.

    PubMed

    Liu, K; Shi, Y; Guo, X H; Ouyang, Y B; Wang, S S; Liu, D J; Wang, A N; Li, N; Chen, D X

    2014-01-01

    Increasing autophagy is beneficial for curing hepatocellular carcinoma (HCC). Damage-regulated autophagy modulator (DRAM) was recently reported to induce apoptosis by mediating autophagy. However, the effects of DRAM-mediated autophagy on apoptosis in HCC cells remain unclear. In this study, normal hepatocytes (7702) and HCC cell lines (HepG2, Hep3B and Huh7) were starved for 48 h. Starvation induced apoptosis and autophagy in all cell lines. We determined that starvation also induced DRAM expression and DRAM-mediated autophagy in both normal hepatocytes and HCC cells. However, DRAM-mediated autophagy was involved in apoptosis in normal hepatocytes but not in HCC cells, suggesting that DRAM-mediated autophagy fails to induce apoptosis in hepatoma in response to starvation. Immunoblot and immunofluorescence assays demonstrated that DRAM translocated to mitochondria and induced mitophagy, which led to apoptosis in 7702 cells. In HCC cells, starvation also activated the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which blocks the translocation of DRAM to mitochondria through the binding of p-AKT to DRAM in the cytoplasm. Inactivation of the PI3K/AKT pathway rescued DRAM translocation to mitochondria; subsequently, mitochondrial DRAM induced apoptosis in HCC cells by mediating mitophagy. Our findings open new avenues for the investigation of the mechanisms of DRAM-mediated autophagy and suggest that promoting DRAM-mediated autophagy together with PI3K/AKT inhibition might be more effective for autophagy-based therapy in hepatoma. PMID:24556693

  8. The Akt switch model: Is location sufficient?

    PubMed

    Gray, Catheryn W; Coster, Adelle C F

    2016-06-01

    Akt/PKB is a biochemical regulator that functions as an important cross-talk node between several signalling pathways in the mammalian cell. In particular, Akt is a key mediator of glucose transport in response to insulin. The phosphorylation (activation) of only a small percentage of the Akt pool of insulin-sensitive cells results in maximal translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This enables the diffusion of glucose into the cell. The dysregulation of Akt signalling is associated with the development of diabetes, cancer and cardiovascular disease. Akt is synthesised in the cytoplasm in the inactive state. Under the influence of insulin, it moves to the PM, where it is phosphorylated to form pAkt. Although phosphorylation occurs only at the PM, pAkt is found in many cellular locations, including the PM, the cytoplasm, and the nucleus. Indeed, the spatial distribution of pAkt within the cell appears to be an important determinant of downstream regulation. Here we present a simple, linear, four-compartment ordinary differential equation (ODE) model of Akt activation that tracks both the biochemical state and the physical location of Akt. This model embodies the main features of the activation of this important cross-talk node and is consistent with the experimental data. In particular, it allows different downstream signalling motifs without invoking separate feedback pathways. Moreover, the model is computationally tractable, readily analysed, and elucidates some of the apparent anomalies in insulin signalling via Akt. PMID:26992575

  9. Exendin-4-loaded PLGA microspheres relieve cerebral ischemia/reperfusion injury and neurologic deficits through long-lasting bioactivity-mediated phosphorylated Akt/eNOS signaling in rats.

    PubMed

    Chien, Chiang-Ting; Jou, Ming-Jia; Cheng, Tai-Yu; Yang, Chih-Hui; Yu, Tzu-Ying; Li, Ping-Chia

    2015-11-01

    Glucagon-like peptide-1 (GLP-1) receptor activation in the brain provides neuroprotection. Exendin-4 (Ex-4), a GLP-1 analog, has seen limited clinical usage because of its short half-life. We developed long-lasting Ex-4-loaded poly(D,L-lactide-co-glycolide) microspheres (PEx-4) and explored its neuroprotective potential against cerebral ischemia in diabetic rats. Compared with Ex-4, PEx-4 in the gradually degraded microspheres sustained higher Ex-4 levels in the plasma and cerebrospinal fluid for at least 2 weeks and improved diabetes-induced glycemia after a single subcutaneous administration (20 μg/day). Ten minutes of bilateral carotid artery occlusion (CAO) combined with hemorrhage-induced hypotension (around 30 mm Hg) significantly decreased cerebral blood flow and microcirculation in male Wistar rats subjected to streptozotocin-induced diabetes. CAO increased cortical O2(-) levels by chemiluminescence amplification and prefrontal cortex edema by T2-weighted magnetic resonance imaging analysis. CAO significantly increased aquaporin 4 and glial fibrillary acidic protein expression and led to cognition deficits. CAO downregulated phosphorylated Akt/endothelial nitric oxide synthase (p-Akt/p-eNOS) signaling and enhanced nuclear factor (NF)-κBp65/intercellular adhesion molecule-1 (ICAM-1) expression, endoplasmic reticulum (ER) stress, and apoptosis in the cerebral cortex. PEx-4 was more effective than Ex-4 to improve CAO-induced oxidative injury and cognitive deficits. The neuroprotection provided by PEx-4 was through p-Akt/p-eNOS pathways, which suppressed CAO-enhanced NF-κB/ICAM-1 signaling, ER stress, and apoptosis. PMID:26058696

  10. Total saponin from Korean Red Ginseng inhibits binding of adhesive proteins to glycoprotein IIb/IIIa via phosphorylation of VASP (Ser157) and dephosphorylation of PI3K and Akt

    PubMed Central

    Kwon, Hyuk-Woo; Shin, Jung-Hae; Cho, Hyun-Jeong; Rhee, Man Hee; Park, Hwa-Jin

    2015-01-01

    Background Binding of adhesive proteins (i.e., fibrinogen, fibronectin, vitronectin) to platelet integrin glycoprotein IIb/IIIa (αIIb/β3) by various agonists (thrombin, collagen, adenosine diphosphate) involve in strength of thrombus. This study was carried out to evaluate the antiplatelet effect of total saponin from Korean Red Ginseng (KRG-TS) by investigating whether KRG-TS inhibits thrombin-induced binding of fibrinogen and fibronectin to αIIb/β3. Methods We investigated the effect of KRG-TS on phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and dephosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt, affecting binding of fibrinogen and fibronectin to αIIb/β3, and clot retraction. Results KRG-TS had an antiplatelet effect by inhibiting the binding of fibrinogen and fibronectin to αIIb/β3 via phosphorylation of VASP (Ser157), and dephosphorylation of PI3K and Akt on thrombin-induced platelet aggregation. Moreover, A-kinase inhibitor Rp-8-Br-cyclic adenosine monophosphates (cAMPs) reduced KRG-TS-increased VASP (Ser157) phosphorylation, and increased KRG-TS-inhibited fibrinogen-, and fibronectin-binding to αIIb/β3. These findings indicate that KRG-TS interferes with the binding of fibrinogen and fibronectin to αIIb/β3 via cAMP-dependent phosphorylation of VASP (Ser157). In addition, KRG-TS decreased the rate of clot retraction, reflecting inhibition of αIIb/β3 activation. In this study, we clarified ginsenoside Ro (G-Ro) in KRG-TS inhibited thrombin-induced platelet aggregation via both inhibition of [Ca2+]i mobilization and increase of cAMP production. Conclusion These results strongly indicate that KRG-TS is a beneficial herbal substance inhibiting fibrinogen-, and fibronectin-binding to αIIb/β3, and clot retraction, and may prevent platelet αIIb/β3-mediated thrombotic disease. In addition, we demonstrate that G-Ro is a novel compound with antiplatelet characteristics of KRG-TS. PMID:26843825

  11. Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway.

    PubMed

    Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Hui-Young; Hong, Suntaek; Kim, Seong-Jin; Kim, Byung-Chul

    2013-06-14

    Hydrogen peroxide (H2O2) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H2O2 are less understood. Here we report an important mechanism for antagonistic effects of H2O2 on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H2O2 (0.05-0.2 mM) completely blocked TGF-β1-mediated induction of p15(INK4B) expression and increase of its promoter activity. Interestingly, H2O2 selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H2O2 increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H2O2 on TGF-β1-induced increase of p15(INK4B)-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H2O2 as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing a potential mechanism whereby H2O2 antagonizes the cytostatic function of TGF-β1. PMID:23685151

  12. Transforming growth factor-β1 regulated phosphorylated AKT and interferon gamma expressions are associated with epithelial cell survival in rhesus macaque colon explants.

    PubMed

    Pahar, Bapi; Pan, Diganta; Lala, Wendy; Kenway-Lynch, Carys S; Das, Arpita

    2015-05-01

    Transforming growth factor-β1 (TGF-β1) is an important immunoregulatory cytokine that plays an obligate role in regulating T-cell functions. Here, we demonstrated the role of TGF-β1 in regulating the survival of intestinal epithelial cells (ECs) in rhesus colon explant cultures using either anti-TGF-β1 antibody or recombinant TGF-β1 proteins. Neutralization of endogenous TGF-β1 using anti-TGF-β1 antibodies induced apoptosis of both intestinal ECs and lamina propria (LP) cells. Additionally, endogenous TGF-β1 blocking significantly increased expression of IFNγ, TNFα, CD107a and Perforin in LP cells compared to media and isotype controls. A significant decrease in pAKT expression was detected in anti-TGF-β1 MAbs treated explants compared to isotype and rTGF-β1 protein treated explants. Our results demonstrated TGF-β1 regulated pAKT and IFNγ expressions were associated with epithelial cell survival in rhesus macaque colon explants and suggest a potential role of mucosal TGF-β1 in regulating intestinal homeostasis and EC integrity. PMID:25769244

  13. Transforming growth factor-β1 regulated phosphorylated AKT and interferon gamma expressions are associated with epithelial cell survival in rhesus macaque colon explants

    PubMed Central

    Pahar, Bapi; Pan, Diganta; Lala, Wendy; Kenway-Lynch, Carys S.; Das, Arpita

    2015-01-01

    Transforming growth factor-β1 (TGF-β1) is an important immunoregulatory cytokine that plays an obligate role in regulating T-cell functions. Here, we demonstrated the role of TGF-β1 in regulating the survival of intestinal epithelial cells (ECs) in rhesus colon explant cultures using either anti-TGF-β1 antibody or recombinant TGF-β1 proteins. Neutralization of endogenous TGF-β1 using anti-TGF-β1 antibodies induced apoptosis of both intestinal ECs and lamina propria (LP) cells. Additionally, endogenous TGF-β1 blocking significantly increased expression of IFNγ, TNFα, CD107a and Perforin in LP cells compared to media and isotype controls. A significant decrease in pAKT expression was detected in anti-TGF-β1 MAbs treated explants compared to isotype and rTGF-β1 protein treated explants. Our results demonstrated TGF-β1 regulated pAKT and IFNγ expressions were associated with epithelial cell survival in rhesus macaque colon explants and suggest a potential role of mucosal TGF-β1 in regulating intestinal homeostasis and EC integrity. PMID:25769244

  14. DSePA Antagonizes High Glucose-Induced Neurotoxicity: Evidences for DNA Damage-Mediated p53 Phosphorylation and MAPKs and AKT Pathways.

    PubMed

    Wang, Kun; Fu, Xiao-Yan; Fu, Xiao-Ting; Hou, Ya-Jun; Fang, Jie; Zhang, Shuai; Yang, Ming-Feng; Li, Da-Wei; Mao, Lei-Lei; Sun, Jing-Yi; Yuan, Hui; Yang, Xiao-Yi; Fan, Cun-Dong; Zhang, Zong-Yong; Sun, Bao-Liang

    2016-09-01

    Hyperglycemia as the major hallmark of diabetic neuropathy severely limited its therapeutic efficiency. Evidences have revealed that selenium (Se) as an essential trace element could effectively reduce the risk of neurological diseases. In the present study, 3,3'-diselenodipropionic acid (DSePA), a derivative of selenocystine, was employed to investigate its protective effect against high glucose-induced neurotoxicity in PC12 cells and evaluate the underlying mechanism. The results suggested that high glucose showed significant cytotoxicity through launching mitochondria-mediated apoptosis in PC12 cells, accompanied by poly (ADP-ribose) polymerase (PARP) cleavage, caspase activation, and mitochondrial dysfunction. Moreover, high glucose also triggered DNA damage and dysregulation of MAPKs and AKT pathways through reactive oxygen species (ROS) overproduction. p53 RNA interference partially suppressed high glucose-induced cytotoxicity and apoptosis, indicating the role of p53 in high glucose-induced signal. However, DSePA pretreatment effectively attenuated high glucose-induced cytotoxicity, inhibited the mitochondrial dysfunction through regulation of Bcl-2 family, and ultimately reversed high glucose-induced apoptotic cell death in PC12 cells. Attenuation of caspase activation, PARP cleavage, DNA damage, and ROS accumulation all confirmed its protective effects. Moreover, DSePA markedly alleviated the dysregulation of AKT and MAPKs pathways induced by high glucose. Our findings revealed that the strategy of using DSePA to antagonize high glucose-induced neurotoxicity may be a highly effective strategy in combating high glucose-mediated neurological diseases. PMID:26232068

  15. Akt2 and Akt3 play a pivotal role in malignant gliomas

    PubMed Central

    Mure, Hideo; Matsuzaki, Kazuhito; Kitazato, Keiko T.; Mizobuchi, Yoshifumi; Kuwayama, Kazuyuki; Kageji, Teruyoshi; Nagahiro, Shinji

    2010-01-01

    Akt, one of the major downstream effectors of phosphatidylinositol 3-kinase, is hyper-expressed and activated in a variety of cancers including glioblastoma. However, the expression profiles of the Akt isoforms Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ and their functional roles in malignant glioma are not well understood. Therefore, we examined the protein and mRNA expression patterns of Akt isoforms in tissues from human astrocytomas, glioblastomas, and non-neoplastic regions. We also explored the biological role of each Akt isoform in malignant glioma cells using RNA interference-mediated knock-down and the over-expression of plasmid DNA of each isoform. The expression of Akt1 protein and mRNA was similar in glioma and normal control tissues. Although the protein and mRNA level of Akt2 increased with the pathological grade of malignancy, the expression of Akt3 mRNA and protein decreased as the malignancy grade increased. In U87MG, T98G, and TGB cells, the down-regulation of Akt2 or Akt3 by RNA interference reduced the expression of the phosphorylated form of Bad, resulting in the induction of caspase-dependent apoptosis. Akt1 knock-down did not affect cell growth or survival. We first demonstrate that the over-expression of Akt2 or Akt3 down-regulated the expression of the other protein and that endogenous Akt3 protein showed high kinase activity in U87MG cells. Our data suggest that Akt2 and Akt3 play an important role in the viability of human malignant glioma cells. Targeting Akt2 and Akt3 may hold promise for the treatment of patients with gliomas. PMID:20167810

  16. miR-150 Deficiency Protects against FAS-Induced Acute Liver Injury in Mice through Regulation of AKT

    PubMed Central

    Chen, Weina; Han, Chang; Zhang, Jinqiang; Song, Kyoungsub; Wang, Ying; Wu, Tong

    2015-01-01

    Although miR-150 is implicated in the regulation of immune cell differentiation and activation, it remains unknown whether miR-150 is involved in liver biology and disease. This study was performed to explore the potential role of miR-150 in LPS/D-GalN and Fas-induced liver injuries by using wild type and miR-150 knockout (KO) mice. Whereas knockout of miR-150 did not significantly alter LPS/D-GalN-induced animal death and liver injury, it protected against Fas-induced liver injury and mortality. The Jo2-induced increase in serum transaminases, apoptotic hepatocytes, PARP cleavage, as well as caspase-3/7, caspase-8, and caspase-9 activities were significantly attenuated in miR-150 KO mice. The liver tissues from Jo2-treated miR-150 KO mice expressed higher levels of Akt1, Akt2, total Akt, as well as p-Akt(Ser473) compared to the wild type livers. Pretreatment with the Akt inhibitor V reversed Jo2-induced liver injury in miR-150 KO mice. The primary hepatocytes isolated from miR-150 KO mice also showed protection against Fas-induced apoptosis in vitro (characterized by less prominent PARP cleavage, less nuclear fragmentation and less caspase activation) in comparison to hepatocytes from wild type mice. Luciferase reporter assays in hepatocytes transfected with the Akt1 or Akt2 3’-UTR reporter constructs (with or without mutation of miR-150 binding site) established Akt1 and Akt2 as direct targets of miR-150. Tail vein injection of lentiviral particles containing pre-miR-150 enhanced Jo2-induced liver injury in miR-150 KO mice. These findings demonstrate that miR-150 deficiency prevents Fas-induced hepatocyte apoptosis and liver injury through regulation of the Akt pathway. PMID:26196694

  17. Low dose of IGF-I increases cell size of skeletal muscle satellite cells via Akt/S6K signaling pathway.

    PubMed

    Gao, Chun-qi; Zhi, Rui; Yang, Zhou; Li, Hai-chang; Yan, Hui-chao; Wang, Xiu-qi

    2015-11-01

    The objective of this study was to investigate the effect of insulin growth factor-I (IGF-I) on the size of pig skeletal muscle satellite cells (SCs). Using microarray, real-time RT-PCR, radioimmunoassay analysis and western blot, we first showed that supplementation of low-dose of IGF-I in culture medium resulted in enlarged cell size of Lantang SCs, only Akt and S6K were up-regulated at both the mRNA and protein levels among almost all of the mTOR pathway key genes, but had no effect on cell number. To elucidate the signaling mechanisms responsible for regulating cell size under low-dose of IGF-I treatment, we blocked Akt and S6K activity with the specific inhibitors MK2206 and PF4708671, respectively. Both inhibitors caused a decrease in cell size. In addition, MK2206 lowered the protein level of p-Akt (Ser473), p-S6K (Thr389), and p-rpS6 (Ser235/236), whereas PF4708671 lowered the protein level of p-S6K (Thr389) and p-rpS6 (Ser235/236). However, low dose of IGF-I didn't affect the protein level of p-mTOR (Ser2448) and p-mTOR (Ser2481). When both inhibitors were applied simultaneously, the effect was the same as that of the Akt inhibition alone. Taken together, we report for the first time that low-dose of IGF-I treatment increases cell size via Akt/S6K signaling pathway. PMID:25923195

  18. COMP-Ang1 enhances DNA synthesis and cell cycle progression in human periodontal ligament cells via Tie2-mediated phosphorylation of PI3K/Akt and MAPKs.

    PubMed

    Lim, Shin-Saeng; Kook, Sung-Ho; Lee, Jeong-Chae

    2016-05-01

    Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1), and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP) can stimulate multiple cellular processes. Proliferative capacity of periodontal ligament (PDL) fibroblasts (PLFs) is important for maintaining PDL integrity and homeostasis. In this study, we explored whether exogenous COMP-Ang1 addition enhances proliferation of human PLFs and the cellular mechanisms therein. We initially isolated and characterized PLFs, where the cells showed highly positive staining for surface markers, CD90, CD105, and CD146. COMP-Ang1 treatment increased proliferation of PLFs by stimulating migration of cells into S and G2/M phases. This increase was coupled with decreased p21(Cip) and p27(Kip) levels and enhanced cyclin D1, cyclin-dependent kinase (CDK) 2, and CDK4 induction. Transfection with si-Tie2 near completely blocked COMP-Ang1-stimulated cell cycle progression in PLFs. Tie2 knockdown also inhibited COMP-Ang1-induced phosphorylation of mitogen-activated protein kinases (MAPKs). In addition, COMP-Ang1-mediated activation of Akt and c-Jun was suppressed by treating each of pharmacological inhibitors specific to phosphoinositide 3-kinase (PI3K) (LY294002 and Wortmannin) or MAPKs (PD98059, SB203580, and SP600125). Similarly, COMP-Ang1-mediated increases in DNA synthesis and cyclin D1 induction were prevented by treating inhibitor of MAPKs and PI3K or by c-Jun knockdown. These results suggest that COMP-Ang1 enhances survival and proliferation of human PLFs through the activation of Tie2-mediated signaling, where PI3K/Akt and MAPK-c-Jun signaling pathways act as downstream effectors. Collectively, COMP-Ang1 may be a useful as a stimulator of human PLFs and therefore improves PDL integrity and homeostasis. PMID:27107990

  19. Cafestol, a coffee-specific diterpene, induces apoptosis in renal carcinoma Caki cells through down-regulation of anti-apoptotic proteins and Akt phosphorylation.

    PubMed

    Choi, Min Jung; Park, Eun Jung; Oh, Jung Hwa; Min, Kyoung-Jin; Yang, Eun Sun; Kim, Young Ho; Lee, Tae Jin; Kim, Sang Hyun; Choi, Yung Hyun; Park, Jong-Wook; Kwon, Taeg Kyu

    2011-04-25

    Cafestol, one of the major compounds in coffee beans, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity, although the mechanism of action is poorly understood. In the present study, we investigated the effect of cafestol on the apoptotic pathway in human renal Caki cells and other cancer cell lines. Cafestol treatment inhibited Caki cells viability a dose-dependent manner by inducing apoptosis, as evidenced by DNA fragmentation and the accumulation of sub-G1 phase. Cafestol-induced apoptosis is associated with the reduction of mitochondrial membrane potential (MMP), activation of caspase 3, cytochrome c release, and down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and cFLIP). Cafestol-induced apoptosis was blocked by pretreatment with broad caspase inhibitor z-VAD-fmk, showing its dependence on caspases. Ectopic expression of Bcl-2 or Mcl-1 in Caki cells attenuates cafestol-induced apoptosis. In addition, we have also shown that cafestol inhibits phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, and PI3K inhibitor LY29004 significantly increases cafestol-induced apoptosis in Caki cells. Taken together, our results show the activity of cafestol to modulate multiple components in apoptotic response of human renal Caki cells and a potential as a therapeutic agent for preventing cancers such as renal carcinoma. PMID:21334318

  20. Akt Specific Activator SC79 Protects against Early Brain Injury following Subarachnoid Hemorrhage.

    PubMed

    Zhang, Dingding; Zhang, Huasheng; Hao, Shuangying; Yan, Huiying; Zhang, Zihuan; Hu, Yangchun; Zhuang, Zong; Li, Wei; Zhou, Mengliang; Li, Kuanyu; Hang, Chunhua

    2016-06-15

    A growing body of evidence demonstrates that Akt may serve as a therapeutic target for treatment of early brain injury following subarachnoid hemorrhage (SAH). The purpose of the current study was to evaluate the neuroprotective effect of Akt specific activator SC79 in an experimental rat model of SAH. SAH was induced by injecting 300 μL of blood into the prechiasmatic cistern. Intracerebroventricular (ICV) injection of SC79 (30 min post-SAH) induced the p-Akt (Ser473) expression in a dose-dependent manner. A single ICV dose treatment of SC79 (100 μg/rat) significantly increased the expression of Bcl-2 and p-GSK-3β (Ser9), decreased the protein levels of Bax, cytoplasm cytochrome c, and cleaved caspase-3, indicating the antiapoptotic effect of SC79. As a result, the number of apoptotic cells was reduced 24 h post SAH. Moreover, SC79 treatment alleviated SAH-induced oxidative stress, restored mitochondrial morphology, and improved neurological deficits. Strikingly, treatment of SC79 provided a beneficial outcome against neurologic deficit with a therapeutic window of at least 4 h post SAH by ICV injection and 30 min post SAH by intraperitoneal injection. Collectively, SC79 exerts its neuroprotective effect likely through the dual activities of antioxidation and antiapoptosis. These data provide a basic platform to consider SC79 as a novel therapeutic agent for treatment of SAH. PMID:26983552

  1. Akt-mediated regulation of NFκB and the essentialness of NFκB for the oncogenicity of PI3K and Akt

    PubMed Central

    Bai, Dong; Ueno, Lynn; Vogt, Peter K.

    2009-01-01

    The serine/threonine kinase Akt (cellular homolog of murine thymoma virus akt8 oncogene), also known as PKB (protein kinase B), is activated by lipid products of phosphatidylinositol 3-kinase (PI3K). Akt phosphorylates numerous protein targets that control cell survival, proliferation and motility. Previous studies suggest that Akt regulates transcriptional activity of the nuclear factor-κB (NFκB) by inducing phosphorylation and subsequent degradation of inhibitor of κB (IκB). We show here that NFκB-driven transcription increases in chicken embryonic fibroblasts (CEF) transformed by myristylated Akt (myrAkt). Accordingly, both a dominant negative mutant of Akt and Akt inhibitors repress NFκB-dependent transcription. The degradation of the IκB protein is strongly enhanced in Akt-transformed cells, and the loss of NFκB activity by introduction of a super-repressor of NFκB, IκBSR, interferes with PI3K- and Akt-induced oncogenic transformation of CEF. The phosphorylation of the p65 subunit of NFκB at serine 534 is also upregulated in Akt-transformed cells. Our data suggest that the stimulation of NFκB by Akt is dependent on the phosphorylation of p65 at S534, mediated by IKK (IκB kinase) α and β. Akt phosphorylates IKKα on T23, and this phosphorylation event is a prerequisite for the phosphorylation of p65 at S534 by IKKα and β. Our results demonstrate two separate functions of the IKK complex in NFκB activation in cells with constitutive Akt activity: the phosphorylation and consequent degradation of IκB and the phosphorylation of p65. The data further support the conclusion that NFκB activity is essential for PI3K- and Akt-induced oncogenic transformation. PMID:19609947

  2. Myostatin inhibits IGF-I-induced myotube hypertrophy through Akt

    PubMed Central

    Morissette, Michael R.; Cook, Stuart A.; Buranasombati, Cattleya; Rosenberg, Michael A.

    2009-01-01

    Myostatin is a highly conserved negative regulator of skeletal muscle growth. Loss of functional myostatin in cattle, mice, sheep, dogs, and humans results in increased muscle mass. The molecular mechanisms responsible for this increase in muscle growth are not fully understood. Previously, we have reported that phenylephrine-induced cardiac muscle growth and Akt activation are enhanced in myostatin knockout mice compared with controls. Here we report that skeletal muscle from myostatin knockout mice show increased Akt protein expression and overall activity at baseline secondary to an increase in Akt mRNA. We examined the functional role of myostatin modulation of Akt in C2C12 myotubes, a well-established in vitro model of skeletal muscle hypertrophy. Adenoviral overexpression of myostatin attenuated the insulin-like growth factor-I (IGF-I)-mediated increase in myotube diameter, as well as IGF-I-stimulated Akt phosphorylation. Inhibition of myostatin by overexpression of the NH2-terminal portion of myostatin was sufficient to increase myotube diameter and Akt phosphorylation. Coexpression of myostatin and constitutively active Akt (myr-Akt) restored the increase in myotube diameter. Conversely, expression of dominant negative Akt (dn-Akt) with the inhibitory myostatin propeptide blocked the increase in myotube diameter. Of note, ribosomal protein S6 phosphorylation and atrogin-1/muscle atrophy F box mRNA were increased in skeletal muscle from myostain knockout mice. Together, these data suggest myostatin regulates muscle growth at least in part through regulation of Akt. PMID:19759331

  3. β2-Glycoprotein I Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis by Suppressing the Phosphorylation of Extracellular Signal-Regulated Kinase 1/2, Akt, and Endothelial Nitric Oxide Synthase.

    PubMed

    Chiu, Wen-Chin; Chiou, Tzeon-Jye; Chung, Meng-Ju; Chiang, An-Na

    2016-01-01

    Angiogenesis is the process of new blood vessel formation, and it plays a key role in various physiological and pathological conditions. The β2-glycoprotein I (β2-GPI) is a plasma glycoprotein with multiple biological functions, some of which remain to be elucidated. This study aimed to identify the contribution of 2-GPI on the angiogenesis induced by vascular endothelial growth factor (VEGF), a pro-angiogenic factor that may regulate endothelial remodeling, and its underlying mechanism. Our results revealed that β2-GPI dose-dependently decreased the VEGF-induced increase in endothelial cell proliferation, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bromodeoxyuridine (BrdU) incorporation assays. Furthermore, incubation with both β2-GPI and deglycosylated β2-GPI inhibited the VEGF-induced tube formation. Our results suggest that the carbohydrate residues of β2-GPI do not participate in the function of anti-angiogenesis. Using in vivo Matrigel plug and angioreactor assays, we show that β2-GPI remarkably inhibited the VEGF-induced angiogenesis at a physiological concentration. Moreover, β2-GPI inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and endothelial nitric oxide synthase (eNOS). In summary, our in vitro and in vivo data reveal for the first time that β2-GPI inhibits the VEGF-induced angiogenesis and highlights the potential for β2-GPI in anti-angiogenic therapy. PMID:27579889

  4. AKT serine/threonine protein kinase modulates baicalin-triggered autophagy in human bladder cancer T24 cells.

    PubMed

    Lin, Chingju; Tsai, Shih-Chang; Tseng, Michael T; Peng, Shu-Fen; Kuo, Sheng-Chu; Lin, Meng-Wei; Hsu, Yuan-Man; Lee, Miau-Rong; Amagaya, Sakae; Huang, Wen-Wen; Wu, Tian-Shung; Yang, Jai-Sing

    2013-03-01

    Baicalin is one of the major compounds in the traditional Chinese medicinal herb from Scutellaria baicalensis Georgi. We investigated the molecular mechanisms of cell autophagy induced by baicalin in human bladder cancer T24 cells. Baicalin inhibited cell survival as shown by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. Baicalin did not induce apoptotic cell death in T24 cells by TUNEL and caspase-3 activity assay. Baicalin induced the acidic vesicular organelle cell autophagy marker, manifested by acridine orange (AO) and monodansylcadaverine (MDC) staining and cleavage of microtubule-associated protein 1 light chain 3 (LC3). The protein expression levels of the Atg 5, Atg 7, Atg 12, Beclin-1 and LC3-II were upregulated in T24 cells after baicalin treatment. Inhibition of autophagy by 3-methyl-adenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) reduced the cleavage of LC3 in T24 cells after baicalin treatment. Furthermore, protein expression levels of phospho-AKT (Ser473) and enzyme activity of AKT were downregulated in T24 cells after baicalin treatment. In conclusion, baicalin triggered cell autophagy through the AKT signaling pathway in T24 cells. PMID:23354080

  5. RLN2 Is a Positive Regulator of AKT-2-Induced Gene Expression Required for Osteosarcoma Cells Invasion and Chemoresistance

    PubMed Central

    Ma, Jinfeng; Huang, Hai; Han, Zenggang; Zhu, Changzheng; Yue, Bin

    2015-01-01

    The aim of the study was to determine the effect of H2 relaxin (RLN2) on invasion, migration, and chemosensitivity to cisplatin in human osteosarcoma U2-OS and MG-63 cells and then to investigate the effect of RLN2 on the AKT/NF-κB signaling pathway. The expression of RLN2, p-AKT (Ser473), and p-ERK1/2 (Phospho-Thr202/Tyr204) proteins was detected by western blot in OS tissues from 21 patients with pulmonary metastatic disease, and the correlation between RLN2 and p-AKT or RLN2 and p-ERK1/2 expression was investigated. RLN2 expression was inhibited by RLN2 siRNA transfection in the MG-63 cells. RLN2 was overexpressed in the U2-OS cells by treatment with recombinant relaxin. The results showed that positive relation was found between RLN2 and p-AKT expression in tissues of OS. Silencing RLN2 inhibited cell migratory and invasive ability and angiogenesis formation and increased the chemosensitivity to cisplatin in MG-63 cells. RLN2 overexpression promoted migratory and invasive ability and angiogenesis and increased the chemoresistance to cisplatin in U2-OS cells. Silencing RLN2 inhibited the activity of AKT/NF-κB signaling pathway in MG-63 cells, and vice versa. Blockage of both pathways by specific inhibitors abrogated RLN2-induced survival and invasion of OS cells, and vice versa. Our results indicated RLN2 confers to migratory and invasive ability, angiogenesis, and chemoresistance to cisplatin via modulating the AKT/NF-κB signaling pathway in vitro. PMID:26229955

  6. Temperature sensitivity of phospho-Ser{sup 473}-PKB/AKT

    SciTech Connect

    Oehler-Jaenne, Christoph; Bueren, Andre O. von; Vuong, Van; Hollenstein, Andreas; Grotzer, Michael A.; Pruschy, Martin

    2008-10-24

    The phospho-PKB/Akt status is often used as surrogate marker to measure activation of the PI3K/Akt/mTOR signal transduction pathway. Though, inconsistencies of the p-Ser{sup 473}-PKB/Akt status have raised doubts in the validity of p-Ser{sup 473}-PKB/Akt phosphorylation as endpoint. Here, we determined that p-Ser{sup 473}-PKB/Akt but not p-Thr{sup 308}-PKB/Akt phosphorylation is highly temperature sensitive. p-Ser{sup 473}-PKB/Akt phosphorylation was rapidly reduced to levels below 50% on exposure to 20-25 deg. C in murine and human cell lines including cells expressing constitutively active PI3K or lacking PTEN. Down-regulation of p-Ser{sup 473}-PKB/Akt was reversible and re-exposure to physiological temperature resulted in increased p-Ser{sup 473}-PKB/Akt phosphorylation levels. Phosphatase activity at low temperature was sustained at 75% baseline level and phosphatase inhibition prevented p-Ser{sup 473}-PKB/Akt dephosphorylation induced by the low temperature shift. Interestingly temperature-dependent deregulation of the p-Ser{sup 473}-PKB/Akt status was also observed in response to irradiation. Thus our data demonstrate that minimal additional stress factors deregulate the PI3K/Akt-survival pathway and the p-Ser{sup 473}-PKB/Akt status as experimental endpoint.

  7. Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome

    PubMed Central

    Lindhurst, Marjorie J.; Yourick, Miranda R.; Yu, Yi; Savage, Ronald E.; Ferrari, Dora; Biesecker, Leslie G.

    2015-01-01

    A somatic activating mutation in AKT1, c.49G>A, pGlu17Lys, that results in elevated AKT signaling in mutation-positive cells, is responsible for the mosaic overgrowth condition, Proteus syndrome. ARQ 092 is an allosteric pan-AKT inhibitor under development for treatment in cancer. We tested the efficacy of this drug for suppressing AKT signaling in cells and tissues from patients with Proteus syndrome. ARQ 092 reduced phosphorylation of AKT and downstream targets of AKT in a concentration-dependent manner in as little as two hours. While AKT signaling was suppressed with ARQ 092 treatment, cells retained their ability to respond to growth factor stimulation by increasing pAKT levels proportionally to untreated cells. At concentrations sufficient to decrease AKT signaling, little reduction in cell viability was seen. These results indicate that ARQ 092 can suppress AKT signaling and warrants further development as a therapeutic option for patients with Proteus syndrome. PMID:26657992

  8. The phosphoinositide-3-kinase (PI3K)-delta and gamma inhibitor, IPI-145 (Duvelisib), overcomes signals from the PI3K/AKT/S6 pathway and promotes apoptosis in CLL.

    PubMed

    Balakrishnan, K; Peluso, M; Fu, M; Rosin, N Y; Burger, J A; Wierda, W G; Keating, M J; Faia, K; O'Brien, S; Kutok, J L; Gandhi, V

    2015-09-01

    The functional relevance of the B-cell receptor (BCR) and the evolution of protein kinases as therapeutic targets have recently shifted the paradigm for treatment of B-cell malignancies. Inhibition of p110δ with idelalisib has shown clinical activity in chronic lymphocytic leukemia (CLL). The dynamic interplay of isoforms p110δ and p110γ in leukocytes support the hypothesis that dual blockade may provide a therapeutic benefit. IPI-145, an oral inhibitor of p110δ and p110γ isoforms, sensitizes BCR-stimulated and/or stromal co-cultured primary CLL cells to apoptosis (median 20%, n=57; P<0.0001) including samples with poor prognostic markers, unmutated IgVH (n=28) and prior treatment (n=15; P<0.0001). IPI-145 potently inhibits the CD40L/IL-2/IL-10 induced proliferation of CLL cells with an IC50 in sub-nanomolar range. A corresponding dose-responsive inhibition of pAKT(Ser473) is observed with an IC50 of 0.36 nM. IPI-145 diminishes the BCR-induced chemokines CCL3 and CCL4 secretion to 17% and 37%, respectively. Pre-treatment with 1 μM IPI-145 inhibits the chemotaxis toward CXCL12; reduces pseudoemperipolesis to median 50%, inferring its ability to interfere with homing capabilities of CLL cells. BCR-activated signaling proteins AKT(Ser473), BAD(Ser112), ERK(Thr202/Tyr204) and S6(Ser235/236) are mitigated by IPI-145. Importantly, for clinical development in hematological malignancies, IPI-145 is selective to CLL B cells, sparing normal B- and T-lymphocytes. PMID:25917267

  9. Regulation of Bax/mitochondria interaction by AKT.

    PubMed

    Simonyan, Lilit; Renault, Thibaud T; Novais, Maria João da Costa; Sousa, Maria João; Côrte-Real, Manuela; Camougrand, Nadine; Gonzalez, Cécile; Manon, Stéphen

    2016-01-01

    Bax-dependent mitochondrial permeabilization during apoptosis is controlled by multiple factors, including the phosphorylation by the protein kinase AKT. We used the heterologous co-expression of human Bax and AKT1 in yeast to investigate how the kinase modulates the different steps underlying Bax activation. We found that AKT activated Bax and increased its cellular content. Both effects were dependent on Ser184, but a phosphorylation of this residue did not fully explain the effects of AKT. Additional experiments with mutants substituted on Ser184 suggested that the regulation of Bax dynamic equilibrium between the cytosol and mitochondria might be more tightly regulated by Bcl-xL when Bax is phosphorylated. PMID:26763134

  10. Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor, ARQ 092

    PubMed Central

    Yu, Yi; Savage, Ronald E.; Eathiraj, Sudharshan; Meade, Justin; Wick, Michael J.; Hall, Terence; Abbadessa, Giovanni; Schwartz, Brian

    2015-01-01

    As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies. PMID:26469692

  11. Hydrogen peroxide/ceramide/Akt signaling axis play a critical role in the antileukemic potential of sanguinarine.

    PubMed

    Rahman, Anees; Thayyullathil, Faisal; Pallichankandy, Siraj; Galadari, Sehamuddin

    2016-07-01

    Dysregulation of apoptosis is a prime hallmark of leukemia. Therefore, drugs which restore the sensitivity of leukemic cells to apoptotic stimuli are promising candidates in the treatment of leukemia. Recently, we have demonstrated that sanguinarine (SNG), a benzophenanthridine alkaloid, isolated from Sanguinaria canadensis induces ROS-dependent ERK1/2 activation and autophagic cell death in human malignant glioma cells (Pallichankandy et al., 2015; [43]). In this study, we investigated the antileukemic potential of SNG in vitro, and further examined the molecular mechanisms of SNG-induced cell death. In human leukemic cells, SNG activated apoptotic cell death pathway characterized by activation of caspase cascade, DNA fragmentation and down-regulation of anti-apoptotic proteins. Importantly, we have identified a crucial role for hydrogen peroxide (H2O2)-dependent ceramide (Cer) generation in the facilitation of SNG-induced apoptosis. Additionally, we have found that SNG inhibits Akt, a key anti-apoptotic protein kinase by dephosphorylating it at Ser(473), leading to the dephosphorylation of its downstream targets, GSK3β and mTOR. Interestingly, inhibition of Cer generation, using acid sphingomyelinase inhibitor, significantly reduced the SNG-induced Akt dephosphorylation and apoptosis, whereas, activation of Cer generation using inhibitors of acid ceramidase and glucosylceramide synthase enhanced it. Furthermore, using a group of ceramide activated protein phosphatases (CAPPs) inhibitor (calyculin A, Okadaic acid, and phosphatidic acid), the involvement of protein phosphatase 1 form of CAPP in SNG-induced Akt dephosphorylation and apoptosis was demonstrated. Altogether, these results underscore a critical role for H2O2-Cer-Akt signaling axis in the antileukemic action of SNG. PMID:27154977

  12. Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming.

    PubMed

    Chae, Young Chan; Vaira, Valentina; Caino, M Cecilia; Tang, Hsin-Yao; Seo, Jae Ho; Kossenkov, Andrew V; Ottobrini, Luisa; Martelli, Cristina; Lucignani, Giovanni; Bertolini, Irene; Locatelli, Marco; Bryant, Kelly G; Ghosh, Jagadish C; Lisanti, Sofia; Ku, Bonsu; Bosari, Silvano; Languino, Lucia R; Speicher, David W; Altieri, Dario C

    2016-08-01

    Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism toward glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an "actionable" therapeutic target in cancer. PMID:27505672

  13. Essential role of AKT in tumor cells addicted to FGFR.

    PubMed

    Hu, Yi; Lu, Huiru; Zhang, Jinchao; Chen, Jun; Chai, Zhifang; Zhang, Jingxin

    2014-02-01

    Tumor cells with genetic amplifications or mutations in the fibroblast growth factor receptor (FGFR) family are often addicted to FGFR and heavily dependent on its signaling to survive. Although it is critical to understand which signaling pathway downstream of FGFR plays an essential role to guide the research and development of FGFR inhibitors, it has remained unclear partly because the tool compounds used in the literature also hit many other kinases, making the results difficult to interpret. With the development of a potent FGFR-specific inhibitor, BGJ398, we are now able to dissect various pathways with low drug concentrations to minimize multiple-target effects. Importantly, here, we show that inhibition of FGFR signaling by BGJ398 leads to only transient inhibition of ERK1/2 phosphorylation, whereas the inhibitory effect on AKT phosphorylation is sustainable, indicating that AKT, not ERK as commonly believed, serves as an appropriate pharmacodynamic biomarker for BGJ398. Although AKT inhibition by a pan-PI3K inhibitor alone has almost no effect on cell growth, heterologous expression of myr-AKT, an active form of AKT, rescues BGJ398-mediated suppression of tumor cell proliferation. These results indicate that AKT is an essential component downstream of FGFR. Finally, combination of the FGFR inhibitor BGJ398 with rapamycin significantly inhibits AKT phosphorylation and enhances their antiproliferative effects in FGFR-addicted cells, suggesting an effective combination strategy for clinical development of FGFR inhibitors. PMID:24100276

  14. The Paradox of Akt-mTOR Interactions

    PubMed Central

    Vadlakonda, Lakshmipathi; Dash, Abhinandita; Pasupuleti, Mukesh; Anil Kumar, Kotha; Reddanna, Pallu

    2013-01-01

    The serine threonine protein kinase, Akt, is at the central hub of signaling pathways that regulates cell growth, differentiation, and survival. The reciprocal relation that exists between the two activating phosphorylation sites of Akt, T308 and S473, and the two mTOR complexes, C1 and C2, forms the central controlling hub that regulates these cellular functions. In our previous review “PI3Kinase (PI3K)-AKT-mTOR and Wnt signaling pathways in cell cycle” we discussed the reciprocal relation between mTORC1 and C2 complexes in regulating cell metabolism and cell cycle progression in cancer cells. We present in this article, a hypothesis that activation of Akt-T308 phosphorylation in the presence of high ATP:AMP ratio promotes the stability of its phosphorylations and activates mTORC1 and the energy consuming biosynthetic processes. Depletion of energy leads to inactivation of mTORC1, activation of AMPK, FoxO, and promotes constitution of mTORC2 that leads to phosphorylation of Akt S473. Akt can also be activated independent of PI3K; this appears to have an advantage under situations like dietary restrictions, where insulin/insulin growth factor signaling could be a casualty. PMID:23802099

  15. Synthetic sulfoglycolipids targeting the serine-threonine protein kinase Akt.

    PubMed

    Costa, Barbara; Dangate, Milind; Vetro, Maria; Donvito, Giulia; Gabrielli, Luca; Amigoni, Loredana; Cassinelli, Giuliana; Lanzi, Cinzia; Ceriani, Michela; De Gioia, Luca; Filippi, Giulia; Cipolla, Laura; Zaffaroni, Nadia; Perego, Paola; Colombo, Diego

    2016-08-15

    The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a β-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-β-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors. PMID:27316541

  16. Akt isoform-dependent regulation of ATP-Binding cassette A1 expression by apolipoprotein E.

    PubMed

    Okoro, Emmanuel U; Guo, Zhongmao; Yang, Hong

    2016-08-12

    We previously reported that apolipoprotein E (apoE) upregulates ATP-binding cassette transporter A1 (ABCA1) transcription through phosphatidylinositol 3-kinase (PI3K). Here we demonstrate that treatment of murine macrophages with human apoE3 enhanced Akt phosphorylation, and upregulated ABCA1 protein and mRNA expression. Inhibition of PI3K weakened apoE3-induced Akt phosphorylation, and ABCA1 protein and mRNA increase. In contrast, inhibition of Akt only diminished apoE-induced ABCA1 protein but not the mRNA level. Suppression of protein synthesis did not erase the ability of apoE3 to increase ABCA1 protein level. Further, apoE3 increased the resistance of ABCA1 protein to calpain-mediated degradation without affecting calpain activity. Treatment of macrophages with apoE3 selectively enhanced the phosphorylation of Akt1 and Akt2, but not Akt3. Knockdown of Akt1 or Akt2 increased and decreased ABCA1 protein level, respectively; while overexpression of these Akt isoenzymes caused changes in ABCA1 protein level opposite to those induced by knockdown of the corresponding Akt. These data imply that apoE3 guards against calpain-mediated ABCA1 degradation through Akt2. PMID:27297104

  17. [Relationship between PTEN mutations and protein kinase B phosphorylation caused by insulin or recombinant human epidermal growth factor stimulation].

    PubMed

    Zhong, Hailan; Hu, Xianfu; Lin, Jianhua

    2016-08-01

    Objective To study the effect of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) mutations on protein kinase B (Akt) phosphorylation of CNE-1 nasopharyngeal carcinoma cell line. Methods CNE-1 cells were cultured in RPMI1640 medium containing 100 mL/L fetal calf serum, and then transfected with wild-type PTEN (wtPTEN), mutant PTEN C124S and mutant PTEN G129E plasmid separately. After overnight serum starvation, the cells were stimulated with 0.15 IU/mL insulin or 0.3 μg/mL recombinant human epidermal growth factor (rhEGF). At last, Akt phosphorylation was evaluated by Western blotting. Results Insulin or rhEGF stimulation led to Akt activation in CNE-1 cells. The wtPTEN inhibited insulin- or rhEGF-stimulated phosphorylation of Akt. PTEN C124S mutant activated insulin-stimulated phosphorylation of Akt, but not rhEGF-stimulated phosphorylation of Akt. PTEN G129E mutant inhibited insulin-stimulated phosphorylation of Akt. Conclusion The wtPTEN inhibited insulin- or rhEGF-stimulated phosphorylation of Akt, while PTEN C124S and G129E mutants failed to activate the phosphorylation of Akt consistently. This suggested PTEN mutations might not be correlated with activated Akt. PMID:27412938

  18. Inhibitory effects of β-tricalciumphosphate wear particles on osteocytes via apoptotic response and Akt inactivation.

    PubMed

    Zhang, Yun; Yan, Ming; Yu, Aiyue; Mao, Hongjiao; Zhang, Jinping

    2012-07-16

    Wear debris-induced osteolysis, a major contributing factor of orthopedic implant aseptic loosening, affects long-term survival of orthopedic prostheses following joint replacement and revision surgery. Pathogenic effects of wear debris on various cell types including macrophages/monocytes, osteoblasts, and osteoclasts have been well studied. However, the interactions between wear debris particles and osteocytes, which make up over 90% of all bone cells, have not been clearly illustrated. Here, we explored the biological effects of endotoxin-free beta-tricalciumphosphate (β-TCP) wear particles with the average diameter of 1.997 μm (range 1.3-3.2 μm) on osteocytes in vitro. Our results showed that 24 h or 48 h incubation of β-TCP particles dose-dependently inhibited cell viability of osteocytes MLO-Y4. Alternatively, β-TCP particles treatment for 24 h significantly increased the osteocytic marker SOST/sclerostin mRNA expression and the release of inflammatory cytokines including TNF-α and IL-1β into the culture media, but decreased the mRNA expression of another osteocytic marker dentin matrix protein-1 (DMP-1). Furthermore, these osteocytes dysfunctions were accompanied by F-actin disassembly, cell apoptosis, sustained enhancement of intracellular reactive oxygen species (ROS) and mitochondrial injury upon β-TCP particles stimulation. In addition, β-TCP particles also caused Akt inactivation at Ser473 resides with a dose- and time-dependent pattern. Taken together, β-TCP wear particles could cause osteocytes dysfunctions, which may be mediated by apoptotic death and Akt inactivation in MLO-Y4 cells. These findings strongly suggest that osteocytes may play an important role in the β-TCP wear particles-induced osteolysis, and provide valuable insights for understanding the molecular mechanisms of osteocytes death involved in tissue damage during bone cement and intolerance of cemented prostheses. PMID:22522029

  19. Nitric Oxide Synthase and Breast Cancer: Role of TIMP-1 in NO-mediated Akt Activation

    PubMed Central

    Ridnour, Lisa A.; Barasch, Kimberly M.; Windhausen, Alisha N.; Dorsey, Tiffany H.; Lizardo, Michael M.; Yfantis, Harris G.; Lee, Dong H.; Switzer, Christopher H.; Cheng, Robert Y. S.; Heinecke, Julie L.; Brueggemann, Ernst; Hines, Harry B.; Khanna, Chand; Glynn, Sharon A.; Ambs, Stefan; Wink, David A.

    2012-01-01

    Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation. PMID:22957045

  20. Diverse heterocyclic scaffolds as allosteric inhibitors of AKT.

    PubMed

    Kettle, Jason G; Brown, Simon; Crafter, Claire; Davies, Barry R; Dudley, Phillippa; Fairley, Gary; Faulder, Paul; Fillery, Shaun; Greenwood, Hannah; Hawkins, Janet; James, Michael; Johnson, Keith; Lane, Clare D; Pass, Martin; Pink, Jennifer H; Plant, Helen; Cosulich, Sabina C

    2012-02-01

    Wide-ranging exploration of potential replacements for a quinoline-based inhibitor of activation of AKT kinase led to number of alternative, novel scaffolds with potentially improved potency and physicochemical properties. Examples showed predictable DMPK properties, and one such compound demonstrated pharmacodynamic knockdown of phosphorylation of AKT and downstream biomarkers in vivo and inhibition of tumor growth in a breast cancer xenograft model. PMID:22248236

  1. Nitration of JAK-2 at the 1007Y-1008Y activation epitope impedes phosphorylation at this site: defining a GH, AKT/protein kinase B and nitric oxide synthase axis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Generalized liver protein tyrosine nitration (3’-nitrotyrosine, 3’-NT) increases in vivo after GH injection with immunohistocellular patterns strikingly similar to those we observed for a specific nitration of JAK2 at its 1007Y-1008Y regulatory phosphorylation epitope following proinflammatory chall...

  2. Gastrointestinal growth factors and hormones have divergent effects on Akt activation

    PubMed Central

    Berna, Marc J.; Tapia, Jose A.; Sancho, Veronica; Thill, Michelle; Pace, Andrea; Hoffmann, K. Martin; Gonzalez-Fernandez, Lauro; Jensen, Robert T.

    2009-01-01

    Akt is a central regulator of apoptosis, cell growth and survival. Growth factors and some G-protein-coupled receptors (GPCR) regulate Akt. Whereas growth-factor activation of Akt has been extensively studied, the regulation of Akt by GPCR's, especially gastrointestinal hormones/neurotransmitters, remains unclear. To address this area, in this study the effects of GI growth factors and hormones/neurotransmitters were investigate in rat pancreatic acinar cells which are high responsive to these agents. Pancreatic acini expressed Akt and 5 of 7 known pancreatic growth-factors stimulate Akt phosphorylation (T308, S473) and translocation. These effects are mediated by p85 phosphorylation and activation of PI3K. GI hormones increasing intracellular cAMP had similar effects. However, GI-hormones/neurotransmitters[CCK, bombesin,carbachol] activating phospholipase C (PLC) inhibited basal and growth-factor-stimulated Akt activation. Detailed studies with CCK, which has both physiological and pathophysiological effects on pancreatic acinar cells at different concentrations, demonstrated CCK has a biphasic effect: at low concentrations(pM) stimulating Akt by a Src-dependent mechanism and at higher concentrations(nM) inhibited basal and stimulated Akt translocation, phosphorylation and activation, by de-phosphorylating p85 resulting in decreasing PI3K activity. This effect required activation of both limbs of the PLC-pathway and a protein tyrosine phosphatase, but was not mediated by p44/42 MAPK, Src or activation of a serine phosphatase. Akt inhibition by CCK was also found in vivo and in Panc-1 cancer cells where it inhibited serum-mediated rescue from apoptosis. These results demonstrate that GI growth factors as well as gastrointestinal hormones/neurotransmitters with different cellular basis of action can all regulate Akt phosphorylation in pancreatic acinar cells. This regulation is complex with phospholipase C agents such as CCK, because both stimulatory and inhibitory

  3. Angiotensin II Signaling in Human Preadipose Cells: Participation of ERK1,2-Dependent Modulation of Akt

    PubMed Central

    Dünner, Natalia; Quezada, Carolina; Berndt, F. Andrés; Cánovas, José; Rojas, Cecilia V.

    2013-01-01

    The renin-angiotensin system expressed in adipose tissue has been implicated in the modulation of adipocyte formation, glucose metabolism, triglyceride accumulation, lipolysis, and the onset of the adverse metabolic consequences of obesity. As we investigated angiotensin II signal transduction mechanisms in human preadipose cells, an interplay of extracellular-signal-regulated kinases 1 and 2 (ERK1,2) and Akt/PKB became evident. Angiotensin II caused attenuation of phosphorylated Akt (p-Akt), at serine 473; the p-Akt/Akt ratio decreased to 0.5±0.2-fold the control value without angiotensin II (p<0.001). Here we report that the reduction of phosphorylated Akt associates with ERK1,2 activities. In the absence of angiotensin II, inhibition of ERK1,2 activation with U0126 or PD98059 resulted in a 2.1±0.5 (p<0.001) and 1.4±0.2-fold (p<0.05) increase in the p-Akt/Akt ratio, respectively. In addition, partial knockdown of ERK1 protein expression by the short hairpin RNA technique also raised phosphorylated Akt in these cells (the p-Akt/Akt ratio was 1.5±0.1-fold the corresponding control; p<0.05). Furthermore, inhibition of ERK1,2 activation with U0126 prevented the reduction of p-Akt/Akt by angiotensin II. An analogous effect was found on the phosphorylation status of Akt downstream effectors, the forkhead box (Fox) proteins O1 and O4. Altogether, these results indicate that angiotensin II signaling in human preadipose cells involves an ERK1,2-dependent attenuation of Akt activity, whose impact on the biological functions under its regulation is not fully understood. PMID:24098385

  4. Akt1 binds focal adhesion kinase via the Akt1 kinase domain independently of the pleckstrin homology domain.

    PubMed

    Basson, M D; Zeng, B; Wang, S

    2015-10-01

    Akt1 and focal adhesion kinase (FAK) are protein kinases that play key roles in normal cell signaling. Individually, aberrant expression of these kinases has been linked to a variety of cancers. Together, Akt1/FAK interactions facilitate cancer metastasis by increasing cell adhesion under conditions of increased extracellular pressure. Pathological and iatrogenic sources of pressure arise from tumor growth against constraining stroma or direct perioperative manipulation. We previously reported that 15 mmHg increased extracellular pressure causes Akt1 to both directly interact with FAK and to phosphorylate and activate it. We investigated the nature of the Akt1/FAK binding by creating truncations of recombinant FAK, conjugated to glutathione S-transferase (GST), to pull down full-length Akt1. Western blots probing for Akt1 showed that FAK/Akt1 binding persisted in FAK truncations consisting of only amino acids 1-126, FAK(NT1), which contains the F1 subdomain of its band 4.1, ezrin, radixin, and moesin (FERM) domain. Using FAK(NT1) as bait, we then pulled down truncated versions of recombinant Akt1 conjugated to HA (human influenza hemagglutinin). Probes for GST-FAK(NT1) showed Akt1-FAK binding to occur in the absence of the both the Akt1 (N)-terminal pleckstrin homology (PH) domain and its adjacent hinge region. The Akt1 (C)-terminal regulatory domain was equally unnecessary for Akt1/FAK co-immunoprecipitation. Truncations involving the Akt1 catalytic domain showed that the domain by itself was enough to pull down FAK. Additionally, a fragment spanning from the PH domain to half way through the catalytic domain demonstrated increased FAK binding compared to full length Akt1. These results begin to delineate the Akt1/FAK interaction and can be used to manipulate their force-activated signal interactions. Furthermore, the finding that the N-terminal half of the Akt1 catalytic domain binds so strongly to FAK when cleaved from the rest of the protein may suggest a means

  5. Somatic Activation of AKT3 Causes Hemispheric Developmental Brain Malformations

    PubMed Central

    Poduri, Annapurna; Evrony, Gilad D.; Cai, Xuyu; Elhosary, Princess Christina; Beroukhim, Rameen; Lehtinen, Maria K.; Hills, L. Benjamin; Heinzen, Erin L.; Hill, Anthony; Hill, R. Sean; Barry, Brenda J.; Bourgeois, Blaise F.D.; Riviello, James J.; Barkovich, A. James; Black, Peter M.; Ligon, Keith L.; Walsh, Christopher A.

    2012-01-01

    Summary Hemimegalencephaly (HMG) is a developmental brain disorder characterized by an enlarged, malformed cerebral hemisphere, typically causing epilepsy that requires surgical resection. We studied resected HMG tissue to test whether the condition might reflect somatic mutations affecting genes critical to brain development. We found that 2/8 HMG samples showed trisomy of chromosome 1q, encompassing many genes, including AKT3, which is known to regulate brain size. A third case showed a known activating mutation in AKT3 (c.49G→A, creating p.E17K) that was not present in the patient’s blood cells. Remarkably, the E17K mutation in AKT3 is exactly paralogous to E17K mutations in AKT1 and AKT2 recently discovered in somatic overgrowth syndromes. We show that AKT3 is the most abundant AKT paralogue in brain during neurogenesis and that phosphorylated AKT is abundant in cortical progenitor cells. Our data suggest that somatic mutations limited to brain could represent an important cause of complex neurogenetic disease. PMID:22500628

  6. Histone phosphorylation

    PubMed Central

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-01-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes. PMID:22948226

  7. Crosstalk Between MAPK/ERK and PI3K/AKT Signal Pathways During Brain Ischemia/Reperfusion

    PubMed Central

    Zhou, Jing; Du, Ting; Li, Baoman; Rong, Yan; Verkhratsky, Alexei

    2015-01-01

    The epidermal growth factor receptor (EGFR) is linked to the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Raf/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2) signaling pathways. During brain ischemia/reperfusion, EGFR could be transactivated, which stimulates these intracellular signaling cascades that either protect cells or potentiate cell injury. In the present study, we investigated the activation of EGFR, PI3K/AKT, and Raf/MAPK/ERK1/2 during ischemia or reperfusion of the brain using the middle cerebral artery occlusion model. We found that EGFR was phosphorylated and transactivated during both ischemia and reperfusion periods. During ischemia, the activity of PI3K/AKT pathway was significantly increased, as judged from the strong phosphorylation of AKT; this activation was suppressed by the inhibitors of EGFR and Zn-dependent metalloproteinase. Ischemia, however, did not induce ERK1/2 phosphorylation, which was dependent on reperfusion. Coimmunoprecipitation of Son of sevenless 1 (SOS1) with EGFR showed increased association between the receptor and SOS1 in ischemia, indicating the inhibitory node downstream of SOS1. The inhibitory phosphorylation site of Raf-1 at Ser259, but not its stimulatory phosphorylation site at Ser338, was phosphorylated during ischemia. Furthermore, ischemia prompted the interaction between Raf-1 and AKT, while both the inhibitors of PI3K and AKT not only abolished AKT phosphorylation but also restored ERK1/2 phosphorylation. All these findings suggest that Raf/MAPK/ERK1/2 signal pathway is inhibited by AKT via direct phosphorylation and inhibition at Raf-1 node during ischemia. During reperfusion, we observed a significant increase of ERK1/2 phosphorylation but no change in AKT phosphorylation. Inhibitors of reactive oxygen species and phosphatase and tensin homolog restored AKT phosphorylation but abolished ERK1/2 phosphorylation, suggesting that the reactive oxygen species

  8. Activation of Akt by the Bacterial Inositol Phosphatase, SopB, is Wortmannin Insensitive

    PubMed Central

    Cooper, Kendal G.; Winfree, Seth; Malik-Kale, Preeti; Jolly, Carrie; Ireland, Robin; Knodler, Leigh A.; Steele-Mortimer, Olivia

    2011-01-01

    Salmonella enterica uses effector proteins translocated by a Type III Secretion System to invade epithelial cells. One of the invasion-associated effectors, SopB, is an inositol phosphatase that mediates sustained activation of the pro-survival kinase Akt in infected cells. Canonical activation of Akt involves membrane translocation and phosphorylation and is dependent on phosphatidyl inositide 3 kinase (PI3K). Here we have investigated these two distinct processes in Salmonella infected HeLa cells. Firstly, we found that SopB-dependent membrane translocation and phosphorylation of Akt are insensitive to the PI3K inhibitor wortmannin. Similarly, depletion of the PI3K regulatory subunits p85α and p85ß by RNAi had no inhibitory effect on SopB-dependent Akt phosphorylation. Nevertheless, SopB-dependent phosphorylation does depend on the Akt kinases, PDK1 and rictor-mTOR. Membrane translocation assays revealed a dependence on SopB for Akt recruitment to Salmonella ruffles and suggest that this is mediated by phosphoinositide (3,4) P2 rather than phosphoinositide (3,4,5) P3. Altogether these data demonstrate that Salmonella activates Akt via a wortmannin insensitive mechanism that is likely a class I PI3K-independent process that incorporates some essential elements of the canonical pathway. PMID:21779406

  9. Activation of the PI3K/AKT Pathway in Merkel Cell Carcinoma

    PubMed Central

    Baeurle, Anne; Ritter, Cathrin; Schrama, David; Landthaler, Michael; Becker, Juergen C.

    2012-01-01

    Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with an increasing incidence. The understanding of the molecular carcinogenesis of MCC is limited. Here, we scrutinized the PI3K/AKT pathway, one of the major pathways activated in human cancer, in MCC. Immunohistochemical analysis of 41 tumor tissues and 9 MCC cell lines revealed high levels of AKT phosphorylation at threonine 308 in 88% of samples. Notably, the AKT phosphorylation was not correlated with the presence or absence of the Merkel cell polyoma virus (MCV). Accordingly, knock-down of the large and small T antigen by shRNA in MCV positive MCC cells did not affect phosphorylation of AKT. We also analyzed 46 MCC samples for activating PIK3CA and AKT1 mutations. Oncogenic PIK3CA mutations were found in 2/46 (4%) MCCs whereas mutations in exon 4 of AKT1 were absent. MCC cell lines demonstrated a high sensitivity towards the PI3K inhibitor LY-294002. This finding together with our observation that the PI3K/AKT pathway is activated in the majority of human MCCs identifies PI3K/AKT as a potential new therapeutic target for MCC patients. PMID:22363598

  10. Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3-kinase activation.

    PubMed

    Bijur, Gautam N; Jope, Richard S

    2003-12-01

    We describe here a new component of the phosphatidylinositol 3-kinase/Akt signaling pathway that directly impacts mitochondria. Akt (protein kinase B) was shown for the first time to be localized in mitochondria, where it was found to reside in the matrix and the inner and outer membranes, and the level of mitochondrial Akt was very dynamically regulated. Stimulation of a variety of cell types with insulin-like growth factor-1, insulin, or stress (induced by heat shock), induced translocation of Akt to the mitochondria within only several minutes of stimulation, causing increases of nearly eight- to 12-fold, and the mitochondrial Akt was in its phosphorylated, active state. Two mitochondrial proteins were identified to be phosphorylated following stimulation of mitochondrial Akt, the beta-subunit of ATP synthase and glycogen synthase kinase-3beta. The finding that mitochondrial glycogen synthase kinase-3beta was rapidly and substantially modified by Ser9 phosphorylation, which inhibits its activity, following translocation of Akt to the mitochondria is the first evidence for a regulatory mechanism affecting mitochondrial glycogen synthase kinase-3beta. These results demonstrate that signals emanating from plasma membrane receptors or generated by stress rapidly modulate Akt and glycogen synthase kinase-3beta in mitochondria. PMID:14713298

  11. PP2A inhibition results in hepatic insulin resistance despite Akt2 activation.

    PubMed

    Galbo, Thomas; Perry, Rachel J; Nishimura, Erica; Samuel, Varman T; Quistorff, Bjørn; Shulman, Gerald I

    2013-10-01

    In the liver, insulin suppresses hepatic gluconeogenesis by activating Akt, which inactivates the key gluconeogenic transcription factor FoxO1 (Forkhead Box O1). Recent studies have implicated hyperactivity of the Akt phosphatase Protein Phosphatase 2A (PP2A) and impaired Akt signaling as a molecular defect underlying insulin resistance. We therefore hypothesized that PP2A inhibition would enhance insulin-stimulated Akt activity and decrease glucose production. PP2A inhibitors increased hepatic Akt phosphorylation and inhibited FoxO1in vitro and in vivo, and suppressed gluconeogenesis in hepatocytes. Paradoxically, PP2A inhibition exacerbated insulin resistance in vivo. This was explained by phosphorylation of both hepatic glycogen synthase (GS) (inactivation) and phosphorylase (activation) resulting in impairment of glycogen storage. Our findings underline the significance of GS and Phosphorylase as hepatic PP2A substrates and importance of glycogen metabolism in acute plasma glucose regulation. PMID:24150286

  12. Design, synthesis and biological evaluation of pyrazol-furan carboxamide analogues as novel Akt kinase inhibitors.

    PubMed

    Zhan, Wenhu; Xu, Lei; Dong, Xiaowu; Dong, Jun; Yi, Xiao; Ma, Xiaodong; Qiu, Ni; Li, Jia; Yang, Bo; Zhou, Yubo; Hu, Yongzhou

    2016-07-19

    A series of novel pyrazol-furan carboxamide analogues were designed, synthesized and biologically evaluated for their Akt1 inhibitory activities, as well as anti-proliferative efficacies against HCT116 and OVCAR-8 cell lines. Most compounds exhibited moderate to excellent Akt1 inhibitory activities, together with favorable cytotoxicities. Further kinase selectivity assay of the most promising compound 25e illustrated that it was also potent against the structurally related AGC kinases, including Akt2, Akt3, ROCK1 and PKA, but was specific over kinases from other subfamilies. In addition, the Western blot analysis indicated that 25e could significantly suppress the phosphorylation level of Akt substrate GSK3β in PC-3 cell. Moreover, 25e demonstrated a concentration-dependent inhibition of phosphorylation of PRAS40 in LNCaP cell, with IC50 value of 30.4 nM. PMID:27089211

  13. Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA

    PubMed Central

    Nguyen, Le Xuan Truong; Mitchell, Beverly S.

    2013-01-01

    Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation. PMID:24297901

  14. Isoform-specific regulation of adipocyte differentiation by Akt/protein kinase B{alpha}

    SciTech Connect

    Yun, Sung-Ji; Kim, Eun-Kyoung; Tucker, David F.; Kim, Chi Dae; Birnbaum, Morris J.; Bae, Sun Sik

    2008-06-20

    The phosphatidylinositol 3-kinase (PI3K)/Akt pathway tightly regulates adipose cell differentiation. Here we show that loss of Akt1/PKB{alpha} in primary mouse embryo fibroblast (MEF) cells results in a defect of adipocyte differentiation. Adipocyte differentiation in vitro and ex vivo was restored in cells lacking both Akt1/PKB{alpha} and Akt2/PKB{beta} by ectopic expression of Akt1/PKB{alpha} but not Akt2/PKB{beta}. Akt1/PKB{alpha} was found to be the major regulator of phosphorylation and nuclear export of FoxO1, whose presence in the nucleus strongly attenuates adipocyte differentiation. Differentiation-induced cell division was significantly abrogated in Akt1/PKB{alpha}-deficient cells, but was restored after forced expression of Akt1/PKB{alpha}. Moreover, expression of p27{sup Kip1}, an inhibitor of the cell cycle, was down regulated in an Akt1/PKB{alpha}-specific manner during adipocyte differentiation. Based on these data, we suggest that the Akt1/PKB{alpha} isoform plays a major role in adipocyte differentiation by regulating FoxO1 and p27{sup Kip1}.

  15. mTORC2/Rictor Signaling Disrupts Dopamine-Dependent Behaviors via Defects in Striatal Dopamine Neurotransmission

    PubMed Central

    Dadalko, Olga I.; Siuta, Michael; Poe, Amanda; Erreger, Kevin; Matthies, Heinrich J.G.; Niswender, Kevin

    2015-01-01

    Disrupted neuronal protein kinase B (Akt) signaling has been associated with dopamine (DA)-related neuropsychiatric disorders, including schizophrenia, a devastating mental illness. We hypothesize that proper DA neurotransmission is therefore dependent upon intact neuronal Akt function. Akt is activated by phosphorylation of two key residues: Thr308 and Ser473. Blunted Akt phosphorylation at Ser473 (pAkt-473) has been observed in lymphocytes and postmortem brains of schizophrenia patients, and psychosis-prone normal individuals. Mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multiprotein complex that is responsible for phosphorylation of Akt at Ser473 (pAkt-473). We demonstrate that mice with disrupted mTORC2 signaling in brain exhibit altered striatal DA-dependent behaviors, such as increased basal locomotion, stereotypic counts, and exaggerated response to the psychomotor effects of amphetamine (AMPH). Combining in vivo and ex vivo pharmacological, electrophysiological, and biochemical techniques, we demonstrate that the changes in striatal DA neurotransmission and associated behaviors are caused, at least in part, by elevated D2 DA receptor (D2R) expression and upregulated ERK1/2 activation. Haloperidol, a typical antipsychotic and D2R blocker, reduced AMPH hypersensitivity and elevated pERK1/2 to the levels of control animals. By viral gene delivery, we downregulated mTORC2 solely in the dorsal striatum of adult wild-type mice, demonstrating that striatal mTORC2 regulates AMPH-stimulated behaviors. Our findings implicate mTORC2 signaling as a novel pathway regulating striatal DA tone and D2R signaling. PMID:26063917

  16. Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

    SciTech Connect

    Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won; Bae, Jin Ung; Kim, Gyu Hee; Park, So Youn; Kim, Chi Dae

    2012-04-01

    Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways led to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.

  17. Illuminating the phosphatidylinositol 3-kinase/Akt pathway

    NASA Astrophysics Data System (ADS)

    Ni, Qiang; Fosbrink, Matthew; Zhang, Jin

    2008-02-01

    Genetically encodable fluorescent biosensors based on fluorescence resonance energy transfer (FRET) are being developed for analyzing spatiotemporal dynamics of various signaling events in living cells, as these events are often dynamically regulated and spatially compartmentalized within specific signaling context. In particular, to investigate the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in the cellular context, we have developed a series of such biosensors that enable dynamic visualization of several key signaling events in this pathway, namely InPAkt for lipid second messenger dynamics, BAKR for Akt activity, and ReAktion for the action of Akt during its multi-step activation process. Discussed here are several studies that have been carried out with these novel biosensors. First, we examined nuclear phosphatidylinositol-3,4,5-triphosphate (PIP 3) in living cells using nucleus-targeted InPAkt. Second, we analyzed signal propagation from the plasma membrane to the nucleus by using plasma membrane-targeted InPAkt and nucleus-targeted BKAR to simultaneously monitor PIP 3 dynamics and Akt activity in the same cell. Of note, results from these co-imaging experiments suggest that active Akt can dissociate from the plasma membrane and translocate into the nucleus in the presence of high levels of PIP 3 at the plasma membrane. This finding has led to a further study of the action of Akt during its activation process, particularly focusing on how Akt dissociates from the membrane. In this regard, a live-cell molecular analysis using ReAktion reveals a conformational change in Akt that is critically dependent on the existence of a phosphorylatable T308 in the activation loop. Subsequently this has led to the discovery of new regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.

  18. Calpain-2 activates Akt via TGF-β1-mTORC2 pathway in pulmonary artery smooth muscle cells.

    PubMed

    Abeyrathna, Prasanna; Kovacs, Laszlo; Han, Weihong; Su, Yunchao

    2016-07-01

    Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 small interfering RNA attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308), as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable-specific transforming growth factor (TGF)-β receptor inhibitor SB431542 attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB-431452 and knocking down activin receptor-like kinase-5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF-β1 using a cell-impermeable TGF-β1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Furthermore, inhibition of mammalian target of rapamycin complex 2 (mTORC2) by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 upregulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGF-β1/mTORC2 mechanism. PMID:27099352

  19. TRAIL-induced caspase/p38 activation is responsible for the increased catalytic and invasive activities of Akt

    PubMed Central

    SUN, BO K.; KIM, JOO-HANG; NGUYEN, HOAN N.; KIM, SO Y.; OH, SEEUN; LEE, YONG J.; SONG, JAE J.

    2010-01-01

    We previously observed that TRAIL induces acquired TRAIL resistance coinciding with increased Akt phosphorylation brought about by the Src-PI3K-Akt signaling pathways and mediated by c-Cbl. c-Cbl, a ubiquitously expressed cytoplasmic adaptor protein, is simultaneously involved in the rapid degradation of TRAIL receptors and Akt phosphorylation during TRAIL treatment. Here, we show that Akt phosphorylation is not exclusively responsible for acquired TRAIL resistance. Akt catalytic activation is known to increase during metabolic oxidative stress, but we show that TRAIL also dramatically induces the catalytic activation of Akt in TRAIL-sensitive cells, but not in TRAIL-resistant cells. This suggests that Akt catalytic activation during TRAIL-induced apoptosis is likely to play a compensatory role in the maintenance of cell homeostasis. In addition, activated p38 and phosphorylated HSP27 were found to act as downstream effector molecules of p38 during TRAIL treatment and were shown to be responsible for increased Akt catalytic and invasive activities. PMID:21109947

  20. BRAFV600E Negatively Regulates the AKT Pathway in Melanoma Cell Lines

    PubMed Central

    Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F.; Niu, Huifeng

    2012-01-01

    Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways. PMID:22880048

  1. BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

    PubMed

    Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F; Niu, Huifeng

    2012-01-01

    Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways. PMID:22880048

  2. Interplay between Akt and p38 MAPK pathways in the regulation of renal tubular cell apoptosis associated with diabetic nephropathy

    PubMed Central

    Song, Ye; Jin, Shunying; Barati, Michelle T.; Wu, Rui; Kausar, Hina; Tan, Yi; Wang, Yuehui; Zhou, Guihua; Klein, Jon B.; Li, Xiaokun

    2010-01-01

    Hyperglycemia induces p38 MAPK-mediated renal proximal tubular cell (RPTC) apoptosis. The current study hypothesized that alteration of the Akt signaling pathway by hyperglycemia may contribute to p38 MAPK activation and development of diabetic nephropathy. Immunoblot analysis demonstrated a hyperglycemia-induced increase in Akt phosphorylation in diabetic kidneys at 1 mo, peaking at 3 mo, and dropping back to baseline by 6 mo. Immunohistochemical staining with anti-pAkt antisera localized Akt phosphorylation to renal tubules. Maximal p38 MAPK phosphorylation was detected concomitant with increase in terminal uridine deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and caspase-3 activity in 6-mo diabetic kidneys. Exposure of cultured RPTCs to high glucose (HG; 22.5 mM) significantly increased Akt phosphorylation at 3, 6, and 9 h, and decreased thereafter. In contrast, p38 MAPK phosphorylation was detected between 9 and 48 h of HG treatment. Increased p38 MAPK activation at 24 and 48 h coincided with increased apoptosis, demonstrated by increased caspase-3 activity at 24 h and increased TUNEL-positive cells at 48 h of HG exposure. Blockade of p38 cascade with SB203850 inhibited HG-induced caspase-3 activation and TUNEL-positive cells. Overexpression of constitutively active Akt abrogated HG-induced p38 MAPK phosphorylation and RPTC apoptosis. In addition, blockade of the phosphatidylinositol-3 kinase/Akt pathway with LY294002 and silencing of Akt expression with Akt small interfering RNA induced p38 MAPK phosphorylation in the absence of HG. These results collectively suggest that downregulation of Akt activation during long-term hyperglycemia contributes to enhanced p38 MAPK activation and RPTC apoptosis. Mechanism of downregulation of Akt activation in 6-mo streptozotocin diabetic kidneys was attributed to decreased Akt-heat shock protein (Hsp) 25, Akt-p38 interaction, and decreased PTEN activity. Thus PTEN or Hsp25 could serve

  3. Interplay between Akt and p38 MAPK pathways in the regulation of renal tubular cell apoptosis associated with diabetic nephropathy.

    PubMed

    Rane, Madhavi J; Song, Ye; Jin, Shunying; Barati, Michelle T; Wu, Rui; Kausar, Hina; Tan, Yi; Wang, Yuehui; Zhou, Guihua; Klein, Jon B; Li, Xiaokun; Cai, Lu

    2010-01-01

    Hyperglycemia induces p38 MAPK-mediated renal proximal tubular cell (RPTC) apoptosis. The current study hypothesized that alteration of the Akt signaling pathway by hyperglycemia may contribute to p38 MAPK activation and development of diabetic nephropathy. Immunoblot analysis demonstrated a hyperglycemia-induced increase in Akt phosphorylation in diabetic kidneys at 1 mo, peaking at 3 mo, and dropping back to baseline by 6 mo. Immunohistochemical staining with anti-pAkt antisera localized Akt phosphorylation to renal tubules. Maximal p38 MAPK phosphorylation was detected concomitant with increase in terminal uridine deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and caspase-3 activity in 6-mo diabetic kidneys. Exposure of cultured RPTCs to high glucose (HG; 22.5 mM) significantly increased Akt phosphorylation at 3, 6, and 9 h, and decreased thereafter. In contrast, p38 MAPK phosphorylation was detected between 9 and 48 h of HG treatment. Increased p38 MAPK activation at 24 and 48 h coincided with increased apoptosis, demonstrated by increased caspase-3 activity at 24 h and increased TUNEL-positive cells at 48 h of HG exposure. Blockade of p38 cascade with SB203850 inhibited HG-induced caspase-3 activation and TUNEL-positive cells. Overexpression of constitutively active Akt abrogated HG-induced p38 MAPK phosphorylation and RPTC apoptosis. In addition, blockade of the phosphatidylinositol-3 kinase/Akt pathway with LY294002 and silencing of Akt expression with Akt small interfering RNA induced p38 MAPK phosphorylation in the absence of HG. These results collectively suggest that downregulation of Akt activation during long-term hyperglycemia contributes to enhanced p38 MAPK activation and RPTC apoptosis. Mechanism of downregulation of Akt activation in 6-mo streptozotocin diabetic kidneys was attributed to decreased Akt-heat shock protein (Hsp) 25, Akt-p38 interaction, and decreased PTEN activity. Thus PTEN or Hsp25 could serve

  4. Neutrophil AKT2 regulates heterotypic cell-cell interactions during vascular inflammation.

    PubMed

    Li, Jing; Kim, Kyungho; Hahm, Eunsil; Molokie, Robert; Hay, Nissim; Gordeuk, Victor R; Du, Xiaoping; Cho, Jaehyung

    2014-04-01

    Interactions between platelets, leukocytes, and activated endothelial cells are important during microvascular occlusion; however, the regulatory mechanisms of these heterotypic cell-cell interactions remain unclear. Here, using intravital microscopy to evaluate mice lacking specific isoforms of the serine/threonine kinase AKT and bone marrow chimeras, we found that hematopoietic cell-associated AKT2 is important for neutrophil adhesion and crawling and neutrophil-platelet interactions on activated endothelial cells during TNF-α-induced venular inflammation. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under shear conditions. In particular, neutrophil AKT2 was critical for membrane translocation of αMβ2 integrin, β2-talin1 interaction, and intracellular Ca2+ mobilization. We found that the basal phosphorylation levels of AKT isoforms were markedly increased in neutrophils and platelets isolated from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow rates. Our results provide evidence that neutrophil AKT2 regulates αMβ2 integrin function and suggest that AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions under thromboinflammatory conditions such as SCD. PMID:24642468

  5. Polyunsaturated fatty acids affect the localization and signaling of PIP3/AKT in prostate cancer cells.

    PubMed

    Gu, Zhennan; Wu, Jiansheng; Wang, Shihua; Suburu, Janel; Chen, Haiqin; Thomas, Michael J; Shi, Lihong; Edwards, Iris J; Berquin, Isabelle M; Chen, Yong Q

    2013-09-01

    AKT is a serine-threonine protein kinase that plays important roles in cell growth, proliferation and apoptosis. It is activated after binding to phosphatidylinositol phosphates (PIPs) with phosphate groups at positions 3,4 and 3,4,5 on the inositol ring. In spite of extensive research on AKT, one aspect has been largely overlooked, namely the role of the fatty acid chains on PIPs. PIPs are phospholipids composed of a glycerol backbone with fatty acids at the sn-1 and sn-2 position and inositol at the sn-3 position. Here, we show that polyunsaturated fatty acids (PUFAs) modify phospholipid content. Docosahexaenoic acid (DHA), an ω3 PUFA, can replace the fatty acid at the sn-2 position of the glycerol backbone, thereby changing the species of phospholipids. DHA also inhibits AKT(T308) but not AKT(S473) phosphorylation, alters PI(3,4,5)P3 (PIP3) and phospho-AKT(S473) protein localization, decreases pPDPK1(S241)-AKT and AKT-BAD interaction and suppresses prostate tumor growth. Our study highlights a potential novel mechanism of cancer inhibition by ω3 PUFA through alteration of PIP3 and AKT localization and affecting the AKT signaling pathway. PMID:23633519

  6. Regulation of Akt signaling by Sirtuins: Its implication in cardiac hypertrophy and aging

    PubMed Central

    Pillai, Vinodkumar B.; Sundaresan, Nagalingam R.; Gupta, Mahesh P.

    2014-01-01

    Cardiac hypertrophy is a multifactorial disease characterized by multiple molecular alterations. One of these alterations is change in activity of Akt, which plays a central role in regulating a variety of cellular processes ranging from cell survival to aging. Akt activation is mainly achieved by its binding to phosphatidylinositol 3,4,5 triphosphate (PIP3). This results in a conformational change that exposes the kinase domain of Akt for phosphorylation and activation by its upstream kinase PDK1 in the cell membrane. Recent studies have shown that sirtuin isoforms SIRT1, SIRT3 and SIRT6 play an essential role in the regulation of Akt activation. While SIRT1 deacetylates Akt to promote PIP3 binding and activation, SIRT3 controls ROS-mediated Akt activation and SIRT6 transcriptionally represses Akt at the level of chromatin. In the first part of this review, we discuss the mechanisms by which sirtuins regulate Akt activation and how they influence other post-translational modifications of Akt. In the latter part of the review, we summarize the implications of sirtuin-dependent regulation of Akt signaling in the control of major cellular processes like cellular growth, angiogenesis, apoptosis, autophagy and aging; which are involved in the initiation and progression of several diseases. PMID:24436432

  7. The basal level of intracellular calcium gates the activation of phosphoinositide 3-kinase - Akt signaling by brain-derived neurotrophic factor in cortical neurons

    PubMed Central

    Zheng, Fei; Soellner, Deborah; Nunez, Joseph; Wang, Hongbing

    2008-01-01

    Brain derived neurotrophic factor (BDNF) mediates survival and neuroplasticity through the activation of phosphoinositide 3-kinase (PI3K)-Akt pathway. Although previous studies suggested the roles of MAPK, PLC-γ-mediated intra-cellular calcium ([Ca2+]i) increase, and extra-cellular calcium influx in regulating Akt activation, the cellular mechanisms are largely unknown. We demonstrated that sub-nanomolar BDNF significantly induced Akt activation in developing cortical neurons. The TrkB-dependent Akt phosphorylation at S473 and T308 required only PI3K, but not PLC and MAPK activity. Blocking NMDA receptors, L-type voltage-gated calcium channels, and chelating extra-cellular calcium by EGTA failed to block BDNF-induced Akt phosphorylation. In contrast, chelating [Ca2+]i by BAPTA-AM abolished Akt phosphorylation. Interestingly, sub-nanomolar BDNF did not stimulate [Ca2+]i increase under our culture conditions. Together with that NMDA- and membrane depolarization-induced [Ca2+]i increase did not activate Akt, we conclude that the basal level of [Ca2+]i gates BDNF function. Furthermore, inhibiting calmodulin by W13 suppressed Akt phosphorylation. On the other hand, inhibition of protein phosphatase 1 by okadaic acid and tautomycin rescued Akt phosphorylation in BAPTA- and W13-treated neurons. We further demonstrated that the phosphorylation of PDK1 did not correlate with Akt phosphorylation at T308. Our results suggested novel roles of basal [Ca2+]i, rather than activity-induced calcium elevation, in BDNF-Akt signaling. PMID:18485103

  8. Spatiotemporal dynamics of phosphorylation in lipid second messenger signaling.

    PubMed

    Antal, Corina E; Newton, Alexandra C

    2013-12-01

    The plasma membrane serves as a dynamic interface that relays information received at the cell surface into the cell. Lipid second messengers coordinate signaling on this platform by recruiting and activating kinases and phosphatases. Specifically, diacylglycerol and phosphatidylinositol 3,4,5-trisphosphate activate protein kinase C and Akt, respectively, which then phosphorylate target proteins to transduce downstream signaling. This review addresses how the spatiotemporal dynamics of protein kinase C and Akt signaling can be monitored using genetically encoded reporters and provides information on how the coordination of signaling at protein scaffolds or membrane microdomains affords fidelity and specificity in phosphorylation events. PMID:23788531

  9. The Akt1/IL-6/STAT3 pathway regulates growth of lung tumor initiating cells.

    PubMed

    Malanga, Donatella; De Marco, Carmela; Guerriero, Ilaria; Colelli, Fabiana; Rinaldo, Nicola; Scrima, Marianna; Mirante, Teresa; De Vitis, Claudia; Zoppoli, Pietro; Ceccarelli, Michele; Riccardi, Miriam; Ravo, Maria; Weisz, Alessandro; Federico, Antonella; Franco, Renato; Rocco, Gaetano; Mancini, Rita; Rizzuto, Antonia; Gulletta, Elio; Ciliberto, Gennaro; Viglietto, Giuseppe

    2015-12-15

    Here we report that the PI3K/Akt1/IL-6/STAT3 signalling pathway regulates generation and stem cell-like properties of Non-Small Cell Lung Cancer (NSCLC) tumor initiating cells (TICs). Mutant Akt1, mutant PIK3CA or PTEN loss enhances formation of lung cancer spheroids (LCS), self-renewal, expression of stemness markers and tumorigenic potential of human immortalized bronchial cells (BEAS-2B) whereas Akt inhibition suppresses these activities in established (NCI-H460) and primary NSCLC cells. Matched microarray analysis of Akt1-interfered cells and LCSs identified IL-6 as a critical target of Akt signalling in NSCLC TICs. Accordingly, suppression of Akt in NSCLC cells decreases IL-6 levels, phosphorylation of IkK and IkB, NF-kB transcriptional activity, phosphorylation and transcriptional activity of STAT3 whereas active Akt1 up-regulates them. Exposure of LCSs isolated from NSCLC cells to blocking anti-IL-6 mAbs, shRNA to IL-6 receptor or to STAT3 markedly reduces the capability to generate LCSs, to self-renew and to form tumors, whereas administration of IL-6 to Akt-interfered cells restores the capability to generate LCSs. Finally, immunohistochemical studies in NSCLC patients demonstrated a positive correlative trend between activated Akt, IL-6 expression and STAT3 phosphorylation (n = 94; p < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC. PMID:26486080

  10. HDAC Inhibition Elicits Myocardial Protective Effect through Modulation of MKK3/Akt-1

    PubMed Central

    Zhao, Ting C.; Du, Jianfeng; Zhuang, Shugang; Liu, Paul; Zhang, Ling X.

    2013-01-01

    We and others have demonstrated that HDAC inhibition protects the heart against myocardial injury. It is known that Akt-1 and MAP kinase play an essential role in modulation of myocardial protection and cardiac preconditioning. Our recent observations have shown that Akt-1 was activated in post-myocardial infarction following HDAC inhibition. However, it remains unknown whether MKK3 and Akt-1 are involved in HDAC inhibition-induced myocardial protection in acute myocardial ischemia and reperfusion injury. We sought to investigate whether the genetic disruption of Akt-1 and MKK3 eliminate cardioprotection elicited by HDAC inhibition and whether Akt-1 is associated with MKK3 to ultimately achieve protective effects. Adult wild type and MKK3−/−, Akt-1−/− mice received intraperitoneal injections of trichostatin A (0.1mg/kg), a potent inhibitor of HDACs. The hearts were subjected to 30 min myocardial ischemia/30 min reperfusion in the Langendorff perfused heart after twenty four hours to elicit pharmacologic preconditioning. Left ventricular function was measured, and infarct size was determined. Acetylation and phosphorylation of MKK3 were detected and disruption of Akt-1 abolished both acetylation and phosphorylation of MKK3. HDAC inhibition produces an improvement in left ventricular functional recovery, but these effects were abrogated by disruption of either Akt-1 or MKK3. Disruption of Akt-1 or MKK3 abolished the effects of HDAC inhibition-induced reduction of infarct size. Trichostatin A treatment resulted in an increase in MKK3 phosphorylation or acetylation in myocardium. Taken together, these results indicate that stimulation of the MKK3 and Akt-1 pathway is a novel approach to HDAC inhibition -induced cardioprotection. PMID:23762381

  11. The Akt1/IL-6/STAT3 pathway regulates growth of lung tumor initiating cells

    PubMed Central

    Malanga, Donatella; De Marco, Carmela; Guerriero, Ilaria; Colelli, Fabiana; Rinaldo, Nicola; Scrima, Marianna; Mirante, Teresa; De Vitis, Claudia; Zoppoli, Pietro; Ceccarelli, Michele; Riccardi, Miriam; Ravo, Maria; Weisz, Alessandro; Federico, Antonella; Franco, Renato; Rocco, Gaetano; Mancini, Rita; Rizzuto, Antonia; Gulletta, Elio; Ciliberto, Gennaro; Viglietto, Giuseppe

    2015-01-01

    Here we report that the PI3K/Akt1/IL-6/STAT3 signalling pathway regulates generation and stem cell-like properties of Non-Small Cell Lung Cancer (NSCLC) tumor initiating cells (TICs). Mutant Akt1, mutant PIK3CA or PTEN loss enhances formation of lung cancer spheroids (LCS), self-renewal, expression of stemness markers and tumorigenic potential of human immortalized bronchial cells (BEAS-2B) whereas Akt inhibition suppresses these activities in established (NCI-H460) and primary NSCLC cells. Matched microarray analysis of Akt1-interfered cells and LCSs identified IL-6 as a critical target of Akt signalling in NSCLC TICs. Accordingly, suppression of Akt in NSCLC cells decreases IL-6 levels, phosphorylation of IkK and IkB, NF-kB transcriptional activity, phosphorylation and transcriptional activity of STAT3 whereas active Akt1 up-regulates them. Exposure of LCSs isolated from NSCLC cells to blocking anti-IL-6 mAbs, shRNA to IL-6 receptor or to STAT3 markedly reduces the capability to generate LCSs, to self-renew and to form tumors, whereas administration of IL-6 to Akt-interfered cells restores the capability to generate LCSs. Finally, immunohistochemical studies in NSCLC patients demonstrated a positive correlative trend between activated Akt, IL-6 expression and STAT3 phosphorylation (n = 94; p < 0.05). In conclusion, our data indicate that aberrant Akt signalling contributes to maintaining stemness in lung cancer TICs through a NF-kB/IL-6/STAT3 pathway and provide novel potential therapeutic targets for eliminating these malignant cells in NSCLC. PMID:26486080

  12. Glutaredoxin exerts an antiapoptotic effect by regulating the redox state of Akt.

    PubMed

    Murata, Hiroaki; Ihara, Yoshito; Nakamura, Hajime; Yodoi, Junji; Sumikawa, Koji; Kondo, Takahito

    2003-12-12

    Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro assay revealed that GRX reduced oxidized Akt in concert with glutathione, NADPH, and glutathione-disulfide reductase. Thus, GRX plays an important role in protecting cells from apoptosis by regulating the redox state of Akt. PMID:14522978

  13. Morin mitigates acetaminophen-induced liver injury by potentiating Nrf2 regulated survival mechanism through molecular intervention in PHLPP2-Akt-Gsk3β axis.

    PubMed

    Rizvi, Fatima; Mathur, Alpana; Kakkar, Poonam

    2015-10-01

    Acetaminophen (APAP) is frequently taken to relieve pain. Staggered APAP overdoses have been reported to cause acetaminophen-induced liver injury (AILI). Identification of efficacious therapeutic modalities to address complications imposed by accidental/intentional long-term APAP ingestion is needed. Morin, a plant-derived phytochemical, possesses a multitude of pharmacological properties including hepatoprotective action; however, the underlying mechanisms have been inadequately explored. Our present report demonstrates significant attenuation of APAP-mediated liver injury by morin supplementation in vivo as indicated by reduction in histological and serum markers of hepatotoxicity. Morin not only limited necroinflammation as revealed by reduced HMGB1 release, NALP3 and caspase-1 maturation, but also suppressed oxidative stress and mitochondrial dysfunction. This suggests that morin may have exerted its cytoprotective role by way of early intervention in the pathway leading to perpetuation of AILI. Morin reinforced cellular defenses by suppressing Nrf2 ubiquitination and promoting nuclear Nrf2 retention as well as ARE-Nrf2 binding affinity. The effects were observed to be a result of molecular intervention in the activity of PHLPP2, a phosphatase previously reported by us to subdue cellular Nrf2 responses via Fyn kinase activation. Morin was observed to inhibit APAP-induced increase in PHLPP2 activity ex vivo as well as its association with cellular target Akt1. As a result, morin prevented oxidative stress induced deactivation of Akt (Ser473) leading to suppression in GSK3β and Fyn kinase activation. The study supports the inhibitory action of morin against PHLPP2-regulated Nrf2-suppression and hence indentifies Nrf2-potentiating property of morin that may be exploited in developing novel therapeutic strategy to address AILI. PMID:26286854

  14. Preclinical pharmacology, antitumor activity and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930

    PubMed Central

    Yap, Timothy A.; Walton, Mike I.; Hunter, Lisa-Jane K.; Valenti, Melanie; de Haven Brandon, Alexis; Eve, Paul D.; Ruddle, Ruth; Heaton, Simon P.; Henley, Alan; Pickard, Lisa; Vijayaraghavan, Gowri; Caldwell, John J.; Thompson, Neil T.; Aherne, Wynne; Raynaud, Florence I.; Eccles, Suzanne A.; Workman, Paul; Collins, Ian; Garrett, Michelle D.

    2016-01-01

    AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G1 arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective and potent AKT inhibitor, which blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being employed in clinical trials. PMID:21191045

  15. A Switch in Akt Isoforms Is Required for Notch-Induced Snail1 Expression and Protection from Cell Death

    PubMed Central

    Frías, Alex; Lambies, Guillem; Viñas-Castells, Rosa; Martínez-Guillamon, Catalina; Dave, Natàlia

    2015-01-01

    Notch activation in aortic endothelial cells (ECs) takes place at embryonic stages during cardiac valve formation and induces endothelial-to-mesenchymal transition (EndMT). Using aortic ECs, we show here that active Notch expression promotes EndMT, resulting in downregulation of vascular endothelial cadherin (VE-cadherin) and upregulation of mesenchymal genes such as those for fibronectin and Snail1/2. In these cells, transforming growth factor β1 exacerbates Notch effects by increasing Snail1 and fibronectin activation. When Notch-downstream pathways were analyzed, we detected an increase in glycogen synthase kinase 3β (GSK-3β) phosphorylation and inactivation that facilitates Snail1 nuclear retention and protein stabilization. However, the total activity of Akt was downregulated. The discrepancy between Akt activity and GSK-3β phosphorylation is explained by a Notch-induced switch in the Akt isoforms, whereby Akt1, the predominant isoform expressed in ECs, is decreased and Akt2 transcription is upregulated. Mechanistically, Akt2 induction requires the stimulation of the β-catenin/TCF4 transcriptional complex, which activates the Akt2 promoter. Active, phosphorylated Akt2 translocates to the nucleus in Notch-expressing cells, resulting in GSK-3β inactivation in this compartment. Akt2, but not Akt1, colocalizes in the nucleus with lamin B in the nuclear envelope. In addition to promoting GSK-3β inactivation, Notch downregulates Forkhead box O1 (FoxO1), another Akt2 nuclear substrate. Moreover, Notch protects ECs from oxidative stress-induced apoptosis through an Akt2- and Snail1-dependent mechanism. PMID:26711268

  16. Preclinical pharmacology, antitumor activity, and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930.

    PubMed

    Yap, Timothy A; Walton, Mike I; Hunter, Lisa-Jane K; Valenti, Melanie; de Haven Brandon, Alexis; Eve, Paul D; Ruddle, Ruth; Heaton, Simon P; Henley, Alan; Pickard, Lisa; Vijayaraghavan, Gowri; Caldwell, John J; Thompson, Neil T; Aherne, Wynne; Raynaud, Florence I; Eccles, Suzanne A; Workman, Paul; Collins, Ian; Garrett, Michelle D

    2011-02-01

    AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G(1) arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials. PMID:21191045

  17. PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

    SciTech Connect

    Hinoi, Eiichi; Iezaki, Takashi; Fujita, Hiroyuki; Watanabe, Takumi; Odaka, Yoshiaki; Ozaki, Kakeru; Yoneda, Yukio

    2014-07-18

    Highlights: • Akt is preferentially phosphorylated in BAT and sWAT of aP2-GDF5 mice. • PI3K/Akt signaling is involved in GDF5-induced brown adipogenesis. • PI3K/Akt signaling regulates GDF5-induced Smad5 phosphorylation. - Abstract: We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5{sup Rgsc451} mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.

  18. Up-regulation of PI3K/Akt signaling by 17{beta}-estradiol through activation of estrogen receptor-{alpha}, but not estrogen receptor-{beta}, and stimulates cell growth in breast cancer cells

    SciTech Connect

    Lee, Young-Rae; Park, Jinny; Kim, Jong-Suk; Youn, Hyun Jo; Jung, Sung Hoo . E-mail: shjung@chonbuk.ac.kr

    2005-11-04

    Estrogen stimulates cell proliferation in breast cancer. The biological effects of estrogen are mediated through two intracellular receptors, estrogen receptor-{alpha} (ER{alpha}) and estrogen receptor-{beta} (ER{beta}). However, the role of ERs in the proliferative action of estrogen is not well established. Recently, it has been known that ER activates phosphatidylinositol-3-OH kinase (PI3K) through binding with the p85 regulatory subunit of PI3K. Therefore, possible mechanisms may include ER-mediated phosphoinositide metabolism with subsequent formation of phosphatidylinositol-3,4,5-trisphosphate (PIP{sub 3}), which is generated from phosphatidylinositol 4,5-bisphosphate via PI3K activation. The present study demonstrates that 17{beta}-estradiol (E2) up-regulates PI3K in an ER{alpha}-dependent manner, but not ER{beta}, and stimulates cell growth in breast cancer cells. In order to study this phenomenon, we have treated ER{alpha}-positive MCF-7 cells and ER{alpha}-negative MDA-MB-231 cells with 10 nM E2. Treatment of MCF-7 cells with E2 resulted in a marked increase in PI3K (p85) expression, which paralleled an increase in phospho-Akt (Ser-473) and PIP{sub 3} level. These observations also correlated with an increased activity to E2-induced cell proliferation. However, these effects of E2 on breast cancer cells were not observed in the MDA-MB-231 cell line, indicating that the E2-mediated up-regulation of PI3K/Akt pathway is ER{alpha}-dependent. These results suggest that estrogen activates PI3K/Akt signaling through ER{alpha}-dependent mechanism in MCF-7 cells.

  19. TRIB3 suppresses tumorigenesis by controlling mTORC2/AKT/FOXO signaling

    PubMed Central

    Salazar, María; Lorente, Mar; García-Taboada, Elena; Gómez, Eduardo Pérez; Dávila, David; Zúñiga-García, Patricia; Flores, Juana M; Rodríguez, Antonio; Hegedus, Zoltan; Mosén-Ansorena, David; Aransay, Ana M; Hernández-Tiedra, Sonia; López-Valero, Israel; Quintanilla, Miguel; Sánchez, Cristina; Iovanna, Juan L; Dusetti, Nelson; Guzmán, Manuel; Francis, Sheila E; Carracedo, Arkaitz; Kiss-Toth, Endre; Velasco, Guillermo

    2015-01-01

    In a recent article, we found that Tribbles pseudokinase 3 (TRIB3) plays a tumor suppressor role and that this effect relies on the dysregulation of the phosphorylation of v-akt murine thymoma viral oncogene homolog (AKT) by the mammalian target of rapamycin complex 2 (mTORC2 complex), and the subsequent hyperphosphorylation and inactivation of the transcription factor Forkhead box O3 (FOXO3). PMID:27308456

  20. Oncogenic AKT1(E17K) mutation induces mammary hyperplasia but prevents HER2-driven tumorigenesis

    PubMed Central

    Mancini, Maria L.; Lien, Evan C.; Toker, Alex

    2016-01-01

    One of the most frequently deregulated signaling pathways in breast cancer is the PI 3-K/Akt cascade. Genetic lesions are commonly found in PIK3CA, PTEN, and AKT, which lead to excessive and constitutive activation of Akt and downstream signaling that results in uncontrolled proliferation and increased cellular survival. One such genetic lesion is the somatic AKT1(E17K) mutation, which has been identified in 4-8% of breast cancer patients. To determine how this mutation contributes to mammary tumorigenesis, we constructed a genetically engineered mouse model that conditionally expresses human AKT1(E17K) in the mammary epithelium. Although AKT1(E17K) is only weakly constitutively active and does not promote proliferation in vitro, it is capable of escaping negative feedback inhibition to exhibit sustained signaling dynamics in vitro. Consistently, both virgin and multiparous AKT1(E17K) mice develop mammary gland hyperplasia that do not progress to carcinoma. This hyperplasia is accompanied by increased estrogen receptor expression, although exposure of the mice to estrogen does not promote tumor development. Moreover, AKT1(E17K) prevents HER2-driven mammary tumor formation, in part through negative feedback inhibition of RTK signaling. Analysis of TCGA breast cancer data revealed that the mRNA expression, total protein levels, and phosphorylation of various RTKs are decreased in human tumors harboring AKT1(E17K). PMID:27004402

  1. Mutual inhibition of insulin signaling and PHLPP-1 determines cardioprotective efficiency of Akt in aged heart.

    PubMed

    Xing, Yuan; Sun, Wanqing; Wang, Yishi; Gao, Feng; Ma, Heng

    2016-05-01

    Insulin protects cardiomyocytes from myocardial ischemia/reperfusion (MI/R) injury through activating Akt. However, phosphatase PHLPP-1 (PH domain leucine-rich repeat protein phosphatase-1) dephosphorylates and inactivates Akt. The balanced competitive interaction of insulin and PHLPP-1 has not been directly examined. In this study, we have identified the effect of mutual inhibition of insulin signaling and PHLPP-1 on the cardioprotective efficiency of Akt in aged heart. Young (3 months) and aged (20 months) Sprague Dawley (SD) rats were subjected to MI/Rin vivo. The PHLPP-1 level was higher in aged vs. young hearts at base. But, insulin treatment failed to decrease PHLPP-1 level during reperfusion in the aged hearts. Consequently, the cardioprotection of insulin-induced Akt activation was impaired in aged hearts, resulting in more susceptible to MI/R injury. In cultured rat ventricular myocytes, PHLPP-1 knockdown significantly enhanced insulin-induced Akt phosphorylation and reduced simulated hypoxia/reoxygenation-induced apoptosis. Contrary, PHLPP-1 overexpression terminated Akt phosphorylation and deteriorated myocytes apoptosis. Using in vivo aged animal models, we confirmed that cardiac PHLPP-1 knockdown or enhanced insulin sensitivity by exercise training dramatically increased insulin-induced Akt phosphorylation. Specifically, MI/R-induced cardiomyocyte apoptosis and infarct size were decreased and cardiac function was increased. More importantly, we found that insulin regulated the degradation of PHLPP-1 and insulin treatment could enhance the binding between PHLPP-1 and β-transducin repeat-containing protein (β-TrCP) to target for ubiquitin-dependent degradation. Altogether, we have identified a new mechanism by which insulin suppresses PHLPP-1 to enhance Akt activation. But, aged heart possesses lower insulin effectiveness and fails to decrease PHLPP-1 during MI/R, which subsequently limited Akt activity and cardioprotection. PHLPP-1 could be a

  2. Mutual inhibition of insulin signaling and PHLPP-1 determines cardioprotective efficiency of Akt in aged heart

    PubMed Central

    Xing, Yuan; Sun, Wanqing; Wang, Yishi; Gao, Feng; Ma, Heng

    2016-01-01

    Insulin protects cardiomyocytes from myocardial ischemia/reperfusion (MI/R) injury through activating Akt. However, phosphatase PHLPP-1 (PH domain leucine-rich repeat protein phosphatase-1) dephosphorylates and inactivates Akt. The balanced competitive interaction of insulin and PHLPP-1 has not been directly examined. In this study, we have identified the effect of mutual inhibition of insulin signaling and PHLPP-1 on the cardioprotective efficiency of Akt in aged heart. Young (3 mon) and aged (20 mon) Sprague Dawley (SD) rats were subjected to MI/R in vivo. The PHLPP-1 level was higher in aged vs. young hearts at base. But, insulin treatment failed to decrease PHLPP-1 level during reperfusion in the aged hearts. Consequently, the cardioprotection of insulin-induced Akt activation was impaired in aged hearts, resulting in more susceptible to MI/R injury. In cultured rat ventricular myocytes, PHLPP-1 knockdown significantly enhanced insulin-induced Akt phosphorylation and reduced simulated hypoxia/reoxygenation-induced apoptosis. Contrary, PHLPP-1 overexpression terminated Akt phosphorylation and deteriorated myocytes apoptosis. Using in vivo aged animal models, we confirmed that cardiac PHLPP-1 knockdown or enhanced insulin sensitivity by exercise training dramatically increased insulin-induced Akt phosphorylation. Specifically, MI/R-induced cardiomyocyte apoptosis and infarct size were decreased and cardiac function was increased. More importantly, we found that insulin regulated the degradation of PHLPP-1 and insulin treatment could enhance the binding between PHLPP-1 and β-transducin repeat-containing protein (β-TrCP) to target for ubiquitin-dependent degradation. Altogether, we have identified a new mechanism by which insulin suppresses PHLPP-1 to enhance Akt activation. But, aged heart possesses lower insulin effectiveness and fails to decrease PHLPP-1 during MI/R, which subsequently limited Akt activity and cardioprotection. PHLPP-1 could be a promising

  3. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

    SciTech Connect

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.

  4. Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes

    PubMed Central

    Tian, Jin; Zhang, Xiaozhan; Wu, Hongxia; Liu, Chunguo; Li, Zhijie; Hu, Xiaoliang; Su, Shuo; Wang, Lin-Fa; Qu, Liandong

    2015-01-01

    Many host cellular signaling pathways were activated and exploited by virus infection for more efficient replication. The PI3K/Akt pathway has recently attracted considerable interest due to its role in regulating virus replication. This study demonstrated for the first time that the mammalian reovirus strains Masked Palm Civet/China/2004 (MPC/04) and Bat/China/2003 (B/03) can induce transient activation of the PI3K/Akt pathway early in infection in vitro. When UV-treated, both viruses activated PI3K/Akt signaling, indicating that the virus/receptor interaction was sufficient to activate PI3K/Akt. Reovirus virions can use both clathrin- and caveolae-mediated endocytosis, but only chlorpromazine, a specific inhibitor of clathrin-mediated endocytosis, or siRNA targeting clathrin suppressed Akt phosphorylation. We also identified the upstream molecules of the PI3K pathway. Virus infection induced phosphorylation of focal adhesion kinase (FAK) but not Gab1, and blockage of FAK phosphorylation suppressed Akt phosphorylation. Blockage of PI3K/Akt activation increased virus RNA synthesis and viral yield. We also found that reovirus infection activated the IFN-stimulated response element (ISRE) in an interferon-independent manner and up-regulated IFN-stimulated genes (ISGs) via the PI3K/Akt/EMSY pathway. Suppression of PI3K/Akt activation impaired the induction of ISRE and down-regulated the expression of ISGs. Overexpression of ISG15 and Viperin inhibited virus replication, and knockdown of either enhanced virus replication. Collectively, these results demonstrate that PI3K/Akt activated by mammalian reovirus serves as a pathway for sensing and then inhibiting virus replication/infection. PMID:26388843

  5. The effect of photoinitiators on intracellular AKT signaling pathway in tissue engineering application

    PubMed Central

    Xu, Leyuan; Sheybani, Natasha; Yeudall, W. Andrew; Yang, Hu

    2015-01-01

    Free-radical photopolymerization initiated by photoinitiators is an important method to make tissue engineering scaffolds. To advance understanding of photoinitiator cytocompatibility, we examined three photoinitiators including 2,2-dimethoxy-2-phenylacetophenone (DMPA), Irgacure 2959 (I-2959), and eosin Y photoinitiating system (EY) in terms of their effects on viability of HN4 cells and expression levels of intracellular AKT and its phosphorylated form p-AKT. Our results show that the photoinitiators and their UV-exposed counterparts affect intracellular AKT signaling, which can be used in conjunction with cell viability for cytocompatibility assessment of photoinitiators. PMID:25709809

  6. Reactivation of AKT signaling following treatment of cancer cells with PI3K inhibitors attenuates their antitumor effects

    SciTech Connect

    Dufour, Marc; Dormond-Meuwly, Anne; Pythoud, Catherine; Demartines, Nicolas; Dormond, Olivier

    2013-08-16

    Highlights: •PI3K inhibitors inhibit AKT only transiently. •Re-activation of AKT limits the anti-cancer effect of PI3K inhibitors. •The results suggest to combine PI3K and AKT inhibitors in cancer therapy. -- Abstract: Targeting the phosphatidylinositol-3-kinase (PI3K) is a promising approach in cancer therapy. In particular, PI3K blockade leads to the inhibition of AKT, a major downstream effector responsible for the oncogenic activity of PI3K. However, we report here that small molecule inhibitors of PI3K only transiently block AKT signaling. Indeed, treatment of cancer cells with PI3K inhibitors results in a rapid inhibition of AKT phosphorylation and signaling which is followed by the reactivation of AKT signaling after 48 h as observed by Western blot. Reactivation of AKT signaling occurs despite effective inhibition of PI3K activity by PI3K inhibitors. In addition, wortmannin, a broad range PI3K inhibitor, did not block AKT reactivation suggesting that AKT signals independently of PI3K. In a therapeutical perspective, combining AKT and PI3K inhibitors exhibit stronger anti-proliferative and pro-apoptotic effects compared to AKT or PI3K inhibitors alone. Similarly, in a tumor xenograft mouse model, concomitant PI3K and AKT blockade results in stronger anti-cancer activity compared with either blockade alone. This study shows that PI3K inhibitors only transiently inhibit AKT which limits their antitumor activities. It also provides the proof of concept to combine PI3K inhibitors with AKT inhibitors in cancer therapy.

  7. Platelet-derived growth factor-BB-mediated glycosaminoglycan synthesis is transduced through Akt.

    PubMed

    Cartel, Nicholas J; Wang, Jinxia; Post, Martin

    2002-04-01

    Previously we have demonstrated that the phosphoinositide 3-kinase (PI-3K) signal-transduction pathway mediates platelet-derived growth factor (PDGF)-BB-induced glycosaminoglycan (GAG) synthesis in fetal lung fibroblasts. In the present study we further investigated the signal-transduction pathway(s) that results in PDGF-BB-induced GAG synthesis. Over-expression of a soluble PDGF beta-receptor as well as a mutated form of the beta-receptor, unable to bind PI-3K, diminished GAG synthesis in fetal lung fibroblasts subsequent to PDGF-BB stimulation. The PI-3K inhibitor wortmannin blocked PDGF-BB-induced Akt activity as well as significantly diminishing PDGF-BB-mediated GAG synthesis. Expression of dominant-negative PI-3K also abrogated Akt activity and GAG synthesis. Furthermore, expression of dominant-negative Akt abrogated endogenous Akt activity, Rab3D phosphorylation and GAG synthesis, whereas expression of constitutively activated Akt stimulated Rab3D phosphorylation and GAG synthesis in the absence of PDGF-BB. Over-expression of wild-type PTEN (phosphatase and tensin homologue deleted in chromosome 10) inhibited Akt activity and concomitantly attenuated GAG synthesis in fibroblasts stimulated with PDGF-BB. These data suggest that Akt is an integral protein involved in PDGF-BB-mediated GAG regulation in fetal lung fibroblasts. PMID:11903042

  8. Exendin-4 induces myocardial protection through MKK3 and Akt-1 in infarcted hearts.

    PubMed

    Du, Jianfeng; Zhang, Ling; Wang, Zhengke; Yano, Naohiro; Zhao, Yu Tina; Wei, Lei; Dubielecka-Szczerba, Patrycja; Liu, Paul Y; Zhuang, Shougang; Qin, Gangjian; Zhao, Ting C

    2016-02-15

    We have demonstrated that glucagon like peptide-1 (GLP-1) protects the heart against ischemic injury. However, the physiological mechanism by which GLP-1 receptor (GLP-1R) initiates cardioprotection remains to be determined. The objective of this study is to elucidate the functional roles of MAPK kinase 3 (MKK3) and Akt-1 in mediating exendin-4-elicited protection in the infarcted hearts. Adult mouse myocardial infarction (MI) was created by ligation of the left descending artery. Wild-type, MKK3(-/-), Akt-1(-/-), and Akt-1(-/-);MKK3(-/-) mice were divided into one of several groups: 1) sham: animals underwent thoracotomy without ligation; 2) MI: animals underwent MI and received a daily dose of intraperitoneal injection of vehicle (saline); 3) MI + exendin-4: infarcted mice received daily injections of exendin-4, a GLP-1R agonist (0.1 mg/kg, ip). Echocardiographic measurements indicate that exendin-4 treatment resulted in the preservation of ventricular function and increases in the survival rate, but these effects were diminished in MKK3(-/-), Akt-1(-/-), and Akt-1(-/-);MKK3(-/-) mice. Exendin-4 treatments suppressed cardiac hypotrophy and reduced scar size and cardiac interstitial fibrosis, respectively, but these beneficial effects were lost in genetic elimination of MKK3, Akt-1, or Akt-1(-/-);MKK3(-/-) mice. GLP-1R stimulation stimulated angiogenic responses, which were also mitigated by deletion of MKK3 and Akt-1. Exendin-4 treatment increased phosphorylation of MKK3, p38, and Akt-1 at Ser129 but decreased levels of active caspase-3 and cleaved poly (ADP-ribose) polymerase; these proteins were diminished in MKK3(-/-), Akt-1(-/-), and Akt-1(-/-);MKK3(-/-) mice. These results reveal that exendin-4 treatment improves cardiac function, attenuates cardiac remodeling, and promotes angiogenesis in the infarcted myocardium through MKK3 and Akt-1 pathway. PMID:26739490

  9. Drosophila tribbles antagonizes insulin signaling-mediated growth and metabolism via interactions with Akt kinase.

    PubMed

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  10. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    SciTech Connect

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa; Shin, Incheol

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  11. Prediabetes Linked to Excess Glucagon in Transgenic Mice with Pancreatic Active AKT1

    PubMed Central

    Albury-Warren, Toya M.; Pandey, Veethika; Spinel, Lina P.; Masternak, Michal M.; Altomare, Deborah A

    2015-01-01

    Protein Kinase B/AKT, has three isoforms (AKT1-3) and is renowned for its central role in the regulation of cell growth and proliferation, due to its constitutive activation in various cancers. AKT2, which is highly expressed in insulin responsive tissues, has been identified as a primary regulator of glucose metabolism as Akt2 knockout mice (Akt2−/−) are glucose intolerant and insulin resistant. However, the role of AKT1 in glucose metabolism is not as clearly defined. We previously showed that mice with myristoylated Akt1 (AKT1Myr) expressed through a bicistronic Pdx1-TetA and TetO-MyrAkt1 system were susceptible to islet cell carcinomas, and in this study we characterized an early onset, prediabetic phenotype. Beginning at weaning (3 weeks of age), the glucose intolerant AKT1Myr mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. In contrast, treatment with doxycycline diet to turn-off the transgene, caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. Collectively, this model highlights a novel glucagon-mediated mechanism by which AKT1 hyperactivation affects glucose homeostasis, and provides an avenue to better delineate the molecular mechanisms responsible for diabetes mellitus and the potential association with pancreatic cancer. PMID:26487674

  12. Prediabetes linked to excess glucagon in transgenic mice with pancreatic active AKT1.

    PubMed

    Albury-Warren, Toya M; Pandey, Veethika; Spinel, Lina P; Masternak, Michal M; Altomare, Deborah A

    2016-01-01

    Protein kinase B/AKT has three isoforms (AKT1-3) and is renowned for its central role in the regulation of cell growth and proliferation, due to its constitutive activation in various cancers. AKT2, which is highly expressed in insulin-responsive tissues, has been identified as a primary regulator of glucose metabolism as Akt2 knockout mice (Akt2(-/-)) are glucose-intolerant and insulin-resistant. However, the role of AKT1 in glucose metabolism is not as clearly defined. We previously showed that mice with myristoylated Akt1 (AKT1(Myr)) expressed through a bicistronic Pdx1-TetA and TetO-MyrAkt1 system were susceptible to islet cell carcinomas, and in this study we characterized an early onset, prediabetic phenotype. Beginning at weaning (3 weeks of age), the glucose-intolerant AKT1(Myr) mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. In contrast, treatment with doxycycline diet to turn off the transgene caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. Collectively, this model highlights a novel glucagon-mediated mechanism by which AKT1 hyperactivation affects glucose homeostasis and provides an avenue to better delineate the molecular mechanisms responsible for diabetes mellitus and the potential association with pancreatic cancer. PMID:26487674

  13. Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

    PubMed Central

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L.

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  14. DNA-PK mediates AKT activation and apoptosis inhibition in clinically acquired platinum resistance.

    PubMed

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-11-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  15. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance12

    PubMed Central

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-01-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  16. Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro

    SciTech Connect

    Wang, Tao Fang; Wang, Heng; Peng, Ai Fen; Luo, Qing Feng; Liu, Zhi Li; Zhou, Rong Ping; Gao, Song; Zhou, Yang; Chen, Wen Zhao

    2013-10-18

    Highlights: •We investigate the relationship between FASN and HER2 or p-HER2 by IHC in OS tissues. •We construct FASN-specific RNAi plasmid. •Inhibiting FASN down-regulates HER2/PI3K/AKT cell signaling in U-2 OS. •Inhibiting FASN blocks U-2 OS cell invasion and migration. -- Abstract: FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the “HER2/PI3K/AKT” axis in vitro. FASN blocker may be a new therapeutic strategy in OS management.

  17. The AKT/mTOR signaling pathway plays a key role in statin-induced myotoxicity.

    PubMed

    Bonifacio, Annalisa; Sanvee, Gerda M; Bouitbir, Jamal; Krähenbühl, Stephan

    2015-08-01

    Statins are drugs that lower blood cholesterol levels and reduce cardiovascular morbidity and mortality. They are generally well-tolerated, but myopathy is a potentially severe adverse reaction of these compounds. The mechanisms by which statins induce myotoxicity are not completely understood, but may be related to inhibition of the AKT signaling pathway. The current studies were performed to explore the down-stream effects of the statin-associated inhibition of AKT within the AKT signaling pathway and on myocyte biology and morphology in C2C12 myotubes and in mice in vivo. We exposed C2C12 myotubes to 10 μM or 50 μM simvastatin, atorvastatin or rosuvastatin for 24 h. Simvastatin and atorvastatin inhibited AKT phosphorylation and were cytotoxic starting at 10 μM, whereas similar effects were observed for rosuvastatin at 50 μM. Inhibition of AKT phosphorylation was associated with impaired phosphorylation of S6 kinase, ribosomal protein S6, 4E-binding protein 1 and FoxO3a, resulting in reduced protein synthesis, accelerated myofibrillar degradation and atrophy of C2C12 myotubes. Furthermore, impaired AKT phosphorylation was associated with activation of caspases and PARP, reflecting induction of apoptosis. Similar findings were detected in skeletal muscle of mice treated orally with 5 mg/kg/day simvastatin for 3 weeks. In conclusion, this study highlights the importance of the AKT/mTOR signaling pathway in statin-induced myotoxicity and reveals potential drug targets for treatment of patients with statin-associated myopathies. PMID:25913013

  18. The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

    SciTech Connect

    Xiao Wei; Yang Yi; Weng Qingbei; Lin Tiehao; Yuan Meijin; Yang Kai; Pang Yi

    2009-08-15

    Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-specific inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

  19. The role of mouse Akt2 in insulin-dependent suppression of adipocyte lipolysis in vivo

    PubMed Central

    Koren, Shlomit; DiPilato, Lisa M.; Emmett, Matthew J.; Shearin, Abigail L.; Chu, Qingwei; Monks, Bob; Birnbaum, Morris J.

    2015-01-01

    Aim/hypothesis The release of fatty acids from adipocytes, i.e. lipolysis, is maintained under tight control, primarily by the opposing actions of catecholamines and insulin. A widely accepted model is that insulin antagonises catecholamine-dependent lipolysis through phosphorylation and activation of cAMP phosphodiesterase 3B (PDE3B) by the serine-threonine protein kinase Akt (protein kinase B). Recently, this hypothesis has been challenged, as in cultured adipocytes insulin appears, under some conditions, to suppress lipolysis independently of Akt. Methods To address the requirement for Akt2, the predominant isoform expressed in classic insulin target tissues, in the suppression of fatty acid release in vivo, we assessed lipolysis in mice lacking Akt2. Results In the fed state and following an oral glucose challenge, Akt2 null mice were glucose intolerant and hyperinsulinaemic, but nonetheless exhibited normal serum NEFA and glycerol levels, suggestive of normal suppression of lipolysis. Furthermore, insulin partially inhibited lipolysis in Akt2 null mice during an insulin tolerance test (ITT) and hyperinsulinaemic–euglycaemic clamp, respectively. In support of these in vivo observations, insulin antagonised catecholamine-induced lipolysis in primary brown fat adipocytes from Akt2-deficient nice. Conclusion These data suggest that suppression of lipolysis by insulin in hyperinsulinaemic states can take place in the absence of Akt2. PMID:25740694

  20. SIRT1 at the crossroads of AKT1 and ERβ in malignant pleural mesothelioma cells.

    PubMed

    Pinton, Giulia; Zonca, Sara; Manente, Arcangela G; Cavaletto, Maria; Borroni, Ester; Daga, Antonio; Jithesh, Puthen V; Fennell, Dean; Nilsson, Stefan; Moro, Laura

    2016-03-22

    In this report, we show that malignant pleural mesothelioma (MPM) patients whose tumors express high levels of AKT1 exhibit a significantly worse prognosis, whereas no significant correlation with AKT3 expression is observed. We provide data that establish a phosphorylation independent role of AKT1 in affecting MPM cell shape and anchorage independent cell growth in vitro and highlight the AKT1 isoform-specific nature of these effects.We describe that AKT1 activity is inhibited by the loss of SIRT1-mediated deacetylation and identify, by mass spectrometry, 11 unique proteins that interact with acetylated AKT1.Our data demonstrate a role of the AKT1/SIRT1/FOXM1 axis in the expression of the tumor suppressor ERβ. We further demonstrate an inhibitory feedback loop by ERβ, activated by the selective agonist KB9520, on this axis both in vitro and in vivo.Our data broaden the current knowledge of ERβ and AKT isoform-specific functions that could be valuable in the design of novel and effective therapeutic strategies for MPM. PMID:26885609

  1. SIRT1 at the crossroads of AKT1 and ERβ in malignant pleural mesothelioma cells

    PubMed Central

    Pinton, Giulia; Zonca, Sara; Manente, Arcangela G.; Cavaletto, Maria; Borroni, Ester; Daga, Antonio; Jithesh, Puthen V.; Fennell, Dean; Nilsson, Stefan; Moro, Laura

    2016-01-01

    In this report, we show that malignant pleural mesothelioma (MPM) patients whose tumors express high levels of AKT1 exhibit a significantly worse prognosis, whereas no significant correlation with AKT3 expression is observed. We provide data that establish a phosphorylation independent role of AKT1 in affecting MPM cell shape and anchorage independent cell growth in vitro and highlight the AKT1 isoform-specific nature of these effects. We describe that AKT1 activity is inhibited by the loss of SIRT1-mediated deacetylation and identify, by mass spectrometry, 11 unique proteins that interact with acetylated AKT1. Our data demonstrate a role of the AKT1/SIRT1/FOXM1 axis in the expression of the tumor suppressor ERβ. We further demonstrate an inhibitory feedback loop by ERβ, activated by the selective agonist KB9520, on this axis both in vitro and in vivo. Our data broaden the current knowledge of ERβ and AKT isoform-specific functions that could be valuable in the design of novel and effective therapeutic strategies for MPM. PMID:26885609

  2. Sphingosine-1-Phosphate Protects Intestinal Epithelial Cells from Apoptosis Through the Akt Signaling Pathway

    PubMed Central

    Greenspon, Jose; Li, Ruiyun; Xiao, Lan; Rao, Jaladanki N.; Marasa, Bernard S.; Strauch, Eric D.; Wang, Jian-Ying; Turner, Douglas J.

    2009-01-01

    Objective The regulation of apoptosis of intestinal mucosal cells is important in maintenance of normal intestinal physiology. Summary Sphingosine-1-phosphate (S1P) has been shown to play a critical role in cellular protection to otherwise lethal stimuli in several nonintestinal tissues. Methods The current study determines whether S1P protected normal intestinal epithelial cells (IECs) from apoptosis and whether Akt activation was the central pathway for this effect. Results S1P demonstrated significantly reduced levels of apoptosis induced by tumor necrosis factor-alpha (TNF-α)/cycloheximide (CHX). S1P induced increased levels of phosphorylated Akt and increased Akt activity, but did not affect total amounts of Akt. This activation of Akt was associated with decreased levels of both caspase-3 protein levels and of caspase-3 activity. Inactivation of Akt by treatment with the PI3K chemical inhibitor LY294002 or by overexpression of the dominant negative mutant of Akt (DNMAkt) prevented the protective effect of S1P on apoptosis. Additionally, silencing of the S1P-1 receptor by specific siRNA demonstrated a lesser decrease in apoptosis to S1P exposure. Conclusion These results indicate that S1P protects intestinal epithelial cells from apoptosis via an Akt-dependent pathway. PMID:18654850

  3. Phospholipase D2 Mediates Survival Signaling through Direct Regulation of Akt in Glioblastoma Cells*♦

    PubMed Central

    Bruntz, Ronald C.; Taylor, Harry E.; Lindsley, Craig W.; Brown, H. Alex

    2014-01-01

    The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipase D (PLD) is a novel regulator of Akt in GBM. Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase. PMID:24257753

  4. Human Dynactin-Associated Protein Transforms NIH3T3 Cells to Generate Highly Vascularized Tumors with Weak Cell-Cell Interaction.

    PubMed

    Kunoh, Tatsuki; Wang, Weixiang; Kobayashi, Hiroaki; Matsuzaki, Daisuke; Togo, Yuki; Tokuyama, Masahiro; Hosoi, Miho; Koseki, Koichi; Wada, Shu-Ichi; Nagai, Nobuo; Nakamura, Toshinobu; Nomura, Shintaro; Hasegawa, Makoto; Sasaki, Ryuzo; Mizukami, Tamio

    2015-01-01

    Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes AktSer473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchorage-deficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell-cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy. PMID:26284361

  5. Human Dynactin-Associated Protein Transforms NIH3T3 Cells to Generate Highly Vascularized Tumors with Weak Cell-Cell Interaction

    PubMed Central

    Kunoh, Tatsuki; Wang, Weixiang; Kobayashi, Hiroaki; Matsuzaki, Daisuke; Togo, Yuki; Tokuyama, Masahiro; Hosoi, Miho; Koseki, Koichi; Wada, Shu-ichi; Nagai, Nobuo; Nakamura, Toshinobu; Nomura, Shintaro; Hasegawa, Makoto; Sasaki, Ryuzo; Mizukami, Tamio

    2015-01-01

    Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes AktSer473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchorage-deficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell—cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy. PMID:26284361

  6. PI3K/Akt signalling pathway and cancer.

    PubMed

    Fresno Vara, Juan Angel; Casado, Enrique; de Castro, Javier; Cejas, Paloma; Belda-Iniesta, Cristóbal; González-Barón, Manuel

    2004-04-01

    Phosphatidylinositol-3 kinases, PI3Ks, constitute a lipid kinase family characterized by their ability to phosphorylate inositol ring 3'-OH group in inositol phospholipids to generate the second messenger phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P(3)). RPTK activation results in PI(3,4,5)P(3) and PI(3,4)P(2) production by PI3K at the inner side of the plasma membrane. Akt interacts with these phospholipids, causing its translocation to the inner membrane, where it is phosphorylated and activated by PDK1 and PDK2. Activated Akt modulates the function of numerous substrates involved in the regulation of cell survival, cell cycle progression and cellular growth. In recent years, it has been shown that PI3K/Akt signalling pathway components are frequently altered in human cancers. Cancer treatment by chemotherapy and gamma-irradiation kills target cells primarily by the induction of apoptosis. However, the development of resistance to therapy is an important clinical problem. Failure to activate the apoptotic programme represents an important mode of drug resistance in tumor cells. Survival signals induced by several receptors are mediated mainly by PI3K/Akt, hence this pathway may decisively contribute to the resistant phenotype. Many of the signalling pathways involved in cellular transformation have been elucidated and efforts are underway to develop treatment strategies that target these specific signalling molecules or their downstream effectors. The PI3K/Akt pathway is involved in many of the mechanisms targeted by these new drugs, thus a better understanding of this crossroad can help to fully exploit the potential benefits of these new agents. PMID:15023437

  7. MECHANISMS OF SPHINGOSINE-1-PHOSPHATE INDUCED AKT DEPENDENT SMOOTH MUSCLE CELL MIGRATION

    PubMed Central

    Roztocil, Elisa; Nicholl, Suzanne M.; Davies, Mark G.

    2008-01-01

    Background Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid released from activated platelets, which stimulates migration of vascular smooth muscle cells (VSMC) in vitro. S-1-P will activate akt, which can regulate multiple cellular functions including cell migration. Akt activation is downstream of phosphatidyl-inositol 3′ kinase (PI3-K) and Phosphoinositide-dependent protein kinase-1 (PDK1). Objective To examine the regulation of akt signaling during smooth muscle cell migration in response to S-1-P. Methods Murine arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P with and without an akt inhibitor (aktI). Assays were performed for PI3-K, PDK1, akt and GSK3β activation in the presence of various inhibitors and after transfection with the Gβγ inhibitor. βARKCT. Results S-1-P induced time dependent PI3-K, PDK1 and akt activation. The migratory responses in both assays to S-1-P were blocked by akt inhibitor (aktI). Activation of akt and dephosphorylation of its downstream kinase, GSK3 β, were inhibited by aktI. Inhibition of PI3-K with LY294002 significantly reduced both PI3-K and akt activation. Inhibition of G βγ inhibited akt activation through a reduction in both PI3-K and PDK1 activation. While inhibition of the ras with manumycin A had no effect, inhibition of rho with C3 limited both PI3K and akt activation. PDK1 responses were unchanged by inhibition of GTPases. Inhibition of reactive oxygen species generation with N-acetylcysteine and of EGFR with AG1478 inhibited PDK1 activation in response to S-1-P. Conclusion S-1-P mediated migration is akt dependent. S-1-P mediated akt phosphorylation is controlled by G βγ dependent, PI3-K activation, which requires the GTPase rho and Gβγ. PDK1 activation requires Gβγ reactive oxygen species generation and EGFR activation. PMID:19081473

  8. Testosterone regulation of Akt/mTORC1/FoxO3a Signaling in Skeletal Muscle

    PubMed Central

    White, James P.; Gao, Song; Puppa, Melissa J.; Sato, Shuichi; Welle, Stephen L.; Carson, James A.

    2012-01-01

    Low endogenous testosterone production, known as hypogonadism is commonly associated with conditions inducing muscle wasting. Akt signaling can control skeletal muscle mass through mTOR regulation of protein synthesis and FoxO regulation of protein degradation, and this pathway has been previously identified as a target of androgen signaling. However, the testosterone sensitivity of Akt/mTOR signaling requires further understanding in order to grasp the significance of varied testosterone levels seen with wasting disease on muscle protein turnover regulation. Therefore, the purpose of this study is to determine the effect of androgen availability on muscle Akt/mTORC1/FoxO3a regulation in skeletal muscle and cultured C2C12 myotubes. C57BL/6 mice were either castrated for 42 days or castrated and treated with the nandrolone decanoate (ND) (6 mg/kg bw/wk). Testosterone loss (TL) significantly decreased volitional grip strength, body weight, and gastrocnemius (GAS) muscle mass, and ND reversed these changes. Related to muscle mass regulation, TL decreased muscle IGF-1 mRNA, the rate of myofibrillar protein synthesis, Akt phosphorylation, and the phosphorylation of Akt targets, GSK3β, PRAS40 and FoxO3a. TL induced expression of FoxO transcriptional targets, MuRF1, atrogin1 and REDD1. Muscle AMPK and raptor phosphorylation, mTOR inhibitors, were not altered by low testosterone. ND restored IGF-1 expression and Akt/mTORC1 signaling while repressing expression of FoxO transcriptional targets. Testosterone (T) sensitivity of Akt/mTORC1 signaling was examined in C2C12 myotubes, and mTOR phosphorylation was induced independent of Akt activation at low T concentrations, while a higher T concentration was required to activate Akt signaling. Interestingly, low concentration T was sufficient to amplify myotube mTOR and Akt signaling after 24h of T withdrawal, demonstrating the potential in cultured myotubes for a T initiated positive feedback mechanism to amplify Akt

  9. Testosterone regulation of Akt/mTORC1/FoxO3a signaling in skeletal muscle.

    PubMed

    White, James P; Gao, Song; Puppa, Melissa J; Sato, Shuichi; Welle, Stephen L; Carson, James A

    2013-01-30

    Low endogenous testosterone production, known as hypogonadism is commonly associated with conditions inducing muscle wasting. Akt signaling can control skeletal muscle mass through mTOR regulation of protein synthesis and FoxO regulation of protein degradation, and this pathway has been previously identified as a target of androgen signaling. However, the testosterone sensitivity of Akt/mTOR signaling requires further understanding in order to grasp the significance of varied testosterone levels seen with wasting disease on muscle protein turnover regulation. Therefore, the purpose of this study is to determine the effect of androgen availability on muscle Akt/mTORC1/FoxO3a regulation in skeletal muscle and cultured C(2)C(12) myotubes. C57BL/6 mice were either castrated for 42 days or castrated and treated with the nandrolone decanoate (ND) (6 mg/kg bw/wk). Testosterone loss (TL) significantly decreased volitional grip strength, body weight, and gastrocnemius (GAS) muscle mass, and ND reversed these changes. Related to muscle mass regulation, TL decreased muscle IGF-1 mRNA, the rate of myofibrillar protein synthesis, Akt phosphorylation, and the phosphorylation of Akt targets, GSK3β, PRAS40 and FoxO3a. TL induced expression of FoxO transcriptional targets, MuRF1, atrogin1 and REDD1. Muscle AMPK and raptor phosphorylation, mTOR inhibitors, were not altered by low testosterone. ND restored IGF-1 expression and Akt/mTORC1 signaling while repressing expression of FoxO transcriptional targets. Testosterone (T) sensitivity of Akt/mTORC1 signaling was examined in C(2)C(12) myotubes, and mTOR phosphorylation was induced independent of Akt activation at low T concentrations, while a higher T concentration was required to activate Akt signaling. Interestingly, low concentration T was sufficient to amplify myotube mTOR and Akt signaling after 24 h of T withdrawal, demonstrating the potential in cultured myotubes for a T initiated positive feedback mechanism to amplify Akt

  10. Optimal Classes of Chemotherapeutic Agents Sensitized by Specific Small-Molecule Inhibitors of Akt In Vitro and In Vivo

    PubMed Central

    Shi, Yan; Liu, Xuesong; Han, Edward K.; Guan, Ran; Shoemaker, Alexander R.; Oleksijew, Anatol; Woods, Keith W.; Fisher, John P.; Klinghofer, Vered; Lasko, Loren; McGonigal, Thomas; Li, Qun; Rosenberg, Saul H.; Giranda, Vincent L.; Luo, Yan

    2005-01-01

    Abstract Akt is a serine/threonine kinase that transduces survival signals from survival/growth factors. Deregulation and signal imbalance in cancer cells make them prone to apoptosis. Upregulation or activation of Akt to aid the survival of cancer cells is a common theme in human malignancies. We have developed small-molecule Akt inhibitors that are potent and specific. These Akt inhibitors can inhibit Akt activity and block phosphorylation by Akt on multiple downstream targets in cells. Synergy in apoptosis induction was observed when Akt inhibitors were combined with doxorubicin or camptothecin. Akt inhibitor–induced enhancement of topoisomerase inhibitor cytotoxicity was also evident in long-term cell survival assay. Synergy with paclitaxel in apoptosis induction was evident in cells pretreated with paclitaxel, and enhancement of tumor delay by paclitaxel was demonstrated through cotreatment with Akt inhibitor Compound A (A-443654). Combination with other classes of chemotherapeutic agents did not yield any enhancement of cytotoxicity. These findings provide important guidance in selecting appropriate classes of chemotherapeutic agents for combination with Akt inhibitors in cancer treatment. PMID:16331885

  11. Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7.

    PubMed

    Mohammad, Dara K; Nore, Beston F; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

    2016-09-01

    The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK's interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK's binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK's interaction with Importin 7. PMID:27381982

  12. Akt kinase-mediated checkpoint of cGAS DNA sensing pathway

    PubMed Central

    Seo, Gil Ju; Yang, Aerin; Tan, Brandon; Kim, Sungyoon; Liang, Qiming; Choi, Younho; Yuan, Weiming; Feng, Pinghui; Park, Hee-Sung; Jung, Jae U.

    2015-01-01

    SUMMARY Upon DNA stimulation, cyclic GMP-AMP synthetase (cGAS) synthesizes the second messenger cyclic GMP-AMP (cGAMP) that binds to the STING, triggering antiviral interferon-β (IFN-β) production. However, it has remained undetermined how hosts regulate cGAS enzymatic activity after the resolution of DNA immunogen. Here, we show that Akt kinase plays a negative role in cGAS-mediated anti-viral immune response. Akt phosphorylated the S291 or S305 residue of the enzymatic domain of mouse or human cGAS, respectively, and this phosphorylation robustly suppressed its enzymatic activity. Consequently, expression of activated Akt led to the reduction of cGAMP and IFN-β production and the increase of herpes simplex virus 1 replication, whereas treatment with Akt inhibitor augmented cGAS-mediated IFN-β production. Furthermore, expression of the phosphorylation-resistant cGAS S291A mutant enhanced IFN-β production upon DNA stimulation, HSV-1 infection, and vaccinia virus infection. Our study identifies an Akt kinase-mediated checkpoint to fine-tune hosts’ immune responses to DNA stimulation. PMID:26440888

  13. Effects of streptozotocin-induced type 1 maternal diabetes on PI3K/AKT signaling pathway in the hippocampus of rat neonates.

    PubMed

    Hami, Javad; Kerachian, Mohammad-Amin; Karimi, Razieh; Haghir, Hossein; Sadr-Nabavi, Ariane

    2016-06-01

    Diabetes in pregnancy impairs hippocampus development in offspring, leading to behavioral problems and learning deficits. Phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway plays a pivotal role in the regulation of neuronal proliferation, survival and death. The present study was designed to examine the effects of maternal diabetes on PKB/Akt expression and phosphorylation in the developing rat hippocampus. Wistar female rats were maintained diabetic from a week before pregnancy through parturition and male offspring was killed at first postnatal day (P1). The hippocampal expression and phosphorylation level of PKB/Akt, one of the key molecules in PI3K/AKT signaling pathway, was evaluated using real-time polymerase chain reaction (PCR) and western blot analysis. We found a significant bilateral downregulation of AKT1 gene expression in the hippocampus of pups born to diabetic mothers (p < 0.05). Interestingly, our results revealed a marked upregulation of Akt1 gene in insulin-treated group compared with other groups (p < 0.05). The western blot analysis also showed the reduction of phosphorylation level of all AKT isoforms in both diabetic and insulin-treated groups compared with control (p < 0.05). Moreover, the results showed a significant increase in phosphorylation level of AKT in insulin-treated group compared with the diabetic group. These results represent that diabetes during pregnancy strongly influences the regulation of PKB/AKT in the developing rat hippocampus. Furthermore, although the control of glycemia by insulin administration is not sufficient to prevent the alterations in PKB/Akt expression, it modulates the phosphorylation process, thus ultimately resulting in a situation comparable to that found in the normal condition. PMID:26479041

  14. Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells*

    PubMed Central

    Patel, Satyananda; Djerdjouri, Bahia; Raoul-Des-Essarts, Yannick; Dang, Pham My-Chan; El-Benna, Jamel; Périanin, Axel

    2010-01-01

    Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (Km 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67phox and p47phox, ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47phox, p67phox, and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase. PMID:20693286

  15. Evaluation of novel Akt1 inhibitors as anticancer agents using virtual co-crystallized pharmacophore generation.

    PubMed

    Al-Sha'er, Mahmoud A; Mansi, Iman; Almazari, Inas; Hakooz, Nancy

    2015-11-01

    The pharmacophoric features of the virtual co-crystallized protein of 17 Akt1 proteins were downloaded from the protein data bank, and explored to end up with 132 generated pharmacophores that had been evaluated using the decoy list composed of 1724 compounds. The areas under the curve of the Receiver-Operating Characteristic (ROC-AUC) were sorted, and the highest ranked pharmacophore 3MV5_2_01 was selected to be used as a searching tool in the National Cancer Institute (NCI) database. The captured hits were mapped based on successful hypotheses and the best fitted compounds were selected. The inhibition of Akt1 was measured and expressed as a percentage of inhibition. 24 out of the 40 compounds showed inhibition of Akt1, out of which 13 compounds showed more than 50% inhibition. Compound 1 showed 93.3% inhibition at 100 μM concentration. To confirm the inhibition of Akt1 phosphorylation, MCF10A cell line was co-treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and 100 μM of each of the most potent 13 Akt inhibitors (1-13). It was found that compounds 1 exert 91.6% inhibition of Akt1 phosphorylation in MCF10A cell line. PMID:26485540

  16. Acidic Fibroblast Growth Factor Promotes Endothelial Progenitor Cells Function via Akt/FOXO3a Pathway

    PubMed Central

    Wang, Yuqiang; Cao, Qing; Sang, Tiantian; Liu, Fang; Chen, Shuyan

    2015-01-01

    Acidic fibroblast growth factor (FGF1) has been suggested to enhance the functional activities of endothelial progenitor cells (EPCs). The Forkhead homeobox type O transcription factors (FOXOs), a key substrate of the survival kinase Akt, play important roles in regulation of various cellular processes. We previously have shown that FOXO3a is the main subtype of FOXOs expressed in EPCs. Here, we aim to determine whether FGF1 promotes EPC function through Akt/FOXO3a pathway. Human peripheral blood derived EPCs were transduced with adenoviral vectors either expressing a non-phosphorylable, constitutively active triple mutant of FOXO3a (Ad-TM-FOXO3a) or a GFP control (Ad-GFP). FGF1 treatment improved functional activities of Ad-GFP transduced EPCs, including cell viability, proliferation, antiapoptosis, migration and tube formation, whereas these beneficial effects disappeared by Akt inhibitor pretreatment. Moreover, EPC function was declined by Ad-TM-FOXO3a transduction and failed to be attenuated even with FGF1 treatment. FGF1 upregulated phosphorylation levels of Akt and FOXO3a in Ad-GFP transduced EPCs, which were repressed by Akt inhibitor pretreatment. However, FGF1 failed to recover Ad-TM-FOXO3a transduced EPCs from dysfunction. These data indicate that FGF1 promoting EPC function is at least in part mediated through Akt/FOXO3a pathway. Our study may provide novel ideas for enhancing EPC angiogenic ability and optimizing EPC transplantation therapy in the future. PMID:26061278

  17. A positive feedback loop involving Erk5 and Akt turns on mesangial cell proliferation in response to PDGF.

    PubMed

    Bera, Amit; Das, Falguni; Ghosh-Choudhury, Nandini; Li, Xiaonan; Pal, Sanjay; Gorin, Yves; Kasinath, Balakuntalam S; Abboud, Hanna E; Ghosh Choudhury, Goutam

    2014-06-01

    Platelet-derived growth factor BB and its receptor (PDGFRβ) play a pivotal role in the development of renal glomerular mesangial cells. Their roles in increased mesangial cell proliferation during mesangioproliferative glomerulonephritis have long been noted, but the operating logic of signaling mechanisms regulating these changes remains poorly understood. We examined the role of a recently identified MAPK, Erk5, in this process. PDGF increased the activating phosphorylation of Erk5 and tyrosine phosphorylation of proteins in a time-dependent manner. A pharmacologic inhibitor of Erk5, XMD8-92, abrogated PDGF-induced DNA synthesis and mesangial cell proliferation. Similarly, expression of dominant negative Erk5 or siRNAs against Erk5 blocked PDGF-stimulated DNA synthesis and proliferation. Inhibition of Erk5 attenuated expression of cyclin D1 mRNA and protein, resulting in suppression of CDK4-mediated phosphorylation of the tumor suppressor protein pRb. Expression of cyclin D1 or CDK4 prevented the dominant negative Erk5- or siErk5-mediated inhibition of DNA synthesis and mesangial cell proliferation induced by PDGF. We have previously shown that phosphatidylinositol 3-kinase (PI3-kinase) contributes to PDGF-induced proliferation of mesangial cells. Inhibition of PI3-kinase blocked PDGF-induced phosphorylation of Erk5. Since PI3-kinase acts through Akt, we determined the role of Erk5 on Akt phosphorylation. XMD8-92, dominant negative Erk5, and siErk5 inhibited phosphorylation of Akt by PDGF. Interestingly, we found inhibition of PDGF-induced Erk5 phosphorylation by a pharmacological inhibitor of Akt kinase and kinase dead Akt in mesangial cells. Thus our data unfold the presence of a positive feedback microcircuit between Erk5 and Akt downstream of PI3-kinase nodal point for PDGF-induced mesangial cell proliferation. PMID:24740537

  18. Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

    SciTech Connect

    Li, Lingzhi; Qiu, Ping; Chen, Bailing; Lu, Yongju; Wu, Kai; Thakur, Chitra; Chang, Qingshan; Sun, Jiaying; Chen, Fei

    2014-05-01

    Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.

  19. Role of PECAM-1 in the shear-stress-induced activation of Akt and the endothelial nitric oxide synthase (eNOS) in endothelial cells.

    PubMed

    Fleming, Ingrid; Fisslthaler, Beate; Dixit, Madhulika; Busse, Rudi

    2005-09-15

    The application of fluid shear stress to endothelial cells elicits the formation of nitric oxide (NO) and phosphorylation of the endothelial NO synthase (eNOS). Shear stress also elicits the enhanced tyrosine phosphorylation of endothelial proteins, especially of those situated in the vicinity of cell-cell contacts. Since a major constituent of these endothelial cell-cell contacts is the platelet endothelial cell adhesion molecule-1 (PECAM-1) we assessed the role of PECAM-1 in the activation of eNOS. In human endothelial cells, shear stress induced the tyrosine phosphorylation of PECAM-1 and enhanced the association of PECAM-1 with eNOS. Endothelial cell stimulation with shear stress elicited the phosphorylation of Akt and eNOS as well as of the AMP-activated protein kinase (AMPK). While the shear-stress-induced tyrosine phosphorylation of PECAM-1 as well as the serine phosphorylation of Akt and eNOS were abolished by the pre-treatment of cells with the tyrosine kinase inhibitor PP1 the phosphorylation of AMPK was unaffected. Down-regulation of PECAM-1 using a siRNA approach attenuated the shear-stress-induced phosphorylation of Akt and eNOS, as well as the shear-stress-induced accumulation of cyclic GMP levels while the shear-stress-induced phosphorylation of AMPK remained intact. A comparable attenuation of Akt and eNOS (but not AMPK) phosphorylation and NO production was also observed in endothelial cells generated from PECAM-1-deficient mice. These data indicate that the shear-stress-induced activation of Akt and eNOS in endothelial cells is modulated by the tyrosine phosphorylation of PECAM-1 whereas the shear-stress-induced phosphorylation of AMPK is controlled by an alternative signaling pathway. PMID:16118242

  20. RUNX2 and the PI3K/AKT axis reciprocal activation as a driving force for tumor progression.

    PubMed

    Cohen-Solal, Karine A; Boregowda, Rajeev K; Lasfar, Ahmed

    2015-01-01

    From the first reported role of the transcription factor RUNX2 in osteoblast and chondrocyte differentiation and migration to its involvement in promigratory/proinvasive behavior of breast, prostate, and thyroid cancer cells, osteosarcoma, or melanoma cells, RUNX2 currently emerges as a key player in metastasis. In this review, we address the interaction of RUNX2 with the PI3K/AKT signaling pathway, one of the critical axes controlling cancer growth and metastasis. AKT, either by directly phosphorylating/activating RUNX2 or phosphorylating/inactivating regulators of RUNX2 stability or activity, contributes to RUNX2 transcriptional activity. Reciprocally, the activation of the PI3K/AKT pathway by RUNX2 regulation of its different components has been described in non-transformed and transformed cells. This mutual activation in the context of cancer cells exhibiting constitutive AKT activation and high levels of RUNX2 might constitute a major driving force in tumor progression and aggressiveness. PMID:26204939

  1. B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing

    PubMed Central

    Mohammad, Dara K.; Ali, Raja H.; Turunen, Janne J.; Nore, Beston F.; Smith, C. I. Edvard

    2016-01-01

    Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins. PMID:27487157

  2. B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing.

    PubMed

    Mohammad, Dara K; Ali, Raja H; Turunen, Janne J; Nore, Beston F; Smith, C I Edvard

    2016-01-01

    Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins. PMID:27487157

  3. Greatwall promotes cell transformation by hyperactivating AKT in human malignancies

    PubMed Central

    Vera, Jorge; Lartigue, Lydia; Vigneron, Suzanne; Gadea, Gilles; Gire, Veronique; Del Rio, Maguy; Soubeyran, Isabelle; Chibon, Frederic; Lorca, Thierry; Castro, Anna

    2015-01-01

    The PP2A phosphatase is often inactivated in cancer and is considered as a tumour suppressor. A new pathway controlling PP2A activity in mitosis has been recently described. This pathway includes the Greatwall (GWL) kinase and its substrates endosulfines. At mitotic entry, GWL is activated and phosphorylates endosulfines that then bind and inhibit PP2A. We analysed whether GWL overexpression could participate in cancer development. We show that GWL overexpression promotes cell transformation and increases invasive capacities of cells through hyperphosphorylation of the oncogenic kinase AKT. Interestingly, AKT hyperphosphorylation induced by GWL is independent of endosulfines. Rather, GWL induces GSK3 kinase dephosphorylation in its inhibitory sites and subsequent SCF-dependent degradation of the PHLPP phosphatase responsible for AKT dephosphorylation. In line with its oncogenic activity, we find that GWL is often overexpressed in human colorectal tumoral tissues. Thus, GWL is a human oncoprotein that promotes the hyperactivation of AKT via the degradation of its phosphatase, PHLPP, in human malignancies. DOI: http://dx.doi.org/10.7554/eLife.10115.001 PMID:26613407

  4. All Akt Isoforms (Akt1, Akt2, Akt3) Are Involved in Normal Hearing, but Only Akt2 and Akt3 Are Involved in Auditory Hair Cell Survival in the Mammalian Inner Ear

    PubMed Central

    Brand, Yves; Levano, Soledad; Radojevic, Vesna; Naldi, Arianne Monge; Setz, Cristian; Ryan, Allen F.; Pak, Kwang; Hemmings, Brian A.; Bodmer, Daniel

    2015-01-01

    The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear—especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear. PMID:25811375

  5. MYOCARDIAL AKT: THE OMNIPRESENT NEXUS

    PubMed Central

    Sussman, Mark A.; Völkers, Mirko; Fischer, Kimberlee; Bailey, Brandi; Cottage, Christopher T.; Din, Shabana; Gude, Natalie; Avitabile, Daniele; Alvarez, Roberto; Sundararaman, Balaji; Quijada, Pearl; Mason, Matt; Konstandin, Mathias H.; Malhowski, Amy; Cheng, Zhaokang; Khan, Mohsin; McGregor, Michael

    2013-01-01

    One of the greatest examples of integrated signal transduction is revealed by examination of effects mediated by AKT kinase in myocardial biology. Positioned at the intersection of multiple afferent and efferent signals, AKT exemplifies a molecular sensing node that coordinates dynamic responses of the cell in literally every aspect of biological responses. The balanced and nuanced nature of homeostatic signaling is particularly essential within the myocardial context, where regulation of survival, energy production, contractility, and response to pathological stress all flow through the nexus of AKT activation or repression. Equally important, the loss of regulated AKT activity is primarily the cause or consequence of pathological conditions leading to remodeling of the heart and eventual decompensation. This review presents an overview compendium of the complex world of myocardial AKT biology gleaned from more than a decade of research. Summarization of the widespread influence that AKT exerts upon myocardial responses leaves no doubt that the participation of AKT in molecular signaling will need to be reckoned with as a seemingly omnipresent regulator of myocardial molecular biological responses. PMID:21742795

  6. Polydatin improves glucose and lipid metabolism in experimental diabetes through activating the Akt signaling pathway.

    PubMed

    Hao, Jie; Chen, Cheng; Huang, Kaipeng; Huang, Junying; Li, Jie; Liu, Peiqing; Huang, Heqing

    2014-12-15

    Recently, the effect of polydatin on lipid regulation has gained considerable attention. And previous study has demonstrated that polydatin has hypoglycemic effect on experimental diabetic rats. Repressed Akt pathway contributes to glucose and lipid disorders in diabetes. Thus, whether polydatin regulates glucose and lipid metabolism in experimental diabetic models through the Akt pathway arouses interest. The purpose was to explore the regulatory mechanism of polydain on glucose and lipid through Akt pathway. We used a diabetic rat model induced by high-fat and -sugar diet with low-dose of streptozocin and an insulin resistant HepG2 cell model induced by palmitic acid to clarify the role of polydatin on glucose and lipid metabolism. Here, we found that polydatin significantly attenuated fasting blood–glucose, glycosylated hemoglobin, glycosylated serum protein, total cholesterol, triglyceride, and low-density lipoprotein cholesterol in diabetic rats. Furthermore, polydatin significantly increased glucose uptake and consumption and decreased lipid accumulation in insulin resistant HepG2 cells. Polydatin markedly increased serum insulin levels in diabetic rats, and obviously activated the Akt signaling pathway in diabetic rat livers and insulin resistant HepG2 cells. Polydatin markedly increased phosphorylated GSK-3β, decreased the protein levels of G6Pase and SREBP-1c, and increased protein levels of GCK, LDLR, and phosphorylated IRS in livers and HepG2 cells. Overall, the results indicate that polydatin regulates glucose and lipid metabolism in experimental diabetic models, the underlying mechanism is probably associated with regulating the Akt pathway. The effect of polydatin on increased Akt phosphorylation is independent of prompting insulin secretion, but dependent of increasing IRS phosphorylation. PMID:25310908

  7. Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals.

    PubMed

    Nicholson-Fish, Jessica C; Cousin, Michael A; Smillie, Karen J

    2016-03-01

    The efficient retrieval of synaptic vesicle membrane and cargo in central nerve terminals is dependent on the efficient recruitment of a series of endocytosis modes by different patterns of neuronal activity. During intense neuronal activity the dominant endocytosis mode is activity-dependent endocytosis (ADBE). Triggering of ADBE is linked to calcineurin-mediated dynamin I dephosphorylation since the same stimulation intensities trigger both. Dynamin I dephosphorylation is maximised by a simultaneous inhibition of its kinase glycogen synthase kinase 3 (GSK3) by the protein kinase Akt, however it is unknown how increased neuronal activity is transduced into Akt activation. To address this question we determined how the activity-dependent increases in intracellular free calcium ([Ca(2+)]i) control activation of Akt. This was achieved using either trains of high frequency action potentials to evoke localised [Ca(2+)]i increases at active zones, or a calcium ionophore to raise [Ca(2+)]i uniformly across the nerve terminal. Through the use of either non-specific calcium channel antagonists or intracellular calcium chelators we found that Akt phosphorylation (and subsequent GSK3 phosphorylation) was dependent on localised [Ca(2+)]i increases at the active zone. In an attempt to determine mechanism, we antagonised either phosphatidylinositol 3-kinase (PI3K) or calmodulin. Activity-dependent phosphorylation of both Akt and GSK3 was arrested on inhibition of PI3K, but not calmodulin. Thus localised calcium influx in central nerve terminals activates PI3K via an unknown calcium sensor to trigger the activity-dependent phosphorylation of Akt and GSK3. PMID:26198194

  8. Combined inhibition of the EGFR/AKT pathways by a novel conjugate of quinazoline with isothiocyanate.

    PubMed

    Tarozzi, Andrea; Marchetti, Chiara; Nicolini, Benedetta; D'Amico, Massimo; Ticchi, Nicole; Pruccoli, Letizia; Tumiatti, Vincenzo; Simoni, Elena; Lodola, Alessio; Mor, Marco; Milelli, Andrea; Minarini, Anna

    2016-07-19

    Epidermal growth factor receptor inhibitors (EGFR-TKIs) represent a class of compounds widely used in anticancer therapy. An increasing number of studies reports on combination therapies in which the block of the EGFR-TK activity is associated with inhibition of its downstream pathways, as PI3K-Akt. Sulforaphane targets the PI3K-Akt pathway whose dysregulation is implicated in many functions of cancer cells. According to these considerations, a series of multitarget molecules have been designed by combining key structural features derived from an EGFR-TKI, PD168393, and the isothiocyanate sulforaphane. Among the obtained molecules 1-6, compound 6 emerges as a promising lead compound able to exert antiproliferative and proapoptotic effects in A431 epithelial cancer cell line by covalently binding to EGFR-TK, and reducing the phosphorylation of Akt without affecting the total Akt levels. PMID:27135370

  9. Direct reversal of glucocorticoid resistance by AKT inhibition in acute lymphoblastic leukemia.

    PubMed

    Piovan, Erich; Yu, Jiyang; Tosello, Valeria; Herranz, Daniel; Ambesi-Impiombato, Alberto; Da Silva, Ana Carolina; Sanchez-Martin, Marta; Perez-Garcia, Arianne; Rigo, Isaura; Castillo, Mireia; Indraccolo, Stefano; Cross, Justin R; de Stanchina, Elisa; Paietta, Elisabeth; Racevskis, Janis; Rowe, Jacob M; Tallman, Martin S; Basso, Giuseppe; Meijerink, Jules P; Cordon-Cardo, Carlos; Califano, Andrea; Ferrando, Adolfo A

    2013-12-01

    Glucocorticoid resistance is a major driver of therapeutic failure in T cell acute lymphoblastic leukemia (T-ALL). Here, we identify the AKT1 kinase as a major negative regulator of the NR3C1 glucocorticoid receptor protein activity driving glucocorticoid resistance in T-ALL. Mechanistically, AKT1 impairs glucocorticoid-induced gene expression by direct phosphorylation of NR3C1 at position S134 and blocking glucocorticoid-induced NR3C1 translocation to the nucleus. Moreover, we demonstrate that loss of PTEN and consequent AKT1 activation can effectively block glucocorticoid-induced apoptosis and induce resistance to glucocorticoid therapy. Conversely, pharmacologic inhibition of AKT with MK2206 effectively restores glucocorticoid-induced NR3C1 translocation to the nucleus, increases the response of T-ALL cells to glucocorticoid therapy, and effectively reverses glucocorticoid resistance in vitro and in vivo. PMID:24291004

  10. Direct reversal of glucocorticoid resistance by AKT inhibition in acute lymphoblastic leukemia

    PubMed Central

    Tosello, Valeria; Herranz, Daniel; Ambesi-Impiombato, Alberto; Da Silva, Ana Carolina; Sanchez-Martin, Marta; Perez-Garcia, Arianne; Rigo, Isaura; Castillo, Mireia; Indraccolo, Stefano; Cross, Justin R; de Stanchina, Elisa; Paietta, Elisabeth; Racevskis, Janis; Rowe, Jacob M; Tallman, Martin S; Basso, Giuseppe; Meijerink, Jules P; Cordon-Cardo, Carlos; Califano, Andrea; Ferrando, Adolfo A.

    2013-01-01

    SUMMARY Glucocorticoid resistance is a major driver of therapeutic failure in T-cell acute lymphoblastic leukemia (T-ALL). Here we identify the AKT1 kinase as a major negative regulator of the NR3C1 glucocorticoid receptor protein activity driving glucocorticoid resistance in T-ALL. Mechanistically, AKT1 impairs glucocorticoid-induced gene expression by direct phosphorylation of NR3C1 at position S134 and blocking glucocorticoid-induced NR3C1 translocation to the nucleus. Moreover, we demonstrate that loss of PTEN and consequent AKT1 activation can effectively block glucocorticoid induced apoptosis and induce resistance to glucocorticoid therapy. Conversely, pharmacologic inhibition of AKT with MK2206 effectively restores glucocorticoid-induced NR3C1 translocation to the nucleus, increases the response of T-ALL cells to glucocorticoid therapy and effectively reverses glucocorticoid resistance in vitro and in vivo. PMID:24291004

  11. Interference with Akt signaling pathway contributes curcumin-induced adipocyte insulin resistance.

    PubMed

    Zhang, Deling; Zhang, Yemin; Ye, Mao; Ding, Youming; Tang, Zhao; Li, Mingxin; Zhou, Yu; Wang, Changhua

    2016-07-01

    Previous study has shown that curcumin directly or indirectly suppresses insulin signaling in 3T3-L1 adipocytes. However, the underlying mechanism remains unclear. Here we experimentally demonstrate that curcumin inhibited the ubiquitin-proteasome system (UPS) function, activated autophagy, and reduced protein levels of protein kinase B (Akt) in a dose- and time-dependent manner in 3T3-L1 adipocytes, accompanied with attenuation of insulin-stimulated Akt phosphorylation, plasma membrane translocation of glucose transporter type 4 (GLUT4), and glucose uptake. These in vitro inhibitory effects of curcumin on Akt protein expression and insulin action were reversed by pharmacological and genetic inhibition of autophagy but not by inhibition of the UPS and caspases. In addition, Akt reduction in adipose tissues of mice treated with curcumin could be recovered by administration of autophagy inhibitor bafilomycin A1 (BFA). This new finding provides a novel mechanism by which curcumin induces insulin resistance in adipocytes. PMID:27113027

  12. Akt-1 mediates survival of chondrocytes from endoplasmic reticulum-induced stress.

    PubMed

    Price, Jeremy; Zaidi, Asifa K; Bohensky, Jolene; Srinivas, Vickram; Shapiro, Irving M; Ali, Hydar

    2010-03-01

    The unfolded protein response (UPR) is an evolutionary conserved adaptive mechanism that permits cells to react and adjust to conditions of endoplasmic reticulum (ER) stress. In addition to UPR, phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal regulated kinase (ERK) signaling pathways protect a variety of cells from ER stress. The goal of the present study was to assess the susceptibility of chondrocytes to ER stress and to determine the signaling pathways involved in their survival. We found that low concentration of thapsigargin (10 nM) reduced the viability of a chondrocyte cell line (N1511 cells) and that these cells were approximately 100 fold more susceptible to thapsigargin-induced stress than fibroblasts. Interestingly, in thapsigargin and tunicamycin-stressed chondrocytes induction of the proapoptotic transcription factor CHOP preceded that of the anti-apoptotic BiP by 12 h. Although both of these agents caused sustained Akt and ERK phosphorylation; inhibition of Akt phosphorylation sensitized chondrocytes to ER stress, while blocking ERK signaling by U0126 had no effect. We found that Akt-1, but not Akt-2 or Akt-3, is predominantly expressed in N1511 chondrocytes. Furthermore, siRNA-mediated knockdown of Akt-1 sensitized chondrocytes to ER stress, which was associated with increased capsase-3 activity and decreased Bcl(XL) expression. These data suggest that under condition of ER stress, multiple signaling processes regulate chondrocyte's survival-death decisions. Thus, rapid upregulation of CHOP likely contributes to chondrocyte death, while Akt-1-mediated inactivation of caspase 3 and induction of BclXL promotes survival. PMID:20020442

  13. KCTD20, a relative of BTBD10, is a positive regulator of Akt

    PubMed Central

    2013-01-01

    Background BTBD10 binds to Akt and protein phosphatase 2A (PP2A) and inhibits the PP2A-mediated dephosphorylation of Akt, thereby keeping Akt activated. Previous studies have suggested that BTBD10 plays an important role in preventing motor neuronal death and accelerating the growth of pancreatic beta cells. Because levels of BTBD10 expression are much lower in many non-nervous tissues than nervous tissues, there may be a relative of BTBD10 that has BTBD10-like function in non-neuronal cells. Results A 419-amino-acid BTBD10-like protein, named KCTD20 (potassium channel tetramerization protein domain containing 20), was to found to bind to all Akt isoforms and PP2A. Overexpression of KCTD20 increased Akt phosphorylation at Thr308, as BTBD10 did, which suggests that KCTD20 as well as BTBD10 positively regulates the function of Akt. KCTD20 was ubiquitously expressed in non-nervous as well as nervous tissues. Conclusions KCTD20 is a positive regulator of Akt and may play an important role in regulating the death and growth of some non-nervous and nervous cells. PMID:24156551

  14. AKT activation controls cell survival in response to HDAC6 inhibition.

    PubMed

    Kaliszczak, M; Trousil, S; Ali, T; Aboagye, E O

    2016-01-01

    HDAC6 is emerging as an important therapeutic target for cancer. We investigated mechanisms responsible for survival of tumor cells treated with a HDAC6 inhibitor. Expression of the 20 000 genes examined did not change following HDAC6 treatment in vivo. We found that HDAC6 inhibition led to an increase of AKT activation (P-AKT) in vitro, and genetic knockdown of HDAC6 phenocopied drug-induced AKT activation. The activation of AKT was not observed in PTEN null cells; otherwise, PTEN/PIK3CA expression per se did not predict HDAC6 inhibitor sensitivity. Interestingly, HDAC6 inhibitor treatment led to inactivating phosphorylation of PTEN (P-PTEN Ser380), which likely led to the increased P-AKT in cells that express PTEN. Synergy was observed with phosphatidylinositol 3'-kinases (PI3K) inhibitor treatment in vitro, accompanied by increased caspase 3/7 activity. Furthermore, combination of HDAC6 inhibitor with a PI3K inhibitor caused substantial tumor growth inhibition in vivo compared with either treatment alone, also detectable by Ki-67 immunostaining and (18)F-FLT positron emission tomography (PET). In aggregate AKT activation appears to be a key survival mechanism for HDAC6 inhibitor treatment. Our findings indicate that dual inhibition of HDAC6 and P-AKT may be necessary to substantially inhibit growth of solid tumors. PMID:27362804

  15. AKT inhibition overcomes rapamycin resistance by enhancing the repressive function of PRAS40 on mTORC1/4E-BP1 axis

    PubMed Central

    Mi, Wenting; Ye, Qing; Liu, Side; She, Qing-Bai

    2015-01-01

    The mTORC1 inhibitors, rapamycin and its analogs, are known to show only modest antitumor activity in clinic, but the underlying mechanisms remain largely elusive. Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1. Treatment of tumor cells with rapamycin or the AKT inhibitor MK2206 showed a limited activity in inhibiting 4E-BP1 phosphorylation, cap-dependent translation, cell growth and motility. However, treatment with both drugs resulted in profound effects in vitro and in vivo. Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation. Knockdown of PRAS40 or 4E-BP1 expression markedly reduced the dependence of tumor cells on AKT/mTORC1 signaling for translation and survival. Together, these findings reveal a critical role of PRAS40 as an integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor cell growth and motility, and highlight PRAS40 phosphorylation as a potential biomarker to evaluate the therapeutic response to mTOR/AKT inhibitors. PMID:25961827

  16. VEGF signaling, mTOR complexes, and the endoplasmic reticulum: Towards a role of metabolic sensing in the regulation of angiogenesis

    PubMed Central

    Karali, Evdoxia; Bellou, Sofia; Stellas, Dimitris; Klinakis, Apostolos; Murphy, Carol; Fotsis, Theodore

    2014-01-01

    Vascular endothelial growth factor (VEGF) activates unfolded protein response sensors in the endoplasmic reticulum through phospholipase C gamma (PLCγ)-mediated crosstalk with mammalian target of rapamycin complex 1 (mTORC1). Activation of transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) activate mTORC2, ensuring maximal endothelial cell survival and angiogenic activity through phosphorylation of AKT on Ser473. As mTORC1 is a metabolic sensor, metabolic signals may be integrated with signals from VEGF in the regulation of angiogenesis.

  17. Amelioration of carbon tetrachloride-induced cirrhosis and portal hypertension in rat using adenoviral gene transfer of Akt

    PubMed Central

    Deng, Gang; Huang, Xiang-Jun; Luo, Hong-Wu; Huang, Fei-Zhou; Liu, Xun-Yang; Wang, Yong-Heng

    2013-01-01

    AIM: To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride (CCl4). METHODS: Using cre-loxp technique, we created an Ad-myr-HA-Akt virus, in which Akt is labeled by a HA tag and its expression is driven by myr promoter. Further, through measuring enzyme levels and histological structure, we determined the efficacy of this Ad-myr-HA-Akt virus in inhibiting the development of cirrhosis induced by CCl4 in rats. Lastly, using western blotting, we examined the expression levels and/or phosphorylation status of Akt, apoptotic mediators, endothelial nitric oxide synthase (eNOS), and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus. RESULTS: The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment, which was consistent with the sequence reported in the GenBank. The concentrations of Ad-myr-HA-Akt and adenoviral enhanced green fluorescent protein (Ad-EGFP) virus used in the current study were 5.5 × 1011 vp/mL. The portal vein diameter, peak velocity of blood flow, portal blood flow and congestion index were significantly increased in untreated, saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection. In contrast, these parameters in the Akt cirrhosis group were comparable to normal control group. Compared to the normal control, the liver function (Alanine aminotransferase, Aspartate aminotransferase and Albumin) was significantly impaired in the untreated, saline and Ad-EGFP cirrhosis groups. The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated, saline and Ad-EGFP cirrhosis groups. The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups

  18. Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity

    NASA Astrophysics Data System (ADS)

    Shen, Duanwen; Bai, Mingfeng; Tang, Rui; Xu, Baogang; Ju, Xiaoming; Pestell, Richard G.; Achilefu, Samuel

    2013-04-01

    Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

  19. Dynamic Adipocyte Phosphoproteome Reveals that Akt Directly Regulates mTORC2

    PubMed Central

    Humphrey, Sean J.; Yang, Guang; Yang, Pengyi; Fazakerley, Daniel J.; Stöckli, Jacqueline; Yang, Jean Y.; James, David E.

    2013-01-01

    Summary A major challenge of the post-genomics era is to define the connectivity of protein phosphorylation networks. Here, we quantitatively delineate the insulin signaling network in adipocytes by high-resolution mass spectrometry-based proteomics. These data reveal the complexity of intracellular protein phosphorylation. We identified 37,248 phosphorylation sites on 5,705 proteins in this single-cell type, with approximately 15% responding to insulin. We integrated these large-scale phosphoproteomics data using a machine learning approach to predict physiological substrates of several diverse insulin-regulated kinases. This led to the identification of an Akt substrate, SIN1, a core component of the mTORC2 complex. The phosphorylation of SIN1 by Akt was found to regulate mTORC2 activity in response to growth factors, revealing topological insights into the Akt/mTOR signaling network. The dynamic phosphoproteome described here contains numerous phosphorylation sites on proteins involved in diverse molecular functions and should serve as a useful functional resource for cell biologists. PMID:23684622

  20. Lithium protects against methamphetamine-induced neurotoxicity in PC12 cells via Akt/GSK3β/mTOR pathway

    SciTech Connect

    Wu, Jintao; Zhu, Dexiao; Zhang, Jing; Li, Guibao; Liu, Zengxun; Sun, Jinhao

    2015-09-25

    Methamphetamine (MA) is neurotoxic, especially in dopaminergic neurons. Long-lasting exposure to MA causes psychosis and increases the risk of Parkinson's disease. Lithium (Li) is a known mood stabilizer and has neuroprotective effects. Previous studies suggest that MA exposure decreases the phosphorylation of Akt/GSK3β pathway in vivo, whereas Li facilitates the phosphorylation of Akt/GSK3β pathway. Moreover, GSK3β and mTOR are implicated in the locomotor sensitization induced by psychostimulants and mTOR plays a critical role in MA induced toxicity. However, the effect of MA on Akt/GSK3β/mTOR pathway has not been fully investigated in vitro. Here, we found that MA exposure significantly dephosphorylated Akt/GSK3β/mTOR pathway in PC12 cells. In addition, Li remarkably attenuated the dephosphorylation effect of MA exposure on Akt/GSK3β/mTOR pathway. Furthermore, Li showed obvious protective effects against MA toxicity and LY294002 (Akt inhibitor) suppressed the protective effects of Li. Together, MA exposure dephosphorylates Akt/GSK3β/mTOR pathway in vitro, while lithium protects against MA-induced neurotoxicity via phosphorylation of Akt/GSK3β/mTOR pathway. - Highlights: • Lithium protects against methamphetamine-induced neurotoxicity in vitro. • Methamphetamine exposure dephosphorylates Akt/GSK3β/mTOR pathway. • Lithium attenuates methamphetamine-induced toxicity via phosphorylating Akt/GSK3β/mTOR pathway.

  1. A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt

    PubMed Central

    Aoki, Masahiro; Blazek, Erik; Vogt, Peter K.

    2001-01-01

    The oncoproteins P3k (homolog of the catalytic subunit of class IA phosphoinositide 3-kinase) and Akt (protein kinase B) induce oncogenic transformation of chicken embryo fibroblasts. The transformed cells show constitutive phosphorylation of the positive regulator of translation p70S6 kinase (S6K) and of the eukaryotic initiation factor 4E-BP1 binding protein (4E-BP1), a negative regulator of translation. Phosphorylation activates S6K and inactivates 4E-BP1. A mutant of Akt that retains kinase activity but does not induce phosphorylation of S6K or of 4E-BP1 fails to transform chicken embryo fibroblasts, suggesting a correlation between the oncogenicity of Akt and phosphorylation of S6K and 4E-BP1. The macrolide antibiotic rapamycin effectively blocks oncogenic transformation induced by either P3k or Akt but does not reduce the transforming activity of 11 other oncoproteins. Rapamycin inhibits the kinase mTOR, an important regulator of translation, and this inhibition requires binding of the antibiotic to the immunophilin FKBP12. Displacement of rapamycin from FKBP12 relieves the inhibition of mTOR and also restores P3k-induced transformation. These data are in accord with the hypothesis that transformation by P3k or Akt involves intervention in translational controls. PMID:11134523

  2. Phosphorylated p-70S6K predicts tamoxifen resistance in postmenopausal breast cancer patients randomized between adjuvant tamoxifen versus no systemic treatment

    PubMed Central

    2014-01-01

    Introduction Activation of the phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathways results in anti-estrogen resistance in vitro, but a biomarker with clinical validity to predict intrinsic resistance has not been identified. In metastatic breast cancer patients with previous exposure to endocrine therapy, the addition of a mammalian target of rapamycine (mTOR) inhibitor has been shown to be beneficial. Whether or not patients on adjuvant endocrine treatment might benefit from these drugs is currently unclear. A biomarker that predicts intrinsic resistance could potentially be used as companion diagnostic in this setting. We tested the clinical validity of different downstream-activated proteins in the PI3K and/or MAPK pathways to predict intrinsic tamoxifen resistance in postmenopausal primary breast cancer patients. Methods We recollected primary tumor tissue from patients who participated in a randomized trial of adjuvant tamoxifen (1–3 years) versus observation. After constructing a tissue micro-array, cores from 563 estrogen receptor α positive were immunostained for p-AKT(Thr308), p-AKT(Ser473), p-mTOR, p-p706SK and p-ERK1/2. Cox proportional hazard models for recurrence free interval were used to assess hazard ratios and interactions between these markers and tamoxifen treatment efficacy. Results Interactions were identified between tamoxifen and p-AKT(Thr308), p-mTOR, p-p70S6K and p-ERK1/2. Applying a conservative level of significance, p-p70S6K remained significantly associated with tamoxifen resistance. Patients with p-p70S6K negative tumors derived significant benefit from tamoxifen (HR 0.24, P < 0.0001), while patients whose tumor did express p-p70S6K did not (HR = 1.02, P =0.95), P for interaction 0.004. In systemically untreated breast cancer patients, p-p70S6K was associated with a decreased risk for recurrence. Conclusions Patients whose tumor expresses p-p70S6K, as a marker of downstream PI3K and

  3. Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

    SciTech Connect

    Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.; Burgos, Patricia V.; Villanueva, Carolina I.; Figueroa, Carlos D.; González, Carlos B.

    2013-11-29

    Highlights: •AVP induces mTOR phosphorylation in A-10 cells cultured in high glucose concentration. •The mTOR phosphorylation is mediated by the PI3K/Akt pathway activation. •The AVP-induced mTOR phosphorylation inhibited autophagy and stimulated cell proliferation. -- Abstract: Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.

  4. Avian reovirus σA and σNS proteins activate the phosphatidylinositol 3-kinase-dependent Akt signalling pathway.

    PubMed

    Xie, Liji; Xie, Zhixun; Huang, Li; Fan, Qing; Luo, Sisi; Huang, Jiaoling; Deng, Xianwen; Xie, Zhiqin; Zeng, Tingting; Zhang, Yanfang; Wang, Sheng

    2016-08-01

    The present study was conducted to identify avian reovirus (ARV) proteins that can activate the phosphatidylinositol 3-kinase (PI3K)-dependent Akt pathway. Based on ARV protein amino acid sequence analysis, σA, σNS, μA, μB and μNS were identified as putative proteins capable of mediating PI3K/Akt pathway activation. The recombinant plasmids σA-pcAGEN, σNS-pcAGEN, μA-pcAGEN, μB-pcAGEN and μNS-pcAGEN were constructed and used to transfect Vero cells, and the expression levels of the corresponding genes were quantified by immunofluorescence and Western blot analysis. Phosphorylated Akt (P-Akt) levels in the transfected cells were measured by flow cytometry and Western blot analysis. The results showed that the σA, σNS, μA, μB and μNS genes were expressed in Vero cells. σA-expressing and σNS-expressing cells had higher P-Akt levels than negative control cells, pcAGEN-expressing cells and cells designed to express other proteins (i.e., μA, μB and μNS). Pre-treatment with the PI3K inhibitor LY294002 inhibited Akt phosphorylation in σA- and σNS-expressing cells. These results indicate that the σA and σNS proteins can activate the PI3K/Akt pathway. PMID:27233800

  5. Calorie restriction leads to greater Akt2 activity and glucose uptake by insulin-stimulated skeletal muscle from old rats.

    PubMed

    Wang, Haiyan; Arias, Edward B; Cartee, Gregory D

    2016-03-01

    Skeletal muscle insulin resistance is associated with many common age-related diseases, but moderate calorie restriction (CR) can substantially elevate glucose uptake by insulin-stimulated skeletal muscle from both young and old rats. The current study evaluated the isolated epitrochlearis muscle from ∼24.5-mo-old rats that were either fed ad libitum (AL) or subjected to CR (consuming ∼65% of ad libitum, AL, intake beginning at ∼22.5 mo old). Some muscles were also incubated with MK-2206, a potent and selective Akt inhibitor. The most important results were that in isolated muscles, CR vs. AL resulted in 1) greater insulin-stimulated glucose uptake 2) that was accompanied by significantly increased insulin-mediated activation of Akt2, as indicated by greater phosphorylation on both Thr(309) and Ser(474) along with greater Akt2 activity, 3) concomitant with enhanced phosphorylation of several Akt substrates, including an Akt substrate of 160 kDa on Thr(642) and Ser(588), filamin C on Ser(2213) and proline-rich Akt substrate of 40 kDa on Thr(246), but not TBC1D1 on Thr(596); and 4) each of the CR effects was eliminated by MK-2206. These data provide compelling new evidence linking greater Akt2 activation to the CR-induced elevation of insulin-stimulated glucose uptake by muscle from old animals. PMID:26739650

  6. Akt and MAPK signaling mediate pregnancy-induced cardiac adaptation.

    PubMed

    Chung, Eunhee; Yeung, Fan; Leinwand, Leslie A

    2012-05-01

    Although the signaling pathways underlying exercise-induced cardiac adaptation have been extensively studied, little is known about the molecular mechanisms that result in the response of the heart to pregnancy. The objective of this study was to define the morphological, functional, and gene expression patterns that define the hearts of pregnant mice, and to identify the signaling pathways that mediate this response. Mice were divided into three groups: nonpregnant diestrus control, midpregnancy, and late pregnancy. Both time points of pregnancy were associated with significant cardiac hypertrophy. The prosurvival signaling cascades of Akt and ERK1/2 were activated in the hearts of pregnant mice, while the stress kinase, p38, was decreased. Given the activation of Akt in pregnancy and its known role in cardiac hypertrophy, the hypertrophic response to pregnancy was tested in mice expressing a cardiac-specific activated (myristoylated) form of Akt (myrAkt) or a cardiac-specific constitutively active (antipathologic hypertrophic) form of its downstream target, glycogen synthase kinase 3β (caGSK3β). The pregnancy-induced hypertrophic responses of hearts from these mice were significantly attenuated. Finally, we tested whether pregnancy-associated sex hormones could induce hypertrophy and alter signaling pathways in isolated neonatal rat ventricular myocytes (NRVMs). In fact, progesterone, but not estradiol treatment increased NRVM cell size via phosphorylation of ERK1/2. Inhibition of MEK1 effectively blocked progesterone-induced cellular hypertrophy. Taken together, our study demonstrates that pregnancy-induced cardiac hypertrophy is mediated by activation of Akt and ERK1/2 pathways. PMID:22345431

  7. Silencing p110{beta} prevents rapid depletion of nuclear pAkt

    SciTech Connect

    Ye, Zhi-wei; Ghalali, Aram; Hoegberg, Johan; Stenius, Ulla

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer p110{beta} was essential for the statin- and ATP-induced depletion of nuclear pAkt and an associated inhibition of growth. Black-Right-Pointing-Pointer p110{beta} knock-out inhibited statin-induced changes in binding between FKBP51, pAkt and PTEN. Black-Right-Pointing-Pointer Data supports the hypothesis that nuclear pAkt is important for anti-cancer effects of statins. -- Abstract: The p110{beta} subunit in the class IA PI3K family may act as an oncogene and is critical for prostate tumor development in PTEN knockout mice. We tested the possible involvement of p110{beta} in a recently described rapid depletion of phosphorylated Akt (pAkt) in the nucleus. Previous work showed that this down-regulation is induced by extracellular ATP or by statins and is mediated by the purinergic receptor P2X7. Here, we used p110{beta} knock out mouse embryonic fibroblasts (MEFs) and siRNA-treated cancer cells. We found that p110{beta} is essential for ATP- or statin-induced nuclear pAkt depletion in MEFs and in several cancer cell lines including prostate cancer cells. ATP, statin or the selective P2X7 agonist BzATP also inhibited cell growth, and this inhibition was not seen in p110{beta} knock out cells. We also found that p110{beta} was necessary for statin-induced changes in binding between FKBP51, pAkt and PTEN. Our data show that p110{beta} is essential for the ATP- and statin-induced effects and support a role of nuclear pAkt in cancer development. They also provide support for a chemopreventive effect of statins mediated by depletion of nuclear pAkt.

  8. HEATR1 Negatively Regulates Akt to Help Sensitize Pancreatic Cancer Cells to Chemotherapy.

    PubMed

    Liu, Tongzheng; Fang, Yuan; Zhang, Haoxing; Deng, Min; Gao, Bowen; Niu, Nifang; Yu, Jia; Lee, SeungBaek; Kim, JungJin; Qin, Bo; Xie, Fang; Evans, Debra; Wang, Liewei; Lou, Wenhui; Lou, Zhenkun

    2016-02-01

    Elucidating mechanisms of chemoresistance is critical to improve cancer therapy, especially for the treatment of pancreatic ductal adenocarcinoma (PDAC). Genome-wide association studies have suggested the less studied gene HEAT repeat-containing protein 1 (HEATR1) as a possible determinant of cellular sensitivity to different chemotherapeutic drugs. In this study, we assessed this hypothesized link in PDAC, where HEATR1 expression is downregulated significantly. HEATR1 silencing in PDAC cells increased resistance to gemcitabine and other chemotherapeutics, where this effect was associated with increased AKT kinase phosphorylation at the Thr308 regulatory site. Mechanistically, HEATR1 enhanced cell responsiveness to gemcitabine by acting as a scaffold to facilitate interactions between AKT and the protein phosphatase PP2A, thereby promoting Thr308 dephosphorylation. Consistent with these findings, treatment with the AKT inhibitor triciribine sensitized HEATR1-depleted PDAC cells to gemcitabine, suggesting that this therapeutic combination may overcome gemcitabine resistance in patients with low HEATR1 expression. Clinically, we found that HEATR1 downregulation in PDAC patients was associated with increased AKT phosphorylation, poor response to tumor resection plus gemcitabine standard-of-care treatment, and shorter overall survival. Collectively, our findings establish HEATR1 as a novel regulator of AKT and a candidate predictive and prognostic indicator of drug responsiveness and outcome in PDAC patients. PMID:26676747

  9. Hirsutenone in Alnus extract inhibits akt activity and suppresses prostate cancer cell proliferation.

    PubMed

    Kang, Soouk; Kim, Jong-Eun; Li, Yan; Jung, Sung Keun; Song, Nu Ry; Thimmegowda, N R; Kim, Bo Yeon; Lee, Hyong Joo; Bode, Ann M; Dong, Zigang; Lee, Ki Won

    2015-11-01

    Although specific compounds found in some East Asian traditional medicines have been shown to exhibit bioactive properties, their molecular mechanisms of action remain elusive. The bark of the Alnus species has been used for the treatment of various pathological conditions including hemorrhage, alcoholism, fever, diarrhea, skin diseases, inflammation, and cancer in East Asia for centuries. In this study, we show that hirsutenone, a bioactive compound in Alnus japonica, exhibits anti-cancer effects against prostate cancer through a direct physical inhibition of Akt1/2. Hirsutenone suppressed anchorage-dependent and independent cell growth of PC3 and LNCaP human prostate cancer cells. Annexin V and Propidium iodide (PI) staining results demonstrated that hirsutenone strongly induces apoptotic cell death in both PC3 and LNCaP cells. Furthermore, treatment of hirsutenone attenuated phosphorylation of mammalian target of rapamycin (mTOR), a downstream substrate of Akt, without affecting Akt phosphorylation. Kinase and pull-down assay results clearly show that hirsutenone inhibits Akt1 and 2 by direct binding in an adenosine triphosphate (ATP)-noncompetitive manner in vitro and ex vivo. Our results show that hirsutenone suppresses human prostate cancer by targeting Akt1 and 2 as a key component to explain for anti-cancer activity of Alnus species. PMID:25213146

  10. Two weeks of metformin treatment induces AMPK-dependent enhancement of insulin-stimulated glucose uptake in mouse soleus muscle

    PubMed Central

    Kristensen, Jonas Møller; Treebak, Jonas T.; Schjerling, Peter; Goodyear, Laurie

    2014-01-01

    Metformin-induced activation of the 5′-AMP-activated protein kinase (AMPK) has been associated with enhanced glucose uptake in skeletal muscle, but so far no direct causality has been examined. We hypothesized that an effect of in vivo metformin treatment on glucose uptake in mouse skeletal muscles is dependent on AMPK signaling. Oral doses of metformin or saline treatment were given to muscle-specific kinase dead (KD) AMPKα2 mice and wild-type (WT) littermates either once or chronically for 2 wk. Soleus and extensor digitorum longus muscles were used for measurements of glucose transport and Western blot analyses. Chronic treatment with metformin enhanced insulin-stimulated glucose uptake in soleus muscles of WT (∼45%, P < 0.01) but not of AMPK KD mice. Insulin signaling at the level of Akt protein expression or Thr308 and Ser473 phosphorylation was not changed by metformin treatment. Insulin signaling at the level of Akt and TBC1D4 protein expression as well as Akt Thr308/Ser473 and TBC1D4 Thr642/Ser711 phosphorylation were not changed by metformin treatment. Also, protein expressions of Rab4, GLUT4, and hexokinase II were unaltered after treatment. The acute metformin treatment did not affect glucose uptake in muscle of either of the genotypes. In conclusion, we provide novel evidence for a role of AMPK in potentiating the effect of insulin on glucose uptake in soleus muscle in response to chronic metformin treatment. PMID:24644243

  11. Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation.

    PubMed

    Zheng, Bing; Mao, Jia-Hui; Li, Xiao-Qing; Qian, Lin; Zhu, Hua; Gu, Dong-Hua; Pan, Xiao-Dong

    2016-01-01

    Here, we demonstrated that DNA-PKcs is over-expressed in multiple human renal cell carcinoma (RCC) tissues and in primary/established human RCCs. Pharmacological or genetic inhibition of DNA-PKcs suppressed proliferation of RCC cells. DNA-PKcs was in the complex of mTOR and SIN1, mediating mTORC2 activation and HIF-2α expression in RCC cells. Inhibiting or silencing DNA-PKcs suppressed AKT Ser-473 phosphorylation and HIF-2α expression. In vivo, DNA-PKcs knockdown or oral administration of the DNA-PKcs inhibitor NU-7441 inhibited AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 RCC xenograft growth in nude mice. We showed that miRNA-101 level was decreased in RCC tissues/cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in RCC cells. We show that DNA-PKcs over-expression regulates mTORC2-AKT activation, HIF-2α expression and RCC cell proliferation. PMID:27412013

  12. Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation

    PubMed Central

    Zheng, Bing; Mao, Jia-Hui; Li, Xiao-Qing; Qian, Lin; Zhu, Hua; Gu, Dong-hua; Pan, Xiao-dong

    2016-01-01

    Here, we demonstrated that DNA-PKcs is over-expressed in multiple human renal cell carcinoma (RCC) tissues and in primary/established human RCCs. Pharmacological or genetic inhibition of DNA-PKcs suppressed proliferation of RCC cells. DNA-PKcs was in the complex of mTOR and SIN1, mediating mTORC2 activation and HIF-2α expression in RCC cells. Inhibiting or silencing DNA-PKcs suppressed AKT Ser-473 phosphorylation and HIF-2α expression. In vivo, DNA-PKcs knockdown or oral administration of the DNA-PKcs inhibitor NU-7441 inhibited AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 RCC xenograft growth in nude mice. We showed that miRNA-101 level was decreased in RCC tissues/cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in RCC cells. We show that DNA-PKcs over-expression regulates mTORC2-AKT activation, HIF-2α expression and RCC cell proliferation. PMID:27412013

  13. Phosphatidylinositol 3-kinase/Akt signaling enhances nuclear localization and transcriptional activity of BRCA1

    SciTech Connect

    Hinton, Cimona V.; Fitzgerald, Latricia D.; Thompson, Marilyn E. . E-mail: methompson@mmc.edu

    2007-05-15

    Signaling pathways involved in regulating nuclear-cytoplasmic distribution of BRCA1 have not been previously reported. Here, we provide evidence that heregulin {beta}1-induced activation of the Akt pathway increases the nuclear content of BRCA1. First, treatment of T47D breast cancer cells with heregulin {beta}1 results in a two-fold increase in nuclear BRCA1 as assessed by FACS analysis, immunoblotting and immunofluorescence. This heregulin-induced increase in nuclear BRCA1 is blocked by siRNA-mediated down-regulation of Akt. Second, mutation of threonine 509 in BRCA1, the site of Akt phosphorylation, to an alanine, attenuates the ability of heregulin to induce BRCA1 nuclear accumulation. These data suggest that Akt-catalyzed phosphorylation of BRCA1 is required for the heregulin-regulated nuclear concentration of BRCA1. Because most functions ascribed to BRCA1 occur within the nucleus, we postulated that phosphorylation-dependent nuclear accumulation of BRCA1 would result in enhanced nuclear activity, specifically transcriptional activity, of BRCA1. This postulate is affirmed by our observation that the ability of BRCA1 to transactivate GADD45 promoter constructs was enhanced in T47D cells treated with heregulin {beta}1. Furthermore, the heterologous expression of BRCA1 in HCC1937 human breast cancer cells, which have constitutively active Akt, also induces GADD45 promoter activity, whereas the expression of BRCA1 in which threonine 509 has been mutated to an alanine is able to only minimally induce promoter activity. These findings implicate Akt in upstream events leading to BRCA1 nuclear localization and function.

  14. Phosphatidylinositol 3-kinase/Akt signaling enhances nuclear localization and transcriptional activity of BRCA1

    PubMed Central

    Hinton, Cimona V.; Fitzgerald, Latricia D.; Thompson, Marilyn E.

    2007-01-01

    Signaling pathways involved in regulating nuclear-cytoplasmic distribution of BRCA1 have not been previously reported. Here, we provide evidence that heregulin β1-induced activation of the Akt pathway increases the nuclear content of BRCA1. First, treatment of T47D breast cancer cells with heregulin β1 results in a two-fold increase in nuclear BRCA1 as assessed by FACS analysis, immunoblotting and immunofluorescence. This heregulin-induced increase in nuclear BRCA1 is blocked by siRNA-mediated down-regulation of Akt. Second, mutation of threonine 509 in BRCA1, the site of Akt phosphorylation, to an alanine, attenuates the ability of heregulin to induce BRCA1 nuclear accumulation. These data suggest that Akt-catalyzed phosphorylation of BRCA1 is required for the heregulin-regulated nuclear concentration of BRCA1. Because most functions ascribed to BRCA1 occur within the nucleus, we postulated that phosphorylation-dependent nuclear accumulation of BRCA1 would result in enhanced nuclear activity, specifically transcriptional activity, of BRCA1. This postulate is affirmed by our observation that the ability of BRCA1 to transactivate GADD45 promoter constructs was enhanced in T47D cells treated with heregulin β1. Furthermore, the heterologous expression of BRCA1 in HCC1937 human breast cancer cells, which have constitutively active Akt, also induces GADD45 promoter activity, whereas the expression of BRCA1 in which threonine 509 has been mutated to an alanine is able to only minimally induce promoter activity. These findings implicate Akt in upstream events leading to BRCA1 nuclear localization and function. PMID:17428466

  15. IL-13 Induces YY1 through the AKT Pathway in Lung Fibroblasts

    PubMed Central

    Guo, Jia; Yao, Hongwei; Lin, Xin; Xu, Haodong; Dean, David; Zhu, Zhou; Liu, Gang; Sime, Patricia

    2015-01-01

    A key feature of lung fibrosis is the accumulation of myofibroblasts. Interleukin 13 (IL-13) is a pro-fibrotic mediator that directly and indirectly influences the activation of myofibroblasts. Transforming growth factor beta (TGF-β) promotes the differentiation of fibroblasts into myofibroblasts, and can be regulated by IL-13. However, IL-13’s downstream signaling pathways are not completely understood. We previously reported that the transcription factor Yin Yang 1 (YY1) is upregulated in fibroblasts treated with TGF-β and in the lungs of mice and patients with pulmonary fibrosis. Moreover, YY1 directly regulates collagen and alpha smooth muscle actin (α-SMA) expression in fibroblasts. However, it is not known if IL-13 regulates fibroblast activation through YY1 expression. We hypothesize that IL-13 up-regulates YY1 expression through regulation of AKT activation, leading to fibroblast activation. In this study we found that YY1 was upregulated by IL-13 in lung fibroblasts in a dose- and time-dependent manner, resulting in increased α-SMA. Conversely, knockdown of YY1 blocked IL-13-induced α-SMA expression in fibroblasts. Furthermore, AKT phosphorylation was increased in fibroblasts treated with IL-13, and AKT overexpression upregulated YY1, whereas blockade of AKT phosphorylation suppressed the induction of YY1 by IL-13 in vitro. In vivo YY1 was upregulated in fibrotic lungs from CC10-IL-13 transgenic mice compared to that from wild-type littermates, which was associated with increased AKT phosphorylation. Taken together, these findings demonstrate that IL-13 is a potent stimulator and activator of fibroblasts, at least in part, through AKT-mediated YY1 activation. PMID:25775215

  16. Akt activation is a common event in pediatric malignant gliomas and a potential adverse prognostic marker: a report from the children’s oncology group

    PubMed Central

    Hamilton, Ronald L.; Murdoch, Geoffrey H.; Burger, Peter C.; Brat, Daniel J.; Rosenblum, Marc K.; Nikiforova, Marina N.; Holmes, Emiko J.; Zhou, Tianni; Cohen, Kenneth J.; Jakacki, Regina I.

    2010-01-01

    Aberrant activation of Akt is a common finding in adult malignant gliomas, resulting in most cases from mutations or deletions involving PTEN, which allows constitutive Akt phosphorylation. In contrast, we have previously reported that pediatric malignant gliomas, which are morphologically similar to lesions arising in adults, have a substantially lower incidence of genomic alterations of PTEN. The objective of this study was to determine whether Akt activation was also an uncommon finding in childhood malignant gliomas and whether this feature was associated with survival. To address this issue, we examined the frequency of Akt activation, determined by overexpression of the activated phosphorylated form of Akt (Se473) on immunohistochemical analysis, in a series of 53 childhood malignant gliomas obtained from newly diagnosed patients treated on the Children’s Oncology Group ACNS0126 and 0423 studies. The relationship between Akt activation and p53 over-expression, MIB1 labeling, and tumor histology was evaluated. The association between Akt activation and survival was also assessed. Overexpression of activated Akt was observed in 42 of 53 tumors, far in excess of the frequency of PTEN mutations we have previously observed. There was no association between Akt activation and either histology, p53 overexpression, or MIB1 proliferation indices. Although tumors that lacked Akt overexpression had a trend toward more favorable event-free survival and overall survival (p = 0.06), this association reflected that non-overexpressing tumors were significantly more likely to have undergone extensive tumor removal, which was independently associated with outcome. Activation of Akt is a common finding in pediatric malignant gliomas, although it remains uncertain whether this is an independent adverse prognostic factor. In view of the frequency of Akt activation, the evaluation of molecularly targeted therapies that inhibit this pathway warrants consideration for these tumors

  17. Evidence for AKT-independent regulation of FOXO1 and FOXO3 in haematopoietic stem and progenitor cells.

    PubMed

    Liang, Raymond; Rimmelé, Pauline; Bigarella, Carolina L; Yalcin, Safak; Ghaffari, Saghi

    2016-03-18

    Transcription factors FOXOs (1, 3, 4) are essential for the maintenance of haematopoietic stem cells. FOXOs are evolutionary conserved substrates of the AKT serine threonine protein kinase that are also phosphorylated by several kinases other than AKT. Specifically, phosphorylation by AKT is known to result in the cytosolic localization of FOXO and subsequent inhibition of FOXO transcriptional activity. In addition to phosphorylation, FOXOs are regulated by a number of other post-translational modifications including acetylation, methylation, redox modulation, and ubiquitination that altogether determine these factors' output. Cumulating evidence raises the possibility that in stem cells, including in haematopoietic stem cells, AKT may not be the dominant regulator of FOXO. To address this question in more detail, we examined gene expression, subcellular localization, and response to AKT inhibition of FOXO1 and FOXO3, the main FOXO expressed in HSPCs (haematopoietic stem and progenitor cells). Here we show that while FOXO1 and FOXO3 transcripts are expressed at similar levels, endogenous FOXO3 protein is mostly nuclear compared to the cytoplasmic localization of FOXO1 in HSPCs. Furthermore, inhibition of AKT does not enhance nuclear localization of FOXO1 nor FOXO3. Nonetheless AKT inhibition in the context of loss of NAD-dependent SIRT1 deacetylase modulates FOXO3 localization in HSPCs. Together, these data suggest that FOXO3 is more active than FOXO1 in primitive haematopoietic stem and multipotent progenitor cells. In addition, they indicate that upstream regulators other than AKT, such as SIRT1, maintain nuclear FOXO localization and activity in HSPCs. PMID:26929388

  18. Akt inactivation induces endoplasmic reticulum stress-independent autophagy in fibroblasts from patients with Pompe disease.

    PubMed

    Nishiyama, Yurika; Shimada, Yohta; Yokoi, Takayuki; Kobayashi, Hiroshi; Higuchi, Takashi; Eto, Yoshikatsu; Ida, Hiroyuki; Ohashi, Toya

    2012-11-01

    Pompe disease (glycogen storage disease type II) is an autosomal recessive neuromuscular disorder arising from a deficiency of lysosomal acid α-glucosidase (GAA). Accumulation of autophagosomes is a key pathological change in skeletal muscle fibers and fibroblasts from patients with Pompe disease and is implicated in the poor response to enzyme replacement therapy (ERT). We previously found that mutant GAA-induced endoplasmic reticulum (ER) stress initiated autophagy in patient fibroblasts. However, the mechanism of induction of autophagy in fibroblasts from Pompe disease patients lacking ER stress remains unclear. In this study, we show that inactivated Akt induces ER stress-independent autophagy via mTOR suppression in patient fibroblasts. Activated autophagy as evidenced by increased levels of LC3-II and autophagic vesicles was observed in patient fibroblasts, whereas PERK phosphorylation reflecting the presence of ER stress was not observed in them. These patient fibroblasts showed decreased levels of not only phosphorylated Akt, but also phosphorylated p70 S6 kinase. Treatment with insulin, which acts as an activator of the Akt signaling pathway, resulted in increased phosphorylation of both Akt and p70 S6 kinase and suppression of autophagy in patient fibroblasts. In addition, following combination treatment with recombinant human GAA plus insulin, enhanced localization of the enzymes with lysosomes was observed in patient fibroblasts. These findings define a critical role of Akt suppression in the induction of autophagy in fibroblasts from patients with Pompe disease carrying an ER stress non-inducible mutation, and they provide evidence that insulin may potentiate the effect of ERT. PMID:23041259

  19. Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis

    PubMed Central

    Fritsche-Guenther, Raphaela; Witzel, Franziska; Kempa, Stefan; Brummer, Tilman; Sers, Christine; Blüthgen, Nils

    2016-01-01

    Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell. PMID:26799289

  20. Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis.

    PubMed

    Fritsche-Guenther, Raphaela; Witzel, Franziska; Kempa, Stefan; Brummer, Tilman; Sers, Christine; Blüthgen, Nils

    2016-02-16

    Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell. PMID:26799289

  1. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

    SciTech Connect

    Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.

  2. Resveratrol augments ER stress and the cytotoxic effects of glycolytic inhibition in neuroblastoma by downregulating Akt in a mechanism independent of SIRT1

    PubMed Central

    Graham, Regina M; Hernandez, Fiorela; Puerta, Nataly; De Angulo, Guillermo; Webster, Keith A; Vanni, Steven

    2016-01-01

    Cancer cells typically display increased rates of aerobic glycolysis that are correlated with tumor aggressiveness and a poor prognosis. Targeting the glycolytic pathway has emerged as an attractive therapeutic route mainly because it should spare normal cells. Here, we evaluate the effects of combining the inhibition of glycolysis with application of the polyphenolic compound resveratrol (RSV) in neuroblastoma (NB) cancer cell lines. Inhibiting glycolysis with 2-deoxy-D-glucose (2-DG) significantly reduced NB cell viability and was associated with increased endoplasmic reticulum (ER) stress and Akt activity. Administration of 2-DG increased the expression of the ER molecular chaperones GRP78 and GRP94, the prodeath protein C/EBP homology protein (CHOP) and the phosphorylation of Akt at S473, T450 and T308. Combined treatment with both RSV and 2-DG reduced GRP78, GRP94 and Akt phosphorylation but increased CHOP and NB cell death when compared with the administration of 2-DG alone. The selective inhibition of Akt activity also decreased 2-DG-induced GRP78 and GRP94 expression and increased CHOP expression, suggesting that Akt can modulate ER stress. Protein phosphatase 1α (PP1α) was activated by RSV, as indicated by a reduction in PP1α phosphorylation at T320. Pretreatment of cells with tautomycin, a selective PP1α inhibitor, prevented the RSV-mediated decrease in Akt phosphorylation, suggesting that RSV enhances 2-DG-induced cell death by activating PP1 and downregulating Akt. The RSV-mediated inhibition of Akt in the presence of 2-DG was not prevented by the selective inhibition of SIRT1, a known target of RSV, indicating that the effects of RSV on this pathway are independent of SIRT1. We propose that RSV inhibits Akt activity by increasing PP1α activity, thereby potentiating 2-DG-induced ER stress and NB cell death. PMID:26891914

  3. Sustained Akt Activity Is Required to Maintain Cell Viability in Seborrheic Keratosis, a Benign Epithelial Tumor.

    PubMed

    Neel, Victor A; Todorova, Kristina; Wang, Jun; Kwon, Eunjeong; Kang, Minjeong; Liu, Qingsong; Gray, Nathanael; Lee, Sam W; Mandinova, Anna

    2016-03-01

    Seborrheic keratoses (SKs) are common benign skin tumors that share many morphological features with their malignant counterpart, squamous cell carcinoma. SKs frequently have acquired oncogenic mutations in the receptor tyrosine kinase/phosphatidylinositol 3-kinase/Akt signaling cascade. We developed a reliable culture system to study SKs in vitro and screened these cells using a library of selective kinase inhibitors to evaluate effects on cell survival. These benign tumors are sensitive to inhibition by ATP-competitive Akt inhibitors, including A-443654 and GSK690693. RNA interference-mediated Akt suppression mimicked the effects of enzyme inhibition in cultured cells. Akt inhibition suppressed phosphorylation of downstream targets of Akt kinase that are critical for cell survival, including MDM2 and FOXO3a, and induced apoptosis. Cell death was also dependent on p53, mutations in which, although common in cutaneous squamous cell carcinoma, have not been identified in SKs. Intact explants of SKs were also sensitive to Akt inhibition. In addition to the obvious therapeutic implications of these findings, identifying the signaling characteristics that differentiate benign and malignant tumors may inform our understanding of the malignant state. PMID:26739095

  4. New insights on PI3K/AKT pathway alterations and clinical outcomes in breast cancer.

    PubMed

    Yang, Sherry X; Polley, Eric; Lipkowitz, Stanley

    2016-04-01

    PI3K/AKT signaling pathway plays an important role in tumorigenesis and regulates critical cellular functions including survival, proliferation and metabolism. PIK3CA mutations and AKT activation by phosphorylation (pAKT) are often detected in many cancers and especially at high frequencies in breast cancer. Mounting data suggest that PIK3CA mutations or pAKT are mostly associated with better or insignificant outcomes in estrogen receptor-positive (ER+) early stage breast cancer and tend to be with worse prognosis in ER- disease. pAKT expression has been identified to predict paclitaxel chemotherapy benefit in node-positive breast cancer. Preclinical and neoadjuvant trial data suggest that PIK3CA alterations confer resistance to HER2-targeted therapy and are associated with lower pathological complete response (pCR) rate in HER2-positive breast cancer. However, recent results from randomized clinical trials of adjuvant and metastatic settings show that patients with mutant and wildtype PIK3CA tumors derived similar benefit from anti-HER2 therapy. This article, with our new insights, aims to decipher the mixed data and discusses the influence of the potential confounding factors in the assessments. We also share our views for validation of PI3K/AKT alterations in relation to clinical outcome in the context of specific breast cancer subtypes and treatment modalities towards further advance of the precision medicine for breast cancer treatment. PMID:26995633

  5. Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric AKT Inhibitor.

    PubMed

    Lapierre, Jean-Marc; Eathiraj, Sudharshan; Vensel, David; Liu, Yanbin; Bull, Cathy O; Cornell-Kennon, Susan; Iimura, Shin; Kelleher, Eugene W; Kizer, Darin E; Koerner, Steffi; Makhija, Sapna; Matsuda, Akihisa; Moussa, Magdi; Namdev, Nivedita; Savage, Ronald E; Szwaya, Jeff; Volckova, Erika; Westlund, Neil; Wu, Hui; Schwartz, Brian

    2016-07-14

    The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumor growth in a human xenograft mouse model of endometrial adenocarcinoma. PMID:27305487

  6. Ketamine affects the neurogenesis of rat fetal neural stem progenitor cells via the PI3K/Akt-p27 signaling pathway

    PubMed Central

    Dong, Chaoxuan; Rovnaghi, Cynthia R.; Anand, KJS

    2014-01-01

    Ketamine is widely used as an anesthetic, analgesic, or sedative in pediatric patients. We reported that ketamine alters the normal neurogenesis of rat fetal neural stem progenitor cells (NSPCs) in the developing brain, but the underlying mechanisms remain unknown. The PI3K-PKB/Akt (Phosphatidylinositide 3-kinases/protein kinase B) signaling pathway plays many important roles in cell survival, apoptosis, and proliferation. We hypothesized that PI3K-PKB/Akt signaling may be involved in ketamine-altered neurogenesis of cultured NSPCs in vitro. NSPCs were isolated from Sprague-Dawley rat fetuses on gestational day 17. BrdU (bromodeoxyuridine) incorporation, Ki67 staining, and differentiation tests were utilized to identify primary cultured NSPCs. Immunofluorescent staining was used to detect Akt expression, whereas, Western blots measured phosphorylated Akt and p27 expression in NSPCs exposed to different treatments. We report that cultured NSPCs had properties of neurogenesis: proliferation and neural differentiation. PKB/Akt was expressed in cultured rat fetal cortical NSPCs. Ketamine inhibited the phosphorylation of Akt and further enhanced p27 expression in cultured NSPCs. All ketamine-induced PI3K/Akt signaling changes could be recovered by NMDA (N-Methyl-D-aspartate) receptor agonist, NMDA. These data suggest that inhibition of PI3K/Akt-p27 signaling may be involved in ketamine-induced neurotoxicity in the developing brain, whereas excitatory NMDA receptor activation may reverse these effects. PMID:25231110

  7. mTORC1 is necessary but mTORC2 and GSK3β are inhibitory for AKT3-induced axon regeneration in the central nervous system

    PubMed Central

    Miao, Linqing; Yang, Liu; Huang, Haoliang; Liang, Feisi; Ling, Chen; Hu, Yang

    2016-01-01

    Injured mature CNS axons do not regenerate in mammals. Deletion of PTEN, the negative regulator of PI3K, induces CNS axon regeneration through the activation of PI3K-mTOR signaling. We have conducted an extensive molecular dissection of the cross-regulating mechanisms in axon regeneration that involve the downstream effectors of PI3K, AKT and the two mTOR complexes (mTORC1 and mTORC2). We found that the predominant AKT isoform in CNS, AKT3, induces much more robust axon regeneration than AKT1 and that activation of mTORC1 and inhibition of GSK3β are two critical parallel pathways for AKT-induced axon regeneration. Surprisingly, phosphorylation of T308 and S473 of AKT play opposite roles in GSK3β phosphorylation and inhibition, by which mTORC2 and pAKT-S473 negatively regulate axon regeneration. Thus, our study revealed a complex neuron-intrinsic balancing mechanism involving AKT as the nodal point of PI3K, mTORC1/2 and GSK3β that coordinates both positive and negative cues to regulate adult CNS axon regeneration. DOI: http://dx.doi.org/10.7554/eLife.14908.001 PMID:27026523

  8. The antioxidant compound tert-butylhydroquinone activates Akt in myocardium, suppresses apoptosis and ameliorates pressure overload-induced cardiac dysfunction

    PubMed Central

    Zhang, Yongtao; Fang Liu, Fang; Bi, Xiaolei; Wang, Shuangxi; Wu, Xiao; Jiang, Fan

    2015-01-01

    Tert-butylhydroquinone (TBHQ) is an antioxidant compound which shows multiple cytoprotective actions. We evaluated the effects of TBHQ on pathological cardiac remodeling and dysfunction induced by chronic overload. Pressure overload was created by transverse aortic constriction (TAC) in male C57BL/6 mice. TBHQ was incorporated in the diet and administered for 4 weeks. TBHQ treatment prevented left ventricular dilatation and cardiac dysfunction induced by TAC, and decreased the prevalence of myocardial apoptosis. The beneficial effects of TBHQ were associated with an increase in Akt activation, but not related to activations of Nrf2 or AMP-activated protein kinase. TBHQ-induced Akt activation was accompanied by increased phosphorylation of Bad, glycogen synthase kinase-3β (GSK-3β) and mammalian target of rapamycin (mTOR). Mechanistically, we showed that in cultured H9c2 cells and primary cardiac myocytes, TBHQ stimulated Akt phosphorylation and suppressed oxidant-induced apoptosis; this effect was abolished by wortmannin or an Akt inhibitor. Blockade of the Akt pathway in vivo accelerated cardiac dysfunction, and abrogated the protective effects of TBHQ. TBHQ also reduced the reactive aldehyde production and protein carbonylation in stressed myocardium. We suggest that TBHQ treatment may represent a novel strategy for timely activation of the cytoprotective Akt pathway in stressed myocardium. PMID:26260024

  9. Tetrandrine suppresses metastatic phenotype of prostate cancer cells by regulating Akt/mTOR/MMP-9 signaling pathway.

    PubMed

    Kou, Bo; Liu, Wei; He, Wenbo; Zhang, Yuanyuan; Zheng, Jianjie; Yan, Yang; Zhang, Yongjian; Xu, Suochun; Wang, Haichen

    2016-05-01

    Tetrandrine (TET), a bisbenzylisoquinoline alkaloid found in traditional Chinese medicines, exerts anticancer activity in vitro and in vivo. However, its potential role in the prostate cancer metastatic process has not yet been elucidated. Thus, we investigated the inhibition effect of tetrandrine on prostate cancer migration and invasion and the corresponding molecular basis underlying its anticancer activity. Cell migration and invasion were determined using the Transwell chamber model. The protein expression of Akt, phosphorylated Akt, the mammalian target of rapamycin (mTOR), phosphorylated mTOR and matrix metalloproteinases 9 (MMP-9) was detected by western blot in the presence or absence of tetrandrine or in the group tetrandrine combination with LY294002 (inhibitor of Akt) and rapamycin (inhibitor of mTOR). Our studies showed that excluding the effect of tetrandrine on cell proliferation, tetrandrine significantly inhibited cell migration and invasion in prostate cancer DU145 and PC3 cells. Furthermore, tetrandrine decreased the protein levels of p-Akt, p-mTOR, and MMP-9. While the inhibition of Akt or mTOR by the respective inhibitors could potentiate this effect of tetrandrine on prostate cancer cells, the studies indicate that tetrandrine inhibits the metastasis process by negatively regulating the Akt/mTOR/MMP-9 signaling pathway. These results suggest that tetrandrine might serve as a potential metastasis suppressor to treat cancer cells that have escaped surgical removal or that have disseminated widely. PMID:26935264

  10. Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation

    PubMed Central

    Tobias, Irene S.; Kaulich, Manuel; Kim, Peter K.; Simon, Nitya; Jacinto, Estela; Dowdy, Steven F.; King, Charles C.; Newton, Alexandra C.

    2016-01-01

    Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ. PMID:26635352

  11. Insulin Signaling and Glucose Uptake in the Soleus Muscle of 30-Month-Old Rats After Calorie Restriction With or Without Acute Exercise.

    PubMed

    Wang, Haiyan; Sharma, Naveen; Arias, Edward B; Cartee, Gregory D

    2016-03-01

    Exercise and calorie restriction (CR) can each improve insulin sensitivity in older individuals, but benefits of combining these treatments on skeletal muscle insulin signaling and glucose uptake are poorly understood, especially in predominantly slow-twitch muscles (eg, soleus). Accordingly, our purpose was to determine independent and combined effects of prior acute exercise and CR (beginning at 14 weeks old) on insulin signaling and glucose uptake in insul