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Sample records for akt phosphorylation levels

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  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. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  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. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  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. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  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. 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

  20. 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

  1. 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

  2. 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

  3. MAT2B promotes adipogenesis by modulating SAMe levels and activating AKT/ERK pathway during porcine intramuscular preadipocyte differentiation.

    PubMed

    Zhao, Cunzhen; Chen, Xiaochang; Wu, Wenjing; Wang, Wusu; Pang, Weijun; Yang, Gongshe

    2016-05-15

    Intramuscular fat (IMF) has been demonstrated as one of the crucial factors of livestock meat quality. The MAT2B protein with MAT2α catalyzes the formation of methyl donor S- adenosylmethionine (SAMe) to mediate cell metabolism including proliferation and apoptosis. However, the regulatory effect of MAT2B on IMF deposition is still unclear. In this study, the effect of MAT2B on adipogenesis and its potential mechanism during porcine intramuscular preadipocyte differentiation was studied. The results showed that overexpression of MAT2B promoted adipogenesis and significantly up-regulated the mRNA and protein levels of adipogenic marker genes including FASN, PPARγ and aP2, consistently, knockdown of MAT2B inhibited lipid accumulation and down-regulated the mRNA and protein levels of the above genes. Furthermore, flow cytometry and EdU-labeling assay indicated that MAT2B regulate adipogenesis was partly due to influence intracellular SAMe levels and further affect cell clonal expansion. Also, increased expression of MAT2B activated the phosphorylations of AKT and ERK1/2, whereas knockdown of MAT2B blocked AKT signaling and repressed the phosphorylation of ERK1/2. Moreover, the inhibitory effect of LY294002 (a specific PI3K inhibitor) on the activities of AKT and ERK1/2 was partially recovered by overexpression of MAT2B in porcine intramuscular adipocytes. Finally, Co-IP experiments showed that MAT2B can directly interact with AKT. Taken together, our findings suggested that MAT2B acted as a positive regulator through modifying SAMe levels as well as activating AKT/ERK signaling pathway to promote porcine intramuscular adipocyte differentiation. PMID:26940012

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. Toward a systems-level view of dynamic phosphorylation networks

    PubMed Central

    Newman, Robert H.; Zhang, Jin; Zhu, Heng

    2014-01-01

    To better understand how cells sense and respond to their environment, it is important to understand the organization and regulation of the phosphorylation networks that underlie most cellular signal transduction pathways. These networks, which are composed of protein kinases, protein phosphatases and their respective cellular targets, are highly dynamic. Importantly, to achieve signaling specificity, phosphorylation networks must be regulated at several levels, including at the level of protein expression, substrate recognition, and spatiotemporal modulation of enzymatic activity. Here, we briefly summarize some of the traditional methods used to study the phosphorylation status of cellular proteins before focusing our attention on several recent technological advances, such as protein microarrays, quantitative mass spectrometry, and genetically-targetable fluorescent biosensors, that are offering new insights into the organization and regulation of cellular phosphorylation networks. Together, these approaches promise to lead to a systems-level view of dynamic phosphorylation networks. PMID:25177341

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. Paclitaxel and beta-lapachone synergistically induce apoptosis in human retinoblastoma Y79 cells by downregulating the levels of phospho-Akt.

    PubMed

    D'Anneo, Antonella; Augello, Giuseppa; Santulli, Andrea; Giuliano, Michela; di Fiore, Riccardo; Messina, Concetta; Tesoriere, Giovanni; Vento, Renza

    2010-02-01

    Paclitaxel (PTX) and beta-lapachone (LPC) are naturally occurring compounds that have shown a large spectrum of anticancer activity. In this article we show for the first time that PTX/LPC combination induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and LPC (1.5 microM) caused biochemical and morphological signs of apoptosis at 48 h of treatment. These effects were accompanied by potent lowering in inhibitor of apoptosis proteins and by activation of Bid and caspases 3 and 6 with lamin B and PARP breakdown. PTX/LPC combination acted by favoring p53 stabilization through a lowering in p-Akt levels and in ps166-MDM2, the phosphorylated-MDM2 form that enters the nucleus and induces p53 export and degradation. Treatment with wortmannin or transfection with a dominant negative form of Akt anticipated at 24 h the effects induced by PTX/LPC, suggesting a protective role against apoptosis played by Akt in Y79 cells. In line with these results, we demonstrated that Y79 cells contain constitutively active Akt, which forms a cytosolic complex with p53 and MDM2 driving p53 degradation. PTX/LPC treatment induced a weakness of Akt-MDM2-p53 complex and increased nuclear p53 levels. Our results suggest that phospho-Akt lowering is at the root of the apoptotic action exerted by PTX/LPC combination and provide strong validation for a treatment approach that targets survival signals represented by phospho-Akt and inhibitor of apoptosis proteins. PMID:19918798

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. UV light induces premature senescence in Akt1-null mouse embryonic fibroblasts by increasing intracellular levels of ROS

    SciTech Connect

    Jee, Hye Jin; Kim, Hyun-Ju; Kim, Ae Jeong; Bae, Yoe-Sik; Bae, Sun Sik; Yun, Jeanho

    2009-06-05

    Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2{sup -/-} mouse embryonic fibroblasts (MEFs) while Akt1{sup -/-} MEFs show cell cycle arrest. Here, we find that Akt1{sup -/-} MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated {beta}-galactosidase (SA {beta}-gal) staining indicate that Akt1{sup -/-} MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1{sup -/-} MEFs suppressed SA {beta}-gal activity, indicating that UV-induced senescence is due to the absence of Akt1 function. Notably, levels of ROS were rapidly increased upon UV-irradiation and the ROS scavenger NAC inhibits UV-induced senescence of Akt1{sup -/-} MEFs, suggesting that UV light induces premature senescence in Akt1{sup -/-} MEFs by modulating intracellular levels of ROS. In conjunction with our previous work, this indicates that different isoforms of Akt have distinct function in response to UV-irradiation.

  19. 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

  20. 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

  1. 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

  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. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism.

    PubMed

    Puebla, Carlos; Cisterna, Bruno A; Salas, Daniela P; Delgado-López, Fernando; Lampe, Paul D; Sáez, Juan C

    2016-05-01

    Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt. PMID:26869446

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. Higher Levels of c-Met Expression and Phosphorylation Identify Cell Lines With Increased Sensitivity to AMG-458, a Novel Selective c-Met Inhibitor With Radiosensitizing Effects

    SciTech Connect

    Li Bo; Torossian, Artour; Sun, Yunguang; Du, Ruihong; Dicker, Adam P.; Lu Bo

    2012-11-15

    Purpose: c-Met is overexpressed in some non-small cell lung cancer (NSCLC) cell lines and tissues. Cell lines with higher levels of c-Met expression and phosphorylation depend on this receptor for survival. We studied the effects of AMG-458 on 2 NSCLC cell lines. Methods and Materials: 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium assays assessed the sensitivities of the cells to AMG-458. Clonogenic survival assays illustrated the radiosensitizing effects of AMG-458. Western blot for cleaved caspase 3 measured apoptosis. Immunoblotting for c-Met, phospho-Met (p-Met), Akt/p-Akt, and Erk/p-Erk was performed to observe downstream signaling. Results: AMG-458 enhanced radiosensitivity in H441 but not in A549. H441 showed constitutive phosphorylation of c-Met. A549 expressed low levels of c-Met, which were phosphorylated only in the presence of exogenous hepatocyte growth factor. The combination of radiation therapy and AMG-458 treatment was found to synergistically increase apoptosis in the H441 cell line but not in A549. Radiation therapy, AMG-458, and combination treatment were found to reduce p-Akt and p-Erk levels in H441 but not in A549. H441 became less sensitive to AMG-458 after small interfering RNA knockdown of c-Met; there was no change in A549. After overexpression of c-Met, A549 became more sensitive, while H441 became less sensitive to AMG-458. Conclusions: AMG-458 was more effective in cells that expressed higher levels of c-Met/p-Met, suggesting that higher levels of c-Met and p-Met in NSCLC tissue may classify a subset of tumors that are more sensitive to molecular therapies against this receptor.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. Inhibitory effect of ethanol on AMPK phosphorylation is mediated in part through elevated ceramide levels

    PubMed Central

    Sozio, Margaret S.; Shin, Eric; Zhao, Zhenwen; Xu, Yan; Ross, Ruth A.; Zeng, Yan; Crabb, David W.

    2010-01-01

    Ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMP-activated protein kinase (AMPK). This study shows that the inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of the phosphorylation of upstream kinases and the activation of protein phosphatase 2A (PP2A).Inhibition of AMPK phosphorylation by palmitate was attributed to ceramide-dependent PP2A activation. We hypothesized that the inhibitory effect of ethanol on AMPK phosphorylation was mediated partly through the generation of ceramide. The effect of ethanol and inhibitors of ceramide synthesis on AMPK phosphorylation, ceramide levels, and PP2A activity were assessed in rat hepatoma cells (H4IIEC3). The effect of ethanol on hepatic ceramide levels was also studied in C57BL/6J mice fed the Lieber-DeCarli diet. In H4IIEC3 cells, ceramide reduced AMPK phosphorylation when they were treated for between 4 and 12 h. The basal level of AMPK phosphorylation in hepatoma cells was increased with the treatment of ceramide synthase inhibitor, fumonisin B1. Ethanol treatment significantly increased cellular ceramide content and PP2A activity by ∼18–23%, when the cells were treated with ethanol for between 4 and 12 h. These changes in intracellular ceramide concentrations and PP2A activity correlated with the time course over which ethanol inhibited AMPK phosphorylation. The activation of PP2A and inhibition of AMPK phosphorylation caused by ethanol was attenuated by fumonisin B1 and imipramine, an acid sphingomyelinase (SMase) inhibitor. There was a significant increase in the levels of ceramide and acid SMase mRNA in the livers of ethanol-fed mice compared with controls. We concluded that the effect of ethanol on AMPK appears to be mediated in part through increased cellular levels of ceramide and activation of PP2A. PMID:20224005

  5. 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.

  6. Galangin attenuates airway remodelling by inhibiting TGF-β1-mediated ROS generation and MAPK/Akt phosphorylation in asthma

    PubMed Central

    Liu, Ya-Nan; Zha, Wang-Jian; Ma, Yuan; Chen, Fei-Fei; Zhu, Wen; Ge, Ai; Zeng, Xiao-Ning; Huang, Mao

    2015-01-01

    Galangin, a natural flavonol, has attracted much attention for its potential anti-inflammatory properties. However, its role in the regulation of airway remodelling in asthma has not been explored. The present study aimed to elucidate the effects of galangin on chronic inflammation and airway remodelling and to investigate the underlying mechanisms both in vivo and in vitro. Ovalbumin (OVA)-sensitised mice were administered with galangin 30 min before challenge. Our results showed that severe inflammatory responses and airway remodelling occurred in OVA-induced mice. Treatment with galangin markedly attenuated the leakage of inflammatory cells into bronchoalveolar lavage fluid (BALF) and decreased the level of OVA-specific IgE in serum. Galangin significantly inhibited goblet cell hyperplasia, collagen deposition and α-SMA expression. Lowered level of TGF-β1 and suppressed expression of VEGF and MMP-9 were observed in BALF or lung tissue, implying that galangin has an optimal anti-remodelling effect in vivo. Consistently, the TGF-β1-induced proliferation of airway smooth muscle cells was reduced by galangin in vitro, which might be due to the alleviation of ROS levels and inhibition of MAPK pathway. Taken together, the present findings highlight a novel role for galangin as a promising anti-remodelling agent in asthma, which likely involves the TGF-β1-ROS-MAPK pathway. PMID:26156213

  7. 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

  8. Membrane-tethered AKT kinase regulates basal synaptic transmission and early phase LTP expression by modulation of post-synaptic AMPA receptor level.

    PubMed

    Pen, Y; Borovok, N; Reichenstein, M; Sheinin, A; Michaelevski, I

    2016-09-01

    The serine/threonine kinase AKT/PKB plays a fundamental role in a wide variety of neuronal functions, including neuronal cell development, axonal growth, and synaptic plasticity. Multiple evidence link AKT signaling pathways to regulation of late phase long-term synaptic plasticity, synaptogenesis, and spinogenesis, as well as long-term memory formation. Nevertheless, the downstream effectors mediating the effects of AKT on early phase long-term potentiation (eLTP) are currently unknown. Here we report that using different regimes of pharmacological activation and inhibition of AKT activity in acute hippocampal slices, we found that AKT regulates the post-synaptic expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) receptors affecting solely the expression of eLTP, with no effect on its induction and maintenance. We further show that both maintenance of basal synaptic activity and expression of eLTP require plasma membrane tethering by activated AKT and that basal synaptic activity may be regulated via the direct effects of AKT1 on the expression level of post-synaptic AMPA receptors bypassing the canonical AKT signaling. Finally, we establish that eLTP expression requires the involvement of both the canonical AKT signaling pathways and the direct effect of AKT1 on AMPA receptor activity/expression in the post-synaptic membrane. © 2016 Wiley Periodicals, Inc. PMID:27068236

  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. Activation of the PI3K/Akt signal transduction pathway and increased levels of insulin receptor in protein repair-deficient mice.

    PubMed

    Farrar, Christine; Houser, Carolyn R; Clarke, Steven

    2005-02-01

    Protein L-isoaspartate (D-aspartate) O-methyltransferase is an enzyme that catalyses the repair of isoaspartyl damage in proteins. Mice lacking this enzyme (Pcmt1-/- mice) have a progressive increase in brain size compared with wild-type mice (Pcmt1+/+ mice), a phenotype that can be associated with alterations in the PI3K/Akt signal transduction pathway. Here we show that components of this pathway, including Akt, GSK3beta and PDK-1, are more highly phosphorylated in the brains of Pcmt1-/- mice, particularly in cells of the hippocampus, in comparison with Pcmt1+/+ mice. Examination of upstream elements of this pathway in the hippocampus revealed that Pcmt1-/- mice have increased activation of insulin-like growth factor-I (IGF-I) receptor and/or insulin receptor. Western blot analysis revealed an approximate 200% increase in insulin receptor protein levels and an approximate 50% increase in IGF-I receptor protein levels in the hippocampus of Pcmt1-/- mice. Higher levels of the insulin receptor protein were also found in other regions of the adult brain and in whole tissue extracts of brain, liver, heart and testes of both juvenile and adult Pcmt1-/- mice. There were no significant differences in plasma insulin levels for adult Pcmt1-/- mice during glucose tolerance tests. However, they did show higher peak levels of blood glucose, suggesting a mild impairment in glucose tolerance. We propose that Pcmt1-/- mice have altered regulation of the insulin pathway, possibly as a compensatory response to altered glucose uptake or metabolism or as an adaptive response to a general accumulation of isoaspartyl protein damage in the brain and other tissues. PMID:15659208

  12. 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

  13. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52

    NASA Astrophysics Data System (ADS)

    Jo, Chulman; Gundemir, Soner; Pritchard, Susanne; Jin, Youngnam N.; Rahman, Irfan; Johnson, Gail V. W.

    2014-03-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

  14. 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.

  15. Retinoids regulate TGFβ signaling at the level of Smad2 phosphorylation and nuclear accumulation

    PubMed Central

    Hoover, Loretta L.; Burton, Elizabeth G.; O'Neill, Megan L.; Brooks, Bonnie A.; Sreedharan, Shilpa; Dawson, Nineveh A.; Kubalak, Steven W.

    2008-01-01

    Indirect regulation of transforming growth factor (TGF)-β signaling by retinoids occurs on a long-term timescale, secondary to transcriptional events. Studies by our group show loss of retinoid X receptor (RXR) alpha results in increased TGFβ2 in the midgestational heart, which may play a role in the cardiac defects seen in this model[1]. Acute and direct interactions between retinoid and TGFβ signaling, however, are not clearly understood. Treatment of dispersed hearts and NIH3T3 cells for one-hour with TGFβ and retinoids (dual treatment) resulted in increased phosphorylated Smad2 and Smad3 when compared to treatment with TGFβ alone. Of all dual treatments, those with the RXR agonist Bexarotene, resulted in the highest level of phosphorylated Smad2, a 7-fold increase over TGFβ2 alone. Additionally, during dual treatment phosphorylation of Smad2 occurs via the TGFβ type I receptor but not by increased activation of the receptor. As loss of RXRα results in increased levels of Smad2 phosphorylation in response to TGFβ treatment and since nuclear accumulation of phosphorylated Smad2 is decreased during dual treatment, we propose that RXRα directly regulates the activities of Smad2. These data show retinoid signaling influences the TGFβ pathway in an acute and direct manner that has been unappreciated until now. PMID:18773928

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  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. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  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. Sustained high levels of neuroprotective, high molecular weight, phosphorylated tau in the longest-lived rodent

    PubMed Central

    Orr, Miranda E.; Garbarino, Valentina R.; Salinas, Angelica; Buffenstein, Rochelle

    2016-01-01

    Tau protein is primarily expressed in neuronal axons and modulates microtubule stability. Tau phosphorylation, aggregation and subcellular mislocalization coincide with neurodegeneration in numerous diseases, including Alzheimer's disease [AD]. During AD pathogenesis, tau misprocessing accompanies Aß accumulation; however, AD animal models, despite elevated Aß, fail to develop tauopathy. To assess whether lack of tau pathology is linked to short lifespan common to most AD models, we examined tau processing in extraordinarily long-lived, mouse-sized naked mole-rats (NMR; ~32 years), which express appreciable levels of Aß throughout life. NMRs, like other mammals, displayed highest tau phosphorylation during brain development. While tau phosphorylation decreased with aging, unexpectedly adult NMRs had higher levels than transgenic mice overexpressing mutant human tau. However, in sharp contrast with the somatodendritic accumulation of misprocessed tau in the transgenic mice, NMRs maintain axonal tau localization. Intriguingly, the adult NMR tau protein is 88kDa, much larger than 45-68kDa tau expressed in other mammals. We propose that this 88kDa tau protein may offer exceptional microtubule stability and neuroprotection against lifelong elevated Aß. PMID:25576082

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  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. Global Analysis of Protein Expression and Phosphorylation Levels in Nicotine-Treated Pancreatic Stellate Cells.

    PubMed

    Paulo, Joao A; Gaun, Aleksandr; Gygi, Steven P

    2015-10-01

    Smoking is a risk factor in pancreatic disease; however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large-scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12 h in triplicate and compared alterations in protein expression and phosphorylation levels to mock-treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8100 proteins were quantified across all nine samples, of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease, and lysosomal proteins. In addition, we measured differences for ∼16 000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  11. Global analysis of protein expression and phosphorylation levels in nicotine-treated pancreatic stellate cells

    PubMed Central

    Paulo, Joao A.; Gaun, Aleksandr; Gygi, Steven P.

    2016-01-01

    Smoking is a risk factor in pancreatic disease, however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12hr in triplicate and compared alterations in protein expression and phosphorylation levels to mock treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8,100 proteins were quantified across all nine samples of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease and lysosomal proteins. In addition, we measured differences for ∼16,000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. Fasting upregulates adipose triglyceride lipase and hormone-sensitive lipase levels and phosphorylation in mouse kidney.

    PubMed

    Marvyn, Phillip M; Bradley, Ryan M; Button, Emily B; Mardian, Emily B; Duncan, Robin E

    2015-06-01

    Circulating non-esterified fatty acids (NEFA) rise during fasting and are taken up by the kidneys, either directly from the plasma or during re-uptake of albumin from glomerular filtrate, and are stored as triacylglycerol (TAG). Subsequent utilization of stored fatty acids requires their hydrolytic release from cellular lipid droplets, but relatively little is known about renal lipolysis. We found that total [(3)H]triolein hydrolase activity of kidney lysates was significantly increased by 15% in the fasted state. Adipose triglyceride lipase (Atgl) and hormone-sensitive lipase (Hsl) mRNA expression was time-dependently increased by fasting, along with other fatty acid metabolism genes (Pparα, Cd36, and Aox). ATGL and HSL protein levels were also significantly induced (by 239 ± 7% and 322 ± 8%, respectively). Concomitant with changes in total protein levels, there was an increase in ATGL phosphorylation at the AMPK-regulated serine 406 site in the 14-3-3 binding motif, and an increase in HSL phosphorylation at serines 565 and 660 that are regulated by AMPK and PKA, respectively. Using immunofluorescence, we further demonstrate nearly ubiquitous expression of ATGL in the renal cortex with a concentration on the apical/lumenal surface of some cortical tubules. Our findings suggest a role for ATGL and HSL in kidney lipolysis. PMID:25879679

  2. Dichloromethane fraction of Cimicifuga heracleifolia decreases the level of melanin synthesis by activating the ERK or AKT signaling pathway in B16F10 cells.

    PubMed

    Jang, Ji Yeon; Lee, Jun Hyuk; Kang, Byoung Won; Chung, Kyung Tae; Choi, Yung Hyun; Choi, Byung Tae

    2009-03-01

    Cimicifuga rhizoma has long been used in traditional Korean medicine. In particular, a Cimicifuga heracleifolia extract (CHE) was reported to inhibit the formation of glutamate and the glutamate dehydrogenase activity in cultured rat islet. Glutamate activates melanogenesis by activating tyrosinase. Accordingly, it was hypothesized that a CHE might inhibit the melanogenesis-related signal pathways including the inhibition of microphthalmia-associated transcription factor (MITF)-tyrosinase signaling and/or the activation of extracellular signal-regulated kinase (ERK)-Akt signaling. The results showed that CHE inhibits the cellular melanin contents, tyrosinase activity and expression of melanogenesis-related proteins including MITF, tyrosinase and tyrosinase-related protein (TRP)s in alpha-melanocyte-stimulating hormone-stimulated B16 cells. Moreover, CHE phosphorylates MEK, ERK1/2 and Akt, which are melanogenesis inhibitory proteins. The data suggest that CHE inhibits melanogenesis signaling by both inhibiting the tyrosinase directly and activating the MEK-ERK or Akt signal pathways-mediated suppression of MITF and its downstream signal pathway, including tyrosinase and TRPs. Therefore, C. heracleifolia would be a useful therapeutic agent for treating hyperpigmentation and an effective component in whitening and/or lightening cosmetics. PMID:18803655

  3. 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

  4. 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

  5. LRRK2 phosphorylation level correlates with abnormal motor behaviour in an experimental model of levodopa-induced dyskinesias.

    PubMed

    Stanic, Jennifer; Mellone, Manuela; Cirnaru, Maria Daniela; Perez-Carrion, Maria; Zianni, Elisa; Di Luca, Monica; Gardoni, Fabrizio; Piccoli, Giovanni

    2016-01-01

    Levodopa (L-DOPA)-induced dyskinesias (LIDs) represent the major side effect in Parkinson's disease (PD) therapy. Leucine-rich repeat kinase 2 (LRRK2) mutations account for up to 13 % of familial cases of PD. LRRK2 N-terminal domain encompasses several serine residues that undergo phosphorylation influencing LRRK2 function. This work aims at investigating whether LRRK2 phosphorylation/function may be involved in the molecular pathways downstream D1 dopamine receptor leading to LIDs. Here we show that LRRK2 phosphorylation level at serine 935 correlates with LIDs induction and that inhibition of LRRK2 induces a significant increase in the dyskinetic score in L-DOPA treated parkinsonian animals. Our findings support a close link between LRKK2 functional state and L-DOPA-induced abnormal motor behaviour and highlight that LRRK2 phosphorylation level may be implicated in LIDs, calling for novel therapeutic strategies. PMID:27169991

  6. 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

  7. 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

  8. Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors

    PubMed Central

    González, Natalia; Moresco, James J.; Bustos, Francisco; Yates, John R.; Olguín, Hugo C.

    2016-01-01

    Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate. PMID:27144531

  9. Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors.

    PubMed

    González, Natalia; Moresco, James J; Cabezas, Felipe; de la Vega, Eduardo; Bustos, Francisco; Yates, John R; Olguín, Hugo C

    2016-01-01

    Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate. PMID:27144531

  10. Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels.

    PubMed

    Williams, Claire M; El Mohsen, Manal Abd; Vauzour, David; Rendeiro, Catarina; Butler, Laurie T; Ellis, Judi A; Whiteman, Matthew; Spencer, Jeremy P E

    2008-08-01

    Phytochemical-rich foods have been shown to be effective at reversing age-related deficits in memory in both animals and humans. We show that a supplementation with a blueberry diet (2% w/w) for 12 weeks improves the performance of aged animals in spatial working memory tasks. This improvement emerged within 3 weeks and persisted for the remainder of the testing period. Memory performance correlated well with the activation of cAMP-response element-binding protein (CREB) and increases in both pro- and mature levels of brain-derived neurotrophic factor (BDNF) in the hippocampus. Changes in CREB and BDNF in aged and blueberry-supplemented animals were accompanied by increases in the phosphorylation state of extracellular signal-related kinase (ERK1/2), rather than that of calcium calmodulin kinase (CaMKII and CaMKIV) or protein kinase A. Furthermore, age and blueberry supplementation were linked to changes in the activation state of Akt, mTOR, and the levels of Arc/Arg3.1 in the hippocampus, suggesting that pathways involved in de novo protein synthesis may be involved. Although causal relationships cannot be made among supplementation, behavior, and biochemical parameters, the measurement of anthocyanins and flavanols in the brain following blueberry supplementation may indicate that changes in spatial working memory in aged animals are linked to the effects of flavonoids on the ERK-CREB-BDNF pathway. PMID:18457678

  11. Tripeptidyl Peptidase II Mediates Levels of Nuclear Phosphorylated ERK1 and ERK2*

    PubMed Central

    Wiemhoefer, Anne; Stargardt, Anita; van der Linden, Wouter A.; Renner, Maria C.; van Kesteren, Ronald E.; Stap, Jan; Raspe, Marcel A.; Tomkinson, Birgitta; Kessels, Helmut W.; Ovaa, Huib; Overkleeft, Herman S.; Florea, Bogdan; Reits, Eric A.

    2015-01-01

    Tripeptidyl peptidase II (TPP2) is a serine peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide mediated signaling. The underlying mechanisms of how a peptidase can influence this multitude of processes still remain unknown. We identified rapid proteomic changes in neuroblastoma cells following selective TPP2 inhibition using the known reversible inhibitor butabindide, as well as a new, more potent, and irreversible peptide phosphonate inhibitor. Our data show that TPP2 inhibition indirectly but rapidly decreases the levels of active, di-phosphorylated extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus, thereby down-regulating signal transduction downstream of growth factors and mitogenic stimuli. We conclude that TPP2 mediates many important cellular functions by controlling ERK1 and ERK2 phosphorylation. For instance, we show that TPP2 inhibition of neurons in the hippocampus leads to an excessive strengthening of synapses, indicating that TPP2 activity is crucial for normal brain function. PMID:26041847

  12. 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

  13. 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

  14. Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase

    PubMed Central

    Tong, Chao; Wang, Bing; Chen, Yongbin; Jiang, Jin

    2016-01-01

    Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR)-family protein Smoothened (Smo). Upon stimulation, Smo accumulates on the cell surface in Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish)/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II) in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail). We find that CL-II phosphorylation is promoted by protein kinase A (PKA)-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh) pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo. PMID:27280464

  15. Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase.

    PubMed

    Li, Shuangxi; Li, Shuang; Han, Yuhong; Tong, Chao; Wang, Bing; Chen, Yongbin; Jiang, Jin

    2016-06-01

    Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR)-family protein Smoothened (Smo). Upon stimulation, Smo accumulates on the cell surface in Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish)/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II) in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail). We find that CL-II phosphorylation is promoted by protein kinase A (PKA)-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh) pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo. PMID:27280464

  16. 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

  17. Daily intermittent hypoxia augments spinal BDNF levels, ERK phosphorylation and respiratory long-term facilitation

    PubMed Central

    Wilkerson, Julia E.R.; Mitchell, Gordon S.

    2009-01-01

    Acute intermittent hypoxia (AIH) elicits a form of respiratory plasticity known as long-term facilitation (LTF). We hypothesized that: 1) daily AIH (dAIH) preconditioning enhances phrenic and hypoglossal (XII) LTF in a rat strain with low constitutive LTF expression; 2) dAIH induces brain-derived neurotrophic factor (BDNF), a critical protein for phrenic LTF (pLTF) in the cervical spinal cord; and 3) dAIH increases post-AIH extracellular regulated kinase (ERK) activation. Phrenic and XII motor output were monitored in anesthetized dAIH- or sham-treated Brown Norway rats with and without acute AIH. pLTF was observed in both sham (18 ± 9% baseline; 60 min post-hypoxia; p < 0.05; n = 18) and dAIH treated rats (37 ± 8%; p < 0.05; n = 14), but these values were not significantly different (p = 0.13). XII LTF was not observed in sham-treated rats (4 ± 5%), but was revealed in dAIH pretreated rats (48 ± 18%; p < 0.05). dAIH preconditioning increased basal ventral cervical BDNF protein levels (24 ± 8%; p < 0.05), but had no significant effect on ERK phosphorylation. AIH increased BDNF in sham (25 ± 8%; p < 0.05), but not dAIH-pretreated rats (−7 ± 4%), and had complex effects on ERK phosphorylation (ERK2 increased in shams whereas ERK1 increased in dAIH-treated rats). Thus, dAIH elicits metaplasticity in LTF, revealing XII LTF in a rat strain with no constitutive XII LTF expression. Increased BDNF synthesis may no longer be necessary for phrenic LTF following dAIH preconditioning since BDNF concentration is already elevated. PMID:19416672

  18. 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

  19. Loss of PTEN stabilizes the lipid modifying enzyme cytosolic phospholipase A2α via AKT in prostate cancer cells

    PubMed Central

    Vignarajan, Soma; Xie, Chanlu; Yao, Mu; Sun, Yuting; Simanainen, Ulla; Sved, Paul; Liu, Tao; Dong, Qihan

    2014-01-01

    Aberrant increase in pAKT, due to a gain-of-function mutation of PI3K or loss-of-function mutation or deletion of PTEN, occurs in prostate cancer and is associated with poor patient prognosis. Cytosolic phospholipase A2α (cPLA2α) is a lipid modifying enzyme by catalyzing the hydrolysis of membrane arachidonic acid. Arachidonic acid and its metabolites contribute to survival and proliferation of prostate cancer cells. We examined whether AKT plays a role in promoting cPLA2α action in prostate cancer cells. We found a concordant increase in pAKT and cPLA2α levels in prostate tissue of prostate epithelial-specific PTEN-knockout but not PTEN-wide type mice. Restoration of PTEN expression or inhibition of PI3K action decreased cPLA2α expression in PTEN-mutated or deleted prostate cancer cells. An increase in AKT by Myr-AKT elevated cPLA2α protein levels, which could be diminished by inhibition of AKT phosphorylation without noticeable change in total AKT levels. pAKT levels had no influence on cPLA2α at mRNA levels but reduced cPLA2α protein degradation. Anti-AKT antibody co-immunoprecipitated cPLA2α and vice versa. Hence, AKT plays a role in enhancing cPLA2α protein stability in PTEN-null prostate cancer cells, revealing a link between oncogenic pathway and lipid metabolism. PMID:25026288

  20. 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

  1. Determinants of hepcidin levels in sepsis-associated acute kidney injury: Impact on pAKT/PTEN pathways?

    PubMed

    Schaalan, Mona F; Mohamed, Walid A

    2016-09-01

    The antimicrobial β-defensin-like role of hepcidin (HEPC) has been increasingly investigated for its potential role in acute kidney injury (AKI). In sepsis-induced AKI, there is a complex interplay between positive and negative regulation of HEPC, with consequently altered distributions of iron caused by changes in HEPC levels. The aim of the current research was to assess serum HEPC levels in a cohort of septic patients with AKI and investigate the regulatory impact of hypoxia-inducing factor (HIF)-1α, erythropoietin (EPO) and inflammation on HEPC levels and related signal cascades in these patients. Baseline, higher levels of SCr (2.3-fold), blood urea nitrogen (BUN) (1.8-fold), uric acid (2.3-fold) and white blood cell (2.3-fold) were noted in septic AKI patients, along with decreased levels of albumin (15.7%), creatinine (44.7%) and BUN/creatinine ratios (23.8%), compared to in normal subjects. These hosts also had increased serum levels of TNFα (4.4-times) and TGFβ1 (3.2-times) compared to controls (p < 0.05). Further, HEPC and HIF-1α levels were also increased (8.8- and 3.6-times control levels), while EPO levels were decreased (77.8%) from control levels. After 12 weeks of antibiotic therapy, all septic AKI patients showed significant improvement of the altered markers of kidney dysfunction. In line with significant reductions in serum TNFα and TGFβ1 (25.5% and 26.2%, respectively), HEPC and HIF-1α levels were significantly decreased (31.6% and 19.3%), and EPO levels increased (1.9-fold) compared to pretreatment values. There was a significant positive correlation between HEPC levels and kidney function markers (SCr and BUN), inflammatory TNFα and TGFβ1 and serum HIF-1α and pAKT in septic AKI patients before and after treatment. Based on the results here, we conclude that HEPC, EPO and HIF-1α are involved in the pathogenesis of sepsis-induced AKI and confirm the dominating effects of inflammatory determinants over hypoxia

  2. 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

  3. Phosphorylated neurofilament subunit levels in the serum of cervical compressive myelopathy patients.

    PubMed

    Kato, So; Chikuda, Hirotaka; Ohya, Junichi; Hayakawa, Kentaro; Takeshita, Katsushi; Tanaka, Sakae; Ogata, Toru

    2015-10-01

    We investigated the serum levels of the phosphorylated form of the high molecular weight neurofilament subunit (pNF-H) in patients with cervical compressive myelopathy. pNF-H is becoming increasingly recognized as a biomarker for axonal injury, however, it remains unclear whether serum pNF-H is elevated in chronic spinal cord compression. We examined 26 patients who underwent surgery for cervical compressive myelopathy. Peripheral blood samples were obtained both preoperatively and 1 week after surgery to evaluate the serum pNF-H levels using an enzyme-linked immunosorbent assay. A history of recent aggravation of myelopathy was also investigated. Of the 26 myelopathy patients, the preoperative serum pNF-H level was negative in 20 patients and moderately elevated in six. Patients who were positive for pNF-H were more likely to have had a recent aggravation of myelopathy compared with the pNF-H negative patients (83 versus 25%; p=0.02). All patients who were positive for pNF-H before surgery remained positive after surgery. Two patients who became positive after surgery demonstrated a neurologic deterioration associated with the surgery. In conclusion, the serum pNF-H level was negative in the majority of patients with cervical compressive myelopathy. Our results suggest that an elevated serum level of pNF-H is associated with an acute worsening of myelopathy and that a positive conversion of pNF-H after surgery is a marker of perioperative neural damage. PMID:26195334

  4. Cofilin/Twinstar Phosphorylation Levels Increase in Response to Impaired Coenzyme A Metabolism

    PubMed Central

    Siudeja, Katarzyna; Grzeschik, Nicola A.; Rana, Anil; de Jong, Jannie; Sibon, Ody C. M.

    2012-01-01

    Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. Here we use Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture – a process that is strongly dependent on actin remodeling. Our results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics. PMID:22912811

  5. 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

  6. 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

  7. 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

  8. 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

  9. Thiamine deficiency degrades the link between spatial behavior and hippocampal synapsin I and phosphorylated synapsin I protein levels.

    PubMed

    Resende, Leticia S; Ribeiro, Angela M; Werner, David; Hall, Joseph M; Savage, Lisa M

    2012-07-01

    The links between spatial behavior and hippocampal levels of synapsin I and phosphosynapsin I were assessed in normal rats and in the pyrithiamine-induced thiamine deficiency (PTD) rat model of Wernicke-Korsakoff's syndrome. Synapsin I tethers small synaptic vesicles to the actin cytoskeleton in a phosphorylation-dependent manner, is involved in neurotransmitter release and has been implicated in hippocampal-dependent learning. Positive correlations between spontaneous alternation behavior and hippocampal levels of both synapsin I and phosphorylated synapsin I were found in control rats. However, spontaneous alternation performance was impaired in PTD rats and was accompanied by a significant reduction (30%) in phosphorylated synapsin I. Furthermore, no correlations were observed between either form of synapsin I and behavior in PTD rats. These data suggest that successful spontaneous alternation performance is related to high levels of hippocampal synapsin I and phosphorylated synapsin I. These results not only support the previous findings that implicate impaired hippocampal neurotransmission in the spatial learning and memory deficits associated with thiamine deficiency, but also suggest a presynaptic mechanism. PMID:22507301

  10. Chronic resistance training activates autophagy and reduces apoptosis of muscle cells by modulating IGF-1 and its receptors, Akt/mTOR and Akt/FOXO3a signaling in aged rats.

    PubMed

    Luo, Li; Lu, A-Ming; Wang, Yan; Hong, An; Chen, Yulan; Hu, Juan; Li, Xiaoning; Qin, Zheng-Hong

    2013-04-01

    Resistance exercise training (RET) remains the most effective treatment for the loss of muscle mass and strength in elderly people. However, the underlying cellular and molecular mechanisms are not well understood. Recent evidence suggests that autophagic signaling is altered in aged skeletal muscles. This study aimed to investigate if RET affects IGF-1 and its receptors, the Akt/mTOR, and Akt/FOXO3a signaling pathways and regulates autophagy and apoptosis in the gastrocnemius muscles of 18-20 month old rats. The results showed that 9 weeks of RET prevented the loss of muscle mass and improved muscle strength, accompanied by reduced LC3-II/LC3-I ratio, reduced p62 protein levels, and increased levels of autophagy regulatory proteins, including Beclin 1, Atg5/12, Atg7, and the lysosomal enzyme cathepsin L. RET also reduced cytochrome c level in the cytosol but increased its level in mitochondrial fraction, and inhibited cleaved caspase 3 production and apoptosis. Furthermore, RET upregulated the expression of IGF-1 and its receptors but downregulated the phosphorylation of Akt and mTOR. In addition, RET upregulated the expression of total AMPK, phosphorylated AMPK, and FOXO3a. Taken together, these results suggest that the benefits of RET are associated with increased autophagy activity and reduced apoptosis of muscle cells by modulating IGF-1 and its receptors, the Akt/mTOR and Akt/FOXO3a signaling pathways in aged skeletal muscles. PMID:23419688

  11. 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

  12. Induction of apoptosis by the ginsenoside Rh2 by internalization of lipid rafts and caveolae and inactivation of Akt

    PubMed Central

    Park, E-K; Lee, EJ; Lee, S-H; Koo, KH; Sung, JY; Hwang, EH; Park, JH; Kim, C-W; Jeong, K-C; Park, B-K; Kim, Y-N

    2010-01-01

    Background and purpose: Lipid rafts and caveolae are membrane microdomains with important roles in cell survival signalling involving the Akt pathway. Cholesterol is important for the structure and function of these microdomains. The ginsenoside Rh2 exhibits anti-tumour activity. Because Rh2 is structurally similar to cholesterol, we investigated the possibility that Rh2 exerted its anti-tumour effect by modulating rafts and caveolae. Experimental approach: A431 cells (human epidermoid carcinoma cell line) were treated with Rh2 and the effects on cell apoptosis, raft localization and Akt activation measured. We also examined the effects of over-expression of Akt and active-Akt on Rh2-induced cell death. Key results: Rh2 induced apoptosis concentration- and time-dependently. Rh2 reduced the levels of rafts and caveolae in the plasma membrane and increased their internalization. Furthermore, Akt activity was decreased and consequently, Akt-dependent phosphorylation of Bad, a pro-survival protein, was decreased whereas the pro-apoptotic proteins, Bim and Bax, were increased upon Rh2 treatment. Unlike microdomain internalization induce by cholesterol depletion, Rh2-mediated internalization of rafts and caveolae was not reversed by cholesterol addition. Also, cholesterol addition did not restore Akt activation or rescue cells from Rh2-induced cell death. Rh2-induced cell death was attenuated in MDA-MB-231 cells over-expressing either wild-type or dominant-active Akt. Conclusions and implications: Rh2 induced internalization of rafts and caveolae, leading to Akt inactivation, and ultimately apoptosis. Because elevated levels of membrane rafts and caveolae, and Akt activation have been correlated with cancer development, internalization of these microdomains by Rh2 could potentially be used as an anti-cancer therapy. PMID:20590613

  13. Action of methyl-, propyl- and butylparaben on GPR30 gene and protein expression, cAMP levels and activation of ERK1/2 and PI3K/Akt signaling pathways in MCF-7 breast cancer cells and MCF-10A non-transformed breast epithelial cells.

    PubMed

    Wróbel, Anna Maria; Gregoraszczuk, Ewa Łucja

    2015-10-14

    In the present study, we examined cAMP levels and activation of the MAPK/ERK1/2 and PI3K/Akt signaling pathways in response to the actions of parabens on GPR30 in MCF-7 and MCF-10A cells. Cells were exposed to methyl-, propyl- or butylparaben at a concentration of 20nM; 17-β-estradiol (10nM) was used as a positive control. 17β-estradiol and all tested parabens increased GPR30 gene and protein expression in MCF-7 and MCF-10A cells. No parabens affected cAMP levels in either cell line, with the exception of propylparaben in MCF-10A cells. 17β-estradiol, propylparaben, and butylparaben increased phosphorylation of ERK1/2 in MCF-7 cells, whereas 17β-estradiol, methyl- and butylparaben, but not propylparaben, increased phosphorylation of ERK1/2 in MCF-10A cells. Akt activation was noted only in MCF-7 cells and only with propylparaben treatment. Collectively, the data presented here point to a nongenomic mechanism of action of parabens in activation GPR30 in both cancer and non-cancer breast cell lines through βγ dimer-mediated activation of the ERK1/2 pathway, but not the cAMP/PKA pathway. Moreover, among investigated parabens, propylparaben appears to inhibit apoptosis in cancer cells through activation of Akt kinases, confirming conclusions suggested by our previously published data. Nevertheless, continuing research on the carcinogenic action of parabens is warranted. PMID:26253279

  14. PRAS40 deregulates apoptosis in Ewing sarcoma family tumors by enhancing the insulin receptor/Akt and mTOR signaling pathways

    PubMed Central

    Lv, Dan; Liu, Jinye; Guo, Lianying; Wu, Dawei; Matsumoto, Ken; Huang, Lin

    2016-01-01

    EWS expression in Ewing sarcoma family tumors (ESFTs) is decreased due to the haploinsufficiency elicited by chromosomal translocation. The abnormal expression levels of EWS and its downstream factors contribute to the manifestation of ESFTs. Previously, we reported that increased Proline-rich Akt substrate of 40 kDa (PRAS40), which is encoded by an EWS mRNA target, promotes the development of ESFTs. However, the mechanism remains elusive. To clarify the role of PRAS40 in ESFTs, we silenced PRAS40 expression in ESFT cells using siRNAs and found increased levels of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. Cleaved caspase 3 levels and cytochrome C release were increased simultaneously. Furthermore, with PRAS40 knockdown, the phosphorylation of Akt and mTOR downstream factors, i.e., S6K and S6, was attenuated notably. Ectopic expression of PRAS40 increased Akt and S6 phosphorylation. Activation of Akt only partially reversed the apoptosis induced by PRAS40 knockdown, and downregulation of S6 phosphorylation by PRAS40 silencing could not be sufficiently restored via Akt activation. Searching the upstream factors in this pathway, the autophosphorylation of insulin receptor (IR) was found to be inhibited significantly by PRAS40 silencing but increased by PRAS40 overexpression. Therefore, PRAS40 may enhance IR phosphorylation to facilitate Akt and mTOR signaling leading to the apoptosis deregulation in ESFTs. Moreover, in vivo results confirmed that PRAS40 deletion suppressed the growth of ESFT xenografts and downregulated IR and S6 phosphorylation. Our findings suggest a novel functioning model for PRAS40, which represents a novel therapeutic target for ESFTs. PMID:27186418

  15. Nucleophosmin protein expression level, but not threonine 198 phosphorylation, is essential in growth and proliferation

    PubMed Central

    Brady, SN; Maggi, LB; Winkeler, CL; Toso, EA; Gwinn, AS; Pelletier, CL; Weber, JD

    2010-01-01

    Nucleophosmin (NPM), an oligomeric phosphoprotein and nucleolar target of the ARF tumor suppressor, contributes to several critical cellular processes. Previous studies have shown that the human NPM’s phosphorylation by cyclin E–cyclin-dependent kinase 2 (cdk2) on threonine (Thr) 199 regulates its translocation from the centrosome during cell cycle progression. Given our previous finding that ARF directly binds NPM, impeding its transit to the cytoplasm and arresting cells before S-phase entry, we hypothesized that ARF might also inhibit NPM phosphorylation. However, ARF induction did not impair phosphorylation of the cdk2 target residue in murine NPM, Thr198. Furthermore, phosphorylation of Thr198 occurred throughout the cell cycle and was concomitant with increases in overall NPM expression. To investigate the cell’s presumed requirement for NPM-Thr198 phosphorylation in promoting the processes of growth and proliferation, we examined the effects of a non-phosphorylatable NPM mutant, T198A, in a clean cell system in which endogenous NPM had been removed by RNA interference. Here, we show that the T198A mutant is fully capable of executing NPM’s described roles in nucleocytoplasmic shuttling, ribosome export and cell cycle progression. Moreover, the proliferative defects observed with stable NPM knockdown were restored by mutant NPM-T198A expression. Thus, we demonstrate that the reduction in NPM protein expression blocks cellular growth and proliferation, whereas phosphorylation of NPM-Thr198 is not essential for NPM’s capacity to drive cell cycle progression and proliferation. PMID:19561638

  16. 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

  17. Akt1 signaling coordinates BMP signaling and β-catenin activity to regulate second heart field progenitor development.

    PubMed

    Luo, Wen; Zhao, Xia; Jin, Hengwei; Tao, Lichan; Zhu, Jingai; Wang, Huijuan; Hemmings, Brian A; Yang, Zhongzhou

    2015-02-15

    Second heart field (SHF) progenitors exhibit continued proliferation and delayed differentiation, which are modulated by FGF4/8/10, BMP and canonical Wnt/β-catenin signaling. PTEN-Akt signaling regulates the stem cell/progenitor cell homeostasis in several systems, such as hematopoietic stem cells, intestinal stem cells and neural progenitor cells. To address whether PTEN-Akt signaling is involved in regulating cardiac progenitors, we deleted Pten in SHF progenitors. Deletion of Pten caused SHF expansion and increased the size of the SHF derivatives, the right ventricle and the outflow tract. Cell proliferation of cardiac progenitors was enhanced, whereas cardiac differentiation was unaffected by Pten deletion. Removal of Akt1 rescued the phenotype and early lethality of Pten deletion mice, suggesting that Akt1 was the key downstream target that was negatively regulated by PTEN in cardiac progenitors. Furthermore, we found that inhibition of FOXO by Akt1 suppressed the expression of the gene encoding the BMP ligand (BMP7), leading to dampened BMP signaling in the hearts of Pten deletion mice. Cardiac activation of Akt also increased the Ser552 phosphorylation of β-catenin, thus enhancing its activity. Reducing β-catenin levels could partially rescue heart defects of Pten deletion mice. We conclude that Akt signaling regulates the cell proliferation of SHF progenitors through coordination of BMP signaling and β-catenin activity. PMID:25670795

  18. [Phosphorylation and dephosphorylation of rna polymerase III holoenzyme are modifications regulating the level of transcription in vitro].

    PubMed

    Nikitina, T V; Tishchenko, L I; Sedova, V M

    2002-01-01

    Two subforms of RNA polymerase III-IIIa and IIIb--were identified in human placenta nuclei. These subforms differed in molecular weight of one subunit, and in buoyant density in glycerol concentration gradient. Protein kinase activity, which phosphorylates at least four subunits of RNA polymerase IIIa and three subunits of RNA polymerase IIIb in vitro, was copurified with both the subforms. Protein kinase activity was inhibited by wortmannin, a specific PI3-kinase inhibitor. RNA polymerase III dephosphorylation by alkaline phosphatase in vitro decrease the transcription level on specific Alu-template. The associated protein kinase was not able to phosphorylate dephosphorylated RNA polymerase IIIa and to restore the transcription level up to the control one. PMID:12094766

  19. Inhibition of TYRO3/Akt signaling participates in hypoxic injury in hippocampal neurons

    PubMed Central

    Zhu, Yan-zhen; Wang, Wei; Xian, Na; Wu, Bing

    2016-01-01

    In this study, we investigated the role of the TYRO3/Akt signaling pathway in hypoxic injury to hippocampal neurons. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that hypoxia inhibited the proliferation and viability of hippocampal neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated that hypoxia induced neuronal apoptosis in a time-dependent manner, with a greater number of apoptotic cells with longer hypoxic exposure. Immunofluorescence labeling revealed that hypoxia suppressed TYRO3 expression. Western blot assay showed that hypoxia decreased Akt phosphorylation levels in a time-dependent manner. Taken together, these findings suggest that hypoxia inhibits the proliferation of hippocampal neurons and promotes apoptosis, and that the inhibition of the TYRO3/Akt signaling pathway plays an important role in hypoxia-induced neuronal injury. PMID:27335558

  20. Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization

    SciTech Connect

    Kim, Chung Kwon; Lee, Sang Bae; Nguyen, Truong L.X.; Lee, Kyung-Hoon; Um, Sung Hee; Kim, Jihoe; Ahn, Jee-Yin

    2012-01-15

    p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.

  1. 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

  2. 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

  3. Synthesis and biological evaluation of a novel class of isatin analogs as dual inhibitors of tubulin polymerization and Akt pathway

    PubMed Central

    Krishnegowda, Gowdahalli; Gowda, A. S. Prakasha; Tagaram, Hephzibah Rani S.; Staveley-O’ Carroll, Kevin F; Irby, Rosalyn B.; Sharma, Arun K.; Amin, Shantu

    2011-01-01

    A novel series of 5,7-dibromoisatin analogs were synthesized and evaluated for their cytotoxicities against four human cancer cell lines including colon HT29, breast MCF-7, lung A549 and melanoma UACC903. Analogs 6, 11 and 13 displayed good in vitro anticancer activity on the HT29 human colon cancer cell line in the 1 µM range. Analogs 5, 9 and 12, containing a selenocyanate group in the alkyl chain were the most promising compounds on the breast cancer MCF-7 cell line. Biological assays relating to apoptosis were performed to understand the mechanism of action of these analogs. Compounds 5 and 6 were found to inhibit tubulin polymerization to the same extent as the anticancer drug vinblastine sulfate, but compounds 11 and 13 inhibited significantly better than vinblastine. Further western blot analysis suggested that compound 6 at 2 µM reduced both levels and phosphorylation state of Akt. Compounds 11 and 13 at 1 µM caused reduced Akt protein levels and strongly suppressed the phosphorylation of Akt. Therefore, 11 and 13 were demonstrated as efficient dual inhibitors of both tubulin polymerization and the Akt pathway and good candidates for further study. More importantly, the strategy of microtubule and Akt dual inhibitors might be a promising direction for developing novel drugs for cancer. PMID:21920762

  4. 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.

  5. Microwave radiation, in the absence of hyperthermia, has no detectable effect on synapsin I levels or phosphorylation

    SciTech Connect

    Browning, M.D.; Haycock, J.W.

    1988-09-01

    Recent reports have indicated that microwave radiation can produce effects on a variety of cell types in vitro. To determine whether microwave radiation might be neurotoxic, the effects of microwave radiation on synapsin I have been examined. Synapsin I is a neuron-specific phosphoprotein that is present in all neurons, where it is localized to the presynaptic terminal and is associated with synaptic vesicles. O'Callaghan and Miller have demonstrated that studies of such neuron-specific proteins can provide reliable indices of neurotoxicity. We have used a radioimmunoassay for synapsin I to determine whether microwave irradiation has any effect on the levels of synapsin I. Neither acute nor chronic exposure to microwave irradiation had any detectable effect on synapsin I levels. We have also examined the calcium-dependent phosphorylation of synapsin I in synaptosomes isolated from rats that had been subjected to microwave radiation. The phosphorylation of synapsin I in synaptosomes reflects numerous components of the presynaptic aspect of neuronal transmission. At intensities below that required to produce mild hyperthermia, no effects of microwave irradiation were seen on synapsin I phosphorylation.

  6. Modulatory role of garlicin in migration and invasion of intrahepatic cholangiocarcinoma via PI3K/AKT pathway

    PubMed Central

    Xie, Kun; Nian, Jianze; Zhu, Xingyang; Geng, Xiaoping; Liu, Fubao

    2015-01-01

    Increasing evidences have indicated the role of garlicin in inhibiting the progression of various tumors including glioma, pulmonary carcinoma and pancreatic carcinoma, via mediating cell apoptosis or cell cycle. The regulatory effect and related molecular mechanism of garlicin in intrahepatic cholangiocarcinoma, however, remained unknown. This study thus aimed to investigate this scientific issue. HCCC-9810 cell line was treated with serially diluted garlicin, followed by cell proliferation assay using MTT approach. Transwell migration and invasion assays were further employed the regulatory effect of garlicin. The expression level of p-AKT and AKT proteins in tumor cells was quantified by Western blot. The growth of tumor cells was significantly inhibited by high concentration of garlicin (> 1.5 μM). Lower concentration of garlicin showed dose-dependent inhibition of tumor cell invasion and migration. After using specific agonist IGF-1 (50 ng/mL) of PI3K/AKT signaling pathway, such facilitating effects of garlicin were depressed (P < 0.05). Western blotting showed significantly decreased phosphorylation level of AKT after treated with gradient concentrations of garlicin, while leaving the total AKT protein level unchanged. Garlicin may inhibit the invasion and migration of intrahepatic cholangiocarcinoma cells via inhibiting PI3K/AKT signaling pathway. PMID:26823715

  7. Cross-talk between the Akt and NF-κB Signaling Pathways Inhibits MEHP-Induced Germ Cell Apoptosis

    PubMed Central

    Rogers, Rachel; Ouellet, Gregory; Moyer, Ben; Rasoulpour, Teresa; Hixon, Mary

    2008-01-01

    Phthalates are ubiquitous contaminants that target the testis during in utero and postnatal development. The PI3K/Akt and nuclear factor kappa B (NF-κB) signaling pathways have been implicated in germ cell survival following testicular injury. Here we observe that Akt kinase activity increases in the testes of postnatal day 28 wild-type mice following exposure to 500 mg/kg mono-(2-ethylhexyl) phthalate (MEHP), and that loss of Akt1 results in the premature onset of germ cell apoptosis. To further determine the basis for this sensitivity, we investigated the potential for cross-talk between the PI3K/Akt and NF-κB signaling pathways. We found a twofold increase in Akt1-dependent phosphorylation of the IκBα subunit following exposure to 500 mg/kg MEHP and decreased levels of the total IκBα protein. Examination of the expression of the NF-κB subunits, p50 and p65, in Akt1 wild-type testes following MEHP exposure revealed a twofold increase in p50 mRNA at 6 h. Interestingly, in Akt1-deficient testes, basal expression of both the p50 and p65 subunits was elevated 1.6- and 4-fold, respectively. This was due, at least in part, to increased levels of oxidative stress as measured by both superoxide anion formation and increased expression of SMAC/DIABLO, a proapoptotic mitochondrial protein. In wild-type testes, MEHP-induced Akt1-dependent transcription of the antiapoptotic mitochondrial target gene, Bcl-xL. Together, these results indicate that Akt1 plays a role in the initial protection of germ cells following MEHP-induced germ cell apoptosis and that this response is partially mediated by cross-talk with the NF-κB signaling pathway and an increased sensitivity to oxidative stress. PMID:18755736

  8. 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

  9. Akt Activation Correlates with Snail Expression and Potentially Determines the Recurrence of Prostate Cancer in Patients at Stage T2 after a Radical Prostatectomy

    PubMed Central

    Chen, Wei-Yu; Hua, Kuo-Tai; Lee, Wei-Jiunn; Lin, Yung-Wei; Liu, Yen-Nien; Chen, Chi-Long; Wen, Yu-Ching; Chien, Ming-Hsien

    2016-01-01

    Our previous work demonstrated the epithelial-mesenchymal transition factor, Snail, is a potential marker for predicting the recurrence of localized prostate cancer (PCa). Akt activation is important for Snail stabilization and transcription in PCa. The purpose of this study was to retrospectively investigate the relationship between the phosphorylated level of Akt (p-Akt) in radical prostatectomy (RP) specimens and cancer biochemical recurrence (BCR). Using a tissue microarray and immunohistochemistry, the expression of p-Akt was measured in benign and neoplastic tissues from RP specimens in 53 patients whose cancer was pathologically defined as T2 without positive margins. Herein, we observed that the p-Akt level was higher in PCa than in benign tissues and was significantly associated with the Snail level. A high p-Akt image score (≥8) was significantly correlated with a higher histological Gleason sum, Snail image score, and preoperative prostate-specific antigen (PSA) value. Moreover, the high p-Akt image score and Gleason score sum (≥7) showed similar discriminatory abilities for BCR according to a receiver-operator characteristic curve analysis and were correlated with worse recurrence-free survival according to a log-rank test (p < 0.05). To further determine whether a high p-Akt image score could predict the risk of BCR, a Cox proportional hazard model showed that only a high p-Akt image score (hazard ratio (HR): 3.12, p = 0.05) and a high Gleason score sum (≥7) (HR: 1.18, p = 0.05) but not a high preoperative PSA value (HR: 0.62, p = 0.57) were significantly associated with a higher risk of developing BCR. Our data indicate that, for localized PCa patients after an RP, p-Akt can serve as a potential prognostic marker that improves predictions of BCR-free survival. PMID:27455254

  10. Sex-Specific and Estrous Cycle-Dependent Antidepressant-Like Effects and Hippocampal Akt Signaling of Leptin.

    PubMed

    Carrier, Nicole; Wang, Xuezhen; Sun, Linshan; Lu, Xin-Yun

    2015-10-01

    Sex differences in the incidence of depression and antidepressant treatment responses are well documented. Depression is twice as common in women as in men. Recent studies indicate that low levels of leptin, an adipocyte-derived hormone, are associated with increased symptoms of depression in women. Leptin has been shown to produce antidepressant-like effects in male rodents. In the present study, we examined sex differences and estrous cycle variations in antidepressant-like responses to leptin. Leptin administration significantly reduced immobility, a putative measure of behavioral despair, in the forced swim test in intact female mice in the proestrus phase but not in the diestrus phase of the estrous cycle. Moreover, leptin administration stimulated Akt phosphorylation in the hippocampus of female mice in proestrus but not in diestrus, in correlation with its differential behavioral effects in these two phases of the cycle. Leptin-induced behavioral responses and stimulation of hippocampal Akt phosphorylation in female mice were abolished by ovariectomy. By contrast, the antidepressant-like effect of leptin in male mice was not affected by gonadectomy (castration). Pretreatment with 17β-estradiol restored sensitivity to the effects of leptin on behavior and hippocampal Akt phosphorylation in ovariectomized female mice. These results suggest leptin regulates depression-like behavior and hippocampal Akt signaling in a sex-specific and estrous cycle-dependent manner. PMID:26181103

  11. Crosstalking between Androgen and PI3K/AKT Signaling Pathways in Prostate Cancer Cells*

    PubMed Central

    Lee, Suk Hyung; Johnson, Daniel; Luong, Richard; Sun, Zijie

    2015-01-01

    Both androgen action and PI3K medicated signaling pathways have been implicated in prostate tumorigenesis. Our androgen receptor (AR) conditional transgenic mice developed murine prostatic intraepithelial neoplasia (mPIN) and prostatic adenocarcinoma lesions recapitulating human prostate cancer development and progression. Role of transgenic AR contributing to malignancy was demonstrated by high degree of transgenic AR expression in atypical and tumor cells in mPIN as well as prostatic adenocarcinoma lesions of the transgenic mice, but not in adjacent normal tissue. Interestingly, reduced PI3K/Akt activation also appeared in these mouse atypical and tumor cells, suggesting an interaction between androgen and PI3K/AKT pathways. In this study, we further investigated this interaction. We showed that the androgen depletion or knockdown of AR expression results in elevated levels of active phosphorylated AKT in prostate cancer cells. Castration of conditional Pten knock-out mice showed increased Akt, phosphorylated Akt, and pS6 expression in the mouse prostate. Using a series of newly generated Ar reporter and Pten knock-out compound mice, we showed that Pten loss directly represses endogenous Ar expression in prostatic epithelial cells. Moreover, Pten loss and PI3K/Akt activation reduced Ar-mediated transcription in purified Pten-null cells. This study provides novel evidence demonstrating interplay between androgen and PI3K pathways, as well as introduces unique and relevant mouse models for further studies of PI3K and AR pathways in the context of prostate tumorigenesis. PMID:25527506

  12. Role of Akt and Ca2+ on cell permeabilization via connexin43 hemichannels induced by metabolic inhibition.

    PubMed

    Salas, Daniela; Puebla, Carlos; Lampe, Paul D; Lavandero, Sergio; Sáez, Juan C

    2015-07-01

    Connexin hemichannels are regulated under physiological and pathological conditions. Metabolic inhibition, a model of ischemia, promotes surface hemichannel activation associated, in part, with increased surface hemichannel levels, but little is known about its underlying mechanism. Here, we investigated the role of Akt on the connexin43 hemichannel's response induced by metabolic inhibition. In HeLa cells stably transfected with rat connexin43 fused to EGFP (HeLa43 cells), metabolic inhibition induced a transient Akt activation necessary to increase the amount of surface connexin43. The increase in levels of surface connexin43 was also found to depend on an intracellular Ca2+ signal increase that was partially mediated by Akt activation. However, the metabolic inhibition-induced Akt activation was not significantly affected by intracellular Ca2+ chelation. The Akt-dependent increase in connexin43 hemichannel activity in HeLa43 cells also occurred after oxygen-glucose deprivation, another ischemia-like condition, and in cultured cortical astrocytes (endogenous connexin43 expression system) under metabolic inhibition. Since opening of hemichannels has been shown to accelerate cell death, inhibition of Akt-dependent phosphorylation of connexin43 hemichannels could reduce cell death induced by ischemia/reperfusion. PMID:25779082

  13. Cancer Stem Cells in Small Cell Lung Cancer Cell Line H446: Higher Dependency on Oxidative Phosphorylation and Mitochondrial Substrate-Level Phosphorylation than Non-Stem Cancer Cells

    PubMed Central

    Jin, Fang; Miao, Yajing; Qiu, Xiaofei

    2016-01-01

    Recently, targeting cancer stem cells (CSCs) metabolism is becoming a promising therapeutic approach to improve cancer treatment outcomes. However, knowledge of the metabolic state of CSCs in small cell lung cancer is still lacking. In this study, we found that CSCs had significantly lower oxygen consumption rate and extracellular acidification rate than non-stem cancer cells. Meanwhile, this subpopulation of cells consumed less glucose, produced less lactate and maintained lower ATP levels. We also revealed that CSCs could produce more ATP through mitochondrial substrate-level phosphorylation during respiratory inhibition compared with non-stem cancer cells. Furthermore, they were more sensitive to suppression of oxidative phosphorylation. Therefore, oligomycin (inhibitor of oxidative phosphorylation) could severely impair sphere-forming and tumor-initiating abilities of CSCs. Our work suggests that CSCs represent metabolically inactive tumor subpopulations which sustain in a state showing low metabolic activity. However, mitochondrial substrate-level phosphorylation of CSCs may be more active than that of non-stem cancer cells. Moreover, CSCs showed preferential use of oxidative phosphorylation over glycolysis to meet their energy demand. These results extend our understanding of CSCs metabolism, potentially providing novel treatment strategies targeting metabolic pathways in small cell lung cancer. PMID:27167619

  14. Cancer Stem Cells in Small Cell Lung Cancer Cell Line H446: Higher Dependency on Oxidative Phosphorylation and Mitochondrial Substrate-Level Phosphorylation than Non-Stem Cancer Cells.

    PubMed

    Gao, Cuicui; Shen, Yao; Jin, Fang; Miao, Yajing; Qiu, Xiaofei

    2016-01-01

    Recently, targeting cancer stem cells (CSCs) metabolism is becoming a promising therapeutic approach to improve cancer treatment outcomes. However, knowledge of the metabolic state of CSCs in small cell lung cancer is still lacking. In this study, we found that CSCs had significantly lower oxygen consumption rate and extracellular acidification rate than non-stem cancer cells. Meanwhile, this subpopulation of cells consumed less glucose, produced less lactate and maintained lower ATP levels. We also revealed that CSCs could produce more ATP through mitochondrial substrate-level phosphorylation during respiratory inhibition compared with non-stem cancer cells. Furthermore, they were more sensitive to suppression of oxidative phosphorylation. Therefore, oligomycin (inhibitor of oxidative phosphorylation) could severely impair sphere-forming and tumor-initiating abilities of CSCs. Our work suggests that CSCs represent metabolically inactive tumor subpopulations which sustain in a state showing low metabolic activity. However, mitochondrial substrate-level phosphorylation of CSCs may be more active than that of non-stem cancer cells. Moreover, CSCs showed preferential use of oxidative phosphorylation over glycolysis to meet their energy demand. These results extend our understanding of CSCs metabolism, potentially providing novel treatment strategies targeting metabolic pathways in small cell lung cancer. PMID:27167619

  15. 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

  16. Up-regulation of survivin by AKT and hypoxia-inducible factor 1α contributes to cisplatin resistance in gastric cancer.

    PubMed

    Sun, Xue-Pu; Dong, Xuesong; Lin, Lele; Jiang, Xian; Wei, Zheng; Zhai, Bo; Sun, Bo; Zhang, Qiang; Wang, Xiaolong; Jiang, Hongchi; Krissansen, Geoffrey W; Qiao, Haiquan; Sun, Xueying

    2014-01-01

    This study investigated the contribution of survivin and its upstream regulators, AKT and hypoxia-inducible factor 1α (HIF-1α), to the resistance of gastric cancer cells to cisplatin (CDDP). We found that over-expression of survivin increased the resistance of SGC7901 and BGC823 gastric cancer cells to CDDP. Its over-expression abrogated CDDP-induced inhibition of cell proliferation and CDDP-induced cell apoptosis. In contrast, down-regulation of survivin expression using small hairpin RNA (shRNA) vectors and the small-molecule inhibitor YM155, or inhibition of survivin function using a recombinant cell-permeable dominant-negative survivin protein (dNSur9), promoted CDDP-induced apoptosis. CDDP-resistant sub-lines generated from the parental SGC7901 and BGC823 cells by exposure to increasing concentrations of CDDP expressed higher levels of HIF-1α and survivin in response to hypoxia, and higher levels of phosphorylated AKT (pAKT). Specific inhibition of AKT reduced the expression of HIF-1α and survivin, whereas specific inhibition or depletion of HIF-1α reduced survivin expression but had no effect on the expression of phosphorylated AKT. The expression levels of survivin affected the therapeutic efficacy of CDDP in treating gastric tumors in mice. Specific inhibition of survivin, AKT and HIF-1α enhanced the sensitivity of CDDP-resistant cells to CDDP. Specific inhibition of survivin, AKT and HIF-1α synergized with CDDP to suppress the growth of gastric tumors that had been engineered to overexpress survivin. In summary, the results provide evidence that up-regulation of survivin by AKT and HIF-1α contributes to CDDP resistance, indicating that inhibition of these pathways may be a potential strategy for overcoming CDDP resistance in the treatment of gastric cancer. PMID:24165223

  17. Dysregulation of AKT Pathway by SMYD2-Mediated Lysine Methylation on PTEN1,2

    PubMed Central

    Nakakido, Makoto; Deng, Zhenzhong; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2015-01-01

    Phosphatase and tensin homologue (PTEN), one of the well-characterized tumor suppressor proteins, counteracts the phosphatidylinositol 3-kinase-AKT pathway through its unique lipid phosphatase activity. The functions of PTEN are regulated by a variety of posttranslational modifications such as acetylation, oxidation, ubiquitylation, phosphorylation, and SUMOylation. However, methylation of PTEN has not been reported so far. In this study, we demonstrated that the oncogenic protein lysine methyltransferase SET and MYND domain containing 2 (SMYD2) methylates PTEN at lysine 313 in vitro and in vivo. Knockdown of SMYD2 suppressed the cell growth of breast cancer cells and attenuated phosphorylation levels of AKT, indicating that SMYD2-mediated methylation negatively regulates PTEN tumor suppressor activity and results in activation of the phosphatidylinositol 3-kinase-AKT pathway. Furthermore, PTEN protein with lysine 313 substitution diminished phosphorylation of PTEN at serine 380, which is known to inactivate tumor suppressor functions of PTEN. Taken together, our findings unveil a novel mechanism of PTEN dysregulation regulated by lysine methylation in human cancer. PMID:25925379

  18. Dysregulation of AKT Pathway by SMYD2-Mediated Lysine Methylation on PTEN.

    PubMed

    Nakakido, Makoto; Deng, Zhenzhong; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2015-04-01

    Phosphatase and tensin homologue (PTEN), one of the well-characterized tumor suppressor proteins, counteracts the phosphatidylinositol 3-kinase-AKT pathway through its unique lipid phosphatase activity. The functions of PTEN are regulated by a variety of posttranslational modifications such as acetylation, oxidation, ubiquitylation, phosphorylation, and SUMOylation. However, methylation of PTEN has not been reported so far. In this study, we demonstrated that the oncogenic protein lysine methyltransferase SET and MYND domain containing 2 (SMYD2) methylates PTEN at lysine 313 in vitro and in vivo. Knockdown of SMYD2 suppressed the cell growth of breast cancer cells and attenuated phosphorylation levels of AKT, indicating that SMYD2-mediated methylation negatively regulates PTEN tumor suppressor activity and results in activation of the phosphatidylinositol 3-kinase-AKT pathway. Furthermore, PTEN protein with lysine 313 substitution diminished phosphorylation of PTEN at serine 380, which is known to inactivate tumor suppressor functions of PTEN. Taken together, our findings unveil a novel mechanism of PTEN dysregulation regulated by lysine methylation in human cancer. PMID:25925379

  19. Metformin and rapamycin have distinct effects on the AKT pathway and proliferation in breast cancer cells.

    PubMed

    Zakikhani, Mahvash; Blouin, Marie-José; Piura, Esther; Pollak, Michael N

    2010-08-01

    Rapamycin and its analogues inhibit mTOR, which leads to decreased protein synthesis and decreased cancer cell proliferation in many experimental systems. Adenosine 5'- monophosphate-activated protein kinase (AMPK) activators such as metformin have similar actions, in keeping with the TSC2/1 pathway linking activation of AMPK to inhibition of mTOR. As mTOR inhibition by rapamycin is associated with attenuation of negative feedback to IRS-1, rapamycin is known to increase activation of AKT, which may reduce its anti-neoplastic activity. We observed that metformin exposure decreases AKT activation, an action opposite to that of rapamycin. We show that metformin (but not rapamycin) exposure leads to increased phosphorylation of IRS-1 at Ser(789), a site previously reported to inhibit downstream signaling and to be an AMPK substrate phosphorylated under conditions of cellular energy depletion. siRNA methods confirmed that reduction of AMPK levels attenuates both the IRS-1 Ser(789) phosphorylation and the inhibition of AKT activation associated with metformin exposure. Although both rapamycin and metformin inhibit mTOR (the former directly and the latter through AMPK signaling), our results demonstrate previously unrecognized differences between these agents. The data are consistent with the observation that maximal induction of apoptosis and inhibition of proliferation are greater for metformin than rapamycin. PMID:20135346

  20. Correlation between phosphorylation level of a hippocampal 86kDa protein and extinction of a behaviour in a model of Wernicke-Korsakoff syndrome.

    PubMed

    Pires, Rita G W; Pereira, Sílvia R C; Carvalho, Fabiana M; Oliveira-Silva, Ieda F; Ferraz, Vany P; Ribeiro, Angela M

    2007-06-01

    The effects of chronic ethanol and thiamine deficiency, alone or associated, on hippocampal protein phosphorylation profiles ranging in molecular weight from 30 to 250kDa molecular weight, in stimulated (high K(+) concentration) and unstimulated (basal) conditions were investigated. These treatments significantly changed the phosphorylation level of an 86kDa phosphoprotein. Thiamine deficiency, but not chronic ethanol, induced a decrease in a behavioural extinction index, which is significantly correlated to the phosphorylation level of the p86 protein. These data add to and extend previous findings by our laboratory implicating the involvement of hippocampal neurotransmission components in extinction of a behaviour which involves learning of environmental spatial cues. PMID:17395279

  1. 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

  2. Leucine minimizes denervation-induced skeletal muscle atrophy of rats through akt/mtor signaling pathways

    PubMed Central

    Ribeiro, Carolina B.; Christofoletti, Daiane C.; Pezolato, Vitor A.; de Cássia Marqueti Durigan, Rita; Prestes, Jonato; Tibana, Ramires A.; Pereira, Elaine C. L.; de Sousa Neto, Ivo V.; Durigan, João L. Q.; da Silva, Carlos A.

    2015-01-01

    The aim of the present study was to evaluate the effect of leucine treatment (0.30 mM) on muscle weight and signaling of myoproteins related to synthesis and degradation pathways of soleus muscle following seven days of complete sciatic nerve lesion. Wistar rats (n = 24) of 3–4 months of age (192 ± 23 g) were used. The animals were randomly distributed into four experimental groups (n = 6/group): control, treated with leucine (L), denervated (D) and denervated treated with leucine (DL). Dependent measures were proteins levels of AKT, AMPK, mTOR, and ACC performed by Western blot. Leucine induced a reduction in the phosphorylation of AMPK (p < 0.05) by 16% in the L and by 68% in the DL groups as compared with control group. Denervation increased AMPK by 24% in the D group as compared with the control group (p < 0.05). AKT was also modulated by denervation and leucine treatment, highlighted by the elevation of AKT phosphorylation in the D (65%), L (98%) and DL (146%) groups as compared with the control group (p < 0.05). AKT phosphorylation was 49% higher in the D group as compared with the DL group. Furthermore, denervation decreased mTOR phosphorylation by 29% in the D group as compared with the control group. However, leucine treatment induced an increase of 49% in the phosphorylation of mTOR in the L group as compared with the control group, and an increase of 154% in the DL as compared with the D group (p < 0.05). ACC phosphorylation was 20% greater in the D group than the control group. Furthermore, ACC in the soleus was 22% lower in the in the L group and 50% lower in the DL group than the respective control group (p < 0.05). In conclusion, leucine treatment minimized the deleterious effects of denervation on rat soleus muscle by increasing anabolic (AKT and mTOR) and decreasing catabolic (AMPK) pathways. These results may be interesting for muscle recovery following acute denervation, which may contribute to musculoskeletal rehabilitation after denervation

  3. Notch1 Receptor Regulates AKT Protein Activation Loop (Thr308) Dephosphorylation through Modulation of the PP2A Phosphatase in Phosphatase and Tensin Homolog (PTEN)-null T-cell Acute Lymphoblastic Leukemia Cells*

    PubMed Central

    Hales, Eric C.; Orr, Steven M.; Larson Gedman, Amanda; Taub, Jeffrey W.; Matherly, Larry H.

    2013-01-01

    Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr308 phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr308 phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr308 phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr308 by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr172 and p70S6K-Thr389, both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K. PMID:23788636

  4. Roles of Akt and SGK1 in the Regulation of Renal Tubular Transport

    PubMed Central

    Satoh, Nobuhiko; Nakamura, Motonobu; Suzuki, Masashi; Suzuki, Atsushi; Seki, George; Horita, Shoko

    2015-01-01

    A serine/threonine kinase Akt is a key mediator in various signaling pathways including regulation of renal tubular transport. In proximal tubules, Akt mediates insulin signaling via insulin receptor substrate 2 (IRS2) and stimulates sodium-bicarbonate cotransporter (NBCe1), resulting in increased sodium reabsorption. In insulin resistance, the IRS2 in kidney cortex is exceptionally preserved and may mediate the stimulatory effect of insulin on NBCe1 to cause hypertension in diabetes via sodium retention. Likewise, in distal convoluted tubules and cortical collecting ducts, insulin-induced Akt phosphorylation mediates several hormonal signals to enhance sodium-chloride cotransporter (NCC) and epithelial sodium channel (ENaC) activities, resulting in increased sodium reabsorption. Serum- and glucocorticoid-inducible kinase 1 (SGK1) mediates aldosterone signaling. Insulin can stimulate SGK1 to exert various effects on renal transporters. In renal cortical collecting ducts, SGK1 regulates the expression level of ENaC through inhibition of its degradation. In addition, SGK1 and Akt cooperatively regulate potassium secretion by renal outer medullary potassium channel (ROMK). Moreover, sodium-proton exchanger 3 (NHE3) in proximal tubules is possibly activated by SGK1. This review focuses on recent advances in understanding of the roles of Akt and SGK1 in the regulation of renal tubular transport. PMID:26491696

  5. Roles of Akt and SGK1 in the Regulation of Renal Tubular Transport.

    PubMed

    Satoh, Nobuhiko; Nakamura, Motonobu; Suzuki, Masashi; Suzuki, Atsushi; Seki, George; Horita, Shoko

    2015-01-01

    A serine/threonine kinase Akt is a key mediator in various signaling pathways including regulation of renal tubular transport. In proximal tubules, Akt mediates insulin signaling via insulin receptor substrate 2 (IRS2) and stimulates sodium-bicarbonate cotransporter (NBCe1), resulting in increased sodium reabsorption. In insulin resistance, the IRS2 in kidney cortex is exceptionally preserved and may mediate the stimulatory effect of insulin on NBCe1 to cause hypertension in diabetes via sodium retention. Likewise, in distal convoluted tubules and cortical collecting ducts, insulin-induced Akt phosphorylation mediates several hormonal signals to enhance sodium-chloride cotransporter (NCC) and epithelial sodium channel (ENaC) activities, resulting in increased sodium reabsorption. Serum- and glucocorticoid-inducible kinase 1 (SGK1) mediates aldosterone signaling. Insulin can stimulate SGK1 to exert various effects on renal transporters. In renal cortical collecting ducts, SGK1 regulates the expression level of ENaC through inhibition of its degradation. In addition, SGK1 and Akt cooperatively regulate potassium secretion by renal outer medullary potassium channel (ROMK). Moreover, sodium-proton exchanger 3 (NHE3) in proximal tubules is possibly activated by SGK1. This review focuses on recent advances in understanding of the roles of Akt and SGK1 in the regulation of renal tubular transport. PMID:26491696

  6. Mechanism of Akt1 inhibition of breast cancer cell invasionreveals a protumorigenic role for TSC2

    SciTech Connect

    Liu, Hong; Radisky, Derek C.; Nelson, Celeste M.; Zhang, Hui; Fata, Jimmie; Roth, Richard A.; Bissell, Mina J.

    2006-02-07

    Akt1 is frequently upregulated in human tumors, and has been shown to accelerate cell proliferation and to suppress programmed cell death; consequently, inhibiting the activity of Akt1 has been seen as an attractive target for therapeutic intervention. Paradoxically, hyperactivation of the Akt1 oncogene can also prevent the invasive behavior that underlies progression to metastasis. Here we show that overexpression of activated myr-Akt1 in human breast cancer cells phosphorylates and thereby targets the tumor suppressor tuberous sclerosis complex 2 (TSC2) for degradation, leading to reduced Rho-GTPase activity, decreased actin stress fibers and focal adhesions, and reduced motility and invasion. Overexpression of TSC2 rescues the migration phenotype of myr-Akt1-expressing tumor cells, and high levels of TSC2 in breast cancer patients correlate with increased metastasis and reduced survival. These data indicate that the functional properties of genes designated as oncogenes or tumor suppressor genes depends on the context of the cell type and the tissues studied, and suggest the need for caution in designing therapies targeting the function of individual genes in epithelial tissues.

  7. Lipid phosphorylation in isolated rat liver nuclei. Synthesis of polyphosphoinositides at subnuclear level.

    PubMed

    Capitani, S; Bertagnolo, V; Mazzoni, M; Santi, P; Previati, M; Antonucci, A; Manzoli, F A

    1989-08-28

    Isolated rat liver nuclei and subnuclear fractions synthesize polyphosphoinositides in vitro in a mode dependent on the presence of nuclear membrane, detergent and exogenous substrates. The nuclear membrane is not essential as a source of lipid kinases, since the addition of exogenous phosphatidylinositol or phosphatidylinositol monophosphate to reaction mixtures lacking membranes restores the synthesis of phosphatidylinositol mono- and bisphosphate, respectively. Inositide phosphorylation is best accomplished by high-salt extracted nuclei and pre-detergent lamina. These data suggest that the nucleus, and especially the nuclear periphery, is a cell compartment in which polyphosphoinositide synthesis occurs; this might be related to the progression of phosphatidylinositol metabolism-dependent signals to the genetic apparatus. PMID:2550277

  8. Upregulation of AKT3 Confers Resistance to the AKT Inhibitor MK2206 in Breast Cancer.

    PubMed

    Stottrup, Casey; Tsang, Tiffany; Chin, Y Rebecca

    2016-08-01

    Acquired resistance to molecular targeted therapy represents a major challenge for the effective treatment of cancer. Hyperactivation of the PI3K/AKT pathway is frequently observed in virtually all human malignancies, and numerous PI3K and AKT inhibitors are currently under clinical evaluation. However, mechanisms of acquired resistance to AKT inhibitors have yet to be described. Here, we use a breast cancer preclinical model to identify resistance mechanisms to a small molecule allosteric AKT inhibitor, MK2206. Using a step-wise and chronic high-dose exposure, breast cancer cell lines harboring oncogenic PI3K resistant to MK2206 were established. Using this model, we reveal that AKT3 expression is markedly upregulated in AKT inhibitor-resistant cells. Induction of AKT3 is regulated epigenetically by the bromodomain and extra terminal domain proteins. Importantly, knockdown of AKT3, but not AKT1 or AKT2, in resistant cells restores sensitivity to MK2206. AKT inhibitor-resistant cells also display an epithelial to mesenchymal transition phenotype as assessed by alterations in the levels of E-Cadherin, N-Cadherin, and vimentin, as well as enhanced invasiveness of tumor spheroids. Notably, the invasive morphology of resistant spheroids is diminished upon AKT3 depletion. We also show that resistance to MK2206 is reversible because upon drug removal resistant cells regain sensitivity to AKT inhibition, accompanied by reexpression of epithelial markers and reduction of AKT3 expression, implying that epigenetic reprogramming contributes to acquisition of resistance. These findings provide a rationale for developing therapeutics targeting AKT3 to circumvent acquired resistance in breast cancer. Mol Cancer Ther; 15(8); 1964-74. ©2016 AACR. PMID:27297869

  9. Sequence and phosphorylation level determination of two donkey beta-caseins by mass spectrometry.

    PubMed

    Cunsolo, Vincenzo; Cairone, Elisa; Saletti, Rosaria; Muccilli, Vera; Foti, Salvatore

    2009-07-01

    Two coeluting components, with experimentally measured M(r) values of 25529 and 24606 Da, were identified by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometric analysis in the dephosphorylated casein fraction of a milk sample collected from an individual donkey belonging to the Ragusano breed of the east of Sicily. By coupling enzymatic digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and RP-HPLC/nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) analysis, the two proteins were identified as donkey beta-CNs and their sequences characterized completely, using the two known beta-CNs from mare as references. The two donkey beta-CNs, showing a mass difference of 923 Da, differ by the presence of the domain E(27)SITHINK(34) in the full-length component (M(r) 25529 Da). In comparison with the mare's beta-CNs used as reference, they present nine amino acid substitutions: L-->S(37), R-->H(52), S-->N(81), P-->V(84), L-->V(91), R-->Q(203), P-->L/I(206), L-->F(210) and A-->P(219). Together, these substitutions account for the increase of 18 Da in the M(r) of the donkey beta-CNs with respect to the counterparts from the mare. The molecular mass determination by ESI-MS for the phosphorylated proteins showed that the full-length component was composed of highly multi-phosphorylated isoforms with five to seven phosphate groups. By analogy with the homologous mare's beta-CNs, the full-length (226 amino acids) beta-CN was termed variant A, whereas the shorter (218 amino acids) beta-CN was termed variant A(Delta5). PMID:19462407

  10. 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

  11. A Hot-spot of In-frame Duplications Activates the Oncoprotein AKT1 in Juvenile Granulosa Cell Tumors

    PubMed Central

    Bessière, Laurianne; Todeschini, Anne-Laure; Auguste, Aurélie; Sarnacki, Sabine; Flatters, Delphine; Legois, Bérangère; Sultan, Charles; Kalfa, Nicolas; Galmiche, Louise; Veitia, Reiner A.

    2015-01-01

    Background Ovarian granulosa cell tumors are the most common sex-cord stromal tumors and have juvenile (JGCTs) and adult forms. In a previous study we reported the occurrence of activating somatic mutations of Gαs, which transduces mitogenic signals, in 30% of the analyzed JGCTs. Methods We have searched for alterations in other proteins involved in ovarian mitogenic signaling. We focused on the PI3K–AKT axis. As we found mutations in AKT1, we analyzed the subcellular localization of the mutated proteins and performed functional explorations using Western-blot and luciferase assays. Findings We detected in-frame duplications affecting the pleckstrin-homology domain of AKT1 in more than 60% of the tumors occurring in girls under 15 years of age. The somatic status of the mutations was confirmed when peritumoral DNA was available. The JGCTs without duplications carried point mutations affecting highly conserved residues. Several of these substitutions were somatic lesions. The mutated proteins carrying the duplications had a non-wild-type subcellular distribution, with a marked enrichment at the plasma membrane. This led to a striking degree of AKT1 activation demonstrated by a strong phosphorylation level and by reporter assays. Interpretation Our study incriminates somatic mutations of AKT1 as a major event in the pathogenesis of JGCTs. The existence of AKT inhibitors currently tested in clinical trials opens new perspectives for targeted therapies for these tumors, which are currently treated with standard non-specific chemotherapy protocols. PMID:26137586

  12. IKVAV regulates ERK1/2 and Akt signalling pathways in BMMSC population growth and proliferation

    PubMed Central

    Li, B; Qiu, T; Zhang, P; Wang, X; Yin, Y; Li, S

    2014-01-01

    Objectives The molecular mechanism of bone marrow mesenchymal stem cell (BMMSC) population growth and proliferation, induced by Isoleucyl-lysyl-valyl-alanyl-valine (IKVAV), was explored in this study. Materials and methods IKVAV peptides were synthesized by the solid-phase method. Influence of IKVAV on BMMSC population growth and proliferation were investigated by assays of CCK-8, flow cytometry, real-time PCR and western blotting. Results IKVAV peptide was found to induce proliferation and proliferating cell nuclear antigen (PCNA) synthesis of BMMSC in a dose- and time-dependent manner. Cell cycle analysis showed that the proportion of IKVAV-treated BMMSC in S phase in was higher than controls. Western blot results suggested that mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathways were activated by IKVAV by enhancing phosphorylation levels of ERK1/2 and Akt in the BMMSCs. Meanwhile, phosphorylation levels of ERK1/2 and Akt were partially blocked by ERK1/2 inhibitor (PD98059) and Akt inhibitor (wortmannin), respectively. Conclusions Our results demonstrated that IKVAV stimulated BMMSC population growth and proliferation by activating MAPK/ERK1/2 and PI3K/Akt signalling pathways. This study is the first to reveal an enhancement effect of IKVAV peptide on BMMSC at the signal transduction level, and the outcome could provide experimental evidence for application of IKVAV-grafted scaffolds in the field of BMMSC-based tissue engineering. PMID:24617901

  13. Accelerated Tumor Growth Mediated by Sub-lytic Levels of Antibody-Induced Complement Activation is Associated with Activation of the PI3K/AKT Survival Pathway

    PubMed Central

    Wu, Xiaohong; Ragupathi, Govind; Panageas, Katherine; Hong, Feng; Livingston, Philip O.

    2013-01-01

    Purpose We addressed the possibility that low levels of tumor cell bound antibodies targeting gangliosides might accelerate tumor growth. Experimental Design To test this hypothesis, we treated mice with a range of mAb doses against GM2, GD2, GD3 and CD20 after challenge with tumors expressing these antigens and tested the activity of the same mAbs in-vitro. We also explored the mechanisms behind the complement-mediated tumor growth acceleration that we observed and an approach to overcome it. Results Serologically detectable levels of IgM-mAb against GM2 are able to delay or prevent tumor growth of high GM2-expressing cell lines both in-vitro and in a SCID mouse model, while very low levels of this mAb resulted in slight but consistent acceleration of tumor growth in both settings. Surprisingly, this is not restricted to IgM antibodies targeting GM2 but consistent against IgG-mAb targeting GD3 as well. These findings were mirrored by in-vitro studies with antibodies against these antigens as well as GD2 and CD20 (with Rituxan), and shown to be complement-dependent in all cases. Complement-mediated accelerated growth of cultured tumor cell lines initiated by low mAb levels was associated with activation of the PI3K/AKT survival pathway and significantly elevated levels of both p-AKT and p-PRAS40. This complement-mediated PI3K-activation and accelerated tumor growth in-vitro and in-vivo are eliminated by PI3K-inhibitors NVP-BEZ235 and Wortmannin. These PI3K-inhibitors also significantly increased efficacy of high doses of these 4 mAbs. Conclusion Our findings suggest that manipulation of the PI3K/AKT pathway and its signaling network can significantly increase the potency of passively administered mAbs and vaccine-induced-antibodies targeting a variety of tumor-cell-surface-antigens. PMID:23833306

  14. Impairments in Site-Specific AS160 Phosphorylation and Effects of Exercise Training

    PubMed Central

    Consitt, Leslie A.; Van Meter, Jessica; Newton, Christopher A.; Collier, David N.; Dar, Moahad S.; Wojtaszewski, Jørgen F.P.; Treebak, Jonas T.; Tanner, Charles J.; Houmard, Joseph A.

    2013-01-01

    The purpose of this study was to determine if site-specific phosphorylation at the level of Akt substrate of 160 kDa (AS160) is altered in skeletal muscle from sedentary humans across a wide range of the adult life span (18–84 years of age) and if endurance- and/or strength-oriented exercise training could rescue decrements in insulin action and skeletal muscle AS160 phosphorylation. A euglycemic-hyperinsulinemic clamp and skeletal muscle biopsies were performed in 73 individuals encompassing a wide age range (18–84 years of age), and insulin-stimulated AS160 phosphorylation was determined. Decrements in whole-body insulin action were associated with impairments in insulin-induced phosphorylation of skeletal muscle AS160 on sites Ser-588, Thr-642, Ser-666, and phospho-Akt substrate, but not Ser-318 or Ser-751. Twelve weeks of endurance- or strength-oriented exercise training increased whole-body insulin action and reversed impairments in AS160 phosphorylation evident in insulin-resistant aged individuals. These findings suggest that a dampening of insulin-induced phosphorylation of AS160 on specific sites in skeletal muscle contributes to the insulin resistance evident in a sedentary aging population and that exercise training is an effective intervention for treating these impairments. PMID:23801578

  15. Identification of 4-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-{[(3S)-3-piperidinylmethyl]oxy}-1H-imidazo[4,5-c]pyridin-4-yl)-2-methyl-3-butyn-2-ol (GSK690693), a Novel Inhibitor of AKT Kinase

    SciTech Connect

    Heerding, Dirk A.; Rhodes, Nelson; Leber, Jack D.; Clark, Tammy J.; Keenan, Richard M.; Lafrance, Louis V.; Li, Mei; Safonov, Igor G.; Takata, Dennis T.; Venslavsky, Joseph W.; Yamashita, Dennis S.; Choudhry, Anthony E.; Copeland, Robert A.; Lai, Zhihong; Schaber, Michael D.; Tummino, Peter J.; Strum, Susan L.; Wood, Edgar R.; Duckett, Derek R.; Eberwein, Derek; Knick, Victoria B.; Lansing, Timothy J.; McConnell, Randy T.; Zhang, ShuYun; Minthorn, Elisabeth A.; Concha, Nestor O.; Warren, Gregory L.; Kumar, Rakesh

    2009-07-22

    Overexpression of AKT has an antiapoptotic effect in many cell types, and expression of dominant negative AKT blocks the ability of a variety of growth factors to promote survival. Therefore, inhibitors of AKT kinase activity might be useful as monotherapy for the treatment of tumors with activated AKT. Herein, we describe our lead optimization studies culminating in the discovery of compound 3g (GSK690693). Compound 3g is a novel ATP competitive, pan-AKT kinase inhibitor with IC{sub 50} values of 2, 13, and 9 nM against AKT1, 2, and 3, respectively. An X-ray cocrystal structure was solved with 3g and the kinase domain of AKT2, confirming that 3g bound in the ATP binding pocket. Compound 3g potently inhibits intracellular AKT activity as measured by the inhibition of the phosphorylation levels of GSK3{beta}. Intraperitoneal administration of 3g in immunocompromised mice results in the inhibition of GSK3{beta} phosphorylation and tumor growth in human breast carcinoma (BT474) xenografts.

  16. Gardenamide A Protects RGC-5 Cells from H2O2-Induced Oxidative Stress Insults by Activating PI3K/Akt/eNOS Signaling Pathway

    PubMed Central

    Wang, Rikang; Peng, Lizhi; Zhao, Jiaqiang; Zhang, Laitao; Guo, Cuiping; Zheng, Wenhua; Chen, Heru

    2015-01-01

    Gardenamide A (GA) protects the rat retinal ganglion (RGC-5) cells against cell apoptosis induced by H2O2. The protective effect of GA was completely abrogated by the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the specific protein kinase B (Akt) inhibitor Akt VIII respectively, indicating that the protective mechanism of GA is mediated by the PI3K/Akt signaling pathway. The specific extracellular signal-regulated kinase (ERK1/2) inhibitor PD98059 could not block the neuroprotection of GA. GA attenuated the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) induced by H2O2. Western blotting showed that GA promoted the phosphorylation of ERK1/2, Akt and endothelial nitric oxide synthase (eNOS), respectively, and effectively reversed the H2O2-inhibited phosphorylation of these three proteins. LY294002 completely inhibited the GA-activated phosphorylation of Akt, while only partially inhibiting eNOS. This evidence implies that eNOS may be activated directly by GA. PD98059 attenuated only partially the GA-induced phosphorylation of ERK1/2 with/without the presence of H2O2, indicating that GA may activate ERK1/2 directly. All these results put together confirm that GA protects RGC-5 cells from H2O2 insults via the activation of PI3K/Akt/eNOS signaling pathway. Whether the ERK1/2 signaling pathway is involved requires further investigations. PMID:26389892

  17. Dissecting the EGFR-PI3K-AKT pathway in oral cancer highlights the role of the EGFR variant III and its clinical relevance

    PubMed Central

    2013-01-01

    Background Dysregulated epidermal growth factor receptor (EGFR)-phosphoinositide-3-kinase (PI3K)-AKT signaling is considered pivotal for oral cancer, and the pathway is a potential candidate for therapeutic targeting. Results A total of 108 archival samples which were from surgically resected oral cancer were examined. Immunohistochemical staining showed the protein expression of membranous wild-type EGFR and cytoplasmic phosphorylated AKT was detected in 63.9% and 86.9% of the specimens, respectively. In 49.1% of the samples, no phosphatase and tensin homolog (PTEN) expression was detected. With regard to the EGFR variant III (EGFRvIII), 75.0% of the samples showed positive expression for moderate to severe staining, 31.5% of which had high expression levels. Real-time polymerase chain reaction assays for gene copy number assessment of PIK3CA revealed that 24.8% of the samples had alterations, and of EGFR showed that 49.0% had amplification. Direct sequencing of PIK3CA gene showed 2.3% of the samples had a hotspot point mutation. Statistical assessment showed the expression of the EGFRvIII correlated with the T classification and TNM stage. The Kaplan-Meier analyses for patient survival showed that the individual status of phosphorylated AKT and EGFRvIII led to significant differences in survival outcome. The multivariate analysis indicated that phosphorylated AKT, EGFRvIII expression and disease stage were patient survival determinants. Conclusions Aberrations in the EGFR-PI3K-AKT pathway were frequently found in oral cancers. EGFRvIII and phosphorylated AKT were predictors for the patient survival and clinical outcome. PMID:23806066

  18. 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

  19. PI 3-kinase-dependent phosphorylation of Plk1–Ser99 promotes association with 14-3-3γ and is required for metaphase–anaphase transition

    PubMed Central

    Kasahara, Kousuke; Goto, Hidemasa; Izawa, Ichiro; Kiyono, Tohru; Watanabe, Nobumoto; Elowe, Sabine; Nigg, Erich A; Inagaki, Masaki

    2013-01-01

    Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1–Thr210 phosphorylation. Plk1–Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1–Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1–Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1–Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1–Ser99 phosphorylation downstream of the PI3K–Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase. PMID:23695676

  20. Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk

    PubMed Central

    2012-01-01

    Background The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-β/γ1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1α induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells. Results Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1α solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling. Conclusions Here we show that hybrid cells could evolve exhibiting a differential active RAF-AKT

  1. Glutamine starvation enhances PCV2 replication via the phosphorylation of p38 MAPK, as promoted by reducing glutathione levels.

    PubMed

    Chen, Xingxiang; Shi, Xiuli; Gan, Fang; Huang, Da; Huang, Kehe

    2015-01-01

    Glutamine has a positive effect on ameliorating reproductive failure caused by porcine circovirus type 2 (PCV2). However, the mechanism by which glutamine affects PCV2 replication remains unclear. This study was conducted to investigate the effects of glutamine on PCV2 replication and its underlying mechanisms in vitro. The results show that glutamine promoted PK-15 cell viability. Surprisingly, glutamine starvation significantly increased PCV2 replication. The promotion of PCV2 replication by glutamine starvation disappeared after fresh media with 4 mM glutamine was added. Likewise, promotion of PCV2 was observed after adding buthionine sulfoximine (BSO). Glutamine starvation or BSO treatment increased the level of p38 MAPK phosphorylation and PCV2 replication in PK-15 cells. Meanwhile, p38 MAPK phosphorylation and PCV2 replication significantly decreased in p38-knockdown PK-15 cells. Promotion of PCV2 replication caused by glutamine starvation could be blocked in p38-knockdown PK-15 cells. Therefore, glutamine starvation increased PCV2 replication by promoting p38 MAPK activation, which was associated with the down regulation of intracellular glutathione levels. Our findings may contribute toward interpreting the possible pathogenic mechanism of PCV2 and provide a theoretical reference for application of glutamine in controlling porcine circovirus-associated diseases. PMID:25879878

  2. 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.

  3. 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

  4. 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

  5. Fucoidan/FGF-2 induces angiogenesis through JNK- and p38-mediated activation of AKT/MMP-2 signalling

    SciTech Connect

    Kim, Beom Su; Park, Ji-Yun; Kang, Hyo-Jin; Kim, Hyung-Jin; Lee, Jun

    2014-08-08

    Graphical abstract: Schematic diagram of the angiogenic activity mechanism by FGF-2/fucoidan treatment in HUVECs. Fucoidan enhances the FGF-2-induced phosphorylation of p38, JNK, and ERK MAPKs. However, p38 and JNK were involved in AKT phosphorylation and MMP-2 activation and resulted in enhanced angiogenic activity, such as tube formation and migration, in HUVECs. - Highlights: • The angiogenic activity of fucoidan in HUVECs was explored. • Fucoidan enhanced HUVEC proliferation, migration, and tube formation. • Fucoidan enhanced angiogenesis through p38 and JNK but not ERK in HUVECs. • Fucoidan targeted angiogenesis-mediated AKT/MMP-2 signalling in HUVECs. - Abstract: Angiogenesis is an important biological process in tissue development and repair. Fucoidan has previously been shown to potentiate in vitro tube formation in the presence of basic fibroblast growth factor (FGF-2). However, the underlying molecular mechanism remains largely unknown. This study was designed to investigate the action of fucoidan in angiogenesis in human umbilical vein endothelial cells (HUVECs) and to explore fucoidan-signalling pathways. First, we evaluated the effect of fucoidan on cell proliferation. Matrigel-based tube formation and wound healing assays were performed to investigate angiogenesis. Matrix metalloproteinase-2 (MMP-2) mRNA expression and activity levels were analysed by reverse transcription polymerase chain reaction (RT-PCR) and zymography, respectively. Additionally, phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) was detected by Western blot. The results indicate that fucoidan treatment significantly increased cell proliferation in the presence of FGF-2. Moreover, compared to the effect of FGF-2 alone, fucoidan and FGF-2 had a greater effect on tube formation and cell migration, and this effect was found to be synergistic. Furthermore, fucoidan enhanced the phosphorylation of extracellular signal-regulated kinase (ERK

  6. 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

  7. 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

  8. Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells.

    PubMed

    Kuo, Po-Lin; Hsu, Ya-Ling; Cho, Chien-Yu

    2006-12-01

    This study is the first to investigate the anticancer effect of plumbagin in human breast cancer cells. Plumbagin exhibited cell proliferation inhibition by inducing cells to undergo G2-M arrest and autophagic cell death. Blockade of the cell cycle was associated with increased p21/WAF1 expression and Chk2 activation, and reduced amounts of cyclin B1, cyclin A, Cdc2, and Cdc25C. Plumbagin also reduced Cdc2 function by increasing the association of p21/WAF1/Cdc2 complex and the levels of inactivated phospho-Cdc2 and phospho-Cdc25C by Chk2 activation. Plumbagin triggered autophagic cell death but not predominantly apoptosis. Pretreatment of cells with autophagy inhibitor bafilomycin suppressed plumbagin-mediated cell death. We also found that plumbagin inhibited survival signaling through the phosphatidylinositol 3-kinase/AKT signaling pathway by blocking the activation of AKT and downstream targets, including the mammalian target of rapamycin, forkhead transcription factors, and glycogen synthase kinase 3beta. Phosphorylation of both of mammalian target of rapamycin downstream targets, p70 ribosomal protein S6 kinase and 4E-BP1, was also diminished. Overexpression of AKT by AKT cDNA transfection decreased plumbagin-mediated autophagic cell death, whereas reduction of AKT expression by small interfering RNA potentiated the effect of plumbagin, supporting the inhibition of AKT being beneficial to autophagy. Furthermore, suppression of AKT by plumbagin enhanced the activation of Chk2, resulting in increased inactive phosphorylation of Cdc25C and Cdc2. Further investigation revealed that plumbagin inhibition of cell growth was also evident in a nude mouse model. Taken together, these results imply a critical role for AKT inhibition in plumbagin-induced G2-M arrest and autophagy of human breast cancer cells. PMID:17172425

  9. Statins and ATP regulate nuclear pAkt via the P2X7 purinergic receptor in epithelial cells

    SciTech Connect

    Mistafa, Oras; Hoegberg, Johan; Stenius, Ulla

    2008-01-04

    Many studies have documented P2X7 receptor functions in cells of mesenchymal origin. P2X7 is also expressed in epithelial cells and its role in these cells remains largely unknown. Our data indicate that P2X7 regulate nuclear pAkt in epithelial cells. We show that low concentration of atorvastatin, a drug inhibiting HMG-CoA reductase and cholesterol metabolism, or the natural agonist extracellular ATP rapidly decreased the level of insulin-induced phosphorylated Akt in the nucleus. This effect was seen within minutes and was inhibited by P2X7 inhibitors. Experiments employing P2X7 siRNA and HEK293 cells heterologously expressing P2X7 and in vivo experiments further supported an involvement of P2X7. These data indicate that extracellular ATP and statins via the P2X7 receptor modulate insulin-induced Akt signaling in epithelial cells.

  10. 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

  11. Akt signaling in platelets and thrombosis

    PubMed Central

    Woulfe, Donna S

    2010-01-01

    Akt is a Ser–Thr kinase with pleiotropic effects on cell survival, growth and metabolism. Recent evidence from gene-deletion studies in mice, and analysis of human platelets treated with Akt inhibitors, suggest that Akt regulates platelet activation, with potential consequences for thrombosis. Akt activation is regulated by the level of phosphoinositide 3-phosphates, and proteins that regulate concentrations of this lipid also regulate Akt activation and platelet function. Although the effectors through which Akt contributes to platelet activation are not definitively known, several candidates are discussed, including endothelial nitric oxide synthase, glycogen synthase kinase 3β, phosphodiesterase 3A and the integrin β3 tail. Selective inhibitors of Akt isoforms or of proteins that contribute to its activation, such as individual PI3K isoforms, may make attractive targets for antithrombotic therapy. This review summarizes the current literature describing Akt activity and its regulation in platelets, including speculation regarding the future of Akt or its regulatory pathways as targets for the development of antithrombotic therapies. PMID:20352060

  12. Increased Hepatic Fatty Acids Uptake and Oxidation by LRPPRC-Driven Oxidative Phosphorylation Reduces Blood Lipid Levels

    PubMed Central

    Lei, Shi; Sun, Run-zhu; Wang, Di; Gong, Mei-zhen; Su, Xiang-ping; Yi, Fei; Peng, Zheng-wu

    2016-01-01

    Hyperlipidemia is one of the major risk factors of atherosclerosis and other cardiovascular diseases. This study aimed to investigate the impact of leucine rich pentatricopeptide repeat containing protein (LRPPRC)-driven hepatic oxidative phoshorylation on blood lipid levels. The hepatic LRPPRC level was modulated by liver-specific transgenic or adeno-associated virus 8 carried shRNA targeting Lrpprc (aav-shLrpprc). Mice were fed with a high fat diet to induce obesity. Gene expression was analyzed by quantitative real-time PCR and / or western blot. The hepatic ATP level, hepatic and serum lipids contents, and mitochondria oxidative phosphorylation (OxPhos) complex activities were measured using specific assay kits. The uptake and oxidation of fatty acid by hepatocytes were assessed using 14C-palmitate. LRPPRC regulated the expression of genes encoded by mitochondrial genome but not those by nuclear genome involved in mitochondria biogenesis, OxPhos, and lipid metabolism. Increased OxPhos in liver mediated by LRPPRC resulted in the increase of hepatic ATP level. Lrpprc promoted palmitate uptake and oxidation by hypatocytes. The hepatic and serum triglyceride and total cholesterol levels were inversely associated with the hepatic LRPPRC level. These data demonstrated that LRPPRC-driven hepatic OxPhos could promote fatty acids uptake and oxidation by hepatocytes and reduce both hepatic and circulating triglyceride and cholesterol levels. PMID:27462273

  13. Increased Hepatic Fatty Acids Uptake and Oxidation by LRPPRC-Driven Oxidative Phosphorylation Reduces Blood Lipid Levels.

    PubMed

    Lei, Shi; Sun, Run-Zhu; Wang, Di; Gong, Mei-Zhen; Su, Xiang-Ping; Yi, Fei; Peng, Zheng-Wu

    2016-01-01

    Hyperlipidemia is one of the major risk factors of atherosclerosis and other cardiovascular diseases. This study aimed to investigate the impact of leucine rich pentatricopeptide repeat containing protein (LRPPRC)-driven hepatic oxidative phoshorylation on blood lipid levels. The hepatic LRPPRC level was modulated by liver-specific transgenic or adeno-associated virus 8 carried shRNA targeting Lrpprc (aav-shLrpprc). Mice were fed with a high fat diet to induce obesity. Gene expression was analyzed by quantitative real-time PCR and / or western blot. The hepatic ATP level, hepatic and serum lipids contents, and mitochondria oxidative phosphorylation (OxPhos) complex activities were measured using specific assay kits. The uptake and oxidation of fatty acid by hepatocytes were assessed using (14)C-palmitate. LRPPRC regulated the expression of genes encoded by mitochondrial genome but not those by nuclear genome involved in mitochondria biogenesis, OxPhos, and lipid metabolism. Increased OxPhos in liver mediated by LRPPRC resulted in the increase of hepatic ATP level. Lrpprc promoted palmitate uptake and oxidation by hypatocytes. The hepatic and serum triglyceride and total cholesterol levels were inversely associated with the hepatic LRPPRC level. These data demonstrated that LRPPRC-driven hepatic OxPhos could promote fatty acids uptake and oxidation by hepatocytes and reduce both hepatic and circulating triglyceride and cholesterol levels. PMID:27462273

  14. SchA-p85-FAK complex dictates isoform-specific activation of Akt2 and subsequent PCBP1-mediated post-transcriptional regulation of TGFβ-mediated epithelial to mesenchymal transition in human lung cancer cell line A549.

    PubMed

    Xue, Xinying; Wang, Xin; Liu, Yuxia; Teng, Guigen; Wang, Yong; Zang, Xuefeng; Wang, Kaifei; Zhang, Jinghui; Xu, Yali; Wang, Jianxin; Pan, Lei

    2014-08-01

    A post-transcriptional pathway by which TGF-β modulates expression of specific proteins, Disabled-2 (Dab2) and Interleukin-like EMT Inducer (ILEI), inherent to epithelial to mesenchymal transition (EMT) in murine epithelial cells through Akt2-mediated phosphorylation of poly r(C) binding protein (PCBP1), has been previously elucidated. The aims of the current study were to determine if the same mechanism is operative in the non-small cell lung cancer (NSCLC) cell line, A549, and to delineate the underlying mechanism. Steady-state transcript and protein expression levels of Dab2 and ILEI were examined in A549 cells treated with TGF-β for up to 48 h. Induction of translational de-repression in this model was quantified by polysomal fractionation followed by qRT-PCR. The underlying mechanism of isoform-specific activation of Akt2 was elucidated through a combination of co-immunoprecipitation studies. TGF-β induced EMT in A549 cells concomitant with translational upregulation of Dab2 and ILEI proteins through isoform-specific activation of Akt2 followed by phosphorylation of PCBP1 at serine-43. Our experiments further elucidated that the adaptor protein SchA is phosphorylated at tyrosine residues following TGF-β treatment, which initiated a signaling cascade resulting in the sequential recruitment of p85 subunit of PI3K and focal adhesion kinase (FAK). The SchA-FAK-p85 complex subsequently selectively recruited and activated Akt2, not Akt1. Inhibition of the p85 subunit through phosphorylated 1257 peptide completely attenuated EMT in these cells. We have defined the underlying mechanism responsible for isoform-specific recruitment and activation of Akt2, not Akt1, during TGF-β-mediated EMT in A549 cells. Inhibition of the formation of this complex thus represents an important and novel therapeutic target in metastatic lung carcinoma. PMID:24819169

  15. [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

  16. Sirt1 decreased adipose inflammation by interacting with Akt2 and inhibiting mTOR/S6K1 pathway in mice.

    PubMed

    Liu, Zhenjiang; Gan, Lu; Liu, Guannv; Chen, Yizhe; Wu, Tianjiao; Feng, Fei; Sun, Chao

    2016-08-01

    Sirtuin type 1 (Sirt1) and protein kinase B (Akt2) are associated with development of obesity and inflammation, but the molecular mechanisms of Sirt1 and Akt2 interaction on adipose inflammation remain unclear. To explore these mechanisms, a mouse model was used. Mice were fed with a high-fat diet (HFD) for 8 weeks, with interventions of resveratrol (RES) or nicotinamide (NAM) during the last 15 days. The HFD reduced Sirt1 mRNA in adipose tissue and elevated interleukin-6 (IL-6) expression. RES reduced the adipose tissue weight, increased the Sirt1 mRNA level, and reduced both mRNA and protein levels of IL-6, MCP-1, inducible nitric oxide synthase, and TNF-α by inhibiting phosphorylation of Akt2 in adipose tissue. Additionally, macrophage type I marker genes were reduced while macrophage type II marker genes were elevated by RES addition. Moreover, activation of Akt2 signal by using insulin significantly blunted the inhibitory effect of RES on adipose inflammation. Immunoprecipitation assay demonstrated that RES enhances the protein-protein interaction between Sirt1 and Akt2, but NAM inhibits this interaction. Furthermore, Sirt1 significantly reduced the levels of raptor and inactivated mammalian target of rapamycin (mTOR)C1 signal by interacting with Akt2, and confirmed that RES attenuated adipose inflammation by inhibiting the mTOR/S6K1 pathway via rapamycin. PMID:27317762

  17. Melatonin Attenuates Her-2, p38 MAPK, p-AKT, and mTOR Levels in Ovarian Carcinoma of Ethanol-Preferring Rats

    PubMed Central

    Ferreira, Grazielle M.; Martinez, Marcelo; Camargo, Isabel Cristina C.; Domeniconi, Raquel F.; Martinez, Francisco Eduardo; Chuffa, Luiz Gustavo A.

    2014-01-01

    Epidermal growth factor receptors 2 (Her-2) and 4 (Her-4) are closely associated with ovarian cancer (OC) progression and metastasis, and a more complete understanding of these signaling pathways allow the development of new therapeutic strategies. Melatonin (Mel) is recognized as having several anticancer properties and has been reported to modulate Her-2 system in aggressive tumors. Here, we investigated OC and the role of Mel therapy on the Her-2- and Her-4-signaling pathway related to downstream molecules in an ethanol-preferring rat model. To induce OC, the left ovary was injected directly with a single dose of 100 µg 7,12-dimethylbenz(a)anthracene (DMBA) dissolved in 10 µL of sesame oil under the bursa. Right ovaries were used as sham-surgery controls. After developing OC, half of the animals received i.p. injections of Mel (200 µg/100 g b.w./day) for 60 days. While Mel therapy was unable to reduce Her-4 and phosphoinositide 3-kinase (PI3K) levels, it was able to suppress the OC-related increase in the levels of the Her-2, p38 mitogen-activated protein kinases (p38 MAPK), protein kinase B (phospho-AKT), and mammalian target of rapamycin (mTOR). In addition, Mel significantly attenuated the expression of Her-2, p38 MAPK, and p-AKT, which are involved in OC signaling during ethanol intake. Collectively, our results suggest that Mel attenuates the Her-2-signaling pathway in OC of ethanol-preferring rats, providing an effective contribution for further development of adjuvant therapies. PMID:25368672

  18. Regulation of cell apoptosis and proliferation in pancreatic cancer through PI3K/Akt pathway via Polo-like kinase 1

    PubMed Central

    MAO, YONGHUAN; XI, LING; LI, QUAN; CAI, ZELING; LAI, YIMEI; ZHANG, XINHUA; YU, CHUNZHAO

    2016-01-01

    Pancreatic cancer has a poor prognosis. It is reported that the PI3K/Akt pathway is activated in many cancers, and inhibition of the PI3K/Akt pathway can induce cell apoptosis in most cancers. Polo-like kinase 1 (Plk1) is also overexpressed in most malignancies, and it controls multiple aspects of mitosis and apoptosis. Previous studies identified that PI3K/Akt-dependent phosphorylation of Plk1-Ser99 is required for metaphase-anaphase transition. In this study, we aimed to investigate the molecular mechanism of PI3K/Akt pathway regulating cell proliferation and apoptosis in pancreatic cancer cell lines (AsPC-1, BxPC-3, PANC-1). Immunohistochemistry (IHC) was used to assess Akt levels in human pancreatic tissues and pancreatic cancer tissues. MTT assay was used to detect cell proliferation. The mRNA was quantified by quantitative reverse transcription-PCR. Western blot analysis was used to detect the protein levels of p-Akt, Akt, Plk1, BAX, Bcl-2, XIAP, cleaved caspase-3 and caspase-3. Recombinant adenovirus vector containing Plk1-shRNA was constructed to inhibit Plk1 expression. Cell apoptosis was detected by flow cytometry and the apoptosis of tumor xenograft was assessed by TUNEL assay. The study showed that inhibition of PI3K/Akt pathway can induce cell apoptosis and reduce cell proliferation by downregulating Plk1 in vitro and in vivo. Additionally, Plk1 inhibition can lead to cancer cell apoptosis through inactivating XIAP, activating caspase-3, upregulating BAX and downregulating Bcl-2. Therefore, this study provided the molecular mechanism of PI3K/Akt pathway and Plk1 in the pancreatic cancer cell proliferation and apoptosis, which may benefit for the therapy of pancreatic cancer. PMID:27220401

  19. Targeted Apoptotic Effects of Thymoquinone and Tamoxifen on XIAP Mediated Akt Regulation in Breast Cancer

    PubMed Central

    Rajput, Shashi; Kumar, B. N. Prashanth; Sarkar, Siddik; Das, Subhasis; Azab, Belal; Santhekadur, Prasanna K.; Das, Swadesh K.; Emdad, Luni; Sarkar, Devanand; Fisher, Paul B.; Mandal, Mahitosh

    2013-01-01

    X-linked inhibitor of apoptosis protein (XIAP) is constitutively expressed endogenous inhibitor of apoptosis, exhibit its antiapoptotic effect by inactivating key caspases such as caspase-3, caspase-7 and caspase-9 and also play pivotal role in rendering cancer chemoresistance. Our studies showed the coadministration of TQ and TAM resulting in a substantial increase in breast cancer cell apoptosis and marked inhibition of cell growth both in vitro and in vivo. Anti-angiogenic and anti-invasive potential of TQ and TAM was assessed through in vitro studies. This novel combinatorial regimen leads to regulation of multiple cell signaling targets including inactivation of Akt and XIAP degradation. At molecular level, TQ and TAM synergistically lowers XIAP expression resulting in binding and activation of caspase-9 in apoptotic cascade, and interfere with cell survival through PI3-K/Akt pathway by inhibiting Akt phosphorylation. Cleaved caspase-9 further processes other intracellular death substrates such as PARP thereby shifting the balance from survival to apoptosis, indicated by rise in the sub-G1 cell population. This combination also downregulates the expression of Akt-regulated downstream effectors such as Bcl-xL, Bcl-2 and induce expression of Bax, AIF, cytochrome C and p-27. Consistent with these results, overexpression studies further confirmed the involvement of XIAP and its regulatory action on Akt phosphorylation along with procaspase-9 and PARP cleavage in TQ-TAM coadministrated induced apoptosis. The ability of TQ and TAM in inhibiting XIAP was confirmed through siRNA-XIAP cotransfection studies. This novel modality may be a promising tool in breast cancer treatment. PMID:23613836

  20. 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

  1. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

    PubMed Central

    Lima, V.V.; Lobato, N.S.; Filgueira, F.P.; Webb, R.C.; Tostes, R.C.; Giachini, F.R.

    2014-01-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  2. Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

    PubMed

    Lima, V V; Lobato, N S; Filgueira, F P; Webb, R C; Tostes, R C; Giachini, F R

    2014-10-01

    O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction. PMID:25140811

  3. JNK/PI3K/Akt signaling pathway is involved in myocardial ischemia/reperfusion injury in diabetic rats: effects of salvianolic acid A intervention.

    PubMed

    Chen, Qiuping; Xu, Tongda; Li, Dongye; Pan, Defeng; Wu, Pei; Luo, Yuanyuan; Ma, Yanfeng; Liu, Yang

    2016-01-01

    Recent studies have demonstrated that diabetes impairs the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway, while insulin resistance syndrome has been associated with alterations of this pathway in diabetic rats after ischemia/reperfusion (I/R), and activation of C-jun N-terminal kinase (JNK) is involved. The present study was designed to investigate whether inhibiting JNK activity would partially restore the PI3K/Akt signaling pathway and protect against myocardial I/R injury in diabetic rats, and to explore the effect of intervention with salvianolic acid A (Sal A). The inhibitor of JNK (SP600125) and Sal A were used in type 2 diabetic (T2D) rats, outcome measures included heart hemodynamic data, myocardial infarct size, the release of lactate dehydrogenase (LDH), SERCA2a activity, cardiomyocyte apotosis, expression levels of Bcl-2, Bax and cleaved caspase-3, and the phosphorylation status of Akt and JNK. The p-Akt levels were increased after myocardial I/R in non-diabetic rats, while there was no change in diabetic rats. Pretreatment with the SP600125 and Sal A decreased the p-JNK levels and increased the p-Akt levels in diabetic rats with I/R, and heart hemodynamic data improved, infarct size and LDH release decreased, SERCA2a activity increased, Bax and cleaved caspase-3 expression levels decreased, and the expression of Bcl-2 and the Bcl-2/Bax ratio increased. Our results suggest that the JNK/PI3K/Akt signaling pathway is involved in myocardial I/R injury in diabetic rats and Sal A exerts an anti-apoptotic effect and improves cardiac function following I/R injury through the JNK/PI3K/Akt signaling pathway in this model. PMID:27398138

  4. JNK/PI3K/Akt signaling pathway is involved in myocardial ischemia/reperfusion injury in diabetic rats: effects of salvianolic acid A intervention

    PubMed Central

    Chen, Qiuping; Xu, Tongda; Li, Dongye; Pan, Defeng; Wu, Pei; Luo, Yuanyuan; Ma, Yanfeng; Liu, Yang

    2016-01-01

    Recent studies have demonstrated that diabetes impairs the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway, while insulin resistance syndrome has been associated with alterations of this pathway in diabetic rats after ischemia/reperfusion (I/R), and activation of C-jun N-terminal kinase (JNK) is involved. The present study was designed to investigate whether inhibiting JNK activity would partially restore the PI3K/Akt signaling pathway and protect against myocardial I/R injury in diabetic rats, and to explore the effect of intervention with salvianolic acid A (Sal A). The inhibitor of JNK (SP600125) and Sal A were used in type 2 diabetic (T2D) rats, outcome measures included heart hemodynamic data, myocardial infarct size, the release of lactate dehydrogenase (LDH), SERCA2a activity, cardiomyocyte apotosis, expression levels of Bcl-2, Bax and cleaved caspase-3, and the phosphorylation status of Akt and JNK. The p-Akt levels were increased after myocardial I/R in non-diabetic rats, while there was no change in diabetic rats. Pretreatment with the SP600125 and Sal A decreased the p-JNK levels and increased the p-Akt levels in diabetic rats with I/R, and heart hemodynamic data improved, infarct size and LDH release decreased, SERCA2a activity increased, Bax and cleaved caspase-3 expression levels decreased, and the expression of Bcl-2 and the Bcl-2/Bax ratio increased. Our results suggest that the JNK/PI3K/Akt signaling pathway is involved in myocardial I/R injury in diabetic rats and Sal A exerts an anti-apoptotic effect and improves cardiac function following I/R injury through the JNK/PI3K/Akt signaling pathway in this model. PMID:27398138

  5. Filamin A Expression Negatively Regulates Sphingosine-1-Phosphate-Induced NF-κB Activation in Melanoma Cells by Inhibition of Akt Signaling

    PubMed Central

    Campos, Ludmila S.; Rodriguez, Yamila I.; Leopoldino, Andreia M.; Hait, Nitai C.; Lopez Bergami, Pablo; Castro, Melina G.; Sanchez, Emilse S.; Maceyka, Michael

    2015-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that regulates many processes in inflammation and cancer. S1P is a ligand for five G-protein-coupled receptors, S1PR1 to -5, and also has important intracellular actions. Previously, we showed that intracellular S1P is involved in tumor necrosis factor alpha (TNF)-induced NF-κB activation in melanoma cell lines that express filamin A (FLNA). Here, we show that extracellular S1P activates NF-κB only in melanoma cells that lack FLNA. In these cells, S1P, but not TNF, promotes IκB kinase (IKK) and p65 phosphorylation, IκBα degradation, p65 nuclear translocation, and NF-κB reporter activity. NF-κB activation induced by S1P was mediated via S1PR1 and S1PR2. Exogenous S1P enhanced the phosphorylation of protein kinase Cδ (PKCδ), and its downregulation reduced S1P-induced the phosphorylation of IKK and p65. In addition, silencing of Bcl10 also inhibited S1P-induced IKK phosphorylation. Surprisingly, S1P reduced Akt activation in melanoma cells that express FLNA, whereas in the absence of FLNA, high phosphorylation levels of Akt were maintained, enabling S1P-mediated NF-κB signaling. In accord, inhibition of Akt suppressed S1P-mediated IKK and p65 phosphorylation and degradation of IκBα. Hence, these results support a negative role of FLNA in S1P-mediated NF-κB activation in melanoma cells through modulation of Akt. PMID:26552704

  6. Filamin A Expression Negatively Regulates Sphingosine-1-Phosphate-Induced NF-κB Activation in Melanoma Cells by Inhibition of Akt Signaling.

    PubMed

    Campos, Ludmila S; Rodriguez, Yamila I; Leopoldino, Andreia M; Hait, Nitai C; Lopez Bergami, Pablo; Castro, Melina G; Sanchez, Emilse S; Maceyka, Michael; Spiegel, Sarah; Alvarez, Sergio E

    2015-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that regulates many processes in inflammation and cancer. S1P is a ligand for five G-protein-coupled receptors, S1PR1 to -5, and also has important intracellular actions. Previously, we showed that intracellular S1P is involved in tumor necrosis factor alpha (TNF)-induced NF-κB activation in melanoma cell lines that express filamin A (FLNA). Here, we show that extracellular S1P activates NF-κB only in melanoma cells that lack FLNA. In these cells, S1P, but not TNF, promotes IκB kinase (IKK) and p65 phosphorylation, IκBα degradation, p65 nuclear translocation, and NF-κB reporter activity. NF-κB activation induced by S1P was mediated via S1PR1 and S1PR2. Exogenous S1P enhanced the phosphorylation of protein kinase Cδ (PKCδ), and its downregulation reduced S1P-induced the phosphorylation of IKK and p65. In addition, silencing of Bcl10 also inhibited S1P-induced IKK phosphorylation. Surprisingly, S1P reduced Akt activation in melanoma cells that express FLNA, whereas in the absence of FLNA, high phosphorylation levels of Akt were maintained, enabling S1P-mediated NF-κB signaling. In accord, inhibition of Akt suppressed S1P-mediated IKK and p65 phosphorylation and degradation of IκBα. Hence, these results support a negative role of FLNA in S1P-mediated NF-κB activation in melanoma cells through modulation of Akt. PMID:26552704

  7. Metformin inhibits the proliferation of A431 cells by modulating the PI3K/Akt signaling pathway

    PubMed Central

    LIU, YINGSHAN; ZHANG, YAN; JIA, KUN; DONG, YUHAO; MA, WEIYUAN

    2015-01-01

    The ability of metformin, an antidiabetic drug with wide applications, to inhibit tumor cell growth has recently been discovered. The PI3K/Akt signaling pathway has been found to play an important role in the survival, proliferation and apoptosis of tumor cells. The aim of the present study was to explore the effect of metformin on the proliferation of A431 human squamous cell carcinoma cells and the underlying molecular mechanisms. A431 cells in the logarithmic growth phase were treated with 0, 15, 30, 45 and 60 mM metformin for 12, 24 and 36 h, respectively. Cell morphology with 45 mM metformin treatment for 24 h was observed under a microscope. The proliferation of A431 cells was detected by the Cell Counting kit-8 colorimetric method. The mRNA expression levels of PI3K and Akt were detected by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression levels of PI3K, Akt and phosphorylated (p)-Akt were detected by western blot analysis. Metformin treatment caused morphological change in A431 cells and inhibited their proliferation in a significant time- and dose-dependent manner. RT-PCR results showed that the mRNA expression of PI3K was inhibited by metformin in a time- and dose-dependent manner (P<0.05). However, there was no significant change in the mRNA expression of Akt following metformin treatment (P>0.05). Western blotting results showed that the protein expression levels of PI3K and p-Akt were inhibited by metformin in a time- and dose-dependent manner (P<0.05). In conclusion, metformin significantly inhibited the proliferation of A431 cells in the current study, which may be strongly associated with the inhibition of the PI3K/Akt signaling pathway. PMID:25780442

  8. Curcumin Attenuated Bupivacaine-Induced Neurotoxicity in SH-SY5Y Cells Via Activation of the Akt Signaling Pathway.

    PubMed

    Fan, You-Ling; Li, Heng-Chang; Zhao, Wei; Peng, Hui-Hua; Huang, Fang; Jiang, Wei-Hang; Xu, Shi-Yuan

    2016-09-01

    Bupivacaine is widely used for regional anesthesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway. PMID:27233246

  9. 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

  10. Irisin inhibits hepatic gluconeogenesis and increases glycogen synthesis via the PI3K/Akt pathway in type 2 diabetic mice and hepatocytes.

    PubMed

    Liu, Tong-Yan; Shi, Chang-Xiang; Gao, Run; Sun, Hai-Jian; Xiong, Xiao-Qing; Ding, Lei; Chen, Qi; Li, Yue-Hua; Wang, Jue-Jin; Kang, Yu-Ming; Zhu, Guo-Qing

    2015-11-01

    Increased glucose production and reduced hepatic glycogen storage contribute to metabolic abnormalities in diabetes. Irisin, a newly identified myokine, induces the browning of white adipose tissue, but its effects on gluconeogenesis and glycogenesis are unknown. In the present study, we investigated the effects and underlying mechanisms of irisin on gluconeogenesis and glycogenesis in hepatocytes with insulin resistance, and its therapeutic role in type 2 diabetic mice. Insulin resistance was induced by glucosamine (GlcN) or palmitate in human hepatocellular carcinoma (HepG2) cells and mouse primary hepatocytes. Type 2 diabetes was induced by streptozotocin/high-fat diet (STZ/HFD) in mice. In HepG2 cells, irisin ameliorated the GlcN-induced increases in glucose production, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression, and glycogen synthase (GS) phosphorylation; it prevented GlcN-induced decreases in glycogen content and the phosphoinositide 3-kinase (PI3K) p110α subunit level, and the phosphorylation of Akt/protein kinase B, forkhead box transcription factor O1 (FOXO1) and glycogen synthase kinase-3 (GSK3). These effects of irisin were abolished by the inhibition of PI3K or Akt. The effects of irisin were confirmed in mouse primary hepatocytes with GlcN-induced insulin resistance and in human HepG2 cells with palmitate-induced insulin resistance. In diabetic mice, persistent subcutaneous perfusion of irisin improved the insulin sensitivity, reduced fasting blood glucose, increased GSK3 and Akt phosphorylation, glycogen content and irisin level, and suppressed GS phosphorylation and PEPCK and G6Pase expression in the liver. Irisin improves glucose homoeostasis by reducing gluconeogenesis via PI3K/Akt/FOXO1-mediated PEPCK and G6Pase down-regulation and increasing glycogenesis via PI3K/Akt/GSK3-mediated GS activation. Irisin may be regarded as a novel therapeutic strategy for insulin resistance and type 2 diabetes. PMID

  11. Morphine Suppresses T helper Lymphocyte Differentiation to Th1 Type Through PI3K/AKT Pathway.

    PubMed

    Mao, Mao; Qian, Yanning; Sun, Jie

    2016-04-01

    To investigate the effect of morphine on T helper lymphocyte differentiation and PI3K/AKT pathway mechanism, CD4+ lymphocytes were treated by phorbol-myristate-acetate (25 ng/ml) (PMA) plus ionomycin (1 μg/ml) in the presence of various concentrations of morphine (25, 50, 100, 200 ng/ml) for 4 h. Th1 and Th2 subsets, supernatant cytokines, and PI3K, AKT, and protein kinase C-theta (PKC-θ) levels were detected. The Th1 cell percentage, Th1-derived cytokines, and ratio of Th1/Th2 decreased in the presence of morphine in a concentration-dependent manner. However, Th2 cell percentage kept stable after morphine treatment. The phosphorylation of PI3K and AKT decreased, but the phosphorylation of PKC-θ did not change in the presence of morphine. The decreased percentage of Th1 cells and ratio of Th1/Th2 was recovered by naloxone concentration-dependently. Morphine can inhibit the differentiation of Th1 lymphocytes and decrease the ratio of Th1/Th2 via the pathway of PI3K/AKT. The effect can be inhibited by naloxone. PMID:26883517

  12. Fragile Histidine Triad (FHIT) Suppresses Proliferation and Promotes Apoptosis in Cholangiocarcinoma Cells by Blocking PI3K-Akt Pathway

    PubMed Central

    Huang, Qiang; Liu, Zhen; Xie, Fang; Liu, Chenhai; Shao, Feng; Zhu, Cheng-lin; Hu, Sanyuan

    2014-01-01

    Fragile histidine triad (FHIT) is a tumor suppressor protein that regulates cancer cell proliferation and apoptosis. However, its exact mechanism of action is poorly understood. Phosphatidylinositol 3-OH kinase (PI3K)-Akt-survivin is an important signaling pathway that was regulated by FHIT in lung cancer cells. To determine whether FHIT can regulate this pathway in cholangiocarcinoma QBC939 cells, we constructed an FHIT expression plasmid and used it to transfect QBC939 cells. Protein and mRNA expression were measured by western blotting and qRT-PCR, respectively. The viability and apoptosis of QBC939 cells were then assessed using MTT assays and flow cytometry. Our results revealed that the expression of survivin and Bcl-2 was downregulated, and caspase 3 was upregulated, in cells overexpressing FHIT. In addition, FHIT suppressed the phosphorylation of Akt. The changes in cell proliferation and apoptosis were obvious in cells overexpressing FHIT which parallels that of treatment with LY294002, a potent inhibitor of phosphoinositide 3-kinases. Treatment with LY294002 further decreased the expression of survivin and Bcl-2 and increased caspase-3 levels. These results suggest that FHIT can block the PI3K-Akt-survivin pathway by suppressing the phosphorylation of Akt and the expression of survivin and Bcl-2 and upregulating caspase 3. PMID:24757411

  13. 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

  14. Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

    PubMed

    Ejaz, Asim; Mitterberger, Maria C; Lu, Zhen; Mattesich, Monika; Zwierzina, Marit E; Hörl, Susanne; Kaiser, Andreas; Viertler, Hans-Peter; Rostek, Ursula; Meryk, Andreas; Khalid, Sana; Pierer, Gerhard; Bast, Robert C; Zwerschke, Werner

    2016-04-01

    Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells. PMID:27211557

  15. Vitamin E succinate induces apoptosis via the PI3K/AKT signaling pathways in EC109 esophageal cancer cells

    PubMed Central

    Yang, Peng; Zhao, Jiaying; Hou, Liying; Yang, Lei; Wu, Kun; Zhang, Linyou

    2016-01-01

    Esophageal cancer is the fourth most common gastrointestinal cancer, it generally has a poor prognosis and novel strategies are required for prevention and treatment. Vitamin E succinate (VES) is a potential chemical agent for cancer prevention and therapy as it exerts anti-tumor effects in a variety of cancers. However, the role of VES in tumorigenesis and progression of cancer remains to be elucidated. The present study aimed to determine the effects of VES in regulating the survival and apoptosis of human esophageal cancer cells. EC109 human esophageal cancer cells were used to investigate the anti-proliferative effects of VES. The MTT and Annexin V-fluorescein isothiocyanate/propidium iodide assays demonstrated that VES inhibited cell proliferation and induced apoptosis in esophageal cancer cells. Furthermore, VES downregulated constitutively active basal levels of phosphorylated (p)-serine-threonine kinase AKT (AKT) and p-mammalian target of rapamycin (mTOR), and decreased the phosphorylation of AKT substrates Bcl-2-associated death receptor and caspase-9, in addition to mTOR effectors, ribosomal protein S6 kinase β1 and eIF4E-binding protein 1. Phosphoinositide-3-kinase (PI3K) inhibitor, LY294002 suppressed p-AKT and p-mTOR, indicating PI3K is a common upstream mediator. The apoptosis induced by VES was increased by inhibition of AKT or mTOR with their respective inhibitor in esophageal cancer cells. The results of the present study suggested that VES targeted the PI3K/AKT signaling pathways and induced apoptosis in esophageal cancer cells. Furthermore, the current study suggests that VES may be useful in a combinational therapeutic strategy employing an mTOR inhibitor. PMID:27357907

  16. κ-Opioid Receptor Stimulation Improves Endothelial Function via Akt-stimulated NO Production in Hyperlipidemic Rats

    PubMed Central

    Tian, Fei; Zheng, Xu-Yang; Li, Juan; Zhang, Shu-Miao; Feng, Na; Guo, Hai-Tao; Jia, Min; Wang, Yue-Min; Fan, Rong; Pei, Jian-Ming

    2016-01-01

    This study was designed to investigate the effect of U50,488H (a selective κ-opioid receptor agonist) on endothelial function impaired by hyperlipidemia and to determine the role of Akt-stimulated NO production in it. Hyperlipidemic model was established by feeding rats with a high-fat diet for 14 weeks. U50,488H and nor-BNI (a selective κ-opioid receptor antagonist) were administered intraperitoneally. In vitro, the involvement of the PI3K/Akt/eNOS pathway in the effect of U50,488H was studied using cultured endothelial cells subjected to artificial hyperlipidemia. Serum total cholesterol and low-density lipoprotein cholesterol concentrations dramatically increased after high-fat diet feeding. Administration of U50,488H significantly alleviated endothelial ultrastructural destruction and endothelium-dependent vasorelaxation impairment caused by hyperlipidemia. U50,488H also increased Akt/eNOS phosphorylation and serum/medium NO level both in vivo and in vitro. U50,488H increased eNOS activity and suppressed iNOS activity in vivo. The effects of U50,488H were abolished in vitro by siRNAs targeting κ-opioid receptor and Akt or PI3K/Akt/eNOS inhibitors. All effects of U50,488H were blocked by nor-BNI. These results demonstrate that κ-opioid receptor stimulation normalizes endothelial ultrastructure and function under hyperlipidemic condition. Its mechanism is related to the preservation of eNOS phosphorylation through activation of the PI3K/Akt signaling pathway and downregulation of iNOS expression/activity. PMID:27226238

  17. κ-Opioid Receptor Stimulation Improves Endothelial Function via Akt-stimulated NO Production in Hyperlipidemic Rats.

    PubMed

    Tian, Fei; Zheng, Xu-Yang; Li, Juan; Zhang, Shu-Miao; Feng, Na; Guo, Hai-Tao; Jia, Min; Wang, Yue-Min; Fan, Rong; Pei, Jian-Ming

    2016-01-01

    This study was designed to investigate the effect of U50,488H (a selective κ-opioid receptor agonist) on endothelial function impaired by hyperlipidemia and to determine the role of Akt-stimulated NO production in it. Hyperlipidemic model was established by feeding rats with a high-fat diet for 14 weeks. U50,488H and nor-BNI (a selective κ-opioid receptor antagonist) were administered intraperitoneally. In vitro, the involvement of the PI3K/Akt/eNOS pathway in the effect of U50,488H was studied using cultured endothelial cells subjected to artificial hyperlipidemia. Serum total cholesterol and low-density lipoprotein cholesterol concentrations dramatically increased after high-fat diet feeding. Administration of U50,488H significantly alleviated endothelial ultrastructural destruction and endothelium-dependent vasorelaxation impairment caused by hyperlipidemia. U50,488H also increased Akt/eNOS phosphorylation and serum/medium NO level both in vivo and in vitro. U50,488H increased eNOS activity and suppressed iNOS activity in vivo. The effects of U50,488H were abolished in vitro by siRNAs targeting κ-opioid receptor and Akt or PI3K/Akt/eNOS inhibitors. All effects of U50,488H were blocked by nor-BNI. These results demonstrate that κ-opioid receptor stimulation normalizes endothelial ultrastructure and function under hyperlipidemic condition. Its mechanism is related to the preservation of eNOS phosphorylation through activation of the PI3K/Akt signaling pathway and downregulation of iNOS expression/activity. PMID:27226238

  18. 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

  19. Interleukin-6 upregulates paraoxonase 1 gene expression via an AKT/NF-κB-dependent pathway

    SciTech Connect

    Cheng, Chi-Chih; Hsueh, Chi-Mei; Chen, Chiu-Yuan; Chen, Tzu-Hsiu; Hsu, Shih-Lan

    2013-07-19

    Highlights: •IL-6 could induce PON1 gene expression. •IL-6 increased NF-κB protein expression and NF-κB-p50 and -p65 subunits nuclear translocation. •IL-6-induced PON1 up-regulation was through an AKT/NF-κB pathway. -- Abstract: The aim of this study is to investigate the relationship between paraoxonase 1 (PON1) and atherosclerosis-related inflammation. In this study, human hepatoma HepG2 cell line was used as a hepatocyte model to examine the effects of the pro-inflammatory cytokines on PON1 expression. The results showed that IL-6, but not TNF-α and IL-1β, significantly increased both the function and protein level of PON1; data from real-time RT-PCR analysis revealed that the IL-6-induced PON1 expression occurred at the transcriptional level. Increase of IκB kinase activity and IκB phosphorylation, and reduction of IκB protein level were also observed in IL-6-treated HepG2 cells compared with untreated culture. This event was accompanied by increase of NF-κB-p50 and -p65 nuclear translocation. Moreover, treatment with IL-6 augmented the DNA binding activity of NF-κB. Furthermore, pharmacological inhibition of NF-κB activation by PDTC and BAY 11-7082, markedly suppressed the IL-6-mediated PON1 expression. In addition, IL-6 increased the levels of phosphorylated protein kinase B (PKB, AKT). An AKT inhibitor LY294002 effectively suppressed IKK/IκB/NF-κB signaling and PON1 gene expression induced by IL-6. Our findings demonstrate that IL-6 upregulates PON1 gene expression through an AKT/NF-κB signaling axis in human hepatocyte-derived HepG2 cell line.

  20. 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...

  1. The transplantation of Akt-overexpressing amniotic fluid-derived mesenchymal stem cells protects the heart against ischemia-reperfusion injury in rabbits

    PubMed Central

    WANG, YAN; LI, YIGANG; SONG, LEI; LI, YANYAN; JIANG, SHAN; ZHANG, SONG

    2016-01-01

    Amniotic fluid-derived mesenchymal stem cells (AFMSCs) are an attractive cell source for applications in regenerative medicine, due to characteristics such as proliferative capacity and multipotency. In addition, Akt, a serine-threonine kinase, maintains stem cells by promoting viability and proliferation. Whether the transplantation of Akt-overexpressing AFMSCs protects the heart against ischemia-reperfusion (I/R) injury has yet to be elucidated. Accordingly, the Akt gene was overexpressed in AFMSCs using lentiviral transduction, and Akt-AFMSCs were transplanted into the ischemic myocardium of rabbits prior to reperfusion. Any protective effects resulting from this procedure were subsequently sought after three weeks later. A histological examination revealed that there was a decrease in intramyocardial inflammation and ultrastructural damage, and an increase in capillary density and in the levels of GATA binding protein 4, connexin 43 and cardiac troponin T in the Akt-AFMSC group compared with the control group. A significant decrease in cardiomyocyte apoptosis, accompanying an increase in phosphorylated Akt and B-cell lymphoma 2 (Bcl-2) and a decrease in caspase-3, was also observed. Furthermore, the left ventricular function was markedly augmented in the Akt-AFMSC group compared with the control group. These observations suggested that the protective effect of AFMSCs may be due to the delivery of secreted cytokines, promotion of neoangiogenesis, prevention of cardiomyocyte apoptosis, transdifferentiation into cardiomyocytes and promotion of the viability of AFMSCs, which are assisted by Akt gene modification. Taken together, the results of the present study have indicated that transplantation of Akt-AFMSCs is able to alleviate myocardial I/R injury and improve cardiac function. PMID:27151366

  2. Attenuated Levels of Hippocampal Connexin 43 and its Phosphorylation Correlate with Antidepressant- and Anxiolytic-Like Activities in Mice

    PubMed Central

    Quesseveur, Gaël; Portal, Benjamin; Basile, Jean-Arnaud; Ezan, Pascal; Mathou, Alexia; Halley, Hélène; Leloup, Corinne; Fioramonti, Xavier; Déglon, Nicole; Giaume, Christian; Rampon, Claire; Guiard, Bruno P.

    2015-01-01

    a lower level of phosphorylation in the hippocampus. PMID:26733815

  3. The Phosphorylation Flow of the Vibrio harveyi Quorum-Sensing Cascade Determines Levels of Phenotypic Heterogeneity in the Population

    PubMed Central

    Plener, Laure; Lorenz, Nicola; Reiger, Matthias; Ramalho, Tiago; Gerland, Ulrich

    2015-01-01

    ABSTRACT Quorum sensing (QS) is a communication process that enables a bacterial population to coordinate and synchronize specific behaviors. The bioluminescent marine bacterium Vibrio harveyi integrates three autoinducer (AI) signals into one quorum-sensing cascade comprising a phosphorelay involving three hybrid sensor kinases: LuxU; LuxO, an Hfq/small RNA (sRNA) switch; and the transcriptional regulator LuxR. Using a new set of V. harveyi mutants lacking genes for the AI synthases and/or sensors, we assayed the activity of the quorum-sensing cascade at the population and single-cell levels, with a specific focus on signal integration and noise levels. We found that the ratios of kinase activities to phosphatase activities of the three sensors and, hence, the extent of phosphorylation of LuxU/LuxO are important not only for the signaling output but also for the degree of noise in the system. The pools of phosphorylated LuxU/LuxO per cell directly determine the amounts of sRNAs produced and, consequently, the copy number of LuxR, generating heterogeneous quorum-sensing activation at the single-cell level. We conclude that the ability to drive the heterogeneous expression of QS-regulated genes in V. harveyi is an inherent feature of the architecture of the QS cascade. IMPORTANCE V. harveyi possesses one of the most complex quorum-sensing (QS) cascades known, using three different autoinducers (AIs) to control the induction of, e.g., bioluminescence, virulence factors, and biofilm and exoprotease production. We constructed various V. harveyi mutants to study the impact of each component and subsystem of the QS signaling cascade on QS activation at the population and single-cell levels. We found that the output was homogeneous only in the presence of all AIs. In the absence of any one AI, QS activation varied from cell to cell, resulting in phenotypic heterogeneity. This study elucidates a molecular design principle which enables a tightly integrated signaling

  4. Increased CSF Levels of Phosphorylated Neurofilament Heavy Protein following Bout in Amateur Boxers

    PubMed Central

    Neselius, Sanna; Zetterberg, Henrik; Blennow, Kaj; Marcusson, Jan; Brisby, Helena

    2013-01-01

    Introduction Diagnosis of mild TBI is hampered by the lack of imaging or biochemical measurements for identifying or quantifying mild TBI in a clinical setting. We have previously shown increased biomarker levels of protein reflecting axonal (neurofilament light protein and tau) and glial (GFAP and S-100B) damage in cerebrospinal fluid (CSF) after a boxing bout. The aims of this study were to find other biomarkers of mild TBI, which may help clinicians diagnose and monitor mild TBI, and to calculate the role of APOE ε4 allele genotype which has been associated with poor outcome after TBI. Materials and Methods Thirty amateur boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in a prospective cohort study. CSF and blood were collected at one occasion between 1 and 6 days after a bout, and after a rest period for at least 14 days (follow up). The controls were tested once. CSF levels of neurofilament heavy (pNFH), amyloid precursor proteins (sAPPα and sAPPβ), ApoE and ApoA1 were analyzed. In blood, plasma levels of Aβ42 and ApoE genotype were analyzed. Results CSF levels of pNFH were significantly increased between 1 and 6 days after boxing as compared with controls (p<0.001). The concentrations decreased at follow up but were still significantly increased compared to controls (p = 0.018). CSF pNFH concentrations correlated with NFL (r =  0.57 after bout and 0.64 at follow up, p<0.001). No significant change was found in the other biomarkers, as compared to controls. Boxers carrying the APOE ε4 allele had similar biomarker concentrations as non-carriers. Conclusions Subconcussive repetitive trauma in amateur boxing causes a mild TBI that may be diagnosed by CSF analysis of pNFH, even without unconsciousness or concussion symptoms. Possession of the APOE ε4 allele was not found to influence biomarker levels after acute TBI. PMID:24260563

  5. Sustained Activation of Akt Elicits Mitochondrial Dysfunction to Block Plasmodium falciparum Infection in the Mosquito Host

    PubMed Central

    Drexler, Anna L.; Antonova-Koch, Yevgeniya; Sakaguchi, Danielle; Napoli, Eleonora; Wong, Sarah; Price, Mark S.; Eigenheer, Richard; Phinney, Brett S.; Pakpour, Nazzy; Pietri, Jose E.; Cheung, Kong; Georgis, Martha; Riehle, Michael

    2013-01-01

    The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3–5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the

  6. Sustained activation of Akt elicits mitochondrial dysfunction to block Plasmodium falciparum infection in the mosquito host.

    PubMed

    Luckhart, Shirley; Giulivi, Cecilia; Drexler, Anna L; Antonova-Koch, Yevgeniya; Sakaguchi, Danielle; Napoli, Eleonora; Wong, Sarah; Price, Mark S; Eigenheer, Richard; Phinney, Brett S; Pakpour, Nazzy; Pietri, Jose E; Cheung, Kong; Georgis, Martha; Riehle, Michael

    2013-02-01

    The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3-5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the

  7. Soluble epoxide hydrolase inhibition ameliorates proteinuria-induced epithelial-mesenchymal transition by regulating the PI3K-Akt-GSK-3β signaling pathway.

    PubMed

    Liang, Yaoxian; Jing, Ziyang; Deng, Hui; Li, Zhengqian; Zhuang, Zhen; Wang, Song; Wang, Yue

    Soluble epoxide hydrolase (sEH) plays an essential role in chronic kidney disease by hydrolyzing renoprotective epoxyeicosatrienoic acids to the corresponding inactive dihydroxyeicosatrienoic acids. However, there have been few mechanistic studies elucidating the role of sEH in epithelial-mesenchymal transition (EMT). The present study investigated, in vitro and in vivo, the role of sEH in proteinuria-induced renal tubular EMT and the underlying signaling pathway. We report that urinary protein (UP) induced EMT in cultured NRK-52E cells, as evidenced by decreased E-cadherin expression, increased alpha-smooth muscle actin (α-SMA) expression, and the morphological conversion to a myofibroblast-like phenotype. UP incubation also resulted in upregulated sEH, activated phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) signaling and increased phosphorylated glycogen synthase kinase-3β (GSK-3β). The PI3K inhibitor LY-294002 inhibited phosphorylation of Akt and GSK-3β as well as blocking EMT. Importantly, pharmacological inhibition of sEH with 12-(3-adamantan-1-yl- ureido)-dodecanoic acid (AUDA) markedly suppressed PI3K-Akt activation and GSK-3β phosphorylation. EMT associated E-cadherin suppression, α-SMA elevation and phenotypic transition were also attenuated by AUDA. Furthermore, in rats with chronic proteinuric renal disease, AUDA treatment inhibited PI3K-Akt activation and GSK-3β phosphorylation, while attenuating levels of EMT markers. Overall, our findings suggest that sEH inhibition ameliorates proteinuria-induced renal tubular EMT by regulating the PI3K-Akt-GSK-3β signaling pathway. Targeting sEH might be a potential strategy for the treatment of EMT and renal fibrosis. PMID:25986738

  8. 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

  9. 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

  10. 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

  11. 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

  12. Akt signaling dynamics in individual cells

    PubMed Central

    Gross, Sean M.; Rotwein, Peter

    2015-01-01

    ABSTRACT The protein kinase Akt (for which there are three isoforms) is a key intracellular mediator of many biological processes, yet knowledge of Akt signaling dynamics is limited. Here, we have constructed a fluorescent reporter molecule in a lentiviral delivery system to assess Akt kinase activity at the single cell level. The reporter, a fusion between a modified FoxO1 transcription factor and clover, a green fluorescent protein, rapidly translocates from the nucleus to the cytoplasm in response to Akt stimulation. Because of its long half-life and the intensity of clover fluorescence, the sensor provides a robust readout that can be tracked for days under a range of biological conditions. Using this reporter, we find that stimulation of Akt activity by IGF-I is encoded into stable and reproducible analog responses at the population level, but that single cell signaling outcomes are variable. This reporter, which provides a simple and dynamic measure of Akt activity, should be compatible with many cell types and experimental platforms, and thus opens the door to new insights into how Akt regulates its biological responses. PMID:26040286

  13. Partial Loss of the Glutamate Transporter GLT-1 Alters Brain Akt and Insulin Signaling in a Mouse Model of Alzheimer’s Disease

    PubMed Central

    Meeker, Kole D.; Meabon, James S.; Cook, David G.

    2016-01-01

    The glutamate transporter GLT-1 (also called EAAT2 in humans) plays a critical role in regulating extracellular glutamate levels in the central nervous system (CNS). In Alzheimer’s disease (AD),EAAT2 loss is associated with neuropathology and cognitive impairment. In keeping with this, we have reported that partial GLT-1 loss (GLT-1+/−) causes early-occurring cognitive deficits in mice harboring familial AD AβPPswe/PS1ΔE9 mutations. GLT-1 plays important roles in several molecular pathways that regulate brain metabolism, including Akt and insulin signaling in astrocytes. Significantly, AD pathogenesis also involves chronic Akt activation and reduced insulin signaling in the CNS. In this report we tested the hypothesis that GLT-1 heterozygosity (which reduces GLT-1 to levels that are comparable to losses in AD patients) in AβPPswe/PS1ΔE9 mice would induce sustained activation of Akt and disturb components of the CNS insulin signaling cascade. We found that partial GLT-1 loss chronically increased Akt activation (reflected by increased phosphorylation at serine 473), impaired insulin signaling (reflected by decreased IRβ phosphorylation of tyrosines 1150/1151 and increased IRS-1 phosphorylation at serines 632/635 –denoted as 636/639 in humans), and reduced insulin degrading enzyme (IDE) activity in brains of mice expressing familial AβPPswe/PS1ΔE9 AD mutations. GLT-1 loss also caused an apparent compensatory increase in IDE activity in the liver, an organ that has been shown to regulate peripheral amyloid-β levels and expresses GLT-1. Taken together, these findings demonstrate that partial GLT-1 loss can cause insulin/Akt signaling abnormalities that are in keeping with those observed in AD. PMID:25589729

  14. Knockdown of sphingosine kinase 1 inhibits the migration and invasion of human rheumatoid arthritis fibroblast-like synoviocytes by down-regulating the PI3K/AKT activation and MMP-2/9 production in vitro.

    PubMed

    Yuan, Hongxia; Yang, Pingting; Zhou, Dun; Gao, Wei; Qiu, Zhenyu; Fang, Fang; Ding, Shuang; Xiao, Weiguo

    2014-08-01

    To investigate the potential regulation of sphingosine kinase 1 (SPHK1) on the migration, invasion, and matrix metalloproteinase (MMP) expression in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). RA-FLS were transfected control siRNA or SPHK1 siRNA. The migration and invasion of unmanipulated control, control siRNA or SPHK1 siRNA- transfected RA-FLS in vitro were measured by the transwell system. The relative levels of SPHK1, PI3K, and AKT as well as AKT phosphorylation in RA-FLS were determined by Western blot. The levels of MMP-2/9 secreted by RA-FLS were detected by ELISA. Knockdown of SPHK1 significantly inhibited the spontaneous migration and invasion of RA-FLS, accompanied by significantly reduced levels of PI3K expression and AKT phosphorylation. Similarly, treatment with LY294002, an inhibitor of the PI3K/AKT pathway, inhibited the migration and invasion of RA-FLS. Knockdown of SPHK1 and treatment with the inhibitor synergistically inhibited the migration and invasion of RA-FLS, by further reducing the levels of PI3K expression and AKT phosphorylation. In addition, knockdown of SPHK1 or treatment with LY294002 inhibited the secretion of MMP-2 and MMP-9, and both synergistically reduced the production of MMP-2 and MMP-9 in RA-FLS in vitro. Knockdown of SPHK1 expression inhibits the PI3K/AKT activation, MMP-2 and MMP-9 expression, and human RA-FLS migration and invasion in vitro. Potentially, SPHK1 may be a novel therapeutic target for RA. PMID:24816639

  15. 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

  16. Mitochondrial diaphorases as NAD+ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition

    PubMed Central

    Kiss, Gergely; Konrad, Csaba; Pour-Ghaz, Issa; Mansour, Josef J.; Németh, Beáta; Starkov, Anatoly A.; Adam-Vizi, Vera; Chinopoulos, Christos

    2014-01-01

    Substrate-level phosphorylation mediated by succinyl-CoA ligase in the mitochondrial matrix produces high-energy phosphates in the absence of oxidative phosphorylation. Furthermore, when the electron transport chain is dysfunctional, provision of succinyl-CoA by the α-ketoglutarate dehydrogenase complex (KGDHC) is crucial for maintaining the function of succinyl-CoA ligase yielding ATP, preventing the adenine nucleotide translocase from reversing. We addressed the source of the NAD+ supply for KGDHC under anoxic conditions and inhibition of complex I. Using pharmacologic tools and specific substrates and by examining tissues from pigeon liver exhibiting no diaphorase activity, we showed that mitochondrial diaphorases in the mouse liver contribute up to 81% to the NAD+ pool during respiratory inhibition. Under these conditions, KGDHC's function, essential for the provision of succinyl-CoA to succinyl-CoA ligase, is supported by NAD+ derived from diaphorases. Through this process, diaphorases contribute to the maintenance of substrate-level phosphorylation during respiratory inhibition, which is manifested in the forward operation of adenine nucleotide translocase. Finally, we show that reoxidation of the reducible substrates for the diaphorases is mediated by complex III of the respiratory chain.—Kiss, G., Konrad, C., Pour-Ghaz, I., Mansour, J. J., Németh, B., Starkov, A. A., Adam-Vizi, V., Chinopoulos, C. Mitochondrial diaphorases as NAD+ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition. PMID:24391134

  17. 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

  18. 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

  19. Melatonin modulates endoplasmic reticulum stress and Akt/GSK3-beta signaling pathway in a rat model of renal warm ischemia reperfusion.

    PubMed

    Hadj Ayed Tka, Kaouther; Mahfoudh Boussaid, Asma; Zaouali, Mohamed Amine; Kammoun, Rym; Bejaoui, Mohamed; Ghoul Mazgar, Sonia; Rosello Catafau, Joan; Ben Abdennebi, Hassen

    2015-01-01

    Melatonin (Mel) is widely used to attenuate ischemia/reperfusion (I/R) injury in several organs. Nevertheless, the underlying mechanisms remain unclear. This study was conducted to explore the effect of Mel on endoplasmic reticulum (ER) stress, Akt and MAPK cascades after renal warm I/R. Eighteen Wistar rats were randomized into three groups: Sham, I/R, and Mel + I/R. The ischemia period was 60 min followed by 120 min of reperfusion. Mel (10 mg/kg) was administrated 30 min prior to ischemia. The creatinine clearance, MDA, LDH levels, and histopathological changes were evaluated. In addition, Western blot was performed to study ER stress and its downstream apoptosis as well as phosphorylation of Akt, GSK-3β, VDAC, ERK, and P38. Mel decreased cytolysis and lipid peroxidation and improved renal function and morphology compared to I/R group. Parallely, it significantly reduced the ER stress parameters including GRP 78, p-PERK, XBP 1, ATF 6, CHOP, and JNK. Simultaneously, p-Akt level was significantly enhanced and its target molecules GSK-3β and VDAC were inhibited. Furthermore, the ERK and P38 phosphorylation were evidently augmented after Mel administration in comparison to I/R group. In conclusion, Mel improves the recovery of renal function by decreasing ER stress and stimulating Akt pathway after renal I/R injury. PMID:26229743

  20. Melatonin Modulates Endoplasmic Reticulum Stress and Akt/GSK3-Beta Signaling Pathway in a Rat Model of Renal Warm Ischemia Reperfusion

    PubMed Central

    Hadj Ayed Tka, Kaouther; Mahfoudh Boussaid, Asma; Zaouali, Mohamed Amine; Kammoun, Rym; Bejaoui, Mohamed; Ghoul Mazgar, Sonia; Rosello Catafau, Joan; Ben Abdennebi, Hassen

    2015-01-01

    Melatonin (Mel) is widely used to attenuate ischemia/reperfusion (I/R) injury in several organs. Nevertheless, the underlying mechanisms remain unclear. This study was conducted to explore the effect of Mel on endoplasmic reticulum (ER) stress, Akt and MAPK cascades after renal warm I/R. Eighteen Wistar rats were randomized into three groups: Sham, I/R, and Mel + I/R. The ischemia period was 60 min followed by 120 min of reperfusion. Mel (10 mg/kg) was administrated 30 min prior to ischemia. The creatinine clearance, MDA, LDH levels, and histopathological changes were evaluated. In addition, Western blot was performed to study ER stress and its downstream apoptosis as well as phosphorylation of Akt, GSK-3β, VDAC, ERK, and P38. Mel decreased cytolysis and lipid peroxidation and improved renal function and morphology compared to I/R group. Parallely, it significantly reduced the ER stress parameters including GRP 78, p-PERK, XBP 1, ATF 6, CHOP, and JNK. Simultaneously, p-Akt level was significantly enhanced and its target molecules GSK-3β and VDAC were inhibited. Furthermore, the ERK and P38 phosphorylation were evidently augmented after Mel administration in comparison to I/R group. In conclusion, Mel improves the recovery of renal function by decreasing ER stress and stimulating Akt pathway after renal I/R injury. PMID:26229743

  1. 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

  2. CLOCK-BMAL1 regulate the cardiac L-type calcium channel subunit CACNA1C through PI3K-Akt signaling pathway.

    PubMed

    Chen, Yanhong; Zhu, Didi; Yuan, Jiamin; Han, Zhonglin; Wang, Yao; Qian, Zhiyong; Hou, Xiaofeng; Wu, Tingting; Zou, Jiangang

    2016-09-01

    The heterodimerized transcription factors CLOCK-BMAL1 regulate the cardiomyocyte circadian rhythms. The L-type calcium currents play important role in the cardiac electrogenesis and arrhythmogenesis. Whether and how the CLOCK-BMAL1 regulate the cardiac L-type calcium channels are yet to be determined. The functions of the L-type calcium channels were evaluated with patch clamping techniques. Recombinant adenoviruses of CLOCK and BMAL1 were used in the expression experiments. We reported that the expressions and functions of CACNA1C (the α-subunit of the L-type calcium channels) showed circadian rhythms, with the peak at zeitgeber time 3 (ZT3). The endocardial action potential durations 90 (APD90) were correspondingly longer at ZT3. The protein levels of the phosphorylated Akt at threonine 308 (pAkt T308) also showed circadian rhythms. Overexpressions of CLOCK-BMAL1 significantly reduced the levels of CACNA1C while increasing the levels of pAkt T308 and pik3r1. Furthermore, the inhibitory effects of CLOCK-BMAL1 on CACNA1C could be abolished by the Akt inhibitor MK2206 or the PDK1 inhibitor GSK2334470. Collectively, our findings suggested that the expressions of the cardiac CACNA1C were under the CLOCK-BMAL1 regulation, probably through the PI3K-Akt signal pathway. PMID:27376484

  3. 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

  4. 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.

  5. Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels

    SciTech Connect

    Mertins, Philipp; Yang, Feng; Liu, Tao; Mani, DR; Petyuk, Vladislav A.; Gillette, Michael; Clauser, Karl; Qiao, Jana; Gritsenko, Marina A.; Moore, Ronald J.; Levine, Douglas; Townsend, Reid; Erdmann-Gilmore, Petra; Snider, Jacqueline E.; Davies, Sherri; Ruggles, Kelly; Fenyo, David; Kitchens, R. T.; Li, Shunqiang; Olvera, Narcisco; Dao, Fanny; Rodriguez, Henry; Chan, Daniel W.; Liebler, Daniel; White, Forest; Rodland, Karin D.; Mills, Gordon; Smith, Richard D.; Paulovich, Amanda G.; Ellis, Matthew; Carr, Steven A.

    2014-07-01

    Advances in quantitative mass spectrometry (MS)-based proteomics have sparked efforts to characterize the proteomes of tumor samples to provide complementary and unique information inaccessible by genomics. Tumor samples are usually not accrued with proteomic characterization in mind, raising concerns regarding effects of undocumented sample ischemia on protein abundance and phosphosite stoichiometry. Here we report the effects of cold ischemia time on clinical ovarian cancer samples and patient-derived basal and luminal breast cancer xenografts. Tumor tissues were excised and collected prior to vascular ligation, subjected to accurately defined ischemia times up to 60 min, and analyzed by quantitative proteomics and phosphoproteomics using isobaric tags and high-performance, multidimensional LC-MS/MS. No significant changes were detected at the protein level in each tumor type after 60 minutes of ischemia, and the majority of the >25,000 phosphosites detected were also stable. However, large, reproducible increases and decreases in protein phosphorylation at specific sites were observed in up to 24% of the phosphoproteome starting as early as 5 minutes post-excision. Early and sustained activation of stress response, transcriptional regulation and cell death pathways were observed in common across tumor types. Tissue-specific changes in phosphosite stability were also observed suggesting idiosyncratic effects of ischemia in particular lineages. Our study provides insights into the information that may be obtained by proteomic characterization of tumor samples after undocumented periods of ischemia, and suggests caution especially in interpreting activation of stress pathways in such samples as they may reflect sample handling rather than tumor physiology.

  6. Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis

    PubMed Central

    Takagi, Satoshi; Takemoto, Ai; Takami, Miho; Oh-hara, Tomoko; Fujita, Naoya

    2014-01-01

    The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co-culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho-antibody arrays revealed that co-culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas. PMID:24974736

  7. Cooperation between Shh and IGF-I in Promoting Myogenic Proliferation and Differentiation via the MAPK/ERK and PI3K/Akt Pathways Requires Smo Activity

    PubMed Central

    Madhala-Levy, D; Williams, VC; Hughes, SM; Reshef, R; O, Halevy

    2012-01-01

    Sonic hedgehog (Shh) has been shown to promote adult myoblast proliferation and differentiation and affect Akt phosphorylation via its effector Smoothened (Smo). Here, the relationship between Shh and IGF-I was examined with regard to myogenic differentiation via signaling pathways which regulate this process. Each factor enhanced Akt and MAPK/ERK (p42/44) phosphorylation and myogenic factor expression levels in a dose-responsive manner, while combinations of Shh and IGF-I showed additive effects. Blockage of the IGF-I effects by neutralizing antibody partially reduced Shh’s effects on signaling pathways, suggesting that IGF-I enhances, but is not essential for Shh effects. Addition of cyclopamine, a Smo inhibitor, reduced Shh- and IGF-I-induced Akt phosphorylation in a similar manner, implying that Shh affects gain of the IGF-I signaling pathway. This implication was also examined via a genetic approach. In cultures derived from Smomut (MCre;Smoflox/flox) mice lacking Smo expression specifically in hindlimb muscles, IGF-I-induced Akt and p42/44 phosphorylation was significantly reduced compared to IGF-I’s effect on Smocont cells. Moreover, remarkable inhibition of the stimulatory effect of IGF-I on myogenic differentiation was observed in Smomut cultures, implying that intact Smo is required for IGF-I effects in myoblasts. Immunoprecipitation assays revealed that p-Tyr proteins, including the regulatory unit of PI3K (p85), are recruited to Smo in response to Shh. Moreover, IGF-IR was found to associate with Smo in response to Shh and to IGF-I, suggesting that Shh and IGF-I are already integrated at the receptor level, a mechanism by which their signaling pathways interact in augmenting their effects on adult myoblasts. PMID:21618536

  8. Progesterone is neuroprotective against ischemic brain injury through its effects on the PI3K/Akt signaling pathway

    PubMed Central

    Ishrat, Tauheed; Sayeed, Iqbal; Atif, Fahim; Hua, Fang; Stein, Donald G.

    2012-01-01

    We tested the hypothesis that the phosphatidylinositol-3 kinase (PI3K/Akt) pathway mediates some of the neuroprotective effects of progesterone (PROG) after ischemic stroke. We examined whether PROG acting through the PI3K/Akt pathway could affect the expression of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Rats underwent permanent focal cerebral ischemia (pMCAO) by electro-coagulation and received intraperitoneal injections of PROG (8mg/kg) or vehicle at 1h post-occlusion and subcutaneous injections at 6, 24, and 48h. PAkt/Akt levels, apoptosis and apoptosis-related proteins (pBAD, BAD, caspase-3, and cleaved caspase-3) were analyzed by TUNEL assays, Western blotting and immunohistochemistry at 24h post-pMCAO. VEGF and BDNF were analyzed at 24, 72h and 14 days post-pMCAO with Western blots. Following pMCAO, PROG treatment significantly (p<0.05) reduced ischemic lesion size and edema. Treatment with PROG significantly (p<0.05) decreased VEGF at 24 and 72h but increased VEGF expression 14d after injury. The treatment also increased BDNF, and attenuated apoptosis by increasing Akt phosphorylation compared to vehicle-alone. The selective PI3K inhibitor Wortmannin compromised PROG-induced neuroprotective effects and reduced the elevation of pAkt levels in the ischemic penumbra. Our findings lead us to suggest that the PI3K/Akt pathway can play a role in mediating the neuroprotective effects of PROG after stroke by altering the expression of trophic factors in the brain. PMID:22450229

  9. 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

  10. Gender differences in cardioprotection against ischemia/reperfusion injury in adult rat hearts: focus on Akt and protein kinase C signaling.

    PubMed

    Bae, Soochan; Zhang, Lubo

    2005-12-01

    Previous studies have reported the sex differences in heart susceptibility to ischemia/reperfusion (I/R) injury, but the mechanisms are not understood. The present study tested the hypothesis that Akt and protein kinase C (PKC)epsilon play an important role in the sexual dimorphism of heart susceptibility to I/R injury. Isolated hearts from 2-month-old male and female rats were subjected to I/R in the Langendorff preparation. The postischemic recovery of left ventricular function was significantly better, and infarct size was significantly smaller in female (37.1 +/- 1.9%) than in male (48.3 +/- 2.3%) hearts after 25-min ischemia followed by 2-h reperfusion. Inhibition of phosphatidylinositol 3-kinase/Akt pathway by wortmannin or PKC by chelerythrine chloride before ischemia significantly reduced postischemic recovery and increased infarct size in female but not male hearts. There were no differences in myocardial protein levels of heat shock protein 70, Akt, and PKCepsilon, respectively, between male and female rats. However, the ratio of phosphorylated (p)-Akt/Akt (0.58 +/- 0.05 versus 0.22 +/- 0.04; P < 0.05) and p-PKCepsilon/PKCepsilon (0.35 +/- 0.03 versus 0.22 +/- 0.02; P < 0.05) was significantly higher in female than in male hearts. In addition, there were significant increases in p-Akt and p-PKCepsilon levels during reperfusion in female but not in male hearts. The results suggest that increased p-Akt and p-PKCepsilon levels in female hearts contribute to the gender-related differences in heart susceptibility to I/R and play an important role in cardioprotection against I/R injury in females. PMID:16099927

  11. A post-MI power struggle: adaptations in cardiac power occur at the sarcomere level alongside MyBP-C and RLC phosphorylation

    PubMed Central

    Sikkel, Markus B.; Caorsi, Valentina; Vydyanath, Anupama; Torre, Iratxe; Copeland, O'Neal; Lyon, Alexander R.; Marston, Steven B.; Luther, Pradeep K.; Macleod, Kenneth T.; West, Timothy G.; Ferenczi, Michael A.

    2016-01-01

    Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic “compensation” and congestive “decompensation.” Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae. PMID:27233767

  12. A post-MI power struggle: adaptations in cardiac power occur at the sarcomere level alongside MyBP-C and RLC phosphorylation.

    PubMed

    Toepfer, Christopher N; Sikkel, Markus B; Caorsi, Valentina; Vydyanath, Anupama; Torre, Iratxe; Copeland, O'Neal; Lyon, Alexander R; Marston, Steven B; Luther, Pradeep K; Macleod, Kenneth T; West, Timothy G; Ferenczi, Michael A

    2016-08-01

    Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic "compensation" and congestive "decompensation." Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae. PMID:27233767

  13. Extrapancreatic roles of glimepiride on osteoblasts from rat manibular bone in vitro: Regulation of cytodifferentiation through PI3-kinases/Akt signalling pathway.

    PubMed

    Ma, Pan; Xiong, Wei; Liu, Hongchen; Ma, Junli; Gu, Bin; Wu, Xia

    2011-04-01

    Glimepiride, a third-generation sulfonylurea, has also been reported to have extrapancreatic functions including activation of PI3-kinase (PI3K) and Akt in rat adipocytes, skeletal muscle and endothelial cells. It is tempting to speculate that glimepiride would improve bone-implant contact in diabetic patients by mediating the activity of GLUT1 and 3 via the PI3K/Akt pathway. In this study, we investigated the effects of glimepiride on rat mandible osteoblasts cultured under two different levels of glucose. Cell proliferation was determined by the MTT assay. The supernatant was used to measure alkaline phosphatase (ALP) activity. Glucose uptake was determined by measuring the rate of 2-deoxy-d-glucose (2-DG) uptake. Western blotting was performed used to determine collagen I and PI3K/Akt expression. RT-PCR was performed used to determine osteocalcin (OCN) mRNA expression. We found that hyperglycemia down-regulated proliferation, ALP activity, OCN mRNA and GLUT3 protein expression in rat osteoblasts, and upregulated collagen I and GLUT1 protein expressions. Glimepiride enhanced the proliferation, ALP activity and OCN mRNA levels, and upregulated collagen I and GLUT1 and 3 protein expressions of rat osteoblasts at two different glucose concentrations. This study also provides the first evidence that glimepiride stimulates the phosphorylation of PI3K/Akt in osteoblasts and ameliorated the damage caused by high concentrations of glucose through the PI3K/Akt pathway. PMID:21055727

  14. 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

  15. NecroX-5 protects mitochondrial oxidative phosphorylation capacity and preserves PGC1α expression levels during hypoxia/reoxygenation injury.

    PubMed

    Thu, Vu Thi; Kim, Hyoung Kyu; Long, Le Thanh; Nyamaa, Bayalagmaa; Song, In-Sung; Thuy, To Thanh; Huy, Nguyen Quang; Marquez, Jubert; Kim, Soon Ha; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo; Han, Jin

    2016-03-01

    Although the antioxidant and cardioprotective effects of NecroX-5 on various in vitro and in vivo models have been demonstrated, the action of this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment (10 µM) and non-treatment were employed on isolated rat hearts during hypoxia/reoxygenation treatment using an ex vivo Langendorff system. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and non-labeling peptide count protein quantification. Real-time PCR, western blot, citrate synthases and mitochondrial complex activity assays were then performed to assess heart function. Treatment with NecroX-5 during hypoxia significantly preserved electron transport chain proteins involved in oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V function. Additionally, markedly higher peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC1α) expression levels were observed in NecroX-5-treated rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving PGC1α during cardiac HR injuries. PMID:26937217

  16. NecroX-5 protects mitochondrial oxidative phosphorylation capacity and preserves PGC1α expression levels during hypoxia/reoxygenation injury

    PubMed Central

    Thu, Vu Thi; Kim, Hyoung Kyu; Long, Le Thanh; Nyamaa, Bayalagmaa; Song, In-Sung; Thuy, To Thanh; Huy, Nguyen Quang; Marquez, Jubert; Kim, Soon Ha; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo

    2016-01-01

    Although the antioxidant and cardioprotective effects of NecroX-5 on various in vitro and in vivo models have been demonstrated, the action of this compound on the mitochondrial oxidative phosphorylation system remains unclear. Here we verify the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity during hypoxia-reoxygenation (HR). Necrox-5 treatment (10 µM) and non-treatment were employed on isolated rat hearts during hypoxia/reoxygenation treatment using an ex vivo Langendorff system. Proteomic analysis was performed using liquid chromatography-mass spectrometry (LC-MS) and non-labeling peptide count protein quantification. Real-time PCR, western blot, citrate synthases and mitochondrial complex activity assays were then performed to assess heart function. Treatment with NecroX-5 during hypoxia significantly preserved electron transport chain proteins involved in oxidative phosphorylation and metabolic functions. NecroX-5 also improved mitochondrial complex I, II, and V function. Additionally, markedly higher peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC1α) expression levels were observed in NecroX-5-treated rat hearts. These novel results provide convincing evidence for the role of NecroX-5 in protecting mitochondrial oxidative phosphorylation capacity and in preserving PGC1α during cardiac HR injuries. PMID:26937217

  17. The mammalian target of rapamycin signaling pathway regulates myocyte enhancer factor-2C phosphorylation levels through integrin-linked kinase in goat skeletal muscle satellite cells.

    PubMed

    Wu, Haiqing; Ren, Yu; Pan, Wei; Dong, Zhenguo; Cang, Ming; Liu, Dongjun

    2015-11-01

    Mammalian target of rapamycin (mTOR) signaling pathway plays a key role in muscle development and is involved in multiple intracellular signaling pathways. Myocyte enhancer factor-2 (MEF2) regulates muscle cell proliferation and differentiation. However, how the mTOR signaling pathway regulates MEF2 activity remains unclear. We isolated goat skeletal muscle satellite cells (gSSCs) as model cells to explore mTOR signaling pathway regulation of MEF2C. We inhibited mTOR activity in gSSCs with PP242 and found that MEF2C phosphorylation was decreased and that muscle creatine kinase (MCK) expression was suppressed. Subsequently, we detected integrin-linked kinase (ILK) using MEF2C coimmunoprecipitation; ILK and MEF2C were colocalized in the gSSCs. We found that inhibiting mTOR activity increased ILK phosphorylation levels and that inhibiting ILK activity with Cpd 22 and knocking down ILK with small interfering RNA increased MEF2C phosphorylation and MCK expression. In the presence of Cpd 22, mTOR activity inhibition did not affect MEF2C phosphorylation. Moreover, ILK dephosphorylated MEF2C in vitro. These results suggest that the mTOR signaling pathway regulates MEF2C positively and regulates ILK negatively and that ILK regulates MEF2C negatively. It appears that the mTOR signaling pathway regulates MEF2C through ILK, further regulating the expression of muscle-related genes in gSSCs. PMID:26041412

  18. Concomitant activation of the PI3K/Akt and ERK1/2 signalling is involved in cyclic compressive force-induced IL-6 secretion in MLO-Y4 cells.

    PubMed

    Yin, Jian; Hao, Zhichao; Ma, Yuanyuan; Liao, Shuang; Li, Xianxian; Fu, Jing; Wu, Yeke; Shen, Jiefei; Zhang, Ping; Li, Xiaoyu; Wang, Hang

    2014-05-01

    IL-6 has a dual role in bone remodelling. The ERK1/2 pathway partially upregulated IL-6 secretion in osteocyte-like MLO-Y4 cells exposed to CCF. We have now investigated the possible role of phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway in the CCF-induced IL-6 expression. MLO-Y4 cells were treated with CCF 2,000 µstrain, 2 Hz, or 10, 30 min, 1, 3 and 6 h. IL-6 expression, Akt and ERK1/2 and PI3K/Akt phosphorylation were determined by RT-PCR, ELISA and Western blotting. Inhibition of PI3K/Akt with LY294002 or ERK1/2 with PD98059 significantly attenuated IL-6 upregulation, and IL-6 expression was abolished by inhibiting both pathways. Inhibition of one pathway downregulated the other's phosphorylation level. In conclusion, concomitant activation of PI3K/Akt and ERK1/2 pathways mediated IL-6 expression in MLO-Y4 cells under CCF. PMID:24375569

  19. PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis.

    PubMed

    Lopes-Pires, M Elisa; Naime, Ana C Antunes; Almeida Cardelli, Nádia J; Anjos, Débora J; Antunes, Edson; Marcondes, Sisi

    2015-01-01

    Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg, i.p.) and blood was collected after 6 h. Platelet aggregation was induced by ADP (10 μM). Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act

  20. PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis

    PubMed Central

    Lopes-Pires, M. Elisa; Naime, Ana C. Antunes; Almeida Cardelli, Nádia J.; Anjos, Débora J.; Antunes, Edson; Marcondes, Sisi

    2015-01-01

    Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg, i.p.) and blood was collected after 6 h. Platelet aggregation was induced by ADP (10 μM). Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act

  1. Oleanolic acid supplement attenuates liquid fructose-induced adipose tissue insulin resistance through the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt signaling pathway in rats

    SciTech Connect

    Li, Ying; Wang, Jianwei; Gu, Tieguang; Yamahara, Johji; Li, Yuhao

    2014-06-01

    Oleanolic acid, a triterpenoid contained in more than 1620 plants including various fruits and foodstuffs, has numerous metabolic effects, such as hepatoprotection. However, its underlying mechanisms remain poorly understood. Adipose tissue insulin resistance (Adipo-IR) may contribute to the development and progress of metabolic abnormalities through release of excessive free fatty acids from adipose tissue. This study investigated the effect of oleanolic acid on Adipo-IR. The results showed that supplement with oleanolic acid (25 mg/kg, once daily, by oral gavage) over 10 weeks attenuated liquid fructose-induced increase in plasma insulin concentration and the homeostasis model assessment of insulin resistance (HOMA-IR) index in rats. Simultaneously, oleanolic acid reversed the increase in the Adipo-IR index and plasma non-esterified fatty acid concentrations during the oral glucose tolerance test assessment. In white adipose tissue, oleanolic acid enhanced mRNA expression of the genes encoding insulin receptor, insulin receptor substrate (IRS)-1 and phosphatidylinositol 3-kinase. At the protein level, oleanolic acid upregulated total IRS-1 expression, suppressed the increased phosphorylated IRS-1 at serine-307, and restored the increased phosphorylated IRS-1 to total IRS-1 ratio. In contrast, phosphorylated Akt to total Akt ratio was increased. Furthermore, oleanolic acid reversed fructose-induced decrease in phosphorylated-Akt/Akt protein to plasma insulin concentration ratio. However, oleanolic acid did not affect IRS-2 mRNA expression. Therefore, these results suggest that oleanolic acid supplement ameliorates fructose-induced Adipo-IR in rats via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway. Our findings may provide new insights into the mechanisms of metabolic actions of oleanolic acid. - Highlights: • Adipose insulin resistance (Adipo-IR) contributes to metabolic abnormalities. • We investigated the effect of oleanolic acid (OA) on adipo-IR in

  2. Manipulating the Lateral Diffusion of Surface-Anchored EGF Demonstrates that Receptor Clustering Modulates its Phosphorylation Levels

    SciTech Connect

    Stabley, Daniel; Retterer, Scott T; Marshal, Stephen; Salaita, Khalid

    2013-01-01

    Upon activation, the epidermal growth factor (EGF) receptor becomes phosphorylated and triggers a vast signaling network that has profound effects on cell growth. The EGF receptor is observed to assemble into clusters after ligand binding and tyrosine kinase autophosphorylation, but the role of these assemblies in the receptor signaling pathway remains unclear. To address this question, we measured the phosphorylation of EGFR when the EGF ligand was anchored onto laterally mobile and immobile surfaces. We found that cells generated clusters of ligand-receptor complex on mobile EGF surfaces, and generated a lower ratio of phosphorylated EGFR to EGF than when compared to immobilized EGF that is unable to cluster. This result was verified by tuning the lateral assembly of ligand-receptor complexes on the surface of living cells using patterned supported lipid bilayers. Nanoscale metal lines fabricated into the supported membrane constrained lipid diffusion and EGF receptor assembly into micron and sub-micron scale corrals. Single cell analysis indicated that clustering impacts EGF receptor activation, and larger clusters (> 1 m2) of ligand-receptor complex generated lower EGF receptor phosphorylation per ligand than smaller assemblies (< 1 m2) in HCC1143 cells that were engaged to ligand-functionalized surfaces. We investigated EGFR clustering by treating cells with compounds that disrupt the cytoskeleton (Latrunculin-B), clathrin-mediated endocytosis (Pitstop2), and inhibit EGFR activation (Gefitinib). These results help elucidate the nature of large-scale EGFR clustering, thus underscoring the general significance of receptor spatial organization in tuning function.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. Hypoxia and classical activation limits Mycobacterium tuberculosis survival by Akt-dependent glycolytic shift in macrophages

    PubMed Central

    Matta, S K; Kumar, D

    2016-01-01

    Cellular reactive oxygen species (ROS) is a major antibacterial defense mechanism used by macrophages upon activation. Exposure of Mycobacterium tuberculosis (Mtb)-infected macrophages to hypoxia is known to compromise the survival of the pathogen. Here we report that the hypoxia-induced control of intracellular Mtb load in RAW 264.7 macrophages was mediated by regulating the cellular ROS levels. We show that similar to classical activation, hypoxia incubation of macrophages resulted in decreased mitochondrial outer membrane potential (MOMP) and a concomitant increase in the cellular ROS levels. Mitochondrial depolarization and consequently higher ROS could be blocked by knocking down Akt using siRNAs, which acted by inhibiting the switch to glycolytic mode of metabolism, an essential adaptive response upon classical activation or hypoxic incubation of macrophages. Moreover, in the classically activated macrophages or in the macrophages under hypoxia incubation, supplementation with additional glucose had similar effects as Akt knockdown. Interestingly, in both the cases, the reversal of phenotype was linked with the ability of the mitochondrial F0–F1 ATP synthase activity to maintain the MOMP in the absence of oxidative phosphorylation. Both Akt knockdown and glucose supplementation were also able to rescue Mtb survival in these macrophages upon classical activation or hypoxia incubation. These results provide a framework for better understanding of how the interplay between oxygen supply, which is limiting in the human tubercular granulomas, and nutrient availability could together direct the outcome of infections in vivo. PMID:27551515

  9. Orexin A Affects INS-1 Rat Insulinoma Cell Proliferation via Orexin Receptor 1 and the AKT Signaling Pathway

    PubMed Central

    Chen, Li; Zhao, Yuyan; Zheng, Delu; Ju, Shujing; Shen, Yang; Guo, Lei

    2013-01-01

    Our aim is to investigate the role of the AKT/PKB (protein kinase B) signaling pathway acting via orexin receptor 1 (OX1R) and the effects of orexin A (OXA) on cell proliferation in the insulin-secreting beta-cell line (INS-1 cells). Rat INS-1 cells were exposed to different concentrations of OXA in vitro and treated with OX1R antagonist (SB334867), PI3K antagonist (wortmannin), AKT antagonist (PF-04691502), or negative control. INS-1 amount of cell proliferation, viability and apoptosis, insulin secretion, OX1R protein expression, caspase-3 activity, and AKT protein levels were determined. We report that OXA (10−10 to 10−6 M) stimulates INS-1 cell proliferation and viability, reduces the proapoptotic activity of caspase-3 to protect against apoptotic cell death, and increases insulin secretion. Additionally, AKT phosphorylation was stimulated by OXA (10−10 to 10−6 M). However, the OX1R antagonist SB334867 (10−6 M), the PI3K antagonist wortmannin (10−8 M), the AKT antagonist PF-04691502 (10−6 M), or the combination of both abolished the effects of OXA to a certain extent. These results suggest that the upregulation of OXA-OX1R mediated by AKT activation may inhibit cell apoptosis and promote cell proliferation in INS-1 cells. This finding provides functional evidence of the biological actions of OXA in rat insulinoma cells. PMID:24382962

  10. 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}.

  11. Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch

    PubMed Central

    Yang, Jae-Hyun; Song, Tae-Yang; Jo, Chanhee; Park, Jinyoung; Lee, Han-Young; Song, Ilang; Hong, Suji; Jung, Kwan Young; Kim, Jaehoon; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung

    2016-01-01

    Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis. PMID:27515126

  12. Increased iPLA2 activity and levels of phosphorylated GSK3B in platelets are associated with donepezil treatment in Alzheimer's disease patients.

    PubMed

    Talib, L L; Hototian, S R; Joaquim, H P G; Forlenza, O V; Gattaz, W F

    2015-12-01

    Reduced phospholipase A2 (PLA2) activity and increased phosphorylation of glycogen synthase kinase 3B (GSK3B) participate in the production of beta-amyloid plaques and of neurofibrillary tangles, which are two neuropathological hallmarks of Alzheimer's disease (AD). Experimental evidences suggest a neuroprotective effect of the cholinesterase inhibitor donepezil in the treatment the disease. The aims of the present study were to evaluate in AD patients the effects of treatment with donepezil on PLA2 activity and GSK3B level. Thirty patients with AD were treated during 6 months with 10 mg daily of donepezil. Radio-enzymatic assays were used to measure PLA2 activity and Elisa assays for GSK3B level, both in platelets. Before treatment and after 3 and 6 months on donepezil, AD patients underwent a cognitive assessment and platelet samples were collected. Values were compared to a healthy control group of 42 sex- and age-matched elderly individuals. Before treatment, iPLA2 activity was lower in patients with AD as compared to controls (p < 0.001). At baseline, no differences were found in GSK3B level between both groups. After 3 and 6 months of treatment, we found a significant increase in iPLA2 activity (p = 0.015 and p < 0.001, respectively). iPLA2 increment was related to the cognitive improvement during treatment (p = 0.037). After 6 months, we found an increase in phosphorylated GSK3B (p = 0.02). The present findings suggest two possible mechanisms by which donepezil delays the progression of AD. The increment of iPLA2 activity may reduce the production of beta-amyloid plaques, whereas the phosphorylation of GSK3B inactivates the enzyme, reducing thus the phosphorylation of tau protein. PMID:25920742

  13. 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

  14. Modulation of EZH2 Expression by MEK-ERK or PI3K-AKT Signaling in Lung Cancer Is Dictated by Different KRAS Oncogene Mutations.

    PubMed

    Riquelme, Erick; Behrens, Carmen; Lin, Heather Y; Simon, George; Papadimitrakopoulou, Vassiliki; Izzo, Julie; Moran, Cesar; Kalhor, Neda; Lee, J Jack; Minna, John D; Wistuba, Ignacio I

    2016-02-01

    EZH2 overexpression promotes cancer by increasing histone methylation to silence tumor suppressor genes, but how EZH2 levels become elevated in cancer is not understood. In this study, we investigated the mechanisms by which EZH2 expression is regulated in non-small cell lung carcinoma cells by oncogenic KRAS. In cells harboring KRAS(G12C) and KRAS(G12D) mutations, EZH2 expression was modulated by MEK-ERK and PI3K/AKT signaling, respectively. Accordingly, MEK-ERK depletion decreased EZH2 expression in cells harboring the KRAS(G12C) mutation, whereas PI3K/AKT depletion decreased EZH2 expression, EZH2 phosphorylation, and STAT3 activity in KRAS(G12D)-mutant cell lines. Combined inhibition of EZH2 and MEK-ERK or PI3K/AKT increased the sensitivity of cells with specific KRAS mutations to MEK-ERK and PI3K/AKT-targeted therapies. Our work defines EZH2 as a downstream effector of KRAS signaling and offers a rationale for combining EZH2 inhibitory strategies with MEK-ERK- or PI3K/AKT-targeted therapies to treat lung cancer patients, as stratified into distinct treatment groups based on specific KRAS mutations. PMID:26676756

  15. Akt- and CREB-Mediated Prostate Cancer Cell Proliferation Inhibition by Nexrutine, a Phellodendron amurense Extract1

    PubMed Central

    Garcia, Gretchen E; Nicole, Arevalo; Bhaskaran, Shylesh; Gupta, Ashima; Kyprianou, Natasha; Kumar, Addanki P

    2006-01-01

    Abstract Evidence from epidemiological studies suggests that plant-based diets can reduce the risk of prostate cancer. However, very little information is available concerning the use of botanicals in preventing prostate cancer. As a first step toward developing botanicals as prostate cancer preventives, we examined the effect of Nexrutine on human prostate cancer cells. Nexrutine is a herbal extract developed from Phellodendron amurense. Phellodendron extracts have been used traditionally in Chinese medicine for hundreds of years as an anti-diarrheal, astringent, and anti-inflammatory agent. The present study investigated its potential antitumor effect on human prostate cancer cells. Our results suggest that it inhibits tumor cell proliferation through apoptosis induction and inhibition of cell survival signaling. The results of the present study indicate that Nexrutine treatment 1) inhibits the proliferation of both androgen-responsive and androgen-independent human prostate cancer cells through induction of apoptosis; 2) reduces levels of pAkt, phosphorylated cAMP response-binding protein (pCREB), and CREB DNA-binding activity; and 3) induces apoptosis in prostate cancer cells stably overexpressing Bcl-2. Further Akt kinase activity was reduced in cells treated with Nexrutine, and ectopic expression of myristoylated Akt protected from Nexrutine induced inhibition of proliferation, implicating a role for Akt signaling. PMID:16820098

  16. Anthocyanins protected hearts against ischemic injury by reducing MMP-2 activity via Akt/P38 pathways

    PubMed Central

    Hao, Jie; Du, Hong; Li, Weiwei; Liu, Fan; Lu, Jingchao; Yang, Xiuchun; Cui, Wei

    2016-01-01

    Growing evidences suggest that there are close associations between anthocyanins and cardiac protection. However, little is known about the detailed roles of anthocyanins in regulating extracellular matrix (ECM) remodeling. Incubation of primary cultured fibroblasts with anthocyanins reduced both intracellular collagen expression and extracellular collagen secretion. Down-regulation of collagen production was also shown in infarcted cardiac tissues after permanent coronary artery ligation in mice treated with anthocyanins. The phosphorylation levels of Akt and/or P-38 were significantly increased by anthocyanins supplementation in primary cultured fibroblasts. Gelatin zymography analysis of matrix metalloproteinase-2 (MMP-2) activity in conditioned medium collected from fibroblasts demonstrated that anthocyanins treatment significantly reduced MMP-2 activity. These results demonstrated that anthocyanins play a role in mediating myocardial ECM remodeling and that the Akt/P-38 pathways mediate these protective effects on hearts. PMID:27158396

  17. Downregulation of Rab25 activates Akt1 in head and neck squamous cell carcinoma

    PubMed Central

    SEVEN, DIDEM; DOGAN, SOYDAN; KILIÇ, ERKAN; KARAMAN, EMIN; KOSEOGLU, HIKMET; BUYRU, NUR

    2015-01-01

    Several studies have suggested that Ras-associated binding 25 protein (Rab25) is involved in the pathogenesis of human cancer. Although it has been demonstrated that the development of head and neck squamous cell carcinoma (HNSCC) is the result of an accumulation of multiple sequential genetic and epigenetic alterations in key genes with important functions in cell growth and the cell cycle, recent studies have indicated that HNSCC is a complex and heterogenous disease. To the best of our knowledge, there is no data regarding the regulation of the Rab25 gene at the mRNA or protein level in HNSCC. Furthermore, available data on Rab25 expression in other types of cancer are conflicting. The aim of the present study was to investigate whether Rab25 is involved in the development and/or progression of HNSCC, and to analyze the mechanisms underlying its effects in this type of cancer. The expression of Rab25 mRNA in HNSCC tissues and adjacent non-tumor tissue samples was measured using reverse transcription-quantitative polymerase chain reaction, while the level of the Rab25, Akt1 and phosphorylated-Akt1 proteins was measured using western blotting. Expression of Rab25 mRNA and protein was downregulated in 69.1% and 56.1% of tumor tissue samples, respectively. This downregulation was associated with an increase in p-Akt1 expression, in the absence of a change in total Akt1 protein levels, in tumor tissues compared with normal tissues. The current findings suggest that Rab25 acts as a tumor suppressor in HNSCC. PMID:26622777

  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. The PTEN/PI3K/AKT Pathway in vivo, Cancer Mouse Models

    PubMed Central

    Carnero, Amancio; Paramio, Jesus M.

    2014-01-01

    When PI3K (phosphatidylinositol-3 kinase) is activated by receptor tyrosine kinases, it phosphorylates PIP2 to generate PIP3 and activates the signaling pathway. Phosphatase and tensin homolog deleted on chromosome 10 dephosphorylates PIP3 to PIP2, and thus, negatively regulates the pathway. AKT (v-akt murine thymoma viral oncogene homolog; protein kinase B) is activated downstream of PIP3 and mediates physiological processes. Furthermore, substantial crosstalk exists with other signaling networks at all levels of the PI3K pathway. Because of its diverse array, gene mutations, and amplifications and also as a consequence of its central role in several signal transduction pathways, the PI3K-dependent axis is frequently activated in many tumors and is an attractive therapeutic target. The preclinical testing and analysis of these novel therapies requires appropriate and well-tailored systems. Mouse models in which this pathway has been genetically modified have been essential in understanding the role that this pathway plays in the tumorigenesis process. Here, we review cancer mouse models in which the PI3K/AKT pathway has been genetically modified. PMID:25295225

  20. Coprinus comatus Cap Inhibits Adipocyte Differentiation via Regulation of PPARγ and Akt Signaling Pathway

    PubMed Central

    Jang, Sun-Hee; Kang, Suk Nam; Jeon, Beong-Sam; Ko, Yeoung-Gyu; Kim, Hong-Duck; Won, Chung-Kil; Kim, Gon-Sup; Cho, Jae-Hyeon

    2014-01-01

    This study assessed the effects of Coprinus comatus cap (CCC) on adipogenesis in 3T3-L1 adipocytes and the effects of CCC on the development of diet-induced obesity in rats. Here, we showed that the CCC has an inhibitory effect on the adipocyte differentiation of 3T3-L1 cells, resulting in a significant decrease in lipid accumulation through the downregulation of several adipocyte specific-transcription factors, including CCAAT/enhancer binding protein β, C/EBPδ, and peroxisome proliferator-activated receptor gamma (PPARγ). Moreover, treatment with CCC during adipocyte differentiation induced a significant down-regulation of PPARγ and adipogenic target genes, including adipocyte protein 2, lipoprotein lipase, and adiponectin. Interestingly, the CCC treatment of the 3T3-L1 adipocytes suppressed the insulin-stimulated Akt and GSK3β phosphorylation, and these effects were stronger in the presence of an inhibitor of Akt phosphorylation, LY294002, suggesting that CCC inhibited adipocyte differentiation through the down-regulation of Akt signaling. In the animal study, CCC administration significantly reduced the body weight and adipose tissue weight of rats fed a high fat diet (HFD) and attenuated lipid accumulation in the adipose tissues of the HFD-induced obese rats. The size of the adipocyte in the epididymal fat of the CCC fed rats was significantly smaller than in the HFD rats. CCC treatment significantly reduced the total cholesterol and triglyceride levels in the serum of HFD rats. These results strongly indicated that the CCC-mediated decrease in body weight was due to a reduction in adipose tissue mass. The expression level of PPARγ and phospho-Akt was significantly lower in the CCC-treated HFD rats than that in the HFD obesity rats. These results suggested that CCC inhibited adipocyte differentiation by the down-regulation of major transcription factor involved in the adipogenesis pathway including PPARγ through the regulation of the Akt pathway in 3T3

  1. Coprinus comatus cap inhibits adipocyte differentiation via regulation of PPARγ and Akt signaling pathway.

    PubMed

    Park, Hyoung Joon; Yun, Jisoo; Jang, Sun-Hee; Kang, Suk Nam; Jeon, Beong-Sam; Ko, Yeoung-Gyu; Kim, Hong-Duck; Won, Chung-Kil; Kim, Gon-Sup; Cho, Jae-Hyeon

    2014-01-01

    This study assessed the effects of Coprinus comatus cap (CCC) on adipogenesis in 3T3-L1 adipocytes and the effects of CCC on the development of diet-induced obesity in rats. Here, we showed that the CCC has an inhibitory effect on the adipocyte differentiation of 3T3-L1 cells, resulting in a significant decrease in lipid accumulation through the downregulation of several adipocyte specific-transcription factors, including CCAAT/enhancer binding protein β, C/EBPδ, and peroxisome proliferator-activated receptor gamma (PPARγ). Moreover, treatment with CCC during adipocyte differentiation induced a significant down-regulation of PPARγ and adipogenic target genes, including adipocyte protein 2, lipoprotein lipase, and adiponectin. Interestingly, the CCC treatment of the 3T3-L1 adipocytes suppressed the insulin-stimulated Akt and GSK3β phosphorylation, and these effects were stronger in the presence of an inhibitor of Akt phosphorylation, LY294002, suggesting that CCC inhibited adipocyte differentiation through the down-regulation of Akt signaling. In the animal study, CCC administration significantly reduced the body weight and adipose tissue weight of rats fed a high fat diet (HFD) and attenuated lipid accumulation in the adipose tissues of the HFD-induced obese rats. The size of the adipocyte in the epididymal fat of the CCC fed rats was significantly smaller than in the HFD rats. CCC treatment significantly reduced the total cholesterol and triglyceride levels in the serum of HFD rats. These results strongly indicated that the CCC-mediated decrease in body weight was due to a reduction in adipose tissue mass. The expression level of PPARγ and phospho-Akt was significantly lower in the CCC-treated HFD rats than that in the HFD obesity rats. These results suggested that CCC inhibited adipocyte differentiation by the down-regulation of major transcription factor involved in the adipogenesis pathway including PPARγ through the regulation of the Akt pathway in 3T3

  2. Dissociation of ERK and Akt signaling in endothelial cell angiogenic responses to {beta}-amyloid

    SciTech Connect

    Magrane, Jordi; Christensen, Rial A.; Rosen, Kenneth M.; Veereshwarayya, Vimal; Querfurth, Henry W. . E-mail: hquerf01@granite.tufts.edu

    2006-04-15

    Cerebrovascular deposits of {beta}-amyloid (A{beta}) peptides are found in Alzheimer's disease and cerebral amyloid angiopathy with stroke or dementia. Dysregulations of angiogenesis, the blood-brain barrier and other critical endothelial cell (EC) functions have been implicated in aggravating chronic hypoperfusion in AD brain. We have used cultured ECs to model the effects of {beta}-amyloid on the activated phosphorylation states of multifunctional serine/threonine kinases since these are differentially involved in the survival, proliferation and migration aspects of angiogenesis. Serum-starved EC cultures containing amyloid-{beta} peptides underwent a 2- to 3-fold increase in nuclear pyknosis. Under growth conditions with sublethal doses of {beta}-amyloid, loss of cell membrane integrity and inhibition of cell proliferation were observed. By contrast, cell migration was the most sensitive to A{beta} since inhibition was significant already at 1 {mu}M (P = 0.01, migration vs. proliferation). In previous work, intracellular A{beta} accumulation was shown toxic to ECs and Akt function. Here, extracellular A{beta} peptides do not alter Akt activation, resulting instead in proportionate decreases in the phosphorylations of the MAPKs: ERK1/2 and p38 (starting at 1 {mu}M). This inhibitory action occurs proximal to MEK1/2 activation, possibly through interference with growth factor receptor coupling. Levels of phospho-JNK remained unchanged. Addition of PD98059, but not LY294002, resulted in a similar decrease in activated ERK1/2 levels and inhibition of EC migration. Transfection of ERK1/2 into A{beta}-poisoned ECs functionally rescued migration. The marked effect of extracellular A{beta} on the migration component of angiogenesis is associated with inhibition of MAPK signaling, while Akt-dependent cell survival appears more affected by cellular A{beta}.

  3. MicroRNA-542-3p Suppresses Tumor Cell Invasion via Targeting AKT Pathway in Human Astrocytoma.

    PubMed

    Cai, Junchao; Zhao, JingJing; Zhang, Nu; Xu, Xiaonan; Li, Rong; Yi, Yang; Fang, Lishan; Zhang, Le; Li, Mengfeng; Wu, Jueheng; Zhang, Heng

    2015-10-01

    The molecular mechanism underlying constitutive activation of AKT signaling, which plays essential roles in astrocytoma progression, is not fully characterized. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of astrocytoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we found that microRNA (miR)-542-3p expression was decreased in glioblastoma cell lines and astrocytoma tissues, and reduced levels of miR-542-3p expression correlated with high histopathological grades and poor prognosis of astrocytoma patients. Exogenous miR-542-3p suppressed glioblastoma cell invasion through not only targeting AKT1 itself but also directly down-regulating its two important upstream regulators, namely, integrin-linked kinase and PIK3R1. Notably, overexpressing miR-542-3p decreased AKT1 phosphorylation and directly and indirectly repressed nuclear translocation and transactivation activity of β-catenin to exert its anti-invasive effect. Furthermore, the miR-542-3p expression level negatively correlated with AKT activity as well as levels of integrin-linked kinase and PIK3R1 in human astrocytoma specimens. These findings suggest that miR-542-3p acts as a negative regulator in astrocytoma progression and that miR-542-3p down-regulation contributes to aberrant activation of AKT signaling, leaving open the possibility that miR-542-3p may be a potential therapeutic target for high grade astrocytoma. PMID:26286747

  4. Central insulin signaling is attenuated by long-term insulin exposure via insulin receptor substrate-1 serine phosphorylation, proteasomal degradation, and lysosomal insulin receptor degradation.

    PubMed

    Mayer, Christopher M; Belsham, Denise D

    2010-01-01

    Central insulin signaling is critical for the prevention of insulin resistance. Hyperinsulinemia contributes to insulin resistance, but it is not yet clear whether neurons are subject to cellular insulin resistance. We used an immortalized, hypothalamic, clonal cell line, mHypoE-46, which exemplifies neuronal function and expresses the components of the insulin signaling pathway, to determine how hyperinsulinemia modifies neuronal function. Western blot analysis indicated that prolonged insulin treatment of mHypoE-46 cells attenuated insulin signaling through phospho-Akt. To understand the mechanisms involved, time-course analysis was performed. Insulin exposure for 4 and 8 h phosphorylated Akt and p70-S6 kinase (S6K1), whereas 8 and 24 h treatment decreased insulin receptor (IR) and IR substrate 1 (IRS-1) protein levels. Insulin phosphorylation of S6K1 correlated with IRS-1 ser1101 phosphorylation and the mTOR-S6K1 pathway inhibitor rapamycin prevented IRS-1 serine phosphorylation. The proteasomal inhibitor epoxomicin and the lysosomal pathway inhibitor 3-methyladenine prevented the degradation of IRS-1 and IR by insulin, respectively, and pretreatment with rapamycin, epoxomicin, or 3-methyladenine prevented attenuation of insulin signaling by long-term insulin exposure. Thus, a sustained elevation of insulin levels diminishes neuronal insulin signaling through mTOR-S6K1-mediated IRS-1 serine phosphorylation, proteasomal degradation of IRS-1 and lysosomal degradation of the IR. PMID:19887566

  5. The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway

    PubMed Central

    Calegari-Silva, Teresa C.; Vivarini, Áislan C.; Miqueline, Marina; Dos Santos, Guilherme R. R. M.; Teixeira, Karina Luiza; Saliba, Alessandra Mattos; Nunes de Carvalho, Simone; de Carvalho, Laís; Lopes, Ulisses G.

    2015-01-01

    Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression. PMID:26400473

  6. The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway.

    PubMed

    Calegari-Silva, Teresa C; Vivarini, Áislan C; Miqueline, Marina; Dos Santos, Guilherme R R M; Teixeira, Karina Luiza; Saliba, Alessandra Mattos; Nunes de Carvalho, Simone; de Carvalho, Laís; Lopes, Ulisses G

    2015-09-01

    Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression. PMID:26400473

  7. The protective effects of Chinese herb-Taikong Yangxin Prescription on the atrophic remodeling of cardiac muscle in rats induced by hindlimb unloading through activating Akt/GSK-3beta signaling pathway

    NASA Astrophysics Data System (ADS)

    Ming, Yuan; Min, Yuan; Jianfeng, Zhang; Zhili, Li; Huijuan, Wang; Desheng, Wang; Yinghui, Li; Yongzhi, Li; Shizhong, Jiang

    Objective To test the hypothesis that traditional Chinese herb-TaiKong Yangxin Prescrip-tion can activate the Akt/GSK-3β signaling pathway and alleviate the atrophic remodeling of cardiac muscle in rats induced by hindlimb unloading. Methods The physiological effects of simulated microgravity was induced by 7d hindlimb unloading in rats. TaiKong Yangxin Pre-scription was given daily by gastric irrigation as countermeasure against effects of simulated microgravity. The frozen sections of left ventricular cardiac muscles were stained by FITC la-beled lectin and visualized by laser scanning confocal microscopy, the cross section areas(CSA) of cardiomyocytes were calculated by IPP6.0 Image software. The protein expression of TnI, phosphorylation level of Akt and GSK-3β were measured by Western blot. Results Simulated microgravity decreased the CSA of cardiomyocytes and protein expression of TnI in left ven-tricular cardiac muscles, inhibited the phosphorylation level of Akt at serine 473 and GSK-3β at serine 9. The traditional Chinese herb-TaiKong Yangxin Prescription alleviated the atrophic remodeling of cardiac muscles, reversed the declined protein expression of TnI and phosphoryla-tion levels of Akt at serine 473 and GSK-3β at serine 9 in hindlimb-unloading rats. Conclusion The traditional Chinese herb-TaiKong Yangxin Prescription has significant countermeasure effects on the atrophic remodeling of cardiac muscle induced by hindlimb unloading in rats, in which activating Akt/GSK-3β signaling pathway plays an important role.(Funded by Advanced space medico-engineering research project of China, grant NO. 2005SY5206005 and SJ200801)

  8. Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

    PubMed

    Hunter, Gerald O; Fox, Melanie J; Smith-Kinnaman, Whitney R; Gogol, Madelaine; Fleharty, Brian; Mosley, Amber L

    2016-09-01

    In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes. PMID:27247267

  9. 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

  10. Akt and β-catenin contribute to TMZ resistance and EMT of MGMT negative malignant glioma cell line.

    PubMed

    Yi, Guo-Zhong; Liu, Ya-Wei; Xiang, Wei; Wang, Hai; Chen, Zi-Yang; Xie, Si-di; Qi, Song-Tao

    2016-08-15

    Glioblastoma is one of the most lethal cancers in central nervous system, and some individual cells that cannot be isolated for surgical resection and also show treatment-resistance induce poor prognosis. Hence, in order to research these cells, we treated temozolomide (TMZ)-sensitive U87MG cells with 400μM TMZ in culture media for over 6months and established TMZ-resistant cell line designated as U87/TR. We detected the MGMT status through pyrosequencing and western blotting, and we also assessed the proliferation, migration, EMT-like changes and possible activated signaling pathways in U87/TR cells. Our results demonstrated that U87/TR was MGMT negative, which indicated that MGMT made no contribution for TMZ-resistance of U87/TR. And U87/TR cells displayed cell cycle arrest, higher capacity for migration and EMT-like changes including both phenotype and characteristic proteins. We also revealed that both β-catenin and the phosphorylation level of Akt and PRAS40 were increased in U87/TR, while we did not observe the phosphorylation of mTOR in U87/TR. It indicated that activation of Akt and Wnt/β-catenin pathways may be response for the chemo-resistance and increased invasion of U87/TR cells, and the phosphorylation of PRAS40 and inactivated mTOR may be related to cell cycle arrest in U87/TR cells. PMID:27423571

  11. 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

  12. 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

  13. Protein Kinase Ymr291w/Tda1 Is Essential for Glucose Signaling in Saccharomyces cerevisiae on the Level of Hexokinase Isoenzyme ScHxk2 Phosphorylation*

    PubMed Central

    Kaps, Sonja; Kettner, Karina; Migotti, Rebekka; Kanashova, Tamara; Krause, Udo; Rödel, Gerhard; Dittmar, Gunnar; Kriegel, Thomas M.

    2015-01-01

    The enzyme ScHxk2 of Saccharomyces cerevisiae is a dual-function hexokinase that besides its catalytic role in glycolysis is involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by the phosphorylation of the nuclear fraction of ScHxk2 at serine 15 and the translocation of the phosphoenzyme into the cytosol. Different studies suggest different serine/threonine protein kinases, Ymr291w/Tda1 or Snf1, to accomplish ScHxk2-S15 phosphorylation. The current paper provides evidence that Ymr291w/Tda1 is essential for that modification, whereas protein kinases Ydr477w/Snf1, Ynl307c/Mck1, Yfr014c/Cmk1, and Ykl126w/Ypk1, which are co-purified during Ymr291w/Tda1 tandem affinity purification, as well as protein kinase PKA and PKB homolog Sch9 are dispensable. Taking into account the detection of a significantly higher amount of the Ymr291w/Tda1 protein in cells grown in low-glucose media as compared with a high-glucose environment, Ymr291w/Tda1 is likely to contribute to glucose signaling in S. cerevisiae on the level of ScHxk2-S15 phosphorylation in a situation of limited external glucose availability. The evolutionary conservation of amino acid residue serine 15 in yeast hexokinases and its phosphorylation is illustrated by the finding that YMR291W/TDA1 of S. cerevisiae and the homologous KLLA0A09713 gene of Kluyveromyces lactis allow for cross-complementation of the respective protein kinase single-gene deletion strains. PMID:25593311

  14. Protein kinase Ymr291w/Tda1 is essential for glucose signaling in saccharomyces cerevisiae on the level of hexokinase isoenzyme ScHxk2 phosphorylation*.

    PubMed

    Kaps, Sonja; Kettner, Karina; Migotti, Rebekka; Kanashova, Tamara; Krause, Udo; Rödel, Gerhard; Dittmar, Gunnar; Kriegel, Thomas M

    2015-03-01

    The enzyme ScHxk2 of Saccharomyces cerevisiae is a dual-function hexokinase that besides its catalytic role in glycolysis is involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by the phosphorylation of the nuclear fraction of ScHxk2 at serine 15 and the translocation of the phosphoenzyme into the cytosol. Different studies suggest different serine/threonine protein kinases, Ymr291w/Tda1 or Snf1, to accomplish ScHxk2-S15 phosphorylation. The current paper provides evidence that Ymr291w/Tda1 is essential for that modification, whereas protein kinases Ydr477w/Snf1, Ynl307c/Mck1, Yfr014c/Cmk1, and Ykl126w/Ypk1, which are co-purified during Ymr291w/Tda1 tandem affinity purification, as well as protein kinase PKA and PKB homolog Sch9 are dispensable. Taking into account the detection of a significantly higher amount of the Ymr291w/Tda1 protein in cells grown in low-glucose media as compared with a high-glucose environment, Ymr291w/Tda1 is likely to contribute to glucose signaling in S. cerevisiae on the level of ScHxk2-S15 phosphorylation in a situation of limited external glucose availability. The evolutionary conservation of amino acid residue serine 15 in yeast hexokinases and its phosphorylation is illustrated by the finding that YMR291W/TDA1 of S. cerevisiae and the homologous KLLA0A09713 gene of Kluyveromyces lactis allow for cross-complementation of the respective protein kinase single-gene deletion strains. PMID:25593311

  15. 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