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S6 Kinase (s6 + kinase)
Kinds of S6 Kinase Selected AbstractsCharacterization of p70 S6 kinase 1 in early development of mouse embryosDEVELOPMENTAL DYNAMICS, Issue 12 2009Xiao-Yan Xu Abstract The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes mTORC1 and mTORC2. p70 S6 Kinase 1 (S6K1) is characterized as downstream effector of mTOR. Until recently, the connection between S6K1 and mTORC1 /mTORC2 during the early development of mouse embryos has not been well elucidated. Here, the expression level of total S6K1 and its phosphorylation at Thr389 was determined in four phases of one-cell embryos. S6K1 was active throughout the cell cycle especially with higher activity in G2 and M phases. Rapamycin decreased the activity of M-phase promoting factor (MPF) and delayed the first mitotic cleavage. Down-regulating mTOR and raptor reduced S6K1 phosphorylation at Thr389 in one-cell embryos. Furthermore, rapamycin and microinjection of raptor shRNA decreased the immunofluorescent staining of Thr389 phospho-S6K1. It is proposed that mTORC1 may be involved in the control of MPF by regulating S6K1 during the early development of mouse embryos. Developmental Dynamics 238:3025,3034, 2009. © 2009 Wiley-Liss, Inc. [source] Negative Regulation by p70 S6 Kinase of FGF-2,Stimulated VEGF Release Through Stress-Activated Protein Kinase/c- Jun N-Terminal Kinase in Osteoblasts,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2007Shinji Takai Abstract To clarify the mechanism of VEGF release in osteoblasts, we studied whether p70 S6 kinase is involved in basic FGF-2,stimulated VEGF release in osteoblast-like MC3T3-E1 cells. In this study, we show that p70 S6 kinase activated by FGF-2 negatively regulates VEGF release through SAPK/JNK in osteoblasts. Introduction: Vascular endothelial growth factor (VEGF) plays an important role in bone metabolism. We have previously reported that fibroblast growth factor-2 (FGF-2) stimulates the release of VEGF through p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2,activated p38 MAP kinase negatively regulates VEGF release. However, the mechanism behind VEGF release in osteoblasts is not precisely known. Materials and Methods: The levels of VEGF released from MC3T3-E1 cells were measured by enzyme immunoassay. The phosphorylation of each protein kinase was analyzed by Western blotting. To knock down p70 S6 kinase in MC3T3-E1 cells, the cells were transfected with siRNA to target p70 S6 kinase. Results: FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase. Rapamycin significantly enhanced the FGF-2,stimulated VEGF release and VEGF mRNA expression. The FGF-2,induced phosphorylation of p70 S6 kinase was suppressed by rapamycin. Rapamycin markedly enhanced the FGF-2,induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. SP600125, a specific inhibitor of SAPK/JNK, suppressed the amplification by rapamycin of the FGF-2,stimulated VEGF release similar to the levels of FGF-2 with SP600125. Finally, downregulation of p70 S6 kinase by siRNA significantly enhanced the FGF-2,stimulated VEGF release and phosphorylation of SAPK/JNK. Conclusions: These results strongly suggest that p70 S6 kinase limits FGF-2,stimulated VEGF release through self-regulation of SAPK/JNK, composing a negative feedback loop, in osteoblasts. [source] Differential Extracellular Signal-Regulated Kinases 1 and 2 Activation by the Angiotensin Type 1 Receptor Supports Distinct Phenotypes of Cardiac MyocytesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 5 2007Mark Aplin The biological importance of this, however, remains obscure. Application of the modified analogue [Sar1, Ile4, Ile8]-AngII ([SII] AngII) allowed us to dissect the two pathways of ERK1/2 activation in native cardiac myocytes. Although cytosol-retained, the ,-arrestin2-bound pool of ERK1/2 represents an active signalling component that phosphorylates p90 Ribosomal S6 Kinase, a ubiquitous and versatile mediator of ERK1/2 signal transduction. Moreover, the ,-arrestin2-dependent ERK1/2 signal supports intact proliferation of cardiac myocytes. In contrast to Gq -activated ERK1/2, and in keeping with its failure to translocate to the nucleus, the ,-arrestin2-scaffolded pool of ERK1/2 does not phosphorylate the transcription factor Elk-1, induces no increased transcription of the immediate-early gene c-Fos, and does not entail myocyte hypertrophy. These results clearly demonstrate the biological significance of differential signalling by the AT1R. The opportunity to separate desirable cardiac myocyte division from detrimental hypertrophy holds promise that novel pharmacological approaches will allow targeting of pathway-specific actions. [source] Characterization of p70 S6 kinase 1 in early development of mouse embryosDEVELOPMENTAL DYNAMICS, Issue 12 2009Xiao-Yan Xu Abstract The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes mTORC1 and mTORC2. p70 S6 Kinase 1 (S6K1) is characterized as downstream effector of mTOR. Until recently, the connection between S6K1 and mTORC1 /mTORC2 during the early development of mouse embryos has not been well elucidated. Here, the expression level of total S6K1 and its phosphorylation at Thr389 was determined in four phases of one-cell embryos. S6K1 was active throughout the cell cycle especially with higher activity in G2 and M phases. Rapamycin decreased the activity of M-phase promoting factor (MPF) and delayed the first mitotic cleavage. Down-regulating mTOR and raptor reduced S6K1 phosphorylation at Thr389 in one-cell embryos. Furthermore, rapamycin and microinjection of raptor shRNA decreased the immunofluorescent staining of Thr389 phospho-S6K1. It is proposed that mTORC1 may be involved in the control of MPF by regulating S6K1 during the early development of mouse embryos. Developmental Dynamics 238:3025,3034, 2009. © 2009 Wiley-Liss, Inc. [source] Hypothermia treatment potentiates ERK1/2 activation after traumatic brain injuryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2007Coleen M. Atkins Abstract Traumatic brain injury (TBI) results in significant hippocampal pathology and hippocampal-dependent memory loss, both of which are alleviated by hypothermia treatment. To elucidate the molecular mechanisms regulated by hypothermia after TBI, rats underwent moderate parasagittal fluid-percussion brain injury. Brain temperature was maintained at normothermic or hypothermic temperatures for 30 min prior and up to 4 h after TBI. The ipsilateral hippocampus was assayed with Western blotting. We found that hypothermia potentiated extracellular signal-regulated kinase 1/2 (ERK1/2) activation and its downstream effectors, p90 ribosomal S6 kinase (p90RSK) and the transcription factor cAMP response element-binding protein. Phosphorylation of another p90RSK substrate, Bad, also increased with hypothermia after TBI. ERK1/2 regulates mRNA translation through phosphorylation of mitogen-activated protein kinase-interacting kinase 1 (Mnk1) and the translation factor eukaryotic initiation factor 4E (eIF4E). Hypothermia also potentiated the phosphorylation of both Mnk1 and eIF4E. Augmentation of ERK1/2 activation and its downstream signalling components may be one molecular mechanism that hypothermia treatment elicits to improve functional outcome after TBI. [source] mTOR as a potential therapeutic target for treatment of keloids and excessive scarsEXPERIMENTAL DERMATOLOGY, Issue 5 2007C. T. Ong Abstract:, Keloid is a dermal fibroproliferative disorder characterized by excessive deposition of extracellular matrix (ECM) components such as collagen, glycoproteins and fibronectin. The mammalian target of rapamycin (mTOR) is a serine/theronine kinase which plays an important role in the regulation of metabolic processes and translation rates. Published reports have shown mTOR as regulator of collagen expression and its inhibition induces a decrease in ECM deposition. Our aim was to investigate the role of mTOR in keloid pathogenesis and investigate the effect of rapamycin on proliferating cell nuclear antigen (PCNA), cyclin D1, collagen, fibronectin and alpha-smooth muscle actin (, -SMA) expression in normal fibroblasts (NF) and keloid fibroblasts (KF). Tissue extracts obtained from keloid scar demonstrated elevated expression of mTOR, p70KDa S6 kinase (p70S6K) and their activated forms, suggesting an activated state in keloid scars. Serum stimulation highlighted the heightened responsiveness of KF to mitogens and the importance of mTOR and p70S6K during early phase of wound healing. Application of rapamycin to monoculture NF and KF, dose- and time-dependently downregulates the expression of cytoplasmic PCNA, cyclin D1, fibronectin, collagen and , -SMA, demonstrating the anti-proliferative effect and therapeutic potential of rapamycin in the treatment of keloid scars. The inhibitory effect of rapamycin was found to be reversible following recovery in the expression of proteins following the removal of rapamycin from the culture media. These results demonstrate the important role of mTOR in the regulation of cell cycle and the expression of ECM proteins: fibronectin, collagen and , -SMA. [source] Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytesFEBS JOURNAL, Issue 15 2002Ulrike Krause Certain amino acids, like glutamine and leucine, induce an anabolic response in liver. They activate p70 ribosomal protein S6 kinase (p70S6K) and acetyl-CoA carboxylase (ACC) involved in protein and fatty acids synthesis, respectively. In contrast, the AMP-activated protein kinase (AMPK), which senses the energy state of the cell and becomes activated under metabolic stress, inactivates by phosphorylation key enzymes in biosynthetic pathways thereby conserving ATP. In this paper, we studied the effect of AMPK activation and of protein phosphatase inhibitors, on the amino-acid-induced activation of p70S6K and ACC in hepatocytes in suspension. AMPK was activated under anoxic conditions or by incubation with 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAr) or oligomycin, an inhibitor of mitochondrial oxidative phosphorylation. Incubation of hepatocytes with amino acids activated p70S6K via multiple phosphorylation. It also activated ACC by a phosphatase-dependent mechanism but did not modify AMPK activation. Conversely, the amino-acid-induced activation of both ACC and p70S6K was blocked or reversed when AMPK was activated. This AMPK activation increased Ser79 phosphorylation in ACC but decreased Thr389 phosphorylation in p70S6K. Protein phosphatase inhibitors prevented p70S6K activation when added prior to the incubation with amino acids, whereas they enhanced p70S6K activation when added after the preincubation with amino acids. It is concluded that (a) AMPK blocks amino-acid-induced activation of ACC and p70S6K, directly by phosphorylating Ser79 in ACC, and indirectly by inhibiting p70S6K phosphorylation, and (b) both activation and inhibition of protein phosphatases are involved in the activation of p70S6K by amino acids. p70S6K adds to an increasing list of targets of AMPK in agreement with the inhibition of energy-consuming biosynthetic pathways. [source] Protein kinase B modulates the sensitivity of human neuroblastoma cells to insulin-like growth factor receptor inhibitionINTERNATIONAL JOURNAL OF CANCER, Issue 11 2006Ana S. Guerreiro Abstract The potential of the novel insulin-like growth factor receptor (IGF-IR) inhibitor NVP-AEW541 as an antiproliferative agent in human neuroblastoma was investigated. Proliferation of a panel of neuroblastoma cell lines was inhibited by NVP-AEW541 with IC50 values ranging from 0.15 to 5 ,M. Experiments using an IGF-IR neutralizing antibody confirmed that the IGF-IR was essential to support growth of neuroblastoma cell lines. The expression levels of the IGF-IR in individual neuroblastoma cell lines did not correlate with the sensitivities to NVP-AEW541, while coexpression of the IGF-IR and the insulin receptor (IR) correlated with lower sensitivity to the inhibitor in some cell lines. Intriguingly, high levels of activation of Akt/protein kinase B (PKB) and phosphorylation of the ribosomal S6 protein were observed in neuroblastoma cell lines with decreased sensitivities to NVP-AEW541. Inhibition of Akt/PKB activity restored the sensitivity of neuroblastoma cells to the IGF-IR inhibitor. Transfection of neuroblastoma cells with activated Akt or ribosomal protein S6 kinase (S6K) decreased the sensitivity of the cells to NVP-AEW541. IGF-I-stimulated proliferation of neuroblastoma cell lines was completely blocked by NVP-AEW541, or by a combination of an inhibitor of phosphoinositide 3-kinase and rapamycin. In addition to its antiproliferative effects, NVP-AEW541 sensitized neuroblastoma cells to cisplatin-induced apoptosis. Together, our data demonstrate that NVP-AEW541 in combination with Akt/PKB inhibitors or chemotherapeutic agents may represent a novel approach to target human neuroblastoma cell proliferation. © 2006 Wiley-Liss, Inc. [source] In vivo antitumor effect of the mTOR inhibitor CCI-779 and gemcitabine in xenograft models of human pancreatic cancerINTERNATIONAL JOURNAL OF CANCER, Issue 9 2006Daisuke Ito Abstract Mammalian target of rapamycin (mTOR) is considered to be a major effector of cell growth and proliferation that controls protein synthesis through a large number of downstream targets. We investigated the expression of the phosphatidylinositol 3,-kinase (PI3K)/mTOR signaling pathway in human pancreatic cancer cells and tissues, and the in vivo antitumor effects of the mTOR inhibitor CCI-779 with/without gemcitabine in xenograft models of human pancreatic cancer. We found that the Akt, mTOR and p70 S6 kinase (S6K1) from the PI3K/mTOR signaling pathway were activated in all of the pancreatic cancer cell lines examined. When surgically resected tissue specimens of pancreatic ductal adenocarcinoma were examined, phosphorylation of Akt, mTOR and S6K1 was detected in 50, 55 and 65% of the specimens, respectively. Although CCI-779 had no additive or synergistic antiproliferative effect when combined with gemcitabine in vitro, it showed significant antitumor activity in the AsPC-1 subcutaneous xenograft model as both a single agent and in combination with gemictabine. Furthermore, in the Suit-2 peritoneal dissemination xenograft model, the combination of these 2 drugs achieved significantly better survival when compared with CCI-779 or gemcitabine alone. These results demonstrate promising activity of the mTOR inhibitor CCI-779 against human pancreatic cancer, and suggest that the inhibition of mTOR signaling can be exploited as a potentially tumor-selective therapeutic strategy. © 2005 Wiley-Liss, Inc. [source] Negative Regulation by p70 S6 Kinase of FGF-2,Stimulated VEGF Release Through Stress-Activated Protein Kinase/c- Jun N-Terminal Kinase in Osteoblasts,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2007Shinji Takai Abstract To clarify the mechanism of VEGF release in osteoblasts, we studied whether p70 S6 kinase is involved in basic FGF-2,stimulated VEGF release in osteoblast-like MC3T3-E1 cells. In this study, we show that p70 S6 kinase activated by FGF-2 negatively regulates VEGF release through SAPK/JNK in osteoblasts. Introduction: Vascular endothelial growth factor (VEGF) plays an important role in bone metabolism. We have previously reported that fibroblast growth factor-2 (FGF-2) stimulates the release of VEGF through p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2,activated p38 MAP kinase negatively regulates VEGF release. However, the mechanism behind VEGF release in osteoblasts is not precisely known. Materials and Methods: The levels of VEGF released from MC3T3-E1 cells were measured by enzyme immunoassay. The phosphorylation of each protein kinase was analyzed by Western blotting. To knock down p70 S6 kinase in MC3T3-E1 cells, the cells were transfected with siRNA to target p70 S6 kinase. Results: FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase. Rapamycin significantly enhanced the FGF-2,stimulated VEGF release and VEGF mRNA expression. The FGF-2,induced phosphorylation of p70 S6 kinase was suppressed by rapamycin. Rapamycin markedly enhanced the FGF-2,induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. SP600125, a specific inhibitor of SAPK/JNK, suppressed the amplification by rapamycin of the FGF-2,stimulated VEGF release similar to the levels of FGF-2 with SP600125. Finally, downregulation of p70 S6 kinase by siRNA significantly enhanced the FGF-2,stimulated VEGF release and phosphorylation of SAPK/JNK. Conclusions: These results strongly suggest that p70 S6 kinase limits FGF-2,stimulated VEGF release through self-regulation of SAPK/JNK, composing a negative feedback loop, in osteoblasts. [source] Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responsesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Manchao Zhang Abstract The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin. J. Cell. Biochem. 103: 162,181, 2008. © 2007 Wiley-Liss, Inc. [source] Phosphorylated osteopontin promotes migration of human choriocarcinoma cells via a p70 S6 kinase-dependent pathwayJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Rania Al-Shami Abstract This study examined the role of osteopontin (OPN), a phosphorylated secreted glycoprotein, in the promotion of trophoblastic cell migration, an early event in the embryo implantation process. Three human choriocarcinoma cell lines, namely JAR, BeWo, and JEG-3, were treated with variants of OPN differing in the extent of phosphorylation following sequential dephosphorylation with tartrate-resistant acid phosphatase (TRAP), and their migratory response was measured. The highly phosphorylated human milk form of OPN (OPN-1) strongly triggered migration in all three cell lines, whereas the less phosphorylated variants, OPN-2a and OPN-2b, failed to stimulate migration. JAR cell migration in response to OPN-1 was accompanied by a rapid rearrangement of actin filaments to the cellular membrane. Using broad spectrum protein kinase profiling, we identified p70 S6 kinase as a major signal transduction pathway activated by OPN-1 during the migratory response in JAR cells. Activation was blocked completely by rapamycin and LY294002, thus demonstrating that OPN-1-stimulated migration occurs through mTOR and PI3K pathways, respectively. Conversely, PD98059 did not affect the activation of p70 S6 kinase by OPN-1, therefore, this response does not involve the Ras/ MAPK signaling cascade. Together, these data show that the highly phosphorylated human OPN-1 can stimulate trophoblastic cell migration and provides evidence for the involvement of the PI3K/mTOR/p70 S6 kinase pathway in the JAR cells response. Because both OPN and TRAP are expressed in the uterus during early pregnancy, it is conceivable that extracellular phosphatases such as TRAP may modify OPN charge state and thus modulate cell migration. © 2005 Wiley-Liss, Inc. [source] Lifespan extension by conditions that inhibit translation in Caenorhabditis elegansAGING CELL, Issue 1 2007Malene Hansen Summary Many conditions that shift cells from states of nutrient utilization and growth to states of cell maintenance extend lifespan. We have carried out a systematic lifespan analysis of conditions that inhibit protein synthesis. We find that reducing the levels of ribosomal proteins, ribosomal-protein S6 kinase or translation-initiation factors increases the lifespan of Caenorhabditis elegans. These perturbations, as well as inhibition of the nutrient sensor target of rapamycin (TOR), which is known to increase lifespan, all increase thermal-stress resistance. Thus inhibiting translation may extend lifespan by shifting cells to physiological states that favor maintenance and repair. Interestingly, different types of translation inhibition lead to one of two mutually exclusive outputs, one that increases lifespan and stress resistance through the transcription factor DAF-16/FOXO, and one that increases lifespan and stress resistance independently of DAF-16. Our findings link TOR, but not sir-2.1, to the longevity response induced by dietary restriction (DR) in C. elegans, and they suggest that neither TOR inhibition nor DR extends lifespan simply by reducing protein synthesis. [source] Brain-derived neurotrophic factor stimulates the transcriptional and neuroprotective activity of myocyte-enhancer factor 2C through an ERK1/2-RSK2 signaling cascadeJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Yupeng Wang Abstract Neurotrophin activation of myocyte-enhancer factor (MEF) 2C is one of the strongest pro-survival signaling pathways in developing neurons. To date, neurotrophin stimulation of MEF2C has been largely attributed to its direct phosphorylation by extracellular signal-regulated kinase (ERK) 5. Because MEF2C is not directly phosphorylated by ERK1/2 in vitro, it is generally assumed that the ERK1/2 signaling cascade does not regulate MEF2C. Surprisingly, we discovered that ERK1/2 are required for both the transcriptional and neuroprotective activity of MEF2C in cortical neurons stimulated by brain-derived neurotrophic factor. ERK1/2 stimulation of MEF2C is mediated by p90 ribosomal S6 kinase 2 (RSK2), a Ser/Thr protein kinase downstream of ERK1/2. RSK2 strongly phosphorylates purified recombinant MEF2C protein in vitro. Furthermore, RSK2 can directly phosphorylate MEF2C on S192, a consensus RSK2-phosphorylation site located in the transactivation domain of MEF2C. Substitution of S192 with a non-phosphorylatable alanine diminishes both the transcriptional and neuroprotective activity of MEF2C to an extent similar to mutation on S387, an established activating phosphorylation site. Together, our data identifies ERK1/2-RSK2 signaling as a novel mechanism by which neurotrophins activate MEF2C and promote neuronal survival. [source] Gene and protein expression associated with protein synthesis and breakdown in paraplegic skeletal muscleMUSCLE AND NERVE, Issue 4 2008Micah J. Drummond PhD Abstract Spinal cord injury reduces the rate of skeletal muscle protein synthesis and increases protein breakdown, resulting in rapid muscle loss. The purpose of this study was to determine whether long-term paraplegia would eventually result in a downregulation of muscle mRNA and protein expression associated with both protein synthesis and breakdown. After 10 weeks of spinal cord transection, soleus muscle from 12 rats (6 sham-control, 6 paraplegic) was studied for mRNAs and proteins associated with protein synthesis and breakdown using real-time polymerase chain reaction and immunoblotting techniques. Protein kinase B (PKB/Akt), ribosomal S6 kinase 1 (S6K1), and myogenin mRNA were downregulated, whereas muscle ring finger 1 (MuRF1) and phospho-forkhead transcription factor 4 (FoxO4) protein were increased in paraplegic rats. We conclude that gene and protein expression of pathways associated with protein synthesis are reduced, whereas some markers of protein breakdown remain elevated following chronic paraplegia. Clinical interventions designed to increase muscle protein synthesis may be helpful in preventing excessive muscle loss during long-term paraplegia. Muscle Nerve, 2008 [source] NV-128, a novel isoflavone derivative, induces caspase-independent cell death through the Akt/mammalian target of rapamycin pathwayCANCER, Issue 14 2009Ayesha B. Alvero MD Abstract BACKGROUND: Resistance to apoptosis is 1 of the key events that confer chemoresistance and is mediated by the overexpression of antiapoptotic proteins, which inhibit caspase activation. The objective of this study was to evaluate whether the activation of an alternative, caspase-independent cell death pathway could promote death in chemoresistant ovarian cancer cells. The authors report the characterization of NV-128 as an inducer of cell death through a caspase-independent pathway. METHODS: Primary cultures of epithelial ovarian cancer (EOC) cells were treated with increasing concentration of NV-128, and the concentration that caused 50% growth inhibition (GI50) was determined using a proprietary assay. Apoptotic proteins were characterized by Western blot analyses, assays that measured caspase activity, immunohistochemistry, and flow cytometry. Protein-protein interactions were determined using immunoprecipitation. In vivo activity was measured in a xenograft mice model. RESULTS: NV-128 was able to induce significant cell death in both paclitaxel-resistant and carboplatin-resistant EOC cells with a GI50 between 1 ,g/mL and 5 ,g/mL. Cell death was characterized by chromatin condensation but was caspase-independent. The activated pathway involved the down-regulation of phosphorylated AKT, phosphorylated mammalian target of rapamycin (mTOR), and phosphorylated ribosomal p70 S6 kinase, and the mitochondrial translocation of beclin-1 followed by nuclear translocation of endonuclease G. CONCLUSIONS: The authors characterized a novel compound, NV-128, which inhibits mTOR and promotes caspase-independent cell death. The current results indicated that inhibition of mTOR may represent a relevant pathway for the induction of cell death in cells resistant to the classic caspase-dependent apoptosis. These findings demonstrate the possibility of using therapeutic drugs, such as NV-128, which may have beneficial effects in patients with chemoresistant ovarian cancer. Cancer 2009. © 2009 American Cancer Society. [source] TUSC4/NPRL2, a novel PDK1-interacting protein, inhibits PDK1 tyrosine phosphorylation and its downstream signalingCANCER SCIENCE, Issue 9 2008Atsuo Kurata 3-Phosphoinositide,dependent protein kinase-1 (PDK1) is a key regulator of cell proliferation and survival signal transduction. PDK1 is known to be constitutively active and is further activated by Src-mediated phosphorylation at the tyrosine-9, -373, and -376 residues. To identify novel regulators of PDK1, we performed E. coli -based two-hybrid screening and revealed that tumor suppressor candidate 4 (TUSC4), also known as nitrogen permease regulator-like 2 (NPRL2), formed a complex with PDK1 and suppressed Src-dependent tyrosine phosphorylation and activation of PDK1 in vitro and in cells. The NH2 -terminal 133 amino acid residues of TUSC4 were involved in binding to PDK1. The deletion mutant of TUSC4 that lacked the NH2 -terminal domain showed no inhibitory effects on PDK1 tyrosine phosphorylation or activation. Thus, complex formation is indispensable for TUSC4-mediated PDK1 inactivation. The siRNA-mediated down-regulation of TUSC4 induced cell proliferation, while ectopic TUSC4 expression inactivated the PDK1 downstream signaling pathway, including Akt and p70 ribosomal protein S6 kinase, and increased cancer cell sensitivity to several anticancer drugs. Our results suggest that TUSC4/NPRL2, a novel PDK1-interacting protein, plays a role in regulating the Src/PDK1 signaling pathway and cell sensitivity to multiple cancer chemotherapeutic drugs. (Cancer Sci 2008; 99: 1827,1834) [source] Induction of insulin-like growth factor-I by interleukin-17F in bronchial epithelial cellsCLINICAL & EXPERIMENTAL ALLERGY, Issue 7 2010M. Kawaguchi Summary Cite this as: M. Kawaguchi, J. Fujita, F. Kokubu, G. Ohara, S-K Huang, S. Matsukura, Y. Ishii, M. Adachi, H. Satoh and N. Hizawa, Clinical & Experimental Allergy, 2010 (40) 1036,1043. Background Increased expression of IL-17F has been noted in the airway of asthmatic patients, but its role in asthma has not been fully elucidated. Insulin-like growth factor-I (IGF-I) is known to be involved in airway remodelling and inflammation, while its regulatory mechanisms remain to be defined. Objective To further clarify the biological function of IL-17F, we investigated whether IL-17F is able to regulate the expression of IGF-I in bronchial epithelial cells. Methods Bronchial epithelial cells were stimulated with IL-17F in the presence or absence of T-helper type 2 cytokines. Various kinase inhibitors were added to the culture to identify the key signalling events leading to the expression of IGF-I, in conjunction with the use of short interfering RNAs (siRNAs) targeting mitogen- and stress-activated protein kinase (MSK) 1, p90 ribosomal S6 kinase (p90RSK), and cyclic AMP response element-binding protein (CREB). Results IL-17F significantly induced IGF-I gene and protein expression, and co-stimulation with IL-4 and IL-13 augmented its production. MAP kinase kinase (MEK) inhibitors and the Raf1 kinase inhibitor significantly inhibited IGF-I production, and the combination of PD98059 and Raf1 kinase inhibitor showed further inhibition. Overexpression of Raf1 and Ras dominant-negative mutants inhibited its expression. MSK1 inhibitors significantly blocked IL17F-induced IGF-I expression. Moreover, transfection of the siRNAs targeting MSK1, p90RSK, and CREB blocked its expression. Conclusions In bronchial epithelial cells, IL-17F is able to induce the expression of IGF-I via the Raf1,MEK1/2,ERK1/2,MSK1/p90RSK,CREB pathway in vitro. [source] Sirolimus-induced signaling modifications in Kaposi's sarcoma with resolution in a liver transplant recipientCLINICAL TRANSPLANTATION, Issue 1 2010Cheng-Maw Ho Ho C-M, Huang S-F, Hu R-H, Ho M-C, Wu Y-M, Lee P-H. Sirolimus-induced signaling modifications in Kaposi's sarcoma with resolution in a liver transplant recipient. Clin Transplant 2010: 24: 127,132. © 2009 John Wiley & Sons A/S. Abstract:, Sirolimus is one treatment option in transplant recipients with Kaposi's sarcoma (KS), which involves dysregulation of Akt-mammalian target of rapamycin (mTOR) signaling pathway. Signal modifications after sirolimus therapy in organ recipients with KS are largely unknown and not verified. We reported a case of KS found two yr after liver transplantation in which the immunosuppression was changed from tacrolimus, MMF, and steroid to sirolimus alone. In skin, which was found to have persistent KS after a two-month treatment of sirolimus and was removed completely one yr later, KS was no longer present. The patient went well without graft rejection. Tumor biopsies were performed before, two months, and one yr after the start of sirolimus. Immunohistochemical staining of vascular endothelial growth factor (VEGF), p-Akt, p-mTOR, p-p70 S6 kinase, and Western blot for p-tuberin/ tuberous sclerosis complex (TSC)2 was performed. VEGF was suppressed thoroughly in two-month use of sirolimus. In addition, p-Akt and p-mTOR, which were decreased at two months, could not be detected after one yr of treatment. Moreover, p-p70 S6 kinase, expressed strongly in overlying epidermis initially, was suppressed completely after two months of treatment. However, p-tuberin/TSC2, contrary to suggested theoretically, was not detected through all specimens, implying not to be a significant event. Suppressed expression of VEGF, p-Akt, and p-mTOR was the major event of signaling modification through the long-term use of sirolimus. [source] Drosophila RSK negatively regulates bouton number at the neuromuscular junctionDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2009Matthias Fischer Abstract Ribosomal S6 kinases (RSKs) are growth factor-regulated serine-threonine kinases participating in the RAS-ERK signaling pathway. RSKs have been implicated in memory formation in mammals and flies. To characterize the function of RSK at the synapse level, we investigated the effect of mutations in the rsk gene on the neuromuscular junction (NMJ) in Drosophila larvae. Immunostaining revealed transgenic expressed RSK in presynaptic regions. In mutants with a full deletion or an N-terminal partial deletion of rsk, an increased bouton number was found. Restoring the wild-type rsk function in the null mutant with a genomic rescue construct reverted the synaptic phenotype, and overexpression of the rsk -cDNA in motoneurons reduced bouton numbers. Based on previous observations that RSK interacts with the Drosophila ERK homologue Rolled, genetic epistasis experiments were performed with loss- and gain-of-function mutations in Rolled. These experiments provided evidence that RSK mediates its negative effect on bouton formation at the Drosophila NMJ by inhibition of ERK signaling. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009 [source] | |||||||||||||