Novel Insights into Vascular Endothelial Growth Factor Receptor 2-Mediated Signaling to the Mammalian Target of Rapamycin/Akt Network in SK-N-SH Neuroblastoma Cells
Item
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Title
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Novel Insights into Vascular Endothelial Growth Factor Receptor 2-Mediated Signaling to the Mammalian Target of Rapamycin/Akt Network in SK-N-SH Neuroblastoma Cells
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Identifier
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d_2009_2013:fed9cfb0e203:11012
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identifier
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11412
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Creator
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Edelstein, Jacob,
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Contributor
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Patricia Rockwell
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Date
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2011
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Language
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English
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Publisher
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City University of New York.
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Subject
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Molecular biology | Akt | mTOR | muscarinic | phosphatase | signal transduction | VEGF
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Abstract
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Mammalian target of rapamycin (mTOR) is a central regulator of cell growth and division that exerts many of its effects through regulating protein synthesis. The kinase Akt is a substrate and regulator of mTOR. These proteins are integral to pathological and physiological function in neuronal cells and the Akt/mTOR network is the focus of pharmaceutical interventions. Muscarinic acetylcholine receptors and vascular endothelial growth factor receptor 2 (VEGFR2) can signal protein synthesis but whether they cooperate to mediate mTOR activation has not been demonstrated. Using serum-starved SK-N-SH neuroblastoma cells, we show that the muscarinic receptor agonists carbachol and pilocarpine enhance the activation of the mTOR substrate p70 S6 Kinase (S6K) and its target ribosomal protein S6 (S6) in a VEGFR2-dependent manner. Protein kinase C (PKC) functions in an opposing fashion by positively regulating S6K and S6 phosphorylation and suppressing Akt activation. Treatments with the phosphatase inhibitors sodium orthovanadate and okadaic acid (OA) increase S6, Akt and to a lesser extent S6K phosphorylation, indicating that tyrosine and serine/threonine dephosphorylation also regulates their activity. However, OA elicited a far greater increase in phosphorylation, implicating phosphatase 2A (PP2A) as a critical determinant of their function. Furthermore, PP2A inhibition induces the appearance of novel, high molecular weight, ubiquitinated forms of Akt. The accumulation of phosphorylated Akt induced by PP2A dysfunction causes depletion of total Akt. Rapamycin potentiates Akt phosphorylation and depletion in response to OA through a mechanism regulated by a previously unknown function of VEGFR2. Although hyperactivation of Akt is a common survival mechanism in cancer cells, Akt hyperphosphorylation is associated with induction of a caspase-independent cell death mediated by oxidative stress. Taken together, these results show that the critical role of PP2A in regulating Akt activation also affects Akt ubiquitination, cleavage and removal from the cell. Furthermore, these data indicate the importance of reactive oxygen species in eliciting cell death and that PP2A promotes survival through a suppression of oxidative stress. Finally, VEGFR2 can stimulate mTOR when stimulated by ligand binding, transactivation or an unknown mechanism induced by rapamycin.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Biology
