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Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation.

ABSTRACT: While a plethora of extracellular molecules exist that modulate cellular functions via binding to membrane receptors inside the cell, their actions are mediated by relatively few signalling mechanisms. One of these is activation of phosphatidylinositol 3-kinase (PI-3K), which results in the generation of a membrane-restricted second messenger, polyphosphatidylinositides containing a 3'-phosphate. How these molecules transduced the effects of agonists of PI-3K was unclear until the recent discovery that several protein kinases become activated upon exposure to 3'-phosphorylated inositol lipids. These enzymes include protein kinase B (PKB)/AKT and PtdIns(3,4, 5)P3-dependent kinases 1 and 2, the first two of which interact with 3'-phosphorylated phosphoinositides via pleckstrin homology domains. Once targeted to the membrane by this motif, PKB becomes phosphorylated at two residues, which relieves intermolecular inhibition, allowing the activated complex to dissociate and modify its targets. Identification of these substrates is the subject of intensive research, since at least one must play a key role in suppressing apoptosis, as demonstrated by expression of activated alleles of PKB. The generation of effective transdominant mutants, coupled with genetic analysis of the protein kinase in simpler organisms, should help in elucidating outstanding questions in the functions, targets and regulation of this important mediator of PI-3K signalling.

SUBMITTER: Coffer PJ

PROVIDER: S-EPMC1219745 | biostudies-other | 1998 Oct

REPOSITORIES: biostudies-other

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Project description:Preeclampsia is a hypertensive syndrome that manifests after 20 wk of gestation. Contemporary understanding of the maternal-fetal interface in preeclampsia suggests a major role for placental oxidative stress resulting from ischemia-reperfusion injury. We hypothesized that the pregnancy hormone relaxin would reduce cytotrophoblast apoptosis and necrosis (aponecrosis) and, hence, the export of placental debris into the maternal circulation. If so, then relaxin might be employed as a therapeutic intervention to diminish the activation of the maternal systemic inflammatory response central to the development of clinical disease. HTR-8/SVneo cells, a model for first trimester extravillous trophoblast, were subjected to serum deprivation and hypoxia or hypoxia-reoxygenation. The cells were treated with recombinant human relaxin or vehicle and apoptosis and/or necrosis evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), CellEvent Caspase-3/7 and SYTOX AADvanced kit, and propidium iodide staining as determined by fluorescence microscopy or flow cytometry. To interrogate mechanisms of relaxin cytoprotection, HTR-8/SVneo cells were pretreated with pharmacological inhibitors of PI3-kinase LY294004, Akt/PKB MK-2206, or DMSO vehicle. HTR-8/SVneo cell identity was first confirmed by RT-PCR. The cells expressed placental alkaline phosphatase, aromatase, and human leukocyte antigen G. In addition, the cells expressed the relaxin receptor RXFP1 as well as H1 and H2 relaxins. Serum deprivation and hypoxia increased apoptotic cell death in HTR-8/SVneo cells, which was significantly ameliorated by concurrent treatment with relaxin. Serum deprivation and hypoxia-reoxygenation increased necrotic cell death in HTR-8/SVneo cells, which was also significantly rescued by concurrent treatment with relaxin. Pretreatment with LY294002 or MK-2206, to inhibit the phosphatidylinositol 3-kinase-Akt/protein kinase B cell survival pathway, significantly blunted the cytoprotective effect of relaxin. We demonstrated trophoblast cytoprotection by intervention with supraphysiological concentrations of relaxin, a process in part mediated through the PI3-kinase-Akt/PKB cell survival pathway. These results provide further rationale for clinical investigation of relaxin as a potential therapeutic in preeclampsia.

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Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation.

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