Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation, but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.

Caspases are intracellular proteases that function as initiators and effectors of apoptosis. The kinase Akt and p21-Ras, an Akt activator, induced phosphorylation of pro-caspase-9 (pro-Casp9) in cells. Cytochrome c-induced proteolytic processing of pro-Casp9 was defective in cytosolic extracts from cells expressing either active Ras or Akt. Akt phosphorylated recombinant Casp9 in vitro on serine-196 and inhibited its protease activity. Mutant pro-Casp9(Ser196Ala) was resistant to Akt-mediated phosphorylation and inhibition in vitro and in cells, resulting in Akt-resistant induction of apoptosis. Thus, caspases can be directly regulated by protein phosphorylation.

A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein kinases, the serine-threonine kinase Akt and the p70 ribosomal protein S6 kinase (p70(S6K)). Experiments with pharmacological inhibitors, as well as expression of wild-type and dominant-inhibitory forms of Akt, demonstrated that Akt but not p70(S6K) mediates PI3-K-dependent survival. These findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.

The serine/threonine protein kinase encoded by the Akt proto-oncogene is catalytically inactive in serum-starved primary and immortalized fibroblasts. Here we show that Akt and the Akt-related kinase AKT2 are activated by PDGF. The activation was rapid and specific, and it was abrogated by mutations in the Akt Pleckstrin homology (PH) domain. The Akt activation was also shown to depend on PDGFR beta tyrosines Y740 and Y751, which bind phosphatidylinositol 3-kinase (PI 3-kinase) upon phosphorylation. Moreover, Akt activation was blocked by the PI 3-kinase-specific inhibitor wortmannin and the dominant inhibitory N17Ras. Conversely, Akt activity was induced following the addition of phosphatidylinositol-3-phosphate to Akt immunoprecipitates from serum-starved cells in vitro. These results identify Akt as a novel target of PI 3-kinase and suggest that the Akt PH domain may be a mediator of PI 3-kinase signaling.

It has been found that the expression of miR-877-5p is increased in serum of patients taking NSAIDs drugs. However, whether miR-877-5p play a role in aspirin-induced gastrointestinal mucosal erosion remains largely unknown. In this study, we investigated the effects of miR-877-5p on gastric epithelial cells (GES-1) proliferation and apoptosis in vitro. MiR-877-5p mimic/inhibitor and their oligonucleotides were transfected into GES-1 cells, then GES-1 cells were treated with different concentrations of aspirin (1.25, 2.5, 5 and 10 mmol/L). The bioinformatics software and dual-luciferase reporter assay were used to predict and verify the target gene of miR-877-5p. qRT-PCR and Western Blotting were employed to assess gene and protein expression, and CCK-8 assay and ow cytometry analysis were used to detect cell proliferation and apoptosis, respectively. qRT-PCR data showed that miR-877-5p level was signi cantly increased in aspirin incubated GES-1 cells. The proliferation of GES-1 cells were markedly inhibited and apoptosis was signi cantly induced in the miR-877-5p mimic groups compared to control groups. Using PITA, Targetscan and miRWalk database, the three databases indicated that PDK1 was a target gene of miR-miR-877-5p. Dual luciferase reporter assay con rmed that the existence of a direct interaction between miR-877-5p and PDK1 mRNA. Importantly, miR-877-5p knockdown resulted in a signi cant upregulation of PDK1 mRNA and its encoded protein in GES-1 cells. miR-877-5p plays a role in aspirin-induced gastrointestinal mucosal erosion, which may via down-regulation of targeting PDK1 gene.

The regulation of the serine-threonine kinase Akt by lipid products of phosphoinositide 3-kinase (PI 3-kinase) was investigated. Akt activity was found to correlate with the amount of phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) in vivo, and synthetic PtdIns-3,4-P2 activated Akt both in vitro and in vivo. Binding of PtdIns-3,4-P2 occurred within the Akt pleckstrin homology (PH) domain and facilitated dimerization of Akt. Akt mutated in the PH domain was not activated by PI 3-kinase in vivo or by PtdIns-3, 4-P2 in vitro, and it was impaired in binding to PtdIns-3,4-P2. Examination of the binding to other phosphoinositides revealed that they bound to the Akt PH domain with much lower affinity than did PtdIns-3,4-P2 and failed to increase Akt activity. Thus, Akt is apparently regulated by the direct interaction of PtdIns-3,4-P2 with the Akt PH domain.

Recent research has examined Akt and Akt-related serine-threonine kinases in signaling cascades that regulate cell survival and are important in the pathogenesis of degenerative diseases and in cancer. We seek to recapitulate the research that has helped to define the current understanding of the role of the Akt pathway under normal and pathologic conditions, also in view of genetic models of Akt function. In particular, we will evaluate the mechanisms of Akt regulation and the role of Akt substrates in Akt-dependent biologic responses in the decisions of cell death and cell survival. Here, we hope to establish the mechanisms of apoptosis suppression by Akt kinase as a framework for a more general understanding of growth factor-dependent regulation of cell survival.

The Ca2+-activated protein phosphatase calcineurin induces apoptosis, but the mechanism is unknown. Calcineurin was found to dephosphorylate BAD, a pro-apoptotic member of the Bcl-2 family, thus enhancing BAD heterodimerization with Bcl-xL and promoting apoptosis. The Ca2+-induced dephosphorylation of BAD correlated with its dissociation from 14-3-3 in the cytosol and translocation to mitochondria where Bcl-xL resides. In hippocampal neurons, L-glutamate, an inducer of Ca2+ influx and calcineurin activation, triggered mitochondrial targeting of BAD and apoptosis, which were both suppressible by coexpression of a dominant-inhibitory mutant of calcineurin or pharmacological inhibitors of this phosphatase. Thus, a Ca2+-inducible mechanism for apoptosis induction operates by regulating BAD phosphorylation and localization in cells.