Serum and glucocorticoid‐inducible kinase (SGK) is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated. In this study, we have investigated the regulatory mechanisms that control SGK activity. We have established a peptide kinase assay for SGK and present evidence demonstrating that SGK is a component of the phosphoinositide 3 (PI 3)‐kinase signaling pathway. Treatment of human embryo kidney 293 cells with insulin, IGF‐1 or pervanadate induced a 3‐ to 12‐fold activation of ectopically expressed SGK. Activation was completely abolished by pretreatment of cells with the PI 3‐kinase inhibitor, LY294002. Treatment of activated SGK with protein phosphatase 2A in vitro led to kinase inactivation. Consistent with the similarity of SGK to other second‐messenger regulated kinases, mutation of putative phosphorylation sites at Thr256 and Ser422 inhibited SGK activation. Cotransfection of PDK1 with SGK caused a 6‐fold activation of SGK activity, whereas kinase‐dead PDK1 caused no activation. GST‐pulldown assays revealed a direct interaction between PDK1 and the catalytic domain of SGK. Treatment of rat mammary tumor cells with serum caused hyperphosphorylation of endogenous SGK, and promoted translocation to the nucleus. Both hyperphosphorylation and nuclear translocation could be inhibited by wortmannin, but not by rapamycin.

Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), respectively. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and associated with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a ∼10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.

Protein kinase B (PKB/Akt) is a pivotal regulator of diverse metabolic, phenotypic, and antiapoptotic cellular controls and has been shown to be a key player in cancer progression. Here, using fluorescent reporters, we shown in cells that, contrary to in vitro analyses, 3-phosphoinositide–dependent protein kinase 1 (PDK1) is complexed to its substrate, PKB. The use of Förster resonance energy transfer detected by both frequency domain and two-photon time domain fluorescence lifetime imaging microscopy has lead to novel in vivo findings. The preactivation complex of PKB and PDK1 is maintained in an inactive state through a PKB intramolecular interaction between its pleckstrin homology (PH) and kinase domains, in a “PH-in” conformer. This domain–domain interaction prevents the PKB activation loop from being phosphorylated by PDK1. The interactive regions for this intramolecular PKB interaction were predicted through molecular modeling and tested through mutagenesis, supporting the derived model. Physiologically, agonist-induced phosphorylation of PKB by PDK1 occurs coincident to plasma membrane recruitment, and we further shown here that this process is associated with a conformational change in PKB at the membrane, producing a “PH-out” conformer and enabling PDK1 access the activation loop. The active, phosphorylated, “PH-out” conformer can dissociate from the membrane and retain this conformation to phosphorylate substrates distal to the membrane. These in vivo studies provide a new model for the mechanism of activation of PKB. This study takes a crucial widely studied regulator (physiology and pathology) and addresses the fundamental question of the dynamic in vivo behaviour of PKB with a detailed molecular mechanism. This has important implications not only in extending our understanding of this oncogenic protein kinase but also in opening up distinct opportunities for therapeutic intervention. Regulation of intracellular signaling depends on the precise operation of molecular switches such as kinases and phosphatases. Disruption of their activities leads to inappropriate cellular proliferation, growth, and survival. Protein kinase B (PKB) is a critical kinase that regulates events downstream of growth factor receptors. It is also involved in human cancer, where its overexpression induces malignant transformation. We studied the molecular mechanisms of PKB's interaction with its upstream regulator, 3-phosphoinositide–dependent protein kinase 1 (PDK1). By using a fluorescent probe, we monitored the conformational changes of PKB in cells using Förster resonance energy transfer detected by fluorescence lifetime imaging microscopy. Applying this approach, we show that PKB and PDK1 are found as complexes in the cytoplasm. Despite this proximity to its regulator, we show that PKB remains inactive through an intramolecular interaction of its pleckstrin homology (PH) domain and kinase domain. We refer to this inactive state as the “PH-in” conformer. Following growth factor activation, PKB changes conformation to the “PH-out” conformer and is phosphorylated by PDK1. The active “PH-out” conformer dissociates from the plasma membrane to phosphorylate downstream proteins. Our in vivo studies provide a new model for the mechanism of PKB.

Stroke is a complex disease involved oxidative stress-related pathways in its pathogenesis. The nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway has been considered a potential target for neuroprotection in stroke. Acetyl-11-Keto-β-Boswellic Acid (AKBA) is an active triterpenoid compound from the extract ofBoswellia serrate. The present study was to determine whether AKBA, a novel Nrf2 activator, can protect against cerebral ischemic injury. The stroke model was produced in Sprague–Dawley rats via middle cerebral artery occlusion. To model ischemia-like conditionsin vitro, primary cultured cortical neurons were exposed to transient oxygen and glucose deprivation (OGD). Treatment of AKBA significantly reduced infarct volumes and apoptotic cells and also increased neurologic scores by elevating the Nrf2 and HO-1 expression in brain tissues in middle cerebral artery occlusion (MCAO) rats at 48 hours post reperfusion. In primary cultured neurons, AKBA increased the Nrf2 and HO-1 expression, which provided protection against OGD-induced oxidative insult. Additionally, AKBA treatment increased Nrf2 binding activity to antioxidant-response elements (ARE). The protective effect of AKBA was attenuated by knockdown of Nrf2 or HO-1. In conclusion, these findings provide evidence that AKBA protects neurons against ischemic injury and this neuroprotective effect involves the Nrf2/HO-1 pathway.

We have established that the serum- and glucocorticoid-inducible protein kinase (Sgk) is a new component of the hyperosmotic stress response. Treatment of NMuMg mammary epithelial cells with the organic osmolyte, sorbitol, caused the stable accumulation of Sgk transcripts and protein after an approximately 4-h lag. Transient transfection of a series of sgk-CAT reporter plasmids containing either 5' deletions or continuous 6-base pair substitutions identified a hyperosmotic stress-regulated element that is GC-rich and is necessary for the sorbitol stimulation of sgk gene promoter activity. Gel shift analysis identified four major DNA-protein complexes in the hyperosmotic stress-regulated element that, by competition with excess consensus wild type and mutant oligonucleotides and by antibody supershifts, contains the Sp1 transcription factor. Several lines of evidence suggest that the p38 MAPK signaling pathway mediates the hyperosmotic stress stimulation of sgk gene expression. Treatment with pharmacological inhibitors of p38 MAPK or with a dominant negative form of MKK3, an upstream regulator of p38 MAPK, significantly reduced or ablated the sorbitol induction of sgk promoter activity or protein production. Using an in vitro peptide transphosphorylation assay, sorbitol treatment activates either endogenous or exogenous Sgk that is localized to the cytoplasmic compartment. Thus, we propose that the stimulated expression of enzymatically active Sgk after sorbitol treatment is a newly defined component of the p38 MAPK-mediated response to hyperosmotic stress.