Serum- and glucocorticoid-induced protein kinase 1 (SGK1) is regulated by store-operated Ca2+ entry and mediates cytoprotection against necrotic cell death.

Serum and glucocorticoid-regulated kinase 1 (SGK1) encodes a phosphatidylinositol 3-kinase-dependent serine/threonine kinase that is rapidly induced in response to cellular stressors and is an important cell survival signal. Previous studies have suggested that an increase in cytoplasmic Ca(2+) concentration ([Ca(2+)]c) is required for increased SGK1 expression, but the subcellular source of Ca(2+) regulating SGK1 transcription remains uncertain. Activation of endoplasmic reticulum stress (ERS) with thapsigargin (TG) increased SGK1 mRNA and protein expression in MDA-MB-231 cells. Intracellular Ca(2+) imaging revealed that store-operated Ca(2+) entry played a prominent role in SGK1 induction by TG. Neither ERS nor release of Ca(2+) from the ER was sufficient to activate SGK1. Prolonged elevation of intracellular Ca(2+) levels, however, triggered cell death with a much greater proportion of the cells undergoing necrosis rather than apoptosis. A relative increase in the percentage of cells undergoing necrosis was observed in cells expressing a short hairpin RNA targeted to the SGK1 gene. Necrotic cell death evoked by cytoplasmic Ca(2+) overloading was associated with persistent hyperpolarization of the inner mitochondrial membrane and a modest increase in calpain activation, but did not involve detectable caspase 3 or caspase 7 activation. The effects of cytoplasmic Ca(2+) overloading on mitochondrial membrane potential were significantly reduced in cells expressing SGK1 compared with SGK1-depleted cells. Our findings indicate that store-operated Ca(2+) entry regulates SGK1 expression in epithelial cells and suggest that SGK1-dependent cytoprotective signaling involves effects on maintaining mitochondrial function.

Impact of the New Abbott mPLUS feature on clinical laboratory efficiencies of abbott RealTime assays for detection of HIV-1, Hepatitis C Virus, Hepatitis B Virus, Chlamydia trachomatis, and Neisseria gonorrhoeae.

Diagnostic laboratories are under increasing pressure to improve and expand their services. Greater flexibility in sample processing is a critical factor that can improve the time to results while reducing reagent waste, making laboratories more efficient and cost-effective. The introduction of the Abbott mPLUS feature, with the capacity for extended use of amplification reagents, significantly increases the flexibility of the m2000 platform and enables laboratories to customize their workflows based on sample arrival patterns. The flexibility in sample batch size offered by mPLUS enables significant reductions in processing times. For hepatitis B virus tests, a reduction in sample turnaround times of up to 30% (105 min) was observed for batches of 12 samples compared with those for batches of 24 samples; for Chlamydia trachomatis/Neisseria gonorrhoeae tests, the ability to run batches of 24 samples reduced the turnaround time by 83% (54 min) compared with that for batches of 48 samples. Excellent correlations between mPLUS and m2000 standard condition results were observed for all RealTime viral load assays evaluated in this study, with correlation r values of 0.998 for all assays tested. For the qualitative RealTime C. trachomatis/N. gonorrhoeae assay, the overall agreements between the two conditions tested were >98% for C. trachomatis and 100% for N. gonorrhoeae. Comparable precision results were observed for the two conditions tested for all RealTime assays. The enhanced mPLUS capability provides clinical laboratories with increased efficiencies to meet increasingly stringent turnaround time requirements without increased costs associated with discarding partially used amplification reagents.

Glucocorticoid receptor expression in breast cancer associates with older patient age.

Breast cancer can be classified according to estrogen (ER), progesterone (PR), and HER2 receptor expression. Recent evidence suggests that activation of the glucocorticoid receptor (GR) contributes to breast cell survival, although the incidence of GR expression in primary human breast tumors is not well established. We therefore evaluated ER, PR, HER2, and GR by immunohistochemistry from 231 patients and found that while African American (AA) patient tumors were much more likely to be ER negative compared to tumors from non-AA patients, GR expression was significantly higher in tumors from patients >or=50 regardless of ancestry. Prospective examination of GR expression in tumors should be considered to determine whether GR contributes to long-term clinical outcome.

Ubiquitin-proteasome degradation of serum- and glucocorticoid-regulated kinase-1 (SGK-1) is mediated by the chaperone-dependent E3 ligase CHIP.

SGK-1 (serum- and glucocorticoid-regulated kinase-1) is a stress-induced serine/threonine kinase that is phosphorylated and activated downstream of PI3K (phosphoinositide 3-kinase). SGK-1 plays a critical role in insulin signalling, cation transport and cell survival. SGK-1 mRNA expression is transiently induced following cellular stress, and SGK-1 protein levels are tightly regulated by rapid proteasomal degradation. In the present study we report that SGK-1 forms a complex with the stress-associated E3 ligase CHIP [C-terminus of Hsc (heat-shock cognate protein) 70-interacting protein]; CHIP is required for both the ubiquitin modification and rapid proteasomal degradation of SGK-1. We also show that CHIP co-localizes with SGK-1 at or near the endoplasmic reticulum. CHIP-mediated regulation of SGK-1 steady-state levels alters SGK-1 kinase activity. These data suggest a model that integrates CHIP function with regulation of the PI3K/SGK-1 pathway in the stress response.

Hsp90 regulates the phosphorylation and activity of serum- and glucocorticoid-regulated kinase-1.

SGK-1 (serum- and glucocorticoid-regulated kinase-1), a member of the AGC protein kinase family, plays an important role in regulating ion channel expression and contributes to malignant epithelial cell proliferation and survival. SGK-1 activity is regulated on three levels: transcriptional induction following a variety of environmental and intracellular stresses, proteasomal degradation, and phosphorylation. Here we report that phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of SGK-1 requires formation of a complex between SGK-1 and heat-shock protein 90 (Hsp90). Inactivation of Hsp90 by geldanamycin led to decreased SGK-1 phosphorylation independently of increased proteasomal protein degradation, and inhibition of PI3K activity by LY294002 appeared to eliminate SGK-1 phosphorylation at the same residues as those affected by geldanamycin treatment. Interestingly, geldanamycin-targeted phosphorylation sites were not limited to the known conserved PI3K-dependent sites Thr-256 and Ser-422 in SGK-1 but included additional unknown PI3K-dependent residues. Inhibition of Hsp90 also resulted in a complete loss of SGK-1 kinase activity, suggesting that Hsp90 activity is essential for regulating the PI3K/SGK-1 pathway.