Regulation of Orai1/STIM1 by the kinases SGK1 and AMPK.

STIM and Orai isoforms orchestrate store operated Ca2+ entry (SOCE) and thus cytosolic Ca2+ fluctuations following stimulation by hormones, growth factors and further mediators. Orai1 is a target of Nedd4-2, an ubiquitin ligase preparing several plasma membrane proteins for degradation. Phosphorylation of Nedd4-2 by the serum and glucocorticoid inducible kinase SGK1 leads to the binding of Nedd4-2 to the protein 14-3-3 thus preventing its interaction with Orai1. Nedd4-2 is activated by the energy sensing AMP activated kinase AMPK. Thus, SGK1 disrupts and AMPK fosters degradation of Orai1. New synthesis of both, Orai1 and STIM1, is stimulated by the transcription factor NF-κB (nuclear factor kappa B), which binds to the respective promoter regions of the genes encoding STIM1 and Orai1. SGK1 upregulates and AMPK presumably downregulates NF-κB and thus de novo synthesis of Orai1 and STIM1 proteins. The regulation by SGK1 links SOCE to the signaling of a wide variety of hormones and growth factors, the AMPK dependent regulation of Orai1 and STIM1 may serve to limit inadequate activation of SOCE following energy depletion, which is otherwise expected to activate SOCE by depletion of intracellular Ca2+ stores due to impairment of the ATP consuming sarco/endoplasmatic reticulum Ca2+ ATPase SERCA.

Transcription factor NF-κB regulates expression of pore-forming Ca2+ channel unit, Orai1, and its activator, STIM1, to control Ca2+ entry and affect cellular functions.

The serum and glucocorticoid-inducible kinase SGK1 increases the activity of Orai1, the pore forming unit of store-operated Ca(2+) entry, and thus influences Ca(2+)-dependent cellular functions such as migration. SGK1 further regulates transcription factor nuclear factor κB (NF-κB). This study explored whether SGK1 influences transcription of Orai1 and/or STIM1, the Orai1-activating Ca(2+) sensor. Orai1 and STIM1 transcript levels were decreased in mast cells from SGK1 knock-out mice and increased in HEK293 cells transfected with active (S422D)SGK1 but not with inactive (K127N)SGK1 or in (S422D)SGK1-transfected cells treated with the NF-κB inhibitor Wogonin (100 µm). Treatment with the stem cell factor enhanced transcript levels of STIM1 and Orai1 in sgk1(+/+) but not in sgk1(-/-) mast cells and not in sgk1(+/+) cells treated with Wogonin. Orai1 and STIM1 transcript levels were further increased in sgk1(+/+) and sgk1(-/-) mast cells by transfection with active NF-κB subunit p65 as well as in HEK293 cells by transfection with NF-κB subunits p65/p50 or p65/p52. They were decreased by silencing of NF-κB subunits p65, p50, or p52 or by NF-κB inhibitor Wogonin (100 µm). Luciferase assay and chromatin immunoprecipitation defined NF-κB-binding sites in promoter regions accounting for NF-κB sensitive genomic regulation of STIM1 and Orai1. Store-operated Ca(2+) entry was similarly increased by overexpression of p65/p50 or p65/p52 and decreased by treatment with Wogonin. Transfection of HEK293 cells with p65/p50 or p65/p52 further augmented migration. The present observations reveal powerful genomic regulation of Orai1/STIM1 by SGK1-dependent NF-κB signaling.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes.

Platelets are activated on increase of cytosolic Ca2+ activity ([Ca2+](i)), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+](i) increase are significantly blunted in platelets from SGK1 knockout mice (sgk1(-/-)). Similarly, Ca2+ -dependent degranulation, integrin α(IIb)ß3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1(-/-) platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1(-/-) mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active (S422D)SGK1 but not with inactive (K127N)SGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1µM). Transfection of MEG-01 cells with (S422D)SGK1 significantly increased phosphorylation of IκB kinase α/ß and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of (S422D)SGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10µM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB-dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.

Stimulation of Ca2+-channel Orai1/STIM1 by serum- and glucocorticoid-inducible kinase 1 (SGK1).

Ca(2+) signaling includes store-operated Ca(2+) entry (SOCE) following depletion of endoplasmic reticulum (ER) Ca(2+) stores. On store depletion, the ER Ca(2+) sensor STIM1 activates Orai1, the pore-forming unit of Ca(2+)-release-activated Ca(2+) (CRAC) channels. Here, we show that Orai1 is regulated by serum- and glucocorticoid-inducible kinase 1 (SGK1), a growth factor-regulated kinase. Membrane Orai1 protein abundance, I(CRAC), and SOCE in human embryonic kidney (HEK293) cells stably expressing Orai1 and transfected with STIM1 were each significantly enhanced by coexpression of constitutively active (S422D)SGK1 (by+81, +378, and+136%, respectively) but not by inactive (K127N)SGK1. Coexpression of the ubiquitin ligase Nedd4-2, an established negatively regulated SGK1 target, down-regulated SOCE (by -48%) and I(CRAC) (by -60%), an effect reversed by expression of (S422D)SGK1 (by +175 and +173%, respectively). Orai1 protein abundance and SOCE were significantly lower in mast cells from SGK1-knockout (sgk1(-/-)) mice (by -37% and -52%, respectively) than in mast cells from wild-type (sgk1(+/+)) littermates. Activation of SOCE by sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase-inhibitor thapsigargin (2 µM) stimulated migration, an effect significantly higher (by +306%) in (S422D)SGK1-expressing than in (K127N)SGK1-expressing HEK293 cells, and also significantly higher (by +108%) in sgk1(+/+) than in sgk1(-/-) mast cells. SGK1 is thus a novel key player in the regulation of SOCE.