Abstract 9327: Depletion of Intracellular Ca2+ Stores Induces Phosphorylation of Stromal Interaction Molecular 1 (STIM1), Which Contributes to the Sustained Phase of Store-operated Ca2+ Influx in Vascular Endothelial Cells
Store-operated Ca2+ influx (SOC) is a major Ca2+ entry pathway in vascular endothelial cells, thus contributing to the regulation of endothelial barrier function, nitric oxide production, and angiogenesis. The basic molecular mechanisms underlying SOC have recently been elucidated. Namely, STIM1 serves as a store site Ca2+ sensor and thereby activates SOC channels. However, the mechanisms regulating SOC remains largely unknown. STIM1 has been previously reported to be a phosphoprotein with unknown function. The present study thus investigated the role of STIM1 phosphorylation in the regulation of SOC. STIM1 phosphorylation was evaluated with a new SDS-PAGE technique utilizing a Phos-tagTM compound, which specifically binds to a phosphate group, thus allowing the separation of phosphoproteins from the unphosphorylated form. Thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump, induced a depletion of the Ca2+stores in porcine aortic endothelial cells (PAEC) and HeLa cells in the absence of extracellular Ca2+. The following repletion of the external Ca2+ induced a sustained Ca2+ influx. STIM1 knockdown abolished this influx. Thapsigargin induced >95% phosphorylation of STIM1 in the absence of extracellular Ca2+, which persisted irreversibly even after the repletion of external Ca2+. Thrombin induced Ca2+ depletion and subsequent activation of SOC in PAEC, at a lower level than that seen with thapsigargin. Thrombin also induced a lower level of STIM1 phosphorylation in PAEC. However, it returned to the pre-stimulation level after Ca2+ repletion. Myosin light chain kinase inhibitors, ML-9 and wortmannin, inhibited thapsigargin-induced STIM1 phosphorylation, while Rho kinase inhibitors, Y27632 and H1152, or PKC inhibitor GF109203X had no effect. The effect of the kinase inhibitors were correlated with the effect on the sustained phase of SOC. Analysis of various STIM1 deletion mutants revealed that the C-terminal region is the site of phosphorylation. These results provide the first evidence that STIM1 is phosphorylated following depletion of Ca2+ stores and this phosphorylation contributes to the maintenance of Ca2+ influx via SOC. STIM1 phosphorylation may provide a new therapeutic target to maintain normal endothelial functions.
- © 2011 by American Heart Association, Inc.