Abstract
Store-operated Ca2+ entry (SOCE) mediates the increase in intracellular calcium (Ca2+) in endothelial cells (ECs) that regulates several EC functions including tissue-fluid homeostasis. Stromal-interaction molecule 1 (STIM1), upon sensing the depletion of (Ca2+) from the endoplasmic reticulum (ER) store, organizes as puncta that trigger store-operated Ca2+ entry (SOCE) via plasmalemmal Ca2+-selective Orai1 channels. While the STIM1 and Orai1 binding interfaces have been mapped, signaling mechanisms activating STIM1 recruitment of Orai1 and STIM1-Orai1 interaction remains enigmatic. Here, we show that ER Ca2+-store depletion rapidly induces STIM1 phosphorylation at Y361 via proline-rich kinase 2 (Pyk2) in ECs. Surprisingly, the phospho-defective STIM1-Y361F mutant formed puncta but failed to recruit Orai1, thereby preventing. SOCE Furthermore, studies in mouse lungs, expression of phosphodefective STIM1-Y361F mutant in ECs prevented the increase in vascular permeability induced by the thrombin receptor, protease activated receptor 1 (PAR1). Hence, Pyk2-dependent phosphorylation of STIM1 at Y361 is a critical phospho-switch enabling recruitment of Orai1 into STIM1 puncta leading to SOCE. Therefore, Y361 in STIM1 represents a novel target for limiting SOCE-associated vascular leak.
Highlights
Endothelial barrier function is vital in the regulation of tissue-fluid homeostasis, angiogenesis, and inflammation[1,2]
We show for the first time that while tyrosine phosphorylation of Stromal-interaction molecule 1 (STIM1) at Y361 is not required for STIM1 puncta formation, it is essential for recruiting Orai[1] into STIM1 puncta to trigger Store-operated Ca2+ entry (SOCE)
Human pulmonary aortic endothelial (HPAE) cells were stimulated with thapsigargin at the indicated time points (Fig. 1a) and STIM1 was immunoprecipitated from the cell lysates using an anti-STIM1 antibody
Summary
Endothelial barrier function is vital in the regulation of tissue-fluid homeostasis, angiogenesis, and inflammation[1,2]. A rise in intracellular free calcium concentration ([Ca2+]i) caused by edemagenic agonists such as thrombin induces actin-myosin contraction in endothelial cells (ECs) leaving wide gaps between adjacent ECs5,6 These gaps, if not sealed, lead to elevated vascular permeability resulting in debilitating pulmonary edema. STIM1 has been recently identified as a fundamental sensor of the Ca2+ concentration inside the ER lumen and thereby regulates SOCE in various cell types including ECs9–11 It is a multi-domain protein containing a single transmembrane α-helix[12]. Conformational changes within its EF-SAM domains allowing long-range regulation within the CC1 domain that extends and organizes the SOAR domain to form discrete puncta[23] These puncta are recruited towards the plasma membrane where STIM1 interacts with Orai[1] to trigger SOCE24,25. We established the physiological role of STIM1 phosphorylation in regulating endothelial permeability in vivo by demonstrating that selective transduction of the phospho-defective STIM1-Y361F mutant into the pulmonary endothelium of WT mice blocked the increase in endothelial permeability induced by activation of the thrombin receptor, protease activated receptor 1 (PAR1)
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