Phospho-STIM1 is a downstream effector that mediates the signaling triggered by IGF-1 in HEK293 cells

STIM1 is a Ca2+ sensor of the endoplasmic reticulum (ER) that triggers the activation of plasma membrane Ca2+ channels upon depletion of Ca2+ levels within the ER. During thapsigargin-triggered Ca2+ store depletion, ERK1/2 phosphorylates STIM1 at Ser575, Ser608, and Ser621. This phosphorylation plays a role in the regulation of STIM1 dissociation from the microtubule plus-end binding protein EB1, an essential step for STIM1 activation by thapsigargin. However, little is known regarding the physiological role of this phosphorylation. Because IGF-1 triggers the activation of the RAF–MEK–ERK and the phosphoinositide pathways, the role of STIM1 phosphorylation in IGF-1 stimulation was studied. There was found to be phosphorylation of ERK1/2 in both the presence and the absence of extracellular Ca2+, demonstrating that Ca2+ influx is not essential for ERK1/2 activation. In parallel, IGF-1 triggered STIM1 phosphorylation at the aforementioned sites, an effect that was blocked by PD0325901, a MEK1/2 inhibitor used to block ERK1/2 activation. Also, STIM1-GFP was found in clusters upon IGF-1 stimulation, and STIM1-S575A/S608A/S621A-GFP strongly reduced this multimerization. Interestingly, phospho-STIM1 was mainly found in clusters when cells were treated with IGF-1, and IGF-1 triggered the dissociation of STIM1 from EB1, similarly to what has been observed for thapsigargin, suggesting that STIM1 mediates the IGF-1 signaling pathway. A study of IGF-1-stimulated NFAT translocation was therefore performed, finding that STIM1-S575A/S608A/S621A blocked this translocation, as did the fusion protein STIM1-EB1, confirming that both STIM1 phosphorylation and STIM1–EB1 dissociation are required for IGF-1-triggered Ca2+-dependent signaling, and demonstrating that STIM1 phosphorylation plays a role as a downstream effector of the RAF–MEK–ERK pathway and an upstream activator of Ca2+ entry. •A physiological role for STIM1 phosphorylation is proposed.•IGF-1 triggers the activation of ERK1/2, which phosphorylates STIM1.•ERK1/2-dependent STIM1 phosphorylation releases STIM1 from EB1.•STIM1 phosphorylation mediates IGF-1 downstream signaling.