Summary When cells are activated by calcium-mobilizing agonists at low, physiological concentrations, the resulting calcium signals generally take the form of repetitive regenerative discharges of stored calcium, termed calcium oscillations [1]. These intracellular calcium oscillations have long fascinated biologists as representing a mode of digitized intracellular signaling. Recent work has highlighted the role of calcium influx as an essential component of calcium oscillations [2]. This influx occurs through a process known as store-operated calcium entry which is initiated by calcium sensor proteins in the endoplasmic reticulum, STIM1 and STIM2 [3]. STIM2 is activated by changes in endoplasmic reticulum calcium near the resting level, while a threshold of calcium depletion is required to activate STIM1 [4]. In this study, we show that, surprisingly, it is STIM1 and not STIM2 that is exclusively involved in calcium entry during calcium oscillations. The implication is that each oscillation produces a transient drop in endoplasmic reticulum calcium that is sufficient to transiently activate STIM1. This transient activation of STIM1 can be observed in some cells by total internal reflection fluorescence microscopy. This arrangement nicely provides a clearly defined and unambiguous signaling system, translating a digital calcium release signal into calcium influx that can signal to downstream effectors.
SUMMARY Store-operated Ca2+ entry (SOCE) and Ca2+ release-activated Ca2+ currents (Icrac) are strongly suppressed during mitosis, the only known physiological situation in which Ca2+ store depletion is uncoupled from the activation of Ca2+ influx. We found that the ER Ca2+ sensor, STIM1, failed to rearrange into near-plasma membrane puncta in mitotic cells, a critical step in the SOCE activation pathway. We also found that STIM1 from mitotic cells is recognized by the phosphospecific MPM-2 antibody, suggesting that STIM1 is phosphorylated during mitosis. Removal of 10 MPM-2-recognition sites by truncation at amino acid 482 abolished MPM-2 recognition of mitotic STIM1, and significantly rescued STIM1 rearrangement and SOCE responses in mitosis. We identified S486 and S668 as mitosis-specific phosphorylation sites, and STIM1 containing mutations of these sites to alanine also significantly rescued mitotic SOCE. Therefore, phosphorylation of STIM1 at S486 and S668, and possibly other sites, underlies suppression of SOCE during mitosis.
SummaryStore-operated calcium entry is an almost ubiquitous signaling pathway in eukaryotic cells. The plasma membrane store-operated channels are comprised of subunits of the recently discovered Orai proteins, the major one being Orai1.We have discovered that native Orai1, as well as expressed Orai1, exists in two forms in similar quantities: a longer form (Orai1a) of approximately 33 kDa, and a shorter form (Orai1b) of approximately 23 kDa. The Orai1b form arises from alternative translation initiation from a methionine at position 64, and possibly also 71, in the longer Orai1a form. In the sequence upstream of the initiation site of Orai1b, there is a polyarginine sequence previously suggested to be involved in interaction of Orai1 with plasma membrane phosphatidylinositol-4,5-bisphosphate. The loss of this phospholipid binding domain would be expected to influence the mobility of Orai1 protein in the plasma membrane. Indeed, experiments utilizing fluorescence recovery after photobleaching (FRAP) revealed that the recovery half-time for Orai1b was significantly faster than for Orai1a. Since Orai1 must diffuse to sites of interaction with the Ca 2+ sensor, STIM1, these two mobilities might provide for efficient recruitment of Orai1 subunits to sites of store-operated Ca 2+ entry during agonist-induced Ca 2+ signaling.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.