Activation of STIM1-Orai1 Involves an Intramolecular Switching Mechanism

Section on Molecular Signal Transduction, Program for Developmental Neuroscience, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.
Science Signaling (Impact Factor: 6.28). 11/2010; 3(148):ra82. DOI: 10.1126/scisignal.2001122
Source: PubMed


Stromal interaction molecule 1 (STIM1) stimulates calcium ion (Ca(2+)) entry through plasma membrane Orai1 channels in response to decreased Ca(2+) concentrations in the endoplasmic reticulum lumen. We identified an acidic motif within the STIM1 coiled-coil region that keeps its Ca(2+) activation domain [Ca(2+) release-activated Ca(2+) (CRAC) activation domain/STIM1-Orai activating region (CAD/SOAR)]-a cytoplasmic region required for its activation of Orai1-inactive. The sequence of the STIM1 acidic motif shows substantial similarity to that of the carboxyl-terminal coiled-coil segment of Orai1, which is the postulated site of interaction with STIM1. Mutations within this acidic region rendered STIM1 constitutively active, whereas mutations within a short basic segment of CAD/SOAR prevented Orai1 activation. We propose that the CAD/SOAR domain is released from an intramolecular clamp during STIM1 activation, allowing the basic segment to activate Orai1 channels. This evolutionarily conserved mechanism of STIM1 activation resembles the regulation of protein kinases by intramolecular silencing through pseudosubstrate binding.

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    • "Unfolding of STIM then allows interaction with Orai. SOAR includes an internal basic patch which may interact with an acidic region on Orai to stabilize such folding (Korzeniowski et al., 2010; Kim and Muallem, 2011; Soboloff et al., 2011). To further investigate the potential involvement of SOCs in the generation of [Ca 2+ ] i signals in human sperm, we have used a CPP (KIKKK) that mimics the basic patch on the SOAR region of STIM. "
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    ABSTRACT: Previous work has provided evidence for involvement of store operated channels (SOCs) in [Ca(2+)]i signalling of human sperm, including a contribution to the transient [Ca(2+)]i elevation that occurs upon activation of CatSper, a sperm-specific cation channel localised to the flagellum, by progesterone . To further investigate the potential involvement of SOCs in the generation of [Ca(2+)]i signals in human sperm we have used cell-penetrating peptides containing the important basic sequence KIKKK, part of the STIM-Orai activating region/CRAC activation domain (SOAR/CAD) of the regulatory protein stromal interaction molecule 1 (STIM1). SOAR/CAD plays a key role in controlling the opening of SOCs, which occurs upon mobilisation of stored Ca(2+). Resting [Ca(2+)]i temporarily decreased upon application of KIKKK peptide (3-4 min) but scrambled KIKKK peptide had a similar effect, indicating that this action was not sequence specific. However, in cells pre-treated with KIKKK the transient [Ca(2+)]i elevation induced by stimulation with progesterone decayed significantly more slowly than in parallel controls and in cells pre-treated with scrambled KIKKK peptide. Examination of single cell responses showed that this effect was due, at least in part, to an increase in the proportion of cells in which the initial transient was maintained for an extended period, lasting up to 10 minutes in a sub-population of cells. We hypothesise that SOCs contribute to the progesterone-induced [Ca(2+)]i transient and that interference with the regulatory mechanisms of SOC delays their closure, causing a prolongation of the [Ca(2+)]i transient. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
    Molecular Human Reproduction 04/2015; 21(7). DOI:10.1093/molehr/gav019 · 3.75 Impact Factor
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    • "One of the mechanisms that mediate the activation of STIM1 is the unfolding of the protein in response to store depletion into an open conformation that exposes a STIM1-ORAI1 activating region (SOAR) and allows it to interact with ORAI1 [18] [19]. "
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    ABSTRACT: STIM1 is a key regulator of store-operated calcium entry (SOCE), and therefore a mediator of Ca2+ entry-dependent cellular events. Phosphorylation of STIM1 at ERK1/2 target sites has been described as enhancing STIM1 activation during intracellular Ca2+ emptying triggered by the inhibition of the sarco(endo)plasmic Ca2+-ATPase with thapsigargin. However, no physiological function is known for this specific phosphorylation. The present study examined the role of STIM1 phosphorylation in cell signaling triggered by EGF. Using a human endometrial adenocarcinoma cell line (Ishikawa cells) EGF or H-Ras(G12V), an active mutant of H-Ras, were found to trigger STIM1 phosphorylation at residues Ser575, Ser608, and Ser621, and this process was sensitive to PD0325901, an inhibitor of ERK1/2. Both, ERK1/2 activation and STIM1 phosphorylation took place in the absence of extracellular Ca2+, indicating that both events are upstream steps for Ca2+ entry activation. Also, EGF triggered the dissociation of STIM1 from EB1 (a regulator of microtubule plus-ends) in a manner similar to that reported for the activation of STIM1 by thapsigargin. Migration of the Ishikawa cells was impaired when STIM1 phosphorylation was targeted by Ser-to-Ala substitution mutation of ERK1/2 target sites. This effect was also observed with the Ca2+ channel blocker SKF96365. Phosphomimetic mutation of STIM1 restored the migration to levels similar to that found for STIM1-wild type. Finally, the increased vimentin expression and relocalization of E-cadherin triggered by EGF were largely inhibited by targeting STIM1 phosphorylation, while STIM1-S575E/S608E/S621E normalized the profiles of these two EMT markers.
    Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 01/2015; 1853(1):233. DOI:10.1016/j.bbamcr.2014.10.027 · 5.02 Impact Factor
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    • "The Orai1 cytoplasmic N-and C-termini extend into the cytosol in close proximity to each other [16] [21]. It appears that these Nand C-terminal helices of Orai1 combine to interact with STIM1 [11] [22], and both are required for STIM1-induced activation of the Orai channel [22] [23] [24] [25] [26] [27] [28]. "
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    ABSTRACT: The coupling of ER Ca2+-sensing STIM proteins and PM Orai Ca2+ entry channels generates “store-operated” Ca2+ signals crucial in controlling responses in many cell types. The dimeric derivative of 2-aminoethoxydiphenyl borinate (2-APB), DPB162-AE, blocks functional coupling between STIM1 and Orai1 with an IC50 (200 nM) 100-fold lower than 2-APB. Unlike 2-APB, DPB162-AE does not affect L-type or TRPC channels or Ca2+ pumps at maximal STIM1-Orai1 blocking levels. DPB162-AE blocks STIM1-induced Orai1 or Orai2, but does not block Orai3 or STIM2-mediated effects. We narrowed the DPB162-AE site of action to the STIM-Orai activating region (SOAR) of STIM1. DPB162-AE does not prevent the SOAR-Orai1 interaction but potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores SOAR-Orai binding but only slowly restores Orai1 channel-mediated Ca2+ entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1-Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels.
    Cell Calcium 10/2014; 56(6). DOI:10.1016/j.ceca.2014.10.005 · 3.51 Impact Factor
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