Phosphoregulation of STIM1 Leads to Exclusion of the Endoplasmic Reticulum from the Mitotic Spindle

Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Current biology: CB (Impact Factor: 9.57). 06/2012; 22(16):1487-93. DOI: 10.1016/j.cub.2012.05.057
Source: PubMed


The endoplasmic reticulum (ER) undergoes significant reorganization between interphase and mitosis, but the underlying mechanisms are unknown. Stromal interaction molecule 1 (STIM1) is an ER Ca(2+) sensor that activates store-operated Ca(2+) entry (SOCE) and also functions in ER morphogenesis through its interaction with the microtubule +TIP protein end binding 1 (EB1). We previously demonstrated that phosphorylation of STIM1 during mitosis suppresses SOCE. We now show that STIM1 phosphorylation is a major regulatory mechanism that excludes ER from the mitotic spindle. In mitotic HeLa cells, the ER forms concentric sheets largely excluded from the mitotic spindle. We show that STIM1 dissociates from EB1 in mitosis and localizes to the concentric ER sheets. However, a nonphosphorylatable STIM1 mutant (STIM1(10A)) colocalized extensively with EB1 and drove ER mislocalization by pulling ER tubules into the spindle. This effect was rescued by mutating the EB1 interaction site of STIM1(10A), demonstrating that aberrant association of STIM1(10A) with EB1 is responsible for the ER mislocalization. A STIM1 phosphomimetic exhibited significantly impaired +TIP tracking in interphase but was ineffective at inhibiting SOCE, suggesting different mechanisms of regulation of these two STIM1 functions by phosphorylation. Thus, ER spindle exclusion and ER-dependent Ca(2+) signaling during mitosis require multimodal STIM1 regulation by phosphorylation.

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Available from: Nasser M Rusan, Sep 19, 2015
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    • "As has been reported for other +TIPs, the phosphor-regulation of STIM1-EB1 interaction could be more complex, and other phosphoresidues might trigger a diversity of actions. For instance, the Ser-to-Ala substitution mutation of 10 residues in STIM1 leads to ER mislocalization by pulling ER tubules into the mitotic spindle.60 Because in wild-type STIM1-expressing cells ER tubules are excluded from this mitotic spindle, those results suggest that hyperphosphorylation of STIM1 is required to maintain ER normal structure in mitosis. "
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    ABSTRACT: Calcium ion (Ca(2+)) concentration plays a key role in cell signaling in eukaryotic cells. At the cellular level, Ca(2+) directly participates in such diverse cellular events as adhesion and migration, differentiation, contraction, secretion, synaptic transmission, fertilization, and cell death. As a consequence of these diverse actions, the cytosolic concentration of free Ca(2+) is tightly regulated by the coordinated activity of Ca(2+) channels, Ca(2+) pumps, and Ca(2+)-binding proteins. Although many of these regulators have been studied in depth, other proteins have been described recently, and naturally far less is known about their contribution to cell physiology. Within this last group of proteins, STIM1 has emerged as a major contributor to Ca(2+) signaling by means of its activity as Ca(2+) channel regulator. STIM1 is a protein resident mainly, but not exclusively, in the endoplasmic reticulum (ER), and activates a set of plasma membrane Ca(2+) channels termed store-operated calcium channels (SOCs) when the concentration of free Ca(2+) within the ER drops transiently as a result of Ca(2+) release from this compartment. Knowledge regarding the molecular architecture of STIM1 has grown considerably during the last years, and several structural domains within STIM1 have been reported to be required for the specific molecular interactions with other important players in Ca(2+) signaling, such as Ca(2+) channels and microtubules. Within the modulators of STIM1, phosphorylation has been shown to both activate and inactivate STIM1-dependent Ca(2+) entry depending on the cell type, cell cycle phase, and the specific residue that becomes modified. Here we shall review current knowledge regarding the modulation of STIM1 by phosphorylation.
    Communicative & integrative biology 11/2013; 6(6):e26283. DOI:10.4161/cib.26283
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    • "In a recent paper, Smyth et al. showed that ER targets the mitotic spindle when microtubule plus tip association of the ER protein STIM1 persists during mitosis (Smyth et al., 2012). Importantly , no accumulation of ER membrane at the metaphase plate was observed under these conditions, indicating that solely the aberrant presence of ER in the spindle region does not lead to its premature association with chromatin. "
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    ABSTRACT: Dynamic interactions between membrane-bound organelles and the microtubule cytoskeleton are crucial to establish, maintain, and remodel the internal organization of cells throughout the cell cycle. However, the molecular nature of these interactions remains poorly understood. We performed a biochemical screen for microtubule-membrane linkers and identified REEP4, a previously uncharacterized endoplasmic reticulum (ER) protein. Depletion of REEP4 and the closely related REEP3 from HeLa cells causes defects in cell division and a proliferation of intranuclear membranes derived from the nuclear envelope. This phenotype originates in mitosis, when ER membranes accumulate on metaphase chromosomes. Microtubule binding and mitotic ER clearance from chromosomes both depend on a short, positively charged amino acid sequence connecting the two hydrophobic domains of REEP4. Our results show that REEP3/4 function redundantly to clear the ER from metaphase chromatin, thereby ensuring correct progression through mitosis and proper nuclear envelope architecture.
    Developmental Cell 07/2013; 26(3). DOI:10.1016/j.devcel.2013.06.016 · 9.71 Impact Factor
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    • "Most interestingly, phosphorylation of STIM1 is critical to exclude the ER from the mitotic spindle during cell division [18]. Since EB1 and APC interactions are relevant to microtubule stabilization [19] [20] [21], and given the fact that STIM1 is a microtubule plus end tracking protein [9], we were interested in exploring the role of APC in SOCE. "
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    ABSTRACT: The assembly of STIM1 protein puncta near endoplasmic reticulum-plasma membrane (ER-PM) junctions is required for optimal activation of store-operated channels (SOC). The mechanisms controlling the translocation of STIM1 puncta to ER-PM junctions remain largely unknown. In the present study, we have explored the role of the microtubule binding protein adenomatous polyposis coli (APC), on STIM1 puncta and store-operated calcium entry (SOCE). APC-depleted cells showed reduced STIM1 puncta near ER-PM junctions, instead puncta is found at the ER surrounding the cell nucleus. Reduced STIM1 puncta near ER-PM junctions in APC-depleted cells correlates with a strong inhibition of SOCE and diminished Orai whole-cell currents. Immunoprecipitation and confocal microscopy co-localization studies indicate that, upon depletion of the ER, STIM1 dissociates from EB1 and associates to APC. Deletion analysis identified an APC-binding domain in the carboxyl terminus of STIM1 (STIM1 650-685). These results together position APC as an important element in facilitating the translocation of STIM1 puncta near ER-PM junctions, which in turn is required for efficient SOCE and Orai activation upon depletion of the ER.
    Cell calcium 07/2013; 54(3). DOI:10.1016/j.ceca.2013.06.008 · 3.51 Impact Factor
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