Abstract
Store-operated calcium entry (SOCE) channels composed of Stim and Orai proteins play a critical role in diverse biological processes. Upon endoplasmic reticulum (ER)-mediated calcium (Ca(2+)) depletion, Stim proteins oligomerize with Orai to initiate Ca(2+) influx across the plasma membrane. The ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of ubiquilin 1 are involved in the degradation of presenilin and polyglutamine proteins. Through screening of Orai1 interaction partner(s) that might have an effect on SOCE, ubiquilin 1 was identified as a target of Orai1. However, the UBL and UBA domains of ubiquilin 1 were dispensable for this interaction. Additionally, ubiquilin 1 and Orai1 colocalized in the cytosolic compartment. Ubiquilin 1 increased the ubiquitination of Orai1, resulting in the formation of a high-molecular-weight form. MG132, a proteasome inhibitor, failed to block the degradation of Orai1, whereas bafilomycin A, a lysosome inhibitor, prevented Orai1 degradation. Confocal microscopy studies demonstrated that a fraction of Orai1 colocalized with ubiquilin 1 and the autophagosomal marker LC3. Because Orai1 is a constituent of SOCE, we determined the effect of ubiquilin 1 on Orai1-mediated Ca(2+) influx. As we expected, intracellular Ca(2+) mobilization, a process normally potentiated by Orai1, was downregulated by ubiquilin 1. Taken together, these findings suggest that ubiquilin 1 downregulates intracellular Ca(2+) mobilization and its downstream signaling by promoting the ubiquitination and lysosomal degradation of Orai1.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing
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Autophagy-Related Proteins
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Blotting, Western
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Calcium / metabolism*
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Calcium Channels / genetics
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Calcium Channels / metabolism*
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Calcium Signaling*
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cysteine Proteinase Inhibitors / pharmacology
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Cytosol / drug effects
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Cytosol / metabolism
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Enzyme Inhibitors / pharmacology
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HEK293 Cells
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HeLa Cells
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Humans
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Immunoprecipitation
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Leupeptins / pharmacology
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Lysosomes / drug effects
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Lysosomes / metabolism
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Macrolides / pharmacology
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Neoplasm Proteins / genetics
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Neoplasm Proteins / metabolism*
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ORAI1 Protein
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Phagosomes / drug effects
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Phagosomes / metabolism
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Plasmids / genetics
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Proteasome Endopeptidase Complex / chemistry
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Proteasome Endopeptidase Complex / metabolism
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Proton-Translocating ATPases / antagonists & inhibitors
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Signal Transduction
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Stromal Interaction Molecule 1
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Two-Hybrid System Techniques
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Ubiquitination
Substances
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Adaptor Proteins, Signal Transducing
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Autophagy-Related Proteins
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Calcium Channels
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Carrier Proteins
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Cell Cycle Proteins
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Cysteine Proteinase Inhibitors
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Enzyme Inhibitors
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Leupeptins
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Macrolides
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Membrane Proteins
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Neoplasm Proteins
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ORAI1 Protein
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ORAI1 protein, human
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STIM1 protein, human
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Stromal Interaction Molecule 1
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UBQLN1 protein, human
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bafilomycin A
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Proteasome Endopeptidase Complex
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Proton-Translocating ATPases
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benzyloxycarbonylleucyl-leucyl-leucine aldehyde
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Calcium