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    Keywords: CELLS ; IN-VITRO ; IN-VIVO ; MICROSCOPY ; PROTEIN ; PROTEINS ; ACTIVATION ; DOMAIN ; C-JUN ; CATALYTIC SUBUNIT ; SUBUNIT ; LOCALIZATION ; NUCLEUS ; CRYSTAL-STRUCTURE ; LIVING CELLS ; CALCIUM-DEPENDENT PROTEASE ; DISSOCIATION ; MYOFIBRILLAR PROTEIN-TURNOVER ; NEUTRAL PROTEASE ; NUCLEAR-LOCALIZATION ; RAT CALPAIN ; THIOL PROTEASE
    Abstract: Ubiquitously expressed calpains are Ca2+-dependent, intracellular cysteine proteases comprising a large catalytic subunit (domains DI-DIV) and a noncovalently bound small regulatory subunit (domains DV and DVI). It is unclear whether Ca2+-induced calpain activation is followed by subunit dissociation or not. Here, we have applied advanced fluorescence microscopy techniques to study calpain subunit interactions in living cells using recombinant calpain subunits or domains fused to enhanced cyan and enhanced yellow fluorescent reporter proteins. All of the overexpressed variants of the catalytic subunit (DI-IV, DI-III, and DI-IIb) were active and Ca2+-dependent. The intact large subunit, but not its truncated variants, associates with the small subunit under resting and ionomycin-activated conditions. All of the variants were localized in cytoplasm and nuclei, except DI-IIb, which accumulates in the nucleus and in nucleoli as shown by microscopy and cell fractionation. Localization studies with mutated and chimeric variants indicate that nuclear targeting of the DI-IIb variant is conferred by the two N-terminal helices of DI. Only those variants that contain DIII migrated to membranes upon the addition of ionomycin, suggesting that DIII is essential for membrane targeting. We propose that intracellular localization and in particular membrane targeting of activated calpain, but not dissociation of its intact subunits, contribute to regulate its proteolytic activity in vivo
    Type of Publication: Journal article published
    PubMed ID: 12591934
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