Detection and localization of calpain 3-like protease in a neuronal cell line: possible regulation of apoptotic cell death through degradation of nuclear IkappaBalpha.
Calpains are a family of calcium-dependent cysteine proteases involved in major cellular processes including cell death. Their intracellular localization is essential to the understanding of their biological functions. In a previous confocal microscopy study, we observed the presence of a calpain 3-like protein in the mammalian brain. We thus first identified and confirmed the presence of a calpain 3-like protease in a neuronal cell model (NGF-differentiated PC12 cells). The goal of this study was to determine, for the first time in non-muscular cells, the relation between the subcellular localization, activation and function of this protease. We thus investigated its ability to regulate nuclear IkappaBalpha and therefore NF-kappaB activation after cell death stimulation. The IkappaBalpha/NF-kappaB signalling pathway indeed influences the neurodegenerative process by directly affecting gene expression in neurons. In the present study, we found that calpain 3 is present in the cytoplasm and nucleus of neuron-like PC12 cells and could be activated through autolysis in the nuclei of cells undergoing apoptosis after ionomycin treatment. Moreover, in these conditions, we demonstrated formation of the IkappaBalpha/calpain 3 complex and an increase in calpain-dependent IkappaBalpha cleavage products in cell nuclei. Stimulation of calpain-dependent cell death in neuron activated nuclear calpain 3-like protease and IkappaBalpha proteolysis resulted in the regulation of NF-kappaB activation. These data suggest a new mechanism by which calpain 3 activation is able to regulate the IkappaBalpha/NF-kappaB pathway and thus neurodegenerative processes.
Marcilhac
A
A
EPHE-UMR 5539, University of Montpellier II, CC107, Place Eugene Bataillon, 34000 Montpellier, France. marcilhac@univ-montp2.fr
Raynaud
F
F
Clerc
I
I
Benyamin
Y
Y
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D001329:Autolysis; D002154:Calpain / Q000032:analysis / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006624:Hippocampus / Q000201:enzymology; D020672:I-kappa B Proteins / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism*; D009124:Muscle Proteins / Q000032:analysis / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D016716:PC12 Cells; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051381:Rats
D017304:Annexin A5; D020672:I-kappa B Proteins; D007527:Isoenzymes; D009124:Muscle Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D000072000:NF-KappaB Inhibitor alpha; D015759:Ionomycin; D002154:Calpain; C491372:Capn3 protein, rat; D016650:Fluorescein-5-isothiocyanate