Calpain-mediated activation of NO synthase in human neuroblastoma SK-N-BE cells.
In resting human neuronal cells, nitric oxide synthase (nNOS) is present in its native 160 kDa form in a quiescent state predominantly co-localized on the plasma membrane, via its PDZ (Psd-95/Discs-large/Zona Occludens) domain, with NMDA receptor (NMDA-R) and in tight association with heat shock protein 90 (HSP90). Following exposure of the cells to Ca(2+)-ionophore or to NMDA, nNOS undergoes proteolytic removal of the PDZ domain being converted into a fully active 130 kDa form. The newly generated nNO synthase form dissociates from NMDA-R and extensively diffuses into the cytosol in direct correlation with NO production. Intracellular redistribution and activation of nNOS are completely prevented in cells preloaded with calpain inhibitor-1, indicating that these processes are triggered by a concomitant activation of calpain. The role of calpain has been confirmed by immunoprecipitation experiments revealing that also mu-calpain is specifically recruited into the NMDA-R-nNOS-HSP90 complex following calcium loading. Thus, the formation of clusters containing HSP90, mu-calpain, nNOS and NMDA-R represents the limiting step for the operation of the mechanism that links an efficient synthesis of NO to a local increase in Ca(2+) influx.
Averna
Monica
M
Department of Experimental Medicine (DIMES), Biochemistry Section, and Centre of Excellence for Biomedical Research (CEBR), University of Genoa, Genoa, Italy.
Stifanese
Roberto
R
De Tullio
Roberta
R
Beccaria
Francesco
F
Salamino
Franca
F
Pontremoli
Sandro
S
Melloni
Edon
E
D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D003600:Cytosol / Q000378:metabolism; D004058:Diffusion; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D052248:Nitric Oxide Synthase Type I / Q000187:drug effects / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*
D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D007476:Ionophores; D016194:Receptors, N-Methyl-D-Aspartate; D009569:Nitric Oxide; D052248:Nitric Oxide Synthase Type I; D010447:Peptide Hydrolases; D002154:Calpain; D002118:Calcium