calpain and 4-(4-dimethylaminophenylazo)benzoic-acid

Calpain is a calcium-dependent protease that plays a significant role in synaptic plasticity, cell motility, and neurodegeneration. Two major calpain isoforms are present in brain, with mu-calpain (calpain1) requiring micromolar calcium concentrations for activation and m-calpain (calpain2) needing millimolar concentrations. Recent studies in fibroblasts indicate that epidermal growth factor (EGF) can activate m-calpain independently of calcium via mitogen-activated protein kinase (MAPK)-mediated phosphorylation. In neurons, MAPK is activated by both brain-derived neurotrophic factor (BDNF) and EGF. We therefore examined whether these growth factors could activate m-calpain by MAPK-dependent phosphorylation using cultured primary neurons and HEK-TrkB cells, both of which express BDNF and EGF receptors. Calpain activation was monitored by quantitative analysis of spectrin degradation and by a fluorescence resonance energy transfer (FRET)-based assay, which assessed the truncation of a calpain-specific peptide flanked by the FRET fluorophore pair DABCYL and EDANS. In both cell types, BDNF and EGF rapidly elicited calpain activation, which was completely blocked by MAPK and calpain inhibitors. BDNF stimulated m-calpain but not mu-calpain serine phosphorylation, an effect also blocked by MAPK inhibitors. Remarkably, BDNF- and EGF-induced calpain activation was preferentially localized in dendrites and dendritic spines of hippocampal neurons and was associated with actin polymerization, which was prevented by calpain inhibition. Our results indicate that, in cultured neurons, both BDNF and EGF activate m-calpain by MAPK-mediated phosphorylation. These results strongly support a role for calpain in synaptic plasticity and may explain why m-calpain, although widely expressed in CNS, requires nonphysiological calcium levels for activation.

Topics: Actins; Analysis of Variance; Animals; Axons; Brain-Derived Neurotrophic Factor; Calcium; Calpain; Cells, Cultured; Chelating Agents; Dendritic Spines; Drug Interactions; Egtazic Acid; Embryo, Mammalian; Enzyme Inhibitors; Epidermal Growth Factor; Fluorescence Resonance Energy Transfer; Hippocampus; Humans; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Naphthalenesulfonates; Neocortex; Neurons; p-Dimethylaminoazobenzene; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptor, trkB; RNA, Small Interfering; Transfection

Calpains are intracellular proteases that selectively cleave proteins in response to calcium signals. Although calpains cut many different sequences, residue preferences within peptide substrates were recently determined and incorporated into a superior FRET (fluorescence resonance energy transfer)-based substrate (PLFAER). Here we show PLFAER is cleaved by calpain at the intended F-A scissile bond. Sequential replacement of individual residues by alanine reduced activity except with PLFAAR, which is cleaved 2.3 times faster than PLFAER. The rates of hydrolysis of the alanine-substituted substrates were used to compare substrate preferences of calpain, papain and cathepsins B and L. The preferences of the two major isoforms, calpains 1 and 2, were virtually indistinguishable and were very similar to those of the calpain 1 protease core and papain. However, the activity profiles with the FRET substrate series were significantly different for the cathepsins, particularly cathepsin B.

Topics: Amino Acid Sequence; Amino Acid Substitution; Calpain; Catalytic Domain; Cathepsins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Hydrolysis; In Vitro Techniques; Isoenzymes; Kinetics; Naphthalenesulfonates; Oligopeptides; p-Dimethylaminoazobenzene; Papain; Recombinant Proteins; Substrate Specificity