Calpain-mediated truncation of GSK-3 in post-mortem brain samples.
GSK-3 activity can be regulated by phosphorylation and through interaction with GSK-3-binding proteins. In addition, we have recently demonstrated that calpain activation produces a truncation of GSK-3 that removes the N-terminal inhibitory domain (Goñi-Oliver et al. [2007] J. Biol. Chem. 282:22406). Given that calpain is involved in post-mortem proteolysis in brain samples, the objective of this investigation was to test whether GSK-3 is truncated in post-mortem samples. To achieve this objective, we first investigated the degradation of GSK-3 during different post-mortem intervals in mouse brains and found that the conversion of GSK-3 to proteolytic fragments of 40 and 30 kDa takes place in a way similar that of to p35-CDK-5 subunit and spectrin, two well-known calpain substrates. In addition, we demonstrated that this truncation is mediated by calpain, insofar as pretreatment with MDL 28170, a permeable blood-brain barrier calpain inhibitor, partially inhibited that degradation. When human brain extracts were exposed to calcium, GSK-3 was truncated, generating two fragments of approximately 40 and 30 kDa, a proteolytic process that was inhibited by calpeptin, a specific calpain inhibitor. Thus, this is the first report of calcium-dependent truncation of human GSK-3. These data demonstrate that control samples with similar post-mortem delay are essential to interpret correctly the changes observed in GSK-3 levels in human post-mortem brain, especially when studying human neurodegenerative diseases.
Goñi-Oliver
Paloma
P
Centro de Biología Molecular "Severo Ochoa," CSIC/UAM, Fac. Ciencias, Universidad Autónoma de Madrid, Madrid, Spain.
Avila
Jesús
J
Hernández
Félix
F
D000368:Aged; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D011180:Postmortem Changes
D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D038362:Glycogen Synthase Kinase 3; D002154:Calpain; C058076:calpain inhibitor III