Figure 3
GSK-3β is truncated selectively by calcium-mediated truncation/activation of calpain I.
(A) Western blots show that truncation of GSK-3β coincides with truncation/activation of calpain I in a calcium-dependent manner in human brain extracts. Normal human brain extract was incubated at 30°C for 10 min in the presence of 2.0 mM EDTA and various concentrations (0.00–2.14 mM) of CaCl2. Then the incubated extracts were analyzed by Western blots developed with specific antibodies to calpain I or GSK-3β. (B) Calcium-activated truncations of calpain I and GSK-3β are selectively inhibited by calpain inhibitors. Normal human brain extract was incubated at 30°C for 10 min in the presence of 2.0 mM each of EDTA and CaCl2 plus various selective protease inhibitors, as indicated above the blots, followed by Western blots probed with anti-calpain I or anti-GSK-3β to detect the proteolysis. Apr, aprotinin (a serine protease inhibitor); Pep, pepstatin (an aspartic protease inhibitor); Leu, leupeptin (cysteine and serine protease inhibitor that also inhibits calpain); ALLN, N-acetyl-Leu-Leu-Nle-CHO (a cysteine protease inhibitor that also inhibits calpain); Calp, calpastatin peptide (a specific calpain inhibitor). (C, D) Western blots show calcium concentration-dependent truncation of recombinant GSK-3β from bacteria (C) or from mammalian cells (D) by calpain I. Recombinant GSK-3β was incubated with various concentration of calpain I in the presence of CaCl2 for 10 min at 30°C and the reaction products were subjected to Western blots.
