Figure 6

GSK-3β is truncated at the C-terminus between Ser381 and Ser382 by calpain I in vitro and in AD brain.

(A) Schematic of GSK-3β domain structures and the epitopes of antibodies used to map the truncation. (B) Western blots of GSK-3β proteolysis by calpain I and of control (no calpain I) developed with various antibodies to N-terminal and C-terminal of GSK-3β. Recombinant GSK-3β was incubated without or with 0.2 μg/ml calpain I in the presence of 1 mM CaCl₂ for 10 min at 30°C. The proteolyzed products were analyzed by Western blots with a series of antibodies against different epitopes of GSK-3β. NT, N-terminal region; CT, C-terminal region; HC, Ig G heavy chain; LC, Ig G light chain. (C) Western blots of AD and control brain homogenates developed with various N-terminal and C-terminal anti-GSK-3β. Western blots of frontal cortical homogenates from AD and control cases developed with antibodies against different epitopes of GSK-3β, as labeled in panel A. Arrow indicates truncated GSK-3β; arrow head indicates a non-AD-related truncation of GSK-3β. (D) Western blots of AD and control brain homogenates and of HA-GSK-3β proteolyzed by calpain I in vitro and its control developed with R127. Western blots of frontal cortical homogenates from AD, control cases and immunpuified GSK-3β proteolyzed with or without calpain I as described above developed with antibody R127 to GSK-3β. (E) Amino acid sequences of regions of GSK-3β (β) and GSK-3α (α) where calpain I cleaves GSK-3β (between Ser 381 and Ser 382). GSK-3β-GST recombinant fusion protein was proteolyzed with calpain I in vitro, then subjected to SDS-PAGE and the truncated GSK-3β band recognized by anti-GST was cut out and subjected to N-terminal sequencing. Dotted arrow shows the truncation site between Ile 397-Gln 398 reported in the literature for calpain II²⁹.