Figure 6.

Proteolysis of CREB is catalyzed by calcium-mediated truncation/activation of calpain I. (A) In vitro proteolyses of calpain I and CREB in human brain extracts were activated by calcium. A normal human brain extract was incubated for 10 min at 30°C in the absence (upper two panels) or presence (3th panel) of recombinant MBP–CREB and analyzed by western blots. PP2A catalytic subunit was used as negative control. (B) Calcium-activated proteolysis of calpain I and CREB was inhibited by calpain inhibitors selectively. Recombinant MBP–CREB was incubated with normal human brain extract in the presence of EGTA or CaCl₂ plus various selective protease inhibitors for 10 min at 30°C, followed by western blots to detect the proteolysis. Abbreviations: Apr, aprotinin; Pep, pepstatin; Leu, leupeptin; Calp, calpastatin peptide. (C) Immunopurified CREB from mammalian cells was proteolysed by calpain I. HA-CREB was immunopurified from HEK-293FT cells and incubated with various concentrations of calpain I for 10 min at 30°C. The reaction products were analyzed with western blots. (D–F) Recombinant MBP–CREB was proteolysed by calpain I. Recombinant MBP–CREB was incubated with various concentrations of calpain I for 10 min at 30°C. The proteolytic products were analyzed by Coomassie blue staining (D) and western blots developed with anti-CREB (against amino acids 5–24) (E) or anti-CREB (against middle region) (F).