Figure 4
Proteolysis of GSK-3β by calpain I increases its tau kinase activity.
(A) Proteolysis of GSK-3β by calpain I elevates its kinase activity toward tau in a time dependent manner. Recombinant GSK-3β was incubated with 0.1 μg/ml calpain I for various times at 30°C. The reaction products were subjected to kinase activity assay toward tau441 in presence of a calpain inhibitor, ALLN. (B, C) Immuno-dot-blots developed with phospho-dependent antibodies to various phosphorylation sites of tau and anti-total tau show that proteolysis of GKS-3β by calpain I increases its kinase activity. Recombinant GSK-3β from mammalian cells was incubated without or with 0.2 μg/ml calpain I in presence of 1 mM CaCl2 for 10 min at 30°C. The reaction products were incubated with tau441 for various times (0.5–60 min) in the presence of ALLN. The phosphorylation of tau at individual sites was assayed by immune-dot blots (B) and relative level of tau phosphorylation at individual sites detected in panel B was quantitated by densitometry and plotted against the reaction time (0–60 min) (C).
