Figure 5
Truncation of GSK-3β affects its ability to phosphorylate PKA-primed tau in site-specific manner.
Recombinant GSK-3β was first incubated without or with 0.2 μg/ml calpain I in the presence of 1 mM CaCl2 for 10 min at 30°C. The GSK-3β truncation by calpain I was inhibited by adding ALLN, followed by incubation with tau441 or PKA-prephosphorylated tau441 for various times. The phosphorylation of tau at individual sites was measured by immuno-dot blots developed with phospho-dependent antibodies to various specific phosphorylation sites of tau (A) and the relative levels of tau phosphorylation at individual sites detected in panel A were quantitated by densitometry and plotted against tau phosphorylation reaction times (B). Truncation of GSK-3β by calpain I enhanced its kinase activity toward tau Ser 199, Thr 205, Thr 217 and Ser 396, but like full length GSK-3β (see Fig. S5) the ability of the truncated kinase to phosphorylate PKA-phosphorylated tau at Thr 212 is reduced.
