Fig. 2.

Phosphorylation of free and microtubule-bound tau isoforms (τ3 and τ3L) by a combination of cdk5 (K5) and GSK-3 (K3). τ3 (A) and τ3L (b) as free (open symbols) or microtubule-bound (closed symbols) incubated in the absence (○, •, ▵ and ▴) or presence (□, ▪) of cdk5 as described in Materials and Methods. After 120 min at 30°C, the phosphorylated taus (with non-radioactive ATP) were further phosphorylated by [γ-³²P]ATP and cdk5 (○, •) or GSK-3 (▵, ▴, □ and ▪) for another 120 min at 30°C. Aliquots of the phosphorylation mixture were removed at different times, the reaction was stopped by heating at 95°C for 5 min and denatured tubulin removed by centrifugation (10,000 x g, 10 min). The heat-stable phosphorylated tau in the supernatant was spotted on filter paper and ³²P incorporation was analyzed as described in Materials and Methods; (□, ▪) incubated with cdk5, followed by GSK-3; (▵, ▴) incubated without kinase, followed by GSK-3; (○, •) incubated without kinase followed by cdk5. (C) ³²P-incorporation by second kinase in 120 min at 30°C. Open bars, free taus; closed bars, MT-bound taus.