In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3␤ and tau are parts of an ϳ400 -500 kDa microtubule-associated tau phosphorylation complex (Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940). In this study, we find that when purified brain microtubules are subjected to Superose 12 gel filtration column chromatography, the dimeric scaffold protein 14-3-3 coelutes with the tau phosphorylation complex components tau and GSK3␤. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3, GSK3␤, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3␤, 14-3-3 co-precipitates with GSK3␤, indicating that GSK3␤ binds to 14-3-3. From HEK-293 cells transfected with tau, GSK3␤, and 14-3-3 in different combinations, tau co-immunoprecipitates with GSK3␤ only in the presence of 14-3-3. In vitro, ϳ10-fold more tau binds to GSK3␤ in the presence of than in the absence of 14-3-3. In transfected HEK-293 cells, 14-3-3 stimulates GSK3␤-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3 dimer simultaneously binds and bridges tau and GSK3␤ and stimulates GSK3␤-catalyzed tau phosphorylation.

In Alzheimer's disease, microtubule-associated protein tau is hyperphosphorylated by an unknown mechanism and is aggregated into paired helical filaments. Hyperphosphorylation causes loss of tau function, microtubule instability, and neurodegeneration. Glycogen synthase kinase-3␤ (GSK3␤) has been implicated in the phosphorylation of tau in normal and Alzheimer's disease brain. The molecular mechanism of GSK3␤-tau interaction has not been clarified. In this study, we find that when microtubules are disassembled, microtubuleassociated GSK3␤ dissociates from microtubules. From a gel filtration column, the dissociated GSK3␤ elutes as an ϳ400-kDa complex. When fractions containing the ϳ400-kDa complex are chromatographed through an anti-GSK3␤ immunoaffinity column, tau co-elutes with GSK3␤. From fractions containing the ϳ400-kDa complex, both tau and GSK3␤ co-immunoprecipitate with each other. GSK3␤ binds to nonphosphorylated tau, and the GSK3␤-binding region is located within the N-terminal projection domain of tau. In vitro, GSK3␤ associates with microtubules only in the presence of tau. From brain extract, ϳ6-fold more GSK3␤ co-immunoprecipitates with tau than GSK3␣. These data indicate that, in brain, GSK3␤ is bound to tau within a ϳ400-kDa microtubule-associated complex, and GSK3␤ associates with microtubules via tau.

Neuronal Cdc2-like protein kinase (NCLK), a ϳ58-kDa heterodimer, was isolated from neuronal microtubules (Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T. and Imahori, K. (1992) J. Biol. Chem. 267, 10897-10901). The biochemical nature of NCLK-microtubule association is not known. In this study we found that NCLK is released from microtubules upon microtubule disassembly as a 450-kDa species. The 450-kDa species is an NCLK⅐tau complex, and NCLK-bound tau is in a nonphosphorylated state. Tau phosphorylation causes NCLK⅐tau complex dissociation, and phosphorylated tau does not bind to NCLK. In vitro, the Cdk5 subunit of NCLK binds to the microtubule-binding region of tau and NCLK associates with microtubules only in the presence of tau. Our data indicate that in brain extract NCLK is complexed with tau in a tau phosphorylation-dependent manner and that tau anchors NCLK to microtubules. Recently NCLK has been suggested to be aberrantly activated and to hyperphosphorylate tau in Alzheimer's disease brain (Patrick, G. N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P, and Tsai, L.-H. (1999) Nature 402, 615-622). Our findings may explain why in Alzheimer's disease NCLK specifically hyperphosphorylates tau, although this kinase has a number of protein substrates in the brain.The neuronal Cdc2-like protein kinase (NCLK) 1 (also known as tau kinase II and brain proline-directed protein kinase) is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a neuronal-specific activator p25 subunit (reviewed in Ref. 1). The p25 subunit is a proteolytic fragment of a 35-kDa protein, p35 (1, 2). Targeted disruption of the cdk5 gene in mice results in unique lesions in the central nervous system, lack of cortical laminar structure, as well as cerebellar foliate and prenatal death (3). In differentiating neurons, neurite outgrowth is inhibited by the introduction of a dominant negative Cdk5 mutant and is enhanced by Cdk5 and p35 overexpression (4). Similarly, mice lacking p35 do not show NCLK activity and display cortical lamination defects, seizures, and adult lethality (5). These observations indicate that NCLK plays an important role in brain development, neuronal differentiation, and neurite outgrowth. In neurons NCLK phosphorylates inhibitor 1 (6) and DARPP-32 (7), the two inhibitory proteins that upon phosphorylation suppress phosphoprotein phosphatase 1 activity. Munc18 (8) and synapsin (9), which respectively regulate synaptic vesicle exocytosis and neurotransmitter release, are phosphorylated by NCLK. NCLK also phosphorylates the microtubule-associated protein tau (10, 11) and neurofilaments (12,13). These data indicate that NCLK regulates neural signaling, vesicular exocytosis, neurotransmitter release, and microtubule dynamics.

In mammalian brain, tau, glycogen synthase kinase 3␤ (GSK3␤), and 14-3-3, a phosphoserine-binding protein, are parts of a multiprotein tau phosphorylation complex. Within the complex, 14-3-3 simultaneously binds to tau and GSK3␤ (Agarwal-Mawal, A., Qureshi, H. Y., Cafferty, P. W., Yuan, Z., Han, D., Lin, R., and Paudel, H. K. (2003) J. Biol. Chem. 278, 12722-12728). The molecular mechanism by which 14-3-3 connects GSK3␤ to tau within the complex is not clear. In this study, we find that GSK3␤ within the tau phosphorylation complex is phosphorylated on Ser 9 . From extracts of rat brain and rat primary cultured neurons, Ser 9 -phosphorylated GSK3␤ precipitates with glutathione-agarose beads coated with glutathione S-transferase-14-3-3. Similarly, from rat brain extract, Ser 9 -phosphorylated GSK3␤ coimmunoprecipitates with tau. In vitro, 14-3-3 binds to GSK3␤ only when the kinase is phosphorylated on Ser 9 . In transfected HEK-293 cells, 14-3-3 binds to Ser 9 -phosphorylated GSK3␤ and does not bind to GSK3␤ (S9A). Tau, on the other hand, binds to both GSK3␤ (WT) and GSK3␤ (S9A). Moreover, 14-3-3 enhances the binding of tau with Ser 9 -phosphorylated GSK3␤ by ϳ3-fold but not with GSK3␤ (S9A). Similarly, 14-3-3 stimulates phosphorylation of tau by Ser 9 -phosphorylated GSK3␤ but not by GSK3␤ (S9A). In transfected HEK-293 cells, Ser 9 phosphorylation suppresses GSK3␤-catalyzed tau phosphorylation in the absence of 14-3-3. In the presence of 14-3-3, however, Ser 9 -phosphorylated GSK3␤ remains active and phosphorylates tau. Our data indicate that within the tau phosphorylation complex, 14-3-3 connects Ser 9 -phosphorylated GSK3␤ to tau and Ser 9 -phosphorylated GSK3␤ phosphorylates tau.