Interaction of Neuronal Cdc2-like Protein Kinase with Microtubule-associated Protein Tau

Abstract

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.

Familial FTDP-17 Missense Mutations Inhibit Microtubule Assembly-promoting Activity of Tau by Increasing Phosphorylation at Ser202 in Vitro

“…Proteins and Enzymes-Tau(WT) and various tau mutants were purified from lysates of Escherichia coli overexpressing their respective tau species essentially as described previously (28). Briefly, tau expression was induced by adding isopropyl 1-thio-␤-D-galactopyranoside (0.2 mM) to the overnight bacterial culture.…”

“…Microtubules were purified from fresh bovine brain extract by three cycles of temperature-induced microtubule polymerization and depolymerization as described previously (28,30). Tubulin was purified from purified microtubules through phosphocellulose chromatography (28,30). Monoclonal tau 5 antibody against total tau and tau phosphorylationdependent monoclonal antibodies AT8, PHF-1, MC6, and TG3 have been described previously (29,31).…”

“…Fractions containing tau were pooled, concentrated with Aquacade III (Calbiochem) by dialysis, dialyzed against Hepes buffer (25 mM Hepes (pH 7.2), 0.1 mM EDTA, 0.5 mM dithiothreitol, and 100 mM NaCl), and stored at Ϫ80°C until use. Microtubules were purified from fresh bovine brain extract by three cycles of temperature-induced microtubule polymerization and depolymerization as described previously (28,30). Tubulin was purified from purified microtubules through phosphocellulose chromatography (28,30).…”

“…Cyclin-dependent protein kinase 5 (Cdk5) is one of the major kinases that phosphorylates tau in the brain (27,28). In this study, to determine how FTDP-17 missense mutations affect tau phosphorylation, we phosphorylated four FTDP-17 tau mutants (G272V, P301L, V337M, and R406W) by Cdk5.…”

Microtubule Organization and Microtubule-Associated Proteins (MAPs)

“…Tau also interacts with the neuronal plasma membrane and anchors enzymes to MTs (Brandt et al 1995;Lee et al 1998;Sontag et al 1999). Tau can also associate with spectrin (Carlier et al 1984), actin (Griffith and Pollard 1982), PP1 and PP2A Sontag et al 1999), kinases like CDK5 (Sobue et al 2000), presenilin 1 (Takashima et al 1998), α-synuclein (Jensen et al 1999), phospholipase C (Hwang et al 1996), the Fyn tyrosine kinase (Klein et al 2002;Lee et al 1998), apolipoprotein E (Strittmatter et al 1994), and calmodulin (Baudier et al 1987). It can also bind to chaperones such as Hsp70, Hsp90, and Pin-1 (Dou et al 2003;Lu et al 1999).…”