Phosphorylation of the neurofilament proteins of high and medium relative molecular mass, as well as of the Alzheimer's tau protein, is thought to be catalysed by a protein kinase with Cdc2-like substrate specificity. We have purified a novel Cdc2-like kinase from bovine brain capable of phosphorylating both the neurofilament proteins and tau. The purified enzyme is a heterodimer of cyclin-dependent kinase 5 (Cdk5) and a novel regulatory subunit, p25 (ref. 8). When overexpressed and purified from Escherichia coli, p25 can activate Cdk5 in vitro. Unlike Cdk5, which is ubiquitously expressed in human tissue, the p25 transcript is expressed only in brain. A full-length complementary DNA clone showed that p25 is a truncated form of a larger protein precursor, p35, which seems to be the predominant form of the protein in crude brain extract. Cdk5/p35 is the first example of a Cdc2-like kinase with neuronal function.

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Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and a 25-kDa subunit which is derived from a brain-specific 35-kDa novel protein, p35 (Lew, J., Huang, Q.-Q., Qi, Z., Winkfein, R. J., Aebersold, R., Hunt, T., and Wang, J. H. (1994) Nature 371, 423-426). Three truncated forms of p35 including the one corresponding to the 25-kDa subunit of the kinase have been expressed in Escherichia coli and shown to activate a bacteria-expressed Cdk5 with equal efficacy. The shortest truncated form of p35, p21, spanning amino acid residues 88 to 291, has been used to reconstitute active Cdk5 kinase and to characterize the activation reaction. The purified kinase displays similar specific enzyme activity and similar phosphorylation site specificity as the neuronal Cdc2-like kinase purified from bovine brain. Bovine brain extract contains Cdk5 uncomplexed with p35 or p25 which has also been found to be activated by p21 or p25. The results substantiate the previous suggestion that p35 is a specific Cdk5 activator. Several observations suggest that, unlike other well characterized Cdc2-like kinases whose activities depend on the phosphorylation of the catalytic subunits at a specific site by a distinct kinase, the reconstituted Cdk5/p21 does not depend on the phosphorylation of Cdk5 for activity. The reconstitution of the highly active Cdk5 kinase was achieved without requiring any other kinase in the reconstitution reaction. The possibility of autophosphorylation of Cdk5 on the putative activation site has been ruled out as no phosphorylation occurred on Cdk5 during the enzyme reaction. The rate and extent of the kinase reconstitution were not significantly affected by Mg2+ ATP.

The microtubule-associated protein tau regulates diverse and essential microtubule functions, from the nucleation and promotion of microtubule polymerization to the regulation of microtubule polarity and dynamics, as well as the spacing and bundling of axonal microtubules. Thermodynamic studies show that tau interacts with microtubules in the low-to mid-nanomolar range, implying moderate binding affinity. At the same time, it is well established that microtubule-bound tau does not undergo exchange with the bulk medium readily, suggesting that the taumicrotubule interaction is essentially irreversible. Given this dilemma, we investigated the mechanism of interaction between tau and microtubules in kinetic detail. Stopped-flow kinetic analysis reveals moderate binding affinity between tau and preassembled microtubules and rapid dissociation͞association kinetics. In contrast, when microtubules are generated by copolymerization of tubulin and tau, a distinct population of microtubule-bound tau is observed, the binding of which seems irreversible. We propose that reversible binding occurs between tau and the surface of preassembled microtubules, whereas irreversible binding results when tau is coassembled with tubulin into a tau-microtubule copolymer. Because the latter is expected to be physiologically relevant, its characterization is of central importance.T he microtubule-associated protein (MAP) tau is a critical regulator of microtubule dynamics, and defects in microtubule dynamics have been shown to result in cell death (1). Furthermore, a number of mutations in the tau gene that are associated with frontotemporal dementia with parkinsonism linked to chromosome 17 have proven that tau dysfunction can play a causal role in the death of neurons (2). A characteristic defect associated with these mutations is a weaker interaction between tau and microtubules. Thus, knowledge of the detailed mechanism by which tau binds to microtubules is of crucial importance.To date, no information is available on the kinetic steps involved in the binding process; rather, all studies on the mechanism of tau binding to microtubules have been thermodynamic in nature. Studies by Weingarten et al. (3) demonstrated that tau does not interconvert between differently radioactively tagged tubulin fractions through multiple cycles of polymerization and depolymerization. Thus, binding is inferred to be tight, perhaps irreversible. On the other hand, purified tau binds to microtubules with moderate as opposed to high affinity (4-10), and consequently the microtubule-bound and free forms of tau are expected to be readily exchangeable. Thus, not only is kinetic information lacking, but the available thermodynamic data also seem to be conflicting.To address these issues, we carried out rapid-mixing stoppedflow kinetic experiments as well as equilibrium-competition binding studies to investigate the mechanism of interaction between tau and microtubules. Our data suggest that tau can bind to two distinct sites on microtubules: one that displays ...