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 ...

We have cloned a novel kinase (STY) from an embryonal carcinoma cel line. Sequence analysis of the STY cDNA reveals that it shares sequence homology with serine/threonine-type kinases and yet the bacterial expression product of the STY cDNA appears to have serine-, threonine-, and tyrosine-phosphorylating activities. The predicted STY protein is highly basic and contains a putative nuclear localization signal. During differentiation, two new mRNAs were detected in addition to the embryonic transcript.During embryogenesis there is stringent control over the processes of cellular differentiation and proliferation. Sophisticated networks of regulatory molecules have evolved to sense and transmit signals within and between differentiating cells. The recent demonstration that a number of developmental and cell cycle mutations map to genes encoding protein kinases (3, 4, 6, 11, 12, 27, 30, 32, tially as previously described (37). First-and second-strand cDNAs were prepared from the P19 EC cell poly(A)+ RNA (7) with the cDNA Synthesis System Plus (Amersham) and were subsequently size selected for products larger than 1 kb by agarose gel electorphoresis.Sequencing of cDNA clones. The cDNA inserts were cloned into the EcoRI site of the plasmid pTZ19R (Pharmacia). Nested deletions were generated with an Exo III-mung bean deletion kit (Stratagene). These deletion mutants were sequenced by the dideoxy chain termination method (34).Bacterial expression of STY cDNA. The catalytic region of STY cDNA from the Hindlll site (position 280 bp) to carboxyl-terminal EcoRI was subcloned into the SmaI site of the pATH2 expression vector (31, 36). E. coli JM103 harboring this construct was typically induced to express the trpE-STY fusion protein by growing the bacteria for 12 h in medium lacking tryptophan and containing indoleacrylic acid as described elsewhere (31). Alternatively, E. coli Y1090 was infected with the Agtll STY clone and induced to express STY protein by the addition of 10 mM IPTG (isopropyl-p-D-thiogalactopyranoside) to the bacterial medium for 4 h (20).Phosphoamino acid and tryptic map analysis. Lysates were prepared from bacteria induced to express trpE or trpE-STY by sonication in 20 mM Tris hydrochloride (pH 8.0)-2 mM EDTA-100 mM NaCl-10 mM P-mercaptoethanol-0.075% Nonidet P-40. Lysates were cleared by centrifugation, and the supernatants were immunoprecipitated with either PY 20 or trpE (AB-1) monoclonal antibodies (from ICN Biochemicals and Oncogene Science Inc., respectively) in 10 mM Tris (pH 7.5)-150 mM NaCl-5 mM EDTA-1% Triton X-100-2 mM NaF-2 mM sodium pyrophosphate-500 ,uM ammonium vanadate-200 ,ug of phenylmethylsulfonyl fluoride per ml and assayed for kinase activity in 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7.1)-10 mM MnCl2 for 30 min at 22°C. The reaction products were resolved by 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the dried gels were exposed to Kodak XAR-5 film. Phosphoproteins were electroeluted for phosphoamino acid analysis by t...

Cyclin-dependent kinase 5 (Cdk5) is activated by the neuronal-specific activator protein, p35. In contrast to the activation of typical CDKs by cyclin subunits, p35⅐Cdk5 was not further activated by the CDK-activating kinase (CAK) and was neither phosphorylated nor inhibited by the Tyr-15-specific Wee1 kinase. The previously identified proteolytic active fragment of p35, p25 (residues 91-307) as well as the slightly smaller fragment containing residues 109 -291, was found to be sufficient to bind and activate Cdk5. Other CDKs, including Cdk2, associated weakly with p25. However, their kinase activity was only activated to the low level observed for cyclin A⅐Cdk2 without Thr-160 phosphorylation, and phosphorylation of Thr-160 in Cdk2 did not activate the p25⅐Cdk2 complex further. We have identified distinct regions in p35 required for binding to Cdk5 or activation of Cdk5. Residues ϳ150 -200 of p35 were sufficient for binding to Cdk5, but residues ϳ279 -291 were needed in addition for activation of Cdk5 in vitro.Cyclins and cyclin-dependent kinases (CDKs) 1 are key regulators of the eukaryotic cell cycle (1). Cdc2 is associated with B-type cyclins and regulates M phase (2). Cdk2 is associated with A-and E-type cyclins, and the respective complexes are believed to control the S phase and G 1 -S transition, respectively (3, 4). Cdk4 and Cdk6 are associated with the D-type cyclins and are important for G 1 progression (3).The activity of CDKs is tightly regulated by an intricate system of protein-protein interaction and phosphorylation (5). The activation of CDKs, by definition, requires the association with cyclin partners. Full activation of CDKs requires in addition the phosphorylation of Thr-161/Thr-160, which lies in the activating T-loop in the crystal structure of Cdk2 (6, 7). The Thr-161/Thr-160 residue is phosphorylated by the CDK-activating kinase (CAK), which is composed of a cyclin H-Cdk7 complex and a RING finger protein subunit MAT1 (8). The activity of CDKs can be inhibited by phosphorylation of Thr-14 and Tyr-15 by the Wee1 and Myt1 protein kinases (9). Furthermore, CDKs can be inactivated by binding to CDK inhibitors like those from the p21 cip1/WAF1 family (p21 cip1/WAF1 , p27 kip1 , and p57 kip2 ) and the p16 INK4A family (p16 INK4A , p15 INK4B

Protein aggregation plays a critical role in the pathogenesis of neurodegenerative diseases, and the mechanism of its progression is poorly understood. Here, we examine the structural and dynamic characteristics of transiently evolving protein aggregates under ambient conditions by directly probing protein surface water diffusivity, local protein segment dynamics, and interprotein packing as a function of aggregation time, along the third repeat domain and C terminus of Δtau187 spanning residues 255-441 of the longest isoform of human tau. These measurements were achieved with a set of highly sensitive magnetic resonance tools that rely on sitespecific electron spin labeling of Δtau187. Within minutes of initiated aggregation, the majority of Δtau187 that is initially homogeneously hydrated undergoes structural transformations to form partially structured aggregation intermediates. This is reflected in the dispersion of surface water dynamics that is distinct around the third repeat domain, found to be embedded in an intertau interface, from that of the solvent-exposed C terminus. Over the course of hours and in a rate-limiting process, a majority of these aggregation intermediates proceed to convert into stable β-sheet structured species and maintain their stacking order without exchanging their subunits. The population of β-sheet structured species is >5% within 5 min of aggregation and gradually grows to 50-70% within the early stages of fibril formation, while they mostly anneal block-wisely to form elongated fibrils. Our findings suggest that the formation of dynamic aggregation intermediates constitutes a major event occurring in the earliest stages of tau aggregation that precedes, and likely facilitates, fibril formation and growth.biological water | soluble oligomers | amyloid formation | site-directed spin labeling | Alzheimer's disease