The extracellular aggregation of amyloid b (Ab) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Cdk5 phosphorylates tau at AD-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Ab peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/ Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Ab 1À42 -induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/ Cdk5 activity, nor did it inhibit the other cyclin-dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting 'normal' Cdk5 activity.

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24°C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (,~l = 8 s -1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (,~l = 10-70 s -l) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phospholylation are followed by a fast electrogenic EIP --~ E2P transition. At 24°C and pH 6.2, the rate constant for the EIP --* E2P transition in NaK-ATPase from eel electric organ is > 1,000 s -1.

Cyclin-dependent kinase-5 (Cdk5) is a serine/threonine kinase activated by its neuron-specific activator, p35, or its truncated form, p25. It has been proposed that the deregulation of Cdk5 activity by association with p25 in human brain tissue disrupts the neuronal cytoskeleton and may be involved in neurodegenerative diseases such as Alzheimer's disease. In this study, we demonstrate that a short peptide (amino acid residues 154-279; Cdk5 inhibitory peptide; CIP), derived from p35, specifically inhibits Cdk5 activity in vitro and in HEK293 cells cotransfected with the peptide and Cdk5/p25, but had no effect on endogenous cdc2 kinase activity. Moreover, we demonstrate that the phosphorylation of tau in HEK293 cells, cotransfected with Cdk5/p25 and CIP, is effectively reduced. These results suggest that CIP specifically inhibits both Cdk5/p25 complex activity and the tau hyperphosphorylation induced by Cdk5/ p25. The elucidation of the molecular basis of p25 activation and CIP inhibition of Cdk5 activity may provide insight into mechanisms underlying the pathology of Alzheimer's disease and contribute to therapeutic strategies.Keywords: Cdk5, p35, Cdk5 inhibitory peptide (CIP), Tau phosphorylation, Alzheimer's disease.Cdk5 is a serine/threonine kinase with close homology to the mitotic Cdks [1,2]. It plays a critical role in brain development and neuronal migration [3][4][5]. In contrast to other members of the Cdk family, Cdk5 is activated by binding the neuron-specific noncyclin molecules, p35 or p39 [6][7][8][9]. Mice lacking p35 are viable and fertile but show lamination defects in the cerebral cortex and mild disruption in the hippocampus and cerebellum [10], whereas mice deficient in Cdk5 die perinatally and show severe and widespread defects in neuronal migration [3][4][5]11]. p35/ Cdk5 kinase activity promotes neurite growth and phosphorylates a wide variety of substrates [12]. Deregulation of Cdk5 activity by proteolytic conversion of p35 to p25 has been implicated in neurodegenerative diseases [13,14].Computer modeling and mutagenesis studies have predicted that p35 adopts a cyclin-like tertiary structure [15][16][17]. Although, to produce full activity, in addition to cyclin binding most members of the Cdk family require phosphorylation of an intramolecular domain called the T-loop by another kinase [18]. Cdk5 differs in that full activity can be achieved by binding to p35 in the absence of T-loop phosphorylation [17,19].The p35 activation domain was mapped to the region of amino acid residues 150-291 [16,17]. More recently Amin et al. found that residues 138-291 constitute the smallest fragment (p16) of p35 that fully activates Cdk5 [20] ( Fig 1A). That study found that further truncation of p16, removing either the N-terminal 11 residues (part of the p35 aNT helix, Fig. 1A) or the C-terminal four residues of p16 (the p35 a7 helix, Fig. 1A), produces peptides that bind to Cdk5 with moderate affinity and do not activate it in vitro, but instead competitively inhibit. Remarkably, the peptide that remai...

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by A␤ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or A␤ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys 245 -Ala 277 . p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced A␤(1-42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.The activity of Cdk5, a multifunctional serine/threonine kinase, is critical for neuronal development and synaptic activity; it sustains neurite outgrowth, neuronal migration, cortical lamination, and survival (1-9). Its activity depends on the binding of its neuron-specific, cyclin-related activators, p35 and p39 (10, 11). Cdk5 has also been implicated as a key player in learning and memory (12-15).Normally, Cdk5 activity is tightly regulated, but under conditions of neuronal stress, it is deregulated, leading to hyperactivity, neuronal pathology, and cell death. Accordingly, Cdk5 has been implicated in certain neurodegenerative disorders, such as AD.2 A model of the role of Cdk5 in neurodegeneration suggests that a stress-induced influx of calcium ions into neurons activates calpain, a Ca 2ϩ -activated protease, which cleaves p35 into p25 and a p10 fragment. p25, in turn, forms a more stable Cdk5-p25 hyperactive complex, which hyperphosphorylates Tau and induces cell death (16 -21). Indeed, increased levels of p25 and Cdk5 activity have been reported in AD brains. The finding that p25 may be toxic comes from studies of cortical neurons treated with ␤-amyloid (A␤), a key marker of AD pathology, where p35 is converted to p25 accompanied by activated Cdk5, Tau hyperphosphorylation, and apoptosis (22,23).Expression of the Cdk5-p25 complex seems to be primarily responsible for the Tau pathology ...

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system, where it is involved in neuronal migration, synaptic transmission, and survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates. To explore this possibility, we used a yeast two-hybrid screen of a human cDNA library with p35 as bait and isolated human septin 5 (SEPT5), known also as hCDCrel-1, as an interacting clone. Here we report that p35 associates with SEPT5 in GST (glutathione S-transferase)-pull-down and coimmunoprecipitation assays. We confirmed that Cdk5/p35 phosphorylates SEPT5 in vitro and in vivo and identified S327 of SEPT5 as a major phosphorylation site. A serine (S)-to-alanine (A) 327 mutant of SEPT5 bound syntaxin more efficiently than SEPT5 wild type. Additionally, coimmunoprecipitation from synaptic vesicle fractions and Cdk5 wild-type and knock-out lysates showed that phosphorylation of septin 5 by Cdk5/p35 decreases its binding to syntaxin-1. Moreover, mutant nonphosphorylated SEPT5 potentiated regulated exocytosis more than the wild type when each was expressed in PC12 cells. These data suggest that Cdk5 phosphorylation of human septin SEPT5 at S327 plays a role in modulating exocytotic secretion.

Cyclin-dependent kinase 5 (cdk5), in contrast to other members of the cyclin-dependent kinase family, is not activated by cyclins but instead is activated by complexing with neuron-specific activator molecules (p35, p39, and p67). The most effective activator of cdk5 both in vitro and in vivo is p35. We have taken a kinetic approach to study the interaction between p35, its various truncated forms, and cdk5 to understand better the mechanism of its activation. The cdk5 complexes formed with the truncated forms p25 and p21 produced similar maximum active kinase, whereas the cdk5 complexed with full-length p35 and a further truncated form spanning amino acid residues from 138 to 291, with approximate molecular weight of 16 kDa (p16), produced slightly less (80%) activation than p25. P16 was the smallest fragment of p35 that produced activation equal to or greater than that of full-length p35. By examination of further truncations of p16, we found that a small number of residues, 11 and 4 at the N- and C-termini, respectively, of p16, are essential for cdk5 activation. Further truncation, removing both essential N- and C-terminal domains, produces a peptide with markedly higher affinity for cdk5 compared with the peptides that retain either of these domains. Using these inactive truncated peptides as inhibitors, we examined the kinetics of activation. From these studies we conclude that activation involves at least three cdk5-interacting domains, one located at each end of p16 and at least one located in a central domain. The cdk5 activation process is slow: The second-order rate constant for p16 is about 1.2 microM(-1) hr(-1). On the basis of kinetic data, we suggest that cdk5 exists in two conformations. The inactive kinase conformation predominates in the absence of the activator. Activation occurs in two stages: a rapid and reversible interaction of cdk5 with its activator, which involves only one or two binding domains, followed by a slow stabilization of the active conformation as interaction with all three domains is achieved.

Under normal conditions, the proline-directed serine/threonine residues of neurofilament tail-domain repeats are exclusively phosphorylated in axons. In pathological conditions such as amyotrophic lateral sclerosis (ALS), motor neurons contain abnormal perikaryal accumulations of phosphorylated neurofilament proteins. The precise mechanisms for this compartment-specific phosphorylation of neurofilaments are not completely understood. Although localization of kinases and phosphatases is certainly implicated, another possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine residues in target proteins and isomerizes cis isomers to more stable trans configurations. In this study we show that Pin1 associates with phosphorylated neurofilament-H (p-NF-H) in neurons and is colocalized in ALS-affected spinal cord neuronal inclusions. To mimic the pathology of neurodegeneration, we studied glutamate-stressed neurons that displayed increased p-NF-H in perikaryal accumulations that colocalized with Pin1 and led to cell death. Both effects were reduced upon inhibition of Pin1 activity by the use of an inhibitor juglone and down-regulating Pin1 levels through the use of Pin1 small interfering RNA. Thus, isomerization of lys-ser-pro repeat residues that are abundant in NF-H tail domains by Pin1 can regulate NF-H phosphorylation, which suggests that Pin1 inhibition may be an attractive therapeutic target to reduce pathological accumulations of p-NF-H.

O-METHYLATION has been shown to be the principal pathway for the metabolism of administered adrenaline and noradrenaline (AXELROD, INSCOE, SENOH and WITKOP, 1958; LABROSE, AXELROD and K m , 1958). An enzyme, catechol-0-methyl transferase (COMT),? which catalyses the transfer of a methyl group from S-adenosylmethionine to the 3-hydroxy group of catechois, has been described (AXELROD 1957; AXFLROD and TOMCHICK, 1958). Since the products, metanephrine (3-0-methyladrenaline) and normetanephrine (3-O-methylnoradrenaline), have negligible physiologd activity (EVARTS et a/., 1958), this enzyme may be of major importance in the inactivation of endogenously released catecholamine hormones. The functional role of catecholamines in the autonomic nervous system and the relatively high concentration of these amines in certain regions of the central nervous system (VOGT, 1954) suggested a study of the distribution of COMT in the nervous system and certain organs. METHODS A N D MATERIALS Prepation of tissues. Tissues were obtained from adult monkeys (Macaca mulatt), which were killed by exsanguination under pentobarbital anaesthesia. The tissues were dissected immediately Axaua, J. .ad Touoooc R (19S8)J. W. C k m . 233,702. Axumm J. .nd TOMM~CK R. (unpublbhcd obemtiom). B u m G. L 4 Gnzen16 S. S. (1957) J. Physid. 138, 81. EVANS E. V.. Giusspre L.. FLFMIW T. E. and ~D S M A. (1958) Proc. Sor. exp. Bid., N. r.