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...