The paired helical filament (PHF), which comprises the major tibrous clement of the neurofibrillary tan@ of Aleheimcr's disease, is composed of abnormally phosphorylated microtubule-associated protein tau. Hcrc we show that p42 MAP kinasc phosphorylatcs recombinant tau and converts it to a form which is similar to PHF tau. Of the major scrinelthrconinc protein phosphatases found in mammalian tissues only protein phosphatase 2A (PP2A) could dephosphutylatc tau phosphorylated in this manner, with PP2A, being the most effective form of the enrymc.Alzhcimer's disease; Microtubulc-associated protein tau; Mitogcn-activated protein kinase; Protein phosphatasc 2A 1, INTRODUCTIONAbundant senile plaques and neurofibrillary tangles constitute the major pathological characteristics of Alzheimer's disease. Neurofibrillary tangles appear within the vast majority of nerve cells that degenerate during the course of the disease, where their presence is indicative of dementia (reviewed in [I]). Paired helical filaments (PHFs), which are composed of the microtubuleassociated protein tau, form the principal fibrous component of the neurofibrillary tangle [2][3][4]. In human adult brain, tau comprises six isoforms of 48-67 kDa apparent molecular mass [5], while tau isolated from Alzheimer PHFs runs as three bands of 60, 64 and 68 kDa apparent molecular mass [2,6]. After dephosphorylation with alkaline phosphatasc these bands align with the six rau isoforms expressed in E. cofi, indicating that PHF tau consists of all six isoforms in an abnormally phosphorylatcd state [7]. Abnormal phosphorylation probably produces a change in tau that favours self-association over microtubule binding, resulting in the formation of PHFs.Several sites that are abnormally phosphorylated in PHF tau have been identified through the use of phosphorylation-dependent antibodies which recognize PHF tau but do not recognize normal adult brain tau.Correspondence urldresx M. Goedert, MBC Laboratory oiIvioieouliir Biology, Hills Road, Cambridge, CB2 2QH, UK. Fax: (44) (223) 41 2282. Thus, antiserum T3P recognizes phosphoserine-396 [2] and antibody AT8 recognizes phosphoserine-I99 an& or phosphoserine-202 [S] (using the numbering of the largest human brain tau isoform [S]). Each of these serines is followed by a proline, suggesting that protein kinases or protein phosphatases with specificity for phosphoseryi-proline may be involved. Recently, a protein kinase with the characteristics of a mitogen-activated protein (MAP) kinase was purified from pig brain and shown to phosphorylate recombinant tr.u to en Alzheitner-like state, as evidenced by a reduction i;r gel mobility and the phosphorylation of serine/threonineproline sites [9]. Moreover, two brain protein kinases (PK36 and PK40) phosphorylate tau at serine-396 and other serine/threonine-proline sites [IO] and a kinase called tau protein kinase I also phosphorylates tau at some scrine/threonine-proline sites [l I]. The relationships, if any, of these protein kinases to MAP kinase are not clear. Nothing is kno...

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A platform for specifically modulating kinase-dependent signaling using peptides derived from the catalytic domain of the kinase is presented. This technology, termed KinAce TM , utilizes the canonical structure of protein kinases. The targeted regions (subdomain V and subdomains IX and X) are analyzed and their sequence, threedimensional structure, and involvement in proteinprotein interaction are highlighted. Short myristoylated peptides were derived from the target regions of the tyrosine kinases c-Kit and Lyn and the serine/threonine kinases 3-phosphoinositide-dependent kinase-1 (PDK1) and Akt/protein kinase B (PKB). For each kinase an active designer peptide is shown to selectively inhibit the signaling of the kinase from which it is derived, and to inhibit cancer cell proliferation in the micromolar range. This technology emerges as an applicable tool for deriving sequence-based selective inhibitors for a broad range of protein kinases as hits that may be further developed into drugs. Moreover, it enables identification of novel kinase targets for selected therapeutic indications as demonstrated in the KinScreen application.Protein kinases are important drug targets for oncologic, immunologic, and metabolic disorders. The development of selective protein kinase inhibitors is widely considered a promising approach to drug development. The common strategy, which has led to the development of drugs, such as Glivec and Iressa (1), is to target the ATP binding site (1-3). Other approaches include substrate mimicking inhibitors (4 -9), bi-substrate analogs that target both the ATP and the acceptor binding sites (10), and molecules that target the Src homology 2 domain (11). The KinAce TM (12) approach presented in this article is based upon deriving short peptides from specific regions in the catalytic domain of the kinase that are implicated in kinase-substrate interactions. The KinAce TM peptides mimic regions of the kinase and therefore compete with the kinase for binding to the substrate (or to other modulators of the kinase), and subsequently abrogate the kinase-dependent signaling.Several other groups have used peptides to disrupt proteinprotein interactions and thus modulate kinase signaling. In contrast to our technology, which derives the peptides from the catalytic domain of the kinase, others have derived inhibitory peptides from the substrates (7), pseudosubstrate (13), regulators interacting with the kinase (14 -17), or from non-catalytic domains of the kinase participating in substrate binding (11,18). One of the unique characteristics of the KinAce TM technology is that the regions from which the inhibitory peptides are derived share conserved structural patterns in all kinases. Evidence from the literature supports a potential role for these regions in substrate binding (19 -22). Our technology suggests a general recipe for generating inhibitors of kinase-dependent signaling, applicable to any kinase. To enable permeation of peptides into cells researchers conjugated peptides to membrane penetrat...

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