Truncation of CDK5 Activator p35 Induces Intensive Phosphorylation of Ser202/Thr205 of Human Tau

Mitsuko Hashiguchi,Taro Saito,Shin-Ichi Hisanaga,Toshio Hashiguchi

doi:10.1074/jbc.m207426200

Abstract

Hyperphosphorylated tau is a major component of neurofibrillary tangles, one of the hallmarks of Alzheimer's disease. CDK5 is a kinase that phosphorylates the tau protein, and its endogenous activator, p35, regulates its activity. Recently, calpain was found to digest p35 to its truncated product, p25. Several lines of evidence suggest that p25-CDK5 has much more powerful kinase activity and that it may cause abnormal hyperphosphorylation of tau. In this study, we have examined the kinetic characteristics of in vitro phosphorylation of the longest isoform of human tau by CDK5 and its activators using recombinant proteins. Although the kinase activity of CDK5 in phosphorylating tau was significantly higher in the presence of p25, the affinity of CDK5 for tau was not different. Phosphopeptide mapping revealed enhanced phosphorylation of Ser(202)/Thr(205) residues by p25-CDK5 (amino acid residues of tau are numbered according to the longest isoform of human tau). These results suggest that cleavage of p35 to p25 greatly enhances the kinase activity of CDK5 and increases the phosphorylation of Ser(202)/Thr(205). Considering the fact that phosphorylation of Ser(202)/Thr(205) antagonizes the tau-mediated nucleation of tubulin, p25-CDK5 may play a pivotal role in neuronal cell death in Alzheimer's disease.

Highlights

Hyperphosphorylated tau is a major component of neurofibrillary tangles, one of the hallmarks of Alzheimer’s disease
We have examined the kinetic characteristics of in vitro phosphorylation of the longest isoform of human tau by CDK5 and its activators using recombinant proteins
These results suggest that cleavage of p35 to p25 greatly enhances the kinase activity of CDK5 and increases the phosphorylation of Ser202/Thr205

Summary

EXPERIMENTAL PROCEDURES

Purification of Human Recombinant Tau—Bacterially expressed tau, the longest isoform of human tau protein, was purified as described previously [13] with minor modifications. The kinase assay was initiated by adding the p35-CDK5 or p25-CDK5 complex to the reaction mixtures, and an aliquot of the reaction mixture was withdrawn and placed into a new tube containing SDS sample buffer to terminate the reaction after various incubation times. The assay mixtures were separated by SDS-PAGE, and the bands containing phosphorylated substrate were excised from the gel. Tryptic Phosphopeptide Mapping of Recombinant Tau—Recombinant tau was phosphorylated by the p35-CDK5 or p25-CDK5 complex for a particular time period at 30 °C, and phosphorylated tau was separated by SDS-PAGE (see “In Vitro Kinetic Analysis of Tau Phosphorylation”). The blotted membrane was immunostained with one of the anti-tau antibodies and stripped with stripping buffer (62.5 mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS) for 30 min at 50 °C. Statistics—All data were analyzed by analysis of variance (SYSTAT Version 4, SYSTAT, Evanston, IL), and values were considered significantly different when p Ͻ 0.05

RESULTS

DISCUSSION

Additions and Corrections