Phosphorylation of FTDP-17 Mutant tau by Cyclin-dependent Kinase 5 Complexed with p35, p25, or p39

Abstract

Neurofibrillary tangles are one of the major pathological hallmarks of Alzheimer disease (AD).1 Neurofibrillary tangles are bundles of paired helical filament composed of the microtubule (MT)-associated protein tau in a hyperphosphorylated state (1, 2). Intracellular inclusions made of tau are also found in several other neurodegenerative diseases, including Pick disease, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), collectively called tauopathies (3, 4). Exonic and intronic mutations in the tau gene have recently been identified in familial FTDP-17 (5-7), indicating that dysfunction of the tau protein can cause the above mentioned neurodegenerative diseases. Many of the exonic mutations reduce the ability of tau to promote MT assembly (8 -10), but why these mutations cause the formation of tau inclusions is unknown.The tau protein in inclusions is hyperphosphorylated. Around 25 phosphorylation sites have been identified in paired helical filament tau from AD brains (11-13). Characteristic phosphorylation sites are Ser or Thr residues followed by Pro that are phosphorylated by the proline-directed protein kinase activities of extracellular signal-regulated kinase, glycogen synthase kinase 3␤ (GSK3␤), and cyclin-dependent kinase 5 (Cdk5). Phosphorylation reduces the ability of tau to bind to and polymerize MTs, resulting in an increase in the soluble form of tau dissociated from MTs. However, it is unclear how phosphorylated soluble tau assembles into filamentous aggregates of neurofibrillary tangles and then induces neurodegeneration. Elucidating the relationship between tau phosphorylation and aggregate formation is critical to our understanding of the pathogenesis. Phosphorylation of FTDP-17 mutant tau has been studied mainly with GSK3␤ or in non-neuronal cultured cells (14 -17). Mutant tau proteins were not phosphorylated more than wild-type (WT) tau either in transfected cultured cells or in vitro. Among them, R406W mutant tau showed significantly reduced phosphorylation in those experiments. However, both mutant tau and wildtype tau deposited in FTDP-17 brains are also hyperphosphorylated (18,19). It is important to resolve the discrepancy between the reduced phosphorylation of mutant tau in vitro and in cultured neurons and the high phosphorylation of mutant tau in pathological brains.Cdk5 is a proline-directed Ser/Thr kinase activated by a p35 or p39 Cdk5 activator (20 -22). Cdk5 activity is primarily detected in differentiated neurons because p35 and p39 show limited expression in neurons. As described above, Cdk5 is one of the tau protein kinases that phosphorylate tau in living neurons and is also able to generate several paired helical filament epitopes of tau in AD (23,24). However, the phosphorylation of FTDP-17 mutant tau by Cdk5 has not yet been examined. This question should be addressed because the involvement of Cdk5 in the pathogenic phosphorylation of tau has recently become more evident (25,26). In ...

Suppression of Mutant Huntingtin Aggregate Formation by Cdk5/p35 through the Effect on Microtubule Stability

“…p35 is cleaved to p25 by calpain to generate Cdk5/p25, which has increased kinase activity (Patrick et al, 1999;Hisanaga and Saito, 2003;Sakaue et al, 2005). We observed a similar inhibitory effect of Cdk5/p25 on inclusions (Fig.…”

“…The binding of Cdk5 or p35 with tNhtt-poly(Q)-EGFP was examined by immunoblotting with anti-Cdk5 or anti-p35 antibody after immunoprecipitation of tNhtt-poly(Q)-EGFP from COS-7 cells with anti-GFP antibody . The longer 90Q form was phosphorylated by Cdk5/p25 purified from Sf9 cells in MOPS (3-morpholinepropanesulfonic acid) buffer containing 0.5% Nonidet P-40 and [␥-32 P]ATP (0.1 mM) for 30 min at 35°C (Sakaue et al, 2005). Phosphorylation was estimated by a FLA7000 Image Analyzer (Fuji Film) after SDS-PAGE.…”

“…pFLAG-dynamitin was provided by Dr. Mitsuo Tagaya (Tokyo University of Pharmacy and Life Science, Tokyo, Japan) (Hirose et al, 2004). Atrophin-1-EGFP with 19Q or 81Q (Yamada et al, 2001), pCMV-dominant-negative Cdk5 (K33T or D144N) (Nikolic et al, 1996;Saito et al, 2003), and pSG5-tau (Sakaue et al, 2005) have been described previously.…”

“…Cdk5-positive cells were visualized by immunostaining with antiCdk5 (DC17) antibody. Anti-␤-tubulin staining was performed as described previously (Sakaue et al, 2005).…”

No difference in kinetics of tau or histone phosphorylation by CDK5/p25 versus CDK5/p35 in vitro

“…This is consistent with early work by Kusakawa et al,. who measured the activity of immunoprecipitates of these enzymes (46), and Sakaue et al, who tested both enzymes against several FTDP-17 mutants of tau (47). Recently, Maestras et al (48) reported that CDK5/p25 is significantly more active than CDK5/p35 toward the anaphase-promoting complex factor, cdh1, suggesting that differential enzyme activity may be substrate-dependent.…”