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

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 Ser202/Thr205 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 Ser202/Thr205. Considering the fact that phosphorylation of Ser202/Thr205 antagonizes the tau-mediated nucleation of tubulin, p25-CDK5 may play a pivotal role in neuronal cell death in Alzheimer's disease. 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 Ser202/Thr205 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 Ser202/Thr205. Considering the fact that phosphorylation of Ser202/Thr205 antagonizes the tau-mediated nucleation of tubulin, p25-CDK5 may play a pivotal role in neuronal cell death in Alzheimer's disease. Aberrant phosphorylation of the tau protein is considered to play a decisive role in the pathogenesis of neurodegenerative disorders collectively called tauopathies (1Lee V.M. Goedert M. Trojanowski J.Q. Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2087) Google Scholar). Tau is a neuron-specific, microtubule-associated protein that plays major roles in the assembly and stabilization of microtubules. In the brains of patients with Alzheimer's disease, abnormally phosphorylated tau protein, paired helical filament (PHF) 1The abbreviations used are: PHF, paired helical filament; CDK5, cyclin-dependent kinase-5; PIPES, 1,4-piperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid 1The abbreviations used are: PHF, paired helical filament; CDK5, cyclin-dependent kinase-5; PIPES, 1,4-piperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acidtau, becomes dissociated from neuronal microtubules and accumulates in PHFs, the filamentous network generated by self-aggregation of hyperphosphorylated forms of tau. All six adult isoforms of tau are known to be hyperphosphorylated in PHFs (2Luc B. Thierry B. Valrie B.S. Andre D. Patrick R.H. Brain Res. Rev. 2000; 33: 95-130Crossref PubMed Scopus (1499) Google Scholar). Several lines of evidence suggest that proline-directed serine/threonine kinases, together with protein phosphatases, play an important role in the hyperphosphorylation of tau (3Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Titani K. Ihara Y. J. Biol. Chem. 1995; 270: 823-829Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). Candidate protein kinases include mitogen-activated protein kinase, glycogen synthase kinase-3β, and cyclin-dependent kinase-5 (CDK5). None of these enzymes can generate PHF-tau alone (4Zheng-Fischhofer Q. Biernat J. Mandelkow E.M. Illenberger S. Godemann R. Mandelkow E. Eur. J. Biochem. 1998; 252: 542-552Crossref PubMed Scopus (289) Google Scholar). CDK5, initially known as brain proline-directed protein kinase or neuronal cdc2-like protein kinase, phosphorylates tau at a high stoichiometry (5Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar, 6Paudel H.K J. Biol. Chem. 1997; 272: 28328-28334Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Unlike other tau kinases, the activity of CDK5 is regulated by endogenous regulatory proteins, p35 and p39. In this respect, CDK5 is distinctly different from other tau kinases (7Maccioni R.B. Otth C. Concha I.I Munoz J.P. Eur. J. Biochem. 2001; 268: 1518-1527Crossref PubMed Scopus (158) Google Scholar). Recently, calpain, a calcium-dependent protease, was found to digest both p35 and p39 to their truncated products, p25 and p29, respectively (8Patrick G.N. Zukerberg L. Nikolic M., De La Monte S. Dikkes P. Tsai L.H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1297) Google Scholar, 9Kusakawa G. Saito T. Onuki R. Ishiguro K. Kishimoto T. Hisanaga S. J. Biol. Chem. 2000; 275: 17166-17172Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). Furthermore, truncation of p35 to p25 and the subsequent formation of the p25-CDK5 complex result in neuronal cell death and hyperphosphorylation of tau (10Lee M.S. Kwon Y.T., LI, M. Peng J. Friedlander R.M. Tsai L.H. Nature. 2000; 405: 360-364Crossref PubMed Scopus (896) Google Scholar). From these findings, the following hypothesis emerged. Initially, various stress signals activate calpain by recruiting intracellular Ca2+, and then the activated calpain digests p35 to p25. Finally, the p25-CDK5 complex hyperphosphorylates tau proteins, causing disruption of microtubule integrity and inevitable cell death (11Smith D.S. Greer P.L. Tsai L.H. Cell Growth & Differ. 2001; 12: 277-283PubMed Google Scholar). Several criticisms of this hypothesis have appeared. Among them, the observed accumulation of p25 in the brains of patients with Alzheimer's disease may be accounted for by post-mortem degradation of p35 (12Taniguchi S. Fujita Y. Hayashi S. Kakita A. Takahashi H. Murayama S. Saido T.C. Hisanaga S. Iwatsubo T. Hasegawa M. FEBS Lett. 2001; 489: 46-50Crossref PubMed Scopus (129) Google Scholar). In fact, p35 is notoriously unstable. Most brain-derived p35 is lost or truncated to p25 during purification. Thus, the kinetic activity of tau phosphorylation by p35-CDK5 is only poorly understood. Lack of knowledge about the regulation of CDK5 by its activators, p35 and p25, severely limits our understanding of the possible pathogenesis induced by CDK5. We have examined the kinetic characteristics ofin vitro phosphorylation of the longest isoform of human tau by CDK5 using recombinant proteins. This study clarified not only the kinetics of overall phosphorylation, but also the details of site-specific phosphorylation of human tau by CDK5 complexes. Bacterially expressed tau, the longest isoform of human tau protein, was purified as described previously (13Sobue K. Agarwal-Mawal A., Li, W. Sun W. Miura Y. Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) with minor modifications. After addition of NaCl to 0.5 m, the bacterial extract was boiled in a hot bath for 10 min and centrifuged at 8000 × g. After dialysis in extraction buffer A (50 mm PIPES, pH 6.8, 1 mm EDTA, 1 mm dithiothreitol, and 0.2 mm phenylmethylsulfonyl fluoride), the supernatant was loaded onto a phosphocellulose P-11 column (Whatman) pre-cycled with 0.5 m NaOH and then equilibrated with extraction buffer A. Recombinant tau bound on the column was eluted with a linear gradient of 0–0.5 m NaCl in extraction buffer A. The pooled fractions containing tau were concentrated with Centriprep centrifugal filter devices (Millipore Corp.) and dialyzed against extraction buffer A. After determination by Coomassie protein assay reagent (Pierce) using bovine serum albumin as the standard, tau was stored frozen at −80 °C until used. The cDNA for human CDK5 in pCMV was excised with BamHI and ligated into the BamHI site of a baculovirus transfer vector, BacPAK9 (Clontech, Palo Alto, CA). C-terminally His6-tagged p35 was generated by PCR amplification using pET23a-p35 as the template and oligonucleotides 5′-GGCGAATTCATGGGCACGGTGCTGTCCCT-3′ and 5′-GGGCTTTAGCGGCCGCCGGATCTCAGTG-3′ as the primers. The PCR product was cleaved with EcoRI and NotI and ligated into BacPAK9. His6-tagged p25, a deletion of amino acids 1–98 of His6-tagged p35 in BacPAK9, was generated by PCR-based mutagenesis. BacPAK9 containing CDK5, p35, or p25 cDNA was cotransfected with BacPAK6 virus DNA (Clontech) into Sf9 cells following the manufacturer's instructions. Human CDK5 stock virus was coexpressed with either p35-His6 or p25-His6 virus in Sf9 cells. The Sf9 cell-expressed human recombinant CDK5 complexes were harvested with cold phosphate-buffered saline and centrifuged at 1600 × g for 3 min at 4 °C, and the pellet was frozen in liquid nitrogen. The frozen pellet was thawed at 30 °C, resuspended in cold extraction buffer B (10 mm Tris-HCl, pH 8.0, 0.5 m NaCl, 1% Triton X-100, 10 mmimidazole, 10 mm β-mercaptoethanol, 0.2 mmPefabloc SC, 1 μm E-64, and 10 μg/ml leupeptin), and sonicated three times for 10 s each at 4 °C. The cell suspension was centrifuged at 19,000 × g for 15 min at 4 °C. The supernatant was transferred onto a nickel-nitrilotriacetic acid-agarose column that was prewashed with extraction buffer B and then with wash buffer (10 mm Tris-HCl, pH 7.5, 0.15m NaCl, and 5 mm imidazole) at 4 °C. The p35-CDK5 or p25-CDK5 complex bound on the column was eluted with 10 mm Tris-HCl, pH 7.5, 0.15 m NaCl, and 350 mm imidazole. The pooled fractions containing p35-CDK5 or p25-CDK5 were dialyzed against dialysis buffer (10 mm MOPS, pH 6.8, 5 mm MgCl2, 1 mmdithiothreitol, 100 mm KCl, and 10% glycerol). The concentrations of recombinant CDK5 complexes were determined using the Coomassie protein assay kit (Pierce) with bovine serum albumin as the standard. The purity of the purified human recombinant p35-CDK5 and p25-CDK5 complexes was assessed by double immunostaining with an anti-p35/p25 polyclonal antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-CDK5 polyclonal antibody (C-8; Santa Cruz Biotechnology) at 1:1000 and 1:2000 dilutions, respectively (Fig.1). The kinase activities of the p35-CDK5 and p25-CDK5 complexes were determined as described (14Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), except that the p35-CDK5 and p25-CDK5 complexes were used at a concentration of 15 μg/ml with recombinant tau or histone H1. The kinase assay was initiated by adding the p35-CDK5 or p25-CDK5 complex to the reaction mixtures, and then 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. For the time course analysis,32Pi incorporation into tau or histone H1 was quantified by Cerenkov using a liquid The tau bands were also for A reaction was with the assay mixture containing bovine serum albumin of tau or histone H1. Tau was with the p35-CDK5 or p25-CDK5 complex and 1 mm for at 30 °C. For phosphorylation after by the CDK5 from mixtures were transferred to new and then with p35-CDK5 or p25-CDK5 complex (10 and for at 30 °C. The were by adding sample buffer to the assay mixtures and by Recombinant tau was phosphorylated by the p35-CDK5 or p25-CDK5 complex for a time at 30 °C, and then phosphorylated tau was separated by of Tau The bands containing tau were excised from the three times with for 30 and then with 10% at The tau bands were at °C and with μg/ml in mm pH at 30 °C. The extract was transferred to a new tube and with a were with 100 of After centrifugal the of the was quantified by Cerenkov The was in buffer acid pH aliquot of the containing of was to mapping using The of was in pH at 10 the of was in The were with a The on the separated was then of the and in The of each was quantified by Cerenkov antibody was from and an tau antibody to as the was from was from A antibody was from Santa Cruz were as described (14Hashiguchi M. Sobue K. Paudel H.K. J. Biol. Chem. 2000; 275: 25247-25254Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). tau was separated by and transferred to The was with one of the and then with buffer mm Tris-HCl, pH 100 mm and for 30 min at °C. The was with a different We used antibody the and at and 0.5 μg/ml dilutions, The of tau against was to the of loaded not All were by of and were considered significantly different A concentration of of recombinant tau was in the kinase assay mixture with the p25-CDK5 or p35-CDK5 of the assay mixture were withdrawn at and After of tau by incorporation into tau was using a A result is in of into tau by the p35-CDK5 complex was found to be In the of incorporation into tau was much it was with the p25-CDK5 a the time of the overall phosphorylation be In the in the time of incorporation by p35-CDK5 and p25-CDK5 complexes were and Thus, phosphorylation by the p35-CDK5 complex was times that by the p25-CDK5 phosphorylation of bovine serum albumin was incubation with the CDK5 complexes not of we found that and of were into 1 of tau by the p25-CDK5 and p35-CDK5 respectively Thus, the p25-CDK5 complex at three different of tau. In at be phosphorylated by the p35-CDK5 The results from are in and stoichiometry of phosphorylation of tau and histone H1 by p35-CDK5 or was from at in a new was from at Although tau of phosphorylation, overall incorporation by CDK5 was found to be described by the The kinetic m and were Considering the the of phosphorylation of tau by incorporation with the p35-CDK5 or p25-CDK5 complex was determined after a and reaction the was determined using of during different reaction by both The of phosphorylation as of of was determined incorporation at three different substrate The results were in a The for phosphorylation of tau by the CDK5 complex was the with either p35 or p25 that the affinity of tau for the CDK5 complex was not by the regulatory of of phosphorylation of tau and histone H1 by p35-CDK5 or was from in a new was from the for tau phosphorylation by p25-CDK5 was significantly that by p35-CDK5 from m an of the p25-CDK5 complex was found to be times more in into tau. The of overall phosphorylation of tau by a CDK5 complex was examined using a The of the reaction with the p35-CDK5 and p25-CDK5 complexes were and These or and the was not the phosphorylation of histone a but was as not The time of incorporation by p35-CDK5 and by p25-CDK5 were 10 and of histone H1 by the p35-CDK5 complex was that by the p25-CDK5 The kinetic of histone H1 phosphorylation were determined the characteristics of phosphorylation of each or of tau, mapping of digests was and of phosphorylation were to Considering the fact that the p25-CDK5 complex into tau as as the p35-CDK5 kinase was after 0.5 and 1 for the p25-CDK5 and p35-CDK5 The was to be of the time of each CDK5 complex that of the were phosphorylated in each reaction time of or was to of the A of mapping is in The major and minor after of phosphorylation and In to the of the other the of 1 after 1 of reaction was in the presence of either p35 or p25 A and that 1 was phosphorylated more the other In fact, the of 1 after of reaction with the p25-CDK5 complex was with of the other the of 1 after of reaction with the p35-CDK5 complex was The of the known to be phosphorylated by CDK5 complexes were by the described by P. T. PubMed Scopus Google Scholar). A the observed and revealed that 1 to the phosphorylated Ser202/Thr205 and 3 to the phosphorylated and the phosphorylated 4 and 5 to be from and the phosphorylation of the major incorporation into each was After of the using the each was The on each was then from the and was using a 4 a of 1 was After of the p25-CDK5 complex was found to Ser202/Thr205 to to a In phosphorylation of Ser202/Thr205 by the p35-CDK5 complex was with the other after of the of 1 of the of incorporation into the major The phosphorylation of to alone was found to be much the double phosphorylation of Ser202/Thr205 The p35-CDK5 complex incorporation into a in incorporation into was after These results that the phosphorylation of or is with the overall phosphorylation Furthermore, double phosphorylation of may be a the incorporation into tau by the CDK5 we mapping after incubation with In the in tau was phosphorylated with for a to of of the of and then was Thus, incorporation into tau after was by mapping After an incubation of tau with the p25-CDK5 addition of with p25-CDK5 complex in a in the of The of the only Thus, double phosphorylation of and was not by the p25-CDK5 phosphorylation of was In addition of the p35-CDK5 complex after by the p25-CDK5 complex to the of not Tau was also with the p35-CDK5 complex for with incorporation was then by addition of together with the p25-CDK5 In this both 1 and that was by the p35-CDK5 phosphorylation of tau was using an of to were withdrawn from the reaction and were separated by The were transferred to a that was then with the was and with other was 3 after incubation with the p25-CDK5 during the subsequent incubation After of was by the p25-CDK5 In only a was with the p35-CDK5 complex after of incubation The p25-CDK5 complex a much higher of phosphorylation on the with the p35-CDK5 of Ser202/Thr205 was also by using the After min of incubation with the p25-CDK5 the a in and then the to The 1 after incubation with the p25-CDK5 that The in after incubation with p35-CDK5 was the as for the p25-CDK5 but was the of the to and then The was with the an antibody against phosphorylated tau was phosphorylated by the p35-CDK5 the antibody after 3 of with an of a in and then it of after the p25-CDK5 complex induced a in the of the during the incubation phosphorylation the of the antibody a in the of the antibody after to the p25-CDK5 complex was not with a Thus, the observed in the antibody not result from of the This study determined the kinetic characteristics of in vitro phosphorylation of tau by CDK5 complexes using recombinant proteins. The p25-CDK5 complex was found to tau much the p35-CDK5 the that the time course of phosphorylation can be by a the time of the in was and the results suggest that p25 can the activity of CDK5 by with We determined the stoichiometry of of of tau phosphorylation by CDK5 and found that the p25-CDK5 complex can tau Furthermore, it can more into tau with the p35-CDK5 The stoichiometry of tau phosphorylation by neuronal cdc2-like protein kinase p25-CDK5 was to be of of tau (5Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar). Thus, the recombinant p25-CDK5 complex used in the study is as as the CDK5 Q. K. FEBS Lett. 1998; PubMed Scopus Google Scholar) in of tau at by three different kinases protein kinase protein kinase and and found that into tau by of the three enzymes after 100 min of of overall phosphorylation, protein kinase protein kinase and kinase of of tau. kinase from phosphorylated tau to a stoichiometry of of of tau H.K. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). with these the stoichiometry of overall phosphorylation of tau by the p25-CDK5 complex is the phosphorylation of tau by CDK5 kinetic were determined using the The m for tau phosphorylation by the p35-CDK5 complex were μm and The m for the p25-CDK5 complex was μm and was from that for the p35-CDK5 complex for the p25-CDK5 complex was and was significantly that for the p35-CDK5 m for the p25-CDK5 complex was times that for the p35-CDK5 The kinetic of CDK5 complexes were also determined using histone H1 as a The observed m for the p35-CDK5 complex was in with results P. M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The in the m for the p25-CDK5 and p35-CDK5 complexes was not m for the p25-CDK5 complex was significantly that for the p25-CDK5 Thus, the p25-CDK5 complex is a much more kinase, and the p25 regulatory the of CDK5 its affinity for tau. For tau phosphorylation by kinase, m and were μm and that kinase has a affinity for tau and more of activity with CDK5 complexes H.K. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the stoichiometry of phosphorylation of tau by the p25-CDK5 complex is that for the other tau kinases, the p35-CDK5 a tau kinase, p25-CDK5 to the high phosphorylation stoichiometry of of the phosphorylation of tau revealed a in the activity of CDK5 site-specific phosphorylation be clarified for a understanding of CDK5. We have mapping of the digest of tau and with the for the and of phosphorylation, we found that into 1 was In incorporation into the other was that the time course of overall phosphorylation of tau on the phosphorylation kinetics of and We examined this of site-specific phosphorylation of and We have the following The p35-CDK5 complex incorporation into 1 and the p25-CDK5 complex incorporation into 1 more but only after These results the of phosphorylation of after we mapping after possible phosphorylation with was as an on the After with the p25-CDK5 p25-CDK5 incorporation into 1 incorporation into of the major not These suggest that phosphorylation of was and that only was phosphorylated the time of In the p35-CDK5 complex to both 1 and as by the on the The also that phosphorylation by CDK5 complexes only at the time of phosphorylation of and of tau was The three have with tau only and are phosphorylation of of the residues is not for M. R. E. Neurosci. Lett. 1995; PubMed Scopus Google Scholar, Illenberger S. Mandelkow E.M. Biol. 1995; PubMed Scopus Google Scholar). Thus, is in phosphorylation of Ser202/Thr205 for proline-directed kinases, CDK5 complexes. In an V.M. L. J. Neurosci. Res. 1993; PubMed Scopus Google Scholar). In the study, with recombinant tau phosphorylation of or of The antibody is against phosphorylated of human tau. was the to that and be phosphorylated or be phosphorylated The phosphorylated of the to as as the Although phosphorylation of as a of phosphorylation be the phosphorylation of and the subsequent phosphorylation of to be the to double of the on hyperphosphorylated tau in the brains of Alzheimer's disease patients were to be generated by phosphorylation by different tau For phosphorylation of a amino acid by CDK5 is a for the subsequent kinase of glycogen synthase A., Q. K. T. Biochem. 1997; PubMed Scopus Google Scholar, A. R. A. R.B. FEBS Lett. 1999; PubMed Scopus Google Scholar). of by glycogen synthase is known to protein kinase A on (4Zheng-Fischhofer Q. Biernat J. Mandelkow E.M. Illenberger S. Godemann R. Mandelkow E. Eur. J. Biochem. 1998; 252: 542-552Crossref PubMed Scopus (289) Google Scholar). tau kinases A. C. R. P. J. 1999; PubMed Scopus Google Scholar) it to the of abnormal phosphorylation of tau. We have evidence for the phosphorylation of and of tau by the p25-CDK5 of only after was a phosphorylation of was with phosphorylation of other as and In the p35-CDK5 complex or kinase on suggest that cleavage of p35 to p25 regulates not only the overall kinase activity of CDK5, but also the phosphorylation of and The of CDK5 with a p35 or p25, a of kinase We the of H. K. We L. H. Tsai for human CDK5 cDNA and M. Goedert for the of human tau of CDK5 p35 phosphorylation of Ser202/Thr205 of human of 5 is in PDF