Phosphorylation of Human Microtubule-Associated Protein Tau by Protein Kinases of the AGC Subfamily

FEBS Letters 581 (2007) 2657–2662

Phosphorylation of human microtubule-associated protein tau by protein kinases of the AGC subfamily

Kanwar Virdee, Hirotaka Yoshida, Sew Peak-Chew, Michel Goedert* MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK

Received 13 March 2007; revised 25 April 2007; accepted 4 May 2007

Available online 11 May 2007

Edited by Jesus Avila

the C-terminal region, and no, one or two inserts in the N-ter- Abstract Intraneuronal inclusions made of hyperphosphorylated microtubule-associated protein tau are a defining neuro- minal portion. The repeats constitute the microtubule-binding pathological characteristic of Alzheimer’s disease, and of domains of tau. Tau binds to tubulin through these repeats several other neurodegenerative disorders. Many phosphoryla- and promotes microtubule assembly [10,11]. In developing tion sites in tau are S/TP sites that flank the microtubule-binding brain, only the shortest isoform (three repeats and no amino- repeats. Others are KXGS motifs in the repeats. One site up- terminal inserts) is expressed [8,12]. Phosphorylation of tau stream of the repeats lies in a consensus sequence for AGC is developmentally regulated, in that tau from developing brain kinases. This site (S214) is believed to play an important role is phosphorylated more than tau from adult brain [13–16]. The in the events leading from normal, soluble to filamentous, insol- ability of tau to bind to and stabilise microtubules correlates uble tau. Here, we show that all AGC kinases tested phosphor- inversely with its phosphorylation [17], which may in turn ylated S214. RSK1 and p70 S6 kinase also phosphorylated the facilitate self-assembly. Protein chemical and immunochemical neighbouring T212, a TP site that conforms weakly to the AGC kinase consensus sequence. MSK1 phosphorylated S214, studies have identified more than 25 phosphorylation sites in as well as S262, a KXGS site in the first repeat, and S305 in filamentous tau, about half of which are serine and threonine the second repeat. residues followed by proline [18–20]. Consequently, glycogen Ó 2007 Federation of European Biochemical Societies. Published synthase kinase-3 (GSK3) [21,22], cyclin-dependent kinase 5 by Elsevier B.V. All rights reserved. (cdk5) [23,24], mitogen-activated protein (MAP) kinase [25,26] and stress-activated protein (SAP) kinases [27,28] have Keywords: Tau protein; Neurodegeneration; AGC kinases; been shown to phosphorylate tau protein at sites that are MSKs; MAP kinases hyperphosphorylated in the human tauopathies. Phosphoryla- tion at many of these sites occurs in a significant fraction of fetal tau and a small fraction of tau from adult human brain. However, some sites are phosphorylated in filamentous tau, but not in tau from normal adult human brain. 1. Introduction Phosphorylation at S422 is such an example [29]. This SP site can be phosphorylated by MAP kinase and SAP kinases Many neurodegenerative diseases are characterized by the in vitro [28,29]. Triple phosphorylation of T212, S214 and presence of abnormal filamentous protein inclusions in the T217, as detected by the phosphorylation-dependent anti-tau brain. In Alzheimer’s disease (AD), these inclusions are made antibody AT100, is another example [30]. T212 and T217 are of hyperphosphorylated tau protein. Together with the extra- TP sites, whereas S214 is located within a consensus sequence cellular b-amyloid deposits, they constitute the defining neuro- [RXRXXS/T] for phosphorylation by members of the pathological characteristics of AD [1]. Inclusions made of AGC [PKA (cAMP-dependent protein kinase)/protein kinase hyperphosphorylated tau, in the absence of extracellular G/PKC (protein kinase C)] subfamily of protein kinases. It deposits, are characteristic of progressive supranuclear palsy, comprises more than 50 different members and includes en- corticobasal degeneration, Pick’s disease, argyrophilic grain zymes involved in diverse aspects of cellular regulation [31]. disease and frontotemporal dementia and parkinsonism linked In a transgenic mouse model of human tauopathy, AT100 rec- to chromosome 17 (FTDP-17) [1,2]. The identification of over ognizes only filamentous tau [32], suggesting that phosphoryla- 40 different mutations in Tau in FTDP-17 has firmly estab- tion of T212, S214 and T217 plays an important role in tau lished that dysfunction of tau protein is central to the neurode- assembly. generative process [1,3–5]. Here, we report on the phosphorylation of recombinant In adult human brain, six tau isoforms are produced from a human tau protein by AGC kinases protein kinase B-alpha single gene by alternative mRNA splicing [6–9]. They differ by (PKBa), serum- and glucocorticoid-induced kinase-1 having three or four conserved repeats, located in tandem in (SGK1), p70 S6 kinase (S6K), p90 ribosomal S6 kinases 1 and -2 (RSK1 and RSK2) and mitogen- and stress-activated protein kinase-1 (MSK1). All six kinases phosphorylated S214, with RSK1 and p70S6 kinase also phosphorylating *Corresponding author. Fax: +44 1223 402197. T212. MSK1 phosphorylated S214, as well as S262 and S305 E-mail address: [email protected] (M. Goedert). in the microtubule-binding repeat region of tau.

0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.05.009 2658 K. Virdee et al. / FEBS Letters 581 (2007) 2657–2662

2. Materials and methods A β 2.1. Protein kinases RSK1 GSK3

Active His-p70S6 kinase(1–421)T412E, SGK1(D1–59)S422D, His- RSK2 CaMK II No kinase SGK1 p70S6K PKB PKBa(D1–117)S473E, His-RSK1, His-RSK2, His-MSK1, His-GSK3b MSK1 and calcium/calmodulin-dependent protein kinase II (CaMKII) were 32P purchased from Upstate Biotechnology. For p70 S6 kinase, SGK1, PKBa and MSK1, one unit of activity was that amount of enzyme which incorporated 1 nmol phosphate into 1 nmol CROSStide sub- 83 strate peptide (GRPRTSSFAEG) in 1 min. For RSK1 and RSK2, 1 62 CBB unit of activity was that amount of enzyme which incorporated 1 nmol 47.5 phosphate into 1 nmol long S6 substrate peptide (KEAKEKRQE- QIAKRRRLSSLRASTSKSGGSQK) in 1 min. GSK3b was assayed by the phosphorylation of the phospho-glycogen synthase peptide YRRAAVPPSPSLSRHSSPHQSpEDEEE. One unit of activity was that amount of enzyme which incorporated 1 nmol phosphate into the phosphopeptide in 1 min. For CaMKII puriﬁed from rat forebrain, β one unit of activity was that amount of enzyme which incorporated 1 nmol phosphate into the synthetic peptide KKALRRQETVDAL GSK3 CaMK II RSK2 SGK1 RSK1 Sark. insol. tau No kinase p70S6K PKB in 1 min. B MSK1 pT212 2.2. Tau constructs The 412 amino acid isoform of human brain tau was expressed in E. coli from cDNA clone htau46 and expressed as described [33]. pS214 Site-directed mutagenesis (QuikChange, Stratagene) was used to generate the following 10 tau mutants (using the nomenclature of the 441 amino acid brain tau isoform): T212A; S214A; T212A/S214A; AT100 S262A; S262A/S356A; S262A/S293A/S356A; S262A/S293A/S324A/ S356A; S262A/S293A/S305A/S324A/S356A; T212A/S262A/S293A/ S305A/S324A/S356A and T212A/S214A/S262A/S293A/S305A/S324A/ 12E8 S356A. All constructs were veriﬁed by DNA sequencing.

2.3. Antibodies BR134 The phosphorylation-independent anti-tau antibodies BR134 and BR135 [6] were used. Antibodies pT212 and pS214 were purchased from BioSource International (Camarillo, CA, USA). Antibody Fig. 1. Phosphorylation of recombinant tau by AGC kinases. (A) AT100 [34] was purchased from Innogenetics (Ghent, Belgium). Anti- Recombinant human tau (412 amino acid isoform) was incubated in body 12E8 [35] was a kind gift from Dr. P. Seubert (Elan Pharmaceu- the presence of SGK1, p70S6 kinase, RSK1, RSK2, PKB, MSK1, ticals, San Francisco, CA, USA). Immunoblot analysis was performed GSK3b and CaMKII. Tau incubated in the absence of kinase served as as described [36], using 50 ng of protein per lane. the control. The reactions were separated on SDS–PAGE and the 32P label incorporated into tau visualized by phosphorimaging ( 32P) of 2.4. Phosphorylation assays dried gels stained with Coomassie Brilliant Blue (CBB). Molecular Phosphorylation assays (0.030 ml) were carried out at room temper- weight markers (in kDa) are indicated on the left. (B) Immunoblot ature and comprised 30 mM HEPES, 10 mM magnesium chloride, analysis of recombinant tau phosphorylated by the above kinases. 100 lM[c-32P] ATP (approximately 106 cpm/nmol), 1 mM dithiothre- Antibodies specific for the phosphorylation of tau at T212 (pT212), itol, 10 mM b-glycerophosphate, tau protein (5 lg) and 1 U/ml p70 S6 S214 (pS214), T212, S214 and T217 (AT100), S262 and/or S356 (12E8) kinase, SGK1, PKBa, RSK1, RSK2, MSK1 or CaMKII. When CaM- were used. BR134 recognizes tau in a phosphorylation-independent manner. Sarkosyl-insoluble tau from the brain of mice transgenic for KII was used, 40 lg/ml calmodulin and 1 mM CaCl2 were added. Reactions were initiated by the addition of protein kinase and allowed human P301S tau served as a positive control. to proceed for 6 h. In some experiments, two protein kinases were used in combination or added sequentially; in the latter case, the second enzyme was added 3 h after the first and the reaction allowed to proceed constant amount of protein kinase showed that incorporation for a further 3 h. Phosphorylation was examined by SDS–PAGE and of 32P reached a plateau at approximately 5 lg of tau. Of the autoradiography. The radioactive bands were quantitated using the Typhoon Phosphorimager and analyzed with iQMac2.1 software AGC kinases tested, MSK1 and SGK1 gave the most robust (Molecular Dynamics, Sunnyvale, CA, USA). Alternatively, incorpo- phosphorylation of recombinant tau. Tau protein incubated ration of 32P-radioactivity was measured after adsorption to Whatman with 1 U/ml MSK1 for 6 h incorporated 2.21 ± 0.14 mol phos- P81 paper [26]. Sarkosyl-insoluble tau was extracted from the brains of phate per mol tau (n = 3). This figure was 1.30 ± 0.30 for tau 5-month-old mice transgenic for human P301S tau protein, as described [32]. phosphorylated by SGK1. Phosphorylation by MSK1 also resulted in a reduced mobility of recombinant tau on SDS– PAGE. The AGC kinases p70S6K, RSK1, RSK2 and PKB 3. Results phosphorylated tau less well. Thus, tau protein incubated with 1 U/ml p70 S6 kinase for 6 h only incorporated The relative levels of phosphorylation of wild-type recombi- 0.13 ± 0.04 mol phosphate per mol tau (n = 3). Phosphoryla- nant tau (412 amino acid isoform of human tau) by 1 U/ml tion of tau by CaMKII resulted in strong incorporation of SGK1, p70S6K, RSK1, RSK2, PKB, MSK1, GSK3b and 32P, with lower levels of incorporation following phosphoryla- CaMKII were assessed using the incorporation of 32P tion by GSK3b. (Fig. 1A). Preliminary experiments showed that the incorpora- To identify sites in tau phosphorylated by AGC kinases, tion of 32P into tau had reached a plateau by 6 h. The phos- phosphorylation-dependent antibodies specific for pT212, phorylation reactions were therefore incubated for 6 h. pS214 and pS262/S356 (antibody 12E8) were used (Fig. 1B). Incubation of different amounts of recombinant tau with a The kinases p70S6K and RSK1 generated reactivity with K. Virdee et al. / FEBS Letters 581 (2007) 2657–2662 2659 anti-pT212 antibody. By contrast, SGK1, RSK2, PKB, MSK1 phorylation of T212A tau by RSK1 resulted in a 30% reduc- and CaMKII failed to generate reactivity with antibody tion in 32P incorporation, with no significant reduction pT212. With antibody pS214, kinases SGK1, p70S6K, following phosphorylation by RSK2. Phosphorylation of RSK1, RSK2, CaMKII and, in particular, MSK1 generated S214A tau by RSK1 resulted in a 50% reduction in the incor- strong reactivity, whereas phosphorylation by PKB gave lower poration of 32P, with a 75% reduction following phosphoryla- levels of immunoreactivity. Phosphorylation of S262 and/or tion by RSK2. S356 was assessed using antibody 12E8. Of the kinases tested, Incorporation of 32P into T212A/S214A tau was greatest only MSK1 and CaMKII generated 12E8-reactivity. None of when MSK1 was used, consistent with it phosphorylating a the enzymes tested generated AT100 reactivity when used number of additional sites in tau. The generation of 12E8- alone or in combination with PKA, even in the presence of immunoreactivity upon phosphorylation of htau46 by MSK1 50 lg/ml heparin. The same was true when SGK1 was used indicated phosphorylation of S262 and/or S356, two of four in combination with DYRK1A, JNK1, p70S6 kinase, KXGS sites. We therefore mutated the KXGS sites (S262, SAPK4/p38c or GSK3b (not shown). Sarkosyl-insoluble tau S293, S324, and S356), as well as S305, which was previously from the brains of 5 month-old mice transgenic for human shown to be phosphorylated by MARK/PAR1, a protein ki- P301S tau served as a positive control for the phosphospecific nase known to phosphorylate KXGS sites in tau [37]. Muta- anti-tau antibodies. tion of these five sites from serine to alanine resulted in a To corroborate these findings, tau proteins with mutations 60% reduction in the incorporation of 32P into tau following T212A, S214A and T212A/S214A were phosphorylated by phosphorylation by MSK1 (Fig. 3). Additional mutagenesis SGK1, p70S6K, MSK1, RSK1 or RSK2 and incorporation at T212 gave a similar reduction in incorporation. However, of 32P measured (Fig. 2). SGK1 phosphorylated T212A when S214 was also mutated to alanine, a greater than 90% strongly, with only residual phosphorylation of S214A and reduction in the incorporation of 32P was observed. These find- T212A/S214A tau (15% and 10% of 32P incorporation, respec- ings show that S214, S262 and S305 are major sites in tau tively, with the value for wild-type tau taken as 100%). By con- phosphorylated by MSK1. trast, p70S6 kinase phosphorylated T212A and S214A tau proteins equally well. Incorporation of 32P into T212A/ S214A tau was 30% of that for the wild-type protein. MSK1 strongly phosphorylated T212A/S214A tau, with only a 25% reduction in 32P incorporation compared to wild-type. Phos- 125

100

75 T212A/214A S214A T212A/214A wt T212A S214A wt T212A 50

32 P-Labeling (%)

P 32 25 cont SGK1

32P 0 wt cont p70S6K S262A

32P S262A/S356A

cont MSK1 S262A/S293A/S356A

cont RSK1 RSK2 S262A/S293A/S324A/S356A

32 P S262A/S293A/S305A/S324A/S356A wt wt wt T212A/S262A/S293A/S305A/S324A/S356A T212A T212A T212A S214A S214A S214A T212A/S214A/S262A/S293A/S305A/S324A/S356A T212A/214A T212A/214A T212A/214A Fig. 3. Phosphorylation of wild-type and mutant tau by MSK1. Fig. 2. Phosphorylation of wild-type and mutant tau by SGK1, p70 S6 Recombinant wild-type and mutant tau was incubated in the presence kinase, MSK1, RSK1 and RSK2. Recombinant wild-type and mutant of MSK1. The reactions were separated on SDS–PAGE and the 32P tau was incubated in the presence of SGK1, p70 S6 kinase, MSK1, label incorporated into tau visualized by phosphorimaging. The results RSK1 and RSK2. Tau incubated in the absence of kinase served as the were expressed as % of 32P label of wild-type tau (taken as 100%). Tau control. The reactions were separated on SDS–PAGE and the 32P label was mutated at T212, S214 and S305, as well as at KXGS sites S262, incorporated into tau visualized by phosphorimaging (32P). Tau was S293, S324 and S356 in the repeat region. The results are expressed as mutated at residues T212 and/or S214. the means ± S.E.M. (n = 3). 2660 K. Virdee et al. / FEBS Letters 581 (2007) 2657–2662

4. Discussion therefore appears that the activation of MAP kinase family members can lead to the phosphorylation of tau at most of AGC kinases preferentially phosphorylate serine and threo- the sites that are hyperphosphorylated in the human tauopa- nine residues that lie in RXRXXS/T motifs [31]. They are acti- thies. vated by a variety of different mechanisms. For example, agonists that stimulate adenylate cyclase activity induce the Acknowledgement: This work was supported by the U.K. Alzheimer’s activation of protein kinase A (PKA), whereas those that stim- Research Trust and the Medical Research Council. ulate phosphoinositide 3-kinase (PI3K) activate PKB, p70S6 kinase and SGK. RSK isoforms are activated by the mitogen-activated protein (MAP) kinase family members ERK1 References and ERK2, whereas MSK isoforms are activated by ERK1/ ERK2 and the stress- and cytokine-activated p38 MAP kinase. [1] Goedert, M. and Spillantini, M.G. (2006) A century of Alzhei- In this paper, we show that PKB, SGK1, p70S6 kinase, mer’s disease. Science 314, 777–781. RSK1, RSK2 and MSK1 phosphorylate tau protein at S214, [2] Lee, V.M.Y., Goedert, M. and Trojanowski, J.Q. (2001) Neuro- degenerative tauopathies. Annu. Rev. Neurosci. 24, 1121–1159. in keeping with the consensus sequence RSRTPS (residues [3] Poorkaj, P., Bird, T.D., Wijsman, E., Nemens, E., Garruto, R.M., 209–214). These findings confirm previous work showing that Anderson, L., Andreadis, A., Wiederholt, W.C., Raskind, M. and PKB, SGK and p70S6 kinase phosphorylate tau at S214 [38– Schellenberg, G.D. (1998) Tau is a candidate gene for 41]. They show in addition that RSK1, RSK2 and MSK1 chromosome 17 frontotemporal dementia. Ann. Neurol. 43, phosphorylate S214. RSKs and MSKs are ubiquitously ex- 815–825. [4] Hutton, M., Lendon, C.L., Rizzu, P., Baker, M., Froelich, S., pressed, with many cell types expressing several members of Houlden, H., Pickering-Brown, S., Chakraverty, S., Isaacs, A., each family [42]. Loss-of-function mutations in RSK2 cause Grover, A., Hackett, J., Adamson, J., Lincoln, S., Dickson, D., the Coffin-Lowry syndrome, a syndromic form of mental Davies, P., Petersen, R.C., Stevens, M., de Graaff, E., Wauters, retardation that shows X-linked inheritance [43]. Phosphoryla- E., van Baren, J., Hillebrand, M., Joosse, M., Kwon, J.M., Nowotny, P., Che, L.K., Norton, J., Morris, J.C., Reed, L.A., tion of S214 is known to reduce the ability of tau to interact Trojanowski, J., Basun, H., Lannfelt, L., Neystat, M., Fahn, S., with microtubules [44,45]. 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