Primed phosphorylation of tau at Thr231 by glycogen synthase kinase 3β (GSK3β) plays a critical role in regulating tau's ability to bind and stabilize microtubules

Authors


Address correspondence and reprint requests to Gail V. W. Johnson, Department of Psychiatry, 1720 7th Avenue South, SC1061, University of Alabama at Birmingham, School of Medicine, Birmingham, AL 35294-0017, USA. E-mail: gvwj@uab.edu

Abstract

Site-specific phosphorylation of tau negatively regulates its ability to bind and stabilize microtubule structure. Although tau is a substrate of glycogen synthase kinase 3β (GSK3β), the exact sites on tau that are phosphorylated by this kinase in situ have not yet been established, and the effect of these phosphorylation events on tau–microtubule interactions have not been fully elucidated. GSK3β phosphorylates both primed and unprimed sites on tau, but only primed phosphorylation events significantly decrease the ability of tau to bind microtubules. The focus of the present study is on determining the importance of the GSK3β-mediated phosphorylation of a specific primed site, Thr231, in regulating tau's function. Pre-phosphorylation of Ser235 primes tau for phosphorylation by GSK3β at Thr231. Phosphorylation by GSK3β of wild-type tau or tau with Ser235 mutated to Ala decreases tau–microtubule interactions. However, when Thr231 alone or Thr231 and Ser235 in tau were mutated to Ala, phosphorylation by GSK3β did not decrease the association of tau with the cytoskeleton. Further, T231A tau was still able to efficiently bind microtubules after phosphorylation by GSK3β. Expression of each tau construct alone increased tubulin acetylation, a marker of microtubule stability. However, when cells were cotransfected with wild-type tau and GSK3β, the level of tubulin acetylation was decreased to vector-transfected levels. In contrast, coexpression of GSK3β with mutated tau (T231A/S235A) did not significantly decrease the levels of acetylated tubulin. These results strongly indicate that phosphorylation of Thr231 in tau by GSK3β plays a critical role in regulating tau's ability to bind and stabilize microtubules.

Abbreviations used
BCA

bicinchoninic acid assay

FBS

fetal bovine serum

GSK3β

glycogen synthase kinase 3β

PMSF

phenylmethylsulfonyl fluoride

PBS

phosphate-buffered saline

SDS

sodium dodecyl sulfate

A predominant role of the tau protein is to promote microtubule assembly and stability, primarily in neurons (Johnson and Hartigan 1999; Johnson and Bailey 2002). Tau is a phosphoprotein whose ability to bind and stabilize microtubules is regulated by site-specific phosphorylation. When phosphorylation occurs within its microtubule-binding domains or in the regions flanking them, tau is rendered less able to bind and/or stabilize microtubules (Gustke et al. 1994; Preuss et al. 1997). Although the effects of site-specific phosphorylation on tau function have been studied extensively in vitro (Johnson and Hartigan 1999; Johnson and Jenkins 1999), less is known about how tau's ability to bind and stabilize microtubules is regulated by the phosphorylation of specific residues in situ.

Glycogen synthase kinase 3β (GSK3β), a predominant tau kinase, is found in neurons (Leroy and Brion 1999), associates with microtubules (Ishiguro et al. 1993; Sun et al. 2002), and, when over-expressed in cells (Sperber et al. 1995; Lovestone et al. 1996; Utton et al. 1997) and transgenic mice (Spittaels et al. 2000; Lucas et al. 2001), results in increased tau phosphorylation. GSK3β can phosphorylate both unprimed sites which are in proline-rich regions of a protein (Grimes and Jope 2001), or primed sites where a Ser or Thr is prephosphorylated by another protein kinase at a site that is located four amino acids C-terminal to the GSK3β site (Dajani et al. 2001; Frame et al. 2001; Grimes and Jope 2001; Harwood 2001). Even though GSK3β can phosphorylate both types of sites, there is increasing evidence that the majority of physiologically relevant GSK3β sites are primed (Cohen and Frame 2001; Frame et al. 2001). The physiological relevance of primed GSK3β sites on tau was confirmed by our recent study (Cho and Johnson 2003) employing GSK3β-R96A, a GSK3β mutant capable of efficiently phosphorylating only unprimed sites (Frame et al. 2001). It was already known that tau's association with microtubules was decreased after phosphorylation by GSK3β (Lovestone et al. 1996; Wagner et al. 1996). Our study more specifically showed that, while GSK3β was capable of phosphorylating tau at both primed and unprimed sites (Cho and Johnson 2003), it was the phosphorylation of the primed sites which played the key role in the decrease in tau–microtubule interactions (Cho and Johnson 2003).

Thr231 is one of the more prominent primed sites in tau phosphorylated by GSK3β (Goedert et al. 1994). Thr231's phosphorylation by GSK3β is enhanced by prephosphorylation of Ser235 (Goedert et al. 1994). Interestingly, the prolyl-isomerase Pin-1 specifically binds to the tau phospho-Thr231 epitope, and it has been hypothesized that Pin-1 facilitates the isomerization of the pThr-Pro motif, thereby enabling protein phosphatase 2A to dephosphorylate tau (Lu et al. 1999; Zhou et al. 2000). Such dephosphorylation restores tau's ability to bind microtubules and promote microtubule assembly in vitro (Lu et al. 1999; Zhou et al. 2000), a finding which indirectly suggests that Thr231 plays a major role in regulating tau's function. The goal of our current study is to more directly examine that role.

For these studies we used wild-type tau and tau with either or both of Thr231 and Ser235 mutated to Ala. Phosphorylation of wild-type tau or S235A tau by GSK3β not only significantly reduced tau's association with the cytoskeleton and its binding to microtubules, but also decreased the levels of acetylated tubulin, indicating that tau's ability to stabilize microtubules had been significantly impaired. However, phosphorylation of T231A tau or double-mutated tau (S235A, T231A) by GSK3β did not decrease tau's association with the cytoskeleton nor did it significantly decrease microtubule binding. Further, double-mutated tau proved to be still capable of efficiently stabilizing microtubules after phosphorylation by GSK3β. Taken together, these results clearly demonstrate that phosphorylated Thr231 is a more significant regulator of tau's function than are other GSK3β sites on tau.

Materials and methods

Cell culture

CHO cells were grown in F-12 medium supplemented with 5% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 2 mml-glutamine (Gibco, Rockville, MD, USA), 10 U/mL penicillin (Gibco), and 100 U/mL streptomycin (Gibco). Cells were used at a confluency of 50–80% for all experiments.

Plasmid constructs

The cDNA of full-length tau construct (containing exons 2, 3, and 10) was subcloned into the mammalian expression vector, pcDNA 3.1 (+) (Invitrogen, Carlsbad, CA, USA). pcDNA 3.1-htau40 with a His tag (Tucholski et al. 1999) was used as a template, and PCR was performed to remove the His tag. The PCR reaction product was digested with Bam HI and Not I and ligated into the Bam HI and Not I sites of pcDNA 3.1 (+). The integrity of the tau construct was confirmed by sequence analysis. Thr231 and/or Ser235 in tau were mutated to Ala (T231A, S235A, and T231A/S235A) using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) with the following primer pairs: T231A, forward primer: 5′-TGG CAG TGG TCC GTG CTC CAC CCA AGT CG-3′ and reverse primer: 5′-CGA CTT GGG TGG AGC ACG GAC CAC TGC CA-3′; S235A, forward primer: 5′-GTA CTC CAC CCA AGG CGC CGT CTT CCG CC-3′ and reverse primer: 5′-GGC GGA AGA CGG CGC CTT GGG TGG AGT AC-3′; T231A/S235A, forward primer: 5′-CAG TGG TCC GTG CTC CAC CCA AGG CGC CGT CTT CCG C-3′ and reverse primer: 5′-GCG GAA GAC GGC GCC TTG GGT GGA GCA CGG ACC ACT G-3′. Mutations were verified by DNA sequence analysis.

HA-GSK3β-S9A, referred to as GSK3β throughout the text, was constructed in pcDNA 3.1(–), as previously described (Cho and Johnson 2003).

Transient transfections

Wild-type Tau, T231A, S235A or T231A/S235A and GSKβ were transiently transfected into CHO cells using Fugene-6 (Roche, Indianapolis, IN, USA) transfection reagent according to the manufacturer's protocol. Thirty-three hours after transfection, the cells were washed with ice-cold phosphate-buffered saline (PBS) and then collected and processed as described below for the different assays.

Immunoblotting

Cells were collected in lysis buffer (150 mm NaCl, 10 mm Tris-HCl, 1 mm EGTA, 1 mm EDTA, 0.2 mm sodium vandate, 0.5% Nonidet P40), containing 1 mm PMSF, 0.1 µm okadaic acid, and a 10-µg/mL concentration each of aprotonin, leupeptin, and pepstatin. Lysates were sonicated on ice and centrifuged, and then protein concentrations in the supernatants were determined using the bicinchoninic acid assay (BCA) (Pierce). Samples were diluted with 2 × sodium dodecyl sulfate (SDS) stop buffer (2% SDS, 5 mm EGTA, 5 mm EDTA, 25 mm dithiothreitol, 10% glycerol, 0.01% bromophenol blue and 0.25 m Tris-Cl, pH 6.8) and incubated in a boiling water bath for 5 min Equal amounts of protein from each sample were electrophoresed on 10% SDS–polyacrylamide gels, transferred to nitrocellulose, and probed with the indicated antibodies. The tau antibodies used in this study were: Tau5/5A6 (Tau 5 was from Dr L. Binder), which are phospho-independent tau antibodies (Carmel et al. 1996; Johnson et al. 1997), AT180 (Endogen, Rockford, IL, USA) which recognizes tau when it is phosphorylated at Thr231 (Goedert et al. 1994; Hoffmann et al. 1997; Illenberger et al. 1998), and PHF-1 (from Dr P. Davies) which recognizes tau phosphorylated at Ser396/404 (Otvos et al. 1994). The monoclonal GSK3β antibody was purchased from Transduction Laboratories SDS polyacrylamide gel electrophoresis, the monoclonal actin antibody from Chemicon (Temecula, CA, USA), the monoclonal α-tubulin antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the acetylated-tubulin antibody (6-11B-1) from Sigma (St Louis, MO, USA). 5H1, which is a mouse IgM that recognizes the β-tubulin, was a gift from Dr L. Binder. After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Immuno-Research, West Grove, PA, USA), the blots were developed using ECL (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Tubulin acetylation

For evaluating the effects of tau and/or GSK3β on the stability of the microtubules, cells were transfected with each of the tau constructs in the absence or presence of GSK3β as described above. The extent of acetylation was calculated by using quantitative immunoblotting to determine the amount of acetylated tubulin relative to total tubulin (Fath et al. 2002), with data expressed as the ratio of acetylated α-tubulin/total α-tubulin.

Separation into soluble and cytoskeletal insoluble fractions

Cells were rinsed once with warm PBS, scraped off the plate, collected by centrifugation, re-suspended in 50 µL of prewarmed extraction buffer [0.1% (v/v) Triton X-100, 100 mm 1,4-piperazinediethanesulfonic acid/KOH, 1.0 mm MgCl2, 2.0 mm EGTA, 0.1 mm EDTA, and 30% glycerol, pH 6.75] containing protease inhibitors (0.1 mm PMSF, 10 µg/mL each of leupeptin, pepstatin, and aprotinin) and a phosphatase inhibitor (0.5 µm okadaic acid), and incubated for 8 min at 37°C, before the pellet (cytoskeletal insoluble) and supernatant (soluble) fractions were separated by centrifugation (15 min at 15 000 g and 25°C). Equal amounts of pellet and supernatant fractions were separated by SDS polyacrylamide gel electrophoresis blotted, and processed for immunodetection with anti-tau and anti-actin antibodies.

Microtubule-binding assay

The microtubule-binding assay was carried out as described previously with minor modifications (Cho and Johnson 2003). Cells were collected and re-suspended in 80 mm 1,4-piperazinediethanesulfonic acid/KOH (pH 6.8) containing protease and phosphatase inhibitors. Cell suspensions were briefly sonicated and incubated on ice for 15 min. Samples were brought to 1.5 mm EGTA and centrifuged at 21 000 g for 40 min at 4°C. The supernatant was collected, protein concentrations were determined using the BCA assay and equal amounts of protein supernatant were used in each binding assay. Supernatants were adjusted to 1 mm GTP and 10 μm taxol and incubated with taxol-stabilized microtubules prepared from rat brain (Davis and Johnson 1999) for 10 min at 37°C. The mixtures were centrifuged through 100 µL of 30% (w/v) sucrose cushions in 80 mm 1,4-piperazinediethanesulfonic acid/KOH (pH 6.8) containing 1 mm EGTA, 1 mm GTP and 10 μm taxol, at 100 000 g for 30 min in an airfuge at room temperature. The supernatant (unbound) and pellet (bound) fractions were collected and diluted with 2 × SDS stop buffer and incubated in a boiling water bath for 5 min. Aliquots from each fraction were separated by electrophoresis on a 10% SDS–polyacrylamide gel, transferred to nitrocellulose and probed with the Tau5/5A6 antibodies.

Immunocytochemistry

These procedures were modified from previously described protocols (Leger et al. 1994; Lesort and Johnson 2000). The cells were transiently transfected with wild-type tau and GSK3β using Fugene-6 transfection reagent. Thirty-five hours after transfection, the cells were rinsed with PBS and fixed at room temperature for 1 h in fixation buffer [2% paraformaldehyde, 0.2% glutaraldehyde, 1 mm MgCl2, 1 mm EGTA, 30% (v/v) glycerol in 70 mm 1,4-piperazinediethanesulfonic acid/KOH, pH 6.8]. Cells were then washed three times with PBS before being permeabilized with 0.2% Triton X-100 in PBS for 2 min. After rinsing with PBS, cells were incubated in NaBH4 (10 mg/mL in PBS) for 7 min and again rinsed with PBS. To reduce background before staining, the cells were blocked with 4% BSA in PBS for 30 min. For the Triton X-100 extractions prior to fixation, cells were washed at 37°C with extraction buffer minus Triton X-100, then extracted at room temperature for 1 min with extraction buffer (80 mm 1,4-piperazinediethanesulfonic acid/KOH, pH 6.8, 1 mm MgCl2, 1 mm EGTA, 0.1% Triton X-100, 30% glycerol, 10 mm GTP). The extraction buffer was removed, and the cells were washed with the buffer minus Triton X-100. Cells were fixed, permeabilized, and blocked as described above. The primary antibodies used in this study were a tau rabbit polyclonal antibody (Dako Corporation, Carpinteria, CA, USA), the monoclonal tubulin antibody 5H1, AT180, and/or PHF-1. The cells were incubated for 1.5 h in the primary antibodies diluted in 0.4% BSA. To visualize both tubulin and tau proteins, the cells were extensively rinsed with PBS and then incubated with the secondary antibodies [Texas Red-conjugated donkey anti-mouse IgM and fluorescein (FITC)-conjugated donkey anti-mouse IgG (Jackson Immuno-Research); or, alternatively, Texas red-conjugated donkey anti-mouse IgM and FITC-conjugated donkey anti-rabbit IgG (Jackson Immuno-Research)]. Cells were washed extensively in PBS before the coverslips were mounted and then viewed with a Nikon Diaphot 200 epifluorescence microscope. Images were captured with a Digital spot camera (Diagnostic Instruments, Sterling Heights, MI, USA), digitally stored, and displayed using the accompanying software.

Results

The T231A mutation in tau abolishes AT180 immunoreactivity

When the primed sites on tau are phosphorylated by GSK3β, tau's affinity for microtubules is diminished (Cho and Johnson 2003). To determine how tau's ability not only to bind but also to stabilize microtubules is affected by the phosphorylation of a specific primed epitope, Thr231/Ser235, we mutated these sites on tau to Ala. In all experiments we used HA-GSK3β-S9A (referred to as GSK3β in the text) because it is considered constitutively active, as the mutation of Ser9 to Ala prevents phosphorylation and inactivation of the kinase (Sutherland et al. 1993; Frame et al. 2001). Figure 1 shows that GSK3β phosphorylation of wild-type tau, T231A, S235A or T231A/S235A decreased the electrophoretic mobility of the tau protein and increased reactivity with the PHF-1 antibody, which recognizes phospho-Ser396/404, an unprimed GSK3β site. The wild-type tau was phosphorylated by GSK3β at the AT180 epitope; AT180 has been shown to recognize Thr231 when the site is phosphorylated (Hoffmann et al. 1997). As expected, mutation of Thr231 to Ala abolished AT180 immunoreactivity, and similar results were seen with the T231A/S235A mutant. Interestingly, a decrease in AT180 immunoreactivity was observed when S235A was transfected with GSK3β, likely due to the fact that Thr231 is a primed site. If prior phosphorylation of Ser235 were prevented, the ability of GSK3β to phosphorylate Thr231 would be expected to be less (Fig. 1). In all cases, similar levels of GSK3 were expressed (Fig. 1).

Figure 1.

AT180 immunoreactivity is abolished by the T231A mutation. CHO cells were transiently transfected with vector or wild-type tau alone, or with GSK3β and wild-type tau, T231A, S235A, or T231A/S235A [A/A] constructs and lysates were immunoblotted with the antibodies indicated at the left of each panel. The expression level of all tau constructs was similar and the presence of GSK3β decreased the electrophoretic mobility of all tau constructs. Phosphorylation of wild-type tau by GSK3β resulted in a robust increase in AT180 immunoreactivity, which recognizes the primed site of phospho-Thr231. In contrast, and as expected, no AT180 immunoreactivity was observed in cells transfected with GSK3β and either T231A or T231A/S235A [A/A]. In the cells transfected with GSK3β and S235A, AT180 immunoreactivity was less because prior phosphorylation of Ser235 enhances phosphorylation of Thr231, as this is a primed site. The immunoreactivity of PHF-1 was approximately the same for all of the tau constructs expressed in the presence of GSK3β. The expression level of the GSK3β was the same in all the transiently transfected cells. The actin blots demonstrate that equal amounts of protein were loaded in each lane.

T231A-tau still interacts with the cytoskeleton after phosphorylation by GSK3β

To investigate the effects of the phosphorylation of Thr231 and/or Ser235 on tau's interaction with the cytoskeleton, cells were transfected with each tau construct in the absence or presence of GSK3β. The amount of tau associated with the detergent-insoluble component containing the cytoskeleton was determined by separating it from the soluble component. When expressed alone, all of the tau constructs were enriched in the insoluble fraction, indicating that the mutations themselves do not alter tau's interaction with the cytoskeleton (Fig. 2). Although coexpression with GSK3β did not affect the amount of tau in the soluble fraction of T231A or T231A/S235A (Fig. 2b,d), the amount of wild-type tau (Fig. 2a) and S235A (Fig. 2c) in the soluble fraction was increased relative to the insoluble fraction as determined by densitometric analysis. These data indicate that GSK3β phosphorylation of Thr231 in tau, which is a primed phosphorylation site, plays a critical role in decreasing tau's association with the cytoskeleton.

Figure 2.

The T231A mutation reverses the GSK3β-mediated decrease in tau's association with the cytoskeletal fraction. CHO cells were transiently transfected with (a) wild-type tau, (b) T231A, (c) S235A, or (d) T231A/S235A [A/A] in the absence (–) or presence (+) of GSK3β. Cell lysates were separated into the insoluble cytoskeletal (I) and soluble (S) fractions and immunoblotted with the phospho-independent tau antibodies Tau5/5A6. In the absence of GSK3β, the majority of tau was found in the insoluble cytoskeletal fraction for all the tau constructs. However, when the tau constructs were coexpressed with GSK3β, more wild-type tau (a) and S235A (c) were found in the soluble than in the insoluble fraction. In contrast, T231A (b) and T231A/S235A (d) remained predominantly located in the insoluble cytoskeletal fractions even in the presence of GSK3β. These results demonstrate that phosphorylation of Thr231, which is a primed site, plays a critical role in regulating the interaction of tau with the cytoskeleton. Actin blots are shown as loading controls.

T231A-tau still interacts with microtubules after phosphorylation by GSK3β

To further investigate how the phosphorylation of Thr231 affects the ability of tau to bind microtubules; a microtubule-binding assay was used. Supernatants from cells transfected with tau alone or with each tau construct and GSK3β were incubated with taxol-stabilized microtubules, and the amount of tau bound to the microtubules in the pellet and the amount of tau that remained unbound were measured. In the absence of GSK3β, all of the tau constructs showed an equal degree of microtubule binding (Fig. 3a). However, the phosphorylation of wild-type tau or S235A by GSK3β reduced the affinity of these constructs for microtubules and increased the amount of tau in the supernatant (unbound fraction) (Fig. 3b). In contrast, after phosphorylation by GSK3β, T231A or T231A/S235A remained bound to the microtubules and so appeared at much lower levels in the unbound fractions (Fig. 3b). These data clearly demonstrate that phosphorylation of Thr231 plays a critical role in regulating the binding of tau to microtubules (Fig. 3b).

Figure 3.

The T231A mutation reverses the GSK3β-mediated decrease in tau's association with microtubules. CHO cells were transiently transfected with different tau constructs in the absence or presence of GSK3β, and high speed supernatants were used in a microtubule-binding assay. All samples were immunoblotted with the phospho-independent tau antibodies Tau5/5A6. (a) Wild-type tau, T231A, S235A, and T231A/S235A [A/A] were expressed at the same level (tau input) and showed equivalent microtubule binding (bound tau). (b) When tau alone was expressed, the majority of the tau was bound to microtubules (bound tau); however, coexpression of tau with GSK3β decreased bound tau and increased free tau (unbound tau). The same was true for S235A. In contrast, in cells cotransfected with GSK3β and T231A or T231A/S235A [A/A], the majority of tau was bound to the microtubules, with much less found in the unbound fraction. The amount of tau used in the assay was equivalent for all conditions (input tau).

GSK3β phosphorylation of tau at Thr231 plays an essential role in regulating tau's ability to stabilize microtubules

To determine if phosphorylation of tau at Thr231 plays a critical role in regulating the ability of tau to stabilize microtubules in situ, the effects of the different tau constructs on the acetylation state of microtubules in the absence or presence of GSK3β-mediated phosphorylation were examined. Acetylation of α-tubulin is associated with a more stable microtubule network and consequently serves as marker for stable microtubule subpopulations (Piperno et al. 1987; Takemura et al. 1992). Cells were transfected with each tau construct in the absence or presence of GSK3β and the extent of acetylation was calculated by determining the amount of acetylated tubulin relative to total tubulin levels. When expressed alone, all of the tau constructs significantly increased the level of acetylation to a similar extent compared to what was observed in cells transfected with vector or GSK3β alone. These data indicate that increased tau expression results in increased microtubule stability and that the mutations do not affect this function (Fig. 4). When coexpressed with GSK3β, expression of wild-type tau, T231A and S235A resulted in a far lesser degree of tubulin acetylation than they did when expressed alone (Fig. 4), but the same did not hold true for T231A/S235A, as coexpression with GSK3β did not result in a significant decrease in the extent of tubulin acetylation (Fig. 4). Further, in the presence of GSK3β, the extent of tubulin acetylation tended to be greater in cells expressing T231A/S235A compared to the other tau constructs. Taken together, these data indicate that phosphorylation of Thr231 and Ser235 plays an essential role in regulating the ability of tau to stabilize microtubules.

Figure 4.

Phosphorylation of tau on Thr231 and Ser235 by GSK3β plays an essential role in the modulation of tau's ability to stabilize microtubules. The presence of acetylated tubulin is an indicator of microtubule stability. For each condition, the ratio of acetylated α-tubulin/total α-tubulin was quantitated. Cells were transfected with each tau construct alone (closed bars) or in combination with GSK3β (open bars). Control cells (Con) were transfected with vector alone (closed bar) or GSK3β alone (open bars). Expression of wild-type tau or of the mutated tau constructs significantly increased MT stability, as indicated by an increase in the ratio of acetylated tubulin/total tubulin. Co-expression of tau with GSK3β resulted in acetylation levels that were significantly lower than those seen when wild-type tau alone was expressed. The same was true for T231A and S235A. However, the presence of GSK3β did not significantly alter the level of tubulin acetylation in the cells expressing T231A/S235A [A/A]. Further, in the presence of GSK3β the level of tubulin acetylation was significantly greater in the cells expressing T231A/S235A [A/A] compared to the other tau constructs. These data clearly demonstrate that phosphorylation of Thr231 and Ser235 plays a crucial role in regulating tau's ability to stabilize microtubules in situ. *P < 0.05 when comparing cells transfected with each tau construct in the absence or presence of GSK3β (NS, not significantly different). Quantifications were from three independent experiments. Mean ± SE are shown.

Tau phosphorylated at Thr231 is not tightly associated with microtubules

To evaluate the interaction of tau phosphorylated at different epitopes with the microtubule network, cells were transfected with wild-type tau and GSK3β and the distribution of tau in the cells was examined immunocytochemically. When the cells were fixed and stained without extraction, tau staining was diffuse and specific colocalization with the microtubules was not evident (Fig. 5a–c). PHF-1 (phospho-Ser396/404) and AT180 (phospho-Thr231) staining showed a similar distribution pattern (Fig. 5d–i).

Figure 5.

Cellular localization of tau and tubulin in cells expressing tau and GSK3β. Wild-type tau was coexpressed with GSK3β in CHO cells. After transfection, the cells were fixed, permeabilized, and stained. Total tau, PHF-1 and AT180 are shown in green, and tubulin (Tub) staining in red (a, d, and g). Total tau (b), PHF-1 (e) and AT180 (h) staining are diffused through the cytoplasm, but merging of the tubulin and tau images reveals some colocalization of tau with the microtubules (red + green = yellow/orange) (c, f, and i). Scale bar in lower left panel = 10 µm.

In the next set of experiments cells were transfected with wild-type tau and GSK3β and then extracted with a non-ionic detergent to remove soluble proteins prior to fixation and staining. The microtubule network was clearly evident in these cells (Fig. 6a,d,g) and both total tau staining (Fig. 6b) and PHF-1 staining (Fig. 6e) were colocalized with the microtubules (Fig. 6c,f). In contrast, there is almost no AT180 staining (Fig. 6h), indicating that tau phosphorylated at Thr231 does not strongly interact with microtubules. The only AT180 immunoreactive element after extraction appears to be the microtubule-organizing center (Fig. 6g–i). Interestingly, the presence of tau at the microtubule-organizing center has been reported previously (Lu and Wood 1993; Preuss et al. 1995).

Figure 6.

Tau that is phosphorylated at Thr231 does not colocalize with microtubules. Cells were transfected with wild-type tau and GSK3β and the cells were extracted by Triton X-100 treatment to remove soluble proteins before fixation and permeabilization. Total tau, PHF-1 and AT180 are shown in green, tubulin (Tub) staining in red (a,d and g). When soluble proteins were removed prior to fixation, total tau (b) staining as well as the PHF-1 signal (e) were almost entirely colocalized with tubulin [merged images in c and f (red + green = yellow/orange)]. However, the AT180 signal (h) is almost totally absent after removal of the soluble proteins. The only significant AT180 immunoreactivity appears to be the microtubule-organizing center (h) [and merged AT180 + tubulin image in (i)]. Scale bar in lower left panel = 10 µm.

Discussion

The functioning of tau as a microtubule-associated protein is modulated by its phosphorylation state. In a previous study, we clearly demonstrated that GSK3β phosphorylation of tau at primed sites plays an essential role in decreasing tau's ability to bind microtubules (Cho and Johnson 2003). In our current study, we extend these previous findings and demonstrate that GSK3β-mediated phosphorylation of Thr231, which is a primed site, is key to regulating tau's ability both to bind and stabilize microtubules. Although other phosphorylation sites are likely modulators of tau's function, these are critical findings because they demonstrate that phosphorylation of a single epitope, Thr231, plays an essential, central role in regulating tau's function in situ.

GSK3β phosphorylates both unprimed sites in proline rich regions of a substrate, and primed sites that are prephosphorylated at a site that is located four amino acids to the C-terminal side of the GSK3β target site (Cohen and Frame 2001; Frame et al. 2001). The majority of known substrates of GSK3β must be first phosphorylated by another protein kinase before they are phosphorylated by GSK3β (Cohen and Frame 2001; Frame et al. 2001). In addition, substrates of GSK3β, such as β-catenin, once thought to be unprimed, have recently been found to be primed. It has now become clear that casein kinase Iα primes β-catenin for phosphorylation by GSK3β (Liu et al. 2002). The structural basis for GSK3β's propensity to phosphorylate primed substrates (Dajani et al. 2001; Frame et al. 2001; ter Haar et al. 2001) has led some researchers to speculate that most if not all physiological substrates of GSK3β may actually be primed (Liu et al. 2002).

Tau is an excellent substrate of GSK3βin vitro (Mulot et al. 1994; Godemann et al. 1999) and over-expression of GSK3β in cell model systems results in robust increases in tau phosphorylation at both primed and unprimed epitopes (Lovestone et al. 1994; Wagner et al. 1996; Cho and Johnson 2003). However, it is interesting to note that GSK3β transgenic mice exhibiting a twofold increase in GSK3 activity showed no discernible increase in tau phosphorylation until they reached ∼ 7 months of age (Spittaels et al. 2000). Older GSK3β transgenic animals showed increased tau phosphorylation at the AT180 site (phospho-Thr231) and the AT8 site, but not the PHF-1 site (Spittaels et al. 2000), although Ser396/404, which make up the PHF-1 epitope, was the most prominent site on tau phosphorylated by GSK3βin vitro (Godemann et al. 1999). It is also interesting to note that phospho-Thr231 was the tau phosphorylation site that showed the greatest increase when NT2N cells were infected with GSK3β (Hong et al. 1997). These and other findings clearly indicate that phosphorylation of Thr231 by GSK3β is likely a physiologically relevant event.

Thr231 in tau is a primed GSK3β site as in vitro prephosphorylation of Ser235 enhances Thr231 phosphorylation by GSK3β (Goedert et al. 1994). Our present study has also shown that mutation of Ser235 to Ala reduced the extent of Thr231 phosphorylation by GSK3β. The mutation of Ser235 to Ala is not expected to completely abolish GSK3β phosphorylation of tau at Thr231, since priming phosphorylation, though it increases the efficiency of phosphorylation, is not a required event for GSK3β phosphorylation (Doble and Woodgett 2003).

In the present study, we have shown that phosphorylation of Thr231 plays a significant role in regulating tau's ability both to bind and to stabilize microtubules. Indeed, when both Thr231 and Ser235 were mutated to Ala, phosphorylation by GSK3β no longer reduced tau's ability to stabilize microtubules, demonstrating that tau was still functional, even though other epitopes on tau were phosphorylated (e.g. PHF-1). A previous study examined the effects of tau and GSK3β over-expression on tubulin acetylation (Nuydens et al. 2002). These investigators demonstrated that the levels of acetylated tubulin were elevated in cultured dorsal root ganglion neurons from mice overexpressing human tau; however, the level and distribution of acetylated microtubules in cells from tau X GSK3β double transgenic mice were almost the same as in non-transgenic animals (Nuydens et al. 2002). Our findings are in agreement with these previous results, in that GSK3β could reverse the tau-induced increase in tubulin acetylation. However, GSK3β did not decrease the extent of tubulin acetylation that was induced by the T231A/S235A tau, demonstrating that the effect of tau on microtubule stability is dependent on the phosphorylation status of Thr231/Ser235. Further, tau that is phosphorylated at Thr231 does not efficiently associate with microtubules in a binding assay (Cho and Johnson 2003), and, when cells are extracted to remove soluble proteins, almost all AT180 immunoreactivity is lost. In contrast, there is pronounced staining of the microtubules by an antibody that recognizes total tau as well as PHF-1 after extraction. These data suggest that if tau phosphorylated at Thr231 does interact with microtubules, the interaction is relatively weak. Previous studies have shown that phosphorylation of Thr231 in tau results in a change in conformation (Jicha et al. 1997; Daly et al. 2000) that could negatively impact tau's association with microtubules. It is also interesting to note that it has been hypothesized that phosphorylation of Thr231 in tau affects the rate of prolyl isomerization, resulting in decreased microtubule binding. Furthermore, the prolyl isomerase Pin1 selectively binds phospho Thr231, facilitating the conversion from the cis to trans conformation of the pThr-Pro motif and thus allowing dephosphorylation of the site by the predominant Pro-directed protein phosphatase 2A and the restoration of microtubule binding (Lu et al. 1999; Zhou et al. 2000). Although these findings are intriguing, phosphorylation of peptides from this region of tau did not alter the equilibrium of cis-trans isomers (Daly et al. 2000). Nonetheless, these findings emphasize the importance of the phosphorylation of Thr231 in regulating tau's microtubule- binding function.

Taken together, the results in the present study demonstrate that phosphorylation of Thr231, which is a primed GSK3β site, plays a significant role in regulating tau's function. The extent to which phosphorylation of Thr231 impacts tau's function is quite remarkable given the fact that at least 10 different sites have been reported to be phosphorylated by GSK3βin vitro (Godemann et al. 1999; Reynolds et al. 2000). Although GSK3β-mediated phosphorylation of Thr231 plays a significant role in regulating tau's function, phosphorylation of Ser262 also plays an important role in regulating the function of tau (Trinczek et al. 1995). However, GSK3β does not phosphorylation Ser262, which is a non-Ser-Pro site (Godemann et al. 1999), and therefore the effects of phosphorylation of Ser262 on tau function could not be evaluated in this paradigm. Nevertheless, it is interesting to note that in vitro, phosphorylation of tau at both Thr231 and Ser262 were required for maximal inhibition of microtubule binding (Sengupta et al. 1998). Another caveat that needs to be considered is that the present studies were carried out in transfected non-neuronal cells, and therefore the results may not be fully representative of the phosphorylation events that regulate tau function in vivo. Nonetheless, these findings strongly indicate the importance of phosphorylation of Thr231 in regulating tau's function, and increased phosphorylation at this epitope could be pathological. Indeed, increased phosphorylation of tau at Thr231 appears to be an early event in Alzheimer's disease, as increased TG-3 (which recognizes tau phosphorylate at Thr231) staining precedes paired helical filament formation (Vincent et al. 1997). In addition, tau phosphorylated at Thr231 was found in the CSF of mildly cognitively impaired patients who went on to develop Alzheimer's disease (Arai et al. 2000). Given these and other findings, we can postulate that increased phosphorylation of tau at Thr231 may be an early event in the pathogenic processes of Alzheimer's disease, one which leads to increased levels of ‘free’ tau. In combination with other events (e.g. caspase cleavage of tau or phosphorylation of the PHF-1 epitope (Abraha et al. 2000; Berry et al. 2003)), over-phosphorylation of tau at Thr231 may play a key role in the process of filament formation in Alzheimer's disease brain. However, further research is required to definitively implicate phosphorylation of Thr231 in the development of tau pathology.

Acknowledgements

This work was supported by NIH grant NS35060 and a grant from the Alzheimer's Disease Association. J-H Cho was supported by a fellowship from the John Douglas French Alzheimer's Foundation made possible by support from the Aetna Foundation. We thank Dr L. Binder and Dr P. Davies for their generous gift of antibodies.

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