From the Department of Psychiatry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017
Received for publication, June 22, 2002, and in revised form, October 2, 2002
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ABSTRACT |
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Glycogen synthase kinase 3 Tau is a family of microtubule-associated proteins that are
expressed predominantly in neurons and are produced by alternative splicing of a single gene (1, 2). Although tau likely plays a role in
numerous neuronal processes, such as vesicle transport (3, 4),
microtubule-plasma membrane interactions (5, 6), and the intracellular
localization of protein such as fyn (7, 8), 14-3-3 (9, 10), and protein
phosphatase 2A (11, 12), the best characterized function of tau is the
binding and stabilization of microtubules (13-15). Tau is a
phosphoprotein, and in vitro, numerous studies have
demonstrated that site-specific phosphorylation of tau regulates its
ability to bind and stabilize microtubules. Increased phosphorylation
of tau in situ tends to negatively regulate tau-microtubule
interactions (16-20), although the effects of site-specific tau
phosphorylation on microtubule binding in the cell have not been as
thoroughly studied, as they have been in vitro.
Numerous protein kinases phosphorylate tau in vitro; however
in situ the list is much more abbreviated. Protein kinase A
(21), microtubule affinity regulating kinases (22),
cyclin-dependent protein kinase 5 (cdk5)1/p35[p25] (23, 24),
and glycogen synthase kinase 3 GSK3 GSK3 Although there are data to suggest that tau is both a primed and
unprimed substrate of GSK3 Cell Culture--
CHO cells were grown in F-12 medium
supplemented with 5% fetal bovine serum (Cyclone), 20 mM
L-glutamine (Invitrogen), 10 units/ml penicillin
(Invitrogen), and 100 units/ml streptomycin (Invitrogen). Cells
were used at a confluency of 50-80% for all experiments.
Plasmid Constructs--
The wild type tau (4R-Tau; +exon 10,
The cytomegalovirus expression vectors for p25 and cdk5 were generous
gifts from Dr L.-H. Tsai (24). The cdk5 insert was subcloned into the
BamHI site of pcDNA3.1(+) (48). The wild type HA-GSK3 Transient Transfections--
4R-Tau, wild type HA-GSK3 Immunoblotting--
Cells were collected in lysis buffer (150 mM NaCl, 10 mM Tris-HCl, 1 mM EGTA,
1 mM EDTA, 0.2 mM sodium vanadate, 0.5%
Nonidet P-40), containing 1 mM phenylmethylsulfonyl
fluoride, 0.1 µM okadaic acid, and a 10 µg/ml
concentration each of aprotinin, leupeptin, and pepstatin. Lysates were
sonicated on ice and centrifuged, and protein concentrations in the
supernatants were determined using the bicinchoninic acid assay
(Pierce). 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 (Tau5 from Dr. L. Binder),
which are phospho-independent tau antibodies (49-51), AT180
(Innogenetics), which recognizes tau when it is phosphorylated at
Thr-231 (52, 53), and PHF-1 (from Dr. P. Davies), which recognizes tau
phosphorylated at Ser-396/404 (54). The monoclonal GSK3 Preparation of Primed Recombinant Tau--
To prephosphorylate
(prime) tau to use as a GSK3 Measurement of GSK3
In indicated experiments, the activity of GSK3 Fractionation--
Cell were rinsed once with warm
phosphate-buffered saline and once with warm extraction buffer (80 mM PIPES, pH 6.8, 1.0 mM MgCl2, 2.0 mM EGTA, 30% glycerol, 10 mM benzamidine, 50 µg/ml leupeptin, 1.0 mM phenylmethylsulfonyl fluoride,
and 0.5 µM okadaic acid). Cells were then incubated in
300 µl (for a 60-mm plate) of extraction buffer containing 0.1%
Triton X-100 for 10 min at 37 °C. Cells were transferred into
microcentrifuge tubes and centrifuged for 2 min at 16,000 × g. The supernatant was removed and incubated in a boiling
water bath for 10 min, whereas the pellet was resuspended in 100 µl
of 2× SDS stop buffer, without dithiothreitol or dye, and sonicated.
Protein determinations were made, and samples were diluted with 2× SDS
stop buffer, boiled, electrophoresed on SDS-polyacrylamide gels, and
immunoblotted as described above.
Microtubule-binding Assay Using Cell Lysates--
The
microtubule-binding assay was carried out as described previously with
a few modifications (6). Cells were collected and resuspended in 80 mM PIPES/KOH (pH 6.8) and 1 mM
CaCl2 and protease and phosphatase inhibitors. Cell
suspensions were sonicated briefly on ice and incubated on ice for 15 min. Samples were brought to 1.5 mM EGTA and centrifuged at
100,000 × g for 1 h at 4 °C. Equal amounts of
protein lysate were used in each binding assay. Supernatants were
adjusted to 1 mM GTP and 10 µM taxol and
incubated with taxol-stabilized microtubules (30 µM)
prepared from rat brain (56) in a final volume of 30 µl for 10 min at
37 °C. The mixtures were centrifuged through 100 µl of 30% (w/v)
sucrose cushions in 80 mM PIPES/KOH 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 was collected and diluted with 2× SDS
stop buffer, and the pellet was resuspended in 50 µl of 2× SDS stop
buffer prior to incubation in a boiling water bath for 5 min. The
unbound (supernatant) and bound (pellet) fractions were separated by
electrophoresis on a 10% SDS-polyacrylamide gel, transferred to
nitrocellulose, and immunoblotted as described above.
Microtubule-binding Assay Using Recombinant Tau--
CHO cells
were transfected with HA-GSK3 Wild Type and GSK3 GSK3 Tau Phosphorylated at a Primed Site Is Only Present in the Soluble
Fraction--
To determine whether there is a differential
distribution of tau phosphorylated at primed (AT180) and unprimed
(PHF-1) sites, cells were transfected with tau alone or tau with one of
the GSK3 Primed Phosphorylation Sites of Tau Regulate the Intracellular
Localization of Tau--
The next experiment was to determine how
GSK3 Phosphorylation of Tau at Unprimed Sites by GSK3
To further confirm that the phosphorylation of tau at primed sites
plays an important role in regulating the interaction of tau with
microtubules, recombinant tau was phosphorylated with immunoprecipitated HA-GSK3 The aim of this study was to gain insight into the site-specific
tau phosphorylation by GSK3 It has been demonstrated unequivocally that GSK3 GSK3 There is good evidence that in situ GSK3 (GSK3
)
phosphorylates substrates, including the microtubule-associated protein
tau, at both primed and unprimed epitopes. GSK3
phosphorylation of
tau negatively regulates tau-microtubule interactions; however the
differential effects of phosphorylation at primed and unprimed epitopes
on tau is unknown. To examine the phosphorylation of tau at primed and
unprimed epitopes and how this impacts tau function, the R96A mutant of
GSK3
was used, a mutation that prevents phosphorylation of
substrates at primed sites. Both GSK3
and GSK3
-R96A
phosphorylated tau efficiently in situ. However, expression
of GSK3
-R96A resulted in significantly less phosphorylation of tau
at primed sites compared with GSK3
. Conversely, GSK3
-R96A
phosphorylated unprimed tau sites to a significantly greater extent
than GSK3
. Prephosphorylating tau with cdk5/p25 impaired the ability
of GSK3
-R96A to phosphorylate tau, whereas GSK3
-R96A
phosphorylated recombinant tau to a significantly greater extent than
GSK3
. Moreover, the amount of tau associated with microtubules was
reduced by overexpression of GSK3
but only when tau was
phosphorylated at primed sites, as phosphorylation of tau by
GSK3
-R96A did not negatively regulate the association of tau with
microtubules. These results demonstrate that GSK3
-mediated phosphorylation of tau at primed sites plays a more significant role in
regulating the interaction of tau with microtubules than phosphorylation at unprimed epitopes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(GSK3
) (25-29) have been
shown to phosphorylate tau in situ. Although all these
kinases can phosphorylate tau in situ, there is emerging evidence that GSK3
may play a major role in regulating tau
phosphorylation both in physiological and pathological conditions.
is expressed ubiquitously and found at high levels in brain
where it localizes predominantly to neurons (30). GSK3
is a
critically important protein kinase in brain as it phosphorylates a
number of important substrates including transcription factors such as
-catenin, cAMP-response element-binding protein, and cytoskeletal proteins such as MAP1B and tau, which regulates
their function (31). Although numerous GSK3
substrates have been identified, the consensus sites recognized by GSK3
are not
straightforward. GSK3
can phosphorylate unprimed sites at
Ser/Thr-Pro motifs. Examples of unprimed substrates are axin and
adenomatous polyposis coli gene product (APC) (32-34). In other cases
the substrate must first be phosphorylated at a site that is four amino
acids C-terminal to the target site, which primes the substrate
for phosphorylation by GSK3
. This primed motif
((S/T)XXX(S/T)[P]) often occurs in proline-rich
regions of the substrate (32, 35). Glycogen synthase (36), eukaryotic
protein synthesis initiation factor 2B (37), and
-catenin (38) are
all primed substrates. Indeed, it appears as if the majority of GSK3
substrates are actually primed (35). Interestingly, although GSK3
phosphorylates tau at unprimed sites (39, 40), prior phosphorylation by
cdk5/p25[p25] enhances the phosphorylation of tau at specific sites
(e.g. Thr-231) (41, 42), indicating that tau may be both a
primed and unprimed substrate of GSK3
.
activity is regulated by its phosphorylation state.
Phosphorylation of GSK3
on Tyr-216 increases its activity (43), and
phosphorylation at Ser-9 inhibits kinase activity (44, 45), and both
these events are regulated by growth factor signaling (31). In response
to insulin or insulin-like growth factor, Akt is activated, and this
kinase phosphorylates GSK3
on Ser-9 decreasing its activity (46).
Recently, it was demonstrated that phosphorylation of Ser-9 results in
the N-terminal of GSK3
acting as a pseudosubstrate, occupying the
same binding site used by primed substrates (32, 44, 47). It was also
found that Arg-96 in GSK3
is critically important in forming the
binding pocket for the phosphate group of the primed substrates (or
pseudosubstrate) (32, 44, 47). Mutation of Arg-96 to Ala decreased the
ability of GSK3
to phosphorylate primed substrates significantly but had no effect on its ability to phosphorylate unprimed sites (32).
in situ, this has not been
demonstrated clearly. Further, how phosphorylation of tau by GSK3
on
different sites modulates its association with microtubules has not
been explored thoroughly. By using wild type GSK3
and GSK3
with
the R96A or S9A mutation we demonstrate that GSK3
phosphorylates tau
at both primed sites (pThr-231) and unprimed sites (pSer-396/404). Disruption of the GSK3
phosphate-binding pocket (R96A mutation) results in a significant decrease in tau phosphorylation at the primed
site of Thr-231 and an enhancement of phosphorylation at the unprimed
site of Ser-396/404. Further, GSK3
-R96A phosphorylates recombinant
tau to a significantly greater extent than wild type or GSK3
-S9A;
however if tau is first phosphorylated by cdk5/p25 then GSK3
-R96A is
significantly less effective than wild type or GSK3
-S9A in
phosphorylating tau. Finally, GSK3
-mediated phosphorylation of tau
at only the primed sites decreases tau-microtubule interactions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
exons 2 and 3) construct was a generous gift from Dr. M. Hutton (Mayo
Clinic, Jacksonville, FL). 4R-Tau was subcloned into mammalian
expression vector, pCDNA 3.1(
) (Invitrogen). By using the wild
type tau construct as a template, PCR was performed (forward primer,
5'-GCC GAA TTC TAT GGC TGA GCC CCG CCA G-3'; reverse primer, 5'-CGG GGT
ACC TCA CAA ACC CTG CTT GGC-3'), and the PCR reaction product was
digested with EcoRI and KpnI and ligated into the
EcoRI and KpnI sites of the pCDNA 3.1(
).
The integrity of the 4R-tau construct was confirmed by sequence analysis.
was a generous gift from Dr. J. Woodgett (Ontario Cancer Institute). To
generate HA-GSK3
-S9A and HA-GSK3
-R96A site-directed mutagenesis
using the QuikChangeTM site-directed mutagenesis kit (Stratagene) was
carried out with the following primer pairs: HA-GSK3
-S9A, forward
primer, 5'-GCT CTC CGC AAA GGC GGT TCT GGG C-3'; reverse primer, 5'-GCC
CAG AAC CAC CGC CTT TGC GGA GAG C-3'; HA-GSK3
-R96A, forward primer,
5'-GAC AAG AGA TTT AAG AAT GCA GAG CTC CAG ATC ATG-3'; reverse primer,
5'-CAT GAT CTG GAG CTC TGC ATT CTT AAA TCT CTT GTC-3'. Mutations were verified by DNA sequence analysis.
,
HA-GSK3
-S9A, or HA-GSK
-R96A were transiently transfected into CHO
cells using FuGENE 6 (Roche Molecular Biochemicals) transfection
reagent according to the manufacturer's protocol. 36 h after
transfection, the cells were washed with ice-cold phosphate-buffered
saline and then collected and processed as described below for the
different assays.
was
purchased from Transduction Laboratories, the monoclonal cdk5
antibody was from Santa Cruz Biotechnology, the monoclonal
-tubulin
antibody was from Sigma, the monoclonal
-actin antibody was from
Chemicon, and the monoclonal HA antibody was from Roche Molecular
Biochemicals. After incubation with the appropriate horseradish
peroxidase-conjugated secondary antibody (Jackson ImmunoResearch
Laboratories), the blots were developed using enhanced
chemiluminescence (Amersham Biosciences).
substrate, cdk5 and p25 were
co-expressed in CHO cells, and cdk5 was immunoprecipitated. 120 µg of
the protein extract (1 µg/µl) was incubated with 2 µl of rabbit
polyclonal anti-cdk5 antibody (Santa Cruz Biotechnology) overnight at
4 °C with gentle agitation. Extracts were then incubated with 40 µl of washed protein A-Sepharose (Sigma) for 2 h at 4 °C. The
phosphorylation reaction was carried out by mixing immunoprecipitated cdk5/p25 with 40 µl of kinase buffer containing 20 mM
Tris, pH 7.5, 5 mM MgCl2, 1 mM
dithiothreitol, 250 µM ATP, and 0.2 µg/µl recombinant
tau protein (Takara/Panvera, Madison, WI). For the negative control,
the reaction was performed without ATP in the kinase buffer. The
samples were incubated at 30 °C for 30 min with shaking and then
spun to pellet the cdk5/p25 immune complex. The supernatants containing
the recombinant tau were collected and dialyzed to remove free ATP
using Slide-A-Lyzer mini dialysis units (Pierce). Following dialysis,
protein concentrations were determined by using the bicinchoninic acid
assay. The supernatants were immunoblotted for cdk5 to confirm that
there was no cdk5 present.
Activity--
To immunoprecipitate the
HA-GSK3
constructs, 70 µg of protein from cellular extracts (1 µg/µl) prepared in lysis buffer was incubated with 2 µl of mouse
monoclonal HA antibody overnight at 4 °C with gentle agitation.
Extracts were then incubated with 30 µl of washed protein G-Sepharose
(Sigma) for 2 h at 4 °C. The immobilized immune complexes were
washed twice with lysis buffer and twice with kinase buffer (20 mM Tris, pH 7.5, 5 mM MgCl2, and 1 mM dithiothreitol). Kinase activity was measured by mixing immunoprecipitated GSK3
with 30 µl of kinase buffer containing 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM dithiothreitol, 250 µM ATP, 1.4 µCi of
[
-32P]ATP (Amersham Biosciences), and 0.1 µg/µl
recombinant tau protein (in some experiments prephosphorylated tau was
used). The samples were incubated at 30 °C for 30 min, and 30 µl
of 2× SDS stop buffer was added to each sample to stop the reaction.
Samples were placed in a boiling water bath for 5 min, and proteins
were separated on 10% SDS-polyacrylamide gels. The gels were
vacuum-dried, exposed to a phosphoscreen overnight, and quantitated
using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The
efficiency of HA immunoprecipitation was determined by immunoblotting
for GSK3
.
was measured using a
primed peptide substrate, phosphoglycogen synthase peptide-2 (Upstate
Biotechnology) as described previously (55), except that the peptide
concentration used was 25 µM, and the reaction time was
20 min. A volume of 10 µl of the 30-µl reaction mixture was then
spotted in triplicate on P81 filter paper (Whatman). The filters were
washed three times with 0.5% phosphoric acid for 15 min each and one
time with 95% ethanol for 15 min and dried, and 32P
incorporation into the peptide was measured by scintillation counting.
, HA-GSK3
-S9A, HA-GSK
-R96A, or
vector only as the control. To immunoprecipitate the HA-tagged
constructs, 400 µg of protein from cellular extracts was incubated
with 4 µl of mouse monoclonal HA antibody overnight at 4 °C with
gentle agitation followed by incubation with 40 µl of washed protein
G-Sepharose for 2 h at 4 °C. The immobilized immune complexes
were washed twice with lysis buffer and twice with kinase buffer. The
phosphorylation reaction was carried out by mixing the
immunoprecipitates with 20 µl of kinase buffer and 0.08 µg/µl
recombinant tau protein. The samples were incubated at 30 °C for
1 h with shaking and then spun to pellet the GSK3
immune
complex. Phosphorylated or control tau (100 ng) was diluted in 80 mM PIPES/KOH (pH 6.8), 1.5 mM EGTA. The samples
were adjusted to 1 mM GTP and 10 µM taxol and
incubated with taxol-stabilized microtubules (20 µM) in a
final volume of 30 µl for 10 min at 37 °C. The mixtures were
centrifuged through 100 µl of 30% (w/v) sucrose cushions in 80 mM PIPES/KOH 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 and
pellet were collected and processed as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-R96A Phosphorylate Tau
Differentially--
To determine whether GSK3
differentially
phosphorylates tau when the phosphate-binding pocket of GSK3
is
disrupted, cells were transfected with tau alone or with tau and
HA-GSK3
, HA-GSK3
-R96A, or HA-GSK3
-S9A. Lysates were collected
and immunoblotted with the phosphate-independent tau antibodies
Tau5/5A6, AT180, which recognizes tau when Thr-231 is phosphorylated (a
primed site), PHF-1, which recognizes tau when Ser-396/404 are
phosphorylated (unprimed sites), a GSK3
antibody, or a
-actin
antibody. In the absence of exogenous GSK3
tau migrated as a doublet
that was not recognized by either AT180 or PHF-1 (Fig.
1). Co-expression of either the wild type
or mutant GSK3
resulted in a significant decrease in the mobility of
tau; however in the presence of HA-GSK3
or HA-GSK3
-S9A tau
migrated as a distinct doublet. In contrast, in the presence of
HA-GSK3
-R96A, tau always presented as a blurred band(s); two
distinct tau bands were never observed (also see Fig. 5B).
This differential migration of tau in the presence of HA-GSK3
-R96A
compared with HA-GSK3
or HA-GSK3
-S9A is likely because of the
differential phosphorylation of tau by HA-GSK3
-R96A. Expression of
either HA-GSK3
or HA-GSK3
-S9A with tau resulted in a robust
increase in AT180 immunoreactivity and to a lesser extent increased
PHF-1 staining. In contrast to these findings, expression of
HA-GSK3
-R96A resulted in a large increase in PHF-1 immunoreactivity
and only a minimal increase in AT180 immunoreactivity (Fig. 1). In all
cases wild type and mutant GSK3
were expressed at similar levels
(Fig. 1). These results suggest that tau is both a primed and unprimed
substrate of GSK3
.
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Fig. 1.
GSK3 phosphorylates
tau at both primed and unprimed sites. CHO cells were transiently
transfected with tau alone or tau and either HA-GSK3
(GSK3
), HA-GSK3
-S9A (S9A), or
HA-GSK3
-R96A (R96A). HA-GSK3
-S9A and HA-GSK3
-R96A
were expressed at similar levels compared with wild type HA-GSK3
.
The phospho-independent antibodies Tau5/5A6 show total tau levels in
the transfected cells were approximately the same for all conditions.
Tau5/5A6 immunoblotting revealed that the expression of the GSK3
constructs reduced the electrophoretic mobility of tau significantly,
indicating an increase in phosphorylation. In cells overexpressing
HA-GSK3
or HA-GSK3-S9A AT180 immunoreactivity (primed site,
phosphorylated Thr-231) was increased significantly; in contrast,
expression of HA-GSK3
-R96A resulted in significantly less AT180
immunoreactivity. Expression of HA-GSK3
or HA-GSK3-S9A also
increased PHF-1 immunoreactivity (unprimed sites, phosphorylated
Ser-396/404), but expression of HA-GSK3
-R96A resulted in a much
greater increase in tau phosphorylation at this epitope. Actin blots
are included to show equal protein loading for all the samples.
-R96A Phosphorylates Recombinant Tau Efficiently--
The
in vitro activity of the different GSK3
constructs toward
primed and unprimed substrates was examined. Cells were transfected with HA-GSK3
, HA-GSK3
-R96A, or HA-GSK3
-S9A immunoprecipitated with the HA antibody and used in the in vitro kinase assays.
All the GSK3
constructs efficiently phosphorylated recombinant tau; however HA-GSK3
-R96A phosphorylated recombinant tau ~4-fold more than HA-GSK3
or HA-GSK3
-S9A (Fig.
2A). In contrast, and as
expected (32), HA-GSK3
-R96A was extremely inefficient in
phosphorylating a primed peptide, showing activity that was less than
20% of the activity of HA-GSK3
or HA-GSK3
-S9A (Fig.
2B). To determine whether GSK3
phosphorylates primed tau
in a manner similar to the primed peptide, tau was prephosphorylated
with cdk5/p25 as described under "Experimental Procedures," prior
to use in the in vitro kinase assay. After preparation of
the primed or control tau (no ATP in the reaction), no cdk5 was present
in the tau fractions as confirmed by immunoblotting for cdk5 (data not
shown). Priming of recombinant tau prior to phosphorylation by the
GSK3
constructs demonstrated that HA-GSK3
-R96A was not as
efficient as HA-GSK3
or HA-GSK3
-S9A in phosphorylating primed tau
(Fig. 2C, left panel). In contrast,
HA-GSK3
-R96A phosphorylated the control, mock phosphorylated tau
much more efficiently than HA-GSK3
or HA-GSK3
-S9A (Fig. 2C, right panel). These results demonstrate
clearly that both in situ and in vitro tau is
both a primed and unprimed substrate of GSK3
.
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Fig. 2.
R96A-GSK3
phosphorylates unprimed tau but does not phosphorylate primed
substrates efficiently. HA-GSK3
(GSK3
),
HA-GSK3
-S9A (S9A), or HA-GSK
-R96A (R96A)
were expressed in cells, immunoprecipitated, and used in in
vitro kinase assays. A, HA-GSK3
-R96A phosphorylates
recombinant tau to a significantly greater extent than HA-GSK3
or
HA-GSK3-S9A. Data are presented as a % of HA-GSK3
activity. Shown
is the mean ± S.E. (n = three separate
experiments). *, p < 0.05 compared with HA-GSK3
.
B, HA-GSK3
-R96A phosphorylates a primed peptide to a
significantly lesser extent than HA-GSK3
or HA-GSK3-S9A. Data are
presented as a % of HA-GSK3
activity. Shown is the mean ± S.E. (n = three separate experiments). *,
p < 0.05 compared with HA-GSK3
. C,
HA-GSK3
and HA-GSK3
-S9A phosphorylate primed tau more efficiently
compared with HA-GSK3
-R96A. To prime tau, tau was prephosphorylated
with cdk5/p25 that was immunoprecipitated from cells in which cdk5 and
p25 were co-expressed (left panel). As a control (unprimed
tau), tau was incubated with the cdk5/p25 immunoprecipitate but in the
absence of ATP (right panel). The panels below
each graph show the autoradiographs for the experiment. The
data were expressed as a percent of HA-GSK3
activity and are
representative of results obtained from two to three separate
experiments. These findings demonstrate clearly that priming of tau
with cdk5/p25 diminishes the ability of HA-GSK3
-R96A to
phosphorylate tau.
constructs, and the detergent-insoluble cytoskeleton was
separated from the detergent-soluble fraction. These results (Fig.
3) demonstrate clearly that tau
phosphorylated at primed sites (AT180) is only present in the soluble
fractions, whereas tau phosphorylated at unprimed sites (PHF-1) is
present in both the soluble and insoluble fractions.
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Fig. 3.
Tau that is phosphorylated at AT180 (a primed
site) is in the soluble fraction. CHO cells were transiently
transfected with tau alone or tau and HA-GSK3 (GSK3
),
HA-GSK3
-S9A (S9A), or HA-GSK3
-R96A (R96A).
Cell lysates were separated into cytoskeletal and soluble fractions as
described under "Experimental Procedures." Insoluble cytoskeletal
(Insol) and soluble (Sol) fractions were
electrophoresed and immunoblotted with indicated antibodies. These data
demonstrate that tau phosphorylated at the primed epitope AT180 is only
present in the soluble fractions, whereas PHF-1 immunoreactivity
(unprimed site) is distributed equally between the cytoskeletal and
soluble fractions in cells that were transfected with the GSK3
constructs. Further, AT180 immunoreactivity in cells transfected with
HA-GSK3
-R96A was substantially less than in cells transfected with
HA-GSK3
or HA-GSK3
-S9A, whereas PHF-1 immunoreactivity was
greater.
-mediated phosphorylation of tau at primed and unprimed sites
regulates the association of tau with the cytoskeleton. Cells were
transfected with tau alone or with tau and one of the GSK3
constructs, and the cells were fractionated, and the distribution of
tau between the soluble and insoluble fractions was determined (Fig.
4). Co-expression of HA-GSK3
or
HA-GSK3
-S9A with tau increased the amount of tau in the soluble
fraction compared with what was observed in cells transfected with tau
alone, indicating that GSK3
changed the binding affinity of tau for
the cytoskeleton/microtubules (Fig. 4). In contrast co-expression of
HA-GSK3
-R96A with tau did not result in an obvious increase in
soluble tau levels (Fig. 4). These data suggest that phosphorylation of
tau at primed sites may regulate the association of tau with
microtubules, whereas GSK3
-mediated phosphorylation at unprimed
sites does not significantly impact tau-microtubule interactions.
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Fig. 4.
Phosphorylation of tau at primed sites
results in increased tau levels in the soluble fraction. CHO cells
were transiently transfected with tau alone or tau and HA-GSK3
(GSK3
), HA-GSK3
-S9A (S9A), or
HA-GSK3
-R96A (R96A). Cell lysates were separated into
cytoskeletal insoluble (Insol) and soluble (Sol)
fractions as described under "Experimental Procedures."
A, representative immunoblots showing that expression of
either HA-GSK3
or HA-GSK3
-S9A increases the amount of tau in the
soluble fraction. In contrast expression of HA-GSK3
-R96A does not
increase soluble tau levels significantly compared with that observed
in cells transfected with tau alone. B, quantitative data
demonstrating the increase in soluble tau in cells transfected with
HA-GSK3
or HA-GSK3
-S9A but not with HA-GSK3
-R96A compared with
cells transfected with tau only (control).
-R96A Does Not
Decrease Microtubule Binding--
To further determine the functional
significance of tau phosphorylation by GSK3
at primed and unprimed
sites, a microtubule sedimentation assay was performed. High speed
supernatants from cells transfected with tau alone or tau and
HA-GSK3
, HA-GSK3
-R96A, or HA-GSK3
-S9A 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
was measured. In the absence of GSK3
, all the tau was in the
microtubule-bound pellet. In contrast, when tau was co-expressed with
HA-GSK3
or HA-GSK3
-S9A a significant amount of tau was observed
in the unbound fraction and was recognized by AT180 (Fig.
5A). Interesting, no PHF-1
immunoreactivity was observed in the unbound fractions. Further,
co-expression of tau and HA-GSK3
-R96A did not result in an increase
in unbound tau even though there was a significant increase in PHF-1
immunoreactivity in the bound fraction. Fig. 5B shows that
the expression of tau and GSK3
was equivalent in all conditions.
These results indicate that phosphorylation of tau by GSK3
at primed
sites is necessary to decrease the affinity of tau for microtubules,
whereas phosphorylation at unprimed sites, such as PHF-1, does not
significantly effect tau-microtubule interactions.
View larger version (44K):
[in a new window]
Fig. 5.
Phosphorylation of tau by
GSK3 at primed sites results in decreased
microtubule binding. CHO cells were transiently transfected with
tau alone or tau and HA-GSK3
(GSK3
), HA-GSK3
-S9A
(S9A), or HA-GSK3
-R96A (R96A), and high speed
supernatants from the cells were used in a microtubule-binding assay.
A, tau is presented in soluble unbound fraction when it was
phosphorylated by HA-GSK3
or HA-GSK3
-S9A. Taxol-stabilized
microtubules were added to the high speed supernatants, the
microtubules were pelleted, and the distribution of tau between the
microtubule pellet (Bound) and supernatant
(Unbound) was determined by immunoblotting with several tau
antibodies. These data demonstrate that tau phosphorylated by
GSK3
-R96A remains bound to the microtubules. In contrast, tau
phosphorylated by HA-GSK3
, and to a greater extent GSK3
-S9A,
shifted into the unbound supernatant. Further, AT180 immunoreactivity
was present only in the unbound fractions. PHF-1 immunoreactivity was
present only in the microtubule-bound fractions. B, total
tau and GSK3
expression levels, as well as the phosphorylation state
of tau, were evaluated in the high speed supernatants prior to use in
the microtubule-binding assay. These data show that tau and the GSK3
constructs were all expressed at similar levels.
, HA-GSK3
-S9A, or HA-GSK3
-R96A or with immunoprecipitates from vector-transfected cells. The
phosphorylated recombinant tau was then used in a microtubule-binding
assay, and the amount of bound and free tau was determined. These
results (Fig. 6) again demonstrate that
phosphorylation of tau by HA-GSK3
or HA-GSK3
-S9A increased the
amount of unbound tau compared with controls, whereas tau
phosphorylation by HA-GSK3
-R96A did not increase the level of
unbound tau.
View larger version (22K):
[in a new window]
Fig. 6.
In vitro assay demonstrating that
phosphorylation of tau at primed sites by GSK3
decreases microtubule binding by tau. CHO cells were
transiently transfected HA-GSK3
(GSK3
), HA-GSK3
-S9A
(S9A), HA-GSK3
-R96A (R96A), or vector only
(
). Immunoprecipitations were carried out using an HA antibody, and
the precipitates were used to phosphorylate recombinant tau. A
microtubule-binding assay was carried out using the phosphorylated
recombinant tau and taxol-stabilized microtubules, and the bound and
unbound fractions were immunoblotted for the presence of tau (Tau5/5A6)
or tubulin. These results demonstrate that phosphorylation of tau
in vitro by HA-GSK3
or HA-GSK3
-S9A decreases the
association of tau with microtubule, whereas phosphorylation with
HA-GSK3
-R96A does not alter tau-microtubule interactions
significantly.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and to determine how phosphorylation of
specific epitopes on tau affects its function. Our results clearly
demonstrate that GSK3
phosphorylates tau on both primed and unprimed
sites; however it is the phosphorylation of tau at the primed epitopes
that decreases tau-microtubule interactions.
phosphorylates tau
in situ; however most studies have focused the
phosphorylation of unprimed sites such as PHF-1, Tau-1, and AT8 (25,
28, 57-59), although in a transgenic mouse model that expressed both
human tau and GSK3
-S9A an increase in AT180 immunoreactivity was
noted (59). In vitro it has been demonstrated that AT180
recognition of tau requires Thr-231 to be phosphorylated, and GSK3
can phosphorylate tau on this site only when Ser-235 is already
phosphorylated (53). Therefore, AT180 recognizes a primed GSK3
epitope. In situ we have shown that disruption of the
priming phosphate-binding pocket in GSK3
by making the R96A mutation
(32, 44) impairs the ability of GSK3
to phosphorylate tau at the
AT180 epitope significantly, confirming the in vitro
findings. Further, priming of recombinant tau by prephosphorylating it
with cdk5/p25 (41, 42) results in a decrease in tau phosphorylation by
the GSK3
-R96A mutant compared with wild type GSK3
and GSK3
with the S9A mutation. Interestingly, GSK3
and GSK3
-S9A
phosphorylated tau to a similar extent; however GSK3
-R96A
phosphorylated recombinant tau much more efficiently than both GSK3
and GSK3
-S9A. These data indicate that the enhancement of tau
phosphorylation by the GSK3
-R96A mutant is not because of the
inability of GSK3
-R96A to be inhibited by the pseudosubstrate of the
phosphorylated N-terminal (32, 44). In addition, tau phosphorylation at
the non-primed PHF-1 epitope was much greater when cells were
co-transfected with HA-GSK3
-R96A compared with HA-GSK3
or
HA-GSK3
-S9A. The reason for the enhancement in non-primed tau
phosphorylation by the GSK-R96A mutant is unknown, but may be because
of an increase in the accessibility of tau to the active site. However,
the R96A mutation of GSK3
did not affect phosphorylation of axin or
-catenin (32).
is a constitutively active kinase that is inactivated by Ser-9
phosphorylation. Engagement of signaling pathways that activate Akt
results in GSK3
Ser-9 phosphorylation and down-regulation of GSK3
activity. In addition, other protein kinases such as protein kinase C
and mitogen-activated protein kinase-activated protein kinase 1 can phosphorylate Ser-9 on GSK3
and down-regulate its activity (31).
In almost all experiments in this study, GSK3
-S9A showed slightly,
but not significantly, more activity than GSK3
. The likely reason
for this observation is that all measures were done under basal
conditions and not in the presence of signals that would activate Akt,
and therefore the effects of the S9A mutation on GSK3
activity were
not prominent.
-mediated tau
phosphorylation decreases the affinity of tau for microtubules. For example, microtubule depolymerization in response to colchicine treatment was greater in cells transfected with tau and GSK3
compared with cells transfected with just tau (57, 60), and cells
co-transfected with tau and GSK3
exhibited a reduction in
microtubule bundling compared with cells transfected with tau alone
(26). Further, in mice transgenic for GSK3
-S9A and tau increased tau
phosphorylation occurred, as well a reduction in the axonopathy that
was observed in the mice that just overexpressed human tau (59). The
reduction in the axonopathy in response to increased expression of
GSK3
-S9A is likely because of the fact that the increase in tau
phosphorylation resulted in a decrease in tau-microtubule interactions,
which thus alleviated the inhibition in axonal transport because of tau
overexpression (3, 59). Although it is clear that tau is phosphorylated
by GSK3
on numerous sites, the contribution of these different sites
to the regulation of tau function in situ has not been
explored thoroughly. In this study we show that although GSK3
-R96A
phosphorylates tau in situ at non-primed sites efficiently,
and this results in a decrease in the electrophoretic mobility of tau,
this did not result in an increase in the amount of free,
non-microtubule-bound tau. This is in contrast to wild type GSK3
or
GSK3
-S9A, which did cause an increase in the levels of unbound tau.
Further, AT180 immunoreactivity was only found in the soluble or
unbound fraction, indicating that phosphorylation within this epitope
and/or at other primed sites may play an important role in regulating
the interaction of tau with microtubules. In contrast, PHF-1
immunoreactivity was found exclusively in the bound fractions (Fig.
5A), indicating that phosphorylation of tau at this epitope
and/or at other unprimed GSK3
sites plays less of a role in
regulating tau-microtubule interaction. The finding that GSK3
phosphorylation of primed sites on tau may play a more significant role
in regulating the function of tau than unprimed sites is quite
intriguing given the fact that the majority of GSK3
substrates are
primed (35). Additionally, recent data have shown that
-catenin,
which was believed previously to be an unprimed substrate of GSK3
,
is actually primed by casein kinase I
(61). Further, different
substrates of GSK3
require different priming protein kinases, such
as protein kinase A for cAMP-response element-binding protein and
casein kinase II for glycogen synthase (31) and of course casein kinase I
for
-catenin (61). Therefore one can speculate that the most
functionally relevant GSK3
-mediated tau phosphorylation events are
at primed epitopes and that perhaps protein kinase(s) like cdk5/p25 may
play an essential role in regulating GSK3
-mediated tau
phosphorylation (41, 42). Indeed, GSK3
and cdk5/p35[p25] co-purify
together with tau and microtubules and were referred to originally as
tau protein kinase I and II, respectively (62). A dual kinase system
for the regulation of tau phosphorylation by GSK3
would give a high
degree of control for GSK3
substrate selectivity and provide for the
needed regulation of tau function in the cell. Further studies are
required to fully elucidate the role of GSK3
-mediated tau
phosphorylation at both primed and unprimed sites and how these
site-specific phosphorylation events affect tau function.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. L. Binder and Dr. P. Davies for generous antibody gifts, and Dr. J. Woodgett, Dr. M. Hutton, and Dr. L.-H. Tsai for generous gifts of the constructs.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant NS35060 and by a grant from the Alzheimer's Disease Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a fellowship from the John Douglas French
Alzheimer's Foundation.
§ To whom correspondence should be addressed: Dept. of Psychiatry, 1720 7th Ave. S., SC1061, University of Alabama at Birmingham, School of Medicine, Birmingham, AL 35294-0017. Tel.: 205-934-2465; Fax: 205-934-3709; E-mail: gvwj@uab.edu.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M206236200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
cdk5, cyclin-dependent protein kinase 5;
GSK3, glycogen
synthase kinase 3
;
CHO, Chinese hamster ovary;
HA, hemagglutinin;
PIPES, 1,4-piperazinediethanesulfonic acid.
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