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INTRODUCTION |
Microtubules, the major cytoskeletal structures of eukaryotic
cells, are dynamic structures, and their assembly and disassembly is
regulated by microtubule-associated proteins (1). In neurons, tau is
one of the major microtubule-associated proteins and is mainly found in
the axonal compartment (for reviews, see Refs. 1-3). Tau binds to
microtubules and stabilizes microtubule structure. Studies suggest that
tau regulates microtubule dynamics, axonal transport, and neuronal
morphology by binding and stabilizing the microtubule structure (1-3).
There are six tau isoforms, which migrate with sizes 45-65 kDa
on an SDS-polyacrylamide gel. These isoforms are phosphorylated on
multiple sites in the brain and display a characteristic retarded
mobility on an SDS gel upon phosphorylation (2, 3). Tau phosphorylation
reduces the affinity of tau for microtubules and is one of the
mechanisms that control microtubule structure and dynamics in
vivo (1-3).
In Alzheimer's disease (AD)1
brain, abnormally hyperphosphorylated tau accumulates and forms paired
helical filaments (4, 5). Since abnormally phosphorylated tau does not
bind to microtubules, abnormal tau phosphorylation in AD brain is
thought to cause a loss of tau function, microtubule dysfunction, and
neurodegeneration (2, 3). It is not understood how abnormally
phosphorylated tau accumulates in AD brain, but a defect in the
regulatory mechanism that controls tau
phosphorylation/dephosphorylation is very likely to be involved. The
elucidation of the regulatory mechanism that controls tau
phosphorylation in normal brain and the determination of how this
regulation fails in AD brain are essential steps in understanding
disease ontogeny and developing therapeutic interventions.
Glycogen synthase kinase-3 (GSK3) is an important regulatory enzyme
that phosphorylates numerous substrates and regulates diverse
physiological processes such as glycogen metabolism, gene expression,
apoptosis, signal transduction, and cell fate specification (6-8).
There are two isoforms of GSK3 that are highly expressed in the brain:
~51-kDa GSK3
and ~47-kDa GSK3
(9). In transfected cells and
transgenic mice, enhanced expression of GSK3
leads to tau
phosphorylation and microtubule instability (10-15). In AD brain,
GSK3
is activated in pretangle neurons and accumulates in paired
helical filaments (16, 17). These observations suggest that GSK3
phosphorylates tau in both normal and AD brain. Previous studies have
shown that a large amount of GSK3
in brain is associated with
microtubules (18-20), and microtubule-associated GSK3
is part of an
~400-500-kDa multiprotein complex containing tau and GSK3
(20).
These data indicate that GSK3
phosphorylates tau within a
microtubule-associated multiprotein complex (hereon designated as tau
phosphorylation complex). The enormity of the tau phosphorylation complex suggests that within the complex, there may be proteins other
than tau and GSK3
(20). The identification of all the complex
components and the determination of their functions within the complex
are essential to understanding the mechanism by which GSK3
phosphorylates tau in the brain.
14-3-3 is a family of conserved acidic proteins that are widely
expressed in all eukaryotic tissues (21, 22). There are seven 14-3-3 isoforms, which are products of distinct genes. 14-3-3 is a naturally
dimeric scaffold protein with the size of the monomer being ~30 kDa
(21). Within the 14-3-3 dimer, the ligand binding grooves of each
monomer run in opposite directions, and hence a 14-3-3 dimer can
interconnect and bring two different proteins together. 14-3-3 binds to
diverse cellular proteins, and more than 100 14-3-3 binding proteins
have been identified (21). 14-3-3 is a cofactor of bacterial
toxin Pseudomonas (23). 14-3-3 binds to Raf kinase
and regulates the mitogen-activated protein (MAP) kinase signaling
pathway (24, 25). It also binds cdc25, polyoma virus middle tumor
antigen, p53, protein kinase C, Bcr, PI3 kinase, insulin-like growth
factor, BAD, and p53 (26-37). By binding to its targets, 14-3-3 regulates enzyme activity, stabilizes enzyme conformation, controls
subcellular localization of proteins, and mediates protein-protein
interaction (21, 22). 14-3-3 regulates diverse cellular processes
including cell growth, cell differentiation, cell division, apoptosis,
and neuronal function (21, 22).
In the brain, ~1% of soluble protein is 14-3-3 and has been
suggested to be critical for brain function (21). From bovine brain
extract, 14-3-3
co-immunoprecipitates with tau (36). In
vitro, 14-3-3
binds and changes the tau conformation, thus making tau susceptible for kinase phosphorylation (36). More importantly, a substantial amount of 14-3-3
co-purifies with microtubules from the brain extract (36). These observations suggest
that 14-3-3
is an integral part of brain microtubules and is
involved in the regulation of tau phosphorylation and microtubule dynamics. However, very little information is available about microtubule-associated 14-3-3
. In this study, we have further analyzed microtubule-associated 14-3-3
. Herein we report that brain
microtubule-associated 14-3-3
is part of the tau phosphorylation complex containing GSK3
and tau. Our data indicate that 14-3-3
mediates GSK3
-tau interaction and facilitates tau phosphorylation by
GSK3
within the complex.
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MATERIALS AND METHODS |
cDNA Cloning and Plasmids--
The longest human tau isoform
in a pET-3a vector (38) was amplified by Pfu DNA
polymerase-catalyzed PCR using the forward primer (5'-AAA AAA GAA
TTC AAT GGC TGA GCC CCG C-3') containing EcoR1
(italicized) and reverse primer (5'-AAA AAA GGA TCC TCA CAA
ACC CTG CTT G-3') containing BamHI (italicized) sites.
Adenine overhangs were added to the PCR product by TaqDNA
polymerase, which was then ligated into a pGEX-T Easy vector (Promega)
for amplification (20). After amplification, the insert was
released and ligated into the EcoR1/BamHI
cloning site of FLAG-pcDNA3.1 Zeo vector (Invitrogen, Madison, WI).
Human 14-3-3
cDNA was subcloned into the
BamH1/EcoR1 site of Xpress-pcDNA3.1
(Invitrogen) as described above using 14-3-3
-pGEX-6p (36) as the
template and forward primer (5'-G GAA TTC TAT GAC AAT GGA
TAA AAG T-3') containing the EcoR1 (italicized) and
reverse primer (5'-CG GGA TCC TTA ATT TTC CCC TCC TTC-3')
containing BamHI sites. All cloning procedures were
confirmed by DNA sequencing. pcDNA3.1 containing HA
(hemagglutinin)-tagged human GSK3
was a gift from Dr. James R. Woodgett (The University of Toronto). Other expression vectors,
GSK3
-pGEX-6p, 14-3-3
-pGEX-6p, and tau-pET-3a, are described
previously (20, 36, 38).
Cell Culture and Transfection--
COS-7 and HEK-293
cells were maintained in Dulbecco's modified Eagle's medium (high
glucose) medium (Invitrogen) supplemented with 10% fetal bovine serum.
Cells were plated in 100-mm culture dishes, grown to ~80%
confluency, and transfected by standard calcium phosphate method with
various amounts of the appropriate plasmids. For each 100-mm dish,
5-10 µg of DNA was mixed with 50 µl of CaCl2 (2.5 M) to give a final volume of 500 µl with distilled water.
The mixture of DNA and CaCl2 was added to 500 µl
of 2× HEPES-buffered saline (1.63% NaCl, 1.188% Hepes, 0.02%
Na2HPO4 (pH 7.2)), and the mixture was allowed
to settle at 20 °C for 30 min. DNA mixture was added to the cells
dropwise, and cells were allowed to grow for 12-18 h. The medium was
then changed, and cells were incubated for 48-72 h.
Proteins and GSK3
Activity Assay--
Recombinant tau was
purified from bacterial extract overexpressing the longest human tau
isoform (39). GST-14-3-3
and GST-GSK3
were purified from the
respective bacterial lysates overexpressing the respective proteins
by glutathione-agarose chromatography, and the GST tag was removed as
described previously (20, 40). Polyclonal antibodies against tau,
GSK3
, and 14-3-3
have been described (20, 36). Monoclonal
antibodies against tau and GSK3
were obtained from NeoMarker
(Fremont, CA), and Transduction Laboratories (Lexington, KY),
respectively. Monoclonal anti-HA and anti-FLAG antibodies
were from Sigma. Anti-Xpress monoclonal antibody was purchased from
Invitrogen. Tau phosphorylation-sensitive monoclonal antibodies,
AT8, PHF-1, and 12E8, are described previously (20, 36). GSK3
activity assay was performed essentially as described (20).
Microtubule Assembly/Disassembly and Partial
Purification of 14-3-3
from Microtubule Fractions--
Purification
of microtubules from a fresh bovine brain extract by the
temperature-induced microtubule assembly/disassembly has been described
previously (38). Microtubule pellet obtained by centrifugation after
first, second, third, and fourth cycles of assembly/disassembly were
designated as P1, P2, P3, and P4, and the supernatants were designated
as S1, S2, S3, and S4, respectively.
For a partial purification of 14-3-3
, all procedures were carried
out at 4 °C. Microtubule pellet P3 (~4 mg) was homogenized in
~10 ml of PEM buffer (0.1 M PIPES, 1 mM EGTA,
1 mM MgSO4, and 1 mM DTT)
containing 0.1 mM GTP using a glass homogenizer and then
incubated in ice for 30 min. After incubation, the sample was
centrifuged at 27,000 × g for 20 min, and the
supernatant (~12 ml) was loaded onto a phosphocellulose (Whatman)
column (25 × 5 cm) equilibrated in PEM buffer. The column was
washed extensively, and the column-bound 14-3-3
was eluted with 200 ml of NaCl gradient (0-1 M in PEM buffer). Effluent
fractions were immunoblotted using anti-14-3-3
antibody, and those
containing 14-3-3
were combined and dialyzed against Mops
buffer (25 mM MOPS (pH 7.4), 50 mM
-glycerol phosphate, 0.1 mM EDTA, 1 mM DTT, 0.2 M NaCl, 10 mM NaF, and 15 mM
MgCl2) for 4 h. Dialyzed sample was concentrated by
Aquacide III (Calbiochem) and centrifuged at 27,000 × g for 30 min. The supernatant (~8 ml) was loaded onto an
FPLC Superose 12 (Amersham Biosciences) gel filtration column (2.6 × 50 cm), equilibrated, and eluted with Mops buffer. Effluent
fractions (1 ml each) were collected, and those containing 14-3-3
were pooled and dialyzed against 15 mM MOPS (pH 7.4), 1 mM EDTA, 20 mM NaCl, and 1 mM DTT. The dialyzed sample was loaded onto an FPLC Mono S column (Amersham Biosciences) equilibrated in 25 mM MOPS (pH 7.4), 0.1 mM EDTA, and 0.1 mM DTT. The column was washed
with the equilibration buffer and then eluted with an NaCl gradient
(0-0.5 M) in the equilibration buffer. Fractions (200 µl
each) were collected, and those containing 14-3-3
were combined
(~1.5 ml) and chromatographed through a Sepharose 4B (Sigma) gel
filtration column (2.5 × 60 cm) equilibrated and eluted with Mops
buffer. Fractions (0.5 ml each) were collected.
Immunoprecipitation and GST Pull-down Assay--
Cells in each
culture dish were suspended in 1 ml of lysis buffer (50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 25 mM
-glycerol phosphate, 1 mM EDTA, 1 mM EGTA,
10 mM NaF, 10 mM MgCl2, 1% Nonidet P-40, 100 nM okadaic acid (Sigma), 50 pM
cypermethrane (Calbiochem), 1 mM phenylmethulsulfonyl
fluoride, and 1 µg/ml each of pepstatin, leupeptin, aprotinin). The
cell suspension was incubated in ice for 1 h and then centrifuged
at 4 °C for 15 min. The supernatant was either used for
immunoprecipitation or used for GST pull-down assay.
For immunoprecipitation, the supernatant (~200 µl) was precleared
with ~50 µl of protein G-agarose beads (Sigma) equilibrated in
lysis buffer. The precleared sample was mixed with 10 µg of indicated
antibody, and the mixture was shaken end-over-end for 6 h at
4 °C. After shaking, 30 µl of protein G-agarose beads was added to
the mixture, and the shaking was continued for another 5 h. The
beads were then collected by centrifugation and washed three times (30 min each). The washed beads were dissolved in 50 µl of SDS-PAGE
sample buffer, boiled, and centrifuged, and 20 µl of supernatant was
analyzed by immunoblot analysis using the indicated antibody. The
immunoprecipitation procedure for generating Fig. 4 is essentially as
described (20).
To perform GST pull-down assay, ~50 µl of glutathione-agarose beads
(Sigma) coated with the indicated protein was incubated with 200 µl
of the cell or brain extract with end-over-end shaking for 14 h at
4 °C. After shaking, beads were washed three times with 50 mM Tris-HCl (pH 7.5), 0.5% Nonidet P-40, 1 mM
EDTA, and 1 mM DTT. The washed beads were dissolved in 50 µl of SDS-PAGE sample buffer, boiled, and centrifuged, and 20 µl of
the supernatant was analyzed by immunoblot analysis using the indicated
antibody. To generate Fig. 7, the GST pull-down assay was carried out
as described above, except the brain or cell extract was replaced by
the tau sample (50 mM Tris-HCl (pH 7.5), 100 mM
NaCl, 1 mM EDTA, 1 mM DTT, 0.05% Tween 20, 0.3% bovine serum albumin, and 50 µg/ml tau).
 |
RESULTS |
Microtubule-associated 14-3-3
--
To examine
microtubule-associated 14-3-3
, we purified microtubules from a fresh
bovine brain extract using repeated cycles of temperature-induced
microtubule assembly and disassembly. SDS-PAGE and an immunoblot
analysis showed that microtubules were enriched during each cycle of
assembly and disassembly (Fig. 1,
A and B). An immunoblot analysis using an
anti-14-3-3
antibody indicated that 14-3-3
was present in all the
fractions in a manner similar to tubulin (Fig. 1C). By
quantitating the intensities of various bands in Fig. 1, B
and C, we determined that ~6.6, ~2.8, ~0.96, and
~0.3% of total 14-3-3
in brain extract remained associated with
first (P1), second (P2), third (P3), and fourth (P4) microtubule pellets, respectively. The amount of tubulin was ~29.5, ~12.4, ~7.2, and ~3.0% of the total in P1, P2, P3, and P4, respectively (data not shown). More importantly, the ratio of the amount of 14-3-3
to the amount of tubulin in P1, P2, P3, and P4 was ~0.23, ~0.30, ~0.20, and ~0.16, respectively (Fig. 1D). Thus,
a fraction of 14-3-3
remained stably associated with microtubules
during purification in a manner similar to tubulin. These observations indicated that a significant amount of 14-3-3
is stably bound to
microtubules in the brain.

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Fig. 1.
Co-purification of
14-3-3 with microtubules. Microtubules
were purified from a fresh bovine brain extract (H) by
repeated cycles of microtubule assembly and disassembly. Samples (10 ml
each) were analyzed by SDS-PAGE or immunoblot (IB) analysis
using the indicated antibodies. A, SDS-PAGE showing tubulin
and other microtubule-associated proteins in indicated fractions.
B and C, immunoblots; D,
14-3-3 /tubulin ratio. Blots B and C were
scanned, and the band intensity values of 14-3-3 and tubulin in
various fractions were obtained. The ratio for the indicated fraction
was then determined by dividing the 14-3-3 band intensity value by
the band intensity value of tubulin in that fraction. Values are the
average of three independent determinations. P1,
P2, P3, and P4 indicate pellets,
whereas S1, S2, S3, and S4
indicate supernatants obtained after first, second, third, and fourth
microtubule assembly/disassembly cycles, respectively.
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Microtubule-associated 14-3-3
Is Part of a Large Molecular
Complex--
To further characterize microtubule-associated 14-3-3
,
we depolymerized P3 microtubules by cold incubation and then
subjected them to a phosphocellulose chromatography. 14-3-3
was not
recovered within the flow-through fractions and eluted from the column
with an NaCl gradient along with the other microtubule-associated
proteins (data not shown, but see "Materials and Methods"). We then
combined the column fractions containing 14-3-3
and chromatographed
through an FPLC Superose 12 gel filtration column. Most of 14-3-3
eluted within fractions 40-46 with a size of ~500-kDa (Fig.
2B). Since the size of dimeric
14-3-3
is ~60-kDa (21, 22), these data indicated that 14-3-3
is
bound to another biological molecule within the brain microtubules.

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Fig. 2.
FPLC gel filtration of microtubule-associated
14-3-3 . Microtubules purified by three cycles of assembly and
disassembly were chromatographed through a phosphocellulose column. The
effluent fractions containing 14-3-3 were then analyzed by an FPLC
Superose 12 gel filtration column calibrated previously with the
indicated molecular weight marker proteins. Fractions (1 ml each) were
collected, and 20 µl from each indicated fraction was immunoblotted
using the indicated antibody. A, gel filtration profile.
BSA indicates bovine serum albumin. B,
C, and D, immunoblots. IB indicates
immunoblot.
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Identification of Molecules Bound to 14-3-3
within Brain
Microtubules--
A silver-stained SDS gel of various column
fractions from Fig. 2A showed numerous protein bands
of various sizes within fractions 40-46 (data not shown) and did not
give us any indication as to the identification of the 14-3-3
-bound
protein (s). In a previous study, we found that 14-3-3
is associated
with tau in bovine brain extract and binds to tau in vitro
(36). In a recent study, we showed that within brain microtubules,
GSK3
and tau are parts of a multiprotein complex that elutes from an
FPLC gel filtration column used in this study to generate Fig.
2A with an ~400-500-kDa size (20). We noted a very
similar gel filtration behavior between the high molecular size
14-3-3
present within fractions 40-46 (Fig. 2A) and the
tau phosphorylation complex described by us in a previous study (20).
We therefore analyzed various Fig. 2A column fractions for
the presence of tau and GSK3
. As shown in Fig. 2, C and
D, tau and GSK3
were indeed present within fractions 40-46, indicating that 14-3-3
has co-eluted with the tau
phosphorylation complex from the gel filtration column. We pooled
fractions 40-46 containing 14-3-3
and a portion of the pooled
fraction chromatographed through an FPLC Mono S column. SDS-PAGE and
immunoblot analyses of various effluent fractions indicated that
14-3-3
, tau, and GSK3
had co-eluted from the column (data not
shown). We then pooled column fractions containing 14-3-3
and
chromatographed through a Sepharose 4B gel filtration column. Tau,
GSK3
, and 14-3-3
again co-eluted (data not shown).
An SDS-polyacrylamide gel of the peak Sepharose 4B column fraction
containing tau, GSK3
, and 14-3-3
showed at least 11 prominent protein bands that migrated with various sizes on the gel (Fig. 3A). To find out which of
these bands may represent protein(s) bound to 14-3-3
in brain
microtubules, we determined the intensity value of each prominent
band on the gel and then calculated the molar ratio value (band
intensity divided by molecular size) of each band (Fig. 3B).
The ratios for the ~25-, ~35-, ~100-, ~150-, ~180-, and
~220-kDa bands were ~16, ~14, ~13, ~8, ~9, and ~4, respectively. The ratios for the ~30-, ~47-, ~50-, ~55-, and
~65-kDa bands were ~30, ~28, ~29, and ~28, respectively. Our
immunoblot analysis indicated that the ~30-kDa band corresponds to
14-3-3
, the ~47-kDa band corresponds to GSK3
, and the ~50-,
~55-, and ~65-kDa bands correspond to various tau isoforms. Thus,
in the column fraction containing partially purified 14-3-3
, the
molar ratios of ~14-3-3
, GSK3
, and tau were similar to and
higher than those of ~25-, ~35-, ~100-, ~150-, ~180-, and
~220-kDa proteins. These observations suggested that 14-3-3
may be
bound to GSK3
and/or tau within brain microtubules and may be a
component of tau phosphorylation complex.

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Fig. 3.
SDS-PAGE of the fraction containing partially
purified 14-3-3 . Fractions 40-46 from Fig. 2A were
combined, and a portion of the combined fraction was chromatographed
through an FPLC Mono S column followed by a Sepharose 4B gel filtration
column. An aliquot (20 µl) from the peak effluent gel filtration
column fraction containing 14-3-3 , tau, and GSK3 was
electrophoresed on a 10% SDS gel. The gel was stained with Coomassie
Brilliant Blue for proteins and used to determine the molar ratios.
A, protein-stained gel. B, molar ratio. The gel
in panel A was scanned, and the band intensity values of
various bands were obtained. The molar ratio for the indicated protein
was then determined by dividing the band intensity value by the
molecular weight of that protein.
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To determine whether 14-3-3
is part of the tau phosphorylation
complex, we immunoprecipitated 14-3-3
, tau, or GSK3
from the rest
of the above combined column fractions from Fig. 2A. Each
resulting immune complex was then immunoblotted with anti-tau, anti-GSK3
, or anti-14-3-3
antibody. Tau and GSK3
co-immunoprecipitated with 14-3-3
(Fig.
4A). Similarly, GSK3
and
14-3-3
co-immunoprecipitated with tau (Fig. 4B), and tau
and 14-3-3
co-immunoprecipitated with GSK3
(Fig. 4C).
Thus, 14-3-3
, tau, and GSK3
within Fig. 2A fractions
40-46 could not be separated from each other. Based on these data
and our previous study (20), we concluded that 14-3-3
is very likely
to be one of the components of tau phosphorylation complex.

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Fig. 4.
Co-immunoprecipitation. Combined
fractions from Fig. 2A containing 14-3-3 (A),
tau (B), and GSK3 (C) were used for
immunoprecipitation using the indicated antibodies. Each resulting
immune complex was immunoblotted using the indicated antibody. Similar
results were obtained in four different experiments. IP and
IB indicate immunoprecipitation and immunoblot,
respectively.
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To gain more evidence in support of the above idea and to study the
interactions of 14-3-3
, tau, and GSK3
within the tau phosphorylation complex, we first asked whether or not 14-3-3
could
bind to GSK3
directly. When glutathione-agarose beads coated with
GST-14-3-3
were incubated with a brain extract, GSK3
specifically precipitated with the GST-14-3-3
beads (Fig.
5A). Although this observation
indicated that 14-3-3
associates with GSK3
in the brain extract,
we could not rule out the possibility that tau, which can bind to both
GSK3
(20) and 14-3-3
in vitro (36), may have
influenced observed 14-3-3
and GSK3
association (Fig. 5A). Therefore, we performed a similar GST pull-down assay
as described above by using COS-7 cells that express GSK3
but not tau. As shown in Fig. 5B, GSK3
again came down with
GST-14-3-3
from the cell extract. To confirm that it was GSK3
that came down with GST-14-3-3
and not any other protein of similar
size that may be immunoreactive to our anti-GSK3
antibody used to generate Fig. 5, A and B, we transfected HEK-293
cells with HA-GSK3
. Transfected cells were lysed, and
glutathione-agarose beads coated with GST-14-3-3
were incubated with
the cell lysates. Incubated beads were washed and immunoblotted by
using an anti-HA antibody to test 14-3-3
-GSK3
binding. As
expected, HA-GSK3
bound to GST-14-3-3
(Fig. 5C). Based
on these data, we concluded that 14-3-3
directly binds to GSK3
.

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Fig. 5.
GST pull-down assay. Glutathione-agarose
beads coated with GST-14-3-3 or GST were mixed with an extract from
bovine brain, COS-7 cells, or HEK-293 cells transfected with
HA-GSK3 . Beads were washed and then immunoblotted with the indicated
antibody to detect bead bound GSK3 . These experiments were repeated
three different times with similar results. IP and
IB indicate immunoprecipitation and immunoblot,
respectively. An aliquot (20 µl each) was used from extracts of
brain, COS-7 cells, and HEK-293 cells transfected with HA-GSK3 as
control in A, B, and C,
respectively.
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There are three possible mechanisms by which tau, GSK3
, and
14-3-3
can interact within the tau phosphorylation complex. First,
because in vitro tau binds to GSK3
(20) as well as
14-3-3
(36) and the respective binding sites do not overlap, tau may bridge GSK3
and 14-3-3
within the complex. Second,
14-3-3
is a scaffold protein that can bind two ligands at a same
time (21, 22), and it binds tau (36) and GSK3
(Fig. 5). Therefore, 14-3-3
may anchor GSK3
to tau within the phosphorylation complex. Third, GSK3
can bind tau in vitro (20) and can bind
14-3-3
in vitro (Fig. 5). Thus, GSK3
may be the
central molecule that may hold 14-3-3
and tau simultaneously within
the complex.
To discriminate between the above possibilities, we transfected HEK-293
cells with FLAG-tau, Xpress-14-3-3
, and HA-GSK3
constructs in
various combinations. Transfected cells were lysed, and GSK3
was
immunoprecipitated from each lysate using an anti-HA antibody. Each
immune complex was then immunoblotted with anti-FLAG antibody to detect
tau. FLAG-tau did not co-immunoprecipitate from cells overexpressing
HA-GSK3
and FLAG-tau (Fig.
6A, lane 6),
indicating that GSK3
does not bind to tau directly in
vivo. This means that neither can tau bridge GSK3
to 14-3-3
nor can GSK3
simultaneously bind to tau and 14-3-3
within the tau
phosphorylation complex. Therefore, 14-3-3 must be the central
molecule that holds tau and GSK3
within the complex. Indeed,
FLAG-tau co-immunoprecipitated with HA-GSK3
from cells
overexpressing FLAG-tau and HA-GSK3
only when these cells also
overexpressed Xpress-14-3-3
(Fig. 6A, lanes 8 and 9), indicating that GSK3
associates with tau only in
the presence of 14-3-3
. As discussed above, 14-3-3
binds to tau
(36) and GSK3
(Fig. 5) directly. Taken together, these observations
indicated that 14-3-3
connects GSK3
to tau in
vivo.

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Fig. 6.
Co-immunoprecipitation of tau with
GSK3 in the presence and absence of
14-3-3 . HEK-293 cells transfected with the indicated constructs
were lysed, and the cell lysates were either used for
immunoprecipitation using anti-HA antibody or used for immunoblotting
using the indicated antibody. A, immunoprecipitation. The
resulting anti-HA immune complex was immunoblotted with anti-FLAG
antibody to detect FLAG-tau. B-D, immunoblots. An aliquot
(20 µl) from each cell lysate was immunoblotted with the indicated
antibody to monitor the expressions of indicated gene. Lane
2 represents mock-transfected cells. IP and
IB indicate immunoprecipitation and immunoblot,
respectively. Similar results were obtained in three different
experiments.
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To further confirm the above finding, we performed an in
vitro GST pull-down assay. Glutathione-agarose beads coated with GST-GSK3
were mixed with bacterially expressed recombinant tau in
the presence of a series of 14-3-3
concentrations. Beads were washed, and bead-bound tau was detected by immunoblot analysis using an
anti-tau antibody. Comparatively very little tau bound to beads when
GST-GSK3
was incubated with tau alone (Fig.
7, lane 3). However, when an
increasing amount of 14-3-3
was included in the assay mixture, the
amount of tau binding to GST-GSK3
increased progressively (Fig. 7,
lanes 4-8). When the amount of 14-3-3
was 100 µg/ml in
the assay mixture, ~10-fold more tau bound to GST-GSK3
than in the
absence of 14-3-3
(compare lanes 3 and 7).
Based on these data, we concluded that 14-3-3
promotes in vitro GSK3
-tau binding and is required for a stable association of tau and GSK3
in vivo.

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Fig. 7.
Binding of GSK3 with
tau in the presence of 14-3-3 . Glutathione-agarose beads coated
with GST-GSK3 or GST were incubated with tau solution in the
presence of indicated amounts of 14-3-3 . After incubation, beads
were washed and immunoblotted with anti-tau antibody. This experiment
was repeated three times with similar results. IB indicates
immunoblot.
|
|
14-3-3
Stimulates GSK3
-catalyzed Tau
Phosphorylation--
GSK3
is one of the kinases implicated to
phosphorylate tau in vivo (10-20). Since we find that
14-3-3
is required for a stable association between GSK3
and tau,
we examined the influence of 14-3-3
on GSK3
catalyzed tau
phosphorylation in vivo. We transfected HEK-293 cells in
various combinations with FLAG-tau, Xpress-14-3-3
, and HA-GSK3
constructs. Transfected cells were lysed, and the cell lysates were
analyzed for tau phosphorylation using various tau
phosphorylation-sensitive antibodies: AT8, PHF1, and 12E8, which
recognize tau phosphorylated on Ser199/Ser202,
Ser396/Ser404, and Ser262,
respectively (20, 36). As shown in Fig.
8, A-C, tau was slightly
phosphorylated in cells transfected with FLAG-tau alone (lane
3). This phosphorylation increased in cells co-transfected with
FLAG-tau and HA-GSK3
as expected (lane 5). In cells that were co-transfected with fixed amounts of FLAG-tau and HA-GSK3
but
different amounts of Xpress-14-3-3
, FLAG-tau phosphorylation increased progressively with the increase in the amount of
Xpress-14-3-3
(lanes 7-9). This increase was evident not
only by an increased immunoreactivity against all tau
phosphorylation-sensitive antibodies tested but also by a retarded
mobility of FLAG-tau on the SDS gel, a characteristic feature of
hyperphosphorylated tau (2, 3). Thus, 14-3-3
profoundly stimulated
GSK3
-catalyzed tau phosphorylation in vivo.

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Fig. 8.
Effect of 14-3-3 on
GSK3 -catalyzed tau phosphorylation in
vivo. HEK-293 cells transfected with the indicated
constructs were lysed, and 20 µg protein from each lysate was
immunoblotted using the indicated antibody. A-C, immunoblot
analysis using tau phosphorylation-sensitive monoclonal antibodies,
AT8, PHF1, and 12E8, which only cross-react with phosphorylated
tau. D-F, immunoblots to show expression levels of
FLAG-tau, HA-GSK3 , and Xpress-14-3-3 . Lane 1 represents mock-transfected cells. Similar results were obtained in
three different experiments. IB indicates immunoblot.
|
|
 |
DISCUSSION |
Recently, we reported the existence of a tau phosphorylation
complex containing GSK3
and tau within brain microtubules (20). Since the observed size of the complex is ~400-500 kDa and the sum
of the molecular sizes of tau and GSK3
is ~97 kDa, we suggested that other proteins may also be present within the complex (20). In
this study, we find that microtubule-associated 14-3-3
co-elutes with the tau phosphorylation complex from a gel filtration column, indicating that the size of high molecular size microtubule-associated 14-3-3
is the same as that of tau phosphorylation complex (Fig. 2).
14-3-3
and the tau phosphorylation complex in the microtubule fraction cannot be separated from each other by phosphocellulose, gel filtration, and Mono S chromatographies. Tau, 14-3-3
, and GSK3
co-immunoprecipitate with each other from column fractions containing the phosphorylation complex (Fig. 4). In
vitro, 14-3-3
binds to tau (36) and GSK3
(Fig. 5). These and
other data presented in this study indicate that 14-3-3
is also
a part of the microtubule-associated tau phosphorylation complex.
Tau and GSK3
co-immunoprecipitate with each other from brain
extracts (Fig. 4) (20). In contrast, tau does not co-immunoprecipitate with GSK3
from HEK-293 cell extracts co-transfected with GSK3
and
tau (Fig. 6A, lane 6). These data indicate that
in HEK-293 cells, the interaction of tau with GSK3
is weak, whereas
in the brain, GSK3
stably associates with tau. This in turn suggests that brain contains a factor required for a stable association of
GSK3
with tau, and this factor may be missing in HEK-293 cells.
Our gel filtration data (Fig. 2) and co-immunoprecipitation analysis
(Fig. 4) indicate that within the tau phosphorylation complex, tau,
GSK3
, and 14-3-3
are inseparable. Moreover, in HEK-293 cells, tau
associates with GSK3
only in the presence of 14-3-3
(Fig. 6).
In vitro, ~10-fold more tau binds to GSK3
in the
presence than in the absence of 14-3-3
(Fig. 7). Since 14-3-3
can
bind to tau (36) and GSK3
(Fig. 5) independently, these data
indicate that 14-3-3
is the factor that connects and mediates the
association of GSK3
with tau within the brain. However, as
discussed above, tau does not associate with GSK3
in HEK-293 cells
transfected with only tau and GSK3
, although 14-3-3 is known to be
widely expressed in various cell lines including HEK-293 cells (21). It
is possible that within HEK-293 cells, the endogenous 14-3-3
either
is not sufficient or is not available to mediate the interaction of
transfected GSK3
and tau.
In a previous study, we reported that in vitro GSK3
binds
to the N-terminal region of tau (20). Consistent with that report, we
find that tau comes down with GST-GSK3
in a GST pull-down assay
(Fig. 7, lane 3). However, tau does not co-immunoprecipitate with GSK3
from lysates of HEK-293 cells co-transfected with GSK3
and tau (Fig. 6A, lane 6). These observations
suggest that in the absence of 14-3-3
, GSK3
binds to tau with a
low affinity. It thus appears that in the brain, GSK3
interacts with
tau in two different ways: one with low affinity that does not require 14-3-3
and the other with high affinity that requires 14-3-3
.
The substrate recognition by GSK3 is regulated by two mechanisms. The
first mechanism requires a priming phosphorylation of the substrate
(41, 42). For example, casein kinase 2 phosphorylates glycogen synthase
first and generates a recognition motif for GSK3. GSK3 then
phosphorylates casein kinase 2-phosphoprylated glycogen synthase (42).
The second mechanism does not require priming phosphorylation. Instead,
a scaffold protein bridges GSK3
to its substrate within a
multiprotein complex (42). In the Wnt signaling pathway, GSK3
phosphorylates
-catenin within a
-catenin destruction complex.
-catenin alone is not a good substrate of GSK3
. The scaffold
protein axin connects GSK3
to
-catenin and facilitates
-catenin phosphorylation by GSK3
within the complex (6,
42-44).
Biochemical analyses and studies involving transgenic mice and cultured
mammalian cells have established that GSK3
phosphorylates tau in the
brain (10-20). The mechanism by which GSK3
phosphorylates tau is
not clear. Our recent study (20) and the results presented in this
study indicate that GSK3
, tau, and 14-3-3
are parts of a
microtubule-associated tau phosphorylation complex. Within the complex,
14-3-3
binds to tau and GSK3
simultaneously and assembles the
complex. Thus, the role of 14-3-3
within the tau phosphorylation
complex appears to be similar to that of axin within the
-catenin
destruction complex. Furthermore, 14-3-3
binds to tau and changes
the tau conformation, making tau susceptible for hyperphosphorylation
in vitro (36) and perhaps in vivo (Fig. 8). Since
14-3-3
stimulates tau phosphorylation on Ser199,
Ser198 Ser202, Ser262,
Ser396, and Ser404 (Fig. 8), it appears that
14-3-3
-induced conformational change occurs within a large part of
the C-terminal tau region, which is the main area of in vivo
phosphorylation (45). These observations suggest that 14-3-3
not
only enhances association of tau and GSK3
within the complex but
also prepares tau for GSK3
action.
We have found a unique multiprotein complex containing tau, GSK3
,
and 14-3-3
within brain microtubules. Thus, a pool of GSK3
in the
brain is targeted to microtubules through a stable association with tau
and 14-3-3
. Because the function of this complex is to regulate tau
phosphorylation and microtubule dynamics, we named this complex
the tau phosphorylation complex. It should be noted that the size of
the phosphorylation complex is 400-500-kDa, whereas the sum of the
sizes of tau, GSK3
, and 14-3-3
dimer is ~167-kDa. Therefore, it
is possible that there may be proteins other than, tau, GSK3
, and
14-3-3
within the tau phosphorylation complex. These proteins may
play important roles in regulating tau phosphorylation and interactions
between various phosphorylation complex components. Studies are ongoing
in our laboratory to identify all of the phosphorylation complex components.