 |
INTRODUCTION |
Paired helical filaments
(PHFs),1 the major fibrous
component of the neurofibrillary tangles associated with Alzheimer's
disease (AD), are composed mainly of microtubule-associated protein tau (1, 2; for review, see Ref. 3). PHF-tau (tau isolated from PHFs) has
retarded mobility on an SDS-gel, is highly insoluble, is abnormally
phosphorylated (i.e. contains more phosphate than normal
tau), and is functionally inactive (1-3). After dephosphorylation, PHF-tau migrates as normal tau on an SDS-gel and regains the ability to
bind to, and regulate, microtubule dynamics (4, 5). Abnormal phosphorylation may prevent tau from performing microtubule-related functions, resulting in cytoskeleton instability, loss of axonal transport, and PHF formation (3). Tau is a natural phosphoprotein, and
sites that are phosphorylated in normal adult brain (6) are also
phosphorylated in PHF-tau (2). Fetal tau (6), much like PHF-tau (2), is
also hyperphosphorylated. Strikingly, both normal adult tau and fetal
tau do not form PHFs. Furthermore, PHF-like filaments can be
reconstituted from tau molecules that do not contain any phosphate
(7-9). These observations have raised the possibility that abnormal
phosphorylation alone may not be sufficient, and another factor(s) may
be involved in converting tau to PHFs.
There are 19 phosphorylation sites within PHF-tau (2). Aberrant
activation of tau-specific kinase (s) has been suggested to lead to the
abnormal phosphorylation of tau in AD brain (3), because adult tau is
phosphorylated only on four sites (6). Therefore, considerable effort
is being made by many investigators to identify kinases that
phosphorylate tau. A number of proline-directed and
non-proline-directed kinases phosphorylate tau in vitro
(10-22). Surprisingly, none of these kinases has been shown to be
activated in AD brain. Furthermore, the above-mentioned kinases
normally phosphorylate a diverse group of proteins in neurons, but in
AD brain only phosphorylation of tau is significantly up-regulated (3).
Therefore tau, in AD brain, may be phosphorylated either by a kinase
(s) that is yet to be identified or by a known kinases in the presence
of a tau-specific substrate modulator, which renders tau more
susceptible to phosphorylation.
Sulfated glycosaminoglycans (such as heparin, heparan sulfate,
chondroitin sulfate, and dermatan sulfate) are sulfated copolymers of
glucosamine and uronic acid residues (23). Several studies have
indicated the presence of glycosaminoglycans in senile plaques and
neurofibrillary tangle (9, 24-27). Recently, heparan sulfate was shown
to accumulate in pretangle neurons (9), to stimulate in
vitro tau phosphorylation by various kinases (28-32), to prevent tau from binding to microtubules, and to cause tau to aggregate into
PHF-like filaments (9). An increase in the sulfated glycosaminoglycans within the nerve cells was suggested to trigger the
hyperphosphorylation of tau, destabilization of microtubules, and
assembly of PHFs (9). The interaction of sulfated glycosaminoglycan and
tau was suggested to be the central event in the development of
neuropathology in AD (9, 33). However, the biochemical mechanism by
which glycosaminoglycans enhance tau phosphorylation and cause tau to aggregate into PHFs remains unclear.
In this study, we have investigated the effect of heparin on the
structure and phosphorylation of tau by chemical cross-linking and
phosphopeptide mapping. Herein we report that heparin, in addition to
causing aggregation of tau, also changes tau's conformation, exposing
new sites within the tau molecule for kinase phosphorylation.
 |
MATERIALS AND METHODS |
Proteins and Peptides--
Tau protein used in this study was
purified from extracts of Escherichia coli overexpressing
the longest isoform of human tau (htau 40) as described (19), except
effluent from Q-Sepharose column was chromatographed through an
S-Sepharose column. Neuronal cdc2-like protein kinase (NCLK) was
purified from fresh bovine brain extract as described previously (34).
cAMP-dependent protein kinase (A kinase), catalytic subunit
of A kinase (C subunit), trypsin, and thermolysin (protease type X),
and Kemptide (LRRASLG) were from Sigma. Preparations of polyclonal
antibody against bovine brain tau and synthetic peptide substrate of
NCLK (KTPKKAKKPKTPKKAKKL) were described previously (19). The
concentration of tau was estimated spectrophotometrically (35). Amounts
of A kinase, C subunit, and Kemptide were based on their dry weights.
Concentration of synthetic peptide NCLK substrate was determined by
amino acid analysis. The amount of NCLK was estimated by enzymatic
activity (34).
Kinase Assay--
Unless otherwise stated, NCLK activity was
measured as described previously (19) in an assay mixture containing 25 mM Hepes (pH 7.2), 0.1 mM EDTA, 0.2 mM dithiothreitol, 60 mM NaCl, 0.5 mM [32P]ATP, 10 mM
MgCl2, 50 µM peptide substrate, or 0.5 mg/ml
tau and 400 units/ml NCLK. The assay was initiated by the addition of 5 µl of kinase to a 20-µl mixture containing the rest of the assay
mixture components. After 20 min at 30 °C, aliquots were withdrawn
and analyzed for the amount of radioactivity incorporated into the
substrate by phosphocellulose strip assay. Activity of C subunit was
determined as above, except Kemptide was used as the peptide substrate,
and the concentration of C subunit was 10 µg/ml. A kinase was assayed
in a manner similar to that described above for C subunit, except the
assay mixture also contained 10 µM cAMP.
Chemical Cross-linking--
Chemical cross-linking of tau by
disuccinimidyl suberate (DSS), a homobifunctional chemical cross-linker
with an 11.4-Å spacer arm, was performed essentially as described
previously (36) in a mixture containing 1.5 mg/ml tau, 0.1 mM EDTA, 0.2 mM dithiothreitol, 60 mM NaCl, 1 mM DSS (Pierce) and 2%
N,N-dimethyl formamide (DMF). The reaction was initiated by
the addition of 1 µl of DSS stock solution in DMF to 49 µl of
mixture containing the rest of the cross-linking mixture components.
After various time points at room temperature, aliquots were removed,
mixed with an equal volume of SDS-PAGE sample buffer (0.1 M
Tris-HCl, pH 6.8, 25% glycerol, 0.2% bromphenol blue, 10%
-mercaptoethanol, and 2% SDS), boiled, and electrophoresed on a
7.5% Laemmli SDS-gel. The amounts of cross-linked bands were
quantitated by scanning the gels using a Molecular Dynamics SI personal
densitometer. Band intensities were determined by dividing the optical
density of each cross-linked band with the band intensity of tau
control (treated with the solvent). The amount of tau that was not
recovered in the gel was expressed as the higher molecular size species
that did not enter the gel after DSS cross-linking. Apparent molecular
weights of various cross-linked species were determined essentially as described previously (36).
Phosphopeptide Mapping and Purification of
Phosphopeptides--
Tau (0.5 mg), phosphorylated for 6 h by NCLK
or A kinase, was digested with trypsin and subjected to HPLC
C18 reverse phase chromatography essentially as described
previously (19). To purify phosphopeptide 1, peak 1 fractions depicted
in Fig. 5A were combined, concentrated to ~0.5 ml, and
chromatographed through a Sephadex G-25 column (0.5 × 25 cm)
preequilibrated and eluted with 0.1% trifluoroacetic acid. The
effluent fractions (0.5 ml each) were collected. Only one radioactive
peak eluted from the column. Fractions containing radioactivity were
combined, concentrated, and injected into an HPLC column as described
above. The column was eluted with an acetonitrile gradient of 0-30%
in 50 min. Phosphopeptide 3b was purified from peak 3 (see Fig.
5B) fractions and is shown in Fig. 6B. To purify
phosphopeptide 4, peak 4 fractions (see Fig. 5B) were vacuum
dried and redissolved in 0.2 ml of 50 mM NH4HCO3 (pH 8.0) containing 25 µg/ml
thermolysin. The sample was then incubated at 37 °C for 3 h and
then injected into an HPLC column. The peptide was then eluted from the
column by a linear gradient of acetonitrile (0-40%) in 50 min. To
purify phosphopeptide e, peak e fractions (see Fig. 7B) were
combined, vacuum dried, dissolved in 500 µl of 50 mM
NH4HCO3 containing 25 µg/ml thermolysin, and
incubated at 37 °C for 3 h. After incubation, the sample was loaded onto a ~1-ml DEAE-Sephacel (Sigma) column pre-equilibrated in
25 mM Hepes (pH 7.0). The column was washed with 10 ml of
equilibration buffer and eluted with 0.25 M NaCl in
equilibration buffer. Effluent fractions (0.2 ml each) were collected.
Fractions containing radioactivity were combined and concentrated to
~0.2 ml, and the phosphopeptide was purified by HPLC as above using
acetonitrile gradient 0-40% in 50 min. Phosphopeptides were sequenced
using a gas phase amino acid sequencer (19) at the Department of
Biochemistry and Microbiology, University of Victoria.
 |
RESULTS |
Chemical Cross-linking of tau--
When tau was incubated with DSS
and the product was analyzed by SDS-PAGE, there was a
time-dependent formation of a heavier species with a
concomitant decrease in the tau band intensity (Fig.
1A, lanes 2-4).
The molecular size of the heavier band on an SDS-gel was estimated to
be ~153 kDa. In a previous study we have shown that the tau isoform
used in this study that migrates as a 65-kDa band on the SDS-gel is a
mixture of tau monomers and dimers when purified from bacterial lysate.
These dimers, when cross-linked by DSS, migrate with a size of ~151
kDa on an SDS-gel (36). Thus, the cross-linked band in Fig.
1A, lanes 3 and 4, is the dimeric
tau.

View larger version (85K):
[in this window]
[in a new window]
|
Fig. 1.
Chemical cross-linking of tau by DSS in the
presence and absence of heparin. Tau was preincubated for 30 min
at room temperature with heparin or water in a mixture containing 25 mM Hepes (pH 7.2), 0.1 mM EDTA, 0.2 mM dithiothreitol, 60 mM NaCl, 0.2 mg/ml
heparin, and 1.5 mg/ml tau. DSS was added to each preincubated sample
to initiate the cross-linking. After various time points at room
temperature, 10 µl was removed from each reaction mixture and mixed
with 20 µl of SDS-PAGE sample buffer, and 10 µl was subjected to
SDS-PAGE. Gels were either stained for proteins or subjected to
immunoblotting using antibody against tau. A, protein
stained gel; B, immunoblot; M, molecular size
marker. Lanes 1-4, tau preincubated with water and treated
with DSS for 1, 2.5, 5, and 10 min, respectively; lanes
5-8, tau preincubated with heparin and treated with DSS for 1, 2.5, 5, and 10 min, respectively; lanes 9-12, tau
preincubated with heparin and treated with solvent (DMF) for 1, 2.5, 5, and 10 min, respectively. The immunoblot in B shows that all
the cross-linked bands are derived from tau.
|
|
Cross-linking of tau under identical conditions in the presence of
heparin showed four major differences when compared with tau
cross-linked alone (Fig. 1). First, the intensity of the dimeric band
was higher in tau cross-linked in the presence of heparin (Fig. 1,
compare lanes 3, 4 and 6-8). Second, a band with
size of ~246 kDa was formed in the presence, but not in the absence, of heparin. Because the molecular size of tau is ~65 kDa (Fig. 1A), this 246-kDa band is ~3.8 times heavier than tau and
must therefore be tetrameric tau. Third, a protein band that migrated as a streak on the top portion of the gel was formed only when heparin
was present in the cross-linking mixture (Fig. 1, lanes 7 and 8). This band must be a heavy molecular size tau
aggregate cross-linked by DSS. Fourth, in the presence of heparin, two
species of molecular sizes 72 and 83 kDa were also cross-linked by DSS (Fig. 1, lanes 6-8). When immunoblotted using an
anti-heparin monoclonal antibody (mAb; Chemicon), none of the above
cross-linked bands displayed immunoreactivity (data not shown),
indicating that the DSS cross-linked bands were not formed by
tau-heparin cross-linking. Densitometric quantitation of various bands
in Fig. 1A indicated that at the 15-min time point 3.5%
dimeric tau was cross-linked by DSS in the absence of heparin, whereas
in the presence of heparin 4.1, 6.2, 10.5, 7.5, and 3.0% tau was cross-linked into 72-kDa, 83-kDa, dimer, tetramer, and higher molecular
size species, respectively.
The cross-linking of dimeric, tetrameric, and higher aggregates by DSS
in Fig. 1 suggested that heparin promotes dimerization and causes the
formation of tetrameric and higher molecular sized tau species. These
observations are consistent with previous reports and indicate that
heparin causes tau aggregation (9, 32). To interpret the cross-linking
of 72- and 83-kDa bands by DSS in the presence of heparin (Fig. 1), we
wished to know whether these two cross-linked bands were formed by
cross-linking of tau intermolecularly or intramolecularly. Because DSS
cross-linked dimer and tetramer migrated with molecular sizes of 153 and 246 kDa, respectively, on an SDS-gel (Fig. 1A), the
sizes 72 and 83 kDa are too small to be tau dimer, trimer, or tetramer
that would have been formed if tau were cross-linked intermolecularly.
To substantiate the idea that 72- and 83-kDa bands are formed by
intramolecular cross-linking, we cross-linked tau (0.75 mg) with DSS in
the presence of heparin as described under "Materials and Methods."
The cross-linked tau was then fractionated through a fast protein
liquid chromatography (FPLC) Superose 12 gel filtration column (Fig.
2A), and various column
fractions were immunoblotted using anti-tau antibody (Fig.
2C). Tau tetramers were recovered within fractions 32-36
with peak fraction 34. Tau dimers were present within fractions 36-40
with peak fraction 38. The 72- and 83-kDa species were present within
fractions 40-44 with peak fraction 42. Importantly, tau
chromatographed through the same column under identical conditions
eluted with peak fraction 42 (Fig. 2B). Thus, the sizes of
72- and 83-kDa bands correspond to monomeric tau. As shown in Fig. 1,
72- and 83-kDa species are cross-linked by DSS only in the presence of
heparin. These observations indicated that heparin causes a
conformational change that exposes groups reactive to DSS within tau
molecule leading to intramolecular covalent cross-linking of tau by
DSS. These cross-linked tau species migrate slightly slower than tau on
SDS-gels.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 2.
FPLC gel filtration of tau and tau
preincubated with heparin and treated with DSS. Tau (0.75 mg) was
preincubated with heparin in a total volume of 0.5 ml as described in
the legend to Fig. 1. After 30 min of preincubation, 1 µl of stock
DSS solution was added to initiate the cross-linking. After 15 min at
room temperature, 10 µl of 1.5 M Tris-HCl (pH 8.8) was
added to quench the cross-linking, and then the entire sample injected
into a gel filtration column. Gel filtration was carried out on an
Amersham Pharmacia Biotech FPLC Superose 12 HR 10/30 column (1 × 30 cm) at a flow rate of 0.5 ml/min. Fractions (0.5 ml each) were
collected, and 20 µl from indicated fraction was analyzed by
SDS-PAGE. A, FPLC gel filtration. B and
C, SDS-gels of column fractions representing tau and tau
preincubated with heparin and treated with DSS, respectively.
|
|
Effect of Preincubation Time on Chemical Cross-linking--
In a
previous study (9), heparin-induced aggregation of tau was reported to
be dependent on time of incubation, and PHF-like filaments were formed
after incubating tau with heparin for >48 h. We therefore preincubated
tau with heparin for various time points. Aliquots were removed from
the preincubation mixture, treated with DSS, and subjected to SDS-PAGE.
The intensities of various bands on the gel were quantitated. The
amount of tau that was not recovered on the gel was regarded as the
high molecular size aggregate, which, after cross-linking, did not
enter the SDS-gel.
As shown on Fig. 3, A and
B, the intensities of the dimeric and tetrameric bands
increased with increasing time of preincubation, peaked at 48 h
(Fig. 3A, lane 11) and diminished significantly at 72 and 96 h. The formation of the higher aggregate tau, which was slow until 48 h, sharply increased with concomitant decrease in the intensities of all other cross-linked and monomeric tau bands
when incubated beyond 48 h (Fig. 3B).

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 3.
Chemical cross-linking of tau preincubated
with heparin for various time points. Tau was preincubated with
heparin as described in the legend to Fig. 1. Each preincubated sample
was treated with DSS for 15 min, and the was product analyzed by
SDS-PAGE. A, SDS-gel. Lane 1, tau (7.5 µg)
preincubated with heparin for 96 h; lane 2, tau (7.5 µg) preincubated with DMF for 96 h and treated with DSS;
lanes 3-13, tau (7.5 µg each) preincubated with heparin
for 0.1, 0.17, 0.25, 0.5, 2.5, 6, 24, 30, 48, 72, and 96 h,
respectively, and treated with DSS. B, intensities of
various bands in A. Band intensities were determined as
described under "Materials and Methods" and are expressed as % of
control tau on lane 1.
|
|
Like the dimeric band, 72- and 83-kDa bands were visible within a few
min of preincubation with heparin (Fig. 3A, lane
3) and displayed a biphasic effect with respect to preincubation time (Fig. 3B). However, these two bands peaked at the
30-min time point (Fig. 2A, lanes 6 and
B), remained constant for several hr, and then declined very
slowly until 48 h (lane 11). At 72 and 96 h, both
bands progressively faded.
In conclusion, the dimerization and tetramerization of tau increased
with the increase in heparin incubation time until 48 h. The
formation of higher molecular size aggregate was slow during the
initial period of heparin incubation until 48 h. Incubation with
heparin beyond 48 h converted almost all tau species into the
higher molecular size aggregate. Heparin-induced conformational change
of tau, as detected by DSS cross-linking of 72- and 83-kDa species,
completed within ~30 min of heparin exposure.
Effect of Heparin on tau Phosphorylation--
In addition to
causing aggregation, heparin is known to stimulate tau phosphorylation
by various kinases (28-32). To further investigate this phenomenon, we
measured the activity of NCLK in the presence of heparin. Heparin
stimulated the tau phosphorylation activity of NCLK but not of a
synthetic peptide substrate (Fig. 4A). To test whether heparin
affects tau phosphorylation by other kinases also, we examined tau
phosphorylation by A kinase (Fig. 4B). Heparin was
stimulatory of tau phosphorylation activity of A kinase and had no
effect on the phosphorylation of Kemptide, a synthetic peptide
substrate of A kinase. These results suggested that heparin is a
substrate modulator acting through tau.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of heparin on the activities of NCLK
(A) and A kinase (B). Activities of NCLK
and A kinase were measured for 20 min in the presence of indicated
amounts of heparin using either peptide or tau as the substrate.
|
|
Phosphopeptide Mapping--
To gain further insight into the
effect of heparin on tau phosphorylation, tau was phosphorylated by
NCLK in the presence and absence of heparin under identical conditions.
Phosphorylated tau species were digested with trypsin and fractionated
through an HPLC reverse phase column. As shown in Fig.
5, tau phosphorylated in the absence of
heparin showed three radioactive peaks (Fig. 5A, peaks
1-3). Phosphopeptide map of tau phosphorylated in the presence of
heparin (panel B) showed three differences when compared with that of tau phosphorylated in the absence of heparin (Fig. 5A). First, peak 1 present in Fig. 5A
was absent in Fig. 5B. Second, peak 4 was present
in Fig. 5B but not in Fig. 5A. Third, peak 3 in Fig. 5B was larger than peak 3 in Fig.
5A.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5.
HPLC tryptic phosphopeptide maps of tau
phosphorylated by NCLK in the absence (A) and presence
(B) of heparin. Two tau species (0.5 mg each)
phosphorylated by NCLK in the presence and absence of heparin for
6 h at 30 °C were digested with trypsin under identical
conditions and subjected to HPLC reverse phase chromatography through a
Delta Pak 5-µm 100-A C18 reverse phase column (Millipore)
using a Waters HPLC system at a flow rate of 0.5 ml/min. The column was
eluted with a linear gradient of 0-40% acetonitrile in 50 min.
Fractions (0.5 ml each) were collected, and 10 µl from each fraction
was counted in a liquid scintillation counter. Insets, HPLC
profiles.
|
|
The presence of peak 1 (Fig. 5A) only in the
phosphopeptide map of tau phosphorylated in the absence of heparin
indicated that heparin may have altered the digestibility of tau by
trypsin leading to the disappearance of peak 1, or the site (s)
corresponding to peak 1 was either lost or altered in the presence of
heparin. To determine this site(s), peak 1 fractions were combined,
concentrated, and chromatographed through a Sephadex G 25 followed by
HPLC. Only one phosphopeptide designated as peptide 1 was isolated
(data not shown). This peptide was subjected to 10 cycles of Edman
degradation using a gas phase amino acid sequencer. As shown in Table
I, the amino acid sequence of peptide 1 is TPPKXPSSAK, where X is the unidentified PTH amino acid indicating
that the 5th residue is phosphorylated. This idea is further confirmed
by the release of radioactivity during the fifth cycle. Based on these
results, and the known sequence of tau protein (37), peptide 1 is
determined to extend from residues 231-240 of tau, and
Ser235 (numbered according to the longest isoforms of human
tau; Ref. 37) is the phosphorylation site.
View this table:
[in this window]
[in a new window]
|
Table I
Sequence determination of phosphopeptides
The amino acid sequence of each phosphopeptide was determined by Edman
degradation using a gas phase sequencer. AA, PTH amino acid identified
after each cycle; Yield, pmol of PTH amino acid released after each
cycle; cpm, amount of radioactive released in each cycle; X, amino acid
whose PTH derivative could not be identified. Refer to the text for the
nomenclature of phosphopeptides.
|
|
The presence of peak 4 only in the map of tau phosphorylated
in the presence of heparin (Fig. 5B) indicated that either
heparin affects the cleavage of tau by trypsin, causing the appearance of peak 4 or NCLK phosphorylates tau on novel site (s) in the presence
of heparin. To determine this site (s), fractions containing peak 4 from the phosphopeptide map of tau phosphorylated in the presence of
heparin were combined and digested with thermolysin. The digested
peptide was chromatographed through HPLC. Only one phosphopeptide,
designated peptide 4, was recovered. As shown in Table I, the amino
acid sequence of peptide 4 is VVRXPPKXPSS, where X represents the
unidentified PTH amino acid. This peptide released radioactivity during
the fourth and eighth cycles (Table I). Based on these observations and
the sequence of tau (37), peptide 4 was determined to extend from
residues 228-238 of tau, and Thr231 and Ser235
are the phosphorylation sites.
The above phosphopeptide analysis data showed that NCLK phosphorylates
tau on Thr231 and Ser235 in the presence of
heparin. However, Ser235 is also phosphorylated by NCLK in
the absence of heparin (Table I, phosphopeptide 1). Therefore, in the
presence of heparin a new site, Thr231, is phosphorylated
by NCLK.
Peak 3 in the phosphopeptide map of tau phosphorylated by
NCLK, in the presence of heparin, (Fig. 5B) is larger than
the peak 3 phosphorylated in the absence of heparin (Fig.
5A). We reasoned that in the presence, as opposed to the
absence, of heparin NCLK either phosphorylates the site (s)
corresponding to peak 3 more completely, or NCLK phosphorylates tau on
an additional site(s). To discriminate between these two possibilities,
fractions containing peak 3 from the phosphopeptide maps of tau
phosphorylated in the presence and absence of heparin (Fig. 5,
A and B) were passed through a Sephadex G-25
column and subsequently subjected to HPLC peptide mapping. As shown in
Fig. 6B, peak 3 of tau
phosphorylated in the presence of heparin contained two
phosphopeptides, 3a and 3b. Peak 3 of tau
phosphorylated in the absence of heparin contained only one
phosphopeptide, 3a (Fig. 6A), indicating that
NCLK phosphorylates tau on additional sites in the presence of heparin.
The amino acid sequence of phosphopeptide 3b is shown in Table I. This peptide extends from tau residues 210-221, and Thr212 is
the phosphorylation site. Thus, Thr212 is phosphorylated by
NCLK only in the presence of heparin.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
HPLC peptide maps of peak 3 fractions from
Fig. 5, A and B. Peak 3 fractions from
Fig. 5, A and B were pooled separately and
fractionated through a Sephadex G 25 column. Effluent fractions
containing radioactivity were pooled, lyophilized, redissolved in 0.2 ml of 0.1% trifluoroacetic acid, and rechromatographed by HPLC. All
the chromatographic conditions were same as in Fig. 5, except the
acetonitrile gradient was 0-45% in 50 min. Effluent-containing
peptide peaks were manually collected, and 10 µl was counted in a
scintillation counter. A, HPLC profile of peak 3 from Fig. 5A; B, HPLC profile of peak
3 from Fig. 5B. Note that phosphopeptide 3b
(arrow) is present only in B. This peptide was
subjected to amino acid sequencing.
|
|
An HPLC tryptic phosphopeptide map of tau phosphorylated by A kinase in
the absence of heparin contained four radioactive peaks, designated
a-d (Fig.
7A). The tryptic
phosphopeptide map of tau phosphorylated by A kinase under identical
conditions, but in the presence of heparin, in addition to peaks
a-d also contained peak e (Fig.
7B, arrow). These observations indicated that
peak e may have resulted from heparin either altering the cleavage of
tau by trypsin or A kinase phosphorylating tau on an additional site(s)
in the presence of heparin.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 7.
HPLC tryptic phosphopeptide maps of tau
phosphorylated by A kinase in the absence (A) and presence
(B) of heparin. Tau species (0.5 mg each)
phosphorylated by A kinase in the absence and presence of heparin were
trypsinized and subjected to phosphopeptide mapping as in Fig. 5.
Insets, HPLC profiles.
|
|
To determine the site(s) corresponding to peak e (Fig.
7B), peak e fractions were combined, vacuum dried, and
digested with thermolysin. The digest was subjected to DEAE-Sephacel
followed by HPLC chromatography. Only one phosphopeptide was recovered. This phosphopeptide was designated peptide e. The sequence of peptide e
is IGXTEN (Table I). This peptide is derived from tau residues
260-265, and Ser262 is phosphorylated. Previously,
Ser262 was shown not to be phosphorylated by A kinase (16).
Based on these observations, and complete absence of peak e in the
phosphopeptide map of tau phosphorylated by A kinase in the absence of
heparin (Fig. 7A), we concluded that A kinase phosphorylates
Ser262 only in the presence of heparin.
Our data indicated that NCLK phosphorylates tau on Ser212
and Thr231 only in the presence of heparin. Similarly, A
kinase phosphorylates Ser262 only when heparin is present
in the phosphorylation mixture. To confirm that these effects are
caused specifically by heparin, and not by any other unknown
component(s) present in the phosphorylation mixture, we prepared a
series of tau species phosphorylated under different conditions. Using
heparin from two different sources (Sigma and Fisher), tau was
phosphorylated by NCLK and A kinase. We also phosphorylated tau in the
presence and absence of heparin using NCLK from two different
preparations. Similarly, we prepared tau phosphorylated by A kinase and
catalytic subunit of A kinase in the presence and absence of heparin.
Finally, tau from two independent preparations was phosphorylated by
NCLK and A kinase in the presence and absence of heparin. These
phosphorylated tau species were digested with trypsin, and HPLC
phosphopeptide maps, as in Figs. 5 and 7, were generated and compared.
All tau species phosphorylated by NCLK in the absence of heparin
contained three radioactive peaks corresponding to peaks
1-3 in Fig. 5A, and tau species
phosphorylated by NCLK in the presence of heparin had radioactive peaks
corresponding to peaks 2-4 in Fig. 5B.
On all occasions, the size of peak 3 was larger in the map of tau
phosphorylated in the presence of heparin (data not shown).
Similarly, all tau species phosphorylated by A kinase and C subunit in
the absence of heparin had identical phosphopeptide maps and contained
radioactive peaks corresponding to peaks a-d in
Fig. 7A. Tau species phosphorylated by A kinase or C subunit in the presence of heparin contained five peaks corresponding to
peaks a-e in Fig. 7B (data not shown).
Cross-linking and Activation--
To determine whether stimulation
of tau phosphorylation by heparin is caused by heparin-induced tau
aggregation and/or change in tau conformation, we preincubated tau with
heparin for various time points, and the product was subjected to DSS
cross-linking or phosphorylation by NCLK. Stimulation of tau
phosphorylation rapidly increased within a few min of heparin exposure,
peaked in 30 min, and slowly declined until 48 h (Fig.
8B, filled
circles). At 72 h stimulation of phosphorylation sharply
declined and completely vanished at 96 h. These observations
suggest that the structural change responsible for enhancing tau
phosphorylation (Fig. 8B) is completed within 30 min of
heparin exposure.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of preincubation with heparin on DSS
cross-linking and phosphorylation of tau. Tau was preincubated
with heparin as described in the legend to Fig. 1. After various time
points, aliquots were withdrawn and either subjected to DSS
cross-linking or used as substrate for NCLK activity assay.
A, SDS-gel of DSS cross-linking. To each vial containing 97 µl of tau preincubated for various time points with heparin, 3 µl
of DSS stock solution was added. After 15 min at room temperature, 10 µl was withdrawn from each vial and mixed with an equal volume of
SDS-PAGE sample buffer, and 10 µl was electrophoresed. Lane
1, tau (7.3 µg) preincubated with water for 96 h and
treated with DMF; lanes 2-10, tau (7.3 µg each)
preincubated with heparin for 0.1, 0.17, 0.25, 0.5, 2.5, 24, 48, 72, and 96 h, respectively, and treated with DSS. B,
comparison of phosphorylation and formation of cross-linked dimer,
tetramer, and 72- and 83-kDa species in A. NCLK activity
assay was carried out as described under "Materials and Methods."
To each vial containing 97 µl of tau preincubated with heparin or
water for various time points, an aliquot of 3 µl containing
[ -32P]ATP/Mg2+ and NCLK was added to
initiate the reaction. After 15 min at 30 °C, 20 µl of sample was
withdrawn and analyzed for the amount of 32P incorporated
into tau. NCLK activity is expressed as the fold stimulation
(i.e. activity of NCLK against tau preincubated with heparin
divided by activity of NCLK against tau preincubated with water for
same amount of time as with heparin). Intensities of various bands were
estimated by scanning the gel in A and are expressed as % of control tau (lane 1).
|
|
As shown in Fig. 8, A and B, the dimeric and
tetrameric bands rapidly appeared, and their intensities increased
continuously until 48 h. The intensity profiles of both the 72- and 83-kDa bands increased rapidly and peaked in 30 min (Fig.
8A, lane 5) in a manner similar to
phosphorylation (Fig. 8B). Like phosphorylation, the
intensity profiles of both bands declined slowly until 48 h. Thus
we observed a correlation between formation of 72- and 83-kDa bands and
stimulation in tau phosphorylation by NCLK with respect to heparin
preincubation time (Fig. 8B). These experiments were
repeated two more times using two different tau preparations and
heparin from two different sources. In all occasions identical results
were obtained. These correlative data strongly support the notion that
heparin induces a conformational change, making tau a better substrate
for kinase phosphorylation.
 |
DISCUSSION |
The two characteristic features of PHFs are tau aggregation and
abnormal phosphorylation (1-3). However, the cellular and biochemical
mechanisms by which tau becomes abnormally phosphorylated and
aggregates into PHFs is not clear. The presence of sulfated glycosaminoglycan in pretangle neurons (9, 24-27) and the ability of
heparin to cause tau to aggregate into PHF-like filaments (9, 32) and
to stimulate in vitro tau phosphorylation by various kinases
(28-32) have recently generated considerable interest. The interaction
of tau with sulfated glycosaminoglycan was suggested to be the key
event leading to the abnormal phosphorylation and aggregation of tau
that occurs in degenerating neurons of patients with AD (9, 33). Among
all sulfated glycosaminoglycans tested, heparin was found to be one of
the most effective in causing tau's aggregation (9, 32).
In this study, we investigated the effect of heparin on tau structure.
Our cross-linking data indicate that in the presence of heparin, tau
forms dimers, tetramers, and high molecular size aggregates in a
time-dependent manner. Dimers are the first oligomers formed during the heparin-induced oligomerization of tau, followed by
tetramers and the higher molecular size aggregates (Fig. 3). Although
not analyzed in this study, but based on previous reports (9, 32),
these high molecular size species are likely to be PHF-like filaments.
These observations are consistent with the hypothesis that dimerization
of tau precedes PHF assembly (7).
The optimal aggregation of tau into PHF-like filaments was reported to
require exposure of tau to heparin over 48 h (9). In this study,
we found that when tau was exposed to heparin, dimers followed by
tetramers were formed, and formation of these two species displayed a
biphasic effect with respect to time of heparin exposure. Amounts of
both species increased with exposure time and peaked at exposure time
of 48 h (Fig. 3). Exposure beyond 48 h caused loss of
monomers, dimers, and tetramers. The formation of high molecular size
aggregate was slow during the first part of the exposure period. Even
at 48 h, only ~16% tau aggregated into higher molecular size
species. At exposure time 72 h, however, ~60% tau was lost as
higher molecular size species. Thus, such an abrupt transition of tau
into high molecular size species indicates that heparin-induced
aggregation of tau is a cooperative process as described for protein
aggregation (38, 39), and 48 h is the threshold time point for
this aggregation.
In addition to cross-linking dimers, tetramers, and high molecular size
species, DSS cross-linked tau into 72- and 83-kDa species. FPLC
Superose 12 gel filtration chromatography determined that these two
species are monomeric tau formed by intramolecular cross-linking of tau
molecules. Because 72- and 83-kDa species were not formed when heparin
was excluded from the cross-linking mixture (Fig. 1, lanes
2-4), these observations indicate that heparin causes a
conformational change in the tau molecule in which groups reactive to
DSS become accessible and approach within 11.4 Å, the distance of the
spacer arm of DSS. Because tau is a naturally denatured protein with no
apparent folding (35, 40), such conformational change will induce
polypeptide chain folding within the tau molecule.
As shown in Figs. 3 and 8, heparin-induced dimerization and
tetramerization become maximum at an exposure time of 48 h,
whereas formation of 72- and 83-kDa species plateau at 30 min. Based on the intensities of various bands in Fig. 3, at 15 min, amounts of dimer
and tetramers are ~35% of the maximum, whereas formation of 72- and
83-kDa species are ~80% of the maximum. Thus the polypeptide chain
folding of tau caused by heparin occurs more rapidly than tau's
dimerization and tetramerization. Although yet to be confirmed, heparin-induced polypeptide chain folding of tau may precede tau's oligomerization. If true, heparin-induced aggregation of tau may follow
a sequential mechanism. Polypeptide chain folding may expose docking
surfaces for interchain interaction between tau molecules, leading to
the formation of dimers, tetramers, and high molecular size aggregates.
Such a model has been proposed for aggregation of proteins from
unfolded polypeptides (38).
Heparin, in addition to causing aggregation of tau, also stimulates tau
phosphorylation by various kinases (28-32). Because heparin's
stimulatory effect is not observed when tau is replaced by synthetic
peptide substrates, (Fig. 4), these observations are consistent with
the idea that heparin is a substrate modulator acting through tau (28).
Our cross-linking data show that heparin induces a conformational
change in tau, which can be detected by the DSS cross-linking of 72- and 83-kDa species. More importantly, our data in Fig. 8 show that
cross-linking of 72- and 83-kDa species correlates with the stimulatory
effect of heparin on tau phosphorylation. Furthermore, phosphopeptide
mapping of tau phosphorylated in the presence and absence of heparin
revealed that both NCLK and A kinase phosphorylated tau on sites that
are not accessible for phosphorylation when heparin is excluded from
the phosphorylation mixture (Figs. 5 and 7). Together, these
observations indicate that the heparin-induced conformational change in
tau exposes new sites for phosphorylation by NCLK and A kinase.
Interestingly, these new sites are located within a stretch of 50 amino
acid residues within the tau sequence (37). This 50-amino acid stretch composed of residues 212-262 may be one of the regions that becomes exposed during heparin-induced conformational change of tau.
Tau has been suggested not to have a unique folded conformation but to
exist as a random coil (35, 40). In fact, tau can be boiled and
dissolved in acid without losing its microtubule binding ability,
indicating that folded conformation may not be necessary for
tau-microtubule interaction (41), and, in solution, tau behaves as a
disordered polypeptide without any compact folding (40). In this study,
however, by chemical cross-linking and phosphopeptide mapping we showed
that heparin induces a conformational change in tau, exposing new sites
for kinase phosphorylation. Thus, our data suggest that tau may have a
defined conformation that can be affected by ligands such as heparin.
In a previous study (10), NCLK phosphorylated tau on seven sites,
including a partial phosphorylation of Thr231. In this
study, we found that NCLK phosphorylates Thr231 only when
heparin is included in the phosphorylation mixture. The reason for this
discrepancy is very likely attributable to the type of tau used in the
two studies. In the previous study (10), tau purified form brain
extract was used as the substrate as opposed to the present work, which
used bacterially expressed recombinant tau. Moreover, we found that
phosphorylation by NCLK on this site can be induced by a substrate
modulator such as heparin. A fraction of brain tau used in the previous
study (10) may have contained some substrate modulator, and that may
have led to the phosphorylation of Thr231 by NCLK.
mAb 12E8 was raised against a phosphopeptide corresponding to tau
residues 257-270 containing phosphate on Ser262 (42). This
mAb has been used by various investigators to determine the
phosphorylation of tau on Ser262. Recently, tau
phosphorylated by NCLK in the presence of heparin was reported to be
immunolabeled by this mAb (32). Similarly, this mab was reported to
cross-react with tau phosphorylated by A kinase (43). Based on these
data NCLK was suggested to phosphorylate tau on Ser262 in
the presence of heparin (32), whereas A kinase phosphorylates tau on
Ser262 in the absence of any effector (43). In this study,
we found that NCLK does not phosphorylate tau on Ser262 in
the presence of heparin, and A kinase phosphorylates Ser262
only when heparin is included in the phosphorylation mixture. Consistent with our data, a previous study using direct sequence analysis of phosphopeptides showed that A kinase does not phosphorylate tau on Ser262 (16). Furthermore, protein kinase C and
calcium/calmodulin kinase II (17, 18) do not phosphorylate tau on
Ser262, but tau phosphorylated by both of these kinases
displays immunoreactivity to mAb 12E8 (44). These observations suggest
that mAb 12E8 immunoreactivity may not truly reflect the
phosphorylation state of tau on Ser262. There are other
widely used phosphorylation-sensitive mAbs against tau, such as PHF1,
tau 1, AT8, SMI31, SMI33, SMI34, and AP422. These mAbs have all been
found to be unreliable in determining the site-specific phosphorylation
state of tau (45).