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J. Biol. Chem., Vol. 275, Issue 32, 24977-24983, August 11, 2000
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From the Pharmacia Corporation, Kalamazoo, Michigan
49007
Received for publication, February 2, 2000, and in revised form, May 11, 2000
In Alzheimer's disease, hyperphosphorylated tau
is an integral part of the neurofibrillary tangles that form within
neuronal cell bodies and fails to promote microtubule assembly.
Dysregulation of the brain-specific tau protein kinase II is reported
to play an important role in the pathogenesis of Alzheimer's disease
(Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S.,
Dikkes, P., and Tsai, L.-H. (1999) Nature 402, 615-622).
We report here that in vitro phosphorylation of human tau
by human recombinant tau protein kinase II severely inhibits the
ability of tau to promote microtubule assembly as monitored by tubulin
polymerization. The ultrastructure of tau-mediated polymerized tubulin
was visualized by electron microscopy and compared with phosphorylated
tau. Consistent with the observed slower kinetics of tubulin
polymerization, phosphorylated tau is compromised in its ability to
generate microtubules. Moreover, we show that phosphorylation of
microtubule-associated tau results in tau's dissociation from the
microtubules and tubulin depolymerization. Mutational studies with
human tau indicate that phosphorylation by tau protein kinase II at
serine 396 and serine 404 is primarily responsible for the functional
loss of tau-mediated tubulin polymerization. These in vitro
results suggest a possible role for tau protein kinase II-mediated tau
phosphorylation in initiating the destabilization of microtubules.
Six isoforms of human tau are expressed in adult human brain (1).
These arise from alternate splicing of the mRNA transcribed from a
single gene located on the long arm of chromosome 17 (1-5). Tau
isoforms differ from each other with respect to the presence of three
or four tandem repeats of 31 amino acids each in combination with two
N-terminal inserts of 29 amino acids. The longest form of tau contains
four tandem repeats, and the 58-amino acid N-terminal insert that
yields a full-length protein of 441 amino acids (1). The four 31-amino
acid repeat domains have been shown to bind microtubules, whereas most
of the phosphorylation sites in tau are located outside this
microtubule binding repeat region. One of the functions of the tau
protein is to promote microtubule assembly in vivo and to
stabilize microtubules in the nervous system (6-10).
Neurofibrillary tangles and senile plaques constitute two prominent
neuropathological characteristics of Alzheimer's disease (AD)1 (11, 12). The main
fibrous component of all neurofibrillary lesions is paired helical
filament (PHF), which contains predominantly the abnormally
phosphorylated tau (13-18). It has been hypothesized that aberrant
phosphorylation of tau leads to its aggregation into PHF, resulting in
destabilization of microtubules and the death of neurons (1, 19).
Notably, hyperphosphorylated tau has been detected in other
tau-positive filamentous lesions, in a group of diseases collectively
known as the tauopathies. These neurodegenerative diseases include
progressive supranuclear palsy, corticobasal degeneration, Down's
syndrome, frontotemporal dementia and Parkinsonism linked to chromosome
17, and Pick's disease (20). Recent studies show that these various
phenotypes might be the result of phosphorylation of specific tau
isoforms in different nerve cells in distinct brain regions (21).
Tau-associated with PHF from AD brain is hyperphosphorylated at
several serine/threonine sites that are followed by a proline (22). AD
tau protein in its hyperphosphorylated state fails to promote
microtubule (MT) assembly in vitro (23) and this phosphorylation-dependent tau dysfunction is considered one
of the critical events leading to neuronal degeneration (24). It is not
known which kinase initiates tau hyperphosphorylation and MT
disassembly and which phosphorylated amino acids in tau contribute to
tau's dysfunction. Understanding the molecular basis for tau dysfunction requires identification and availability of highly purified
candidate tau protein kinase, careful mapping of phosphorylation sites,
mutational studies, and functional characterization of the
corresponding phosphorylated tau mutants. There are two
proline-directed kinases, tau protein kinase II (TPK II) and glycogen
synthase kinase 3 One of the hypotheses is that TPK II initiates tau hyperphosphorylation
and MT disassembly. To the best of our knowledge, a highly purified and
well-characterized source of human recombinant TPK II has not been
reported. Therefore, the definition of TPK II-mediated phosphorylation
sites in human tau and their potential role in tau dysfunction remain
unknown. Recently, we have been successful in producing milligram
quantities of highly purified human recombinant TPK II by in
vitro reconstitution using separately expressed regulatory
(Cdk5) and activator (p20) subdomains of this kinase. This critical
step has allowed us to map TPK II phosphorylation sites in human tau by
a combination of mass spectrometric
techniques.2 We found that
the six TPK II-mediated phosphorylation sites in tau are Thr-181,
Thr-205, Thr-212, Thr-217, Ser-396, and Ser-404, based on the numbering
used in the longest form () of human tau (1).
The aims of the present study were to test the TPK II hypothesis for
initiation of tau hyperphosphorylation and MT disassembly and to
understand the molecular basis for phosphorylationdependent tau
dysfunction and its relevance to tauopathies. We report here that
phosphorylation of human tau by TPK II inhibits tau's ability to
promote microtubule assembly and that TPK II-mediated phosphorylation of microtubule-associated tau results in tau's dissociation from the
microtubules and tubulin depolymerization. Based on the knowledge gained from mapping human TPK II-mediated phosphorylation sites in
human tau,2 the molecular basis for this tau dysfunction
has been determined using mutational studies on human tau. We show that
TPK II-mediated tau phosphorylation at Ser-396 and Ser-404 is primarily
responsible for the loss of tau function in MT assembly and
dissociation of tau from microtubules. These results are discussed with
regard to TPK II as a potential target for inhibiting initiation of tau hyperphosphorylation and the implications for tauopathies endowed with
Ser-396 and Ser-404 phosphoepitopes (20, 21).
General Chemicals--
General laboratory chemicals and
molecular biology reagents were purchased from Sigma and Life
Technologies, Inc., respectively. The oligonucleotides were ordered
from Genosys Biotechnologies Inc. The baculovirus gold DNA was obtained
from Pharmingen Inc. The expression vectors were purchased from
Amersham Pharmacia Biotech and Novagen. Competent BL-21 cells were
obtained from Stratagene. The template p35 DNA was kindly provided by
Dr. Tsai (Harvard Medical School). The clone for human tau () was
obtained from Dr. Virginia Lee (University of Pennsylvania). Protein
concentrations were determined using the BCA assay from Pierce. All the
electrophoresis reagents were from Bio-Rad. Recombinant glycogen
synthase kinase 3 In Vitro Reconstitution of TPK II (cdk5/p20)--
The
full-length human cdk5 gene was inserted into the baculovirus genome
using standard baculovirus expression vector technology. A sample
plaque isolate producing the highest level of cdk5 was plaque-purified
three times, and a single virus plaque was used to make stock virus for
cdk5 production. For in vitro reconstitution of TPK II,
human recombinant native cdk5 was partially purified by Q-Sepharose
from baculovirus-infected insect cells. The truncated version of human
p35 activator protein, p20 (G137-R307), was cloned and expressed in
Escherichia coli as a ubiquitin fusion containing an
internal hexa-histidine sequence. For in vitro
reconstitution and purification of human recombinant TPK II (cdk5/p20),
the clone DE-93,4, containing the p20 construct, was grown in
NS-85 medium (29) until the A600 reached 0.4 and
induced for 3 h with 1 mM isopropylthio- Cloning, Expression, and Purification of Tau and Its
Mutants--
Wild type tau () with an octa-histidine C-terminal
tag was cloned and expressed in E. coli by inserting it
in-frame behind the T7 tag of the pET23a expression vector as a
BamHI/EcoRI fragment. Plasmid DNA from the
DE-105,2 clone was isolated and used to transform competent BL-21
(DE-3) cells used for expression with 1 mM
isopropylthio- TPK II Phosphorylation of Tau--
Tau phosphorylation was done
by incubating 5 µM tau with TPK II (100 nM)
for 4 h at 37 °C in a buffer containing 40 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM DTT, and
1 mM ATP. Following incubation the samples were
electrophoresed on 12% SDS-PAGE (31) and stained with Coomassie Blue
R-250 to verify phosphorylation. For tubulin polymerization assays, the
tau samples were dialyzed into 80 mM PIPES, pH 6.8, 1 mM DTT.
Tubulin Polymerization Assay--
The tubulin polymerization
assay was carried out essentially as described by Cytoskeleton. Tubulin
(10 mg/ml) from Cytoskeleton (catalog no. T238) was thawed on ice and
diluted to 2 mg/ml with cold G-PEM buffer (80 mM PIPES, pH
6.8, 0.5 mM MgCl2, 1 mM EGTA, and 1 mM freshly made GTP). The assay was done in 96-well
microtiter plates by incubating 75 µl of 2 mg/ml tubulin (1 mg/ml
final) in a total buffer of 150 µl of G-PEM containing tau at a final concentration of 7 µM. The reaction was initiated with
the addition of tubulin, the plate was incubated at 27 °C in a
Molecular Devices microplate reader, and the
A340 measurement was determined every 1 min for
30 min.
TPK II Phosphorylation of Tau Associated with Polymerized
Tubulin--
Tubulin was polymerized in the presence of 7 µM (1-383 isoform) tau under the conditions described
above. After 30 min, either buffer, TPK II (100 nM final),
ATP (1 mM final), or TPK II + ATP was added to the
preformed microtubules and the A340 was measured every 15 min for the next 3.5 h. Following the turbidity assay, aliquots were taken from the buffer control and TPK II-treated samples,
and 100 ng of tau was subjected to 12% SDS-PAGE and blotted onto
nitrocellulose. The blot was probed with a monoclonal anti-T7 primary
antibody (Novagen) at a dilution of 1:5000 followed by goat anti-(mouse
IgG) peroxidase secondary antibody at a dilution of 1:10,000 and
detected using ECL reagents (Amersham Pharmacia Biotech).
Wild type human tau () was expressed at high levels in
E. coli and purified by IMAC using nickel as the immobilized
metal ion. The 1-383 isoform of human tau used in this work lacked the two N-terminal repeats, but contained the four microtubule binding repeats. It had the same number of proline-directed phosphorylation sites as the longest form () of human tau. Throughout this
discussion, the numbering of phosphorylation sites in human recombinant
tau is based on the longest form () of tau (1). The mass
spectroscopy analysis of IMAC-purified human recombinant tau ()
confirmed that it was devoid of any phosphorylation.
Fig. 1 shows SDS-PAGE of purified
recombinant TPK II, a complex of cdk5, and its p20 activator protein.
The recombinant enzyme was reconstituted on an IMAC column and eluted
with imidazole (lane 1). This partially purified complex was
enzymatically active but required further affinity purification on
immobilized streptavidin (lane 2) and ATP agarose
(lane 3) columns. This highly purified (>90%) and
well-characterized TPK II (lane 3) was used for in vitro tau phosphorylation studies in the presence of 1 mM ATP. The TPK II-phosphorylated tau was subjected to mass
spectrometry analysis, which showed2 phosphorylation of tau
at six proline-directed sites (Thr-181, Thr-205, Thr-212, Thr-217,
Ser-396, and Ser-404). Although the extent of phosphorylation at
each site is unknown, these results were consistent with the
incorporation of 5-6 mol of phosphate/mol of tau.
It is well known that microtubules (MT) are formed in vitro
by noncovalent polymerization of tubulin dimers (23, 32-35). Formation
of MT from tubulin solution can be followed by observing an increase in
absorbance at 340 nm in the presence of GTP and tau (23, 32, 34, 35).
This continuous spectrophotometric tubulin polymerization assay was
used for functional characterization of TPK II-phosphorylated human
recombinant tau. Fig. 2 shows that, under
defined conditions, TPK II-phosphorylated tau failed to promote tubulin
polymerization. Fig. 2 (inset, lane 3) shows that TPK II-phosphorylated tau has a significantly higher molecular mass
than does unphosphorylated tau (inset, lane 2).
There was a requirement for wild type tau, tubulin, and GTP, because no polymerization occurred when tubulin alone was incubated with GTP (data
not shown). The polymerization rate (absorbance units per minute) for
TPK II-phosphorylated tau was 5% compared with wild type tau, and the
level of polymerized tubulin at steady state
(A340 maximum) was 10% compared with wild type
tau. In contrast, tau-dependent tubulin polymerization was
observed when tau was phosphorylated with TPK I (data not shown), in
agreement with a recent report (36). We also studied the effect of tau
concentration on tubulin polymerization under defined assay conditions.
The increase in turbidity (A340) was linear with
tau concentration for unphosphorylated tau, whereas TPK
II-phosphorylated tau inhibited MT assembly at all the tau
concentrations tested (data not shown).
Tau Phosphorylation at Serine 396 and Serine 404 by Human
Recombinant Tau Protein Kinase II Inhibits Tau's Ability to Promote
Microtubule Assembly*
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ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]()
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TPK I), which have been found to be
associated with microtubules (25, 26) and are known to phosphorylate
tau in a cellular environment (27, 28).
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EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TPK I) was from New England BioLabs (5000 units/ml). Tubulin used for the polymerization assays was from
Cytoskeleton (catalog no. T238), and the tubulin polymerization assays
were done using a SPECTRAmax Plus microplate reader from Molecular Devices.
-D-galactoside. The crude E. coli extract was adjusted to pH 8.0 using 2 M Tris and
centrifuged at 12,000 × g for 1 h, and the
supernatant was loaded onto a nickel immobilized metal affinity
chromatography (IMAC) column equilibrated in 50 mM Tris, pH
8.0. The column was washed overnight with 50 mM Tris, pH
8.0, followed by buffer containing 50 mM imidazole and was
then re-equilibrated with 50 mM Tris, pH 8.0. Partially
purified cdk5 was diluted 1:2 with 50 mM Tris, pH 8.0, and
loaded onto the IMAC. The column was washed with buffer containing 50 mM imidazole, and the complex was eluted with buffer
containing 300 mM imidazole. Fractions containing TPK II
(cdk5/p20) by SDS-polyacrylamide gel electrophoresis (PAGE) were pooled
and dialyzed in 20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT). Further purification of
the IMAC-isolated TPK II was carried out using immobilized streptavidin
(Pierce) followed by affinity chromatography on an ATP-agarose column (Sigma).
-D-galactoside for 4 h in NS-85 media
(29). Cells were collected by centrifugation, and the cell pellets were
stored at
80 °C and used for IMAC purification as described
elsewhere (30). The tau protein was eluted with buffer containing 300 mM imidazole, and protein-containing fractions were
determined by absorbance at 280 nm. Samples were run on 12% SDS-PAGE,
and the tau-containing samples were pooled and dialyzed in 50 mM Tris, pH 8.0, 1 mM DTT. All the tau mutants
were obtained using 1-383 tau DNA, mutagenized primers, and the
QuikChange site-directed mutagenesis kit from Stratagene. Expression
and purification of the mutant proteins and truncated tau (244Q-390A)
were carried out as described above for wild type () tau.
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RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

View larger version (45K):
[in a new window]
Fig. 1.
SDS-PAGE (12%) of recombinant TPK II
obtained by in vitro reconstitution from cdk5 and its p20
activator protein. Lane 1, TPK II isolated by in
vitro reconstitution on IMAC; lane 2, TPK II eluted
from immobilized streptavidin in the presence of biotin; lane
3, purified TPK II obtained after elution from an ATP-agarose
column; lane 4, molecular mass markers.

View larger version (22K):
[in a new window]
Fig. 2.
Effect of TPK II phosphorylation on the
ability of wild type human tau (1-383 isoform) to promote microtubule
assembly. The data represent the average of three experiments ± S.D. Open circles, unphosphorylated tau; filled
circles, TPK II-phosphorylated tau. Inset, a 12%
SDS-PAGE of the 1-383 isoform of wild type human tau. Lane
1, molecular mass markers; lane 2, unphosphorylated
wild type tau; lane 3, TPK II-phosphorylated wild type
tau.
As shown in Fig. 2, measurement of turbidity changes allowed us to
differentiate unphosphorylated tau from TPK II-phosphorylated tau by
its ability to promote tubulin polymerization. However, to directly
demonstrate the effect of TPK II-phosphorylated tau on microtubule
assembly, we carried out negative-stain electron microscopy at
steady-state tubulin polymerization. Fig.
3 shows electron micrographs of the MT
assembly reaction at steady state for TPK II-phosphorylated tau and
unphosphorylated tau. As shown, large microtubules were seen in
assembly promoted by unphosphorylated tau (Fig. 3A),
demonstrating that the recombinant human tau is fully functional. In
contrast, MT assembly was significantly impaired in terms of length and
mass in the presence of TPK II-phosphorylated tau (Fig. 3B).
In the tubulin control (data not shown), no microtubule assembly was
observed.
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The above results led us to investigate the effect of TPK II on
MT-associated tau. Fig. 4A
shows that addition of TPK II and ATP to preformed MT caused a decrease
in A340 as a function of time. These results
indicate that, as tau gets phosphorylated on MTs, it disrupts MT
assembly as monitored by a time-dependent decrease in
absorbance. When either TPK II or ATP was added alone either at
t = 0 or t = 30 min, no significant
change in absorbance from the buffer control was observed. Furthermore,
the buffer control sample and TPK II-phosphorylated sample after
dissociation from MTs were analyzed by SDS-PAGE followed by Western
blotting. Tau was probed with anti-T7 monoclonal antibody known to
recognize the N-terminal epitope tag engineered on human tau. As shown
in Fig. 4B, the TPK II-treated sample showed a higher
molecular mass than did the control, suggesting that detachment of tau
from MT is indeed due to phosphorylation of tau by TPK II. In contrast, when a truncated form of tau (244Q-390A), consisting of the four MT
binding repeats but none of the TPK II phosphorylation sites, was used
for MT assembly, the addition of TPK II had no effect on preformed MTs
(Fig. 4A). Taken together, these in vitro results show that TPK II phosphorylation impairs functional activity of tau,
which is critical for stabilizing the microtubule transport system. To
our knowledge, TPK II is the only known example of a proline-directed
kinase that can impair the ability of tau to promote MT assembly and
induce MT disassembly in vitro under defined conditions.
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In the TPK II-phosphorylated tau (Fig. 2), the relative importance of
each of the six phosphorylated sites on tubulin polymerization cannot
be determined. Therefore, in an attempt to understand the molecular
basis for the loss of functional activity of tau upon phosphorylation,
we prepared a number of tau mutants based on identification of TPK II
phosphorylation sites in tau. Five of these mutants (T181A, T205A,
T212A, T217A, and S396A) contain Ala for Thr or Ser, whereas the S404G
mutant contained Gly for Ser. Although the S404A tau mutant was cloned,
expressed, purified, and characterized, this unphosphorylated tau
mutant (S404A) was dysfunctional in the tubulin polymerization assay
for unknown reason(s). Therefore, we were unable to use it as a control
to study the effect of TPK II phosphorylation on tubulin polymerization and decided to prepare and use S404G for our studies. These tau mutants
were phosphorylated with TPK II, and results for T181A, T205A, T212A,
and T217A are shown in Fig.
5A. A similar shift in
molecular mass was observed when the S396A mutant was phosphorylated with TPK II (Fig. 5B). However, as shown in Fig.
5B, the extent of TPK II phosphorylation was relatively less
in single and double mutants containing a mutation at Ser-404. We
interpret these results to mean that TPK II phosphorylation of tau at
Ser-404 is responsible for the observed higher molecular mass in
mutants containing Ser-404. However, other alternative explanations
such as the effect of the S404G or S404A mutation on other TPK II sites
cannot be ruled out from these studies.
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All six unphosphorylated tau mutants were competent to elicit tubulin polymerization (data not shown), and the results were comparable with wild type tau (Fig. 2). Therefore, we studied the effect of TPK II-phosphorylated tau mutants (T181A, T205A, T212A, T217A, S396A, and S404G) on tubulin polymerization. Specifically, we determined the effect of these mutations on nucleation time (lag phase) and extent of polymerization (A340 maximum). These studies showed (Table I) that, like wild type tau (Fig. 2), TPK II phosphorylation of the T181A, T212A, and T217A tau mutants inhibited tau's ability to promote tubulin polymerization as determined by absorbance at steady state. As shown in Table I for these three tau mutants, both the lag time and the extent of polymerization were severely impaired and these results were comparable to the TPK II-phosphorylated tau shown in Fig. 2. These results suggest that phosphorylation at the other TPK II sites (Thr-205, Ser-396, and Ser-404) might play an important role in inhibiting MT assembly. Indeed, the inhibition of tubulin polymerization was less with the TPK II-phosphorylated forms of T205A, S396A, and S404G mutants than with TPK II-phosphorylated WT tau (Table I). Of these three sites, based on mutational studies, phosphorylation at Ser-404 had a more dramatic effect on nucleation time (lag phase). Also, in this case the rate of polymerization after the onset of the nucleation event was significantly higher relative to the other five single tau mutants (data not shown). Taken together, the data suggest that TPK II phosphorylation of tau at Thr-205, Ser-396, and Ser-404 seems to be responsible for impairment of microtubule assembly.
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The above studies led us to prepare double (T205A/S404A; S396A/S404A)
and triple (S396A/S404A/T205A) tau mutants. Their effects on tubulin
polymerization are included in Table I for comparison with single
mutants. Interestingly, the rate and the extent of tubulin
polymerization were both increased using the TPK II-phosphorylated S396A/S404A double mutant relative to the TPK II-phosphorylated wild
type tau (Fig. 6). As shown in Table I,
relative to the phosphorylated forms of S396A/S404A double and S404G
single mutants, an additional T205A mutation in tau does not
significantly alter the lag time or the extent of tubulin
polymerization in the corresponding TPK II-phosphorylated double
(T205A/S404A) and triple (T205A/S396A/S404A) tau mutants. These results
suggest that to a large extent phosphorylation at Ser-396 and Ser-404
is responsible for tau's inability to promote tubulin polymerization.
Table I shows that the extent of tubulin polymerization was increased
with the TPK II-phosphorylated form of the S396A/S404A double mutant,
whereas the lag time was decreased relative to either the TPK
II-phosphorylated S396A or the phosphorylated S404G tau mutants. Taken
together, we conclude that in vitro TPK II phosphorylation
of tau at Ser-396 and Ser-404 is primarily responsible for the
inability of TPK II-phosphorylated tau to promote microtubule
assembly.
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DISCUSSION |
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Bovine brain TPK II (37-39) and activation of cdk5 in the presence of various truncated recombinant forms of the activator protein (p35) from bovine, mice, and human have been reported (40-45). However, there is no report on the purification and characterization of human recombinant TPK II and mapping of the TPK II phosphorylation sites in human tau. Our mapping studies2 show that human TPK II phosphorylates six sites in human tau. These sites are Thr-181, Thr-205, Thr-212, Thr-217, Ser-396, and Ser-404. In contrast, a partially purified protein kinase from bovine brain was shown to phosphorylate proline-directed Ser-144, Thr-147, Ser-177, and Ser-315 in tau from bovine brain extracts (46). These phosphorylation sites in bovine tau corresponded to Ser-202, Thr-205, Ser-235, and Ser-404 in human tau when the sequences of bovine and human tau were aligned (46). This crude kinase fraction from bovine brain was later termed TPK II (25). A similar proline-directed kinase isolated from bovine brain was reported to phosphorylate seven sites in bovine tau. These sites, when aligned with the human tau sequence, corresponded to Ser-195, Ser-202, Thr-205, Thr-231, Ser-235, Ser-396, and Ser-404 (39). Furthermore, it was shown (47) that bovine TPK II phosphorylates human tau at Thr-212 and Thr-231 only in the presence of heparin as an inducer of tau phosphorylation. From these studies (39, 46, 47) it is extremely difficult to directly compare phosphorylation sites and kinases involved, because the outcome of such results depends upon the enzyme source, purity of kinase, source and purity of tau, and presence of inducers like heparin (47). Therefore, it is not surprising to find that these previous studies on bovine and human tau phosphorylation sites (39, 46, 47) were mutually inconsistent. Notably, our mapping studies of human tau phosphorylation sites2 were performed with highly (>90%) purified (Fig. 1) and well-characterized human TPK II without inducers. Thus, prior to our studies, the definition of human TPK II-mediated phosphorylation sites in human tau and the key phosphorylated amino acids that affect microtubule assembly and disassembly were unknown.
Tau phosphorylation and MT disassembly by the proline-directed TPK II may be involved in the etiology of neurofibrillary tangle pathology in AD. Recent studies suggest that TPK II activity is elevated in the AD brain compared with age-matched controls (48). Moreover, the activator protein p35/p25 of TPK II is localized only in central nervous system neurons (40, 41) and has been shown to be accumulated in a truncated form (p25) in the AD brain (49). The accumulation of p25 has also been correlated to an increase in TPK II (cdk5/p25) activity in the AD brain (49), and an overexpression of the p25 activator subunit of TPK II in transgenic mice has been shown to result in hyperphosphorylated tau and cytoskeletal disruption (50). In this context, intracellular tau is undefined and is apt to display microheterogeneity in terms of phosphorylation. We have observed distinct differences in tubulin polymerization in vitro (Table I), depending upon the phosphorylation status of human tau. Therefore, cell and animal studies (27, 28, 49, 50) do not reveal which sites are phosphorylated by which kinase and which phosphorylated amino acids in tau contribute to cytoskeleton disruption. Biochemical studies (Figs. 2-6), using purified human TPK II (Fig. 1) and human tau, are unique and have focused on understanding the functional and molecular basis of initial events that trigger tau phosphorylation and microtubule breakdown.
Axonal transport mechanism relies on microtubules as tracks that are stabilized by tau (51). Our results show that human TPK II-phosphorylated tau is impaired in its ability to promote MT assembly (Figs. 2 and 3). All the proline-directed TPK II phosphorylation sites in tau identified from our mapping studies2 lie outside the MT binding repeat domains. This implies that tau's ability to maintain microtubule tracks seems to depend on the lack of phosphorylation at critical sites outside of the microtubule-binding domain. Our in vitro results indeed show (Fig. 4) that TPK II phosphorylation of tau at critical amino acids detaches tau from microtubules, leading to the breakdown of microtubules.
There are 12 sites in human tau that contain a Ser/Thr-Pro motif (1), and these are canonical sites for proline-directed kinases. These sites are Thr-175, Thr-181, Ser-199, Ser-202, Thr-205, Thr-212, Thr-217, Thr-231, Ser-235, Ser-396, Ser-404, and Ser-422. Human TPK II phosphorylates six of these sites in human tau. Our in vitro tau mutational and functional studies (Figs. 5 and 6, Table I) are the first to demonstrate that of the six sites phosphorylated by TPK II, phosphorylated Ser-396 and Ser-404 are critical in impairing microtubule assembly. To our knowledge, these studies are the first to show that human TPK II, in contrast to TPK I, can initiate tau phosphorylation of MT-associated tau and dissociation of tau from microtubules (Fig. 4).
Studies (52) indicate that TPK I seems to prefer to phosphorylate Ser and Thr residues positioned N-terminal to another phosphoserine and the preferred consensus sequence is (S/T)XXXS-P. Based on the specificity of TPK I, a potential role of TPK II in generating TPK I sites could be envisioned. In earlier studies (53-55), it was predicted that prior phosphorylation of tau by another kinase may be required in the generation of phospho-Thr-231 by TPK I. Recently, it has been shown that phosphorylation of tau by TPK II is required to generate an AD-related TG-3 phosphoepitope by TPK I (55). To inhibit MT-promoting activity by TPK I phosphorylated tau, prior phosphorylation of tau by protein kinase A was required (56). These results are consistent with the notion that TPK I is a secondary kinase. Out of the two proline-directed kinases known to associate with microtubules (25, 26) and to phosphorylate tau in cells (27, 28), phosphorylation at two key amino acids (Ser-396 and Ser-404) by TPK II impairs microtubule assembly (Figs. 2-4, 6, Table I).
A number of phosphorylated epitopes in tau have been observed in tau-based diseases (21, 57, 58) known as tauopathies (Down's syndrome, frontotemporal dementia and Parkinsonism, Pick's disease, progressive supranuclear palsy, and corticobasal degeneration). The most commonly observed proline-directed phosphoepitopes in tau are Ser-202, Thr-205, Thr-212, Ser-214, Thr-231, Ser-235, Ser-396, and Ser-404. Notably, Ser-396 and Ser-404 are a pair of phosphoepitopes found in hyperphosphorylated tau in these diseases. At present, nothing is known about the mechanisms underlying initiation of tau hyperphosphorylation and MT disassembly in these diseases. In the present studies, we have identified the same two sites in human tau (Fig. 6) whose phosphorylation by human TPK II is primarily responsible for tau dysfunction. Although the potential significance of our findings in these tauopathies remains to be elucidated, they suggest that TPK II phosphorylation may be an obligatory step in tau hyperphosphorylation. In addition, identification of phosphorylated Ser-396 and Ser-404 (Fig. 6) is important in developing a unifying molecular theme for the role of TPK II in tau hyperphosphorylation observed in a number of neurodegenerative diseases.
Our studies support the hypothesis that the initial phosphorylation of
tau by TPK II is an essential step in the detachment of tau from
microtubules. These in vitro results are consistent with the
proposed role of TPK II in AD pathogenesis (49) as well as with the
most recent studies in transgenic mice overexpressing the p25 activator
protein of TPK II (50). Accordingly, inhibition of this
proline-directed kinase may prevent initiation of tau hyperphosphorylation and in turn should preserve the MT transport system and delay or prevent the onset of dementia in AD and other related tauopathies.
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ACKNOWLEDGEMENTS |
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We are thankful to the laboratories of Dr. R. L. Heinrikson and Dr. J. Slightom for their help in amino acid analysis and DNA sequencing, respectively. We also thank George Melchior and Irene Abraham for helpful discussions. Electron microscopy studies were performed with the assistance of Dr. Rob Eversole at Western Michigan University.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Pharmacia Corp.,
Protein Science, 7240-267-117, Kalamazoo, MI 49007. Tel.: 616-833-9413; Fax: 616-833-1488; E-mail: satish.k.sharma@am.pnu.com.
Published, JBC Papers in Press, May 18, 2000, DOI 10.1074/jbc.M000808200
2 E. T. Lund, R. McKenna, R., D. B. Evans, S. K. Sharma, and W. R. Mathews, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: AD, Alzheimer's disease; MT, microtubules; TPK II, tau protein kinase II; TPK I, tau protein kinase I; PHF, paired helical filaments; DTT, dithiothreitol; Cdk5, cyclin-dependent kinase 5; IMAC, immobilized metal affinity chromatography; PAGE, polyacrylamide gel electrophoresis; WT, wild type; PIPES, 1,4-piperazinediethanesulfonic acid.
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