(Received for publication, March 9, 1995; and in revised form, December 12, 1995)
From the
Tau proteins isolated from paired helical filaments, the major
building blocks of Alzheimer's disease neurofibrillary tangle,
are abnormally phosphorylated and unable to bind microtubules. To
examine the dynamics of tau phosphorylation and to identify specific
tau phosphorylation sites involved in the stabilization of
microtubules, we treated cultured postmitotic neuron-like cells (NT2N)
derived from a human teratocarcinoma cell line (NTera2/D1) with drugs
that depolymerize microtubules (i.e. colchicine or
nocodazole). This led to the recovery of dephosphorylated tau from the
NT2N cells as monitored by a relative increase in the electrophoretic
mobility of tau and an increase in the turnover of
[P]PO
-labeled tau. However, not all
phosphorylation sites on tau are affected by colchicine or nocodazole.
Ser
/Thr
appears to be completely and
specifically dephosphorylated by protein phosphatase 2A since this
dephosphorylation was blocked by inhibitors of protein phosphatase 2A
but not by inhibitors of protein phosphatase 2B. These findings,
together with the recent observation that protein phosphatase 2A is
normally bound to microtubules in intact cells, suggest that the
polymerization state of microtubules could modulate the phosphorylation
state of tau at specific sites in the normal and Alzheimer's
disease brain.
Paired helical filaments (PHFs) ()are the major
building blocks of neurofibrillary tangles, neuropil threads, and
senile plaque neurites in Alzheimer's disease brains (reviewed in (1, 2, 3) ). PHFs are composed of abnormally
phosphorylated central nervous system (CNS) tau proteins (PHF-tau) (4, 5, 6, 7, 8) that
normally promote and stabilize the assembly of microtubules
(MTs)(9) . Normal adult CNS tau consists of six alternatively
spliced isoforms encoded by one gene(10, 11) , and all
are found in PHF-tau(12) , which differs from normal tau in the
extent and sites of
phosphorylation(2, 8, 13, 14, 15, 16) .
We recently showed that some of the putative ``abnormal''
phosphorylation sites in PHF-tau are normal sites of phosphorylation in
adult rat (17) and adult human brain tau isolated from biopsy
samples (18) . These studies also showed that rat and human
brain tau proteins are phosphorylated to a lesser extent at many of the
same sites as in PHF-tau. Significantly, an increase in tau
phosphorylation decreases MT binding(19, 20) , and
enzymatic dephosphorylation PHF-tau restores its ability to bind
MTs(20) . Several phosphorylation sites in tau (including
Ser
and Ser
) modulate binding to
MTs(20, 21) , and hyperphosphorylation of these sites
may decrease the affinity of PHF-tau for MTs leading to the
depolymerization of MTs, impaired axonal transport, neuronal
degeneration, and the aggregation of tau into PHFs.
The affinity of tau for MTs is regulated by the number of MT binding repeats(22, 23) , proline-rich sequences adjacent to the MT binding repeats(24) , linker sequences between MT binding repeats(25) , and the extent and sites of phosphorylation(19, 20, 21) . The expression of different tau isoforms and their phosphorylation states are developmentally regulated(2, 11, 17, 18, 20) . During human development only the smallest isoform is expressed, and this isoform contains three MT binding repeats and no N-terminal inserts(11) . The six adult human brain tau isoforms differ in the number of MT binding repeats and with respect to the presence or absence of N-terminal inserts(11, 22) , while fetal tau is more phosphorylated than adult tau. Thus, the number of MT binding repeats and phosphorylation may facilitate reorganization of the cytoskeleton during axonal growth and synaptogenesis by modulating the affinity of fetal tau for MTs.
Although several kinases and phosphatases have been implicated in regulating the phosphorylation state of tau in vitro(12, 21, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) , it is not known which of these enzymes performs this function in vivo, and the mechanisms that lead to the abnormal phosphorylation of PHF-tau in the Alzheimer's disease brain are unknown. In part, this reflects the lack of model systems of Alzheimer's disease neurofibrillary lesions. Moreover, biochemical studies of tau in cultured cells are difficult since tau is expressed almost exclusively in postmitotic neurons. Recently, we have obtained nearly pure cultures of neuron-like cells (NT2N) by treating a human teratocarcinoma cell line (NTera2/D1 or NT2) with retinoic acid(39, 40) . Notably, the neuron-like NT2N cells are irreversibly postmitotic and develop the phenotype of late embryonic CNS neurons(40, 41) .
Here, we used the NT2N neurons as a model system to study the regulation of the phosphorylation state of human tau, as well as the affinity of tau for MTs, by perturbing the MT network with colchicine or nocodazole, which lead to the site-specific dephosphorylation of tau. Further, we also demonstrated that the site-specific dephosphorylation of tau is due to the action of protein phosphatase 2A (PP2A).
Figure 2:
Immunoblot analysis of native and
enzymatically dephosphorylated tau isolated from NT2N cells and from
human fetal brain. Heat-stable preparations of high salt extracts from
NT2N cells and isolated human fetal tau were treated with alkaline
phosphatase. Nitrocellulose replicas were probed with the anti-tau mAbs
T14/46 (panel A) and PHF1 (panel B). Lane 1 contains a mixture of all six isoforms of recombinant tau
expressed in E. coli (R); lane 2,
enzymatically dephosphorylated NT2N tau (dP, NT2N, 40
µg); lane 3, native NT2N tau (P, NT2N,
40 µg); lane 4, enzymatically dephosphorylated human fetal
tau (dP, F
, 5 µg); lane 5, native
human fetal tau (P, F
, 5 µg). Note that
native tau from NT2N cells co-migrates with human fetal tau (panel
A, lanes 3 and 5), that dephosphorylated NT2N
tau and dephosphorylated fetal tau co-migrate (panel A, lanes 2 and 4), and that tau from NT2N cells has the
same immunoreactivity for PHF1 as human fetal tau (panel B, lanes 3 and 5). The positions of 66- and 45-kDa
molecular mass standards are shown on the right.
Figure 1: Schematic of tau protein showing epitope location of the antibodies used in this study. The antibodies are identified in boldface, and the numbers in parentheses indicate the amino acid positions of the epitopes on the largest human brain tau isoform. The bars illustrate the relative positions of the epitopes (the figure is not drawn to scale). The relative locations of the N-terminal inserts and C-terminal MT binding repeats are shown. The MT binding repeats are continuous, and the space between each one here is used merely to facilitate the visualization of each repeat. Antibodies 133, ALZ50, T60, T14, and T46 recognize the primary tau sequence; T1, PHF1, T3P, AT27, AT8, and AT18 are phosphorylation-dependent.
Figure 4:
Radiolabeled NT2N cultures treated with
colchicine. A, NT2N cultures were labeled with
[S]Met for 30 min and chased with or without
colchicine (25 µM) in complete medium for 0-4 h. Tau
was immunoprecipitated with T14/46 and analyzed on 10% Tris/Tricine
gel. Lanes 1-3 contain tau from untreated cultures for
0, 2, and 4 h of chase; lanes 4-6 contain tau from
colchicine-treated cultures for 0, 2, and 4 h of chase. Note that tau
from colchicine-treated cultures migrates faster and appears
dephosphorylated (compare lanes 3 and 6). B,
NT2N cultures were labeled with [
P]PO
for 4 h and chased with or without colchicine (25
µM) in complete medium for 0-4 h. Tau was
immunoprecipitated with T14/46 and analyzed on 10% Tris/Tricine gel. Lanes 1-4 contain tau from untreated cultures chased for
0, 1, 2, and 4 h; lanes 5-8 contain tau from
colchicine-treated cultures chased for 0, 1, 2, and 4 h. Note that the
turnover of phosphates was greater for colchicine-treated cultures as
indicated by the decrease in signal (compare lanes 3 and 4 with 7 and 8) and that after 4 h of colchicine
treatment only the fastest migrating tau band is apparent, whereas
after 4 h of chase without colchicine all isoforms are present (compare lanes 4 and 8). The positions of 66- and 45-kDa
molecular mass standards are shown on the right. C,
quantitation of the effect of colchicine on total
S-labeled tau after a 4-h chase. Experiments were
performed as described for panel A. Gels were exposed to
PhosphorImager plates, and tau bands were quantitated using the
ImageQuant software. Colchicine had no significant effect on the amount
of tau detected by quantitative analysis (Student's t test assuming equal variance; significance, p < 0.05). D, quantitation of the effect of colchicine on phosphate
incorporation of tau. Experiments were performed as described for panel B. Gels were exposed to PhosphorImager plates and tau
bands were quantitated using the ImageQuant software. Colchicine
significantly reduced the
P signal for every time point,
and after 4 h the signal was 50% less for colchicine-treated than
control (Student's two-tailed t test assuming equal
variance; significance, p < 0.05). Bars indicate
the standard error of the mean values from five
experiments.
Figure 6:
Western blot analysis of the effects by
colchicine treatment on specific tau phosphorylation sites.
Nitrocellulose replicas of heat-stable, tau-enriched fractions from
NT2N cultures following drug treatment are shown. Human fetal tau (F), tau from untreated NT2N cells (Ctr), and
tau from colchicine-treated cells (Col) were loaded in each panel. Nitrocellulose replicas were probed with anti-tau
antibodies as indicated above each panel. Note that
immunoreactivity for AT8 disappeared following colchicine treatment (panel E, lane 3). Equal protein, as determined by
bicinchoninic acid, was loaded for each lane. The amount of
protein loaded for panels B-D was twice that for panel A, and panels E-G were three times that
for panel A since these phosphorylation-dependent mAbs have
different affinities for tau.
Figure 3:
Synthesis and phosphorylation of tau in
NT2N cells. A, NT2N cultures were labeled with
[S]Met for 15-120 min, and tau was
isolated by immunoprecipitation with T14/46 and electrophoresed on 10%
Tris/Tricine gel. Note that after 15 min of labeling tau appears as a
single band and that following synthesis tau is modified causing an
upward shift in mobility and spreading of the tau band. B,
NT2N cultures were pulse-labeled with [
S]Met for
15 min and chased with complete medium for 1-4 h before
immunoprecipitation with T14/46 and electrophoresis on 10% Tris/Tricine
gel. Note that during the chase periods tau becomes modified as
indicated by an upward shift in mobility and diffusion of the signal
into a wider band. C, NT2N cultures were labeled with
[
P]PO
for 4 h and immunoprecipitated
by different tau antibodies; lane 1, tau immunoprecipitated by
T14/46 (phosphorylation-independent); lane 2, Tau 1 (T1, nonphosphorylation-dependent epitope); lane 3,
PHF1 (phosphorylation-dependent epitope). Note that T1 and PHF1, which
are dependent on the phosphorylation state of the epitope,
immunoprecipitate a subset of tau proteins. The positions of 97- and
66-kDa molecular mass standards are
indicated.
To support the
notion that this post-translational modification is due to
phosphorylation, cultures were radiolabeled with
[P]PO
for 4 h to obtain maximum
labeling, and then tau was immunoprecipitated with several tau mAbs (i.e. T14/46, T1, and PHF1). PHF1 (which recognizes
phosphorylated Ser
) immunoprecipitated only the
slower migrating
P-labeled phosphoisoforms, whereas T1
(which recognizes a nonphosphorylated epitope within residues
189-207) immunoprecipitated the faster migrating
P-labeled phosphoisoforms (Fig. 3C). Since
both T1 and PHF1 immunoprecipitated
P-labeled tau, this
suggests that the tau proteins expressed in the NT2N cells are
phosphorylated at different sites and to varying degrees.
NT2N cells were pulse-labeled with
[S]Met for 30 min and then chased in medium with
or without colchicine for up to 4 h. By the end of the chase period tau
became more phosphorylated in the control cultures, resulting in a
decrease in the electrophoretic mobility of tau and a broadening of the
tau band (Fig. 4A, lanes 1-3). In
contrast, tau from the colchicine-treated NT2N cultures migrated
faster, suggesting that colchicine treatment either led to the
degradation of tau or reduced the level of phosphorylation (or at least
increased the turnover of phosphate groups) in newly synthesized tau in
the NT2N cells (Fig. 4A, compare lane 4 with lanes 5 and 6). To examine this further,
radioactivity in the
S-labeled tau bands was quantified
before and after a 4-h chase. Our data revealed that
S-labeled tau was extremely stable and that there was
negligible turnover or degradation of tau (Fig. 4C).
Indeed, there was no significant difference between the amount of
S-labeled tau that was recovered before or after the 4-h
chase in NT2N cultures that were or were not treated with colchicine.
Other studies using antibodies that recognized epitopes at the N or C
termini of tau (i.e. 133, T1, ALZ50, T60, and T46) revealed no
immunoreactive bands corresponding to tau degradative products in the
NT2N cultures after colchicine treatment (data not shown).
To assess
whether colchicine treatment of NT2N cells resulted in the
dephosphorylation of tau, cultures were labeled with
[P]PO
for 4 h and then chased in the
presence or absence of colchicine. In the absence of colchicine about
40% of the [
P]PO
associated with tau
was turned over by 4 h of chase (Fig. 4B, lanes
1-4, and Fig. 4D). However, colchicine
treatment increased the rate of phosphate turnover such that about 75%
of the [
P]PO
disappeared by 4 h and
most of the [
P]PO
-radiolabeled tau
isoforms migrated more rapidly than their counterparts in the control
NT2N cultures (Fig. 4B, lanes 5-8,
and Fig. 4D). These observations suggest that
colchicine treatment results in the dephosphorylation of tau rather
than proteolysis or some other changes, and this was confirmed in other
studies using quantitative Western blotting (data not shown).
Figure 5:
Time course of the effect of nocodazole on
tau. Western blot analyses of tau and tubulin proteins are shown. NT2N
cultures were treated with 10 µg/ml nocodazole for 0-4 h.
Cultures were processed with MT stabilization buffer to examine the
soluble (S) tau fraction and the fraction of tau associated
with the cytoskeletal pellet (P). An aliquot of the
homogenized cells was removed for tubulin analysis. Equal protein, as
determined by bicinchoninic acid (panel A, 40 µg; panel B, 10 µg), was loaded onto 8.5% polyacrylamide gels
and subjected to SDS-PAGE. Nitrocellulose replicas were probed with
anti-tau mAbs T14/46 (panel A) and with anti--tubulin mAb (panel B). Lanes 1-6, nocodazole-treated; lane 7 shows positive controls. Panel A, human fetal
tau (F
, 15 µg); panel B,
phosphocellulose-purified bovine tubulin (PC, 5 µg).
Protein bands were quantified using
I-labeled IgG
secondary antibody and exposure of the blots to the PhosphorImager
plates. Note that after 2 h of treatment the MTs depolymerized into
soluble tubulin subunits (compare lanes 1 and 2 with lanes 3 and 4, panel B), and most of the tau
was present in the soluble fraction and was downshifted (compare lanes 1 and 2 with lanes 3 and 4, panel A). The positions of 66- and 45-kDa molecular mass
standards are shown on the right.
Figure 7: Site-specific dephosphorylation of tau prevented by phosphatase inhibitors. Nitrocellulose replicas of heat-stable, tau-enriched fractions from NT2N cultures following treatment with various phosphatase inhibitors in the presence or absence of colchicine are shown. Panels A and B (T14/46 and PHF1, respectively): lane 1, untreated; lane 2, 25 µM colchicine; lane 3, 500 nM okadaic acid (Ok); lane 4, 500 nM okadaic acid plus 25 µM colchicine; lane 5, 100 nM calyculin-A (CL-A); lane 6, 100 nM calyculin-A plus 25 µM colchicine; lane 7, 5 µM FK506; lane 8, 5 µM FK506 plus 25 µM colchicine; lane 9, 5 µM FK520; lane 10, 5 µM FK520 plus 25 µM colchicine; lane 11, human fetal tau standard. Equal protein was loaded for each lane (panel A, 40 µg; panel B, 120 µg). Molecular weight markers are indicated by the dashes to the left of each immunoblot.
In this study we used cultured NT2N cells as an effective
model in which to study how the phosphorylation state of tau is
regulated in human CNS neurons. Tau isolated from NT2N cells is
indistinguishable from human fetal tau since both migrate together on
SDS-PAGE gels before and after enzymatic dephosphorylation with
alkaline phosphatase. Further, tau isolated from NT2N cells and human
fetal tau are phosphorylated on the same sites (i.e. Thr, Ser
, Thr
Thr
, Ser
, and Ser
) as
detected by site-specific phosphorylation-dependent mAbs. However, the
phosphorylation and dephosphorylation of tau at these sites in NT2N
cells are dynamic since 40% of the
P-labeled tau turned
over during a 4-h chase period while the amount of
S-labeled tau remained the same. This dynamic turnover of
specific phosphates on tau suggests that phosphorylation may play an
important role in regulating the functions of tau.
One function of
tau that is regulated by phosphorylation is the binding affinity of tau
to MTs. For example, an increase in the phosphorylation of tau reduces
the binding of tau to MTs in vitro(19) . Using an MT
binding assay, we showed here that about 45% of tau in the NT2N neurons
did not bind to MTs and remained in the supernatant. This unbound tau
migrated slower on SDS-PAGE gels and was more highly phosphorylated
than the tau that remained bound to MTs. Thus, it is likely that the
increased phosphorylation of tau at specific sites reduces the binding
of tau to MTs in intact neurons. Indeed, recent studies showed that
Ser and Ser
are two phosphorylation sites
that regulate the binding of tau to MTs(20, 21) .
However, it is unclear at the present time whether
Ser
/Thr
also is involved in regulating the
binding of tau to MTs.
Examination of the effects of depolymerizing
the MT network with colchicine or nocodazole in the NT2N cells showed
that these agents induced the dephosphorylation of tau. This effect was
demonstrated by an acceleration in the electrophoretic mobility of tau
on SDS-PAGE gels, an increase in the turnover of
[P]PO
-labeled tau, and the
site-specific dephosphorylation of tau including the complete
dephosphorylation of tau at Ser
/Thr
. Our
data support the findings of Drubin et al.(64) in
which tau from differentiated PC12 cells treated with colchicine showed
an increased electrophoretic mobility on SDS-PAGE gels. In addition to
the dephosphorylation of tau, treatment of the NT2N cells with
colchicine or nocodazole resulted in the depolymerization of MTs and
the release of tau bound to MTs, as shown by an increase in the level
of soluble tubulin monomers concomitant with an increase in the levels
of dephosphorylated tau in the supernatant. Although this increase in
the level of tau in the supernatant most likely results from the
drug-induced depolymerization of MTs, the concomitant dephosphorylation
of tau may not be due to the direct effects of colchicine or nocodazole
on tau. Instead, it could be due to the indirect activation of
phosphatases that specifically dephosphorylate tau and MAP2C as a
result of the depolymerization of MTs. Indeed, recent studies
demonstrated that substantial amounts of the trimeric PP2A are normally
bound to MTs in intact cells(65) , suggesting that PP2A might
be activated during the depolymerization of MTs. Thus, we speculate
that the dephosphorylation of tau and MAP2C by phosphatases such as
PP2A may reflect an attempt by the neuron to counteract the
drug-induced depolymerization of MTs, since the dephosphorylation of
tau and MAP2C enhances their ability to bind to MTs.
Our data on colchicine- and nocodazole-induced dephosphorylation of tau differ from the results of Mattson(66) , who described an increase in the phosphorylation state of tau in primary cultures of CNS neurons treated with colchicine. The reasons for this discrepancy are unclear, but this may reflect differences between the antibodies and the method used to monitor changes in the phosphorylation state of tau before and after drug treatment.
Our observation that treatment of the NT2N cells
with colchicine and nocodazole leads to the dephosphorylation of a
subset of the phosphate acceptor sites on tau suggests that different
phosphatases may be activated to dephosphorylate tau at different
sites. Indeed, treatment of NT2N cells with a Ca ionophore leads to the selective dephosphorylation of
Thr
, and this dephosphorylation can be inhibited by
inhibitors of PP2B. (
)These results with NT2N cells are in
agreement with previous observations that tau can be dephosphorylated in vitro by highly purified PP2A and
PP2B(32, 67, 68) . Furthermore, our recent
study of biopsy-derived human tau also implicates PP2A as the
phosphatase that dephosphorylates
Ser
/Thr
(18) . Thus, future studies
will identify the functional significance of tau
phosphorylation/dephosphorylation at
Ser
/Thr
.