Article

Liu, F, Grundke-Iqbal, I, Iqbal, K and Gong, CX. Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation. Eur J Neurosci 22: 1942-1950

Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314, USA.
European Journal of Neuroscience (Impact Factor: 3.18). 11/2005; 22(8):1942-50. DOI: 10.1111/j.1460-9568.2005.04391.x
Source: PubMed

ABSTRACT

Abnormal hyperphosphorylation of tau is believed to lead to neurofibrillary degeneration in Alzheimer's disease (AD) and other tauopathies. Recent studies have shown that protein phosphatases (PPs) PP1, PP2A, PP2B and PP5 dephosphorylate tau in vitro, but the exact role of each of these phosphatases in the regulation of site-specific phosphorylation of tau in the human brain was unknown. Hence, we investigated the contributions of these PPs to the regulation of tau phosphorylation quantitatively. We found that these four phosphatases all dephosphorylated tau at Ser199, Ser202, Thr205, Thr212, Ser214, Ser235, Ser262, Ser396, Ser404 and Ser409, but with different efficiencies toward different sites. The K(m) values of tau dephosphorylation catalysed by PP1, PP2A and PP5 were 8-12 microm, similar to the intraneuronal tau concentration of human brain, whereas the K(m) of PP2B was fivefold higher. PP2A, PP1, PP5 and PP2B accounted for approximately 71%, approximately 11%, approximately 10% and approximately 7%, respectively, of the total tau phosphatase activity of human brain. The total phosphatase activity and the activities of PP2A and PP5 toward tau were significantly decreased, whereas that of PP2B was increased in AD brain. PP2A activity negatively correlated to the level of tau phosphorylation at the most phosphorylation sites in human brains. Our findings indicate that PP2A is the major tau phosphatase that regulates its phosphorylation at multiple sites in human brain. The abnormal hyperphosphorylation of tau is partially due to a downregulation of PP2A activity in AD brain.

Full-text

Available from: Cheng-Xin Gong
Contributions of protein phosphatases PP1, PP2A, PP2B
and PP5 to the regulation of tau phosphorylation
Fei Liu, Inge Grundke-Iqbal, Khalid Iqbal and Cheng-Xin Gong
Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road,
Staten Island, New York 10314, USA
Keywords: Alzheimer’s disease, dephosphorylation, human brain, hyperphosphorylation, protein phosphatase 2A
Abstract
Abnormal hyperphosphorylation of tau is believed to lead to neurofibrillary degeneration in Alzheimer’s disease (AD) and other
tauopathies. Recent studies have shown that protein phosphatases (PPs) PP1, PP2A, PP2B and PP5 dephosphorylate tau in vitro,
but the exact role of each of these phosphatases in the regulation of site-specific phosphorylation of tau in the human brain was
unknown. Hence, we investigated the contributions of these PPs to the regulation of tau phosphorylation quantitatively. We found that
these four phosphatases all dephosphorylated tau at Ser199, Ser202, Thr205, Thr212, Ser214, Ser235, Ser262, Ser396, Ser404 and
Ser409, but with different efficiencies toward different sites. The K
m
values of tau dephosphorylation catalysed by PP1, PP2A and
PP5 were 8–12 lm, similar to the intraneuronal tau concentration of human brain, whereas the K
m
of PP2B was fivefold higher. PP2A,
PP1, PP5 and PP2B accounted for 71%, 11%, 10% and 7%, respectively, of the total tau phosphatase activity of human
brain. The total phosphatase activity and the activities of PP2A and PP5 toward tau were significantly decreased, whereas that of
PP2B was increased in AD brain. PP2A activity negatively correlated to the level of tau phosphorylation at the most phosphorylation
sites in human brains. Our findings indicate that PP2A is the major tau phosphatase that regulates its phosphorylation at multiple
sites in human brain. The abnormal hyperphosphorylation of tau is partially due to a downregulation of PP2A activity in AD brain.
Introduction
Alzheimers disease (AD) is characterized by two hallmark brain
lesions ) extracellular deposits of b-amyloid and intracellular neuro-
fibrillary tangles (NFTs) ) the latter of which directly correlates to the
severity of dementia symptoms (Alafuzoff et al., 1987; Arriagada
et al., 1992; Riley et al., 2002). NFTs are composed of bundles of
paired helical filaments (PHFs) and straight filaments, the major
protein component of which is the abnormally hyperphosphorylated
tau (Grundke-Iqbal et al., 1986a,b; Lee et al., 1991). Tau is a
phosphoprotein, normally containing two–three phosphate groups per
molecule. In AD brain, tau is abnormally hyperphosphorylated with
9–10 mol of phosphate per mole of tau (Ksiezak-Reding et al., 1992;
Ko¨pke et al., 1993). More than 30 phosphorylation sites have been
identified in the hyperphosphorylated tau isolated from AD brain (for
review, see Gong et al ., 2005). The abnormal hyperphosphorylation of
tau is believed to be responsible for its loss of biological activity, for
its gain of toxic activity and for its aggregation into PHFs (Iqbal et al.,
1986; Alonso et al., 1994; 2001a,b; Lucas et al., 2001; Fath et al.,
2002; Jackson et al., 2002; Perez et al., 2002). Hence, the abnormal
hyperphosphorylation of tau appears to be critical to the pathogenesis
of AD.
The molecular mechanism leading to the abnormal hyperphosph-
orylation of tau in AD is not well understood. Tau phosphorylation is
catalysed by tau protein kinases and reversed by tau protein
phosphatases (PPs). More than a dozen protein kinases have been
shown to phosphorylate tau in vitro (for review, see Gong et al., 2005).
There are five phosphoserine phosphothreonine PPs, i.e. PP1, PP2A,
PP2B, PP2C and PP5, which are highly expressed in mammalian
brains. All of these phosphatases, except PP2C, dephosphorylate tau
in vitro and possibly in vivo as well (for review, see Gong et al., 2005).
It has been reported that the expressions and activities of some
phosphatases are decreased in the affected areas of AD brain (Gong
et al., 1993, 1995; Lian et al., 2001; Loring et al., 2001; Vogelsberg-
Ragaglia et al., 2001; Sontag et al., 2004; Liu et al., 2005). These
observations suggest that a downregulation of tau phosphatases in AD
brain might underlie the abnormal hyperphosphorylation of tau and
other neuronal proteins. However, the exact contribution of each of
these phosphatases in regulating tau phosphorylation is not known. In
addition, each phosphatase catalyses dephosphorylation of tau at
different phosphorylation sites with different efficiencies. Because the
impact of tau phosphorylation on its biological activity, on its gain of
toxic function and on its polymerization into PHFs is site-specific,
understanding the role of each phosphatase in the regulation of tau
phosphorylation at individual phosphorylation sites is critical.
In the present study, we carried out quantitative and kinetic
analyses, and elucidated for the first time the relative contributions of
PP1, PP2A, PP2B and PP5 in the regulation of tau phosphorylation
and in the abnormal hyperphosphorylation of tau in AD brain.
Materials and methods
Materials
The longest isoform of human brain tau (tau
441
), cyclin-dependent
kinase 5 and its activator p25 (cdk5 p25), and rat PP5 were cloned,
expressed and purified from Escherichia coli, as described previously
Correspondence: Dr C.-X. Gong or Dr F. Liu, as above.
E-mail: cxgong@ultinet.net; feiliu63@hotmail.com
Received 7 July 2005, revised 10 August 2005, accepted 18 August 2005
European Journal of Neuroscience, Vol. 22, pp. 1942–1950, 2005 ª Federation of European Neuroscience Societies
doi:10.1111/j.1460-9568.2005.04391.x
Page 1
(Qi et al., 1995; Skinner et al., 1997; Alonso et al ., 2001a; Liu et al.,
2002a). The catalytic subunit of cAMP-dependent protein kinase
(PKA) was purchased from Sigma (St. Louis, MO, USA). Holoen-
zymes of PP1 and PP2A were from Upstate (Lake Placid, NY, USA).
PP2B holoenzyme was purified from bovine brain according to the
method of Sharma et al. (1983). The activities of all the phosphatases
were standardized by using
32
P-tau as a substrate (see below). Protein
G-agarose beads were purchased from Pierce (Rockford, IL, USA).
All primary antibodies are listed in Table 1. Peroxidase-conjugated
anti-mouse and anti-rabbit IgG was from Jackson ImmunoResearch
Laboratories (West Grove, PA, USA).
125
I-labeled anti-mouse and
anti-rabbit IgG and ECL kit were from Amersham Pharmacia Biotech
(Piscataway, NJ, USA). [c-
32
P]ATP was from ICN Biomedicals (Costa
Mesa, CA, USA). Bradford protein assay reagent was from Bio-Rad
Laboratories (Hercules, CA, USA).
Human brain tissue
The medial temporal gyrus of six AD (one male and five female,
age 86.5 ± 6.5 years [mean ± SD], post-mortem delay 2.6 ± 0.5 h)
and seven age-matched normal human brains (two male and five
female, age 86.6 ± 2.9 years, post-mortem delay 2.9 ± 0.4 h) used
for this study were obtained from the Sun Health Research Institute
Donation Program (Sun City, AZ, USA). Diagnosis of all human
cases was histopathologically confirmed, and the brain tissue
samples were stored at )70 C until used. Frozen human brain
tissue was used in accordance with the US National Institutes of
Health guidelines and approved by our institutional review
committee.
Preparation of phosphorylated tau (P-tau) and
32
P-labeled tau
(
32
P-tau)
Recombinant human brain tau
441
was phosphorylated in vitro with
PKA and cdk5, as described previously (Liu et al., 2005). Under
these conditions, 3 mol of phosphate was incorporated to each
mole of tau
441
.
Immunodepletion and immunoprecipitation of PPs
Human brain tissue was homogenized with 9 · volumes of buffer
containing 50 mm Tris–HCl, pH 7.0, 8.5% sucrose, 10 mm b-merca-
ptoethanol, 2.0 mm EDTA, 2.0 mm benzamidine, and 2.0 lg mL
each of aprotinin, leupeptin and pepstatinin. The 16,000 g extracts
were prepared from the homogenates and the protein concentrations
were determined by the Bradford method. The extracts were then
incubated with antibodies to each PP, which were precoupled to
protein G-agarose beads, at 4 C for 4 h. For negative controls,
protein G-agarose beads without precoupling to the antibodies were
used. The unbound fractions were the specific phosphatase-immuno-
depleted brain extracts. The immunoprecipitated complex was washed
with Tris-buffered saline three times and with 50 mm Tris–HCl
(pH 7.4) twice, and was then used for Western blots and PP activity
assays. The success of the immunodepletion and immunoprecipitation
was examined by Western blot analyses, as described (Liu et al.,
2005).
PP assays
Unless specified, phosphatase activities were assayed in a reaction
mixture containing 50 mm Tris–HCl, pH 7.4, 1.0 mm MnCl
2
,
10 mm b-mercaptoethanol, 0.2 mg mL
32
P-tau and brain extracts.
For PP2B and PP5 assays, 1.5 mm CaCl
2
1.5 lm calmodulin
(CaM) and 5.0 lm arachidonyl-CoA (AA-CoA) were also included,
respectively. For measuring the total tau phosphatase activity of
brain extracts, both CaCl
2
CaM and AA-CoA were included. After
incubation at 30 C for 20 min that was within the linear range of
the reaction, the dephosphorylation reaction was terminated, and the
released
32
Pi was determined after separation from
32
P-tau by
ascending paper chromatography, as described previously (Gong
et al., 1996).
For the kinetic assays, the phosphatase activities were determined
using various concentrations of
32
P-tau (0.5–40 lm)at30C for
20 min. The reaction velocities of tau dephosphorylation were
expressed as pmol Pi released from
32
P-tau per min. The K
m
values
were calculated by using Lineweaver–Burk’s equation.
Table 1. Primary antibodies employed in this study
Antibody Type Specificity Phosphorylation sites* Reference Source
Anti-PP1 Monoclonal PP1 BD Bioscience, Palo Alto, CA, USA
Anti-PP2A Monoclonal PP2A Upstate, Lake Placid, NY, USA
R123d Polyclonal PP2A Pei et al. (1998)
CN-1A Monoclonal PP2B Sigma, St. Louis, MO, USA
Anti-PP5 Polyclonal PP5 Bahl et al. (2001)
pT181 Polyclonal P-tau Thr181 Biosource, Camerillo, CA, USA
pS199 Polyclonal P-tau Ser199 Biosource
pS202 Polyclonal P-tau Ser202 Biosource
pT205 Polyclonal P-tau Thr205 Biosource
pT212 Polyclonal P-tau Thr212 Biosource
pS214 Polyclonal P-tau Ser214 Biosource
pT217 Polyclonal P-tau Thr217 Biosource
pT231 Polyclonal P-tau Thr231 Biosource
pS235 Polyclonal P-tau Ser235 Biosource
pS262 Polyclonal P-tau Ser262 Biosource
pS396 Polyclonal P-tau Ser396 Biosource
pS404 Polyclonal P-tau Ser404 Biosource
pS409 Polyclonal P-tau Ser409 Biosource
pS422 Polyclonal P-tau Ser422 Biosource
R134d Polyclonal Tau Tatebayashi et al. (1999)
*Numbered according to the largest isoform of human brain tau (Goedert et al., 1989).
Protein phosphatases of tau protein 1943
ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1942–1950
Page 2
Dephosphorylation of P-tau by PP
To learn which PPs could dephosphorylate tau at which sites, we
incubated P-tau (0.2 mg mL) with the same unit amount of each
phosphatase at 30 C for up to 2 h in a reaction mixture, as
described above for PP activity assays. The activity units were
defined by phosphatase activity assays, as described above,
with
32
P-tau as a substrate. The reaction was stopped by adding
1 3 volume of fourfold concentrated sample buffer of sodium
dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE).
After heating in boiling water for 5 min, the samples were
subjected to Western blot analysis using site-specific and phos-
phorylation-dependent tau antibodies, as described (Liu et al.,
2005).
For quantitative detection of site-specific dephosphorylation of
tau by phosphatases, P-tau replaced
32
P-tau as a substrate.
The reaction was stopped by addition of stop solution
consisting of 250 mm sodium phosphate, pH 7.5, 5.0 lm okadaic
acid, 50 mm NaF, 5.0 mm EDTA and 5.0 mm EGTA. The
dephosphorylation of P-tau at each specific site was determined
by using a radioimmuno-dot-blot assay, as described previously
(Liu et al., 2002b).
Determination of the level of tau phosphorylation at individual
phosphorylation sites in human brain crude extracts
The level of tau phosphorylation at various individual phosphory-
lation sites was determined by Western blots of the crude extracts
of human temporal cortex, as described (Liu et al., 2005). The
primary antibodies used were site-specific and phosphorylation-
dependent tau antibodies (Table 1). The immunoreactivities of the
blots were then quantified densitometrically, and all the data
obtained with the phosphorylation-dependent tau antibodies were
normalized by the level of total tau protein as determined by R134d
against total tau.
Correlation analysis
Linear correlation between the activity of immunoprecipitated PP2A
and the phosphorylation level of tau at individual phosphorylation
sites of crude extracts from temporal cortices of six AD and seven
control brains, as determined as described above, was analysed, and
the correlation coefficients (r-values) were calculated by using
Microsoft
Excel 2000. The statistic significance (P<0.05) of the
correlation was analysed by t-test.
Results
Site-specific dephosphorylation of tau by PPs
We first studied which phosphorylation sites of tau can be dephos-
phorylated by PP1, PP2A, PP2B and PP5. For this purpose, we used
P-tau as a substrate, which was phosphorylated at all of the 10
abnormal hyperphosphorylation sites of PHF-tau examined (Fig. 1,
lane 2), and has an upward mobility shift in SDS–PAGE (Fig. 1A,
compare lane 2 with lane 1), a feature of the hyperphosphorylated tau
in AD. After incubation of P-tau with the same amount of phosphatase
units of PP1, PP2A, PP2B or PP5 under the standard conditions, we
determined the dephosphorylation of tau at the specific individual
phosphorylation sites by Western blots developed with phosphoryla-
tion-dependent tau antibodies to various individual phosphorylation
sites. We found that the incubation of P-tau with each of the four
phosphatases reduced or abolished the immunoreactivity of tau by all
these antibodies (Fig. 1B–K, lanes 3–6), as well as reversed the gel
mobility of tau to the same level as unphosphorylated tau (Fig. 1A,
compare lanes 3–6 with lane 1). These results indicated that PP1,
PP2A, PP2B and PP5 all could dephosphorylate tau at Ser199,
Ser202, Thr205, Thr212, Ser214, Ser235, Ser262, Ser396, Ser404 and
Ser409 in vitro. However, different phosphorylation sites of tau were
dephosphorylated differentially by the four phosphatases. For
example, Ser199 (B) and Ser202 (C) were most efficiently
Fig. 1. Dephosphorylation of tau at various sites by PP1, PP2A, PP2B and PP5. P-tau after incubation at 30 C for 2 h in dephosphorylation buffer in the absence
(lane 2) or presence of 0.3 U mL of PP1 (lane 3), PP2A (lane 4), PP2B (lane 5) or PP5 (lane 6) was analysed by Western blots using site-specific and
phosphorylation-dependent tau antibodies as indicated under each panel (B–K). Lane 1, recombinant tau
441
before in vitro phosphorylation. Antibody R134d
(A), which recognizes both phosphorylated and non-phosphorylated tau, was used to demonstrate the amount of total tau in each lane and the gel mobility shift.
1944 F. Liu et al.
ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1942–1950
Page 3
dephosphorylated by PP2A and PP5, whereas Ser404 (J) was most
efficiently dephosphorylated by PP2B.
To further investigate the differential dephosphorylation of tau at
each individual phosphorylation site by these phosphatases, we
determined the time kinetics of tau dephosphorylation at each
phosphorylation site with the same amount of phosphatase units of
each phosphatase (defined by catalysing the same amount of
phosphate release from
32
P-tau regardless of phosphorylation sites).
We found that different PPs dephosphorylated each phosphorylation
site differentially (Fig. 2A). After 2 h incubation, the differences
Fig. 2. Comparison of kinetics of tau dephosphorylation at individual phosphorylation sites by various PPs. P-tau was dephosphorylated with 0.3 U mL of PP1,
PP2A or PP2B for various time periods (A), or with various units of PP2A or PP5 for 20 min (B). The phosphorylation level of tau at individual phosphorylation
sites was determined by radioimmuno-dot-blot assays developed with antibodies that react only with tau phosphorylated at the indicated individual sites. Data are
averages of three separate experiments.
Protein phosphatases of tau protein 1945
ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1942–1950
Page 4
among the phosphatases became smaller due to partial saturation at
this time point. Although the trend of the differences in dephosph-
orylation of tau by PP1, PP2A and PP2B as determined by Western
blots (Fig. 1) was the same to that determined by the radioimmuno-
dot-blot assay (Fig. 2A), smaller differences were seen in Fig. 2A as
compared with those in Fig. 1. These small variations probably
resulted from the two different detecting methods used. Because PP5
activity was lost significantly after 30 min incubation under the
conditions used (data not shown), similar time course studies with PP5
could not be carried out. Therefore, we studied the site-specific
dephosphorylation of tau with PP5 by quantitating the dephosphory-
lation of tau after incubation with various amounts of PP5 for 20 min
and included PP2A as a reference for comparison (Fig. 2B).
According to the time or the phosphatase amounts required to reach
50% dephosphorylation (Fig. 2), we compared the dephosphorylation
of tau at various phosphorylation sites with each phosphatase. We
found that each phosphatase dephosphorylated different phosphory-
lation sites of tau in different orders and efficiencies (Table 2A and B).
For example, pThr205, pThr212, pSer214 and pSer409 were the most
preferred substrates of PP1; pThr205, pThr212, pSer262 and pSer409
of PP2A; pSer262, pSer396 and pSer409 of PP2B; and pThr205,
pThr212 and pSer409 of PP5.
Kinetics of tau dephosphorylation with PPs
To learn the affinity of PP1, PP2A, PP2B and PP5 to P-tau, we studied
the kinetic constants (K
m
) of its dephosphorylation by these phospha-
tases. When dephosphorylation of tau by PP1 was assayed at various
concentrations of P-tau, the reaction showed a typical Michaelis–
Menten’s curve (Fig. 3A), and the double reciprocal Lineweaver–Burk
plot of the data demonstrated a very good fit (Fig. 3B). A K
m
of
10.2 lm for the dephosphorylation of P-tau by PP1 was calculated by
using Lineweaver–Burk’s equation. Similar Michaelis–Menten’s and
Lineweaver–Burk’s curves were obtained for dephosphorylation of tau
by PP2A, PP2B and PP5 (data not shown). K
m
values of 11.6 lm,
53.5 lm and 7.6 lm for PP2A, PP2B and PP5, respectively, were
calculated by using the same method (Table 3).
Relative contributions of PP1, PP2A, PP2B and PP5 to
dephosphorylation of tau in human brain
In addition to the affinities of the PPs to phosphorylated tau, as
represented by their K
m
values, how much of a role each PP plays in
the regulation of tau phosphorylation also depends on the relative
abundance of the phosphatases in human brain. We therefore studied
the relative activities of PP1, PP2A, PP2B and PP5 in human brain by
using
32
P-tau as a substrate. Because there is no substrate specific to
each of these phosphatases and the available selective PP inhibitors are
often not specific enough, measuring the phosphatase activities in
crude extracts accurately is difficult. Hence, we first immunodepleted
the individual phosphatase from human brain extracts and then
determined the phosphatase activity of the remaining extracts toward
32
P-tau. The percentage reduction of the tau phosphatase activity after
immunodepletion represented the relative activity of the depleted
phosphatase toward tau. As shown in Fig. 4A, anti-PP2A, anti-PP2B
and anti-PP5 completely depleted the corresponding phosphatases
from human brain extract and did not result in co-depletion of any
other phosphatases. However, anti-PP1 failed to deplete PP1 from the
extract. We also tried several other commercially available antibodies
to PP1, but none of them immunodepleted PP1 from human brain
extract satisfactorily (data not shown). Hence, the relative activity of
PP1 in human brain extracts toward tau was calculated by subtracting
the relative activities of PP2A, PP2B and PP5 from the total tau
Table 2A Site-specific dephosphorylation of tau by various PPs: efficiency of
tau dephosphorylation at specific sites by each PP
Phosphatase Phosphorylation sites of tau
PP1 Thr205 Thr212 Ser214 Ser409 > Ser396
> Ser262 > Ser404 > Ser199 > Ser202
PP2A Thr205 Thr212 Ser262 Ser409 > Ser214
Ser396 > Ser202 > Ser199 > Ser404
PP2B Ser262 Ser396 Ser409 > Ser212 Ser404
> Thr205 Ser214 > Ser202 > Ser199
PP5 Thr205 Thr212 Ser409 > Ser202 Ser214
> Ser199 > Ser396 Ser404 > Ser262
Table 2B Site-specific dephosphorylation of tau by various PPs: efficiency of
PPs in dephosphorylating tau at each specific site
Phosphorylation site Efficiency of PPs
Ser199 PP5 > PP2A > PP1 PP2B
Ser202 PP5 > PP2A > PP2B > PP1
Thr205 PP1 PP2A PP5 > PP2B
Thr212 PP5 > PP1 PP2A > PP2B
Ser214 PP1 > PP2A PP5 > PP2B
Ser262 PP2A PP2B > PP1 > PP5
Ser396 PP2B > PP5 > PP1 PP2A
Ser404 PP2B > PP5 > PP1 > PP2A
Ser409 PP1 > PP2B > PP2A PP5
Fig. 3. Kinetic analysis of tau dephosphorylation by PP1. (A) The velocities
of dephosphorylation catalysed with 0.19 U mL PP1 were determined at
various concentrations of
32
P-tau. (B) Data shown in (A) were plotted by
using the Lineweaver–Burk double-reciprocal method. The x-axis intercept was
used to calculate the K
m
value. The data are representatives of three
independent experiments with similar results.
Table 3. K
m
values of PPs toward P-tau
PP K
m
(lm)*
PP1 10.2
PP2A 11.6
PP2B 53.5
PP5 7.6
*Average of three independent experiments with less than 15% variations.
The K
m
of PP5 has been published previously (Liu et al., 2005).
1946 F. Liu et al.
ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1942–1950
Page 5
phosphatase activity assayed without immunodepletion, assuming that
PP1, PP2A, PP2B and PP5 accounted for the total tau phosphatase
activity. By using this approach, we found that the relative phospha-
tase activities of PP1, PP2A, PP2B and PP5 toward tau in human brain
were 10.4%, 71.4%, 7.3% and 10.8%, respectively (Fig. 4B).
Tau phosphatase activities in AD and control brains
We measured the phosphatase activities of PP2A, PP2B and PP5 that
were immunoprecipitated from equal amounts of extracts of temporal
cortices from six AD and seven control brains by using
32
P-tau as a
substrate. Because we could not immunoprecipitate PP1 satisfactorily
(see above and Fig. 4A), PP1 activity toward tau was not assayed. We
found that the tau phosphatase activities of both PP2A and PP5 were
decreased in AD brain (Fig. 5). Surprisingly, PP2B activity was found
increased by twofold in AD brains as compared with controls (Fig. 5).
However, when PP2B activity was assayed in the brain extracts
without immunoprecipitation or when the immunodepleted brain
extracts were added back into the assay mixtures, no difference in
PP2B activity was found between AD and control group (data not
shown). In addition, we also measured the total tau phosphatase
activity of the brain extracts and found an approximately 50%
decrease in AD brain as compared with age-matched controls (Fig. 5).
These results are in agreement with our finding that PP2B only
accounted for 7% of total tau phosphatase activity in human brain
(Fig. 4B), so that the decreased total tau phosphatase activity may
represent that of PP2A, PP5 and possibly PP1 in AD brain.
Correlation between phosphorylation level of tau at individual
phosphorylation sites and PP2A activity in human brains
Our above results suggest that PP2A is the major tau phosphatase in
human brain, and hyperphosphorylation of tau in AD brain may
partially result from downregulation of PP2A activity. To further
Fig. 4. Relative activity of each PP toward tau in the human brain. (A) PPs were immunodepleted from the same amount of human brain extracts with anti-PP1
(monoclonal), anti-PP2A (polyclonal), anti-PP2B (monoclonal) and anti-PP5 (polyclonal) precoupled to protein G-agarose, as indicated at the top of blots.
Equivalent amounts of the immunodepleted extracts (depleted) and the immunoprecipitates (IP) were analysed by Western blots developed with antibodies against
PP1, PP2A, PP2B and PP5, as indicated under each blot. Arrowheads indicate the immunoprecipitating antibody (IgG) in the immunoprecipitates that was
recognized by the secondary antibodies. Arrows indicate the corresponding PPs. (B) The relative activity of each phosphatase immunodepleted from extracts of
temporal cortices of seven normal human brains was determined by using
32
P-tau as a substrate. The activity of PP1, which could not be successfully
immunodepleted, was calculated by subtracting the activities of PP2A, PP2B and PP5 from the total tau phosphatase activity assayed without immunodepletion. Data
are presented as mean ± SD (n ¼ 7).
Fig. 5. Comparison of tau phosphatase activities between AD and control
brains. Activities of immunoprecipitated PP2A, PP2B and PP5, and of crude
extracts from temporal cortices of six AD and seven control brains were
assayed using
32
P-tau as a substrate. The activities of control cases were defined
as 100. Means ± SD are presented. *P < 0.05, **P < 0.01, as compared with
control group by Student’s t-test.
Protein phosphatases of tau protein 1947
ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1942–1950
Page 6
address the role of PP2A in regulation of site-specific tau phosphory-
lation, we analysed the correlation between the level of tau
phosphorylation and PP2A activity in 13 human brains (six AD and
seven controls). Among the 11 tau phosphorylation sites analysed, we
found a negative correlation between PP2A activity and tau
phosphorylation at all the sites except at Ser214 (Fig. 6). Although
the negative correlation between PP2A activity and tau phosphory-
lation at Ser404 did not reach statistical significance (P > 0.05), the
trend of negative correlation was obvious. These results further
support the role of PP2A in regulation of tau phosphorylation at
multiple phosphorylation sites. The lack of correlation between PP2A
and tau phosphorylation level at Ser214 might suggest that phos-
phorylation of tau at this site might be regulated dominantly by other
phosphatases or kinases than PP2A, although it was dephosphorylated
by PP2A efficiently in vitro (Fig. 2 and Table 2A and B). We did not
do the same analysis for tau phosphorylation at Ser235 and Ser409,
because tau immunoreactivity of crude human brain extracts with
antibodies to pSer235 and pSer409 were too weak (data not shown).
Discussion
Previous studies had suggested a role of PPs in the regulation of tau
phosphorylation (Gong et al., 1994a,b,c; 2000; Goedert et al., 1995;
Sontag et al., 1996, 1999; Kins et al., 2001). However, the relative
contributions of PP1, PP2A, PP2B and PP5, the major brain PPs, in
the regulation of tau phosphorylation in the brain had not been
reported, although this knowledge is crucial to designing therapeutic
strategies for AD via modifying of PP activities. In the present study,
we found that PP1, PP2A and PP5 had K
m
values of approximately
10 lm, and PP2B had a fivefold higher K
m
toward phosphorylated tau.
The intraneuronal tau concentration is estimated to be 5–10 lm on the
basis of tau concentration measured in human brain extracts (Khatoon
et al., 1992). Hence, the K
m
values of PP1, PP2A and PP5 toward tau
are in the range of the intraneuronal tau concentration, suggesting that
these three phosphatases are capable to regulate tau phosphorylation
levels in human brain.
The relative contribution of each PP in the regulation of tau
phosphorylation depends on their affinities to tau, their specific
enzymatic activities toward tau, and their relative abundance in the
brain. The most direct assessment of the relative contribution is to
determine the activity of each phosphatase toward tau in the brain. By
employing immunodepletion technique, we found that PP2A accoun-
ted for 70% of total brain tau phosphatase activity, whereas PP1,
PP2B and PP5 each accounted for only 10% or less. Because PP2A
had a K
m
similar to those of PP1 and PP5 toward tau, our finding that
PP2A accounted for a much higher percentage of the total tau
phosphatase activity than PP1 and PP5 suggests that PP2A might be
much more abundant than PP1 and PP5 in human brain. Negative
correlation between PP2A activity and the level of tau phosphoryla-
tion at most of the phosphorylation sites in human brains further
supports the dominant role of PP2A in regulation of tau phosphory-
lation as compared with other PPs. Our quantitative findings are
consistent with previous non-quantitative studies showing that PP2A
might be the major tau phosphatase in mammalian brain (Goedert
et al., 1995; Sontag et al., 1996, 1999; Gong et al., 2000; Kins et al .,
2001). It is interesting that although PP2B is highly expressed in
mammalian brain, its contribution to total tau phosphatase activity was
only 7%. This may be partially explained by the finding in our
kinetic studies that PP2B had a fivefold higher K
m
than other major
PPs and therefore lower affinity to tau protein in human brain.
Tau isolated from AD brain is phosphorylated at more than 30 sites
(Gong et al., 2005). Each of these phosphorylation sites is differen-
tially dephosphorylated by different PPs. Dephosphorylation of tau at
some of the phosphorylation sites with various PPs has been reported.
The present study is the first to compare the site-specific dephosph-
orylation of tau by four major brain tau phosphatases systematically
and quantitatively. We found that regardless of their relative contri-
butions, each phosphatase had its preferred tau phosphorylated sites as
substrates, and each of these sites had differential sensitivity to various
phosphatases (Table 2A and B). These results suggest that most of the
phosphorylation sites of tau are regulated by more than one
phosphatase, and PP2A is the major overall tau phosphatase. Because
phosphorylation of tau at different sites differentially impacts its
biological activity and its polymerization into NFTs (Biernat et al.,
1993; Sengupta et al., 1998; Wang et al., 1998; Alonso et al., 2004),
elucidation of the site-specific regulation of tau phosphorylation by
each of these PPs is critical to understanding the role of these enzymes
in the neurofibrillary degeneration of AD and may help design the
therapeutic strategies targeting specific PPs.
In the present study, we used phosphorylated tau as a substrate and
determined total tau phosphatase activity and the activities of
individual phosphatases toward tau in AD and control brains that
had very short post-mortem delay (< 3 h). We found a 50%
Fig. 6. Relationship between PP2A activity and phosphorylation level of tau at individual phosphorylation sites in human brains. The activity of
immunoprecipitated PP2A (determined by using
32
P-tau as a substrate) and the phosphorylation level of tau at individual phosphorylation sites (determined by
quantitative Western blots) of crude extracts from temporal cortices of six AD and seven control brains were determined. Linear correlations between PP2A activity
and tau phosphorylation at each phosphorylation site were then analysed, and the correlation coefficients (r-values) are shown. *P<0.05.
1948 F. Liu et al.
ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 1942–1950
Page 7
decrease in total tau phosphatase activity and in PP2A activity, and a
20% decrease in PP5 activity in AD brain. Although we were
unable to measure PP1 activity directly, due to the unsatisfactory
immunoprecipitation, the tau phosphatase activity of PP1 is likely to
be decreased in AD brain also because there was 50% reduction of
total tau phosphatase activity. Our findings are consistent with
previous reports showing that the activities of PP2A, PP1 and PP5
toward other substrates are decreased in AD brain (Gong et al., 1993,
1995; Loring et al., 2001; Vogelsberg-Ragaglia et al., 2001; Sontag
et al., 2004; Liu et al ., 2005). Taken together, these studies indicate
that tau phosphatase activity is downregulated, which may underlie the
abnormal hyperphosphorylation of tau in AD brain.
It was reported that PP2B activity toward
32
P-labeled phosphorylase
kinase and p-nitrophenyl phosphate as substrates is either unchanged
or slightly decreased in AD brain (Gong et al., 1993; Ladner et al.,
1996; Lian et al., 2001). It was to our surprise that we found a
twofold increase rather than a decrease in PP2B activity toward
32
P-tau in AD brain. To understand this apparent contradiction, we
assayed PP2B activity of the same brain extract samples (without
immunoprecipitation) toward
32
P-tau in the presence of 100 nm
okadaic acid to inhibit activities of PP1, PP2A and PP5. No significant
difference in PP2B activity between AD and control groups was found
under these conditions (Liu et al., unpublished work). In another set of
experiments, we added the PP2B-depleted brain extracts back into the
immunoprecipitated PP2B, the activity of which was higher in AD
than in controls, and then found that the PP2B activity in control cases
was activated to the same level as that of AD cases (Liu, et al.,
unpublished work). Further studies suggested that PP2B was truncated
and activated by calpain in AD brain, and that the PP2B truncation and
activation also occurred immediately in the PP2B assay mixtures when
calpain (from brain extracts) and calcium CaM (included in the
reaction buffer) were included, so that PP2B of the control samples
was activated to the same level as that seen in AD samples (Liu, et al.,
unpublished work). The cause and the significance of the upregulation
of PP2B in AD brain are currently unknown. Obviously, this
upregulation does not correlate to tau hyperphosphorylation, because
only 7% of the total tau phosphatase activity is contributed by PP2B
in human brain, and tau is hyperphosphorylated rather than hypo-
phosphorylated in AD brain.
In summary, the present study is the first to compare the
contributions of PP1, PP2A, PP2B and PP5 in regulation of tau
phosphorylation in human brain and in the abnormal hyperphosph-
orylation of tau in AD. In this study, we found that: (i) PP1, PP2A,
PP2B and PP5 all dephosphorylated tau protein at Ser199, Ser202,
Thr205, Thr212, Ser214, Ser235, Ser262, Ser396, Ser404 and
Ser409 in vitro with different efficiencies toward different sites; (ii)
the K
m
values of dephosphorylation of tau by PP1, PP2A and PP5
were in the range of intraneuronal tau concentration, whereas the K
m
of PP2B was fivefold larger; (iii) PP2A, PP5, PP1 and PP2B
accounted for 71%, 11%, 10% and 7% of the total tau
phosphatase activity, respectively, in the human brain; (iv) the total
phosphatase activities and the activities of PP2A and PP5 toward tau
were significantly decreased, whereas that of PP2B increased in AD
brain; and (v) PP2A activity negatively correlated to the level of tau
phosphorylation at most of the phosphorylation sites in the human
brain. These results suggest that in the human brain, PP2A is the
major tau phosphatase, and PP1 and PP5 play a considerably smaller
role than PP2A. The abnormal hyperphosphorylation of tau is likely
caused partially by downregulation of PP2A activity and, to a
considerably lesser degree, of PP1 and PP5 activities. These findings
suggest that recovering PP2A activity is a potential therapeutic target
for AD.
Acknowledgements
We thank Drs T. Beach and L.I. Sue of Sun Health Research Institute Donation
Program, Sun City, AZ, USA, for providing autopsied human brain tissue; Dr E.
El-Akkad of our department for preparing recombinant tau; Dr S. Rossie of
Purdue University, West Lafayette, IN, USA, for providing PP5 and anti-PP5; and
Ms M. Marlow of our institute for editorial help. This work was supported in part
by funds from the New York State Office of Mental Retardation and
Developmental Disabilities; NIH grant AG16760, Alzheimers Association
grant NIRG-03-4721, and an award from the Li Foundation, Inc., NY, USA.
Abbreviations
AA-CoA, arachidonyl-CoA; AD, Alzheimers disease; CaM, calmodulin; cdk5,
cyclin-dependent kinase 5; NFTs, neurofibrillary tangles; PHFs, paired helical
filaments; PKA, cAMP-dependent protein kinase; PP, protein phosphatase;
P-tau, in vitro phosphorylated tau;
32
P-tau, tau in vitro phosphorylated and
labeled with
32
P; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel
electrophoresis.
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  • Source
    • "Several protein kinases, such as glycogen synthase kinase-3b (GSK-3b), extracellular signal-regulated kinases (ERK1/2), cyclin dependent kinases 5 (CDK5), cAMP-dependent protein kinase (PKA), calcium/calmodulin-dependent protein kinase II (CaMK-II), and JNKs, have been implicated in hyperphosphorylation of tau in AD (Gong et al. 2010). Tau phosphorylation is also regulated by protein phosphatase 2A (PP2A) (Gong et al. 2000), which accounts for over 70 % of total tau 104 Cell Mol Neurobiol (2015) 35:101–110 123 Author's personal copy phosphatase activity in the mammalian brain (Liu et al. 2005). Increased tau hyperphosphorylation with concurrent activation of GSK-3b, CDK5, and CaMK-II, as well as inhibition of PP2A is observed in a rat model of CCH, which shows spatial learning/memory deficits (Yao et al. 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Chronic cerebral hypoperfusion (CCH) is a common consequence of various cerebral vascular disorders and hemodynamic and blood changes. Recent studies have revealed an important role of CCH in neurodegeneration and dementia, including vascular dementia and Alzheimer's disease (AD). This article reviews the recent advances in understanding CCH-induced neurodegeneration and AD-related brain pathology and cognitive impairment. We discuss the causes and assessment of CCH, the possible mechanisms by which CCH promotes Alzheimer-like pathology and neurodegeneration, and animal models of CCH. It appears that CCH promotes neurodegeneration and AD through multiple mechanisms, including induction of oxidative stress, Aβ accumulation and aggravation, tau hyperphosphorylation, synaptic dysfunction, neuronal loss, white matter lesion, and neuroinflammation. Better understanding of the mechanisms of CCH will help develop therapeutic strategies for preventing and treating neurodegeneration, including sporadic AD and vascular dementia, caused by CCH.
    Full-text · Article · Oct 2014 · Cellular and Molecular Neurobiology
  • Source
    • "Initially, PP1 activates GSK3β through dephosphorylation of Ser9 (Bennecib et al., 2000; Hernandez et al., 2010), and PP2A dephosphorylates and regulates AKT, inhibiting its activity on GSK3β (Mora et al., 2002; Resjo et al., 2002). Thus, phosphatases influence Tau phosphorylation through several mechanisms, and in a pathological condition such as AD where phosphatase activity is decreased (Liu et al., 2005), these enzymes are key factors in the development of the disease (Figure 1). "
    [Show abstract] [Hide abstract] ABSTRACT: Alzheimer's disease (AD) is the most common cause of dementia worldwide. One of the main pathological changes that occurs in AD is the intracellular accumulation of hyperphosphorylated Tau protein in neurons. Cyclin-dependent kinase 5 (CDK5) is one of the major kinases involved in Tau phosphorylation, directly phosphorylating various residues and simultaneously regulating various substrates such as kinases and phosphatases that influence Tau phosphorylation in a synergistic and antagonistic way. It remains unknown how the interaction between CDK5 and its substrates promotes Tau phosphorylation, and systemic approaches are needed that allow an analysis of all the proteins involved. In this review, the role of the CDK5 signaling pathway in Tau hyperphosphorylation is described, an in silico model of the CDK5 signaling pathway is presented. The relationship among these theoretical and computational models shows that the regulation of Tau phosphorylation by PP2A and glycogen synthase kinase 3β (GSK3β) is essential under basal conditions and also describes the leading role of CDK5 under excitotoxic conditions, where silencing of CDK5 can generate changes in these enzymes to reverse a pathological condition that simulates AD.
    Full-text · Article · Sep 2014 · Frontiers in Aging Neuroscience
  • Source
    • "In AD, total phosphatase activity is reduced by approximately 50% (Liu et al, 2005). PPP2CA is the most efficient phosphatase acting on hyperphosphorylated tau (Liu et al, 2005). Although previous studies reported direct regulation of PPP2CA by miR-125b (Le et al, 2011), PPP2CA protein levels are not downregulated upon miR-125b overexpression in primary rat neurons under our conditions (Fig 3A). "
    [Show abstract] [Hide abstract] ABSTRACT: Sporadic Alzheimer's disease (AD) is the most prevalent form of dementia, but no clear disease-initiating mechanism is known. Aβ deposits and neuronal tangles composed of hyperphosphorylated tau are characteristic for AD. Here, we analyze the contribution of microRNA-125b (miR-125b), which is elevated in AD. In primary neurons, overexpression of miR-125b causes tau hyperphosphorylation and an upregulation of p35, cdk5, and p44/42-MAPK signaling. In parallel, the phosphatases DUSP6 and PPP1CA and the anti-apoptotic factor Bcl-W are downregulated as direct targets of miR-125b. Knockdown of these phosphatases induces tau hyperphosphorylation, and overexpression of PPP1CA and Bcl-W prevents miR-125b-induced tau phosphorylation, suggesting that they mediate the effects of miR-125b on tau. Conversely, suppression of miR-125b in neurons by tough decoys reduces tau phosphorylation and kinase expression/activity. Injecting miR-125b into the hippocampus of mice impairs associative learning and is accompanied by downregulation of Bcl-W, DUSP6, and PPP1CA, resulting in increased tau phosphorylation in vivo. Importantly, DUSP6 and PPP1CA are also reduced in AD brains. These data implicate miR-125b in the pathogenesis of AD by promoting pathological tau phosphorylation.
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