From the Hyogo Institute for Aging Brain and
Cognitive Disorders, Himeji 670-0981 and ¶ Graduate School of
Science, Biosignal Research Center, Kobe University,
Kobe 657-8501, Japan
Received for publication, August 15, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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For the phosphorylation state of
microtubule-associated protein, tau plays a pivotal role in regulating
microtubule networks in neurons. Tau promotes the assembly and
stabilization of microtubules. The potential for tau to bind to
microtubules is down-regulated after local phosphorylation. When we
investigated the effects of PKN activation on tau phosphorylation, we
found that PKN triggers disruption of the microtubule array both
in vitro and in vivo and predominantly
phosphorylates tau in microtubule binding domains (MBDs). PKN has a
catalytic domain highly homologous to protein kinase C (PKC), a kinase
that phosphorylates Ser-313 (= Ser-324, the number used in this study)
in MBDs. Thus, we identified the phosphorylation sites of PKN and PKC
subtypes (PKC- Dynamics and rearrangement of microtubule networks are modulated
by microtubule-associated proteins
(MAPs)1 (1, 2). Isolated MAPs
show varying degrees of phosphorylation (3), and the state of
phosphorylation, balanced by protein kinases and phosphatases, plays a
pivotal role in modulating microtubule networks (4). In neurons, tau, a
neuronal MAP, modulates microtubule organization during morphogenesis
and process outgrowth, and the dynamic nature of tau-microtubule
interactions has been observed even in the axons of mature neurons
(5).
Although it has been shown that tau can be phosphorylated by more than
10 serine/threonine kinases in vitro (6), it has remained to
be determined which kinases modulate tau function through
phosphorylation in vivo. PKN is a fatty acid-activated serine/threonine kinase (7), has a catalytic domain highly homologous
to the protein kinase C (PKC) family (8, 9), and is one of the direct
targets of the small GTP-binding protein Rho (10-12). We reported that
PKN phosphorylates tau both in vitro and in vivo
and colocalizes with neurofibrillary tangles (NFTs), composed of
aberrantly hyperphosphorylated tau in the brains of Alzheimer's
disease patients (13). In the present work, we attempted to identify
the phosphorylation sites on tau by PKN and PKC, and we examined the
role of PKN in tau phosphorylation and microtubule assembly.
Recombinant Proteins--
A cDNA (clone T9) encoding the
human brain tau (383 residues) was kindly provided by Prof. H. Mori
(Osaka City University). All tau constructs used in this study (Fig.
1) were prepared by polymerase chain
reaction amplification using clone T9 as the template. Substitution of
serine with alanine was done using a Quick-Change Site-directed
Mutagenesis Kit (Stratagene). After confirmation of the sequences, they
were subcloned into the pGEX-4T vector (Amersham Pharmacia Biotech).
Expression and purification of GST-tagged proteins were done according
to the instructions of the manufacturer (Amersham Pharmacia Biotech). A
pET23-NHis vector was constructed from the pET-23d vector (Novagen) to
add 8 amino acids (MHHHHHHM) to the N terminus of the protein as a His
tag. M and H indicate methionine and histidine, respectively. A
cDNA (clone T9) was subcloned into pET23-NHis vector. Expression and purification of His-tagged tau protein was done according to the
instructions of the manufacturer (Novagen). The numbering of amino acid
residues was always based on the longest isoform of human tau (441 residues). Recombinant PKN enzyme was obtained as described (14). The
cDNA encoding the open reading frames of rat PKC- In Vitro Assay of Microtubule Assembly--
Tau (50 µg) was
pretreated with either a catalytically active PKN (10 µg) or
catalytically inactive PKN (10 µg) or without PKN in 100 µl of
reaction mixture containing 50 mM Tris/HCl at pH 7.5, 5 mM MgCl2, 100 µM ATP, 1 mM dithiothreitol, 1 mM EDTA for 1 h at
30 °C and then added to 400 µl of assembly buffer (100 mM PIPES at pH 6.8, 1.25 mM EGTA, 1.25 mM GTP) containing 100 µg of tubulin (cytoskeleton) at
37 °C. Polymerization of microtubules was monitored by measuring
absorbance at 350 nm (19, 20).
In Vitro Phosphorylation Assay--
The phosphorylation of
recombinant tau proteins by recombinant enzymes was carried out at
30 °C in 25 µl of reaction mixture containing 20 mM
Tris/HCl at pH 7.5, 4 mM MgCl2, 40 µM ATP, 50 ng of recombinant enzymes, and 61.6 kBq of
[ Mammalian Expression Constructs--
Mammalian expression
construct of tau (Tau) was generated by insertion of
cDNA encoding the open reading frames of tau (383 residues) into
mammalian expression vector pSG5 (Stratagene). TauS320A was
generated using a Quick-Change Site-directed Mutagenesis Kit
(Stratagene) from Tau by mutating the serine residue (amino acid 320) to alanine. pTB701/PKN/AF3 (a catalytically active form of
PKN; aPKN) was constructed by insertion of the cDNA
fragment encoding amino acids 561-942 of PKN into mammalian expression vector pTB701 (21). pTB701/PKN/AF3(K644E) (a catalytically inactive mutant of PKN; iPKN) was generated by Quick-Change
Site-directed Mutagenesis Kit (Stratagene) from aPKN by
mutating the lysine residue (amino acid 644) at the ATP-binding site of
the protein kinase domain to glutamic acid as described (22).
Transfection and Immunoanalysis--
CHO cells seeded at 50%
confluency in a 60-mm dish were transfected with 5 µg of
Tau and 2 µg of pTB701 vector (CHO-tau-Mock), 5 µg of
Tau, and 2 µg of aPKN (CHO-tau-aPKN) and 5 µg
of Tau, 2 µg of iPKN (CHO-tau-iPKN), and 5 µg
of TauS320A and 2 µg of aPKN
(CHO-tauS320A-aPKN) using 15 µl of LipofectAMINE (Life Technologies, Inc.). Twenty one hours after transfection, cells were treated with or
without phosphatase inhibitors FK506 (600 nM), vanadate (100 µM), and okadaic acid (50 nM) for 3 h. Cell lysates were prepared, as described (23), and subjected to
Western blot analysis using appropriate antibodies. Immunoprecipitation
of human neuroblastoma SK-N-MC cells with tau1 and HIA3 were performed
as described (13), and immunoprecipitates were subjected to Western
blot analysis. Human neuroblastoma SH-SY5Y and SK-N-MC cells seeded at
70% confluency on coverslips in a 60-mm dish were transfected with 5 µg of aPKN or iPKN using 10 µl of a SuperFect
Transfection Reagent (Qiagen). Forty to 44 h after transfection,
cells were fixed with 4% paraformaldehyde, and the microtubule
organization was analyzed by immunofluorescence staining using anti-PKN
antibody ( Antibodies--
A polyclonal antibody, Microtubule Assembly Is Inhibited by PKN Activation--
To
examine effects of PKN activation on microtubule assembly, we carried
out the in vitro assay of microtubule assembly. There was a
marked increase in the rate of the turbidity when tau was added,
whereas it was minimal without tau (Fig.
2A). The tau-induced increase
in polymerization of tubulin was almost abolished by adding active PKN,
which indicated that PKN impaired the ability of tau to promote
microtubule assembly. On the other hand, microtubule assembly was
uninhibited when inactive PKN was added to the reaction, indicating
that enzymatic activity is required for inhibition of microtubule
assembly (Fig. 2A).
In human neuroblastoma SH-SY5Y and SK-N-MC cells transfected with
aPKN or iPKN, microtubule organization was
analyzed by double-labeling immunofluorescence histochemistry using an
anti-PKN ( PKN Phosphorylates Tau in Vitro--
We determined that PKN
phosphorylated recombinant His-tagged tau (His-tau) as well as
GST-tagged tau (GST-tau) (13) in vitro. The level of His-tau
phosphorylation reached as high as 4 mol of Pi per monomer
of tau protein (Fig. 3A).
Predominant phosphorylation was seen in GST-tau-MBDs (241-385 aa),
although PKN also phosphorylated GST-tau-FR (191-240 aa). On the
contrary, PKN phosphorylated poorly GST-tau-NT (1-190 aa) and
GST-tau-CT (386-441 aa) (Fig. 3B).
PKN Phosphorylates Ser-214 in Flanking Region (FR) of Tau--
We
first determined PKN phosphorylation sites in the FR of tau. As PKN is
a serine/threonine kinase, a candidate serine or threonine for
phosphorylation site in FR of tau was substituted for alanine (Fig. 1),
and an in vitro phosphorylation assay was performed. When
Ser-214 was substituted by alanine, the phosphorylation of tau was
significantly reduced (Fig. 4),
indicating that Ser-214 is a PKN phosphorylation site in FR of tau.
PKN Phosphorylates Ser-258, Ser-320, and Ser-352 in MBDs of
Tau--
PKN has a catalytic domain highly homologous to protein
kinase C, which phosphorylates tau in MBDs (24). MBDs consist of four
31- or 32-amino acid repeats, 1MBD (244-274 aa), 2MBD (275-305 aa),
3MBD (306-336 aa), and 4MBD (337-378 aa). To identify the phosphorylation site(s) of PKN, every serine residue in each MBD was
substituted by alanine (Fig. 1), and an in vitro
phosphorylation assay was performed. PKN phosphorylated all the
GST-1MBD tau fragments without S258A mutation, indicating that PKN
phosphorylated Ser-258 in 1MBD (Fig.
5A). In the same manner, PKN
phosphorylated Ser-320 in 3MBD and Ser-352 in 4MBD.
PKC Subtypes Phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352
in MBDs of Tau--
To compare the phosphorylation sites of PKN with
those of PKC subtypes, an in vitro phosphorylation assay by
PKC subtypes was done. PKC- PKN Phosphorylates Tau in Vivo--
To examine the phosphorylation
of tau by PKN in vivo, we developed a rabbit polyclonal
antibody (HIA3) directed against the phosphorylated tau at Ser-320, a
PKN-specific site for the phosphorylation of tau. Western blot analysis
revealed that HIA3 did not react with nonphosphorylated recombinant
His-tagged tau (Fig. 6A, lane 1) but did react with PKN-prephosphorylated His-tagged tau (Fig. 6A, lane 2). Moreover, HIA3 did not react with
PKC-phosphorylated His-tagged tau (Fig. 6A, lanes 3 and
4). Thus HIA3 is a PKN-specific phosphorylation-dependent tau antibody. HIA3 did not react
with CHO-tau-Mock (Fig. 6B, lanes 1 and 2) and
CHO-tau-iPKN (Fig. 6B, lane 7) but did react with
CHO-tau-aPKN (Fig. 6B, lanes 3 and 4), indicating
that PKN phosphorylation of tau occurred in vivo. HIA3 did
not react with CHO-tauS320A-aPKN (Fig. 6, lane 5 and 6), indicating that HIA3 recognizes only the phosphorylated
Ser-320 of tau.
HIA3 recognized its antigen among tau protein concentrated by
immunoprecipitation with anti-tau antibody (tau1) from human neuroblastoma SK-N-MC cells. Furthermore, tau1 recognized its antigen
among the protein concentrated by immunoprecipitation with HIA3 (Fig.
6C). These data suggest that Ser-320 phosphorylation occurs
in SK-N-MC cells and would reflect some in vivo cellular processes.
Effects of phosphatase inhibitors on tau phosphorylation at Ser-320 by
PKN were also examined to characterize the phosphorylation of tau by
PKN in vivo. The immunoreactivity of phospho-tau at Ser-320
recognized by HIA3 increased by the treatment with phosphatase inhibitors FK506 (600 nM), orthovanadate (100 µM), and okadaic acid (50 nM) (Fig.
6D). FK506 induced a striking increase in the phosphorylation of tau at Ser-320 by PKN and in vanadate a moderate increase, but the effects of okadaic acid were weak (Fig.
6D). These data indicate that PKN phosphorylates tau at
Ser-320 in vivo, and the phosphorylated Ser-320 is
dephosphorylated by phosphatases, especially calcineurin.
PKN Activation Reduces Phosphorylation Level in Multiple
Proline-directed Phosphorylation Sites in Vivo--
CHO cells
transfected with tau and pTB701 vector (CHO-tau-mock) showed
a hyperphosphorylated tau reacting with AT8, AT180, and AT270 which
recognize with proline-direct phosphorylation sites Ser-202/Thr-205,
Thr-231, and Thr-181, respectively (Fig. 7, lane 1).
Cotransfection of CHO cells with tau and
aPKN (CHO-tau-aPKN) reduced the phosphorylation state
recognized by AT8 almost completely, that recognized by AT180
moderately, and that recognized by AT270 only slightly (Fig. 7,
lane 2). The state of tau phosphorylation was unchanged when
CHO cells were cotransfected with tau and iPKN (CHO-tau-iPKN) (Fig. 7, lane 3), indicating that inhibitory
effects of PKN on tau phosphorylation depend on the kinase activity of PKN. The inhibitory effects of PKN on tau phosphorylation were not
affected by phosphatase inhibitors FK506 (600 nM),
orthovanadate (100 µM), and okadaic acid (50 nM) (Fig. 7, lanes 4-12), although these
treatments did lead to recovery of the phospho-tau recognized by HIA3
(Fig. 6D).
PKN Activation Disrupts Microtubule Organization--
Our data
show that catalytically active PKN disrupts microtubule organization in
neuroblastoma cells and that tau phosphorylated by PKN loses the
potential to promote tubulin assembly in vitro. The
stability of tubulin assembly is modulated by tau (2). PKN did not
phosphorylate tubulin in
vitro,2 thereby
suggesting that PKN does not directly associate with tubulin. These
findings suggest that the phosphorylation of tau by PKN resulted in
disruption of microtubule organization. We reported (25) that PKN
phosphorylates intermediate filament proteins such as neurofilaments,
vimentin, and glial fibrillary acidic protein and that the
phosphorylation by PKN inhibits polymerization. Furthermore, PKN
interacts with PKN Is a Tau Kinase; Identification of Phosphorylation Sites on Tau
by PKN and PKC Subtypes--
We found that PKN phosphorylates
recombinant His-tau as well as GST-tau in vitro. The level
of phosphorylation reached as high as 4 mol of Pi per
monomer of tau protein. In MBDs, PKN phosphorylated Ser-258 in 1MBD,
Ser-320 in 3MBD, and Ser-352 in 4MBD. We also identified
phosphorylation sites by PKC subtypes in MBDs. Therefore, there is no
subtype specificity in phosphorylation of MBDs among the PKC subtypes
examined. All subtypes of PKC examined in this study phosphorylated
Ser-258 in 1MBD, Ser-293 in 2MBD, Ser-324 in 3MBD, and Ser-352 in 4MBD.
Although PKN has a catalytic domain highly homologous to the PKC
family, the phosphorylation patterns differ between PKN and PKC
subtypes. These results indicate that each PKC-related protein kinase
may have a specific function regarding tau phosphorylation. Moreover,
it will be necessary to elucidate the meaning that PKN did not
phosphorylate any of three serines in 2MBD, which is spliced out in the
3-repeat form of tau. PKN also phosphorylated Ser-214 in FR in
vitro. The phosphorylation of Ser-214 has been noted with several
kinases, including protein kinase A, and may play some roles for
tau-microtubule interactions (27).
In Vivo Phosphorylation by PKN and Dephosphorylation by Calcineurin
at Ser-320--
To determine whether PKN induces tau phosphorylation
in vivo, we developed a
phosphorylation-dependent anti-tau antibody (HIA3), which
recognizes phospho-tau at Ser-320, a PKN-specific phosphorylation site.
HIA3 reacted with CHO-tau-aPKN but not with CHO-tau-iPKN, thus indicating that PKN phosphorylated tau at Ser-320 in
vivo. As the immunoreactivity of HIA3 in CHO-tau-aPKN increased by
treatments with phosphatase inhibitors, there is functional involvement
of phosphatase in dephosphorylation of phospho-tau at Ser-320 in vivo. FK506 binds with FKBP12, and the complex of FK506 and FKBP12 inhibits calcineurin (28). Vanadate is an inhibitor of protein tyrosine
phosphatases and also inhibits serine/threonine phosphatase activities
of calcineurin (29). Okadaic acid has been widely used as a protein
phosphatase inhibitor in vitro as well as in cultured cells
and animals. It inhibits protein phosphatase 2A strongly, protein
phosphatase 1 moderately, and calcineurin weakly but does not inhibit
protein phosphatase 2C (30). The dephosphorylation of Ser-320 was
inhibited by these phosphatase inhibitors, which means that the key
phosphatase in question is calcineurin. The findings of colocalization
of PKN and calcineurin with tau in neurons
(13)3 may support the
hypothesis that tau phosphorylation at Ser-320 is regulated by
cross-talk between PKN and calcineurin.
Inhibition of Proline-directed Phosphorylations of Tau by
PKN--
Hyperphosphorylated tau is the major constituent of the
paired helical filament (PHF), the major fibrous component of
neurofibrillary tangles (NFT) seen in Alzheimer brains. Many of the
hyperphosphorylated sites in PHF are proline-directed. We have reported
that PKN is closely associated with NFT at an early stage of PHF
formation (13). The present study revealed that PKN phosphorylates tau in vitro and in vivo. We therefore examined the
effect of PKN activation on proline-directed phosphorylation, and we
found that PKN reduced the level of phosphorylation of multiple
proline-direct phosphorylation sites recognized by AT8, AT180, and
AT270 in vivo. There are at least three possible mechanisms
to explain our observations. First, PKN may activate phosphatases that
dephosphorylate the phosphate from Ser-202/Thr-205, Thr-231, and
Thr-181 recognized by AT8, AT180, and AT270, respectively. FK506,
vanadate, and okadaic acid, however, did not affect the tau
phosphorylation recognized by AT8, AT180, and AT270, although they
effectively recovered the phospho-tau recognized by HIA3. These data
imply that PKN may not activate protein phosphatases. Second, PKN may
inactivate other tau kinases that phosphorylate serine or threonine
recognized by AT8, AT180, and AT270. One of the candidate kinases would
be GSK3, which can phosphorylate Ser-202/Thr-205, Thr-231, and Thr-181 recognized by AT8, AT180, and AT270, respectively (31-33).
Furthermore, GSK3 phosphorylated by mitogen-activated protein
kinase-activated protein kinase-1 (also known as p90Rsk),
p70 ribosomal S6 kinase (p70S6K), and protein kinase B
(also known as AKT/RAC) would be consequently inactivated by
phosphorylation (34). In the same manner, PKN may inhibit GSK3 by
phosphorylation, and the level of phosphorylation recognized by AT8,
AT180, and AT270 might decrease in vivo. We found that PKN
and PKCs directly inhibit GSK3-
The present study revealed that PKN phosphorylates tau at specific
sites and consequently triggers disruption of microtubule organization.
The phosphorylation at Ser-320 in 3MBD is regulated by cross-talk of
PKN with calcineurin. Moreover, phosphorylations recognized by AT8,
AT180, and AT270 and reduced by PKN in vivo are the most
prominent phosphorylations observed in Alzheimer PHFs. On the other
hand, free fatty acids such as arachidonic acid and linoleic acid,
which can activate PKN in vitro (36), stimulate the
polymerization of tau and amyloid , -
I, -
II, -
, -
, -
, -
, and -
) in
MBDs. PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all
PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352.
There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a
novel phosphorylation-dependent antibody recognizing
phosphorylated tau at Ser-320, showed immunoreactivity in Chinese
hamster ovary cells expressing tau and the active form of PKN, but not
in Chinese hamster ovary cells expressing tau and the inactive form of
PKN. The immunoreactivity for phosphorylated tau at Ser-320 increased
in the presence of a phosphatase inhibitor, FK506 treatment, which
means that calcineurin (protein phosphatase 2B) may be involved in
dephosphorylating tau at Ser-320 site. We also noted that PKN reduces
the phosphorylation recognized by the
phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by
specific phosphorylation of tau, which leads to disruption of tubulin assembly.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(15), -
I
(16), -
II (16), -
(15), -
(17), -
(17), -
(18), and
mouse PKC-
(generously provided by Dr. W. Ogawa, Kobe University)
were inserted into the baculovirus transfer vector, and pBlueBacHis/GST
and recombinant enzymes were extracted as described (14).
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Fig. 1.
Schematic representation of various tau
fragments. A schematic drawing of the entire tau protein (clone
T9) is shown at the top of the figures, and His-tagged tau
(His-tau) and GST-tagged deletion mutants and GST-tagged FR and MBD
mutants are aligned below. 1, 2, 3, and 4 indicate 1MBD, 2MBD,
3MBD, and 4MBD, respectively. The numbers preceding and
following each line denote the positions of the terminal aa residue of
each fragment. A in FR and MBD mutants indicates the
substitution of serine for alanine. In all cases, the numbering of the
amino acid residues refers to the longest isoform of human tau (441 residues).
-32P]ATP. After incubation for various times, the
reaction was terminated by adding an equal volume of Laemmli's sample
buffer. The samples were separated by 12% SDS-PAGE, and the
phosphorylation was visualized and quantified using an instant imager
(Packard Instrument Co.). The gels were also stained by Coomassie
Brilliant Blue.
C6) and anti-
tubulin antibody (YL1/2; Sera-Lab) as
described (13).
C6, was raised in
rabbit against a recombinant C-terminal protein corresponding to
residues 863-946 of rat PKN (9). AT8, AT180, and AT270 (Innogenetics)
are phosphorylation-dependent mouse monoclonal antibodies
against tau, which recognize with proline-direct phosphorylation sites
Ser-202/Thr-205, Thr-231, and Thr-181, respectively. tau1 (Roche
Molecular Biochemicals) is a mouse monoclonal antibody against tau. A
monoclonal antibody, HIHT1, was raised in mouse against a recombinant
N-terminal protein corresponding to residues 1-190 without residues
45-102 of human tau. A polyclonal antibody, HIA3, was raised in rabbit
against a synthetic phosphopeptide, CLSKVTS(P)KCGSL, where S(P)
represents phosphoserine. An anti-
-tubulin antibody (YL1/2;
Sera-Lab) is a rat antibody against
-tubulin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
PKN disrupts microtubule
assembly. A, tau phosphorylated by PKN has a reduced
potential to promote microtubule assembly in vitro. Tau not
treated with PKN (+tau only; red line), tau
pretreated with a catalytically active PKN (+tau/aPKN;
green line), or tau pretreated with a catalytically inactive
PKN (+tau/iPKN; blue line) was added to the
assembly buffer containing tubulin. Black line shows tubulin
only. Polymerization of microtubules was monitored by measuring the
absorbance at 350 nm. B, overexpression of PKN breaks down
the microtubule array in SK-N-MC and SH-SY5Y cells. SK-N-MC
(upper panels) cells and SH-SY5Y (lower panels)
were transfected with either aPKN (right panels)
or iPKN (left panels). Forty four hours after
transfection, cells were fixed with 4% paraformaldehyde, and the
microtubule organization was analyzed by double immunofluorescence
staining for PKN (red) and -tubulin (green).
Note that PKN localizes in vesicular structures but is not colocalized
with microtubules (arrow in right upper panel),
which were dramatically lost compared with those in the cells
overexpressing iPKN. Destruction of PKN-positive
microtubules is observed in aPKN-overexpressing cells
(arrowheads in right lower panel). Upper
panels are at the same magnification and bar indicates
50 µm. Bars in lower panels indicate 10 µm.
C6) and an anti-
-tubulin antibody (YL1/2). Many neurons
overexpressing aPKN appeared atrophic compared with the
cells nontransfected, mock-transfected, or overexpressing
iPKN, whereas a subset of aPKN-overexpressing
neurons remained normal in both size and shape and exhibited a dramatic
loss and poorly organized networks of microtubules (Fig.
2B). On the contrary, the microtubule network had normal
appearance in cells transfected with iPKN. Although PKN was
well colocalized with perinuclear microtubules in
iPKN-overexpressing cells, distinct localization of PKN from
microtubules was often seen in the cells transfected with
aPKN (arrow). Microtubules were disrupted to
small fragments in these neurons when doubly immunolabeled
(arrowheads). These data indicate that the cotransfection of
tau and PKN resulted in a disruption of microtubule networks in
neuronal cells.
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Fig. 3.
Tau phosphorylation by PKN in
vitro. A, time course of tau phosphorylation
by PKN. Recombinant His-tau (12.5 pmol) was subjected to in
vitro phosphorylation assay. The reaction was terminated at 0 (lane 1), 5 (lane 2), 10 (lane 3), 30 (lane 4), 60 (lane 5), 90 (lane 6),
120 (lane 7), 150 (lane 8), 180 (lane
9), 240 (lane 10), and 330 min (lane 11).
Incorporated radioactivity was counted, and the stoichiometry of
phosphorylation was calculated. Arrow indicates the time
point when 30 ng of additional PKN was added to the assay mixture.
B, PKN phosphorylates tau in FR and MBDs. GST-tagged
deletion mutants were subjected to in vitro phosphorylation
assay for 30 min. The assay was repeated at least three times. The
representative data are shown. ARG, 32P
incorporation visualized by an imaging analyzer after SDS-PAGE.
CBB, Coomassie Brilliant Blue staining.
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Fig. 4.
PKN phosphorylates Ser-214 in FR of tau
in vitro. GST-tagged FR mutants were subjected to
in vitro phosphorylation assay for 30 min. The assay was
repeated at least three times, and representative data are shown.
ARG, 32P incorporation was visualized using an
imaging analyzer after SDS-PAGE. CBB, Coomassie Brilliant
Blue staining.
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Fig. 5.
Ser-320 in MBDs is phosphorylated by PKN but
not by PKCs. A, PKN phosphorylates Ser-258, Ser-320,
and Ser-352 in MBDs in vitro. GST-tagged MBD mutants were
subjected to in vitro phosphorylation assay for 30 min. The
assay was repeated at least three times, and representative data are
shown. ARG, 32P incorporation was visualized
using an imaging analyzer after SDS-PAGE. CBB, Coomassie
Brilliant Blue staining. B, PKC subtypes phosphorylate
Ser-258, Ser-293, Ser-324, and Ser-352 in MBDs in vitro.
GST-tagged MBD mutants were subjected to in vitro
phosphorylation assay for 30 min. The assay was repeated at least three
times and representative data are shown. ARG,
32P incorporation was visualized using an imaging analyzer
after SDS-PAGE. CBB, Coomassie Brilliant Blue
staining.
phosphorylated Ser-258, Ser-293,
Ser-324, and Ser-352 in MBDs (Fig. 5B). We also determined
the phosphorylation sites of 7 other subtypes, PKC-
I, -
II, -
,
-
, -
, -
, and -
. Of all the subtypes examined- Ser-258,
Ser-293, Ser-324- and Ser-352 were phosphorylated in MBDs. As
summarized in Table I, we found that PKN
does not phosphorylate the Ser-293 in 2MBD, which can be phosphorylated
by PKCs, and that PKN and PKCs phosphorylate a different site, Ser-320
and Ser-324 in 3MBD, respectively.
Phosphorylation sites in each MBD
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Fig. 6.
Immunoblot analysis with a novel antibody,
HIA3. A, HIA3 is a specific antibody for PKN
phosphorylation. Recombinant His-tagged tau (1 ng) without (lane
1) or with prephosphorylation by PKN (lane 2), PKC-
(lane 3), and PKC-
(lane 4) were subjected to
SDS-PAGE followed by Western blotting analysis with HIA3 antibody
(diluted 1:50,000). Arrowhead indicates the position of tau.
B, HIA3 is a phosphorylation- dependent
antibody for Ser-320. Five µg of total cell lysates from CHO-tau-Mock
(lanes 1 and 2), CHO-tau-aPKN (lanes 3 and 4), CHO-tauS320A-aPKN (lanes 5 and
6), and CHO-tau-iPKN (lane 7) were subjected to
SDS-PAGE followed by Western blot analysis with HIA3 antibody (diluted
1:50,000). Arrowhead indicates the position of tau.
C, HIA3 recognizes Ser-320 phospho-tau in SK-N-MC
neuroblastoma cells. Two hundred µg of total cell lysates from
SK-N-MC neuroblastoma cells is immunoprecipitated with control mouse
IgG (lane 1), tau1 antibody (lanes 2-4), control
rabbit IgG (lane 5), or HIA3 antibody (lanes
6-8). Immunoprecipitates were subjected to SDS-PAGE followed by
Western blot analysis with HIA3 antibody (lanes 1-4) or
tau1 antibody (lanes 5-8). * and ** indicate the position
of mouse and rabbit IgG, respectively. D, protease inhibitor
treatments. CHO-tau-Mock (lanes 1, 4, 7, and 10),
CHO-tau-aPKN (lanes 2, 5, 8, and 11), and
CHO-tau-iPKN (lanes 3, 6, 9, and 12) were treated
without (lanes 1-3) or with FK506 (600 nM)
(lanes 4-6), vanadate (100 µM) (lanes
7-9), and okadaic acid (OA) (50 nM)
(lanes 10-12), respectively, and were subjected to SDS-PAGE
followed by Western blotting analysis with HIA3 (diluted 1:10,000).
Arrowhead indicates the position of tau.
View larger version (59K):
[in a new window]
Fig. 7.
PKN suppresses some proline-directed
phosphorylation states of tau in vivo.
CHO-tau-Mock (lanes 1, 4, 7, and 10),
CHO-tau-aPKN (lanes 2, 5, 8, and 11), and
CHO-tau-iPKN (lanes 3, 6, 9, and 12) were treated
without (lanes 1-3) or with FK506 (0.6 µM)
(lanes 4-6), vanadate (100 µM) (lanes
7-9), and okadaic acid (OA) (50 nM)
(lanes 10-12), respectively, and were subjected to SDS-PAGE
followed by Western blotting analysis with HIHT1 (diluted 1:10,000),
AT8 (diluted 1:10,000), AT180 (diluted 1:10,000), AT270 (diluted
1:10,000).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actinin, an actin cytoskeletal-associated protein
(26). These data imply that PKN may be involved in regulation of the
cytoskeleton, such as formation of intermediate or actin filaments and
the microtubule organization.
activity by phosphorylating at least
Ser-9 of GSK3-
and suppress the GSK3-
-induced AT8 and AT270
immunoreactivity of tau, although they did enhance the GSK3-
-induced AT180 immunoreactivity in vitro (35). The discrepancy
between in vitro and in vivo experiments implies
that tau kinases other than GSK3-
may associate with PKN-induced
suppression of the tau phosphorylation at Thr-231 recognized by AT180.
Third, PKN may induce a conformational change of tau by phosphorylation
and consequently make tau inaccessible either by other tau kinases such
as GSK3 or by phosphorylation-dependent tau antibodies such as AT8, AT180, and AT270. Indeed tau prephosphorylated by PKN was less
phosphorylated by GSK3-
in vitro (35).
peptides in vitro
(37). These data suggest that tau phosphorylation by PKN may play
crucial roles in the pathology of Alzheimer's disease by regulating
tau-microtubule interactions. Thus PKN may serve as a point for
therapeutic intervention in neurodegenerative disease including
Alzheimer's disease.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Prof. H. Mori (Osaka City
University) for tau cDNA (clone T9), Dr. W. Ogawa (Kobe University)
for mouse PKC- cDNA, and Dr. M. Hasegawa (Tokyo University) for
technical advice on the in vitro assay of microtubule
assembly. We thank M. Sumida, M. Obana, and A. Hori for their excellent
technical assistance and Y. Kitamura, ASK Co. and D. Akagi (ASK Co.)
for developing the HIHT1 and HIA3 antibody. We also thank M. Ohara for
reading the manuscript.
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FOOTNOTES |
---|
* 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: Hyogo Institute for Aging Brain and Cognitive Disorders, 520 Saisho-ko, Himeji 670-0981, Japan. Tel.: 81-792-95-5511; Fax: 81-792-95-8199; E-mail: tanigu@hiabcd.go.jp.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M007427200
2 H. Mukai, unpublished data.
3 T. Kawamata unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: MAP, microtubule-associated protein; MBD, microtubule binding domain; PKC, protein kinase C; NFT, neurofibrillary tangle; FR, flanking region; PHF, paired helical filament; CHO, Chinese hamster ovary; GST, glutathione S-transferase; PIPES, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; aa, amino acids.
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