From the Turku Centre for Biotechnology, University
of Turku and Åbo Akademi University, P.O. Box 123, FIN-20521 Turku,
Finland, the § Department of Biology, Åbo Akademi
University, BioCity, FIN-20520 Turku, Finland, the
Cancer
Research Center, Kashirskoe sh 24, Moscow 115478, Russia, the
** Department of Cell, Molecular and Structural Biology, Northwestern
University Medical School, Chicago, Illinois 60611, the
Department of Cell and Molecular Biology,
Karolinska Institute, S-17177 Stockholm, Sweden, and the
§§ Department of Biology, Laboratory of Animal
Physiology, University of Turku, FIN-20014 Turku, Finland
Received for publication, October 23, 2000, and in revised form, December 28, 2000
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ABSTRACT |
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The intermediate filament protein nestin is
expressed during early stages of development in the central nervous
system and in muscle tissues. Nestin expression is associated
with morphologically dynamic cells, such as dividing and migrating
cells. However, little is known about regulation of nestin during these
cellular processes. We have characterized the
phosphorylation-based regulation of nestin during different stages of
the cell cycle in a neuronal progenitor cell line, ST15A. Confocal
microscopy of nestin organization and 32P in
vivo labeling studies show that the mitotic reorganization of
nestin is accompanied by elevated phosphorylation of nestin. The
phosphorylation-induced alterations in nestin organization during
mitosis in ST15A cells are associated with partial disassembly of
nestin filaments. Comparative in vitro and in
vivo phosphorylation studies identified cdc2 as the primary
mitotic kinase and Thr316 as a cdc2-specific
phosphorylation site on nestin. We generated a phosphospecific nestin
antibody recognizing the phosphorylated form of this site. By using
this antibody we observed that nestin shows constitutive
phosphorylation at Thr316, which is increased during
mitosis. This study shows that nestin is reorganized during mitosis and
that cdc2-mediated phosphorylation is an important regulator of nestin
organization and dynamics during mitosis.
The intermediate filament
(IF)1 protein nestin is
expressed during early stages of development in progenitor cells of the
central nervous system. Nestin is also found in developing
muscle and myocardial cells (1-4) as well as in developing sertoli
cells (5). Upon differentiation, nestin expression is down-regulated and replaced by other tissue-specific IF proteins, such as glial fibrillary acidic protein (GFAP) in astrocytes, neurofilaments in
neurons, and desmin in muscle tissue, respectively. Interestingly, nestin expression is reinduced during various regenerative and degenerative conditions in the fully differentiated organism (6, 7).
Coexpression of nestin and the class III IF proteins, vimentin, GFAP,
and desmin, has been documented in developing neuronal and embryonic
cells. In these cells, nestin shows a similar intracellular organization as the class III IF proteins (1, 8). Based on these
observations, a possible copolymerization of nestin and the class III
IF proteins, has been postulated. Recent data showed that nestin fails
to assemble into filaments in the absence of a coexpressed IF network
in astrocytes (9). In addition, it was recently shown that nestin
cannot polymerize on its own but is integrated into type III IFs, most
likely as a heterodimer (10).
Nestin has been shown to be associated with dividing and migrating
cells and with cells rapidly changing their morphology. In light of the
fact that nestin is expressed particularly in dynamic cells, it is of
interest to study posttranslational modifications of nestin and the
involvement of such modifications in regulating nestin organization.
Intermediate filament proteins form dynamic structures that change
their intracellular organization during various conditions, such as
mitosis, differentiation, and different pathological situations
(reviewed in Refs. 11-15). The regulatory mechanisms behind these
changes are still not fully understood, but phosphorylation has been
implicated as an important regulator of IFs (reviewed in Refs. 13 and
16-18). The role of phosphorylation in regulating IF organization is
supported by studies showing that several kinases, including protein
kinase C, cAMP-dependent kinase,
Ca2+-calmodulin-dependent protein kinase, and
cdc2 kinase, induce disassembly of IFs in vitro (18-21).
Changes in IF morphology can also be induced by elevating kinase
activities in vivo, for example by expression of
constitutively active forms of protein kinase C or
Ca2+/calmodulin-dependent protein kinase II in
astrocytes (22) or by microinjection of the catalytic subunit of
protein kinase A into fibroblasts (23). Several IF proteins have been
shown to undergo increased phosphorylation with subsequent
reorganization of IF structure during mitosis. These proteins include
vimentin (24, 25), GFAP (26, 27), keratins 8 and 18 (28, 29), and the
nuclear lamins (30, 31). cdc2 kinase has been identified as a key
regulator of IF organization during mitosis (reviewed in Refs. 13 and
18). Phosphorylation may also be involved in regulating the specific
cellular distribution of IFs, as demonstrated by the preferential
localization of phosphorylated neurofilaments in axons (32, 33), and
the localization of K18, phosphorylated on Ser52, to
different cellular domains depending on the tissue (34). In addition to
changes in IF protein phosphorylation during mitosis, there appears to
be a continuous phosphate turnover on IFs in interphase, which could be
involved in regulating the dynamic properties of IFs in interphase
cells (13, 16, 35, 36). Phosphorylation has also been implicated in the
regulation of the interactions of IFs with both cytoskeleton-associated
proteins, e.g. plectin, desmoplakin (37, 38), or signaling
proteins, e.g. 14-3-3 (39-41).
The intracellular organization of nestin has not been characterized in
detail, and little is known about the structural regulation of nestin
during different cellular processes. In view of the previous
observations on mitotic vimentin phosphorylation by cdc2, we have
characterized the role of cdc2 kinase in regulating nestin organization
during mitosis in an immortalized central nervous system precursor rat
cell line, ST15A. In addition, we have identified Thr316 as
an in vitro cdc2-specific nestin phosphorylation site. We have generated a phospho-specific nestin antibody against the phosphorylated Thr316 peptide to characterize
phosphorylation of nestin at this site in vivo. The
phosphorylation of this site is elevated during mitosis but also shows
a certain degree of constitutive phosphorylation in interphase cells.
Cell Culture and Synchronization--
Immortalized rat central
nervous system precursor cells, ST15A (42), were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, 2 mM L-glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin in a humidified incubator in a
5% CO2 atmosphere at 37 °C. For synchronization, cells
were arrested in mitosis with 0.3 µg/ml nocodazole (Sigma) for
12 h. The mitotic cells were harvested by mechanical shake off,
whereas interphase cells were scraped off. Equal amounts of control and
nocodazole-treated cells were harvested. The protein concentrations
were determined by Bradford assay or by densiometric scanning of
SDS-PAGE-separated protein bands, and the samples were normalized
before immunoprecipitation or immunoblotting.
Antibodies, Double Immunofluorescence, and Confocal
Microscopy--
For double label immunofluorescence experiments, cells
were grown on coverslips coated with 0.02 mg/ml
poly-L-lysine (Sigma). Cells were washed in
phosphate-buffered saline (PBS) and fixed in dehydrated methanol for 10 min at
Nestin antiserum 6 was produced using a bacterially expressed fusion
protein (plasmid kindly provided by Martha Marvin, Harvard Medical
School, Boston, MA) containing the bacterial TrpE protein fused with
the last 1197 amino acids of the carboxyl terminus of nestin, as
antigen, according to protocol described elsewhere (43). Affinity
purified rabbit anti-nestin 6 yielded results identical to those
produced with rabbit nestin antibody 130 (1) in immunoblotting with
purified proteins and various cellular extracts (Fig.
1A). Immunofluorescence with
anti-nestin 6 and anti-nestin 130 gave identical microscopic patterns
(Fig. 1B).
A phosphopeptide antibody against the synthezised nestin phosphopeptide
KLEAENSRLQpTPG, anti Thr316 nestin, was generated. The
KLEAENSRLQpTPG peptide was conjugated to keyhole limpet
hemocyanin via glutaraldehyde and used for immunization in
rabbits. The anti-phospho-Thr316 nestin serum was
positively and further negatively affinity purified using prepacked
HiTrap 1-ml columns (Amersham Pharmacia Biotech), to which the
synthesized phosphopeptide and unphosphorylated peptide, respectively,
were covalently coupled. For immunoblotting the double affinity
purified anti-phospho-Thr316 nestin serum was diluted
1:2000 in 5% bovine serum albumin/MOPS buffer (50 mM MOPS,
125 mM NaCl, and 12.5 mM NaOH, pH 7.4) and incubated overnight at 4 °C. The blots were washed four times in
MOPS buffer supplemented with 0.3% Tween, before incubation with an
horseradish peroxidase-conjugated goat anti-rabbit secondary antibody.
Blots were developed using the ECL Western blotting detection system
(Amersham Pharmacia Biotech). To test the specificity of the antiserum,
it was preincubated with excess phosphorylated peptide before
incubating the blots. For immunofluorescence, the anti-Thr316 nestin serum was diluted 1:50 in 1% bovine
serum albumin/PBS and incubated overnight at 4 °C, then washed three
times with PBS, and further incubated with secondary antibody.
Phosphorylation of vimentin on its cdc2-specific site,
Ser55, was detected as previously described by using a
phosphopeptide-specific antibody against vimentin phosphorylated on
Ser55 (44).
Determination of Nestin Phosphorylation Levels and Identification
of Nestin Phosphopeptides--
ST15A cells were metabolically labeled
with 300 µCi/ml [32P]orthophosphate for 4 h in
phosphate-free Dulbecco's modified Eagle's medium supplemented with
10% dialyzed fetal calf serum in the presence of 0.3 µg/ml
nocodazole. This procedure blocked the cells entering mitosis. Mitotic
cells were harvested by mechanical shake-off, centrifuged down at
1500 × g 5 min at +4 °C, and washed twice by
centrifugation in ice-cold PBS. Adherent cells were washed once with
ice-cold PBS and harvested by scraping. Whole cell extracts were
obtained by lysing the cells in SDS lysis buffer (20 mM
Tris-HCl, pH 7.2, 5 mM EGTA, 5 mM EDTA, 0.4%
SDS, 10 mM sodium pyrophosphate, 10 µg/ml leupeptin, 10 µg/ml antipain, and 10 µg/ml pepstatin, respectively, and 1 mM PMSF). The cell extracts were boiled for 5 min and
further sonicated for 20 s with a probe sonicator. Nestin and
vimentin were immunoprecipitated by dilution of whole cell extracts
with RIPA buffer (20 mM Hepes, pH 7.4, 140 mM
NaCl, 10 mM pyrophosphate, 5 mM EDTA, 0.4%
Nonidet P-40, 100 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml antipain) added to either anti-nestin antibody 130 or rabbit
vimentin antibody 264, respectively, and finally recovered with protein
A-Sepharose (Sigma).
The immunoprecipitated proteins were separated on SDS-polyacrylamide
gels (45) and stained with either Coomassie Brilliant Blue or silver to
control for equal loading of proteins. The gels were dried and
autoradiographed at
For phosphopeptide or phosphoamino acid analysis, vimentin and nestin
were separated on 7.5% SDS-PAGE, fixed in 50% methanol, dried, and
autoradiographed. The corresponding nestin and vimentin bands were cut
out from the gels digested twice (9 + 3 h) with trypsin (T-8642,
Sigma, 10 µg/ml in 50 mM ammonium bicarbonate) at
37 °C. The digested peptides were washed with double-distilled water
and dried using a speed vac. Peptide maps of trypsin-digested samples
(46) were obtained by two-dimensional separation on microcrystalline
cellulose TLC plates (Merck). Trypsinized peptides were also subjected
to phosphoamino acid analysis by acid hydrolysis and further
two-dimensional TLC electrophoresis together with Ser(P), Tyr(P), and
Thr(P) standards (46). Phosphoamino acids were visualized with
ninhydrin staining and autoradiography.
Separation of IF Fractions Following Extraction with Triton
X-100--
Triton X-100 buffer (20 mM Hepes, pH 7.6, 100 mM NaCl, 5 mM MgCl2, 5 mM EGTA, 1% Triton X-100, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml antipain) was added to culture
dishes, cells were detached and collected, homogenized on ice, and
centrifuged for 15 min at 10,000 × g (4 °C) to
pellet IF. The supernatant was further centrifuged at 200,000 × g for 30 min at 4 °C to obtain a soluble fraction
(supernatant) and a pellet containing additional, most likely
fragmented IF. All fractions were dissolved in 3× Laemmli sample
buffer (45) for further gel electrophoresis and Western blotting.
In Vitro Phosphorylation of Nestin and Vimentin and
Identification of cdc2-specific Phosphorylation Sites on
Nestin--
For isolation of cellular IF, ST15A cells were harvested
in lysis buffer (0.6 M KCl, 2 mM
MgCl2, 5 mM EGTA, 1% Triton X-100, 1 mM PMSF, and 10 µg/ml of leupeptin, antipain, and
pepstatin in PBS, pH 7.6). The resulting lysate was centrifuged at
3500 × g for 10 min at 4 °C. Pellets were treated
with PBS supplemented with 5 mM EGTA, 1 mM
PMSF, 10 µg/ml leupeptin, 10 µg/ml antipain, 10 µg/ml pepstatin,
and 5 µg/ml DNase I (Sigma) at room temperature for 10 min
and centrifuged 3500 × g at 4 °C for 15 min.
Pellets were sonicated on ice in 8 M urea, 1 mM
EGTA, 10 mM Tris, 2% 2-mercaptoethanol, and 1 mM PMSF and further centrifuged 200,000 × g for 30 min at 20 °C. The supernatant was dialyzed twice
for 2-3 h at 4 °C, in 10 mM Hepes, pH 7.2, 0.2%
2-mercaptoethanol, and 0.2 mM PMSF. Resulting IF
preparations were stored at
IF preparations containing 10 µg of nestin and 100 µg of vimentin
were phosphorylated with ATP (20 µM ATP and 10 µCi of
[ Purification of Trypsinized Nestin Phosphopeptides for
Sequencing--
Dried, trypsin-digested cdc2-phosphorylated nestin
peptides were solubilized in 0.08% trifluoroacetic acid and
fractionated by reverse phase chromatography on a C18 column (Aquapore
OD300, 1 × 250 mm; Applied Biosystems). The chromatography was
preformed at room temperature with a flow rate of 0.1 ml/min. Peptides
were eluted with a continuous gradient of 0-80% acetonitrile
containing 0.08% trifluoroacetic acid for a total of 120 min.
Fractions containing 32P-labeled peptides were collected
and further fractionated on a C8 column (Aquapore RP300, 250 × 1.0 mm; Applied Biosystems). Chromatography was carried out at room
temperature with a flow rate of 0.1 ml/min. Fractions containing
32P-labeled peptides were collected for microseqencing and
further manual Edman degradation.
Amino Acid Sequence Analysis--
The isolated
32P-labeled peptides were subjected to sequence analysis
and mass spectral fingerprinting. Amino-terminal amino acid sequence
analysis was performed with an Applied Biosystems model 477A protein
sequencer equipped with an on-line applied Biosystems model 120A
phenylthiohydantoin amino acid analyzer. Phosphopeptide fractions were
analyzed by mass spectrometry on PerSeptive Biosystems Elite STR
mass spectrometer using Manual Edman Degradation--
Manual Edman degradation was
carried out on the sequenced radioactively labeled nestin peptides as
described (47). The peptides were subjected to manual Edman degradation
for 8-20 cycles. The material released after each cycle and the disc
containing any residual undigested peptides were counted.
The mitotic process involves reorganization of cytoskeletal
structures including the cytoplasmic and nuclear IFs. Because nestin is
expressed predominantly in cells that undergo mitosis or rapidly change
morphology, we examined the regulation of nestin morphology and
organization in comparison with the well characterized class III IF
protein vimentin in a rat immortalized central nervous system precursor
cell line, ST15A, during different stages of the cell cycle.
Morphological Changes of Nestin Organization during
Interphase-Mitosis Transition--
During interphase, ST15A cells
contained IF networks that extended from the perinuclear region to the
cell surface. Nestin and vimentin formed indistinguishable networks in
these cells, as demonstrated by double label confocal microscopy (Fig.
2, A, B,
K, L, and M). During the transition
from late prophase to metaphase, the IF network was reorganized into
cage-like structures surrounding the nucleus (Fig. 2, D and
E). Nestin and vimentin networks underwent identical changes
during mitosis. The colocalization of nestin and vimentin persisted
throughout the cell cycle. During telophase, nestin and vimentin showed
a more diffuse and much less intense fluorescence in the cleavage
furrow, indicating a possible disassembly of the IF networks at this
site (Fig. 2, G and H).
IF networks show variable behavior in different cell types during
mitosis. In some cell types a cage of IF is maintained around the
nucleus, whereas in other cell types IFs break down or disassemble with
a simultaneous increase in the solubility of the IF proteins. It was,
therefore, of interest to determine the solubility state of the IF
proteins in mitotic ST15A cells. Detergent extractions of control and
nocodazole-treated ST15A cells were centrifuged at different speeds to
obtain three different fractions: an insoluble fraction after
10,000 × g and soluble and insoluble fractions after
200,000 × g. Immunoblotting of the fractions
demonstrated that most of the protein stays as filaments (insoluble
fraction after 10,000 × g) in nocodazole-treated
cells. However, there was an increase in the soluble fraction after
centrifugation at 200,000 × g (Fig.
3), indicating the presence of
disassembled soluble subunits. Note the presence of the lower molecular
mass protein bands, representing cleaved forms of nestin. The intensity of these bands increases in the soluble fractions of nocodazole-treated cells, indicating a possible processing of nestin during mitosis.
Mitotic Phosphorylation of Nestin Is Mediated by cdc2
Kinase--
The mitotic reorganization of the cytoplasmic IF networks
in many cell types is known to be regulated by phosphorylation of the
IF proteins. Therefore, the phosphorylation state of nestin in ST15A
cells during interphase-mitosis transition was examined. The mitotic
reorganization of the cytoplasmic IF networks in ST15A cells was
accompanied by an increase in the phosphorylation levels of the IF
proteins, nestin and vimentin (Fig.
4A). In accordance with
previous studies (24, 25), vimentin showed a low level of constitutive
phosphorylation, which was elevated 6-fold in mitotic cells (Fig.
4A, left panel). In comparison with vimentin, nestin had a higher level of constitutive phosphorylation in interphase cells and showed a 3-fold increase in the phosphorylation level during
mitosis (Fig. 4A, right panel). In addition to
the ~220-kDa nestin band, immunoprecipitation with the nestin
antibody also yielded lower molecular mass bands. These bands were
weakly phosphorylated in control cells and showed increased
phosphorylation in nocodazole-treated cells. They most likely represent
cleaved forms of nestin, because immunoblotting of IF preparations from
ST15A cells (Fig. 1) and whole cell extracts of ST15A cells (data not
shown) using antibodies against nestin shows the presence of
corresponding bands. Phosphoamino acid analysis of nestin
immunoprecipitated from in vivo 32P-labeled
mitotic ST15A cells showed phosphorylation of nestin at serine and
threonine residues (Fig. 4C).
cdc2 kinase is involved in the mitotic phosphorylation and consequent
reorganization of various IF networks in different cell lines (13, 25,
47). When IF preparations from ST15A cells were treated with cdc2
kinase, nestin, vimentin, and the nuclear lamins were phosphorylated
(Fig. 4B) (also see 25, 30). The in vitro
phosphorylation of nestin occurred on serine and threonine residues
(Fig. 4C).
Comparative phosphopeptide mapping of nestin immunoprecipitated from
metabolically labeled interphase and mitotic ST15A cells (Fig.
5) showed five mitosis-specific
phosphopeptides (phosphopeptides 1, 2, 3, 4, and 6). There were also
interphase-specific phosphopeptides (phosphopeptides 7, 8, and 9), and
one phosphopeptide that was present in both interphase and mitotic
preparations (5). Although not obvious from Fig. 5, which does not give
a quantititative presentation of the relative labeling intensity on
interphase versus mitotic peptides, peptide 5 showed
consistently elevated phosphorylation in mitotic preparations as
compared with interphase samples. According to quantification by
phosphoimager image analysis, the relative increase
(mitosis/interphase) was 1.9-fold ± 0.3 (mean ± range of
values; measurements made on peptide maps obtained from five different
experiments). The mitosis-specific phosphopeptides 1, 2, 3, 4, and 6 corresponded to cdc2-specific tryptic phosphopeptides (Fig. 5). The
identity of the individual peptides was confirmed by parallel
chromatography of mixed samples (data not shown). The relative
dominance of the mitotic peptides that correlated to peptides
phosphorylated by cdc2 in vitro, indicated that
cdc2-mediated phosphorylation sites are likely to regulate nestin
organization during mitosis. These results identify nestin as a
physiological substrate for cdc2 kinase and suggest that this kinase is
involved in the mitotic reorganization of nestin.
Identification of cdc2-specific Phosphorylation Sites on
Nestin--
To determine cdc2-specific phosphoamino acid residues on
nestin, ST15A IF preparations phosphorylated by cdc2 in
vitro were run on SDS gels, from which the phosphorylated nestin
was cut out and further digested with trypsin. The resulting
32P-labeled tryptic peptides were fractionated by reverse
phase HPLC. Radioactive fractions (radioactive peaks 1-7) were
sequenced and subjected to manual Edman degradation to determine the
cycle at which the radiolabel is released. The nestin-peptide sequence 316LQTPGR was obtained by successful sequencing of
radioactive peak 1. Manual Edman degradation of the fraction confirmed
phosphorylation of the peptide on the third amino acid, corresponding
to the threonine residue (Thr316) of the sequence obtained
(Fig. 6). Sequencing of the other
32P peaks observed in the graph were not successful,
because of the impurity of the fractions. Nestin is a very large
protein (>1800 amino acids), and cleaving nestin with trypsin
generated too many peptides to allow for sufficient purification of all phosphopeptides by the techniques we used.
Because in vitro phosphorylated sites may not reflect the
real phosphorylation sites in vivo, a polyclonal
phosphospecific nestin antibody, anti-phospho-Thr316
nestin, was generated to characterize phosphorylation of nestin at this
site in vivo. The antibody showed high specificity against the phosphopeptide used for immunization, as compared with
unphosphorylated peptide (Fig.
7A). The affinity purified
antibody recognized the full-length nestin protein (~220 kDa) (Fig.
7B, first panel). In addition, the antibody
showed strong immunoreactivity against a ~175-kDa band and
weak reactivity against several bands of lower molecular mass (Fig.
7B, first panel). This ladder pattern can be seen
on Western blots using several different nestin antibodies at high
concentration, Most likely the pattern of fragmented protein represents
processing products of nestin. This kind of fragmentation is seen in
nestin in vivo preparations from many different cell types.2 Western blot of whole
cell extracts of nocodazole-treated and control cells using a
polyclonal nestin antibody raised against the carboxyl terminus of
nestin (rabbit anti-nestin 6) showed a similar pattern (Fig.
7B, third panel). The polyclonal antibody recognized the ~175-kDa band in addition to prominent
immunoreactivity against the ~220-kDa band (Fig. 7B,
third panel). Immunoblotting of whole cell extract of ST15A
cells with a monoclonal nestin antibody also gives immunoreactivity at
~175 kDa (Fig. 7B, fourth panel).
The results by using the phosphopeptide antibody indicated that nestin
was constitutively phosphorylated at Thr316 and that
phosphorylation at this site increased during mitosis, as shown by the
increased intensity of the ~220- and the ~175-kDa bands in
nocodazole-treated cell samples (Fig. 7B, first
panel). The phosphorylation of the ~175-kDa band was especially
pronounced. Experiments on competitive binding inhibition, where the
antibody was preincubated with phosphopeptide in excess, almost
completely abolished the antibody reactivity of the ~220-kDa band and
the band at ~175-kDa (Fig. 7B, second panel
from the left). Immunoblotting of in vitro
cdc2-phosphorylated and unphosphorylated IF preparations showed that
cdc2 kinase increases the phosphorylation of nestin (Fig.
7C).
Immunoblotting with a monoclonal nestin antibody of insoluble and
soluble fractions of control and nocodazole-treated cells after
centrifugation at 10,000 × g and 200,000 × g also revealed the presence of the ~175-kDa band (Fig.
3). This band seems to transfer to the soluble pool after
centrifugation at 200,000 × g during mitosis, because
the intensity of the band increases in the soluble fraction of
nocodazole-treated cells. Another possible explanation for the
increased intensity is that nestin is cleaved during mitosis to yield
an increased amount of a soluble ~175-kDa peptide.
The constitutive phosphorylation of nestin at Thr316 in
interphase cells and the increase in phosphorylation at this site
during mitosis was further supported by immunolabeling of interphase and mitotic cells using the phosphospecific antibody (Fig.
7D). Confocal images showed weak immunoreactivity on nestin
IFs in interphase cells with an elevated reactivity in mitotic cells. In interphase cells there is a punctate staining along the filaments. Toward the cell periphery the staining becomes more diffuse. The immunoreactivity is strongest around the nucleus. In mitotic cells the
staining is more intense, but no clear filaments are seen, compared
with the monoclonal nestin staining, where filaments can be visualized.
In comparison, the one identified cdc2-target on vimentin,
i.e. Ser55, showed complete mitotic specificity
with no phosphorylation in interphase cells, as revealed with a
Ser(P)55-specific antibody (Fig. 7D).
During interphase, nestin and vimentin form indistinguishable
cytoplasmic networks in ST15A cells. This indicates that these two IF
proteins form copolymers. Copolymerization between nestin and class III
IF proteins has been discussed in earlier studies (1, 8) and is
supported by the findings that neither nestin nor vimentin can form
filaments in GFAP-deficient astrocytes without the presence of a
coexpressed IF network (9). The ability of nestin to coassemble with
vimentin in vitro has recently been shown (10). A model for
coassembly of nestin and vimentin was proposed, where nestin-vimentin
heteropolymers are added to a core of vimentin homopolymers, allowing
the long carboxyl-terminal of nestin to stick out of the filament body.
This idea was further developed in a recent review where it was
proposed that nestin could function as a linker protein, where the long
tail of nestin would interact with the microfilaments and the
microtubules, interconnecting the three distinct components of the
cytoskeleton (49). According to our results, there is a dramatic
reorganization of the IF networks into cage-like structures in
normal and nocodazole-arrested mitotic ST15A cells. Coincident with the
morphological changes seen in the IF networks during mitosis in ST15A
cells, there is an increase in the phosphorylation levels of nestin and
vimentin. Nestin shows constitutive phosphorylation in interphase cells
with a 3-fold increase in mitotic cells. In comparison, the elevated
phosphorylation of mitotic vimentin is more dramatic, about 6-fold. The
constitutive phosphorylation of nestin may correspond to specific
phosphorylation sites involved in the tissue-specific functions of
nestin. Other possible functions for phosphorylation of nestin are
regulation of the spatial organization of IF proteins, as has been
shown for neurofilaments, or regulation of protein-protein interactions with other IF proteins, such as vimentin. Assuming that nestin coassembles with type III IF proteins in vivo, it is
possible that the long carboxyl-terminal domain of nestin functions as a spacer or cross-linking element between IFs and other cytoskeletal components, thereby regulating their supramolecular organization as
proposed in Refs. 10 and 49. Phosphorylation could modulate the
configuration of the tail domain, affecting the formation of
protein-protein interactions and thereby the organization of the
cytoskeletal network. In this study, the data suggest a role for cdc2
kinase in the phosphorylation of nestin and its reorganization during
mitosis. Furthermore, we identified Thr316 as a
cdc2-specific in vitro phosphorylation site of nestin.
Interestingly, Thr316 is located in the carboxyl terminus
near the end of the rod domain. The sequences at the carboxyl ends of
the rod domain are highly conserved among the IF proteins. This part of
the rod domain is important for proper IF assembly, and many of the
mutations that have been identified in IF-related diseases, such as
severe cases of epidermolysis bullosa simplex, are located in these
regions (4). Phosphorylation of nestin at Thr316 could,
therefore, have an important impact on the assembly properties of
nestin and the organization of the IF network in the cell. A peptide
nestin antibody directed against a nestin peptide phosphorylated at
Thr316 was generated to characterize phosphorylation of
nestin at this site in vivo. We were able to show that
nestin is constitutively phosphorylated at Thr316 and that
phosphorylation at this site is elevated during mitosis. In addition to
the ~220-kDa nestin band, the phospho-specific nestin peptide
antibody showed very strong immunoreactivity to a ~175-kDa band. The
intensity of the ~175-kDa band was markedly increased in
nocodazole-treated samples. Interestingly, a corresponding ~175-kDa
band could be seen in autoradiographs of nestin immunoprecipitated from
in vivo labeled mitotic ST15A cells. Phosphorylation of this band was increased in nocodazole-treated cells (Fig. 2). There is a
difference between nestin and vimentin when it comes to
proline-directed phosphorylation. The only SP site on vimentin
(Ser55) shows no constitutive phosphorylation, whereas the
TP site on nestin (Thr316) shows significant
phosphorylation in interphase cells.
The kinase responsible for the constitutive phosphorylation at
Thr316 is unknown, but it could be another
cyclin-dependent kinase. For example, cdk5, which is
required for normal neuronal differentiation and muscular development
(50-52), might be a candidate for interphase-specific phosphorylation
of nestin at Thr316. cdk5 together with p35, the protein
activator of cdk5, plays a role in the regulation of the structure of
the neuronal cytoskeleton structure because it has been shown to
phosphorylate cytoskeletal components such as the
microtubule-associated protein tau and the neuronal IF protein,
neurofilament H (53, 54). Considering the requirement for cdk5 activity
for proper neuronal and muscular development and the effect of cdk5
phosphorylation on cytoskeletal organization, we tested whether this
kinase can phosphorylate nestin in vitro, and our
preliminary data shows that nestin is a bona fide substrate
for cdk5.3 cdk5-mediated
phosphorylation could explain the phosphorylation observed in
interphase cells, but further studies are required to elucidate this.
Our data indicate that cdc2 kinase is involved in the regulation of
nestin organization during mitosis. It remains to be determined whether
phosphorylation by cdc2 is sufficient to account for the reorganization
of nestin during mitosis or whether some other kinase is required.
There are other kinases in addition to cdc2 kinase that have been shown
to phosphorylate IF in vivo during mitosis, including p37
kinase, which phosphorylates vimentin at Ser458 and
Thr457 in mitotic BHK-21 cells (55); protein kinase
C, which shows mitosis-specific phosphorylation of lamin B and vimentin
(56, 57); and Rho kinase, which phosphorylates GFAP during late
mitosis (57, 58). Recently, Rho kinase was shown to phosphorylate vimentin at Ser71 at the cleavage furrow in late mitotic
cells (60).
In summary, our interest in IF dynamics has led us to study the
previously uncharacterized regulation of the IF protein nestin during
the cell cycle. We have shown that reorganization of nestin during
mitosis is coupled to increased phosphorylation of nestin and that cdc2
kinase seems to play a major role in this phosphorylation-mediated structural modification of nestin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C. Cells were rinsed in PBS, blocked with 1% bovine
serum albumin, and incubated with nestin antiserum 130 diluted 1:500 or
nestin antiserum 6 diluted 1:200 for 30 min at room temperature. The
cells were then rinsed three times in PBS and incubated with
fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulins
(Molecular Probes). Cells were washed with PBS and further incubated
with a mouse monoclonal anti-vimentin antibody (RPN 1102, Amersham)
diluted 1:30 for 30 min at room temperature, washed three times in PBS,
and incubated for 30 min with Cy5-conjugated goat anti-mouse
immunoglobulins (Zymed Laboratories Inc.) diluted
1:50, and washed three times in PBS before mounting in Mowiol 40-88 (32459-0, Aldrich-Chemie, Steinheim, Germany) supplemented with
100 mg/ml 1.4. diazabicyclo [2.2.2]-octan (2780-2, Aldrich-Chemie).
Samples were analyzed in a Leitz Aristoplan fluorescence microscope and
a Leica TCS40 confocal laser scanning microscope using the program
SCANware 4.2a (Leitz, Heidelberg, Germany).
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Fig. 1.
Characterization of anti-nestin antiserum
6. A, immunoblotting of IF preparations from ST15A
cells or whole cell extracts of primary astrocytes with the nestin
antisera 6 and 130 confirmed that antiserum 6 recognizes the ~220-kDa
nestin band, recognized by antiserum 130. In addition, antiserum 6 gives the same ladder-like pattern as antiserum 130. These lower
molecular mass bands correspond to degradation products of nestin.
B, immunofluorescence labeling of ST15A cells with nestin
antisera 6 and 130 shows identical immunoreactivity patterns.
70 °C using Kodak X-Omat AR or Kodak Biomax MS
films. Specific 32P labeling of proteins was quantified
using a phosphoimager (Bio-Rad).
70 °C.
-32P]ATP) using cdc2 isolated from BHK-21
fibroblasts (15) or recombinant cdc2 kinase (Calbiochem). The kinase
reaction was carried out in 10 mM Hepes, pH 7.2, 60 mM NaCl, 0.5 mM CaCl2, 2.5 mM EGTA, and 2 mM MgCl2 at 37 °C
and stopped after 30 min by addition of 3× Laemmli sample buffer (45).
The in vitro phosphorylated IF preparations were separated
by SDS-PAGE and autoradiographed, cut out of the gel, digested with
trypsin, and further separated by TLC phosphopeptide mapping as
described above.
-cyano-cinnamic acid (10 mg/ml in 50%
acetonitrile, 0.1% trifluoroacetic acid) as the matrix. Spectra were
acquired in the reflector mode, and the mass spectra were internally
mass calibrated. The obtained sequences were confirmed with the
published amino acid sequence of nestin predicted from cDNA
(1).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Reorganization of IF in ST15A cells during
mitosis. ST15A cells were double labeled with nestin
(A, D, and G) and vimentin
(B, E, and H) antibodies.
C, F, and I show DNA staining with
Hoechst. Confocal images show immunolocalization of IFs in ST15A cells
during interphase (A and B), mitosis
(D and E), and telophase (G and
H). The pictures are maximal projections of confocal
images. Higher magnification images (K-M) of nestin
and vimentin immunolabeling of ST15A cells, including an overlay image,
show colocalization of nestin and vimentin.
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Fig. 3.
The effect of the mitotic arrest on the
assembly state of nestin. Blocking ST15A cells in mitosis with
nocodazole increases the solubility of nestin. The figure shows
immunoblotting of Triton-X- insoluble fraction after 10,000 × g, insoluble and soluble fractions after 200,000 × g of control, and nocodazole-treated ST15A cells.
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Fig. 4.
In vivo phosphorylation of nestin
and vimentin during interphase and mitosis. A,
autoradiograph of a SDS 7.5% polyacrylamide gel of nestin and vimentin
immunoprecipitated from in vivo 32P-labeled
interphase (lanes C) and nocodazole-treated (lanes
noc) ST15A cells shows increased phosphorylation of nestin and
vimentin during mitosis. Nestin is constitutively phosphorylated during
interphase but shows in average a 2.8-fold increase in phosphorylation
in mitotic cells (mean value ± S.D.; experimental data from a
minimum of three experiments). The increase in the phosphorylation of
mitotic vimentin is 6.2-fold (mean value ± S.D.). Equal loading
of proteins was controlled by silver staining (data not shown).
B, nestin is phosphorylated in vitro by cdc2. IF
preparations from ST15A cells were phosphorylated in
vitro by cdc2 and separated by SDS-PAGE. The autoradiograph shows
phosphorylation of nestin as well as vimentin and the lamins (double
band at 60 kDa). C, nestin is phosphorylated on serine and
threonine residues in mitotic cells as shown by phosphoamino acid
analysis of nestin immunoprecipitated from metabolically labeled
nocodazole-arrested ST15A cells (Nestin (noc)). Phosphoamino
acid analysis of nestin phosphorylated in vitro by cdc2
phosphorylated nestin shows that cdc2-specific phosphorylation of
nestin occurs on serine and threonine (Nestin (cdc2)).
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Fig. 5.
Comparative phosphopeptide mapping of
in vitro and on vivo phosphorylated
nestin. Two-dimensional tryptic peptide maps of nestin
immunoprecipitated from metabolically labeled interphase cells (I
(in vivo)) or nocodazole-arrested mitotic cells (M (in
vitro)). Tryptic phosphopeptide maps of mitotic nestin show five
(phosphopeptides 1, 2, 3, 4, and 6) mitosis-specific peptides. In
addition phosphorylation of peptide 5 is increased during mitosis.
Comparative tryptic phosphopeptide mapping of nestin phosphorylated
in vivo during mitosis (M (in vivo)) and in
vitro with cdc2 (cdc2(in vitro)) indicates that nestin
is phopshorylated by cdc2 during mitosis. Numbers indicate
corresponding peptides.
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Fig. 6.
Identification of nestin Thr316
as an in vitro cdc2-specific phosphorylation
site. Nestin phosphorylated in vitro by cdc2 was
trypsinized, and phosphopeptides were fractionated by reverse phase
HPLC. 32P-containing fractions were collected separately
and further purified by a second run. Radioactive fractions were
collected, and the amino acid sequence of the tryptic peptide was
determined. Peak 1 yielded the peptide sequence LQTPGR. This
sequence correspond to the nestin sequence starting at amino acid 314 at the end of the rod domain. The fraction was further subjected to
manual Edman degradation to confirm phosphorylation of the threonine
residue.
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Fig. 7.
Characterization of nestin phosphorylation at
Thr316 in vivo. A
phosphopeptide-specific antibody against phosphorylated
Thr316 was generated. A, immunoblotting of 0.5 µg of the Thr316 phosphorylated and unphosphorylated
peptide spotted on nitrocellulose membrane revealed specificity of the
generated antibody. The antibody recognizes the phophorylated peptide
but shows no immunoreactivity against the unphosphorylated peptide.
B, based on immunoblotting of control and nocodazole-treated
cells, nestin is constitutively phosphorylated at this site during
interphase, but the phosphorylation level is increased during mitosis.
The phospho-specific peptide antibody shows strong immunoreactivity
against a ~175-kDa nestin peptide, in addition to reactivity against
the full-length ~220-kDa nestin (P-thr-316 nestin). The
same samples were immunoblotted with a polyclonal nestin antibody
(anti-nestin 6) to confirm the identity of the ~175-kDa band
(polyclonal anti-nestin). Immunoblotting of whole cell
extracts of ST15A cells with a monoclonal nestin antibody also showed
the presence of the ~175-kDa peptide (monoclonal
anti-nestin). Preincubating the antibody with the phosphorylated
peptide abolished immunoreactivity against the ~220-kDa band and the
lower molecular mass band of ~ 175 kDa (P-thr-316 nestin + p-peptide). The additional low molecular bands seen on the blot
were still visible when the antibody was preincubated with excess of
the phosphorylated peptide. Equal numbers of control and
nocodazole-treated cells were harvested, and equal loading of proteins
was further controlled by Coomassie Brilliant Blue staining (data not
shown). C, immunoblotting of in vitro
cdc2-phosphorylated and unphosphorylated IF preparations shows that
cdc2 kinase increases phosphorylation at Thr316.
D, double immunofluorescence labeling of ST15A cells with
the Thr316-nestin phosphopeptide-specific antibody and
monoclonal nestin antibody. Immunofluorescence labeling of interphase
ST15A cells with the nestin phosphopeptide specific antibody
(panel a) shows a fragmented pattern along the filaments.
The intensity of the staining is increased in mitotic cells. As
comparison, panel b shows labeling with the monoclonal
nestin antibody, where the labeling in interphase versus
mitotic cells is of similar intensity. Panel c shows
labeling of ST15A cells with a phosphopeptide antibody which recognizes
vimentin when phosphorylated on the major cdc2-specific site,
Ser55. Panel d shows the whole vimentin IF
network as revealed by a monoclonal vimentin antibody. Immunoreactivity
of the vimentin phosphopeptide-specific antibody is absent in
interphase cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We want to thank Igor Bryzgalov for assistance in generating the antibodies and Minna Poukkula for critical comments on the manuscript. We are also grateful to and Kaija-Liisa Laine and Helena Saarento for technical assistance.
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FOOTNOTES |
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* This work was supported by the Academy of Finland (Research Council for Enviroment and Natural Resources, Grant 44191), the Erna and Victor Hasselblad Foundation, and the Jusélius Foundation.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.
¶ Supported by the Turku Graduate School of Biomedical Sciences.
¶¶ To whom correspondence should be addressed: Dept. of Biology, Laboratory of Animal Physiology, Science Bldg. 1, University of Turku, FIN-20014 Turku, Finland. Tel: 358-2-333-8036; Fax: 358-2-333-8000; E-mail: john.eriksson@utu.fi.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M0096699200
2 C. M. Sahlgren and J. E. Eriksson, unpublished observations.
3 C. M. Sahlgren, A. Mikhailov, J. Hellman, Y.-H. Chou, U. Lendahl, R. D. Goldman, and J. E. Eriksson, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: IF, intermediate filament; GFAP, glial fibrillary acidic protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride..
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---|
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---|
1. | Lendahl, U., Zimmerman, L., and McKay, R. D. G. (1990) Cell 60, 585-595[Medline] [Order article via Infotrieve] |
2. |
Kachinsky, A. M.,
Dominov, J. A.,
and Miller, J. B.
(1995)
J. Histochem. Cytochem.
43,
843-847 |
3. |
Sejersen, T.,
and Lendahl, U.
(1993)
J. Cell Sci.
106,
1291-1300 |
4. | Zimmerman, L. B., Lendahl, U., Cunningham, M., McKay, R. D. G., Pau, B., Gavin, B., Mann, J., Vassileva, G., and McMahon, A. (1994) Neuron 12, 11-24[Medline] [Order article via Infotrieve] |
5. | Fröjdman, K., Pelliniemi, L. J., Lendahl, U., Virtanen, I., and Eriksson, J. E. (1997) Differentiation 61, 243-249[CrossRef][Medline] [Order article via Infotrieve] |
6. | Frisen, J., Johansson, C. B., Török, C., Risling, M., and Lendahl, U. (1995) J. Cell Biol. 131, 453-464[Abstract] |
7. |
Vaittinen, S.,
Lukka, R.,
Sahlgren, C.,
Rantanen, J.,
Hurme, T.,
Lendahl, U.,
Eriksson, J. E.,
and Kalimo, H.
(1999)
Am. J. Pathol.
154,
591-600 |
8. | Sjöberg, G., Jiang, W.-Q., Lendahl, U., Ringertz, N. R., and Sejersen, T. (1994) Exp. Cell Res. 214, 447-458[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Eliasson, C.,
Sahlgren, C., M.,
Berthold, C.-L.,
Stakeberg, J.,
Celis, J. E.,
Bertsholtz, C.,
Eriksson, J. E.,
and Pekny, M.
(1999)
J. Biol. Chem.
274,
23996-24006 |
10. |
Steinert, P. M.,
Chou, Y. H.,
Prahlad, V.,
Parry, D., A,
Marekov, L. N.,
Wu, K. C.,
Jang, S. I.,
and Goldman, R. D.
(1999)
J. Biol. Chem.
274,
9881-90 |
11. | Geisler, N., Hatzfeld, M., and Weber, K. (1989) Eur. J. Biochem. 1, 441-447 |
12. | Klymkowsky, M. W. (1996) Cancer Metastasis Rev. 15, 417-428[Medline] [Order article via Infotrieve] |
13. | Ku, N.-O., Liao, J., Chou, C.-F., and Omary, B. M. (1996) Cancer Metastasis Rev. 15, 429-444[Medline] [Order article via Infotrieve] |
14. |
Fuchs, E.,
and Cleveland, D. W.
(1998)
Science
279,
514-519 |
15. |
Goldman, R. D.,
Chou, Y.-H.,
Prahlad, V.,
and Yoon, M.
(1999)
FASEB. J.
13,
261-265 |
16. | Eriksson, J. E., Brautigan, D. L., Vallee, R., Olmstedt, S., Fujiki, H., and Goldman, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11093-11097[Abstract] |
17. | Skalli, O., Chou, Y.-H., and Goldman, R. D (1992) Trends Cell Biol. 2, 308-312[Medline] [Order article via Infotrieve] |
18. | Inagaki, M., Matsuoka, Y., Tsujimura, K., Ando, S., Tokui, T., Takahashi, T., and Inagaki, N. (1996) BioEssays 18, 481-487 |
19. | Ando, S., Tokui, T., Yano, T., and Inagaki, M. (1996) Biochem. Biophys. Res. Commun. 5, 67-71 |
20. | Geisler, N., and Weber, K. (1988) EMBO J. 7, 15-20[Abstract] |
21. | Skalli, O., and Goldman, R. D. (1991) Cell Motil. Cytoskel. 19, 67-79[Medline] [Order article via Infotrieve] |
22. | Ogawara, M., Inagaki, N., Tsujimura, K., Takai, Y., Sekimata, M., Ha, M. H., Imajoh-Ohmi, S., Hirai, S., Ohno, S., Sugiura, H., Yamauchi, T., and Inagaki, M. (1995) J. Cell Biol. 131, 1055-1066[Abstract] |
23. | Lamb, N. J., Fernandez, A., Feramisco, J. R., and Welch, W. J. (1989) J. Cell Biol. 108, 2409-2422[Abstract] |
24. | Chou, Y.-H., Rosevear, E., and Goldman, R. D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1885-1889[Abstract] |
25. | Chou, Y.-H., Bischoff, J. R., Beach, D., and Goldman, R. D. (1990) Cell 62, 1063-1071[Medline] [Order article via Infotrieve] |
26. | Matsuoka, Y., Nishizawa, K., Yano, T., Shibata, M., Ando, S., Takahashi, T., and Inagaki, M. (1992) EMBO J. 11, 2895-2902[Abstract] |
27. |
Nishizawa, K.,
Yano, T.,
Shibata, M.,
Ando, S.,
Saga, S.,
Takahashi, T.,
and Inagaki, M.
(1991)
J. Biol. Chem.
266,
3074-3079 |
28. | Ku, N.-O., and Omary, B. M. (1994) J. Cell Biol. 127, 161-171[Abstract] |
29. |
Liao, J.,
Ku, N.-O.,
and Omary, B. M.
(1997)
J. Biol. Chem.
272,
17565-17573 |
30. | Peter, M., Nakagawa, J., Dorée, M., Labbe', J. C., and Nigg, E. A. (1990) Cell 61, 591-602[Medline] [Order article via Infotrieve] |
31. | Dessev, G., Iovcheva-Dessev, C., Bishoff, J. R., Beach, D., and Goldman, R. D. (1991) J. Cell Biol. 112, 523-533[Abstract] |
32. | Sternberger, L. A., and Sternberger, N. H. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6126-6130[Abstract] |
33. | Lee, V. M., Carden, M. J., Schlaepfer, W. W., and Trojanowski, J. Q. (1987) J. Neurosci. 7, 3474-3488[Abstract] |
34. | Liao, J., Lowthert, L. A., Ku, N. O., Fernandez, R., and Omary, B. M. (1995) J. Cell Biol. 131, 1291-1301[Abstract] |
35. | Eriksson, J. E., Opal, P., and Goldman, R. D. (1992) Curr. Opin. Cell Biol. 4, 99-104[Medline] [Order article via Infotrieve] |
36. | Eriksson, J. E., and Goldman, R. D. (1993) Adv. Prot. Phosphatases 7, 335-357 |
37. | Foisner, R., Traub, P., and Wiche, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 1, 3812-3816 |
38. |
Toivola, D. M.,
Goldman, R. D.,
Garrod, D. R.,
and Eriksson, J. E.
(1997)
J. Cell Sci.
110,
23-33 |
39. | Liao, J., and Omary, B. M. (1996) J. Cell Biol. 133, 345-357[Abstract] |
40. |
Ku, N.-O.,
Liao, J.,
and Omary, B. M
(1998)
EMBO J.
17,
1892-1906 |
41. |
Tzivion, G.,
Luo, Z. J.,
and Avruch, J.
(2000)
J. Biol. Chem.
275,
29772-29778 |
42. | Frederiksen, K., Jat, P. S., Valtz, N., Levy, D., and McKay, R. (1988) Neuron 1, 439-448[Medline] [Order article via Infotrieve] |
43. | Tohyama, T., Lee, V. M.-Y., Rorke, L., Mann, M., McKay, R. D. G., and Trojanowski, J. Q. (1992) Lab. Invest. 66, 303-313[Medline] [Order article via Infotrieve] |
44. | Eriksson, J. E., Toivola, D. M., Sahlgren, C., Mikhailov, A., and Härmälä-Braske'n, A.-S. (1998) in Methods Enzymol. Part B: Molecular Motors and the Cytoskeleton. (Vallee, R. B, ed) , Academic Press, San Diego, U. S. A. 298, 542-569 |
45. | Laemmli, U., K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
46. | van der Geer, P., Luo, K., Sefton, B. M., and Hunter, T. (1993) in Protein phosphorylation, A practical approach. (Hardie, D. G., ed) , IRL Press Oxford University Press, New York |
47. | Sullivan, S., and Wong, T. W. (1991) Anal. Biochem. 197, 65-68[Medline] [Order article via Infotrieve] |
48. | Tsujimura, K., Ogawara, M., Takeuchi, Y., Imajoh-Ohmi, S., Ha, M. H., and Inagaki, M. (1994) J. Biol. Chem. 264, 31097-31106 |
49. | Herrmann, H., and Aebi, U. (2000) Curr. Opin. Cell Biol. 12, 79-90[CrossRef][Medline] [Order article via Infotrieve] |
50. | Chae, T., Kwon, Y. T., Bronson, R., Dikkes, P., Li, E., and Tsai, L. H. (1997) Neuron 18, 29-42[Medline] [Order article via Infotrieve] |
51. |
Ohshima, T.,
Ward, J. M.,
Huh, C. G.,
Longenecker, G.,
Veeranna,
Pant, H. C.,
Brady, R. O.,
Martin, L. J.,
and Kulkarni, A. B.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
11173-11178 |
52. | Philpott, A., Porro, E. B., Kirschner, M. W., and Tsai, L. H. (1997) Genes Dev. 11, 1409-1421[Abstract] |
53. | Michel, G., Mercken, M., Murayama, M., Noguchi, K., Ishiguro, K., Imahori, K., and Takashima, A. (1998) Biochim. Biophys. Acta 1380, 177-82[Medline] [Order article via Infotrieve] |
54. | Pant, A. C., Veeranna, Pant, H. C., and Amin, N. (1997) Brain Res. 765, 259-266[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Chou, Y.-H.,
Puneet, O.,
Quinlan, R. A.,
and Goldman, R. D.
(1996)
J. Cell Sci.
109,
817-826 |
56. |
Goss, V. L.,
Hocevar, B. A.,
Thompson, L. J.,
Stratton, C. A.,
Burns, D. J.,
and Fields, A. P.
(1994)
J. Biol. Chem.
269,
19074-19080 |
57. | Takai, Y., Ogawara, M., Tomono, Y., Moritoh, C., Imajoh-Ohmi, S., Tsutsumi, O., Taketani, Y., and Inagaki, M. (1996) J. Cell Biol. 133, 141-149[Abstract] |
58. |
Kosako, H.,
Amano, M.,
Yanagida, M.,
Tanabe, K.,
Nishi, Y.,
Kaibuchi, K.,
and Inagaki, M.
(1997)
J. Biol. Chem.
272,
10333-10336 |
59. |
Yasui, Y.,
Amano, M.,
Nagata, K.,
Inagaki, N.,
Nakamura, H.,
Saya, H.,
Kaibuchi, K.,
and Inagaki, M.
(1998)
J. Cell Biol.
143,
1249 |
60. |
Goto, H.,
Kosako, H.,
Tanabe, K.,
Yanagida, M.,
Sakurai, M.,
Amano, M.,
Kaibuchi, K.,
and Inagaki, M.
(1998)
J. Biol. Chem.
273,
11728-11736 |