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The Journal of Neuroscience, February 15, 1998, 18(4):1329-1336
Involvement of p62 Nucleoporin in Angiotensin II-Induced Nuclear
Translocation of STAT3 in Brain Neurons
Di
Lu,
Hong
Yang, and
Mohan K.
Raizada
Department of Physiology, College of Medicine, University of
Florida, Gainesville, Florida 32610
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ABSTRACT |
Chronic stimulation of brain neurons by angiotensin II (Ang II)
results in a increase in norepinephrine (NE) uptake. This involves
stimulation of transcription of NE transporter and tyrosine hydroxylase
genes and is associated with translocation of signaling molecules and
transcription factors from the cytoplasmic compartment into the
neuronal nucleus (Lu et al., 1996a ). We report here that the
phosphorylation of p62, a glycoprotein nucleoporin of the nuclear pore
complex (NPC), by MAP kinase is involved in this process. Ang II caused
a time-dependent translocation of signal transducers and activators of
transcription (STAT3) from the cytoplasmic compartment into the
nucleus. This translocation was attenuated by pretreatment with
antisense oligonucleotide (AON) to MAP kinase. Ang II also stimulated
phosphorylation of p62, and a maximal phosphorylation of 12-fold was
observed with 100 nM Ang II. This stimulation was blocked
by losartan, an AT1 receptor subtype-specific antagonist. The conclusion that MAP kinase is involved in Ang II-induced
phosphorylation of p62 and nuclear translocation of STAT3 is supported
by the following. (1) p62 phosphorylation was blocked by a peptide that competes with p62 as a MAP kinase substrate both in
vitro and in vivo; (2) AON to MAP kinase
attenuated Ang II stimulation of p62 phosphorylation; and (3) in
addition, it also blocked nuclear translocation of STAT3. Intracellular
loading of the peptide containing MAP kinase substrate consensus of the
p62 reduced Ang II stimulation of p62 phosphorylation and nuclear
translocation of STAT3 in both in vivo and in
vitro experiments. These observations suggest that Ang
II-induced phosphorylation of p62 may accelerate the activity of the
NPC, which would result in an increase in the nuclear transport of
transcription factors and signaling molecules. This will stimulate transcriptional processes associated with Ang II regulation of NE
neuromodulation.
Key words:
angiotensin; MAP kinase; nuclear translocation; signal
transduction; p62; nuclear pore complex
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INTRODUCTION |
Ang II exerts chronic norepinephrine
(NE) neuromodulatory actions by stimulating the expression of genes for
norepinephrine transporter (Lu et al., 1996b), tyrosine hydroxylase and
dopamine  -hydroxylase (Yu et al., 1996), key components in the
regulation of turnover, synthesis, and release of catecholamines.
Recent studies have indicated that the interaction of Ang II with the AT1 receptor subtype, a member of the G-protein-coupled
receptor superfamily, initiates a cascade of signaling events involving Ras-Raf-MAP kinase and Jak/signal transducers and activators of transcription (STAT) pathways. Stimulation of these signaling molecules
and transcription factors such as Fos, Jun, and STAT mediates in their
translocation into the nuclear compartment that results in the chronic
actions of this hormone (Marrero et al., 1995; Lu et al., 1996a; Yang
et al., 1996). Despite identification of these signaling molecules,
little is known about the regulatory mechanism by which they are
transported across the nuclear membrane to mediate the chronic effects
of Ang II. In fact, there is paucity of information regarding the
mechanism of translocation of macromolecules across the nuclear
membrane in general (Akey, 1992; Davis, 1995; Melchior and Gerace,
1995).
The transport of large molecules between the cytoplasm and the nucleus
is a key regulatory event in the transmission of chemical signals that
are generated by the activation of cell surface receptors. This
transport is facilitated by the nuclear pore complex (NPC). The NPC is
a large macromolecular assembly consisting of an intricate arrangement
of proteins and cofactors that perforate the nuclear envelope (Akey,
1992; Davis, 1995). Transport takes place along the pore axis and
involves an initial docking step in which an interaction with the NPC
proteins is important (Newmeyer and Fordes, 1988; Richardson et al.,
1988; Moore and Blobel, 1992; Boulikas, 1993; Melchior and Gerace,
1995). This is followed by the translocation through the pore in an
energy-dependent process (Newmeyer and Fordes, 1988; Richardson et al.,
1988; Moore and Blobel, 1992, 1993; Melchior et al., 1993). In recent
years considerable progress has been made in characterizing many
protein components of the NPC (Gerber and Gerace, 1992; Shi and Thomas,
1992; Sukegawa and Blobel, 1993; Miller and Hanover, 1994; Moore and
Blobel, 1994). This has led to the identification of a series of
proteins, collectively called nucleoporins. The p62 nucleoporin is of
particular interest for its involvement in nuclear transport (Starr and
Hanover, 1990; Finlay et al., 1991; Panté and Aebi, 1994). It is
a glycoprotein that contains a large number of O-linked
N-acetylglucosamine residues and has been demonstrated to be
directly involved in the nuclear transport process (Starr and Hanover,
1990; Finlay et al., 1991; Miller and Hanover, 1994; Panté and
Aebi, 1994). Many studies on the structural-functional aspects of this
protein have led to the hypothesis that p62 could be phosphorylated,
and phosphorylation and dephosphorylation of p62 may be a crucial step
in the energy-dependent translocation of macromolecules through the NPC
(Finlay et al., 1991; Kita et al., 1993; Buss and Stewart, 1995). This
study was designed to investigate the phosphorylation of p62 and its
role in nuclear translocation. It shows that Ang II stimulates MAP kinase-dependent phosphorylation of p62 and that Ang II-stimulated translocation of STAT3 into the nucleus is dependent on the
phosphorylation of p62.
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MATERIALS AND METHODS |
One-day-old Wistar Kyoto strain of rats were obtained from our
breeding colony, which originated from Harlan Sprague Dawley (Indianapolis, IN). DMEM, plasma-derived horse serum (PDHS), and 1×
crystalline trypsin (150 U/mg) were obtained from Central Biomedia (Irwin, MO). Ang II was purchased from Sigma (St. Louis, MO). Losartan
potassium was a gift from DuPont Merck (Wilmington, DE), and PD123319
was from RBI (Natick, MA). [32P] orthophosphate (1 mCi = 37 MBq), [ -32P]-ATP (3000 Ci/mmol), and
chemiluminescence assay kit were from DuPont NEN (Boston, MA).
Monoclonal anti-STAT3, anti-phosphotyrosine antibodies,
horseradish-peroxidase-conjugated anti-mouse antibody, and protein A+G
conjugated-agarose beads were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Monoclonal anti-NPC414 antibody was purchased from
BabCo (Richmond, CA), and monoclonal anti-p62 specific antibody was a
gift from Dr. John Hanover (National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
MD). FITC-conjugated anti-mouse antibody was from Boehringer Mannheim
Biochemicals (Indianapolis, IN). BSA-nuclear localization signal (NLS)
fusion protein and its control counterpart were gifts from Dr. Carl
Feldherr (Department of Anatomy and Cell Biology, University of
Florida, Gainesville, FL). BSA-NLS contained NLS of simian virus 40 large T antigen conjugated with rhodamine-labeled BSA. Control fusion
protein was the same as BSA-NLS except NLS sequence was omitted.
Oligonucleotide sense (SON) and antisense (AON) to MAP kinase were
synthesized in the DNA Synthesis Facility of the Interdisciplinary
Center for Biotechnology Research (University of Florida), essentially
as described previously (Yang et al., 1996). Synthetic peptide
corresponding to the p62 amino acid sequence of 189 to 198 (GSPFTPATLA)
and its mutant where the Thr193 was substituted with
Ala were synthesized by Genemed Biotechnologies (San Francisco, CA).
All other biochemicals were from Fisher Scientific (Pittsburgh, PA) and
were of the highest grade available.
Preparation of neuronal cultures from WKY rat brains
Hypothalamus-brainstem areas of 1-d-old rat brains were
dissected, and brain cells were dissociated by trypsin. The
hypothalamic block contained the paraventricular nucleus, the
supraoptic, anterior, lateral, posterior, dorsomedial, and ventromedial
nuclei, whereas the brainstem block contained the medulla oblongata and
pons. Trypsin-dissociated brain cells were plated onto
poly-L-lysine precoated 100 mm tissue culture dishes
(2 × 107 cells/dish) in DMEM containing 10%
PDHS, and the neuronal cultures were established essentially as
described previously (Sumners et al., 1991; Raizada et al., 1993).
Immunohistochemical analysis with the use of neuron-specific antigens
has shown that these cultures contain at least 90% neuronal cells
(Raizada et al., 1994). The remaining cells were of the astroglial type
(Raizada et al., 1994). Neurons established in culture for 15 d
were used throughout this study.
Measurement of STAT3 phosphorylation in cytoplasmic and
nuclear fractions
Isolation of cytoplasmic and nuclear fractions of
neurons. Fractionation of neuronal cells to separate cytoplasmic
and nuclear fractions was performed essentially as described elsewhere
(Yang et al., 1997). This protocol is based on the method of Newmeyer et al. (1986). In brief, neuronal cells were rinsed twice with PBS, pH
7.4, and cells were scraped off the dish; the resulting cell pellet was
suspended in 10 mM HEPES, pH 7.4, containing 10 mM potassium acetate, 1.5 mM magnesium
chloride, 0.5 mM DTT, 0.2 mM PMSF, and 1 µg/ml each aprotinin, leupeptin, and pepstatin for 15 min on ice. The
cells were homogenized with 15 gentle strokes using a type-B pestle in
a Dounce homogenizer. Completion of homogenization was ascertained by
microscopic examination. The cytoplasmic extract was collected by
centrifugation at 3300 × g for 15 min and dialyzed against transport buffer (20 mM HEPES, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 1.5 mM magnesium chloride, 1 mM EGTA, 0.5 mM DTT, and 1 µg/ml each aprotinin, leupeptin, and
pepstatin) overnight at 4°C. This fraction was used for STAT3
immunoprecipitation experiments as described below. The pellet
containing nuclei was lysed by nuclear lysis buffer (25 mM
Tris-Cl, pH 7.4, 25 mM NaCl, 1% Triton X-100, 1%
deoxycholic acid, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and
0.8 µg/ml leupeptin), and centrifuged at 12,000 × g
for 5 min at 4°C. The supernatant was saved as nuclear extract. The
purity of both the cytoplasmic and nuclear fractions was ascertained
with the use of standard markers (Jakob, 1992). The nuclear fraction
was found to be free of the cytoplasmic marker aldolase. The
contamination of cytoplasmic fraction by nuclear markers, as measured
by the levels of lemnin, was only 10%.
Measurement of STAT3 phosphorylation. Cytoplasmic and
nuclear fractions (200 µg protein) were used for immunoprecipitation with the use of anti-phosphotyrosine antibody overnight at 4°C. Immune complexes were collected on agarose beads conjugated with protein A+G and after they were washed three times with the wash buffer
(50 mM Tris-Cl, pH 7.4, 20 mM
MgCl2, and 150 mM NaCl), they were
suspended in Laemmli's buffer, boiled for 3 min, and subjected to
SDS-PAGE (Yang et al., 1996). Samples were transferred to PVDF membrane
and membranes used for immunoblotting with anti-STAT3 monoclonal
antibody as described previously (Yang et al., 1996).
Immunocytochemical staining of STAT3
The protocol used was essentially as described by us previously
except that STAT3 antibody was used and cells were examined by confocal
microscopy (Lu et al., 1996a,b; Yang et al., 1996). Approximately
200-300 cells were examined, and results are representative of such
examination.
MAP kinase AON and SON treatments
AON and SON corresponding to a region for both p44 and p42 cDNAs
of the MAP kinase were synthesized (Yang et al., 1996). The oligonucleotides were made as phosphorothiocate derivatives to enhance
nuclease resistance. Neuronal cultures established for 15 d were
treated with 1 µM AON or SON to MAP kinase dissolved in 2 µg/ml Lipofectin Reagents (Life Technologies) for 24 hr at 37°C,
before the experiments.
Measurement of p62 phosphorylation
Neuronal cells were rinsed in phosphate-free DMEM and incubated
with 2 mCi [32P]-orthophosphate in phosphate-free
DMEM for 2 hr at 37°C to label the intracellular ATP pools. This was
followed by incubation with or without 100 nM Ang II at
37°C. The cells were rinsed six times with ice-cold PBS, pH 7.4, followed by lysis for 20 min on ice in lysis buffer (25 mM
Tris-HCl, pH 7.4, 25 mM NaCl, 1% Triton X-100, 1%
deoxycholic acid, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate,
0.5 mM EGTA, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, and 0.8 µg/ml leupeptin). Samples
were centrifuged at 12,000 × g for 5 min at 4°C, and
supernatants containing 200 µg of protein were used for
immunoprecipitation with either anti-NPC414 (5 µg) or anti-p62 (5 µg) specific antibody overnight at 4°C. Immune complexes were
collected on agarose beads conjugated with protein A+G, suspended in
Laemmli's sample buffer, boiled for 3 min, and subjected to SDS-PAGE
followed by autoradiography. The radiolabeled band corresponding to a
62 kDa protein was quantitated essentially as described previously (Lu
et al., 1996a,b; Yang et al., 1996).
Phosphorylation of p62 by exogenous MAP kinase
Immunoprecipitates of p62 from neuronal cells were obtained
using a protocol described above except the
[32P]-orthophosphate step was excluded. The immune
complex was subject to exogenous MAP kinase assay by a protocol based
on Paxon et al. (1994). Briefly, the pellet containing p62
immunoprecipitate was washed twice and suspended in 20 µl MAP kinase
assay buffer (50 mM HEPES, pH 7.5, 0.1 mM EDTA,
0.015% Triton X-100). This was followed by mixing with 20 µl of MAP
kinase solution (0.3 U MAP kinase containing 0.1 mg BSA/ml and 0.2%
-mercaptoethanol) and 20 µl of ATP mixture (0.3 mM
ATP, 30 mM MgCl, and 200 µCi [-32P]-ATP/ml in kinase assay buffer) to start the
reaction. The reaction was stopped by adding 15 µl of 5× Laemmli's
sample buffer at indicated time periods and boiling for 3 min. The
sample was then subjected to SDS-PAGE electrophoresis and
autoradiography.
Osmotic loading of p62 peptide189-198
Osmotic loading of p62 peptide189-198 was
performed essentially by the method of Ahmad et al. (1995). In brief,
neuronal cells were rinsed with PBS, pH 7.4, and incubated for 10 min
with a loading solution [0.5 M sucrose, 10% polyethylene
glycol 1000, 10% fetal bovine serum, and 200 µg/ml p62
peptide189-198 or its mutant peptide
(Thr193 replaced by Ala) in DMEM buffered with 25 mM HEPES, pH 6.8]. This was followed by a rapid rinse with
a hypotonic solution (6.5 vol H2O:3.5 vol DMEM, buffered
with 25 mM HEPES, pH 6.8), essentially as described
elsewhere (Ahmad et al., 1995). After this treatment, cells were
incubated with DMEM containing 10% PDHS and were subjected to
[32P]-orthophosphate labeling and p62
phosphorylation analysis as described above.
Determination of the effect of peptide189-198
on Ang II-stimulated STAT3 accumulation in isolated nuclei
Neuronal cells were subjected to osmotic loading of p62
peptide189-198 or its mutant form with appropriate
controls. The controls included cells subjected to osmotic shock
without peptide, or with bovine serum albumin or a structurally
unrelated peptide corresponding to the AT1 receptor
sequence (amino acids 295 to 315). These controls acted like mutant
peptide and showed no effect. This was followed by stimulation of cells
with 100 nM Ang II for 10 min at 37°C. Cells were
collected, lysed, and centrifuged at 500 × g for 10 min. The resulting cytoplasmic and nuclear fraction were collected (Yang et al., 1997) and used for in vitro transport assay.
The assay was essentially based on a protocol of Feldherr (1995). In
brief, nuclei (3 × 104) from cells after
various treatments were suspended in a transport buffer containing ATP
and combined with the cytoplasmic fractions containing ~500 µg of
protein. After incubation for 15 min at 30°C, the nuclei were washed
once with the transport buffer, plated on the glass slides, and
subjected to STAT3 immunocytochemistry followed by confocal microscopic
analysis (Lu et al., 1996a,b). In initial recombination experiments we
had established that the nuclear translocation of STAT3 is ATP
sensitive and thus constitutes a specific nuclear transport process in
this preparation. This observation is consistent with other systems
(Feldherr, 1995; Radu et al., 1995a).
Experimental groups and data analysis
Each data point in all experiments was obtained from three
culture dishes, and each experiment was performed at least three times.
Data presented are mean ± SE. Comparisons between data points
were made by using one-way ANOVA and Dunnett's test from the
Statistician software. All immunofluorescent experiments were repeated
at least six times, and 200-300 cells were examined in each staining
and representative images were captured. Bands on autoradiograms were
quantitated with the use of an SW5000 Gel Analysis System (Ultra Violet
Products), as described previously (Lu et al., 1996b; Yang et al.,
1996). The linearity of the absorbance for each band was established in
initial experiments by controlling the exposure times of immunoblot
with the x-ray film. The absorbance of each band was normalized for an
equal amount of protein loading.
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RESULTS |
Effects of Ang II on neuronal STAT3
Previously it has been shown that Ang II stimulates
phosphorylation of STAT3 in vascular smooth muscle cells (Marrero et
al., 1995). In this study, we first determined whether a similar effect of Ang II on STAT3 is also seen in the neurons. Incubation of neuronal
cells with Ang II resulted in a time-dependent stimulation of STAT3
phosphorylation. A maximal, 12-fold stimulation of phosphorylation was
observed in 30 min by 100 nM Ang II. The stimulation was
mediated by the interaction of Ang II with the AT1 receptor
subtype because it was blocked by losartan, its antagonist, and not by
PD123319, an AT2 receptor subtype antagonist. Figure
1 shows that Ang II stimulated
translocation of phosphorylated STAT3 into the nucleus. Initially,
levels of phosphorylated STAT3 were increased in the cytoplasm,
followed by its increase in the nuclear fraction. The nuclear levels
reached optimum in 15 min, and by 30 min the levels in the nucleus
reached a plateau and were fivefold higher than in the cytoplasm.
Confirmation of a nuclear translocation of STAT3 was provided by
immunofluorescence data (Fig. 2). STAT3
was primarily localized in the cytoplasmic compartment in control
neurons (Fig. 2a). After incubation with 100 nM
Ang II for 30 min that results in its phosphorylation, STAT3
localization was shifted from the cytoplasm to the nucleus (Fig.
2b). This translocation was blocked by 10 µM
losartan (Fig. 2c) and not by 10 µM PD123319
(Fig. 2d).
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Figure 1.
Effect of Ang II on STAT3 phosphorylation and its
translocation into the nucleus. Neurons were incubated with 100 nM Ang II for the indicated time period, and cells were
used to isolate cytoplasmic and nuclear fractions as described in
Materials and Methods. The fractions were subjected to
immunoprecipitation with anti-phosphotyrosine antibody followed by
immunoblotting with the use of specific antibody to STAT3 essentially
as described in Materials and Methods. Top, A
representative autoradiogram. Bottom, Bands
corresponding to STAT3 were quantitated with the use of the SW 5000 Gel
Analysis System. Data were normalized for equal protein loading and
presented as mean ± SE (n = 3), as described previously (Yang et al., 1996). *Significantly different;
p < 0.05; nuclear versus cytoplasmic.
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Figure 2.
Immunofluorescent distribution of STAT3 in Ang
II-treated neurons. Experimental conditions were essentially as
described in the legend to Figure 1. a, Control neurons
depicting predominant staining of STAT3 immunoreactivity in the
cytoplasmic compartment. b, Treatment of neurons for 30 min with 100 nM Ang II results in STAT3 redistribution into
the nuclear compartment. c, Treatment of neurons with 10 µM losartan, an AT1 receptor subtype specific antagonist, completely blocks the Ang II-induced nuclear translocation of STAT3. d, Lack of effect of Ang II-induced nuclear
translocation by incubation with 10 µM PD123319, an
AT2 receptor subtype specific antagonist.
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Next, we determined whether MAP kinase is involved in the Ang
II-induced nuclear translocation of STAT3. The rationale for this idea
was based on our earlier observations, which showed that Ang II
stimulates MAP kinase in the neurons and that this stimulation is
important in the neuromodulatory actions of Ang II (Yang et al., 1996).
Treatment of neuronal cells with antisense oligonucleotide (AON) to MAP
kinase results in a ~70-80% depletion of MAP kinase (Yang et al.,
1996). Such a depletion of MAP kinase was associated with a significant
attenuation of Ang II-stimulated accumulation of STAT3 into the nucleus
(Fig. 3A). Sense
oligonucleotide (SON) of MAP kinase showed no effect. Despite
inhibition of nuclear translocation of STAT3, MAP kinase AON showed no
effect on the overall phosphorylation of STAT3 in neuronal cells (Fig.
3B). These data indicate that MAP kinase is involved only in
the translocation of STAT3 into the nucleus and not in its
phosphorylation. This conclusion was supported by the
immunofluorescence experiments presented in Figure
4. Significant staining of STAT3 was
observed in the nuclei of Ang II-treated neurons (Fig. 4b).
Although MAP kinase SON pretreatment showed a comparable
immunoreactivity with the Ang II-treated neurons (Fig. 4d),
AON-treated neurons showed very little STAT3 in the nucleus (Fig.
4c). Thus, MAP kinase AON blocked Ang II-stimulated
transport of STAT3 into the nucleus. As a consequence STAT3 remained
accumulated in the cytoplasmic compartment.
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Figure 3.
Effect of MAP kinase antisense oligonucleotide on
Ang II-induced translocation of STAT3 into the nuclear fraction.
A, Neuronal cultures were incubated without (1,
2) or with 1 µM MAP kinase AON (3,
4) or with 1 µM MAP kinase SON (5,
6) for 24 hr at 37°C. In parallel experiments this
treatment causes a 70-80% decrease in MAP kinase immunoreactivity.
After this, neurons were incubated without (1, 3, 5) or
with 100 nM Ang II (2, 4, 6) for 30 min. Nuclear fractions were isolated and levels of phosphorylated STAT3 were quantitated essentially as described in Materials and Methods. Top, A representative autoradiogram.
Bottom, Data from three separate experiments as
mean ± SE. *Significantly different from 1;
p < 0.005. **Significantly different from
2; p < 0.005. B, The
experimental protocol was identical to the one described in
A, except that the effect of Ang II on levels of STAT3
phosphorylation in whole-cell lysates was analyzed. Data are mean ± SE; n = 3. *Significantly different from
1; p < 0.01.
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Figure 4.
Effect of MAP kinase antisense oligonucleotide on
immunofluorescent distribution of STAT3 in neurons. The
experimental protocol was identical to that described in Figures 2 and
3. Neurons were preincubated without (a, b) or with MAP
kinase AON (c) or with MAP kinase SON
(d) for 24 hr, followed by incubation without
(a) or with (b, c, d) 100 nM Ang II for 30 min.
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Effect of Ang II on p62 phosphorylation
Figure 5A shows
that Ang II stimulated, in a time-dependent manner, phosphorylation of
a ~62 kDa protein that was immunoprecipitated by an anti-NPC antibody
414. Identity of this protein was established to be the p62 on the
basis of specificity of the antibody, its mobility on SDS-PAGE, and its
recognition by another antibody that was specific for the p62 (Starr
and Hanover, 1990). A maximal 12-fold stimulation of p62
phosphorylation was observed in 15 min by 100 nM Ang II.
This stimulation was also AT1 receptor subtype-mediated because it was blocked by losartan (Fig. 5B). The role of
MAP kinase on the phosphorylation of p62 was studied in view of the observations that Ang II stimulates this enzyme and that a putative MAP
kinase substrate consensus sequence is present in the p62 (Cordes et
al., 1991). Immunoprecipitated p62 from neuronal cells was used as a
substrate for exogenous MAP kinase. Figure
6 shows that MAP kinase caused a
time-dependent phosphorylation of p62. The effect of a peptide,
corresponding to amino acids 189-198 of the p62
peptide189-198 that contains the putative
phosphorylation site for MAP kinase, on p62 phosphorylation further
confirmed the role of MAP kinase. Peptide189-198
blocked the phosphorylation of p62 by exogenous MAP kinase in a
dose-dependent manner (Fig. 7). In
contrast, a mutant peptide189-198 in which
Thr193 was replaced with Ala showed no effect (Fig.
7).
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Figure 5.
Ang II stimulation of p62 phosphorylation in
neurons. A, Time course. Neuronal cultures were
prelabeled with [32P]-orthophosphate and incubated
with 100 nM Ang II for 15 min at 37°C; cell lysates were
subjected to immunoprecipitation of p62 as described in Materials and
Methods. Top, A representative autoradiogram showing a
phosphoprotein of ~62 kDa corresponding to p62.
Bottom, Quantitation of p62 bands from three separate experiments and data are mean ± SE. *Significantly different from zero time (p < 0.05). B, Ang
II receptor subtype specificity. Experimental protocol was identical to
that described in A. Cultures were treated with 100 nM Ang II for 15 min in the absence or presence of Ang II
receptor antagonists. 1, Control; 2, 4, 6, 100 nM Ang II; 3, 4, 10 µM losartan; and 5, 6, 10 µM
PD123319. *Significantly different from 1
(p < 0.025). **Significantly different from
2 (p < 0.025).
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Figure 6.
In vitro phosphorylation of p62 by
MAP kinase. Neuronal cells were lysed, and p62 was immunoprecipitated.
Immunoprecipitates were used as substrate for exogenous MAP kinase as
described in Materials and Methods. Phosphorylated p62 bands were
quantitated by SW5000 Gel Analysis System. Top, A
representative autoradiogram. Bottom, Data from three
separate experiments are presented as mean ± SE.
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Figure 7.
Inhibition of MAP kinase-mediated phosphorylation
of p62 by p62 peptide189-198.
Peptide189-198 (GSPFTPATLA) corresponding to amino
acids 189 to 198 of p62 containing a punitive substrate consensus for
MAP kinase (PXTP) was incubated with the immunoprecipitates of p62 from
neurons at the indicated concentrations. In control incubations, mutant
p62 peptide189-198 in which
Thr193 was replaced with Ala was used under
identical conditions. MAP kinase assay and p62 phosphorylation were
performed as described in the legend to Figure 6. Top,
Representative autoradiogram. Bottom, Mean ± SE
(n = 3). *Significantly different from zero time
(p < 0.025).
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Neuronal cultures were pretreated with MAP kinase AON to deplete
them of MAP kinase. This was followed by determining the effect on Ang
II-stimulated p62 phosphorylation. MAP kinase AON treatment caused a
significant blunting of Ang II stimulation of p62 phosphorylation (Fig.
8). It is the same experimental condition in which MAP kinase AON also blocks the effect of Ang II on STAT3 transport into the nucleus (Fig. 3). Treatment with MAP kinase SON
showed no attenuation of the effect of Ang II. Next, neuronal cells
were subjected to osmotic loading to introduce the
peptide189-198 into the neurons to provide an
in vivo evidence for the involvement of MAP kinase in Ang
II-induced p62 phosphorylation. Treatment of neurons with
peptide189-198 resulted in a 85% decrease in the
ability of Ang II to stimulate phosphorylation of endogenous p62 (Fig.
9). This inhibition was specific because
the mutant peptide showed no effect. These data suggest that Ang II
stimulates p62 phosphorylation and this phosphorylation involves MAP
kinase.
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Figure 8.
Effect of MAP kinase antisense oligonucleotide on
Ang II-stimulated phosphorylation of p62 in neurons. Neuronal cultures
were preincubated without (1, 2) or with 1 µM MAP kinase AON (5, 6) or with 1 µM MAP kinase SON (3, 4) for 24 hr
at 37°C to deplete MAP kinase (Yang et al., 1996). This treatment
causes a 70-80% decrease in MAP kinase. Neurons were incubated
without (1, 3, 5) or with 100 nM Ang II
(2, 4, 6) for 15 min. p62 was immunoprecipitated and analyzed by SDS-PAGE as described in Materials and Methods. Top, A representative autoradiogram.
Bottom, The band corresponding to phosphorylated p62 was
quantitated, and data from three experiments are presented as mean ± SE. *Significantly different from 1;
(p < 0.005). **Significantly different from
2; p < 0.01.
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Figure 9.
Effect of intracellular loading of p62
peptide189-198 on Ang II-induced phosphorylation of
p62. Osmotic loading of neuronal cells without (1,
4) or with p62 peptide189-198
(3, 6) or with its mutant (2, 5)
was performed essentially as described in Materials and Methods. This
was followed by incubation without (4-6) or with
100 nM Ang II (1-3) for 15 min. Levels of phosphorylated p62 were quantitated as described in the legend to
Figure 3. Top, A representative autoradiogram of
phosphorylated p62. Bottom, Quantitations of the bands
corresponding to p62 from three separate experiments are presented as
mean ± SE. *Significantly different from 4;
p < 0.05; ¥Significantly different
from 1 and 2; p < 0.05.
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Role of p62 phosphorylation in nuclear translocation of STAT3
Neuronal cells were subjected to osmotic loading protocol to
introduce the peptide189-198 or its mutant
counterpart under the above described condition, which results in
~85% decrease in Ang II stimulation of p62 phosphorylation. This
inhibition of p62 phosphorylation by the
peptide189-198 was associated with a 70% decrease
in STAT3 levels in the nucleus of neurons stimulated by Ang II (Fig.
10). Mutant peptides showed no such
effect. Finally, cytoplasmic and nuclear fractions from control and Ang
II-treated neurons were isolated. They were reconstituted in various
combinations to determine whether the phosphorylation occurring at the
nuclear level is the key in Ang II-induced nuclear translocation of
STAT3. Incubation of cytoplasmic extract from Ang II-treated cells with
the nuclei of Ang II-treated neurons resulted in a high intensity of
STAT3 staining in the nuclei (Fig. 11b). In contrast,
incubation of a cytoplasmic fraction of Ang II-treated neurons with the
nuclei isolated from control neurons showed no nuclear staining of
STAT3 (Fig. 11a). The increased staining in Ang II-treated
nuclei was completely attenuated when the cytoplasmic fraction of Ang
II-treated neurons was incubated with the nuclear fraction from neurons
that were preloaded with the peptide189-198 (Fig.
11d). However, no decrease in nuclear staining was observed when mutant peptide was used (Fig. 11c). These data indicate
that Ang II treatment may have a direct effect on the p62 located on the nuclear membrane.
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Figure 10.
Effect of intracellular loading of p62
peptide189-198 on Ang II-induced nuclear
translocation of STAT3. Cells were collected after loading the p62
peptide189-198 or its mutant counterpart.
Phosphorylated STAT3 levels in the nuclear fraction were quantitated as
described in the legend to Figure 1. Lane 1, 100 nM Ang II; lane 2, 100 nM Ang II + mutant peptide189-198; lane 3, 100 nM Ang II + peptide189-198; lane
4, control; lane 5, control + mutant
peptide189-198; and lane 6, control + peptide189-198. Top, A
representative autoradiogram of phosphorylated STAT3. Bottom, Data from three separate experiments are
mean ± SE. *Significantly different from 4;
p < 0.005. ¥Significantly different
from 1 and 2; p < 0.05.
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Figure 11.
Effect of p62 peptide189-198
on nuclear translocation of STAT3 in isolated nuclei. Cytoplasmic
fractions were prepared from neurons treated with 100 nM
Ang II for 15 min, as described in Materials and Methods. In addition,
nuclear fractions from untreated and Ang II-treated neurons were also
isolated. Cytoplasmic fraction from neurons treated with Ang II were
incubated with the nuclei isolated from the neurons that are treated
without (a) or with (b-d) Ang II
in the absence (a, b) or presence of p62
peptide189-198 (d) or its
mutant analog (c). Nuclei were fixed, and the
accumulation of STAT3 in the nuclei were analyzed by anti-STAT3
antibody and confocal microscopy as described in the legend to Figure
2.
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DISCUSSION |
The observations presented in this study further our understanding
regarding the signal transduction pathway involved in Ang II regulation
of NE neuromodulation in the brain neurons. First, they establish that
the AT1 receptor activation leads to the phosphorylation of
p62, a glycoprotein nucleoporin that is involved in the regulation of
the NPC activity. Second, Ang II stimulation of MAP kinase appears to
be the protein kinase involved in this phosphorylation. Thus,
phosphorylation of p62 constitutes a mechanism by which the transport
activity of the NPC is accelerated. This results in an increased
translocation of transcription factors and signaling molecules such as
STAT3, Fos, and Jun from the cytoplasmic compartment into the nucleus
where they participate in the transcriptional regulation of NE
neuromodulation (Gelband et al., 1997).
Neuronal cells serve as an interesting model for studying the nuclear
transport of proteins because the rate of protein trafficking is
significantly higher in these cells compared with many other cells
(Takahashi, 1992; Schulman, 1995). Additionally, our previous studies
have shown that activation of the AT1 receptor results in
an increased expression of genes relevant to NE synthesis and release
(Lu et al., 1996a; Yu et al., 1996). This involves activation of a
specific signal transduction pathway (Yang et al., 1996) followed by
translocation of signaling molecules and transcription factors into the
nucleus that may be key in regulating the chronic actions of Ang II (Lu
et al., 1996b; Gelband et al., 1997). Coupled together, these studies
suggest that the AT1 receptor stimulation in the neuron
could serve as an excellent model for investigating the nuclear
trafficking of signaling molecules. Finally, neurons provide us with
the opportunity to study the role of various components of nuclear
translocation system in vivo without interruption of the
physiologically relevant actions of Ang II.
Our data show that the AT1 receptor stimulation results in
an increased phosphorylation of STAT3, which is ultimately translocated into the nucleus. They also indicate that the phosphorylation of STAT3
is important for its nuclear translocation. In contrast, our data
indicate that MAP kinase does not play any role in the phosphorylation
of STAT3 in whole cells. This conclusion is based on the observation
that MAP kinase-AON-treated neurons failed to exhibit inhibition of
Ang II-stimulated phosphorylation of STAT3 in whole cells.
Translocation of STAT3 also involves phosphorylation of p62. There are
numerous lines of evidence to support this conclusion. (1) Ang II
stimulates p62 phosphorylation, the characteristics of which are
consistent with the activation of the AT1 receptor signaling cascade. (2) p62 phosphorylation is blocked by a peptide that
competes with p62 for phosphorylation by MAP kinase both in
vitro and in vivo. (3) Immunoprecipitated p62 serves as
an excellent substrate for exogenous MAP kinase; (4) treatment of neurons with AON to MAP kinase attenuates Ang II stimulation of p62
phosphorylation; (5) p62 has substrate consensus sequence for MAP
kinase (Cordes et al., 1991); and (6) intracellular loading of the
peptide containing this consensus sequence significantly reduces Ang II
stimulation of p62 phosphorylation in parallel with the inhibition of
nuclear translocation of STAT3. The magnitude of inhibition of p62
phosphorylation is greater than inhibition of STAT3 translocation. This
discrepancy may very well be a result of the presence of a basal rate
of nuclear translocation of STAT3 that is independent of p62
phosphorylation mechanism, and (7) in vitro Ang II-induced
nuclear accumulation of STAT3 was significantly attenuated by the
addition of p62 peptide189-198 in the reaction
mixture, suggesting that the nuclear structural component p62 is the
phosphorylation site involved in the nuclear translocation of
STAT3.
Finally, although our data provide evidence for the role of
phosphorylation-mediated regulation of nuclear transport of
macromolecules, they also raise interesting questions concerning the
mechanism by which p62 phosphorylation participates in the acceleration of the transport. One could postulate that phosphorylation of p62 may
regulate the docking of the "transport complex" (i.e., a complex of
STAT3-NLS receptor) to the NPC, thus accelerating their translocation
into the nucleus. Evidence for this hypothesis is derived from the
observation showing that repetitive peptide motifs relevant to
potential docking sites have been indicated in nucleoporins, including
p62 (Radu et al., 1995a,b). In addition, p62 has been shown to bind to
NLS receptor with high affinity, providing a basis for physical
interaction between the transport complex and the NPC (Haltiwanger et
al., 1992; Guan et al., 1995; Paschal and Gerace, 1995). Alternatively,
because p62 is a component of the NPC, its phosphorylation could induce
conformational changes in the transport channel of the NPC to regulate
the capacity and speed of the transport through the channel (Akey and
Radermacher, 1993). In conclusion, these data provide strong evidence
for a MAP kinase-dependent phosphorylation of p62 and its role in the transmission of neuromodulatory signals of Ang II from plasma membrane
to the nucleus.
| |
FOOTNOTES |
Received Sept. 12, 1997; revised Dec. 4, 1997; accepted Dec. 5, 1997.
The research was supported by National Institutes of Health Grant
HL-32610. We thank Dr. John Hanover of National Institutes of Health
for providing the p62 antibody and Dr. Carl Feldherr for
rhodamine-labeled BSA-NLS fusion protein and its mutant counterpart. In
addition, we also acknowledge Dr. Feldherr's valuable suggestions throughout this work and his critical review of this manuscript.
Correspondence should be addressed to Dr. Mohan K. Raizada, Department
of Physiology, College of Medicine, University of Florida, P.O. Box
100274, Gainesville, FL 32610.
| |
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Abstract
Introduction
Compound via MeSH
