(Received for publication, June 17, 1996, and in revised form, February 21, 1997)
From the Department of Neurology and Ernest Gallo
Clinic and Research Center, University of California, San
Francisco, California 94110 and the ¶ Department of Molecular
Pharmacology, Stanford University School of Medicine,
Stanford, California 94305-5332
We have studied nerve growth factor (NGF)-induced
differentiation of PC12 cells to identify PKC isozymes important for
neuronal differentiation. Previous work showed that tumor-promoting
phorbol esters and ethanol enhance NGF-induced mitogen-activated
protein (MAP) kinase activation and neurite outgrowth by a
PKC-dependent mechanism. Ethanol also increases expression
of PKC and PKC
, suggesting that one these isozymes regulates
responses to NGF. To examine this possibility, we established PC12 cell
lines that express a fragment encoding the first variable domain of
PKC
(amino acids 2-144), which acts as an isozyme-specific
inhibitor of PKC
in cardiac myocytes. Phorbol ester-stimulated
translocation of PKC
was markedly reduced in these PC12 cell lines.
In addition, phorbol ester and ethanol did not enhance NGF-induced MAP
kinase activation or neurite outgrowth in these cells. In contrast,
phorbol ester and ethanol increased neurite outgrowth and MAP kinase
phosphorylation in cells expressing a fragment derived from the first
variable domain of PKC
. These results demonstrate that PKC
mediates enhancement of NGF-induced signaling and neurite outgrowth by
phorbol esters and ethanol in PC12 cells.
Protein kinase C (PKC)1 is a multigene
family of phospholipid-dependent, serine-threonine kinases
that plays a central role in cell growth and differentiation. Molecular
cloning studies have identified 10 isozymes encoded by 9 different
mRNAs (1, 2). Based on sequence homology and biochemical
properties, the PKC gene family has been divided into three groups:
"conventional" PKCs (,
I,
II, and
) regulated by
calcium and diacylglycerols or phorbol esters; "novel" PKCs (
,
,
, and
), which are calcium-independent but diacylglycerol-
and phorbol ester-sensitive; and "atypical" PKCs (
, and
/
), which are insensitive to calcium, diacylglycerol, and PMA. In
addition, two related phospholipid-dependent kinases, PKCµ and protein kinase D, share sequence homology in their
regulatory domains to novel PKCs and may constitute a new subgroup
(3, 4).
Several studies with tumor-promoting phorbol esters suggest that PKC
modulates neural differentiation. Phorbol esters induce neural tissue
from ectoderm in Xenopus embryos (5) and elicit neurite
outgrowth from chick sensory ganglia (6, 7), chick ciliary ganglion
neurons (8), several human neuroblastoma cell lines (9, 10), and rat
PC12 cells (11, 12). Studies using purified isozymes, kinase-defective
mutants, and transgenic or mutant cell lines have implicated PKC,
-
, -
, -
, and -
in the differentiation of nonneural cells
(13-17). Overexpression of PKC
or -
in Xenopus
embryos enhances neural induction (18), but little else is known about
the identity of specific PKC isozymes that regulate neural
differentiation.
Recent evidence suggests that PKC plays a role in neural
differentiation and plasticity. PKC
is expressed predominantly in
the nervous system and is particularly abundant in the hippocampus, olfactory tubercle, and layers I and II of cerebral cortex (19). Within
immunoreactive neurons, it is localized to the Golgi apparatus and to
axons and presynaptic nerve terminals (19). PKC
is activated by
growth factors that stimulate neural differentiation such as insulin
(20) and NGF (21). In addition, in developing chick brain, it is the
major isozyme found in nondividing, differentiating neurons (22).
Further evidence for involvement of PKC in neural differentiation
has come from studies with PC12 cells. PC12 cells are derived from
neural crest and, when treated with NGF or fibroblast growth factors,
undergo dramatic biochemical and morphological differentiation, developing several characteristics of mature sympathetic neurons (23).
PKC-activating phorbol esters enhance NGF-induced activation of ERK1
and ERK2 mitogen-activated protein (MAP) kinases and neurite outgrowth
in PC12 cells, suggesting that PKC modulates responses to NGF (11, 12,
24). Studies with ethanol-treated PC12 cells helped direct us toward
the PKC isozyme responsible for this effect. Like phorbol esters,
ethanol increases NGF-induced MAP kinase activation and neurite
outgrowth through a PKC-dependent mechanism (11, 24).
Ethanol promotes PKC-mediated phosphorylation in PC12 cells by
increasing levels of messenger RNA and protein for two PKC isozymes,
PKC
and PKC
(25, 26). Recently, we found that overexpression of
PKC
, but not of PKC
, enhances NGF-induced MAP kinase activation
and neurite outgrowth (27). These findings establish PKC
as a
positive modulator of neurite growth. They also suggest that PKC
mediates the neurite-promoting effect of ethanol and phorbol esters in
PC12 cells. However, proof of this hypothesis requires studies with PKC
isozyme-specific inhibitors or cells lacking specific PKC isozymes.
In the current study, we used specific inhibitors of PKC and PKC
to investigate whether PKC
mediates enhancement of neurite outgrowth
by phorbol esters and ethanol. To achieve this goal we used
dominant-negative inhibitors based on the amino acid sequences for
PKC
and PKC
. This approach is based on the observation that upon
activation, PKC isozymes translocate to specific intracellular sites
where they appear to bind anchoring proteins, termed RACKs (receptors for activated
C-kinase) (28). One such protein that has been
cloned is RACK1, which interacts with the C2 domain of conventional
PKCs (29). The sites of interaction between RACK1 and the C2 domain of
PKC
have been mapped, and short peptides derived from these domains
inhibit translocation and activation of PKC
in cardiac myocytes and
Xenopus oocytes (29-31). Homology has been noted between
the unique first variable region of PKC
(
V1) and the C2 domain of
conventional PKCs (32), suggesting that, similar to the C2 domain of
conventional PKCs,
V1 may contain a binding site for an
PKC
-specific RACK. If that is the case, then expression of an
V1
fragment should inhibit PKC
translocation and function. Indeed,
recent work has shown that an
V1 fragment and a peptide
corresponding to amino acids 14-21 in this region prevent phorbol
ester-induced PKC
translocation and inhibition of contraction in
cultured cardiac myocytes (33).
In this paper, we describe studies with PC12 cell lines that stably
express the fragments V1 or
V1, which are derived from the first
variable domains of PKC
or PKC
, respectively. We found that each
fragment selectively inhibited phorbol ester-induced translocation of
its corresponding isozyme, indicating that these fragments can function
as isozyme-selective translocation inhibitors. NGF-induced MAP kinase
phosphorylation and neurite outgrowth were not enhanced by phorbol
esters or ethanol in cells expressing
V1, but they were increased by
these agents in cells expressing
V1 and in cells transfected with
empty vector. These results demonstrate that PKC
specifically
mediates enhancement of MAP kinase activation and neurite growth by
phorbol esters and ethanol in PC12 cells.
NGF (2.5 S) was purchased from Collaborative Research (Bedford, MA). Geneticin (G418), laminin, and poly-L-ornithine (30-70 kDa) were purchased from Sigma. Phorbol 12-myristate 13-acetate (PMA) was from LC Laboratories (Woburn, MA). Antibodies were purchased from the following sources: peroxidase-conjugated goat anti-rabbit IgG from Boehringer Mannheim, fluorescein-conjugated goat anti-rabbit IgG from Cappel (Durham, NC), anti-phospho-MAP kinase antibody from New England Biolabs (Beverly, MA), and anti-Flag M2 antibody from Eastman Kodak Co.
Cell CulturePC12 cells, originally obtained from Dr. John A. Wagner (Cornell University, New York, NY), were cultured in plastic tissue culture flasks at 37 °C in Dulbecco's modified Eagle's medium containing 10% heat-inactivated horse serum, 5% fetal calf serum, 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin, in a humidified atmosphere of 90% air and 10% CO2. For studies of neurite outgrowth, cells were plated at a density of 30-40 × 103 cells/well on 24-well plastic culture plates pretreated for 1 h with poly-L-ornithine (100 µg/ml in 15 mM sodium borate, pH 8.4). In some experiments, cells were plated at a density of 20 × 103 cells/well in 8-well glass chamber slides (Nunc, Naperville, IL) treated first with poly-L-ornithine and then laminin (30 µg/ml) overnight. Cells were cultured in medium containing 50 ng/ml of NGF for 4 days, and neurites were measured as described previously (27). Ethanol-treated cultures were wrapped in Parafilm to prevent evaporation of ethanol, as in prior studies (24, 25).
Immunofluorescence MicroscopyTo detect PKC
immunoreactivity, cells plated on glass chamber slides were incubated
in PBS (137 mM NaCl, 2.7 mM KCl, 1.47 mM KH2PO4 8 mM
Na2HPO4, 0.5 mM MgCl2,
0.9 mM CaCl2, pH 7.2) containing 2%
paraformaldehyde for 30 min and 4% paraformaldehyde for 30 min at
4 °C. Cells were washed three times in PBS and incubated for 2 h in PBS containing 1% normal goat serum and 0.1% Triton X-100. Cells
were incubated 48 h at 4 °C in PBS containing 2 mg/ml of bovine
serum albumin, 0.1% Triton X-100, and 2 µg/ml of rabbit anti-PKC
antibody (34) provided by Dr. Susan C. Kiley (W. Alton Jones Cell
Science Center, Lake Placid, NY). Cells were washed three times in PBS,
and immunoreactivity was detected using fluorescein-conjugated goat
anti-rabbit IgG as described previously (27). Images were detected
using a liquid-cooled CCD camera (Photometrics Ltd., Tucson, AZ) fitted
with a Thompson 7883 chip (384 × 576 pixels) attached to an
Olympus IMT-2 inverted microscope equipped with a × 60 1.3 numerical aperture objective. Exposure times were 0.5 s, and
images were stored on an Apple Macintosh Quadra 950 computer. Fluorescence intensity in growth cones and cytoplasm was measured using
the program BDS Image (Oncor Imaging Systems, Gaithersburg, MD).
To detect PKC immunoreactivity, cells were incubated with rabbit
polyclonal antibody against PKC
(0.2 µg/ml) from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA) as described previously (27).
Immunofluorescence in 0.5-µm optical sections was detected with a
Bio-Rad MRC 1024 confocal laser-scanning microscope equipped with a
Nikon × 60, 1.4 numerical aperture oil immersion objective. Nuclei were stained by incubating cells in PBS containing 2 mg/ml of
bovine serum albumin, 0.1% Triton X-100, and 0.2 µM
TOTO-3 (Molecular Probes, Eugene, OR) for 2 h at 25 °C. After
three washes in PBS, slides were dried, and coverslips were mounted
with Vectashield (Vector, Burlingame, CA). TOTO-3 immunofluorescence
was detected by confocal laser-scanning microscopy. Images were
analyzed in BDS Image to measure the area of each nucleus at its widest
diameter.
The plasmid pDM27 (33), containing
a Flag-epitope tag followed by the sequence encoding amino acids 2-144
of PKC was used to amplify a 480-base pair fragment containing a
NotI site at the 5
-end and a XbaI site at the
3
-end. This amplified fragment, containing an ATG start codon, the
Flag epitope sequence, and the PKC
sequence, was subcloned into the
NotI and XbaI sites of pRc/RSV (Invitrogen, San
Diego, CA) to generate the plasmid pR
V-1. Another plasmid, pDM68
(33), containing the Flag epitope followed by the sequence encoding
amino acids 2-144 of PKC
was used to amplify and subclone a
homologous Flag-tagged PKC
sequence into pRc/RSV to generate the
plasmid pR
V-1. PC12 cells (107) were suspended in 0.5 ml
of Ca2+- and Mg2+-free PBS containing 137 mM NaCl, 2.7 mM KCl, 1.47 mM
KH2PO4, 8 mM
Na2HPO4, pH 7.2, and were electroporated with
80 µg of pR
V-1, pR
V-1, or pRc/RSV as described previously (27).
Geneticin was initially added at 400 µg/ml to select clones and later
was added at 200 µg/ml to maintain cultures. For each vector, 46 clones were selected, expanded, and then examined for expression of
Flag-tagged PKC fragment mRNA using RT-PCR and for Flag
immunoreactivity by Western analysis.
Poly(A) mRNA was isolated from 106
cells using a Micro-Fast Track mRNA isolation kit (Invitrogen, San
Diego, CA). Reverse transcription was carried out with 100 ng of
mRNA using a Stratagene (La Jolla, CA) RT-PCR kit according to the
manufacturer's protocol. Amplification of cDNA was achieved using
the forward primer (5-ACACTGGCGGCCGCATGGACTACAAGGACGACGAT-3
) and the
reverse primers 5
-AGCGAGCTCTAGATCGTTCTTCATTGTCTTTA-3
for pR
V-1,
and 5
-ACAGACCTCTAGAGCGGTTCATAGTTGGGAA-3
for pR
V-1. These primers
were designed to specifically amplify the Flag-V1 sequences and not the
V1 sequences of endogenous PKC
and PKC
in cells. Samples were
heated to 94 °C, and amplification was started by the addition of
Taq DNA polymerase. The amplification cycle was as follows:
annealing for 45 s at 48 °C; elongation for 2 min at 72 °C,
and denaturation for 1 min at 94 °C. Amplification was repeated for
30 cycles.
To detect expression of Flag-tagged peptides, cells were cultured on poly-L-ornithine-coated, 100-mm tissue culture dishes at a density of 6 × 106 cells/dish. Medium was removed, and cells were rinsed twice at 4 °C with buffer A (120 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 0.8 mM MgSO4, 1 mM NaH2PO4, 10 mM glucose, 25 mM HEPES, pH 7.4). Cells were scraped into 1 ml of buffer A containing 40 µg/ml leupeptin, 40 µg/ml soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride and then were frozen on dry ice. Concentrated 5 × sample buffer was added to 400-µl frozen samples to yield a final solution containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 12.5 mg/ml bromphenol blue. Samples were heated at 90 °C for 10 min, passed five times through a 26-gauge needle, and centrifuged at 10,000 × g for 10 min. Samples (80 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis using 14% gels. Proteins were electrophoretically transferred for 2 h at 4 °C to Hybond-C extra membranes (Amersham Corp.). Membranes were blocked for 1 h with 3% nonfat dry milk dissolved in Tris-buffered saline (TBS; 20 mM Tris HCl, pH 7.4, 137 mM NaCl). Blots were then incubated with anti-Flag M2 antibody (10 µg/ml) for 2 h at 25 °C. Blots were washed three times with TBS containing 0.05% Tween-20 (TBS-T) for two minutes and then were incubated with goat anti-mouse IgG-peroxidase-conjugated antibody (1:1000 dilution) in blocking solution overnight at 25 °C. Blots were washed three times for 15 min in TBS-T and once with TBS. Immunoreactive bands were detected with the ECL kit from Amersham.
Activation of ERK1 and ERK2 MAP kinases was assayed with a phospho-specific 42/44-kDa MAP kinase rabbit polyclonal antibody raised against a phosphotyrosine peptide corresponding to residues 196-209 of human ERK1. The antibody detects phosphorylation of ERK1 at tyrosine 204, which is required for ERK1 activation (35). The antibody also detects phosphorylation of the corresponding activating tyrosine of ERK2. Blots from 11% polyacrylamide gels were washed for 5 min with 25 ml of buffer B containing 58 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl, pH 7.4. Membranes were blocked in buffer B containing 0.1% Tween 20 and 5% milk (blocking buffer) for 1 h. Blots were incubated overnight at 4 °C with 1 µg/ml of anti-phospho-MAP kinase antibody in buffer B containing 0.05% Tween 20 and 5% bovine serum albumin (incubation buffer). They were then washed three times for 5 min with 15 ml of blocking buffer and incubated with goat anti-rabbit alkaline phosphatase-conjugated antibody (1:2000 dilution) in incubation buffer for 1 h at 25 °C. Blots were finally washed three times with 15 ml of blocking buffer, and immunoreactive bands were detected using the Western-Light chemiluminescent detection system from Tropix, Inc. (Bedford, MA).
PKC TranslocationCells (3-4 × 106) were
plated on 100-mm plastic tissue culture plates. After 48 h,
cultures were rinsed at 37 °C twice with 10 ml of medium and
incubated with or without 30 nM PMA for 2 min. Cells were
rapidly rinsed twice at 4 °C with Ca2+- and
Mg2+-free PBS and then were scraped into 1 ml of ice-cold
buffer C containing 20 mM Tris HCl, pH 7.5, 2 mM EDTA, 10 mM EGTA, 40 µg/ml leupeptin, 40 µg/ml aprotinin, 20 µg/ml soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride. Cells were homogenized at
4 °C with 10 strokes of a Teflon-glass homogenizer. Sucrose was
added to a final concentration of 250 mM, and the sample
was homogenized with 10 additional strokes. An aliquot of 800 µl was centrifuged at 150,000 × g for 1 h, and the
supernatant was frozen on dry ice. The pellet was dispersed in 800 µl
of buffer C by sonication in a Branson Sonifier 450 for 2 s at a
setting of six. Samples of supernatant and pellet suspension derived
from 100 µg of crude homogenate were separated by SDS-polyacrylamide
gel electrophoresis using 10% gels and analyzed for PKC and PKC
immunoreactivity by Western analysis as described previously (27).
Protein concentrations were measured by the Bradford method (36) using bovine IgG standards. Results are expressed as mean ± S.E. values, and differences between means were analyzed by ANOVA. Where p < 0.05, the significance of differences between means was evaluated by the Scheffe F-test or the Newman Keuls test.
Stably transfected PC12 clones
were tested for expression of the first variable domain of PKC or
PKC
by RT-PCR (Fig. 1A) and Western
analysis (Fig. 1B). Two clones, V1
1 and V1
2,
expressing the V1 region of PKC
(
V1), and two clones, V1
1 and
V1
2, expressing the V1 region of PKC
(
V1), were expanded for
further studies.
To determine whether the V1 and
V1 fragments expressed by our
PC12 clones act as PKC isozyme-selective inhibitors, we measured PMA-induced translocation of PKC
and PKC
to the particulate fraction of these cells. We predicted that if
V1 contains a binding site for a PKC
-specific RACK, it should inhibit translocation of
PKC
. Likewise, if
V1 interacts with an PKC
-specific RACK, then
expression of
V1 should block phorbol ester-mediated translocation of PKC
. In the parent PC12 cell line and in cells transfected with
the pRc/RSV vector alone (clone C1), 30 nM PMA stimulated translocation of PKC
and PKC
to the particulate fraction (Fig. 2). In V1
1 and V1
2 cells, which express the
V1
peptide, 30 nM PMA stimulated translocation of PKC
, but
translocation of PKC
was reduced in these clones (Fig. 2,
A and B). In V1
1 and V1
2 cells, 30 nM PMA stimulated translocation of PKC
, but
translocation of PKC
was reduced compared with control cells (Fig.
2, A and C). These results demonstrate that
V1
and
V1 fragments selectively inhibit PMA-induced translocation of
their corresponding PKC isozymes.
PKC Immunoreactivity in Cells Expressing V1 Fragments
We
attempted to examine cells by indirect immunofluorescence with
anti-Flag antibody to determine the subcellular localization of
expressed V1 and
V1 but were unsuccessful because of high background staining. We next examined the subcellular localization of
endogenous PKC
and PKC
to determine whether their localization was altered by expression of
V1 or
V1. In undifferentiated cells, PKC
and PKC
immunoreactivity was observed throughout the
cytoplasm, as previously reported (27) and was not altered in clones
expressing
V1 or
V1 (data not shown). In PC12 and C1 cells
treated with 50 ng/ml of NGF for 4 days, most PKC
immunoreactivity
was observed in the cytosol, asymmetrically next to the nucleus (Fig.
3). Less intense staining was seen in a thin perinuclear
band and within nuclei. In the majority of V1
1 and V1
2 cells
expressing
V1, most PKC
immunoreactivity appeared in the
perinuclear region (Fig. 3). In addition, nuclei appeared larger in
these cells. This was analyzed further using confocal images of
TOTO-3-stained nuclei at their widest diameter. Nuclei were of similar
size in PC12 (82.8 ± 1.5 µm2; n = 164) and C1 (88.3 ± 2.1 µm2; n = 145) cells but were significantly larger in V1
1 (106.4 ± 3.8 µm2; n = 88) and V1
2 (100.6 ± 2.8 µm2; n = 172) cells
(p < 0.05 compared with PC12 and C1 nuclei; ANOVA and
Scheffe-F test).
Following treatment with NGF, PKC immunoreactivity was observed in
growth cones, neurite shafts, and the cytoplasm of the cell soma in
PC12 and C1 cells (Fig. 4), as noted previously (27). In
V1
1 and V1
2 cells expressing
V1, PKC
immunoreactivity was reduced in growth cones and neurite shafts (Fig. 4). This was examined
further by calculating the ratio of mean fluorescence intensity of each
growth cone and of the cytoplasm at the base of its neurite shaft. In
PC12 (0.73 ± 0.02; n = 71) and C1 (0.76 ± 0.02; n = 55) cells, this ratio was significantly
greater (p < 0.05; ANOVA and Scheffe F-test) than
ratios measured in V1
1 (0.49 ± 0.01; n = 83)
and V1
2 (0.50 ± 0.01; n = 71) cells. These results demonstrate that
V1 and
V1 fragments alter the
localization of their corresponding PKC isozymes in cells undergoing
NGF-induced differentiation. In addition, expression of
V1 is
associated with an increase in nuclear size.
Growth and Neurite Formation in Cell Lines Expressing
The growth rates of V1- and V1
-expressing clones,
parent PC12 cells, and C1 cells were similar before NGF treatment (data not shown). After culture in poly-L-ornithine-treated
culture dishes with 50 ng/ml NGF for 4 days, the number of
neurite-bearing cells and the length of neurites was similar in all
cell lines (Tables I and II). However, in
NGF-treated V1
1 and V1
2 cells, neither ethanol nor PMA (10 nM) increased neurite length or the percentage of cells
that expressed neurites. In contrast, ethanol and PMA increased neurite
length and the percentage of neurite-bearing cells in NGF-treated PC12,
C1, V1
1, and V1
2 cultures. When cells were cultured on glass
slides treated with poly-L-ornithine and coated with
laminin, the results were qualitatively similar but more dramatic
because NGF-induced neurite outgrowth was especially robust following
treatment with ethanol or PMA in all but V1
1 and V1
2 cultures
(Fig. 5, A and B). Therefore,
expression of the
V1 fragment, but not of the
V1 fragment,
appears to prevent enhancement of neurite extension by PMA or ethanol
in NGF-treated cells.
|
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MAP Kinase Phosphorylation in PKC-transfected Cells
NGF and
basic fibroblast growth factor stimulate sustained activation of ERK1
and ERK2 MAP kinases in PC12 cells, which is important for their
neuronal differentiation (37). Previous work has shown that treatment
with either PMA or ethanol increases NGF-induced phosphorylation and
activation of ERK1 and ERK2 (24). Since overexpression of PKC also
enhances NGF-induced MAP kinase phosphorylation (27), we examined
whether expression of
V1 would prevent enhancement of MAP kinase
activation by PMA or ethanol.
ERK1 and ERK2 are activated by dual phosphorylation on neighboring
tyrosine and threonine residues (35). We measured activation of ERK1
and ERK2 by Western analysis using an anti-phospho-MAP kinase antibody
that specifically detects phosphorylation of the activating tyrosine of
each enzyme. As described previously in PC12 cells (24), NGF stimulated
phosphorylation of ERK1 and ERK2 in C1 cells with a biphasic time
course (Fig. 6A). Phosphorylation was maximal
after 5-10 min (peak phase) and then declined to a lower level
(plateau phase) that was maintained for at least 2 h. A similar
pattern of phosphorylation was observed in V11, V1
2, V1
1, and
V1
2 cells (Fig. 6). As previously observed in PC12 cells (24),
co-treatment with 10 nM PMA or pretreatment with 100 mM ethanol for 6 days increased NGF-induced ERK
phosphorylation. This was particularly evident during the plateau phase
of ERK phosphorylation, which was elevated to levels achieved during the peak phase in C1 and V1
2 cells (Fig. 6A). A similar
increase in phosphorylation was also observed in V1
1 cells (Fig. 6,
B and C). In contrast, the plateau phase of ERK
phosphorylation was not increased in V1
1 or V1
2 cells treated
with ethanol or PMA (Fig. 6, A-C). Therefore, expression of
the
V1 fragment specifically inhibits PMA- or ethanol-induced ERK
phosphorylation in NGF-treated cells.
The current results identify PKC as the PKC isozyme responsible
for enhancement of NGF responses by phorbol esters and ethanol in PC12
cells. We previously found that phorbol esters enhance NGF-induced
neurite outgrowth and MAP kinase activation in PC12 cells (11, 24).
Ethanol also increases these responses to NGF by a
PKC-dependent mechanism (11, 24). Recently, we found that
overexpression of PKC
also enhances responses to NGF (27), suggesting that PKC
mediates the neurite-promoting effect of PMA and
ethanol. In this paper, we investigated this issue directly, by
creating PC12 cell lines that express peptides encoding V1 domains of
PKC
or PKC
, which act as isozyme-selective translocation inhibitors (33). We found that expression of the
V1 fragment selectively inhibited PMA-induced translocation of PKC
, whereas expression of the
V1 fragment specifically inhibited translocation of PKC
. Cells expressing these peptides showed no alterations in
cell growth. However, expression of
V1 prevented enhancement of
NGF-induced MAP kinase activation and neurite growth by PMA or ethanol.
In contrast, expression of
V1 did not alter enhancement of NGF
responses by these agents. These findings indicate that PKC
mediates
enhancement of neurite outgrowth and MAP kinase activation by PMA or
ethanol in NGF-treated PC12 cells.
Our data are consistent with a recent study (33) in which a V1
fragment, an
V1 fragment, and an
V1-derived peptide were introduced into cardiac myocytes by transient permeabilization. These
fragments selectively inhibited PMA-induced translocation of their
corresponding PKC isozyme and not translocation of other isozymes
concomitantly activated in these cells. Furthermore, the
V1 fragment
and the short peptide derived from it inhibited phorbol ester-
or hormone-induced regulation of contraction rate, whereas the
V1 fragment or translocation inhibitors of PKC
did not.
Together with the data presented here, these studies reinforce the concept that translocation of PKC is required for its
function (28) and indicate that isozyme-selective inhibitors
of PKC translocation can be used to determine the function of
individual isozymes in a variety of cells.
The ability of the PKC and -
fragments to act as
isozyme-selective translocation inhibitors is also consistent
with the hypothesis that each contains a binding site for a
corresponding, isozyme-specific RACK (28). This hypothesis is
further supported by the finding of structural homology
between
V1 (32) and the C2 domain of conventional PKCs,
which contains a RACK1 binding site (29). Indeed, an
PKC
-specific RACK, RACK2, that has recently been cloned, binds the
V1 fragment in vitro.2
Prominent PKC immunoreactivity was found in neurites and growth
cones of NGF-treated parent PC12 and C1 cells. This was reduced in
processes of cells expressing
V1, suggesting that
V1 displaces endogenous PKC
from binding sites in neurites and growth cones. Localization of PKC
to growth cones is consistent with a role for
this isozyme in regulating neurite outgrowth (38).
Immunoprecipitation-kinase assays indicate that NGF activates PKC
in
PC12 cells (21), suggesting that NGF-induced localization of PKC
to
neurites and growth cones may involve activation of this isozyme. This
suggests that an PKC
-specific RACK may reside in growth cones and
neurites.
In PC12 and C1 control cells, PKC immunoreactivity was most
prominent asymmetrically next to the nucleus. Expression of
V1 was
associated with redistribution of PKC
immunoreactivity to the
perinuclear region and with an increase in nuclear size. It is
difficult to speculate on the physiologic significance of these changes, since the function of PKC
in these cells is not yet known.
However, the findings clearly indicate that
V1 alters the
localization of PKC
and produces a unique change in cell morphology.
NGF-induced activation of ERK1 and ERK2 involves phosphorylation and
binding of phospholipase C and the adapter protein Shc to the NGF
receptor tyrosine kinase TrkA (39). Within minutes of NGF binding,
phosphorylated Shc also forms a complex with another adapter protein,
Grb2, and with the guanine nucleotide exchange factor mSOS (40),
leading to activation of Ras (41) and sequential activation of B-Raf
(42), MAP kinase kinase-1 (43, 44), and ERK1 and ERK2 (45). This
pathway appears essential for NGF-induced neurite outgrowth, since
dominant negative inhibitors of Ras (46) or MAP kinase kinase-1 (47)
block NGF-induced neurite outgrowth in PC12 cells. PKC
could enhance
ERK activation by increasing the activity of members of this pathway.
For example, PKC
appears to phosphorylate and activate Raf-1 (48),
suggesting that other PKC isozymes, such as PKC
, might also modulate
Raf kinases. In addition, NGF stimulates binding of Shc to F-actin in
PC12 cells (49). NGF also activates PKC
(21), and upon activation,
PKC
binds F-actin in nerve terminals (50). This raises the
intriguing possibility that Shc or proteins complexed with Shc interact
with PKC
when both Shc and PKC
are anchored to actin. We do not
yet know if the
V1 fragment prevents binding of PKC
to actin,
since actin binds to PKC
at a site between the first and second
cysteine-rich regions of the C1 domain, which is outside of the
V1
domain (50). Whether binding of PKC
to actin is important for
regulation of MAP kinases and neurite outgrowth also remains to be
determined.
The major effect of phorbol esters or overexpressed PKC is to
increase the late plateau phase of MAP kinase activation rather than
the initial peak phase (24, 27). These kinetics suggest that PKC
may
act by inhibiting dephosphorylation of ERK1 and ERK2 rather than by
promoting ERK activation. MAP kinase phosphatase (MKP)-1, MKP-2, and
hVH3 are dual specificity protein phosphatases that are expressed in
the brain and dephosphorylate and inactivate ERKs (51, 52). MKP-1 and
MKP-2 mRNAs are constitutively expressed at low levels in PC12
cells and are increased by NGF (51). Inhibition of MKP-1 expression in
PC12 cells does not accelerate the early phase of MAP kinase
inactivation following stimulation with growth factors (53). Instead,
protein phosphatase 2A and an unidentified protein-tyrosine phosphatase
appear to mediate the rapid phase of inactivation in these cells (54).
The role of dual specificity phosphatases in regulating the later
plateau phase of NGF-induced MAP kinase activation is not known, but
NGF induction of mRNA for MKP-1 and MKP-2 peaks at 1-2 h (51),
suggesting that they may regulate this phase. If this is the case, then
inhibition of these phosphatases by PKC
may account for enhanced
activation of ERK1 and ERK2.
ERK activity is also regulated by negative feedback inhibition.
Phosphorylation of mSOS by MAP kinase promotes dissociation of mSOS
from tyrosine-phosphorylated Shc and epidermal growth factor receptors
(55, 56). In addition, MAP kinase kinase-dependent phosphorylation of mSOS has been reported to cause dissociation of
mSOS-Grb2 complexes, interrupting mSOS activation of Ras (57). Moreover, ERK1 phosphorylates MAP kinase kinase, and this
phosphorylation appears to reduce MAP kinase kinase activity (58).
Therefore, inhibition of mSOS or MAP kinase kinase retrophosphorylation
may be mechanisms by which PKC could enhance MAP kinase
activation.
Together with studies in primary neurons (20, 22, 59), our findings
suggest that PKC modulates neural differentiation. This may be a
mechanism for enhancement of neurite growth by neurotransmitters that
activate receptors coupled to phospholipase C and could be important
for activity-dependent remodeling of synapses during normal
development (60). Our studies also suggest that excessive activation of
PKC
may contribute to abnormal neurite growth observed in certain
disease states. Chronic abuse of ethanol can damage the nervous system
by disrupting the growth and remodeling of dendrites and axons. In
certain brain regions, ethanol increases the growth of neural processes
and terminals (61-65). This is particularly striking in the
hippocampus, where expression of PKC
is high (19). There prenatal
exposure to ethanol causes marked overgrowth of dentate granule cell
axons (mossy fibers) into the stratum pyramidale and stratum oriens of
CA3, which may contribute to cognitive dysfunction (65). In addition,
abnormal growth of mossy fibers into the supragranular layer of the
dentate gyrus is found in some humans with temporal lobe epilepsy and
in animals following stimuli that induce epilepsy (66). Future studies with transgenic and PKC
mutant mice or with
V1-derived inhibitory peptides may allow us to examine the role of PKC
in normal
development, alcohol-related neurologic disorders, and
epileptogenesis.