From the Department of Cellular and Structural Biology and University of Colorado Cancer Center, University of Colorado School of Medicine, Denver, Colorado 80262
Received for publication, November 20, 2002
, and in revised form, March 26, 2003.
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ABSTRACT |
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INTRODUCTION |
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Members of the protein kinase C (PKC) family are enriched in growth cones
and important for neurite outgrowth and growth cone turning
(9,
10,
11,
12). In neuroblastoma and
pheochromocytoma cells, the isoform in particular has been implicated
in phenomena related to neurite outgrowth
(13,
14,
15,
16). PKC
can localize to
adhesion sites, where it is involved reportedly in the regulation of cell
spreading (17,
18,
19,
20,
21,
22). This raises the question
of its activation. The activation of PKC by phosphatidylserine (PS) and
diacylglycerol (DAG) has been described in detail
(23,
24,
25,
26). A number of reports also
implicate AA and/or its metabolites in PKC stimulation
(27,
28,
29,
30,
31,
32), but they do not provide
evidence for a direct mechanism of activation. Instead, some of them suggest
kinase activation via intermediate steps such as the intracellular release of
DAG and Ca2+ (33,
34). Therefore, the issue has
remained unclear.
In growth cones a major target for PKC is the myristoylated alanine-rich protein kinase C substrate (MARCKS), an adhesion site protein (19, 35, 36, 37, 38). MARCKS can associate reversibly with the membrane in a phosphorylation-dependent manner (39). In its unphosphorylated state MARCKS binds to either the plasma membrane or to F-actin, where it acts as a cross-linker (40). Upon phosphorylation, MARCKS detaches from the membrane and is no longer able to cross-link F-actin (41). Therefore, it is reasonable to postulate that MARCKS phosphorylation may be important for the regulation of adhesion (21).
We have reported that thrombin is a potent repellent for growth cones of
central nervous system neurons
(5). It increases growth cone
levels of AA and of 12- and 15-HETE and also causes growth cone detachment.
These repellent effects have been established for semaphorin 3A as well
(6). The present studies
investigate subsequent signaling steps. They identify PKC isoforms present in
growth cones and test the hypothesis that the eicosanoid 12(S)-HETE
directly activates PKC. Furthermore, our experiments identify MARCKS as
the primary substrate of thrombin- and 12(S)-HETE-activated
phosphorylation in growth cone adhesion sites and show that PKC activation is
necessary for growth cone collapse.
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EXPERIMENTAL PROCEDURES |
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Growth Cone IsolationGrowth cone particles (GCPs) were
prepared as described by Pfenninger et al.
(42) and modified by Lohse
et al. (43). Briefly,
18-day fetal rat brains were homogenized in 8 volumes of 0.32
M sucrose containing 1 mM MgCl2, 2
mM TES buffer, pH 7.3, and 2 µM aprotinin. A low
speed supernatant (1660 x g for 15 min) was loaded onto a
discontinuous sucrose density gradient with steps of 0.83 and 2.66
M sucrose containing MgCl2 and TES. The gradients were
spun to equilibrium at 242,000 x g for 40 min in a vertical
rotor (VTi50, Beckman). The GCP fraction at the 0.32/0.83 M sucrose
interface was collected and used for subsequent experimentation.
Permeabilized GCP Kinase AssayThese assays used endogenous
substrate and kinase, essentially as described by Katz et al.
(37), and were done in the
absence of Ca2+ and exogenous lipids. Briefly, pelleted GCPs (60
µg of protein) were resuspended in 50 µl of cold kinase buffer (20
mM HEPES, pH 7.0, 10 mM MgCl2, 1
mM EGTA), and effector was added for 10 min on ice. GCPs were
permeabilized by adding 0.01% saponin, and reactions were initiated with 50
µM ATP plus 3µCi of -labeled [32P]ATP for
each GCP aliquot. GCPs were incubated for 30 s at 30 °C. Reactions were
stopped by adding 4x Laemmli sample buffer. Under these conditions
reactions were linear for up to 5 min. Samples were resolved by SDS/PAGE, and
phospho-polypeptides were detected by storage phosphor screen or film
autoradiography. Assay results were quantified by densitometry or by
phosphorimaging analysis of the 87-kDa (MARCKS) band. To generate gel-loading
controls, we monitored by Western blot levels of the cytosolic
"marker" LDH (35 kDa) included in the samples. To this end, the
lower portion of the gels (up to about 40 kDa) was cut off and processed for
Western blot as described further below.
Immunoprecipitate Kinase AssayPelleted GCPs (60 µg of
protein) were resuspended in 100 µl of PKC lysis buffer (50 mM
HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5
mM MgCl2, 1 mM EGTA, 10 mM sodium
pyrophosphate, 100 mM sodium orthovanadate, 100 mM NaF,
10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM
phenyl-methylsulfonyl fluoride). Material was allowed to lyse for 30 min at 4
°C with stirring. Lysed material was then precleared by incubation with
rabbit IgG conjugated to agarose. After centrifugation to remove conjugate, 1
µg/ml primary antibody (anti-PKC or
) or control serum was
added to the supernatant, and the supernatant was incubated for 60 min at 4
°C. Next, 20 µl of protein A-conjugated beads was added, and the
supernatant was incubated for 60 min at 4 °C. Immunoprecipitates were
collected by centrifugation, washed 4x in PKC lysis buffer, and
resuspended in 30 µl of kinase buffer (20 mM HEPES, pH 7.4, 5
mM MgCl2, 1 mM MnCl2).
Kinase assays were performed in the presence of Triton X-100 micelles
(containing 123 µM PS and 33 µM DAG) and 200
µg/ml histone H1 or peptide as a substrate. Reactions were initiated by
adding [32P]ATP (3 µCi of [-32P]ATP in a total
of 50 µM ATP) for 5 min at 30 °C and stopped by adding
Laemmli sample buffer. Phospho-polypeptides were resolved by SDS/PAGE and
detected by phosphorimaging.
Recombinant PKC AssayrPKC assays were performed in the
presence of Triton X-100 micelles. rPKC was used at a concentration of 1
ng/µl. rPKC reaction buffer consisted of 20 mM HEPES, pH 7.4, 10
mM MgCl2, 200 µg/ml histone H1 as a substrate, and
0.03% Triton X-100 in the presence of various activators but lacked PS and DAG
(unless noted otherwise). Reactions were initiated by adding
[-32P]ATP (3 µCi in 50 µM ATP) for 5 min at
30 °C and stopped by adding Laemmli sample buffer. Phospho-polypeptides
were resolved by SDS/PAGE and detected by autoradiography. Results were
quantified by densitometry.
Adhesion Site IsolationPetri dishes (35-mm diameter) were coated with 10 µg of mouse laminin in phosphate-buffered saline (PBS) by incubation at room temperature for 60 min with shaking. Dishes were subsequently rinsed 3 times with PBS to remove unbound laminin and then blocked with 5% nonfat dry milk (Carnation) in PBS for 30 min. After three PBS rinses to remove blocking agent, the dishes were ready for the addition of GCPs. Before plating, the GCP fraction was slowly added at 4 °C to an equivalent volume of 2x concentrated, modified Krebs buffer (22 mM sucrose, 50 mM NaCl, 5 mM KCl, 22 mM HEPES, pH 7.4, 10 mM glucose, 1.2 mM NaH2PO4, 1.2 mM MgCl2, and 2 mM CaCl2). After incubation for 5 min at 37 °C, the buffered GCP preparation was added to the laminin-coated Petri dishes. Contact with the substratum was facilitated by centrifuging the dishes for 15 min at 5000 x g (Beckman JS5.2 rotor) at room temperature followed by incubation at 37 °C to allow adhesion site formation. After 10 min, unattached GCPs were removed by rinsing the dishes twice with 1x modified Krebs buffer.
To remove Triton X-100-soluble material, attached GCPs were incubated for 10 min on ice with 1x modified Krebs buffer containing 1% Triton X-100, 0.01% saponin, and for protease inhibition, 10 mg/ml aprotinin, 0.1 mM leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). After incubation, the Triton X-100-soluble fraction was collected, leaving the resistant adhesion site/cytoskeleton fraction attached to the plate. The adhesion complexes were then solubilized in 5% SDS and 2 mM dithiothreitol. Proteins were precipitated in chloroform/methanol and redissolved in a small amount of SDS/dithiothreitol. After a modified Lowry assay to determine total protein, the polypeptides were resolved by SDS-PAGE and analyzed by Western blotting.
Gel Electrophoresis and Western BlottingProtein concentrations were determined by dye binding assay. Polypeptides were resolved by SDS-PAGE. Pre-stained standards were used to determine apparent molecular mass. Resolved proteins were transferred to nitrocellulose essentially as described by Towbin et al. (44) with a semi-dry blotting apparatus (60 min at 300 mA for a 14 x 17-cm gel). Ponceau S staining served to monitor the efficiency of protein transfer. Blots were rinsed in PBS and distilled water and dried. Before incubation with primary antibody, blots were blocked with 5% nonfat milk powder and 0.02% Tween 20 in PBS for 2 h at room temperature.
For detecting PKC isoforms, blots were incubated with the following
antibodies at the indicated concentrations: anti-PKC at 1:500,
anti-PKC
at 1:100, anti-PKC
at 1:1000, and anti-PKC
at
1:1000. All blots were washed five times in blocking solution. This was
followed by incubation with the appropriate secondary antibody
(horseradish-conjugated goat anti-rabbit or anti-mouse antibody at 1:5000) for
1 h in blocking solution. After extensive washing, bound antibody was detected
by enhanced chemiluminescence according to the manufacturer's directions
(PerkinElmer Life Sciences) using contact-exposure of x-ray film (Eastman
Kodak Co. X-Omat BLUE XB-1).
Neuron CultureDorsal root ganglia (DRG) were dissected from embryonic day 15 Sprague-Dawley rats and cultured on laminin-coated coverslips (Assistant brand) in B27 neurobasal medium supplemented with 10% fetal bovine serum and 100 ng/ml nerve growth factor. The cultures were incubated at 37 °C in 5% CO2 in air. After 24 h in culture, the medium was removed and replaced with fresh B27 neurobasal medium without fetal bovine serum and nerve growth factor. Long neurites with spread growth cones were present on day 2 and used for collapse and indirect immunofluorescence experiments as described below.
Growth Cone Collapse AssaysDRG neurons were cultured on laminin-coated coverslips as described above. The coverslips were then mounted in a Sykes-Moore chamber, covered with B27 neurobasal medium medium, and overlaid with mineral oil to maintain pH. The assembled chamber was then placed on the microscope stage under convective heating at 37 °C and allowed to equilibrate before experimentation. Phase contrast images were acquired using a Zeiss Axiovert 200 M microscope and Cooke Sensicam digital camera. Growth cone areas were measured using NIH Image 1.62 software. Student's t test was used to determine statistical significance.
Immunofluorescence Microscopy of Growth Cones in CultureFixation and labeling. DRG cultures were fixed by a slow infusion of fixative (4% paraformaldehyde, 0.1 M phosphate buffer, pH 7.4, 120 mM glucose) as described previously (45). Thereafter, any remaining fixative was removed by rinsing the cultures three times with PBS containing 1 mM glycine. Cultures were then blocked with PBS plus 3% bovine serum albumin and permeabilized by incubation in blocking buffer containing 0.01% Triton X-100 for 5 min. After three washes in PBS/bovine serum albumin the cultures were ready for labeling.
Paxillin, PKC, and MARCKS were visualized by incubating the cultures
with respective antibodies in PBS/bovine serum albumin at room temperature for
1 h. The paxillin antibody was obtained from Transduction Laboratories and the
MARCKS antibody (M-20) was from Santa Cruz Biotechnology, whereas the
PKC
antibody was a generous gift from Dr. Rytis Prekeris. Cultures were
washed again and incubated for 45 min with either Texas Red-conjugated donkey
anti-goat, fluorescein isothiocyanate-conjugated donkey anti-mouse, or
fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody (1:100,
Jackson ImmunoResearch Laboratories). To stain for F-actin, cultures were
incubated with either Texas Red- or fluorescein isothiocyanate-conjugated
phalloidin for 45 min in lieu of secondary antibody. After three washes, the
coverslips were mounted onto slides in an anti-fade mounting medium.
Fluorescence MicroscopyImages were acquired using a Zeiss Axiovert 200 M microscope equipped with a Cooke Sensicam digital camera and SlideBook imaging software (Intelligent Imaging Innovations). Optical sections were taken at 0.20.3 µM spacing and deconvolved using a nearest-neighbor algorithm. Images represent the first optical slice containing immunofluorescence signal as the plane of focus moves from the coverslip into the growth cone.
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RESULTS |
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MARCKS Phosphorylation in Growth ConesThrombin-induced
growth cone collapse is dependent upon the generation of free AA and its
further metabolism to 12(S)-HETE via 12/15-LO
(5). We investigated in
isolated growth cones whether thrombin could increase PKC activity, and if so,
whether this was dependent upon 12(S)-HETE. PKC assays were carried
out with permeabilized GCPs in the absence of Ca2+ and the presence
of -labeled [32P]ATP. Phosphorylated polypeptides were
resolved by SDS-PAGE and detected by phosphorimaging. To ascertain equal
loading, the lower halves of the gels, which were devoid of major
phosphoproteins (data not shown), were blotted and probed for the cytosolic
"marker" LDH. As shown in Fig.
1A, thrombin (100 nM) increased
phosphorylation of a prominent 87-kDa polypeptide compared with untreated
controls. A representative experiment used for quantitative analysis is shown
in Fig. 2, and quantitative
results are presented in Fig.
3A. The measured phosphorylation increase obtained with
thrombin is not large, about 42% above control, but highly significant
(p < 0.006). The non-proteolytic thrombin receptor-activating
peptide (TRAP; 100 µM) also stimulated phosphorylation of this
polypeptide but not as much as thrombin.
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As reported previously, two-dimensional gel electrophoresis revealed that MARCKS is the only major growth cone phosphoprotein in GCPs that migrates at 87 kDa (with a pI of 4.2) (37). To confirm that the 87-kDa phosphoprotein was indeed MARCKS, we performed immunoprecipitations. In controls (no MARCKS antibody), precipitates were free of labeled phosphoprotein (Fig. 1B). However, the MARCKS antibody precipitated most or all of the phosphorylated 87-kDa polypeptide (Fig. 1B). It follows that thrombin challenge stimulates MARCKS phosphorylation in GCPs. The prominent phosphoprotein migrating with an apparent molecular mass of 43 kDa (Figs. 1, A and B) corresponds to the growth cone protein GAP-43 (46).
MARCKS is an abundant GCP protein (see ref. 38). To establish that it associated with growth cone adhesion sites GCPs were allowed to attach to a laminin substratum and form adhesion sites. Such attached growth cones remain functional for at least 2 h and are able to disassemble adhesive contacts after repellent treatment (5). A Triton X-100-resistant fraction nriched for adhesion site proteins was prepared from the laminin-attached growth cones (47). Triton-insoluble proteins were solubilized with SDS and, together with the Triton-soluble fraction and a sample of the GCP parent fraction (low speed supernatant), subjected to Western blot analysis using the anti-MARCKS antibody (Fig. 1C). These blots revealed a substantial amount of MARCKS in the adhesion site fraction together with a larger Triton-soluble (presumably cytosolic) pool of the protein.
The next series of experiments was designed to explore further the roles of PKC and 12(S)-HETE in MARCKS phosphorylation in GCPs. Because control phosphorylation levels in GCPs were prominent and changes in phosphorylation in general relatively small, we included LDH loading controls in all experiments (Fig. 2). Densitometric analysis of LDH immunoreactivity yielded values that varied by only ±7.5% (S.D.). Therefore, the phosphorylation levels measured in each lane were reliable.
To test 12(S)-HETE dependence of MARCKS phosphorylation we pretreated GCPs with CDC, a selective inhibitor of LOs, including 12/15-LO (48). Although CDC alone did not alter control levels of phosphorylation (data not shown), the LO inhibitor attenuated thrombin-stimulated MARCKS phosphorylation significantly (p < 0.015; Figs. 2 and 3A). Conversely, incubation of GCPs with the product of 12/15-LO, 12(S)-HETE, increased phosphorylation of MARCKS significantly over that observed for untreated control (p < 0.0005; Figs. 2 and 3B). At concentrations of 10 nM and 1 µM, 12(S)-HETE increased MARCKS phosphorylation to a level about equal that of thrombin (p > 0.7; Figs. 2 and 3, A and B). Unlike the thrombin effect, however, 12(S)-HETE stimulation was inhibited only partially by CDC (Figs. 2 and 3B). The level of MARCKS phosphorylation achieved with 10 nM eicosanoid was higher than that obtained with 1 µM DAG (Figs. 2 and 3B; p < 0.02). As a positive control, 12-O-tetradecanoylphorbol-13-acetate (1 µM) strongly increased phospho-MARCKS levels in GCPs (Figs. 2 and 3C). The 12(S)-HETE precursor, AA, failed to stimulate MARCKS phosphorylation at 1 µM (data not shown) but increased phosphorylation appreciably at 50 µM (Figs. 2 and 3C). MARCKS phosphorylation was decreased to below control levels by the PKC-specific inhibitor, bisindolylmaleimide I (Bis; 10 nM), as expected, even with HETE stimulation (Figs. 2 and 3C) (49). These results suggest that 12(S)-HETE is a potent and selective activator of PKC and, thus, of MARCKS phosphorylation in growth cones.
Thrombin-induced Growth Cone CollapseWe have demonstrated
elsewhere that (a) thrombin induces growth cone collapse,
(b) thrombin increases the intracellular level of
12(S)-HETE, and (c) 12(S)-HETE is necessary and
sufficient for collapse (5,
6). Here we show that both
thrombin and 12(S)-HETE directly activate PKC (Figs.
2 and
3). Together these results
suggest that PKC
is a downstream mediator of thrombin-induced collapse.
The following experiments test this hypothesis by assessing whether PKC
activity is indeed necessary for collapse. As a measure of collapse we
quantified the areas covered by the same growth cones before and after
treatment (5 min). We compared the changes for growth cones treated with
thrombin (100 nM) alone to those first treated with the PKC
inhibitor Bis (500 nM) and then with thrombin. Although growth
cones treated with thrombin alone exhibited the expected, significant
reduction (p << 0.001) in area, those pretreated with Bis
showed little change in area in response to thrombin challenge
(Fig. 4, A and
B). If anything, there was a marginal increase
(p = 0.07) in growth cone spread. These results indicate that PKC
inhibition protects growth cones from thrombin-induced collapse and, thus,
implicate PKC as a necessary intermediate in thrombin signaling.
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PKC Isoforms Found in Adhesion SitesOur hypothesis predicts that a specific isoform of PKC present at adhesion sites is involved in MARCKS phosphorylation, activated by 12(S)-HETE, and necessary for collapse. Therefore, we determined which PKC isoforms are present in growth cone adhesion sites, operationally defined as the Triton X-100-insoluble fraction of adherent growth cones. Western blots prepared from this fraction as well as from the Triton X-100-soluble supernatant were probed with isoform-specific PKC antibodies.
All PKC isoforms could be detected in the growth cone fraction, but some
were present at very low levels only (data not shown). The isoforms abundant
in fetal brain homogenate were PKC ,
,
, and
and are
shown in Fig. 5. PKC
immunore-activity was prominent in all the fractions tested. Interestingly,
PKC
was abundant in brain homogenate but very sparse in the other
fractions. Because 12(S)-HETE stimulated PKC activity in the absence
of Ca2+, it was logical to suspect that one of the
Ca2+-independent PKC isoforms was involved. PKCs
and
were the only Ca2+-independent PKC isoforms detected at comparably
high levels in the Triton-insoluble fraction
(Fig. 5; TI).
PKC
, present in growth cones, could not be detected in the
Triton-insoluble fraction (data not shown). These results focused our further
analyses on PKC
and
.
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The co-purification of MARCKS and certain PKC isoforms in a
Triton-insoluble fraction of attached GCPs indicates their presence in
adhesion sites and/or their association with the cytoskeleton. To demonstrate
independently that PKC and MARCKS are present in the adhesive area of
the growth cone, we analyzed intact cultured growth cones by
immunofluorescence microscopy and generated optical slice images by digital
deconvolution (Fig. 6). For
reference, growth cones also were labeled with phalloidin to reveal F-actin
and with an antibody to paxillin, a known adhesion site protein. In growth
cones, plasmalemmal adhesive area is defined by a punctate pattern (rather
than plaques) of proteins, such as
1-integrin, paxillin,
talin, and focal adhesion kinase, at least on a laminin substratum
(6,
50). The optical sections
shown in Fig. 6 are immediately
adjacent to the growth substratum and, thus, reveal primarily
membrane-associated proteins. The distributions of MARCKS, PKC
, and
paxillin are co-extensive in this plane, filling the spread-out growth cones
with a punctate pattern. However, relative fluorescence intensities of
PKC
and MARCKS increase about 3-fold over the distal-most 2 µm of the
growth cone lamellipodia (data not shown). Overlap between PKC
and
MARCKS is frequent but not typical. There is very little overlap, however,
between MARCKS and paxillin. F-actin is abundant in growth cones, but its
radial bundles overlap with MARCKS, PKC
, and paxillin only at the distal
edge of the growth cone. Thus, very little MARCKS and PKC
is bound to
the actin cytoskeleton. Instead, these proteins are associated with the
adherent plasma membrane of the growth cone.
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Immunoprecipitated PKC Is Activated by
12(S)-HETEWe immunoprecipitated PKCs
and
from GCPs to
determine whether 12(S)-HETE activated the kinases separated from the
cellular context. The immunoprecipitates were washed extensively and then
tested for kinase activity in a lipid micellar assay with exogenous peptide
substrate (51,
52).
Fig. 7A shows a
Western blot of the parent GCP fraction, an immunoprecipitate control without
primary antibody, and the immunoprecipitate obtained with an antibody to
PKC
. Specific precipitation of PKC
is evident. The precipitates
were divided into equal aliquots and used for PKC assays.
Figs. 7, B and
C, are radioautograms that show the results obtained in
these kinase assays. No kinase activity was detectable in the
immunoprecipitate control (Fig.
7C). In contrast, the immunoprecipitate of PKC
exhibited a dramatic increase in peptide phosphorylation in the presence of
12(S)-HETE (Fig.
7B). We observed a biphasic 12(S)-HETE response
that peaked at 10-8 M 12(S)-HETE. The
stereoisomer 12(R)-HETE failed to stimulate the kinase, indicating
that the 12(S)-HETE effect is stereospecific. Compared with
12(R)-HETE, peptide phosphorylation was even lower in the presence of
Ca2+. Lack of kinase stimulation by Ca2+ was expected
for the Ca2+-independent PKC
. Under these assay conditions no
peptide phosphorylation was observed in the absence of PS/DAG micelles
(Fig. 7B, No
Lipid). Analogous experiments were carried out with immunoprecipitated
PKC
. However, PKC
was not stimulated by 12(S)-HETE (data
not shown).
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Activation of Recombinant PKC by
12(S)-HETETo test the hypothesis that 12(S)-HETE directly
and specifically activates certain isoforms of PKC, we performed kinase assays
with purified rPKC. These assays were done in either a PS/Triton X-100
micellar environment or in pure Triton micelles.
Fig. 8 shows the results of a
representative rPKC assay. For this assay we used rPKC
and, as a
substrate, histone H1 (open arrow). The autoradiogram shows that
10-6 M 12(S)-HETE activated the kinase above
control levels (this assay was done in a PS/Triton micellar environment). Note
the weak phosphorylation of a band of about 90 kDa, probably PKC
. The
presence of the intermediate phosphoprotein is substrate-dependent, but its
identity is not known.
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Experiments were performed to compare PKC activation by various
concentrations of eicosanoid and other reagents.
Fig. 9A is an
autoradiogram of a rPKC assay performed in Triton X-100 micelles lacking
PS/DAG except where indicated. 12(S)-HETE at 10-6 and
10-8 M stimulated rPKC
(>150% of control),
whereas the stereoisomer 12(R)-HETE, at identical levels, did not
(Figs. 9B and
10). Interestingly, the
rPKC
stimulation observed for 10-6 M
12(S)-HETE was much greater than that obtained with identical
concentrations of the known PKC activators, PS and DAG (Figs.
9 and
10). Only at much higher
concentrations and in combination did PS and DAG activate PKC
(Fig. 9A).
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|
rPKC was examined for comparison to determine whether PKC activation
by 12(S)-HETE was specific to PKC
(Fig. 9C). Indeed,
12(S)-HETE failed to activate this isoform. Most PKC isoforms,
including PKC
and
, are known to be maximally stimulated by high
concentrations of PS (123 µM) and DAG (33 µM)
applied in combination (53).
Our assays were no exception, as shown for rPKC
in
Fig. 9A and for
rPKC
in Fig.
9C.
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DISCUSSION |
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Activation of catalytically competent, conventional PKC isoforms involves the stimulation of phospholipase C, the subsequent generation of diacylglycerol, and an increase in cytosolic free Ca2+ (53). Intracellular Ca2+ levels clearly are important for growth cone motility (57, 58). However, repellent-triggered Ca2+ transients and Ca2+ dependence of growth cone collapse have been observed only for some collapsing factors but not for others (59, 60, 61, 62, 63). Ca2+ influx in fact may be more important for attraction than repulsion (64, 65). Although the situation remains unclear, it seems safe to state that Ca2+ transients are not a unifying step of repellent signaling. Neither are there data indicating a repellent-stimulated increase in DAG. Actually, we observed decreased, rather than increased DAG levels after treatment of growth cones with thrombin concentrations that induced rapid and complete growth cone collapse (5). In contrast, levels of AA and 12(S)-HETE were increased after thrombin treatment in the same experiments. These observations led us to test if 12(S)-HETE could activate PKC.
PKC Activity in Growth Cones and Their Adhesion Sites Thrombin stimulation of isolated growth cones increased the phosphorylation of a major polypeptide band at 87 kDa. This band corresponds to a well known PKC substrate (66), the adhesion site protein MARCKS, for the following reasons. (a) In previous experiments, two-dimensional gel analysis revealed a single spot at 87 kDa, with the pI (4.2) of MARCKS (37); (b) the 87-kDa phosphoprotein remained soluble in an acetic acid extraction (67)2; (c) Western blots of GCP proteins probed with an antibody to MARCKS indicated co-migration of the radiolabeled 87-kDa band with MARCKS; (d) immunoprecipitation with a MARCKS antibody specifically isolated the thrombin-stimulated phosphoprotein. These observations indicate that PKC and MARCKS phosphorylation may be involved in growth cone repellent action. The second major phosphoprotein observed at 43 kDa in our radioautograms almost certainly is GAP-43 (37, 68). Interestingly, thrombin and 12(S)-HETE did not stimulate, or potentially even inhibit its phosphorylation (see Figs. 1 and 2); also, treatment of GCPs with the selective PKC inhibitor, Bis, did not abolish its phosphorylation. The observations suggest the action of an insensitive isoform or of another kinase altogether. We reported earlier that the 43-kDa polypeptide may be a substrate of Ca2+/calmodulin-dependent kinase (37). This would be consistent with these findings because lipoxygenase products are potent inhibitors of Ca2+/calmodulin-dependent kinase II (69). Thrombin treatment of GCPs for 30 s increased MARCKS phosphorylation, which could be inhibited by the PKC blocker Bis to below control levels. Thrombin- but not 12(S)-HETE-induced PKC activation was attenuated by pretreating the GCPs with CDC, a selective inhibitor of 12/15-LO. Because the LO inhibitor can be used to block growth cone collapse by thrombin and Sema3A (5, 6), this result further suggests that PKC activation is involved in collapse. Indeed, we found that Bis inhibition of PKC blocks thrombin-induced collapse.
Previous reports suggest that AA may stimulate (or enhance DAG-stimulated) PKC activity (29, 31, 32, 70, 71, 72). Our findings do show increased MARCKS phosphorylation when GCPs are treated with exogenous AA. However, GCPs (as well as other systems analyzed and reported) contain enzymes for the conversion of AA into various eicosanoids. Therefore, these data do not establish direct AA stimulation of PKC. Actually, in GCPs AA is a much less effective activator of PKC than is 12(S)-HETE (Fig. 3) because stimulation of the kinase requires concentrations of 10-5 to 10-4 M AA as opposed to 10-8 to 10-6 M necessary for the eicosanoid. This led us to apply the 12/15-LO product, 12(S)-HETE, directly to GCPs to determine if it could activate PKC. Assays performed on permeabilized GCPs demonstrated a small but significant increase in kinase activity after 12(S)-HETE treatment. Because the assays on permeabilized GCPs were performed for 30 s only and worked in the absence of Ca2+, a direct mechanism of activation of a Ca2+-independent PKC isoform seemed likely.
In Western blots of isolated GCP adhesion site proteins we found
substantial amounts of PKC ,
, and
. Although PKC
was
abundant in growth cones, we were not able to detect it in the adhesion site
fraction. Therefore, the most interesting candidate isoforms for
12(S)-HETE activation were PKC
,
, and
. However,
we did not observe 12(S)-HETE stimulation of immunoprecipitated
PKC
, so that it was unlikely to be involved. Because 12(S)-HETE
activation of PKC is Ca2+-independent but PKC
is a
Ca2+-requiring isoform, we used PKC
for comparison only and
focused our analysis on PKC
, a novel, i.e.
Ca2+-independent, isoform.
Unlike the fibroblast, which forms focal adhesions or adhesion plaques, the
growth cone exhibits a broad belt of adherent spots along its distal rim, as
seen by reflection interference microscopy
(6). As shown here and
elsewhere, this distribution is mirrored by a punctate pattern of adhesion
site proteins, including paxillin, dispersed throughout the plasmalemma
adherent substrate contact area
(6,
50). It is evident in our
images that MARCKS and PKC puncta do not overlap with those of paxillin
but that the three proteins form coextensive patterns in the same optical
section (adhesive plane). This is not surprising in light of the fact that
there is co-distribution but relatively little overlap of
1-integrin and the adhesion site proteins paxillin, talin, or
vinculin in confocal images of growth cones
(50). PKC
and MARCKS
label are concentrated near the distal edge of lamellipodia, a closely
adherent region where a particularly large number of actin filaments are
anchored. Thus, MARCKS and PKC
are indeed associated with the growth
cone adhesive area, an observation that is consistent with earlier reports on
different biological systems
(19,
20). Interestingly, MARCKS and
PKC
are only minimally associated with the growth cone actin
cytoskeleton even though they are known to be able to bind to actin
(18,
25,
36,
73). It follows that PKC
is co-distributed with MARCKS and that it is a highly likely candidate kinase
responsible for MARCKS phosphorylation in growth cones. MARCKS phosphorylation
by PKC
has indeed been reported
(74,
75).
Eicosanoid activation of immunoprecipitated PKC, a preparation
depleted of most other GCP proteins, was much more pronounced than that
observed in GCPs. The increase in activation may be attributed to the
reduction of background PKC activity present in the permeabilized GCPs
(compare Fig. 2,
control, with Fig. 6, No Lipid). Although the maximally stimulating concentration of
12(S)-HETE was somewhat variable in the experiments with
immunoprecipitated PKC
, the dose response always appeared biphasic
(Fig. 6). Biphasic
12(S)-HETE activation of PKC
may be important functionally; it
has been described for a variety of cellular effects attributed to
12(S)-HETE stimulation
(76,
77). Interestingly, the
biphasic activation of the kinase was not observed with rPKC
. This
suggests that the regulation of PKC activity in the cell may involve another
protein. Higher levels of 12(S)-HETE could trigger an inhibitory
effect of an associated protein. As another possibility, at micromolar
concentrations 12(S)-HETE could activate a specific serine
phosphatase to counteract the kinase (phosphatase activation has been
associated with high levels of 12(S)-HETE in epidermoid carcinoma
cells) (34). Alternate
explanations are possible, however, because 12(S)-HETE has been shown
to stimulate other target molecules
(7,
77).
Direct Activation of PKCIt is well
established (for review, see Refs.
25 and
26) that conventional and
novel PKCs must be primed by three phosphorylations to become catalytically
competent. Only then is allosteric activation possible. The initial and
rate-limiting activation loop phosphorylation is catalyzed by
phosphoinositide-dependent kinase-1, and this appears to occur in eukaryotic
expression systems as well. Activation loop phosphorylation triggers
autophosphorylation of the two other sites and, thus, leads to catalytic
competency. These activation steps have been shown for a number of PKC
isoforms, including PKC
(78). Our own results
(Fig. 7) indicate
autophosphorylation of PKC
so that the enzyme is allosterically
activable in our assays. Indeed, stimulation of kinase activity with different
agonists is evident. Our in vitro assays with recombinant kinase and
exogenous substrate show that 12(S)-HETE can directly and
stereospecifically activate rPKC
. Furthermore, this activation appears
selective for PKC
, because PKC
and
were not
stimulated.
As mentioned, previous reports suggest that the effects of
12(S)-HETE on live cells were the result of receptor-mediated
stimulation of phospholipase C, the release of DAG and free Ca2+,
and subsequent kinase activation
(33,
34). Although such a complex
pathway may exist in some systems, it is unlikely to function in growth cones.
Instead, our results indicate that direct 12(S)-HETE stimulation of
PKC is possible and the likely mechanism operating in growth cones. This
activation was observed in the absence of other lipids and is more potent than
the stimulation by identical concentrations of the known PKC activators, PS
and DAG, or of AA. Our results also indicate that 12(S)-HETE can
activate PKC
in the absence of a membrane, at least in
vitro.
In summary, the present report demonstrates the direct stimulation of
PKC by 12(S)-HETE and, at least for the repellent thrombin, the
dependence of growth cone collapse on PKC activity. Our results suggest that
the role of PKC
activation in collapse may be to regulate growth cone
adhesion by phosphorylation of the adhesion site protein MARCKS.
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FOOTNOTES |
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Present address: Dept. of Molecular Medicine, University of Massachusetts
School of Medicine, Worcester, MA 01605.
Guest investigator from the Faculty of Medicine, Prince of Songkla
University, Thailand.
¶ To whom correspondence should be addressed: Dept. of Cellular and Structural Biology and University of Colorado Cancer Center, University of Colorado School of Medicine, 4200 East 9th Ave., Denver, CO 80262. Tel.: 303-315-4704; Fax: 303-315-4729; E-mail: karl.pfenninger{at}uchsc.edu.
1 The abbreviations used are: HETE, hydroxyeicosatetraenoic; AA, arachidonic
acid; 12/15-LO, 12/15-lipoxygenase; PKC, protein kinase C; rPKC, recombinant
PKC; PS, phosphatidylserine; DAG, diacylglycerol; MARCKS, myristoylated
alanine-rich protein kinase C substrate; CDC,
cinnamyl-3,4-dihydroxy--cyanocinnamate; LDH, lactate dehydrogenase;
TRAP, thrombin receptor-activating hexapeptide; GCP, growth cone particle;
TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino} ethanesulfonic acid;
PBS, phosphate-buffered saline; DRG, dorsal root ganglia; Bis,
bisindolylmaleimide I.
2 K. Mikule and K. H. Pfenninger, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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