Eicosanoid Activation of Protein Kinase C {epsilon}

INVOLVEMENT IN GROWTH CONE REPELLENT SIGNALING*

Keith Mikule {ddagger}, Somkiat Sunpaweravong §, Jesse C. Gatlin and Karl H. Pfenninger 

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.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of growing neurons to thrombin or semaphorin 3A stimulates a receptor-mediated signaling cascade that results in collapse of their growth cones. This collapse response necessitates eicosanoid production, as we have shown earlier. The present report investigates whether and which protein kinase C (PKC) isoforms may be activated by such eicosanoids. To examine these questions, we isolated growth cones from fetal rat brain and tested whether thrombin or the eicosanoid, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), could activate endogenous growth cone PKC. We show that both thrombin and 12(S)-HETE stimulate the phosphorylation of the myristoylated alanine-rich protein kinase C substrate, an 87-kDa adhesion site protein. Furthermore, we show both with immunoprecipitated and with recombinant PKC that 12(S)-HETE activation is selective for the {epsilon} isoform and does not require accessory proteins. Last, we demonstrate that PKC activation is necessary for thrombin-induced growth cone collapse. These data indicate that eicosanoid-mediated repellent effects result from the direct and selective activation of PKC{epsilon} and suggest the involvement of myristoylated alanine-rich protein kinase C substrate phosphorylation in growth cone collapse.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The nerve growth cone is the terminal enlargement of the growing neurite, specialized for neurite assembly, advancement, and pathfinding. Growth cones guide neurites to predetermined targets by either turning toward or away from attractive or repulsive pathfinding cues, respectively (1, 2). Growth cone repulsion is the result not only of cytoskeletal rearrangements but also of detachment from the growth substratum (3, 4, 5, 6), suggesting that adhesion sites may be targets of the signaling mechanism involved. Results from our laboratory describe elements of a repellent-activated signaling pathway that causes growth cone detachment and collapse (5, 6). Stimulation of this pathway by thrombin or semaphorin 3A results in the synthesis of 12(S)- and 15(S)-hydroxyeicosatetraenoic acid (HETE)1 from arachidonic acid (AA). This is catalyzed by leukocyte 12/15-lipoxygenase (12/15-LO) (7). 12(S)-HETE is necessary and sufficient for collapse of cortical or dorsal-root-ganglion growth cones as well as for the retraction of cancer cell pseudopods (5, 6, 8). A question of significant interest concerns the mechanism by which 12(S)-HETE causes detachment and collapse.

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 {epsilon} isoform in particular has been implicated in phenomena related to neurite outgrowth (13, 14, 15, 16). PKC{epsilon} 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{epsilon}. 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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials–AA was purchased from Sigma and l-stearoyl-2-arachidonyl-sn-glycerol (DAG), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (PS) from Avanti-Polar Lipids. 12(S)-HETE, 12(R)-HETE, and cinnamyl-3,4-dihydroxy-{alpha}-cyanocinnamate (CDC) were purchased from Biomol. 5(R)-HETE was from Cayman Chemical Co. Horseradish-conjugated goat anti-rabbit antibody was obtained from Vector Laboratories. PKC isoform-specific antibodies were purchased from Transduction Laboratories or Santa Cruz. Anti-PKC{epsilon} used for immunofluorescence was a gift from Dr. Rytis Prekeris, University of Colorado. Lactate dehydrogenase (LDH) antibody was from Abcam. Recombinant PKC (rPKC) isoforms were purchased from Panvera. Thrombin receptor-activating hexapeptide (TRAP) came from Peninsula Laboratories. Polyacrylamide, prestained molecular weight standards, and other reagents for SDS/PAGE were from Invitrogen. Tissue culture dishes were purchased from Fisher. Reagents for chemiluminescence were from PerkinElmer Life Sciences. Aprotinin, PKC{epsilon}- optimized peptide substrate, and histone H1 were from Calbiochem. Other chemicals, unless stated otherwise, were from Sigma and of the highest quality obtainable. Sprague-Dawley rats were from Harlan.

Growth Cone Isolation–Growth 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 Assay–These 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 {gamma}-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 Assay–Pelleted 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 {alpha} or {epsilon}) 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 [{gamma}-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 Assay–rPKC 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 [{gamma}-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 Isolation–Petri 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 Blotting–Protein 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{alpha} at 1:500, anti-PKC{gamma} at 1:100, anti-PKC{tau} at 1:1000, and anti-PKC{epsilon} 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 Culture–Dorsal 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 Assays–DRG 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 Culture–Fixation 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{epsilon}, 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{epsilon} 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 Microscopy–Images 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 0.2–0.3 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The biochemical studies reported here necessitated a preparation enriched in nerve growth cones. We used GCPs isolated by subcellular fractionation from fetal rat forebrain, a preparation highly enriched in re-sealed, primarily axonal growth cones (42, 43). These GCPs are highly responsive to thrombin (5).

MARCKS Phosphorylation in Growth Cones–Thrombin-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 {gamma}-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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FIG. 1.
Protein phosphorylation in growth cones and presence of MARCKS. A, phosphorimage of PKC assays with permeabilized GCPs (incubation 30 s at 30 °C). Lanes contain total GCP proteins (60 µg in each lane) from control and thrombin-treated (100 nM) samples, respectively. The closed arrow points at a prominent, 87-kDa protein whose phosphorylation is increased by thrombin (for quantitative analysis, see Figs. 2 and 3A). The lower phosphorylated band, at about 43 kDa, co-migrates with GAP-43. Western blots below the radioautograms (open arrow) show the cytoplasmic marker, LDH, as a loading control of the same gel lanes. B, in parallel experiments, we immunoprecipitated (IP) MARCKS after the phosphorylation reaction (30 s). The two pairs of lanes show supernatant (left) and precipitate (right) obtained with the MARCKS antibody or control antibody. Western blots (bottom, open arrow) show the presence of LDH. LDH remained in the supernatants. Results indicate immunoprecipitation of the radiolabeled 87-kDa phosphoprotein by anti-MARCKS but not by control antibody. C, Western blot of subcellular fractions from fetal brain probed with an antibody to MARCKS. LSS, low speed supernatant. TS and TI are Triton-soluble and Triton-insoluble fractions, respectively, of GCPs attached to laminin.

 


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FIG. 2.
Phosphorimaging results on MARCKS phosphorylation in GCPs. Permeabilized GCPs (60 µg of protein/lane) were incubated with either thrombin or the indicated effectors and with [32P]ATP for 30 s at 30 °C in the absence or presence of inhibitors and in the absence of Ca2+ and exogenous lipid. A representative experiment is shown. The closed arrow points at MARCKS (87 kDa). The lower phosphorylated band, at about 43 kDa, co-migrates with GAP-43. The open arrow indicates Western blots of the same lanes probed with anti-LDH as a loading control. Densitometrically, LDH loading of each lane was within ± 7.5% (S.D.) and, thus, very consistent. Concentrations: AA, 50 µM; thrombin (Thr), 100 nM; phorbol ester (TPA), 1.6 µM; DAG, 1 µM; 12(S)-HETE, 10-8 M. Inhibitors (pretreatment at 4 °C for 10 min) were CDC, a LO inhibitor (1 µM), and Bis, a PKC inhibitor (10 nM). For numerical data, see Fig. 3.

 


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FIG. 3.
Quantitation of MARCKS phosphorylation assays such as those shown in Fig. 2. Phosphorylation levels determined by phosphorimaging were normalized to controls run on the same gel and averaged. Values are the means ± S.E. A, thrombin (Thr) and the TRAP stimulate MARCKS phosphorylation, and this effect was blocked by the LO inhibitor, CDC. TRAP was used at 100 µM. For other concentrations, see Fig. 2. B, comparison of the effects of DAG and 12(S)-HETE at 10-6 M and of 10-8 M 12(S)-HETE with or without LO inhibitor (CDC) on MARCKS phosphorylation. C, 50 µM AA stimulates MARCKS phosphorylation as does 12-O-tetradecanoylphorbol-13-acetate (TPA) at 10-6 M. The PKC inhibitor Bis strongly reduces MARCKS phosphorylation with or without 12(S)-HETE (10-8 M) challenge. Results are the means ± S.E. for four independent experiments, except for TRAP, CDC/TRAP, and CDC/HETE, which are from single experiments. Note the different scales in A and B versus C. The data indicate that thrombin activation of MARCKS phosphorylation requires LO and that the LO product, 12(S)-HETE, is a PKC activator.

 

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 Collapse–We 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{epsilon} (Figs. 2 and 3). Together these results suggest that PKC{epsilon} 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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FIG. 4.
The effect of PKC inhibition on thrombin-induced growth cone collapse. Growth cones of DRG neurons were challenged with 100 nM thrombin, with (A, bottom row) or without (A, top row) pretreatment with the PKC inhibitor Bis (500 nM). A, representative phase contrast micrographs of growth cones taken at 1-min intervals. B, quantitative analysis. Growth cone areas were measured before and after 5 min of thrombin treatment in both Bis-pretreated and thrombin-only DRG cultures. Results are expressed as the mean change in growth cone area ± S.E. (n > 20). The scale bar in A represents 20 µm.

 

PKC Isoforms Found in Adhesion Sites–Our 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 {alpha}, {gamma}, {tau}, and {epsilon} and are shown in Fig. 5. PKC{alpha} immunore-activity was prominent in all the fractions tested. Interestingly, PKC{gamma} 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 {epsilon} and {tau} were the only Ca2+-independent PKC isoforms detected at comparably high levels in the Triton-insoluble fraction (Fig. 5; TI). PKC{delta}, present in growth cones, could not be detected in the Triton-insoluble fraction (data not shown). These results focused our further analyses on PKC {epsilon} and {tau}.



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FIG. 5.
Western blots of fetal brain fractions probed with PKC isoform-specific antibodies. Equal protein amounts (10 µg) were analyzed throughout. BH, brain homogenate; LSS, low speed supernatant, the parent fraction of GCPs; GC, GCPs; TS, Triton X-100-soluble fraction; TI, Triton X-100-insoluble fraction obtained from laminin-attached GCPs. Note that PKC {alpha}, {epsilon}, and {tau}, but not {gamma}, are prominently present in the Triton X-100-insoluble GCP fraction.

 

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{epsilon} 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 {beta}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{epsilon}, and paxillin are co-extensive in this plane, filling the spread-out growth cones with a punctate pattern. However, relative fluorescence intensities of PKC{epsilon} and MARCKS increase about 3-fold over the distal-most 2 µm of the growth cone lamellipodia (data not shown). Overlap between PKC{epsilon} 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{epsilon}, and paxillin only at the distal edge of the growth cone. Thus, very little MARCKS and PKC{epsilon} is bound to the actin cytoskeleton. Instead, these proteins are associated with the adherent plasma membrane of the growth cone.



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FIG. 6.
Immunofluorescence micrographs of DRG growth cones in culture. Images are optical sections including the adherent plasma membrane and were obtained by digital deconvolution. Top row, growth cone double-labeled with anti-MARCKS and anti-PKC{epsilon}. The panel on the right shows the merger of the two single-channel images (overlap appears yellow). Bottom row, from left to right, double-label images of PKC{epsilon} (PKCeps) and F-actin (labeled with Texas Red-conjugated phalloidin), MARCKS and F-actin (labeled with fluorescein isothiocyanate-conjugated phalloidin), and MARCKS and paxillin. For further explanation, see "Results." Calibration, 10 µm.

 

Immunoprecipitated PKC{epsilon} Is Activated by 12(S)-HETE–We immunoprecipitated PKCs {epsilon} and {tau} 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{epsilon}. Specific precipitation of PKC{epsilon} 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{epsilon} 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{epsilon}. 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{tau}. However, PKC{tau} was not stimulated by 12(S)-HETE (data not shown).



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FIG. 7.
12(S)-HETE activates immunoprecipitated PKC{epsilon} A, GCPs were solubilized in lysis buffer and incubated with control or with anti-PKC{epsilon} antibody for immunoprecipitation. Western blots of the lysate and the precipitates (IP) derived from it were probed with antibody to PKC{epsilon}. The selective precipitation of PKC{epsilon} (arrow) is evident. B, representative autoradiogram of PKC assays performed with equal aliquots of immunoprecipitated PKC{epsilon} using PKC{epsilon}-optimized peptide substrate (arrow). Kinase assays were performed in the presence of Triton X-100 micelles containing PS (123 µM) and DAG (33 µM) for 5 min at 30 °C in Ca2+-free conditions. 12(S)-HETE, but not 12(R)-HETE, stimulates PKC{epsilon} strongly and in a biphasic fashion. C, autoradiogram of a phosphorylation experiment carried out with the control precipitate. Experimental conditions were identical to those in B, except that control antibody was used for precipitation.

 

Activation of Recombinant PKC{epsilon} by 12(S)-HETE–To 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{epsilon} 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{epsilon}. The presence of the intermediate phosphoprotein is substrate-dependent, but its identity is not known.



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FIG. 8.
12(S)-HETE activation of recombinant PKC{epsilon} rPKC{epsilon} assay was performed in the presence of PS-containing lipid micelles (123 µM) for 5 min at 30 °C using histone as a substrate (open arrow) in the absence of Ca2+. Note that 10-6 M 12(S)-HETE stimulated the phosphorylation of the histone substrate over controls (no 12(S)-HETE). Other controls included the omission of rPKC{epsilon}, the omission of substrate, and the omission of Triton X-100 lipid micelles. The closed arrow points at a band that most likely is the kinase. The identity of the intermediate band is unknown.

 

Experiments were performed to compare PKC activation by various concentrations of eicosanoid and other reagents. Fig. 9A is an autoradiogram of a rPKC{epsilon} 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{epsilon} (>150% of control), whereas the stereoisomer 12(R)-HETE, at identical levels, did not (Figs. 9B and 10). Interestingly, the rPKC{epsilon} 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{epsilon} (Fig. 9A).



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FIG. 9.
Stereospecificity and isoform selectivity of PKC activation by 12(S)-HETE. rPKC assays were performed in the absence of PS/DAG (except where indicated) for 5 min at 30 °C using histone as a substrate (arrow). A, stimulation of rPKC{epsilon} with the indicated effectors. At identical concentrations (1 µM) 12(S)-HETE is a more potent activator than PS or DAG. B, rPKC{epsilon} assay with12(R)-HETE at the indicated concentrations. 12(R)-HETE did not stimulate the kinase. C, rPKC{alpha} assay. The kinase was incubated with 12(S)-HETE at the indicated concentrations in the presence of 1 mM Ca2+; PS/DAG (123 µM/33 µM) served as a positive control. 12(S)-HETE was unable to stimulate rPKC{alpha}. For all experiments, controls included all assay components except effector. The closed arrow indicates histone H1 substrate.

 


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FIG. 10.
Quantitation of rPKC{epsilon} assays; densitometric analysis of radioautograms as shown in Fig. 9. All effectors were used at 1 µM.12(S)-HETE increased PKC activity by 150% above non-stimulated control conditions and was the stronger effector than PS or DAG. Results are the means of three independent experiments ±S.E. For reference, maximal stimulation by the combination of PS/DAG (at 123 and 33 µM, respectively) increased histone phosphorylation about 5-fold in the same experiment (not shown).

 

rPKC{alpha} was examined for comparison to determine whether PKC activation by 12(S)-HETE was specific to PKC{epsilon} (Fig. 9C). Indeed, 12(S)-HETE failed to activate this isoform. Most PKC isoforms, including PKC {alpha} and {epsilon}, 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{epsilon} in Fig. 9A and for rPKC{alpha} in Fig. 9C.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Our previous work described elements of a repellent-activated signaling pathway that dissociates adhesion sites in metastatic cancer cells and neuronal growth cones (5, 6, 8). Those studies identified 12(S)-HETE as necessary and sufficient for growth cone collapse and raised the question of subsequent steps that trigger growth cone detachment. PKC is a candidate target of 12(S)-HETE because of its known regulation by lipophilic agents and because its activity has been implicated in neurite outgrowth and amoeboid motility (10, 12, 15, 54, 55, 56).

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 {alpha}, {tau}, and {epsilon}. Although PKC{delta} 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 {alpha}, {epsilon}, and {tau}. However, we did not observe 12(S)-HETE stimulation of immunoprecipitated PKC{tau}, so that it was unlikely to be involved. Because 12(S)-HETE activation of PKC is Ca2+-independent but PKC{alpha} is a Ca2+-requiring isoform, we used PKC{alpha} for comparison only and focused our analysis on PKC{epsilon}, 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{epsilon} 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 {beta}1-integrin and the adhesion site proteins paxillin, talin, or vinculin in confocal images of growth cones (50). PKC{epsilon} 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{epsilon} 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{epsilon} 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{epsilon} 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{epsilon} has indeed been reported (74, 75).

Eicosanoid activation of immunoprecipitated PKC{epsilon}, 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{epsilon}, the dose response always appeared biphasic (Fig. 6). Biphasic 12(S)-HETE activation of PKC{epsilon} 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{epsilon}. 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 PKC{epsilon}It 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{epsilon} (78). Our own results (Fig. 7) indicate autophosphorylation of PKC{epsilon} 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{epsilon}. Furthermore, this activation appears selective for PKC{epsilon}, because PKC {tau} and {alpha} 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{epsilon} 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{epsilon} in the absence of a membrane, at least in vitro.

In summary, the present report demonstrates the direct stimulation of PKC{epsilon} 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{epsilon} activation in collapse may be to regulate growth cone adhesion by phosphorylation of the adhesion site protein MARCKS.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grants 1R01 NS41029 and 5F31 NS44705. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Dept. of Molecular Medicine, University of Massachusetts School of Medicine, Worcester, MA 01605. Back

§ Guest investigator from the Faculty of Medicine, Prince of Songkla University, Thailand. Back

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-{alpha}-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. Back

2 K. Mikule and K. H. Pfenninger, unpublished observations. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Sven Pahlman and his former student, Dr. Sofia Fagerstrom of Lund University, Sweden, for generous help and advice regarding the identification of growth cone PKC isoforms. Further help was provided by B. A. de la Houssaye and Melissa Franco and is herewith acknowledged gratefully.



    REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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