©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence That the Bradykinin-induced Activation of Phospholipase D and of the Mitogen-activated Protein Kinase Cascade Involve Different Protein Kinase C Isoforms (*)

(Received for publication, September 8, 1994; and in revised form, December 7, 1994)

Katherine J. Clark Andrew W. Murray (§)

From the School of Biological Sciences, The Flinders University of South Australia, G. P. O. Box 2100, Adelaide, South Australia 5001

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The effect of alkylglycerol supplementation on protein kinase C (PKC)-mediated signaling events has been studied in fibroblasts from Zellweger patients (SF 3271 cells). Western blotting analysis established that Zellweger fibroblasts express PKC alpha, , and . Incubation with bradykinin induced a rapid transient translocation of PKC alpha and a more sustained translocation of PKC to the particulate fraction; translocation of PKC was unaffected. Bradykinin-induced translocation and activation of PKC alpha, but not translocation of PKC , was blocked in SF 3271 cells which had been incubated with 1-O-hexadecylglycerol (1-O-HDG; 20 µg/ml) for 24 h and then incubated in the absence of 1-O-HDG and serum for a further 24 h. Supplementation with 1-O-HDG increased the mass of ether-linked phospholipid. Bradykinin initiated a transient increase in cytosolic Ca concentration in both control and 1-O-HDG supplemented cells, indicating that the initial receptor linked events were not affected by 1-O-HDG supplementation. Bradykinin also caused a rapid activation of phospholipase D (PLD), measured by phosphatidylbutanol accumulation, and mitogen-activated protein kinase (MAPK) determined by myelin basic protein phosphorylation of Mono Q fractions. Both events were blocked by preincubation of the cells with 12-O-tetradecanoylphorbol-13-acetate for 24 h to deplete PKC protein. 1-O-HDG supplementation prevented the bradykinin-induced activation of PLD, but had no effect on the stimulation of MAPK activity. These results establish that modulation of the ether lipid composition of membranes can alter PKC isozyme translocation and indicate that a PKC isozyme other than PKC alpha, most likely PKC , is involved in MAPK activation.


INTRODUCTION

Protein kinase C (PKC) (^1)is a family of structurally related isozymes that has been shown to participate in the transduction of signals generated by hormones, neurotransmitters, and growth factors(1) . The most widely studied of these signals are diglyceride (DG) and Ca generated as a consequence of phosphatidylinositol 4,5-bisphosphate hydrolysis and DG derived from phosphatidylcholine (PC)(2) .

Molecular cloning has described multiple closely related PKC isozymes which have been divided into groups on the basis of biochemical and structural properties(3) . The classical PKC isozymes alpha, betaI, betaII, and are regulated by phosphatidylserine, DG, and Ca. The novel isozymes such as PKC , , , and lack the Ca binding domain C2 and are Ca-independent, and atypical forms such as PKC are independent of both Ca and DG. PKC isozymes exhibit distinct tissue distribution patterns, with multiple isozymes often found in a single cell type. Together with the different cofactor dependence and activator specificity, this suggests that PKC isotypes participate in distinct signal transduction pathways within the cell, and this has been supported by a number of recent studies.

For the most part these studies have demonstrated changes in signaling pathways following the overexpression of specific PKC isozymes(4, 5, 6, 7) or have used antisense technology to deplete the cells of individual PKC subspecies(8, 9, 10) . However there is very little information on the factors which may be important in regulating individual forms of PKC. The recent report by Ha and Exton(11) , which demonstrated differential translocation of PKC isozymes in response to thrombin in IIC9 fibroblasts, was the first to demonstrate the effect of endogenous factors on PKC translocation. The authors presented evidence that the rapid transient translocation of PKC alpha required both an increase of DG and cytosolic Ca as a result of phosphatidylinositol 4,5-bisphosphate hydrolysis. PKC translocation was rapid and sustained in response to thrombin but could be induced by increases in DG alone and was therefore independent of an increase in cytosolic Ca.

As part of a program to study the role of ether phospholipids in transmembrane signaling, we have used skin fibroblast cells isolated from patients with Zellweger syndrome. This syndrome is characterized by an absence of peroxisomes, organelles that contain the enzymes which catalyze the two initial steps in ether lipid biosynthesis. As a consequence tissues and cells from Zellweger patients have low levels of ether lipids(12) . However incubation of cells with alkylglycerol by-passes the enzyme deficiencies (13) and enables the accumulation of high levels of ether lipids. In the present study we have examined the effect of alkylglycerol supplementation on the translocation of PKC isozymes in bradykinin-stimulated skin fibroblasts and on two responses which can be regulated by PKC, activation of PLD and stimulation of the MAPK cascade. We show differential effects of supplementation on PKC translocation and on the two PKC-mediated responses.


EXPERIMENTAL PROCEDURES

Cell Culture

Human skin fibroblasts isolated from patients with Zellweger syndrome (SF 3271) were the kind gift of Dr. A. Poulos, Women's and Children's Hospital. SF 3271 were cultured in Eagle's modified essential medium (EMEM) with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin. The cells were maintained in 75-cm^3 flasks and were subcultured at 5-7-day intervals with a split ratio of 1:5.

Supplementation with Alkylglycerol

The culture medium was supplemented with 20 µg/ml 1-O-hexadecyl-sn-glycerol (1-O-HDG; Sigma) when near confluent and incubated for 24 h. The cells were washed with phosphate-buffered saline (PBS) and incubated for a further 24 h in EMEM before use. Supplementation did not affect cell number or viability assessed by dextran blue exclusion (data not shown).

Cell Treatment and Subcellular Fractionation

Serum-starved cultures were treated with agonist for the times indicated. After removal of the medium, cells were rinsed with ice-cold PBS, rinsed with sonication buffer (2 ml) containing 25 mM Tris, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 10 mM benzamidine, 10 mM PMSF, and 0.05% leupeptin and then scraped into sonication buffer (400 µl). Cells were then sonicated (Soniprobe, setting 5) for 10 s and the soluble and particulate fractions separated by centrifugation at 100,000 times g for 20 min (Beckman TLA 100.3 fixed angle rotor). The supernatants were collected, and the membrane pellets were suspended in sonication buffer containing 1% Triton X-100, sonicated for 10 s, incubated for 30 min, and then centrifuged at 100,000 times g for 20 min. The resulting supernatants were designated particulate fractions.

Immunoblotting

SDS-polyacrylamide gel electrophoresis was preformed according to the procedure of Laemmli (14) using 10% acrylamide (Mini-Protein II gel system, Bio-Rad). Following electrophoresis, gels were equilibrated for 15 min in transfer buffer (25 mM Tris, 152 mM glycine, 0.02% SDS, and 20% (v/v) methanol). Proteins were transferred onto 0.22-µm nitrocellulose membranes for 2 h at 100 V. The membranes were incubated with a blocking solution of 5% skim milk powder, 0.05% Tween 20 in 40 mM Tris, pH 7.4 for 1 h at room temperature and then with specific antisera for 1 h. Following incubation with alkaline phosphatase-linked anti-rabbit IgG, the blots were developed using enhanced chemiluminescence (Amersham Corp.).

The antisera against PKC alpha, beta, and were raised in this laboratory against the amino acid sequences 313-326, 313-329, and 306-318, respectively (15) and purified against the peptide. Anti-PKC , , and were purchased from Boehringer Mannheim. The MAPK antibody, raised against residues 333-367 of the C terminus of rat 43-kDa MAPK (erk I) which recognizes p42, p43, and p44 MAPK, was a gift from Dr. S. L. Pelech.

Partial Purification and Assay of Protein Kinase C

Confluent 15-cm plates of cells were treated for the times indicated with bradykinin (10 nM) by incubation in EMEM, 10 mM glucose, 1 mg/ml bovine serum albumin, and 10 nM bradykinin. The incubation was terminated by removing the medium and washing the cells with PBS at 4 °C. The cells were then scraped into 500 µl of sonication buffer (100 mM HEPES, pH 7.5, 2 mM EDTA, 5 mM EGTA, 25 mM sucrose, 10 mM dithiothreitol, 10 mM PMSF, 10 mM benzamidine), and sonicated for 10 s. The sonicate was centrifuged at 100,000 times g for 15 min at 4 °C. The particulate fraction was then sonicated as before in sonication buffer containing 0.2% Triton X-100 and allowed to stand for 1 h at 4 °C. PKC was partially purified from particulate extracts by batch elution from DEAE-Sephacel (Pharmacia). Briefly, particulate extracts were applied to DEAE-Sephacel (200 µl, bed volume) that had been equilibrated with column buffer (20 mM Tris, pH 7.4, 2 mM EDTA, 5 mM EGTA, 10 mM dithiothreitol, 10 mM PMSF). After centrifugation in an Eppendorf microcentrifuge (10 s), the unbound material was removed. The DEAE-Sephacel was washed (2 times 2 ml) with column buffer, and the PKC was eluted with column buffer containing 0.2 M NaCl (250 µl). PKC activity was then assayed as described (16) and the activity expressed as Ca/phosphatidylserine (PS)-dependent histone phosphorylation/min.

Determination of Cytosolic Free [Ca]

Cytosolic Ca was measured by a modification of the procedures of Grynkiewicz et al.(17) . Briefly, cells were trypsinized until removed from the surface of the plate, the trypsin was inhibited with trypsin inhibitor (0.2%), and the cells stored on ice for 2 h. The cells were incubated for 1 h at 37 °C with Fura-2AM (5 µM; Calbiochem). After loading, the cells were washed twice with Hanks' buffered salt solution containing 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 0.49 mM MgCl(2), 1.26 mM CaCl(2), 0.44 mM KH(2)PO(4), 3 mM NaHCO(3), and 5 mM glucose. The cells were then subjected to single wavelength spectrofluorometry using a LS 50 luminescence spectrometer and Fluorescence Data Manager software, with excitation wavelength of 340 nM and emission wavelength of 510 nM.

Phospholipid Analysis

Phospholipid extraction, two-dimensional TLC and mild alkaline and acid hydrolysis were carried out as described(18) . Briefly, phospholipids were extracted by the method of Bligh and Dyer (19) and either subjected to alkaline hydrolysis as total phospholipid or separated on aluminum-backed silica gel-60 plates (Merck) by a two-dimensional chromatographic solvent system. This consisted of chloroform:methanol:ammonia (65:25:5, v/v) in the first direction and chloroform:acetone:methanol:acetic acid:water (30:40:10:10:5, v/v) in the second direction. PC was then extracted from the silica with chloroform:methanol (2:1, v/v) and evaporated to dryness. Mild alkaline hydrolysis which releases diacyl-linked PC (incubation with 1 M NaOH at 37 °C for 15 min) and mild acid hydrolysis (incubation with 0.025 M HCl in 12.5 mM HgCl(2) at 37 °C for 15 min) which separates alkenyl- and alkyl-linked PC was then performed. Phospholipid mass analysis was performed by the method of Buss and Stull(20) .

Determination of PLD Activity in Intact Cells

Cells were labeled with [^3H]palmitic acid (1 µCi/ml) for 24 h in EMEM. Treatments, as indicated, were begun by changing medium to EMEM containing 10 mM glucose, 1 mg/ml bovine serum albumin, 0.5% butanol and terminated by the addition of methanol (2 ml) at 4 °C. The lipids were then extracted by the method of Bligh and Dyer (19) and PBut separated by TLC in a solvent containing: ethyl acetate:H(2)O:trimethylpentane:acetic acid 39:30:9:6 (v/v) and identified using an internal marker of unlabeled PBut. Radioactivity associated with PBut was determined by scintillation counting, and the results are expressed as a percentage of the total label incorporated into phospholipid.

Partial Purification and Assay of MAPK

Confluent plates (15 cm diameter) of cells were incubated with bradykinin, DiC(8), TPA, or solvent for the appropriate times. Incubations were stopped by washing with ice-cold PBS and cells scraped into 800 µl of sonication buffer (25 mM Tris-HCl, pH 7.4, 25 mM NaCl, 2 mM EGTA, 38 µM protein kinase A inhibitor (Sigma), 10 mM dithiothreitol, 10 mM PMSF). Samples were sonicated (4 °C) for 10 s and centrifuged at 100,000 times g for 10 min. The supernatant was collected and MAPK partially purified using phenyl-Sepharose 4B (Pharmacia) as described (21) . MAPK activity was assayed as described (18) using myelin basic protein as a substrate.

Mono Q Separation of MAPK Isoforms

Soluble extracts were prepared as described above, but with centrifugation at 100,000 times g for 30 min. The extracts were then applied to a Mono Q (HR5/5) column and eluted with a linear gradient (0-0.5 M NaCl) essentially as described(22) .


RESULTS

Zellweger fibroblasts are deficient in the ability to synthesize ether lipids. The level of ether lipids can be increased by supplementation with alkylglycerol which bypasses the enzyme deficiency (13) . To confirm that supplementation increased ether lipid levels, total phospholipid was separated into diacyl and alkenyl and alkyl subclasses and the mass determined by phosphate analysis. The mass of alkenyl and alkyl phospholipid increased from 45 ± 0.5 nmol/10^3 cells in unsupplemented cells to 65 ± 3 nmol/10^3 cells in cells incubated with 1-O-HDG (20 µg/ml) for 24 h followed by incubation for a further 24 h in the absence of 1-O-HDG. In separate experiments cells were labeled for 24 h with [^3H]choline and the PC separated into diacyl, alkenyl, and alkyl subclasses. The results (data not shown) show that incubation with 1-O-HDG (20 µg/ml) for 24 h followed by incubation for a further 24 h in the absence of 1-O-HDG, increased the radioactivity associated with alkyl-linked PC by approximately 10-fold. Similar results were obtained for alkyl-linked phosphatidylethanolamine when the cells were labeled with [^3H]ethanolamine (data not shown). However, in these experiments it is not known if equilibrium labeling has been reached in each of the separate phospholipid subclasses, and this may be reflected in the difference between the increase in mass and fold increase observed with the labeling data. These results confirm previous findings that supplementation of the tissue culture medium of fibroblasts with alkylglycerol allows its uptake and subsequent incorporation into ether lipids(23, 24) .

Western blotting of whole cell extracts established that Zellweger fibroblasts contained three PKC isozymes (alpha, , and ) and that PKC beta, , or were not present in detectable amounts. The immunoreactivity could be blocked by incubation with the peptide against which the antibody was raised (data not shown). PKC alpha and were located mainly in the cytosol fraction of unstimulated cells, whereas PKC appeared to be predominantly associated with the particulate fraction (data not shown).

Treatment of Zellweger fibroblasts with bradykinin induced a rapid translocation of PKC as detected by measurement of enzyme activity and Western blotting (Fig. 1). The enzyme assay conditions used were specific for Ca-dependent isozymes of PKC. Given that PKC alpha is the only Ca-dependent isozyme expressed by SF 3271, this form is therefore likely to be responsible for the enzyme activity shown in Fig. 1. Analysis of 0.2% Triton X-100 extracts of particulate material by Western blotting with a PKC alpha-specific antibody showed that PKC alpha associated with the membrane 30 s after bradykinin addition. In separate experiments PKC alpha associated with the membrane was shown to have returned to control levels within 2 min (data not shown). In Zellweger fibroblasts supplemented with 1-O-HDG, the translocation of PKC alpha and Ca/PS-dependent histone phosphorylation was dramatically reduced (Fig. 1). The reduction of PKC alpha translocation was not due to a loss of PKC alpha protein in supplemented cells, as total cell extracts of control and supplemented SF 3271 cells contained similar levels of PKC alpha protein determined by Western blotting (data not shown).


Figure 1: Translocation of PKC alpha induced by bradykinin. A, cells were incubated with either carrier for 24 h and then in the absence of serum for 24 h (bullet) or with 20 µg/ml 1-O-HDG for 24 h and then in the absence of 1-O-HDG and serum for a further 24 h (circle). Cells were then incubated with bradykinin (10 nM) for the times indicated. Particulate fractions (extracted with 0.2% Triton X-100) were prepared; and PKC partially purified by batch elution from DEAE-Sephacel and assayed for the Ca/PS-dependent phosphorylation of histone IIIS. B, cells were incubated either with carrier (designated by a -) or 1-O-HDG (designated by a +) as for A. Cells were then activated with bradykinin (10 nM) for the times indicated (times shown are in seconds) and particulate fractions analyzed by Western blotting with PKC alpha antibody. Migration of molecular mass markers (108.5 and 76 kDa) are indicated on the right side of the figure.



Translocation of PKC alpha was also observed in response to the synthetic DG analogue DiC(8) (5 µg/ml) (Fig. 2). Translocation was observed at 5 min and maintained at 20 min, and at both time points 1-O-HDG supplementation inhibited translocation.


Figure 2: Translocation of PKC alpha induced by DiC(8). Cells were incubated with either carrier for 24 h and then in the absence of serum for 24 h (designated by a -) or with 1-O-HDG (20 µg/ml) for 24 h and then in the absence of 1-O-HDG and serum for a further 24 h (designated by a +). Cells were then incubated with DiC(8) (5 µg/ml) for the times indicated (times shown are in minutes). Particulate fractions (extracted with 0.2% Triton X-100) were prepared and analyzed by Western blotting with PKC alpha antibody. Migration of molecular mass markers (108.5 and 76 kDa) are indicated on the right side of the figure.



Western blotting analysis was also used to examine the effect of bradykinin on the translocation of PKC and . In these experiments the extraction conditions were altered from those used to detect PKC alpha as only low levels of PKC was solubilized with 0.2% Triton X-100. However when the Triton X-100 concentration was increased to 1%, translocation of PKC was clearly visible (Fig. 3). PKC alpha translocation was still observed under these extraction conditions. In the time course shown in Fig. 3, PKC alpha translocation had returned to basal levels within 1 min, whereas PKC translocation, which was also rapid, was sustained for at least 5 min. Time points beyond 5 min were not examined. Supplementation had no effect on the translocation of PKC in response to bradykinin. PKC was associated with the particulate fraction at high levels in unstimulated cells, and no change was observed when cells were incubated with either bradykinin or TPA. Similarly, there was no change in the membrane association of PKC in cells supplemented with 1-O-HDG. The higher molecular weight band seen in the TPA-treated tracks corresponds to PKC alpha as the anti-PKC antibody used in these studies cross-reacts with PKC alpha, as has been reported earlier(11) .


Figure 3: Translocation of PKC isozymes alpha, , and induced by bradykinin. Cells were incubated with either carrier for 24 h and then in the absence of serum for 24 h (designated by a -) or with 1-O-HDG (20 µg/ml) for 24 h and then in the absence of 1-O-HDG and serum for a further 24 h (designated by a +). Cells were then incubated with bradykinin (10 nM) or TPA (100 nM) as indicated for the times shown (time is in minutes). Particulate fractions (extracted with 1% Triton X-100) were prepared and analyzed by Western blotting with antibodies specific for the PKC isozyme indicated. Migration of molecular mass markers (108.5 and 76 kDa) are indicated on the right side of the figure.



It is clear from the above results that supplementation with 1-O-HDG selectively blocked the bradykinin-induced translocation of PKC alpha, a Ca-dependent form of PKC. The effect of supplementation on the release of intracellular Ca in response to bradykinin was therefore measured (Fig. 4). Bradykinin caused a very rapid increase in intracellular Ca that returned to control levels within 1 min consistent with previous reports(25, 26) . A second addition of agonist 3 min after the first did not result in a further mobilization of Ca, indicating that the cells became refractory to the agonist. The bradykinin-induced increase in Ca concentration was unaffected by supplementation of cells with 1-O-HDG.


Figure 4: Changes of cytosolic Ca with bradykinin. Cells were incubated with either carrier for 24 h and then in the absence of serum for 24 h (A) or with 1-O-HDG (20 µg/ml) for 24 h and then in the absence of 1-O-HDG and serum for a further 24 h (B). Cells were then trypsinized, loaded with Fura-2AM, followed by the addition of solvent (1 min) or bradykinin (10 nM; 2 and 5 min). The additions are indicated by arrows. Cytosolic Ca measurements were carried out as described under ``Experimental Procedures.''



Phospholipid metabolism in bradykinin-stimulated human fibroblasts has been extensively studied(27, 28, 29) . We have confirmed some of these findings in Zellweger fibroblasts. Bradykinin caused a rapid activation of PLD as measured by PBut accumulation in the presence of butanol. This activation was not observed in cells pretreated with TPA for 24 h (Table 1), consistent with an involvement of PKC. The accumulation of PBut induced by bradykinin was maximal at 5 min (Table 2). In cells supplemented with 1-O-HDG bradykinin-stimulated PLD activation was strongly inhibited, and no PBut accumulation was observed for up to 15 min after bradykinin addition (Table 2). Increased concentrations (up to 1 µM) of bradykinin did not activate PLD in supplemented cells (data not shown). The inhibition observed was not due to a total loss of PLD activity in supplemented cells, as TPA was still able to activate PBut accumulation, although to a lesser extent than in control cells (Table 2). In these experiments, PLD activity was measured by prelabeling the phospholipid pool with [^3H]palmitic acid. It was therefore important to establish that the absence of bradykinin-stimulated [^3H]PBut in supplemented cells was not due to changes in the initial labeling pattern of cellular lipids. Consequently, in separate experiments, the incorporation of radioactivity into diacyl, alkenyl, and alkyl phospholipid subclasses after a 24-h incubation with [^3H]palmitic acid was determined. The counts incorporated into total phospholipid in unsupplemented cells (4296 ± 160 dpm/10^3 cells) were slightly higher than in supplemented cells (3708 ± 125 dpm/10^3 cells), and radioactivity associated with the alkenyl plus alkyl phospholipid pool increased from 5% of that incorporated into total phospholipid in unsupplemented cells to 8.4% in supplemented cells (data not shown). It is therefore clear that a large proportion of [^3H]palmitic acid is incorporated into diacyl phospholipid in both supplemented and unsupplemented cells. It was independently established that the lipids in supplemented cells are substrates for PLD as indicated by the accumulation of labeled PBut in the presence of TPA (Table 2). Consequently we do not believe that the inhibition of bradykinin-induced PBut formation after supplementation can be explained by alterations in phospholipid labeling by [^3H]palmitic acid.





We next examined the effects of supplementation on the bradykinin-induced activation of MAPK. In initial experiments it was shown that bradykinin-induced MAPK activity was blocked by TPA pretreatment for 24 h (Table 3), suggesting an involvement of PKC, whereas EGF-induced activation was unaffected. In these experiments MAPK activity (phosphorylation of myelin basic protein) was measured in soluble extracts, partially purified by phenyl-Sepharose chromatography. A time course of bradykinin-induced MAPK activity established a peak of activity at 1-2 min which returned to control levels by 15 min (data not shown). The isoforms of MAPK activated after 2 min incubation with bradykinin were examined using partial purification by Mono Q chromatography and immunoblotting (Fig. 5). Two peaks of activity were partially resolved on Mono Q. Using Western blotting it was established that the earlier eluting peak corresponded predominantly to p42 and the later peak to p44 protein (see inset in Fig. 5). Both isoforms were activated by bradykinin treatment. Bradykinin-induced activation of both isoforms was unaffected by 1-O-HDG supplementation. The time course of bradykinin induction of MAPK activity was also similar in control and supplemented cells (data not shown).




Figure 5: Activation of mitogen-activated protein kinase isoforms by bradykinin. Cells were incubated with either carrier for 24 h and then in the absence of serum for 24 h (bullet) or with 20 µg/ml 1-O-HDG for 24 h and then in the absence of 1-O-HDG and serum for a further 24 h (circle). Cells were then incubated with bradykinin (10 nM; bullet and circle) or solvent (+) for 2 min. Soluble fractions were prepared and subjected to Mono Q chromatography as described under ``Experimental Procedures.'' The column was eluted with a linear NaCl gradient and fractions analyzed for the ability to phosphorylate myelin basic protein. The inset shows a Western blot of the peak fractions of unsupplemented cells (designated by a -) and 1-O-HDG supplemented cells (designated by a +). The migration of molecular mass markers (49.5 and 32.5 kDa) is indicated on the right side of the figure.




DISCUSSION

This is the first report of PKC signaling related events in fibroblasts isolated from Zellweger patients. Three PKC isozymes (alpha, , and ) were identified in SF 3271 fibroblasts by Western blotting. Bradykinin invoked a rapid transient translocation of PKC alpha, which in timing and duration was closely correlated with an increase in intracellular Ca. PKC translocation was also rapid but sustained for at least 5 min after agonist addition and therefore does not correlate with the increase in intracellular Ca. The membrane association of PKC was not affected by either bradykinin or TPA. These results are consistent with the observations of Ha and Exton (11) who reported similar effects of alpha-thrombin on PKC translocation in IIC9 fibroblasts. The authors also reported that DiC(8) did not induce the translocation of PKC alpha unless cytosolic Ca was increased with ionomycin, whereas in our experiments, DiC(8) alone caused a clear translocation of PKC alpha (Fig. 2). The reason for the different results is likely to be the extraction conditions used. In the earlier work (11) the fraction extracting between 0.02 and 1% Triton X-100 was analyzed, whereas we extracted between 0 and 0.2 or 1% Triton X-100. In preliminary experiments we have shown that a 0.02% Triton X-100 extraction removed a substantial proportion of either DiC(8)- or bradykinin-induced translocation of PKC alpha, indicating that this subspecies is relatively loosely bound to the membrane (data not shown). The translocation of PKC was unaffected by 0.02% Triton X-100, suggesting a tighter membrane association.

The increase in ether phospholipid content caused by 1-O-HDG had a dramatic and unexpected effect on PKC isozyme translocation. 1-O-HDG supplementation inhibited PKC alpha translocation and PLD activation without altering Ca mobilization, PKC or translocation, or MAPK activation. It is therefore clear that modulation of the ether phospholipid content differentially affected PKC signaling events. Several mechanisms could account for the observed results. One possibility is the accumulation of a metabolite of 1-O-HDG which specifically inhibits the translocation of PKC alpha. It has, for example, been reported that alkyl-linked diglycerides inhibit PKC activity in vitro(30) , although possible differential effects on PKC subspecies has not explored. Alternatively, the incorporation of alkylglycerol into the phospholipid pool could cause a physical change in the membrane structure responsible for modulating its interaction with PKC alpha(31) . It will be of considerable interest to extend these studies to cells which express a wider range of PKC subspecies, to determine whether ether lipid supplementation modulates the activity of only the Ca-dependent forms of PKC and whether each of these forms is equally sensitive to supplementation.

Of particular interest is the observation that the differential effect of PKC isozyme translocation is also reflected in PKC-dependent downstream events. It is likely that the bradykinin-induced activation of PLD is mediated by PKC in SF 3271 cells as activation was lost in cells preincubated with TPA for 24 h. Western blotting experiments established that the incubation with TPA resulted in down-regulation of PKC alpha and PKC (data not shown). Although a possible effect of TPA pretreatment on constitutive PLD activity (32) has not been eliminated in these cells, a number of independent studies have implicated PKC in the activation of PLD(33, 34) . The differential effect of 1-O-HDG supplementation on bradykinin-induced PLD activation and translocation of PKC alpha therefore suggests a role for this isozyme in stimulating PLD activity. The results further indicate that PKC is not involved in PLD activation in SF 3271 cells. A number of other studies have linked PKC alpha and activation of PLD. For example, in Madin-Darby canine kidney cells depleted of PKC alpha using antisense technology, activation of PLD by TPA was inhibited(8) . Similarly studies of the regulation of PLD in membranes prepared from CCL39 fibroblasts demonstrated that addition of PKC alpha and PKC beta could activate PLD(35) , although these studies were carried out in the absence of exogenous ATP and assume activation via a phosphorylation independent mechanism. In separate studies we have shown that ceramide specifically inhibits bradykinin-induced activation of PKC alpha and PLD activity in cultured fibroblasts. (^2)There is therefore strong evidence from a number of approaches that PKC alpha is an important regulator of PLD activity. However there are some reports which provide conflicting data. For example in mesangial cells there is evidence that PKC and not PKC alpha can activate PLD. In addition in Swiss 3T3 fibroblasts it has been concluded that overexpression of PKC alpha did not influence the acute stimulation of PLD by TPA but increased the expression of PLD protein. Although we cannot explain the divergent results, it is possible that cell type differences exist resulting, for example, from the different expression patterns of PLD isozymes as suggested earlier(35) .

In contrast to its effect on PLD activation 1-O-HDG supplementation had no effect on the bradykinin-induced activation of MAPK. The MAPK cascade is rapidly activated in response to growth factors which bind tyrosine kinase receptors and agonists which lead to the activation of PKC(36) . The events leading to MAPK activation from tyrosine kinase receptors have been extensively studied and involve the activation of Ras and Raf proteins. A sequential activation of kinases leads to the activation of MAPK by phosphorylation on both tyrosine and threonine residues. Agonists which activate PKC appear to activate the MAPK cascade at the level of either Raf or Ras(37) . In SF 3271, pretreatment with TPA completely blocked bradykinin-induced MAPK activation but did not affect activation by EGF, indicating that the MAPK pathway was functional but that bradykinin requires the action of a PKC isozyme that is down-regulated by TPA. Given the lack of effect of 1-O-HDG supplementation on the translocation of PKC and MAPK activation, it is likely that PKC , and not PKC alpha, is responsible for MAPK activation in SF 3271 cells. However not all known forms of PKC were examined in this study, and we are currently using an antisense approach to deplete cells of the specific subspecies.

The assignment of specific PKC-dependent responses to particular isozymes is an exciting area of research. The possibility that endogenous membrane components control individual PKC isozyme translocation is intriguing. However in this study we have experimentally manipulated the levels of ether-linked phospholipids in a cell line which usually has a low level. It would be of interest to determine if similar effects on PKC signaling are observed in other cells, such as neutrophils(38) , which express naturally high levels of ether lipids.


FOOTNOTES

*
This work was supported by grants from the National Health and Medical Research Council and the Anti-cancer Foundation of the Universities of South Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 61-08-201-2090; Fax: 61-08-201-3015.

(^1)
The abbreviations used are: PKC, protein kinase C; 1-O-HDG, 1-O-hexadecylglycerol; PLD, phospholipase D; MAPK, mitogen-activated protein kinase; DG, diglyceride; PC, phosphatidylcholine; PS, phosphatidylserine; EMEM, Eagle's modified essential medium; PBS, phosphate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; Fura-2AM, pentaacetoxymethyl ester; TPA, 12-O-tetradecanoylphorbol-13-acetate; DiC(8), 1,2,-dioctanoyl-sn-glycerol; PBut, phosphatidylbutanol; EGF, epidermal growth factor.

(^2)
M. J. Jones and A. W. Murray, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Alf Poulos for the provision of the cell line SF 3271 and helpful discussion, Dr. S. L. Pelech for the antibody to MAPK, and Dr. Charles Hii for helpful discussion.


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