©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of Phospholipase D by Epidermal Growth Factor Requires Protein Kinase C Activation in Swiss 3T3 Cells (*)

(Received for publication, September 29, 1994; and in revised form, December 9, 1994)

Eui-Ju Yeo John H. Exton (§)

From the Howard Hughes Medical Institute and the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The proposal that epidermal growth factor (EGF) activates phospholipase D (PLD) by a mechanism(s) not involving phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) hydrolysis was examined in Swiss 3T3 fibroblasts. EGF, basic fibroblast growth factor (bFGF), bombesin, and platelet-derived growth factor (PDGF) activated PLD as measured by transphosphatidylation of butanol to phosphatidylbutanol. The increase in inositol phosphates induced by bFGF, EGF, or bombesin was significantly enhanced by Ro-31-8220, an inhibitor of protein kinase C (PKC), suggesting that PtdIns(4,5)P(2)-hydrolyzing phospholipase is coupled to the receptors for these agonists but that the response is down-regulated by PKC. Activation of PLD by EGF was inhibited dose dependently by the PKC inhibitors bis-indolylmaleimide and Ro-31-8220, which also inhibited the effects of bFGF, bombesin, and PDGF. Down-regulation of PKC by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate also abolished EGF- and PDGF-stimulated phosphatidylbutanol formation. EGF and bombesin induced biphasic translocations of PKC and to the membrane that were detectable at 15 s. In the presence of Ro-31-8220, translocation of PKCalpha became evident, and membrane association of the - and -isozymes was enhanced and/or sustained in response to the two agonists. The inhibitor also enhanced EGF-stimulated [^3H]diacylglycerol formation in cells preincubated with [^3H]arachidonic acid, which labeled predominantly phosphatidylinositol, but inhibited [^3H]diacyl-glycerol production in cells preincubated with [^3H]myristic acid, which labeled mainly phosphatidylcholine. These data support the conclusion that EGF can stimulate diacylglycerol formation from PtdIns(4,5)P(2) and that PKC performs the dual role of down-regulating this response as well as mediating phosphatidylcholine hydrolysis. In summary, all of the results of the study indicate that PLD activation by EGF is downstream of PtdIns(4,5)P(2)-hydrolyzing phospholipase and is dependent upon subsequent PKC activation.


INTRODUCTION

Epidermal growth factor (EGF) (^1)stimulates a number of cellular responses following binding to its specific cell surface receptor and activation of the intrinsic tyrosine kinase(1) . Much evidence has been gathered on the EGF receptor signaling system using the A431 cells. PI-PLC-1 is one of the proteins that is activated by EGF through interaction of the enzyme with autophosphorylation sites on the cytoplasmic tail of the receptor (2, 3, 4) . The activated PLC catalyzes the hydrolysis of PtdIns(4,5)P(2) to generate Ins(1,4,5)P(3) and 1,2-diacylglycerol (DAG), which elevates cytosolic Ca and activates protein kinase C (PKC), respectively.

Recent evidence shows that many agonists, including EGF, also stimulate DAG production through the hydrolysis of PtdCho and that phospholipase D (PLD) is the major enzyme involved(5) . PLD initially produces PtdOH, which may have second messenger roles, but can also be rapidly converted by PtdOH phosphohydrolase to DAG(6) . Most agonists induce a biphasic production of DAG, with PI-PLC being responsible for the initial rapid increase and PtdCho-hydrolyzing phospholipase D (PC-PLD) for the second sustained increase.

Although agonist-induced activation of PLD is a widely occurring phenomenon, the regulatory mechanisms involved are not well defined. Evidence for the involvement of PKC in the regulation of PLD has been obtained in many different cell systems with various agonists. Addition of the phorbol ester, PMA, increases PLD activity in many cells, and treatment of cells with PKC inhibitors or down-regulation of the enzyme abolishes PLD activation by PMA and most agonists(5, 7) . Overexpression of PKCs in fibroblasts (8, 9, 10) also enhances the PLD response. Similarly, when cells are depleted of PKC by antisense methods, PLD activation is inhibited(11) .

Based on the evidence that PKC plays a major role in the regulation of PLD, it has been proposed that DAG produced by initial PtdIns(4,5)P(2) breakdown activates PKCs, which then directly or indirectly activate PLD(12, 13, 14, 15, 16) . However, evidence for this sequential mechanism is limited, and there have been several reports presenting contrary findings. For example, certain agonists in some cell types do not induce detectable increases in InsPs, while they elicit a sustained DAG increase originating from PtdCho(17, 18, 19, 20, 21) . There have also been contradictory results concerning the effect of EGF on PtdIns(4,5)P(2) metabolism. This growth factor induces PtdIns(4,5)P(2) hydrolysis in hepatocytes and A431 cells (22) but produces no detectable increase in InsPs in fibroblast cell lines, although it stimulates mitogenesis(18, 23, 24, 25) . The apparent activation of PLD in the absence of PtdIns(4,5)P(2) hydrolysis suggests the existence of a PKC-independent pathway (19) .

In the present report, we have studied the mechanisms of activation of PC-PLD by EGF in Swiss 3T3 cells. In contrast to the report of Cook and Wakelam(19) , we observed a small but reproducible increase in InsP formation in EGF-stimulated cells, which was enhanced by a PKC inhibitor. In addition, we provide evidence for the involvement of PKC in the activation of PLD by EGF.


EXPERIMENTAL PROCEDURES

Materials

Swiss 3T3 fibroblasts were obtained from American Type Culture Collection (CCL92). Hepes-buffered Dulbecco's modified Eagle's medium (with L-glutamine), penicillin, streptomycin, and polyclonal antibodies recognizing PKCalpha, -, and - were purchased from Life Technologies, Inc. Anti-PKC antibody was from Research and Diagnostic Antibodies. Monoclonal anti-phosphotyrosine antibody (PY-20) was from ICN. Partially purified recombinant PKC isoforms expressed in Sf9 cells were obtained from Sphinx Pharmaceutical Corp. (Durham, NC). Fetal bovine serum, bovine serum albumin (both RIA grade, fraction V, and sterilized 35% solution for cell culture), myelin basic protein (MBP), and 4beta-phorbol 12-myristate 13-acetate (PMA) were from Sigma. PDGF B/B (human recombinant) was from Boehringer Mannheim, and EGF (mouse, receptor grade) was from Upstate Biotechnology Inc. [5,6,8,9,11,12,14,15-^3H]Arachidonic acid (180 Ci/nmol), [9,10-^3H]myristic acid (16 Ci/mmol), EN^3HANCE spray, and [-P]ATP (6000 Ci/mmol) were purchased from DuPont NEN. Ro-31-8220 was generously given by Roche Products Ltd. (Welwyn Garden City, Hertfordshire, United Kingdom), and bis-indolylmaleimide (bIM), also known as GF 109203X, and basic fibroblast growth factor (bFGF) were from CalBiochem. Leupeptin was from Transformation Research Inc. Biotinylated anti-rabbit IgG and Vectastain alkaline phosphatase ABC kit were from Vector Laboratories. ECL detection kit and myo-[2-^3H]inositol (17 Ci/mmol) were from Amersham Corp. Immobilon-P was from Millipore. Bicinchoninic acid protein assay reagents were from Pierce. Silica gel 60 A plates were from Whatman. DAG, PtdOH, phosphatidylserine, and 1,2-dioleoyl-sn-glycero-3-phosphobutanol (PtdBut) were from Avanti (Alabaster, AL).

Cell Cultures

Swiss 3T3 cells were cultured in Hepes-buffered DMEM with 4 mML-glutamine supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units of penicillin/ml, and 100 µg of streptomycin/ml at 37 °C in a humidified, CO(2)-controlled (5%) incubator. For all experiments, cells were grown on a 10-cm dish for 2 days to subconfluency (70-80%). Medium was then replaced with low serum medium (DMEM containing 1% FBS, 0.5% bovine serum albumin (BSA), and antibiotics) for 2 days to allow cells to become quiescent.

Measurement of Total Inositol Phosphates

Cells were serum-starved and labeled with 0.5 µCi/ml of myo-[^3H]inositol overnight in inositol-free DMEM containing 1% FBS, 0.5% BSA, and antibiotics. The cells were then washed three times with the same medium lacking FBS and preincubated in this medium for 1 h at 37 °C prior to incubation with agonists. During the last 10 min of preincubation, 20 mM LiCl was included. Cells were then treated with agonists for 15 min, and the incubations were terminated by removal of medium and washing with ice-cold phosphate-buffered saline. The cells were lysed by a modification of the procedure of Conklin et al.(26) , namely treatment with 3 ml of ice-cold 20 mM formic acid for 30 min on ice. The cells were scraped off and spun in an Eppendorf centrifuge for 10 min. Part of the supernatant (2 ml) was neutralized with 1 ml of 50 mM NH(4)OH and loaded onto a 1-ml column of Bio-Rad AG 1-X8 (formate form, 200-400 mesh). Total inositol phosphates were eluted from the column, according to the procedure of Simpson et al.(27) . The column was washed with 3 times 3 ml of distilled water (free inositol fraction) and then washed with 3 times 3 ml of 60 mM ammonium formate, 5 mM sodium tetraborate (glycerophosphoinositol fraction). Finally, one column was eluted with 6 ml of 1 M ammonium formate, 0.1 M formic acid (total inositol phosphate fraction). 1 ml of each Ins and InsPs fraction was counted in 15 ml of Ready-Safe liquid scintillation mixture.

Measurement of PtdOH, DAG, and PtdBut

The subconfluent cells were starved and then labeled overnight with either 1 µCi/ml [^3H]myristic acid or 0.5 µCi/ml [^3H]arachidonic acid in 10 ml of DMEM containing 1% FBS and 0.5% BSA. Cells were washed and preincubated in DMEM containing 0.5% BSA. In the case of PtdBut formation, 0.3% (v/v) 1-butanol was added for 10 min before agonist treatment. After cells were treated with agonists, cells were washed with ice-cold phosphate-buffered saline and scraped in 3 ml of ice-cold methanol; total lipids were extracted according to Bligh and Dyer(28) . DAG was separated on a thin layer chromatography silica gel 60 A plate using a solvent system of toluene/ether/ethanol/conc. ammonium hydroxide (50:30:2:0.2, v/v), and PtdOH and PtdBut were separated with the upper phase of a solvent system consisting of ethyl acetate/iso-octane/acetic acid/H(2)O (100:50:20:100, v/v). The bands of labeled species from the cells were identified either by their co-migration with authentic standards, which were located by staining with iodine vapor, or by autoradiography of the plate with EN^3HANCE spray. Bands were scraped off the plates, eluted with 500 µl of ethanol/HC1 (100:1), and counted in 7.5 ml of liquid scintillation mixture (Ready Organic).

In Vitro PKC Assay

The effect of PKC inhibitors on phosphorylating activity of recombinant human PKC isoforms, partially purified by DEAE-Sephacel chromatography (29) was determined in a reaction mixture (0.1 ml) containing 25 mM Tris, pH 7.5, 5 mM MgCl(2), 1 mM EGTA, 50 µM [-P]ATP (5 times 10^5 cpm/nmol), 50 µM phosphatidylserine, 0.2 µM PMA, and substrate (0.2 mg/ml MBP). For PKCalpha activity, 0.1 mM CaCl(2) and 0.1 mM EGTA were included in the reaction mixture in place of 1 mM EGTA to give a free Ca concentration of 1.85 µM. Following incubation for 15 min at 30 °C, the reaction was stopped, and the incorporation of [P] into substrate was measured as previously described(20) .

Measurement of Protein Tyrosine Phosphorylation

Quiescent cells were washed and preincubated in the presence or in the absence of PKC inhibitor for 1 h. After treatment with agonists for 5 min, cells were lysed in 2 ml/dish of lysis buffer (50 mM Tris-HCl, pH 7.5, containing 2 mM EDTA, 1 mM EGTA, 1 mM, 0.1 mM 4-amidinophenylmethanesulfonyl fluoride, 100 µg/ml leupeptin, 25 µg/ml aprotinin, 50 mM beta-glycerophosphate, and 0.1 mM sodium orthovanadate) for 30 min on a rocker at 4 °C. The lysates were spun in an Eppendorf centrifuge, and the same amount of soluble protein was analyzed by SDS-polyacrylamide gel electrophoresis. Proteins containing phosphotyrosine were detected by Western blotting against anti-phosphotyrosine antibodies (PY-20).

Translocation of PKCs

Quiescent cells were washed and preincubated in DMEM containing 0.5% BSA for 1 h at 37 °C prior to incubation with agonists. After agonist treatment, the medium was removed, and the cells were quickly washed with phosphate-buffered saline. The cells were then scraped in 2 ml/dish of homogenization buffer containing 25 mM Tris, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 100 µg/ml leupeptin, 25 µg/ml aprotinin, 1 mM APMSF, and 0.02 mg/ml Triton X-100. The cells were homogenized and fractionated as described by Ha and Exton(20) . The protein concentration of each fraction was determined using the bicinchoninic acid assay, and the same amount of protein from each fraction was applied to a Laemmli gel (10% SDS). SDS-polyacrylamide gel electrophoresis, transblotting to Immobilon-P, and immunodetection of the blot were performed as described (20) except that both the Vectastain alkaline phosphatase ABC kit and ECL system were used to detect the immune complexes. Both methods gave the same results, but data using the ECL system are shown since this provided intensified bands yielding better photographs.


RESULTS

Activation of PLD and PI-PLC by Various Agonists Including EGF

We examined agonist-stimulated PLD activity in Swiss 3T3 cells by measuring PtdBut formation in the presence of 1-butanol. We tested the G-protein linked agonist bombesin and the tyrosine-kinase-coupled growth factors bFGF, EGF, and PDGF at concentrations at which they elicit maximal responses on DNA synthesis and PI hydrolysis. As shown in Fig. 1, PDGF was the most efficacious agonist, with bombesin being more efficacious than EGF and bFGF.


Figure 1: Agonist-stimulated PLD activity. Subconfluent, quiescent cells were labeled with 1 µci/ml [^3H]myristic acid overnight, and the cells were preincubated and stimulated with various agonists including EGF (100 nM), bFGF (100 µg/ml), bombesin (Bomb) (100 nM), and PDGF (50 µg/ml) for 15 min in the presence of 0.3% 1-butanol. [^3H]PtdBut accumulation was measured as described under ``Experimental Procedures,'' and net cpm values over the background were calculated and plotted as means ± S.E. of four determinations in a representative experiment. In this and subsequent figures, C refers to control cells incubated without agonist.



A key issue in the present study was to obtain data for or against the hypothesis that PLD activation requires PKC activation and DAG production by PI-PLC as prerequisites. As shown in Fig. 2, PDGF and bombesin were the most potent agonists for PtdIns(4,5)P(2) hydrolysis in Swiss 3T3 cells, whereas bFGF and EGF produced small but reproducible increases, i.e. they were always observed and ranged between 11 and 47% (EGF) and 8 and 45% (bFGF) with averages of 23% (n = 7) (EGF) and 27% (n = 5) (bFGF) (see also Fig. 3).


Figure 2: Agonist-stimulated PI-PLC activity. Subconfluent, quiescent cells were labeled with 0.5 µCi/ml myo-[^3H]inositol overnight, and the cells were preincubated and stimulated with each agonist (100 nM EGF, 100 µg/ml bFGF, 100 nM bombesin (Bomb), and 50 µg/ml PDGF) for 15 min in the presence of 20 mM LiCl. Accumulation of ^3H-labeled InsPs was determined as described under ``Experimental Procedures.'' Each column represents the mean ± S.E. for a representative experiment performed in triplicate.




Figure 3: Effect of a PKC inhibitor, Ro-31-8220, on growth factorstimulated inositol phosphate accumulation in Swiss 3T3 fibroblasts. Cells, prelabeled with myo-[^3H]inositol, were preincubated with 10 µM Ro-31-8220 for 1 h followed by incubation with each agonist (100 µg/ml bFGF, 100 nM EGF, 100 nM bombesin (Bomb), and 50 µg/ml PDGF) for 15 min in the presence of 20 mM LiCl. InsPs were fractionated on a AG 1-X8 anion-exchange column. Data presented as the quotient of InsPs divided by Ins plus InsPs represent means for a typical experiment performed in duplicate.



Effect of PKC Inhibitors on EGF-stimulated Formation of Inositol Phosphates

PKC modulates EGF receptor signaling through phosphorylation of the receptor(30) , resulting in inhibition of PtdIns(4,5)P(2) hydrolysis(22, 31) . If the EGF receptor of Swiss 3T3 cells were down-regulated by PKC in this manner, PKC inhibitors could enhance the EGF-stimulated PtdIns(4,5)P(2) hydrolysis. As shown in Fig. 3, the potent, selective PKC inhibitor, Ro-31-8220, greatly enhanced PtdIns(4,5)P(2) hydrolysis stimulated by bFGF, EGF, or bombesin but did not alter that induced by PDGF. These results are consistent with the idea that PI-PLC is coupled to bFGF and EGF receptors in these cells and that response is small because of PKC-mediated down-regulation.

Involvement of PKC in the Regulation of PLD by EGF

To investigate the role of PKC isozymes in EGF-stimulated PLD, we applied two approaches: inhibition of PKC activity in intact cells and depletion of the enzyme by prolonged exposure to PMA. We tested three compounds, namely bIM, Ro-31-8220, and chelerythrine, which have been reported to be potent, selective PKC inhibitors(32, 33, 34) . Ro-31-8220 and bIM bind to PKC competitively with ATP, whereas chelerythrine is competitive with respect to the phosphate acceptor and noncompetitive with respect to ATP. Cells were preincubated in the presence of increasing concentration of the inhibitors for 60 min prior to EGF stimulation. There were no effects on [^3H]myristic acid incorporation or on the basal level of PtdBut formation (data not shown). However, EGF-stimulated PtdBut accumulation was attenuated by all the inhibitors (Fig. 4). Complete inhibition occurred at 10 µM of bIM, a concentration which maximally affects other PKC-mediated responses in Swiss 3T3 cells(32) . Ro-31-8220 also antagonized EGF-induced PtdBut accumulation with half-maximal inhibition at 4 µM, which is similar to the concentration that reverses PMA effects in platelets(33) . Chelerythrine only partially inhibited PLD activation, and its lower potency has been observed in other cells (11) .


Figure 4: Effect of PKC inhibitors on [^3H]PtdBut accumulation in EGF-treated Swiss 3T3 cells. Cells prelabeled with [^3H]myristic acid were preincubated with increasing concentrations of PKC inhibitors (bIM, Ro-31-8220, and chelerythrine) for 1 h prior to incubation with 100 nM EGF. Control cells received the vehicle (0.1% Me(2)SO) instead of the PKC inhibitors. The generation of PtdBut in response to a 15-min stimulation with EGF was determined. Results, given as percent maximal EGF response on [^3H]PtdBut formation are the means ± S.E. of four determinations in a representative experiment.



Because different PKC isoforms could be involved in the regulation of PLD, we examined the in vitro effects of the two most potent inhibitors, bIM and Ro-31-8220, on the PKC isoforms (alpha, , , and ), which exist in Swiss 3T3 cells. Using recombinant enzymes (29) , MBP phosphorylation activity was measured in the presence of increasing concentrations of the inhibitors. The two compounds showed similar inhibitory patterns on the PKC isoforms, although Ro-31-8220 was slightly more potent (Fig. 5, A and B). Both inhibitors were more potent on PKCalpha and - than on PKC and were almost ineffective on PKC. The concentrations that were effective in vitro were much lower than those required to affect PLD activation in the intact cells (cf.Fig. 4and Fig. 5).


Figure 5: Effect of PKC inhibitors, bIM and Ro-31-8220, on phosphorylating activity of various PKC isozymes. Partially purified recombinant PKC isoforms were incubated with increasing concentrations of bIM (A) or Ro-31-8220 (B) in the PKC assay mixture described under ``Experimental Procedures.'' The incorporation of [P] into substrate (MBP) was measured for 15 min at 30 °C as described, and the percent inhibition of phosphorylation was calculated. Data represent means for a typical experiment performed in duplicate.



Receptor tyrosine kinase activity is necessary for the activation of PLD by EGF and PDGF(19) . (^2)Because bIM and Ro-31-8220 are competitive inhibitors with respect to ATP, it was possible that they attenuated PLD activation by inhibiting EGF receptor tyrosine kinase. We therefore examined the tyrosine phosphorylation of proteins in EGF-stimulated cells in the absence or in the presence of the inhibitors. Western blotting with anti-phosphotyrosine antibody (Fig. 6) demonstrated that EGF stimulated the phosphorylation of the EGF receptor (178 kDa) and other cellular proteins (115, 85, and 45 kDa). The inhibitors did not inhibit the tyrosine phosphorylation of any of those proteins. In fact, bIM and, to a greater extent, Ro-31-8220 enhanced the tyrosine phosphorylation of the EGF receptor (Fig. 6B). This is in accord with the effects of PKC on the receptor, as described earlier.


Figure 6: Effect of PKC inhibitors on tyrosine phosphorylation stimulated by EGF. Subconfluent and quiescent cells were preincubated with PKC inhibitors (A, 5 µM bIM and B, 10 µM Ro-31-8220) for 1 h prior to incubation with 100 nM EGF. Control cells received the vehicle (0.1% Me(2)SO) instead of the PKC inhibitors. Tyrosine phosphorylation in response to a 5-min stimulation with EGF was determined by Western analysis against anti-phosphotyrosine antibodies (PY-20).



PKCalpha, -, -, and - have been reported to be present in Swiss 3T3 cells by Olivier and Parker (35) who observed that treatment of cells with 500 nM PMA for 48 h almost completely down-regulated the alpha, , and isozymes, although the rate of loss of each isoform was not uniform. In contrast, the treatment had no effect on the cellular level of PKC. We tested the effect of PKC down-regulation on EGF-stimulated PtdBut formation. As shown in Fig. 7, the treatment itself significantly enhanced the radioactivity in PtdBut, suggesting that the basal activity of PLD was increased. However, the ability of EGF and PDGF to promote PtdBut accumulation in the PKC down-regulated cells was completely or almost completely abolished. These results strongly support our conclusion that PKC is critically involved in PLD activation by EGF.


Figure 7: Effect of PKC down-regulation on EGF-stimulated [^3H]PtdBut accumulation. Swiss 3T3 cells were pretreated for 48 h with 500 nM PMA. Following a 15-min stimulation with 100 nM EGF in the PtdBut, formation was measured as described under ``Experimental Procedures.'' The results were normalized on the basis of the cpm in the total lipid extract from each dish and are means ± S.E. of four determinations in a representative experiment. Control cells received vehicle (0.1% Me(2)SO) instead of PMA.



Effects of PKC Inhibitors on PLD Activation by Other Growth Factors

We examined the effects of the two most potent PKC inhibitors, Ro-31-8220 and bIM, on PLD activation by other agonists. At 1 µM, bIM diminished PLD activation by bFGF, bombesin, and EGF (Fig. 8A). At 10 µM, Ro-31-8220 also attenuated the stimulation of PtdBut formation by these agonists (Fig. 8B). Higher concentrations of both inhibitors abolished the responses, but there was reduced cell viability (data not shown).


Figure 8: Effect of PKC inhibitors, Ro-31-8220 and bIM, on [^3H]PtdBut accumulation stimulated by various growth factors in Swiss 3T3 cells. Cells, prelabeled with [^3H]myristic acid, were preincubated with 1 µM bIM (A) or 10 µM Ro-31-8220 (B) for 1 h. Control cells received the vehicle (0.1% Me(2)SO) instead of the PKC inhibitors. The generation of PtdBut in response to a 15-min stimulation with each agonist (100 µg/ml bFGF, 100 nM bombesin, and 50 µg/ml PDGF) was determined. Results given as percent control radioactivity in PtdBut obtained from each agonist-stimulated cells are means ± S.E. of four determinations in a representative experiment.



Effects of PKC Inhibitor on DAG and PtdOH Production in Response to EGF

Many agonists induce a biphasic production of DAG in their target cells, e.g. Cook et al.(14) and Plevin et al.(15) . The first increase corresponds with an increase in Ins(1,4,5)P(3) while the second does not, and its source has been shown to be PtdCho in many cell types(5, 6) . To further confirm the involvement of the PI-PLC/PKC pathway in the regulation of PLD, we measured the amount of PtdOH and DAG in cells labeled differentially with [^3H]myristic or [^3H]arachidonic acid (Fig. 9, A and B). Myristic acid was predominantly incorporated into PtdCho, whereas arachidonic acid went mainly into PtdIns (data not shown) in agreement with findings in other cell types(36, 37, 38, 39, 40) . During short-term treatment with EGF for 1 min, the increase in [^3H]PtdOH formation in the [^3H]myristic acidlabeled cells was attributable to PLD activation since the increase in [^3H]DAG was small (Fig. 9A). Due to PtdOH hydrolysis by PtdOH phosphohydrolase, the [^3H]PtdOH level declined at 15 min, and [^3H]DAG accumulated at this time (Fig. 9A). The PKC inhibitor, Ro-31-8220, abolished the [^3H]PtdOH production at 1 min and the subsequent [^3H]DAG production at 15 min induced by EGF, further demonstrating the involvement of PKC in the activation of PLD by EGF.


Figure 9: EGF-stimulated PtdOH and DAG production in Swiss 3T3 cells prelabeled with [^3H]myristic acid or [^3H]arachidonic acid. PtdOH and DAG production was measured in Swiss 3T3 cells stimulated by 100 nM EGF in the absence or presence of 10 µM Ro-31-8220 after prelabeling with different fatty acid precursors. A, [^3H]myristic acid; B, [^3H]arachidonic acid. At the indicated times, the incubations were terminated, lipids were extracted, and the amount of PtdOH and DAG was measured as described under ``Experimental Procedures.'' Control cells were incubated with medium only for each time in the presence or absence of PKC inhibitor. Results are given as percent control value of DAG and PtdOH at each time point and are means ± S.E. of four determinations in a representative experiment.



When PtdOH and DAG were examined in the [^3H]arachidonic acid-labeled cells, the radioactivity in DAG was higher at 1 min than at 15 min (Fig. 9B), consistent with the activation of PI-PLC. The DAG species generated from PtdIns(4,5)P(2) by PI-PLC usually accumulate transiently because of the down-regulation of the response and because the DAG is metabolized by DAG kinase and other enzymes. Thus, the increase in [^3H]PtdOH level at 15 min in Fig. 9B is probably due to the formation of PtdOH from DAG derived from PtdIns(4,5)P(2). Interestingly, in the presence of Ro-31-8220, the increases in [^3H]DAG at 1 min and in [^3H]PtdOH at 15 min in the cells labeled with arachidonic acid were significantly enhanced (Fig. 9B). This is consistent with the effect of the PKC inhibitor to counteract the down-regulation of the activation of PI-PLC by EGF, as observed for InsPs production (Fig. 3). (^3)

Effects of PKC Inhibitor on Translocation of PKC Isozymes in Intact Cells

Translocation provides an index of changes in the amount of DAG and the subsequent activation of PKCs. The Ca increase induced by agonists that activate PI-PLC has been shown to be a critical factor in the differential translocation of Ca-dependent and -independent PKC isozymes in IIC9 cells (20) . Therefore, the translocation of the Cadependent PKCalpha could be considered an index of activation of PI-PLC. PKCalpha was mainly localized to the cytosol in quiescent Swiss 3T3 cells, but significant translocation to the particulate fraction by bombesin or EGF could not be detected at any time. However, with both agonists, enhanced translocation of PKCalpha was observed in the presence of the PKC inhibitor Ro-31-8220 ( Fig. 10and 11). The effect was consistent with the demonstration that the PKC inhibitor enhanced PI hydrolysis by these agonists (Fig. 3), resulting in increased DAG formation (Fig. 9B).


Figure 10: Bombesin-induced PKC translocation. The quiescent cells preincubated with either vehicle (0.1% Me(2)SO) or 10 µM Ro-31-8220 for 1 h were treated with 100 nM bombesin for the indicated times. Cell homogenates were fractionated into cytosolic (C) and particulate (M) fractions. An equal amount of proteins was analyzed by 10% SDS-polyacrylamide gel electrophoresis and Western blotting as described under ``Experimental Procedures.''



Both bombesin and EGF induced a biphasic translocation of PKC ( Fig. 10and Fig. 11) and also caused a rapid translocation of PKC. The membrane association of these isozymes was enhanced and/or prolonged by Ro-31-8220, which also promoted significant association of both isozymes in the absence of the agonists ( Fig. 10and 11). (^4)PKC was not translocated by these agonists at any time (data not shown) in agreement with observations by other investigators(20, 35, 41) .


Figure 11: EGF-induced PKC translocation. Cells were treated with 100 nM EGF for the indicated times in the absence or presence of 10 µM Ro-31-8220. Cytosolic (C) and particulate (M) fractions were prepared, and an equal amount of protein for each fraction was analyzed by 10% SDS-polyacrylamide gel electrophoresis and Western blotting as described under ``Experimental Procedures.''




DISCUSSION

It is becoming clear that PtdCho hydrolysis by PLD is a widespread cellular response to many agonists(5, 6, 7) . Through this pathway, many known or potential signaling molecules such as PtdOH, lysoPtdOH, DAG, monoacylglycerol, and arachidonic acid and its metabolites can be produced. Most G-protein-coupled and receptor tyrosine kinase-coupled agonists have been shown to stimulate PtdCho hydrolysis by PLD by a process that appears to be downstream of the initial Ins(1,4,5)P(3) and DAG formation by PI-PLC and to involve PKC activation(5, 6, 12, 14, 15) .

In A431 cells, EGF has been shown to stimulate the hydrolysis of PtdIns(4,5)P(2) via the activation of PLC due to the tyrosine kinase activity of the EGF receptor(3) . Although Cook and Wakelam (19) have suggested that EGF does not induce the hydrolysis of PtdIns(4,5)P(2) in Swiss 3T3 fibroblasts, the present report provides evidence indicating that PI-PLC is also coupled to the EGF receptor in these cells. We consistently observed a small but reproducible increase in InsPs in EGF-stimulated cells, and this was enhanced by the PKC inhibitor Ro-31-8220 (Fig. 3). The enhancing effect of the inhibitor is probably due to its prevention of down-modulation of the EGF receptor by PKC. Evidence for such down-regulation has been provided by Wahl and Carpenter (42) and is in accordance with the findings of Davis (30) and others(22, 31) . The inability of Cook and Wakelam (19) to observe an increase in InsPs with EGF in Swiss 3T3 cells may relate to their use of cells that had grown to confluency for 48 h. We have observed that such cells show diminished responses to EGF.

In additional support of the involvement of PI-PLC in the activation of PC-PLD in Swiss 3T3 cells is the observation that the relative efficacy of EGF, PDGF, and bombesin in stimulating PtdBut formation (Fig. 1) is the same as for activating PI-PLC (Fig. 2). Similar observations have been made in hepatocytes stimulated by various agonists(43) .

Because of our evidence that EGF is coupled to the PI-PLC signaling system in Swiss 3T3 cells, we expected that the activation of PLD by EGF would be PKC dependent. In contrast to the report of Cook and Wakelam(19) , three potent, selective PKC inhibitors (bIM, Ro-31-8220, and chelerythrine) dose-dependently antagonized EGF-induced PtdBut formation (Fig. 4). The potency of the inhibitors on PLD activation by EGF was similar to that for inhibition of other PKC-mediated cellular responses(32, 33, 34) . The concentrations required to inhibit PLD were much higher than those required to inhibit PKC isozymes in vitro (Fig. 5), reflecting limitations and differences in the permeability of intact cells to the inhibitors. To our knowledge, Fig. 5presents the first data on the effects of bIM and Ro-31-8220 on individual PKC isozymes.

Fig. 8illustrates that 10 µM Ro-31-8220 was unable to completely abolish the stimulation of PLD by four different agonists. Although this could merely be due to an inability to achieve full inhibition of PKC isozymes at this non-toxic dose, it is also possible that PKC-independent mechanisms of PLD activation could exist. However, the fact that down-regulation of PKC eliminated the ability of EGF or PDGF to stimulate PLD (Fig. 7) argues against this possibility. In these experiments, PKC depletion produced a substantial increase in basal PLD activity. The basis for this is unknown, but it could imply a role for PKC in the regulation of the synthesis or degradation of the enzyme. The basal level of PLD activity observed in Fig. 7and Fig. 8is probably due to the existence of multiple PLD isozymes, as has been speculated(44, 45, 46, 47) . Such PKC-independent isozymes could be regulated by other mechanisms, e.g. those involving small G-proteins such as ARF and Rho(48, 49, 50) .

In our studies of the regulation of PLD by PKC, we did not employ the PKC inhibitors, staurosporine and sphingosine, since these have been shown to stimulate PLD (51, 52) via the activation of pertussis toxin-sensitive G-proteins or other mechanisms. This probably accounts for the fact that these inhibitors usually do not produce complete inhibition of the activation of PLD(6) . (^5)Another issue is that PKC is often involved not only in the stimulation of PLD by an agonist but also in the down-regulation of its receptor (22, 31, 42, 43, 53, 54, 55, 56, 57) . Thus, in the presence of a PKC inhibitor, downregulation of the receptor could be blocked, resulting in enhancement of the signal for PtdBut accumulation. As a result of these positive and negative effects, PKC inhibitors could thus have variable effects on PLD activity.

In any consideration of the role of PKC in the regulation of PLD, another point should be recognized. This is that the DAG derived from PtdIns(4,5)P(2) breakdown may be considered to act as a ``trigger'' for PLD activation via PKC, i.e. continuing production of DAG from this source may not be required. This is because, once activated, PLD can produce DAG from PtdCho via PtdOH phosphohydrolase, and this DAG can then sustain PKC activation and hence PLD activity. This would explain why PLD activation can continue despite down-regulation of the PI-PLC response.

The conclusion that the EGF receptor, like the receptor for bombesin, is linked to PI-PLC in Swiss 3T3 cells is further supported by the PKC translocation studies of Fig. 10and Fig. 11. These show a movement of PKCalpha to the membrane in EGF-stimulated cells in the presence of Ro-31-8220. Because we have shown that the inhibitor increases InsPs production (Fig. 3) and [^3H]DAG production in response to EGF in [^3H]arachidonic acid-labeled cells (Fig. 9B), it is likely that the translocation reflects DAG formation from PtdIns(4,5)P(2) in the membrane. In IIC9 fibroblasts, it has been shown that PKCalpha translocation requires concurrent rises in DAG and Ca, i.e. PtdIns(4,5)P(2) hydrolysis(20) .

There have been some efforts to define the role of the various PKC isoforms in the regulation of PLD. In membranes prepared from CCL39 fibroblasts, only PKCalpha and -beta were activators of PLD(58) , but whole cells were not examined. In other studies, PKCalpha and - were implicated in Madin-Darby canine kidney cells (11) and in messangial cells(59) . PKCbeta1 overexpression in rat fibroblasts has been observed to enhance agonist-stimulated PLD activity(8, 9) , and overexpression of PKCalpha enhances it in Swiss 3T3 cells(10) . In contrast, transfection of PKCalpha constructs into Cos-1 cells resulted in partial suppression of the PLD response to PMA(60) . Except for the latter observation, these findings implicate PKC-alpha, -beta, and - in the stimulation of PLD. Since PKC is Ca-independent, it may be the isozyme principally involved in the ``feedback'' activation of PLD that occurs when DAG is derived mainly from PtdCho after the initial phase of agonist stimulation.

Although there is evidence that PLD can be activated by a phosphorylation-independent mechanism in membranes from CCL-39 cells (61) , the stimulation has been shown to require ATP-dependent phosphorylation in many cell types(62, 63) . Since we observed that Ro-31-8220 blocked PLD activation but promoted membrane association of PKCs, it seems that translocation of PKC protein per se does not activate PLD, i.e. it also requires the catalytic activity of the kinase.

Clearly, more work needs to be done to more rigorously define the regulation of PC-PLD by G-protein- and tyrosine kinase-linked agonists. Most important is the need to purify and characterize the PC-PLD isozymes involved and to demonstrate if they are direct substrates for PKC isozymes and if their phosphorylation is correlated with activation. (^6)


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM-40919. 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.

§
Investigator of the Howard Hughes Medical Institute. To whom all correspondence should be addressed. Tel.: 615-322-6494; Fax: 615-322-4381.

(^1)
The abbreviations used are: EGF, epidermal growth factor; PKC, protein kinase C; PI-PLC, PtdIns(4,5)P(2)-hydrolyzing phospholipase C; PC-PLD, PtdCho-hydrolyzing phospholipase D; PtdCho, phosphatidylcholine; DAG, diacylglycerol; PMA, 4beta-phorbol 12-myristate 13-acetate; bIM, bis-indolylmaleimide; bFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor; MBP, myelin basic protein; PtdBut, 1,2-dioleoyl-sn-glycero-3-phosphobutanol; PtdOH, phosphatidic acid; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BSA, bovine serum albumin; Ins(1,4,5)P(3), inositol 1,4,5-trisphosphate; PtdIns(4,5)P(2), phosphatidylinositol 4,5-bisphosphate.

(^2)
E.-J. Yeo and J. H. Exton, unpublished results.

(^3)
The inhibitory effect of Ro-31-8220 on PtdOH formation at 1 min on the [^3H]arachidonic acid-labeled cells (Fig. 9B) probably reflects the fact that some label was incorporated into PtdCho. As shown in Fig. 9A, the PKC inhibitor produced a drastic inhibition of PLD at this time.

(^4)
This may be attributable to the small (approximately 30%) increase in DAG induced by the inhibitor when added alone to cells labeled with [^3H]myristic or [^3H]arachidonic acids (data not shown).

(^5)
Since Song et al.(40) have reported that staurosporine does not inhibit EGF-stimulated PLD activity in A431 cells, we tested the effects of Ro-31-8220 in this system. In accord with our findings in Swiss 3T3 cells, Ro-31-8220 produced a dose-dependent inhibition of the EGF effect in A431 cells. Down-regulation of PKC also abolished the EGF effect in these cells.

(^6)
The general conclusion of this study, namely that EGF activates PC-PLD via PI-PLC and PKC, is supported by studies with TRMP cells expressing mutant PDGF receptors. These showed that only those receptors that could couple to PI-PLC were able to mediate PDGF activation of PLD(64) .


ACKNOWLEDGEMENTS

We thank Jennie Smith for technical support. We also thank Judy Childs for typing this manuscript.


REFERENCES

  1. Carpenter, G. and Cohen, S. (1990) J. Biol. Chem. 265, 7709-7712 [Free Full Text]
  2. Koch, A. C., Anderson, D. A., Moran, M. F., Ellis, C. and Pawson, T. (1991) Science 252, 668-674 [Medline] [Order article via Infotrieve]
  3. Wahl, M. I., Nishibe, S., Suh, P.-G., Rhee, S. G. and Carpenter, G. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1568-1572 [Abstract]
  4. Kim, H. K., Kim, J. W., Zilberstein, A., Margolis, B., Kim, J. G., Schlessinger, J. and Rhee, S. G. (1991) Cell 65, 435-441 [Medline] [Order article via Infotrieve]
  5. Exton, J. H. (1990) J. Biol. Chem. 265, 1-4 [Abstract/Free Full Text]
  6. Exton, J. H. (1994) Biochim. Biophys. Acta 1212, 26-42 [Medline] [Order article via Infotrieve]
  7. Billah, M. M. and Anthes, J. C. (1990) Biochem. J. 269, 281-291 [Medline] [Order article via Infotrieve]
  8. Pai, J.-K., Dobek, E. A. and Bishop, W. R. (1991) Cell Regul. 2, 897-903 [Medline] [Order article via Infotrieve]
  9. Pachter, J. A., Pai, J.-K., Mayer-Ezell, R., Petrin, J. M., Dobek, E. and Bishop, W. R. (1992) J. Biol. Chem. 267, 9826-9830 [Abstract/Free Full Text]
  10. Eldar, H., Ben-Av, P., Schmidt, U.-S., Livneh, E. and Liscovitch, M. (1993) J. Biol. Chem. 268, 12560-12564 [Abstract/Free Full Text]
  11. Balboa, M. A., Firestein, B. L., Godson, C., Bell, K. S. and Insel, P. A. (1994) J. Biol. Chem. 269, 10511-10516 [Abstract/Free Full Text]
  12. Cook, S. J. and Wakelam, M. J. O. (1989) Biochem. J. 263, 581-587 [Medline] [Order article via Infotrieve]
  13. Cook, S. F. and Wakelam, M. J. O. (1991) Biochim. Biophys. Acta 1092, 265-272 [Medline] [Order article via Infotrieve]
  14. Cook, S. J., Palmer, S., Plevin, R. and Wakelam, M. J. O. (1990) Biochem. J. 265, 617-620 [Medline] [Order article via Infotrieve]
  15. Plevin, R., Cook, S. J., Palmer, S. and Wakelam, M. J. O. (1991) Biochem. J. 279, 559-565 [Medline] [Order article via Infotrieve]
  16. Price, B. B., Morris, J. D. H. and Hall, A. (1989) Biochem. J. 264, 509-511 [Medline] [Order article via Infotrieve]
  17. Rosoff, P. M., Savage, N. and Dinarello, C. A. (1988) Cell 54, 73-81 [Medline] [Order article via Infotrieve]
  18. Wright, T. M., Shin, H. S. and Raben, D. M. (1990) Biochem. J. 267, 501-507 [Medline] [Order article via Infotrieve]
  19. Cook, S. J. and Wakelam, M. J. O. (1992) Biochem. J. 285, 247-253 [Medline] [Order article via Infotrieve]
  20. Ha, K.-S. and Exton, J. H. (1993) J. Biol. Chem. 268, 10534-10539 [Abstract/Free Full Text]
  21. Ahmed, A., Plevin, R., Shoaibi, M. A., Fountain, S. A., Ferriani, R. A. and Smith, S. K. (1994) Am. J. Physiol. 266, C206-C212
  22. Pike, L. J. and Eakes, A. T. (1987) J. Biol. Chem. 262, 1644-1651 [Abstract/Free Full Text]
  23. Besterman, J. M., Watson, S. P. and Cuatrecasas, P. (1986) J. Biol. Chem. 261, 723-727 [Abstract/Free Full Text]
  24. L'Allemain, G. and Pouyssegur, J. (1986) FEBS Lett. 197, 344-348 [CrossRef][Medline] [Order article via Infotrieve]
  25. Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380 [Abstract/Free Full Text]
  26. Conklin, B. R., Chabre, O., Wong, Y. H., Federman, A. D. and Bourne, H. R. (1992) J. Biol. Chem. 267, 31-34 [Abstract/Free Full Text]
  27. Simpson, C. M. F., Batty, I. H. and Hawthorne, J. N. (1987) in Neurochemistry (Turner, A. J. and Bachelard, H. S., eds.), pp. 193-224, IRL Press Ltd., Oxford, England
  28. Bligh, E. G. and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
  29. Burns, D. J., Bloomenthal, J., Lee, M.-H. and Bell, R. M. (1990) J. Biol. Chem. 265, 12044-12051 [Abstract/Free Full Text]
  30. Davis, R. J. (1988) J. Biol. Chem. 263, 9462-9469 [Abstract/Free Full Text]
  31. Johnson, R. M. and Garrison, J. C. (1987) J. Biol. Chem. 262, 17285-17293 [Abstract/Free Full Text]
  32. Toullec, D., Pianett, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D. and Kirilovsky, J. (1991) J. Biol. Chem. 266, 15771-15781 [Abstract/Free Full Text]
  33. Davis, P. D., Hill, C. H., Keech, E., Lawton, G., Nixon, J. S., Sedgwick, A. D., Wadsworth, J., Westmacott, D. and Wilkinson, S. E. (1989) FEBS Lett. 259, 61-63 [CrossRef][Medline] [Order article via Infotrieve]
  34. Herbert, J. M., Augereau, J. M., Gleye, J. and Maffrand, J. P. (1990) Biochem. Biophys. Res. Commun. 172, 993-999 [Medline] [Order article via Infotrieve]
  35. Olivier, A. R. and Parker, P. J. (1992) J. Cell. Physiol. 152, 240-244 [Medline] [Order article via Infotrieve]
  36. Swendsen, C. L., Chilton, F. H., O'Flaherty, J. T., Surles, J. R., Piatadosi, C., Waite, M. and Wykle, R. L. (1987) Biochim. Biophys. Acta 919, 79-89 [Medline] [Order article via Infotrieve]
  37. Takayama, H., Gimbrone, M. A. and Schafer, A. I. (1987) Biochim. Biophys. Acta 922, 314-322 [Medline] [Order article via Infotrieve]
  38. Huang, C. and Cabot, M. C. (1990) J. Biol. Chem. 265, 14858-14863 [Abstract/Free Full Text]
  39. Song, J. and Foster, D. A. (1993) Biochem. J. 294, 711-717 [Medline] [Order article via Infotrieve]
  40. Song, J., Jiang, Y.-W. and Foster, D. A. (1994) Cell Growth and Differentiation 5, 79-85 [Abstract]
  41. Olivier, A. R. and Parker, P. J. (1994) J. Biol. Chem. 269, 2758-2763 [Abstract/Free Full Text]
  42. Wahl, M. and Carpenter, G. (1988) J. Biol. Chem. 263, 7581-7590 [Abstract/Free Full Text]
  43. Moehren, G., Gustavsson, L. and Hoek, J. B. (1994) J. Biol. Chem. 269, 838-848 [Abstract/Free Full Text]
  44. Balboa, M. A., Balsinde, J., Aramburu, J., Mollinedo, F. and Lopez-Botet, M. (1992) J. Exp. Med. 176, 9-17 [Abstract]
  45. Agwu, D. E., McCall, C. E. and McPhail, L. C. (1991) J. Immunol. 146, 3895-3903 [Abstract/Free Full Text]
  46. Ben-Av, P., Eli, Y., Schmidt, U. S., Tobias, K., E. and Liscovitch, M. (1993) Eur. J. Biochem. 215, 455-463 [Abstract]
  47. Huang, C., Wykle, R. L., Daniel, L. W. and Cabot, M. C. (1992) J. Biol. Chem. 267, 16859-16865 [Abstract/Free Full Text]
  48. Bowman, E. P., Uhlinger, D. J. and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512 [Abstract/Free Full Text]
  49. Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C. and Sternweis, P. C. (1993) Cell 75, 1137-1144 [Medline] [Order article via Infotrieve]
  50. Cockcroft, S., Thomas, G. M. H., Fensome, A., Geny, B., Cunningham, E., Gout, I., Hiles, I., Totty, N. F., Truong, I. and Hsuan, J. J. (1994) Science 263, 523-526 [Medline] [Order article via Infotrieve]
  51. Lavie, Y. and Liscovitch, M. (1990) J. Biol. Chem. 265, 3868-3872 [Abstract/Free Full Text]
  52. Kanaho, Y., Takahashi, K., Tomita, U., Iiri, T., Katada, T., Ui, M. and Nozawa, Y. (1992) J. Biol. Chem. 267, 23554-23559 [Abstract/Free Full Text]
  53. Lynch, C. J., Charest, R., Bocckino, S. B., Exton, J. H. and Blackmore, P. F. (1985) J. Biol. Chem. 260, 2844-2851 [Abstract]
  54. Leeb-Lundberg, L. M. F., Cotecchia, S., DeBlasi, A., Caron, M. G. and Lefkowitz, R. J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5651-5655 [Abstract]
  55. Lee, L. S. and Weinstein, I. B. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 5168-5172 [Abstract]
  56. Davis, R. J. and Czech, M. P. (1984) J. Biol. Chem. 259, 8545-8549 [Abstract/Free Full Text]
  57. Briscoe, C. P., Plevin, R. and Wakelam, M. J. O. (1994) Biochem. J. 298, 61-67 [Medline] [Order article via Infotrieve]
  58. Conricode, K. M., Smith, J. L., Burns, D. J. and Exton, J. H. (1994) FEBS Lett. 342, 149-153 [CrossRef][Medline] [Order article via Infotrieve]
  59. Pfeilschifter, J. and Huwiler, U. (1993) FEBS Lett. 331, 267-271 [CrossRef][Medline] [Order article via Infotrieve]
  60. McKinnon, M. and Parker, P. (1994) Biochim. Biophys, Acta 1222, 109-112 [Medline] [Order article via Infotrieve]
  61. Conricode, K. M., Brewer, K. A. and Exton, J. H. (1992) J. Biol. Chem. 267, 7199-7202 [Abstract/Free Full Text]
  62. Reinhold, S. L., Prescott, S. M., Zimmerman, G. A. and McIntyre, T. M. (1990) FASEB J. 4, 208-214 [Abstract/Free Full Text]
  63. Olson, S. C., Bowman, E. P. and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242 [Abstract/Free Full Text]
  64. Yeo, E.-J., Kazlauskas, A. and Exton, J. H. (1994) J. Biol. Chem. 269, 27823-27826 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.