Biochimica et Biophysica Acta, 1175 (1992) 88-94 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4889/92/$05.00

88

BBAMCR 13274

Cooperative effects of ethanol and protein kinase C activators on phospholipase-D-mediated hydrolysis of phosphatidylethanolamine in NIH 3T3 fibroblasts Zoltan Kiss The Hormel Institute, University of Minnesota, Austin, MN (USA) (Received 28 January 1992) (Revised manuscript received 1 June 1992)

Key words: Ethanol; Phosphatidylethanolamine; Phospholipase D; Phorbol ester

In a previous study, ethanol was shown to enhance the stimulatory effect of phorbol 12-myristate 13-acetate (PMA), a prominent activator of protein kinase C (PKC), on phospholipase-D (PLD)-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts (Kiss et al. (1991) Eur. J. Biochem. 197, 785-790). Here, the mechanism and possible significance of ethanol-stimulated PtdEtn hydrolysis was further studied. In [14C]ethanolamine-labeled NIH 3T3 fibroblasts, 10 mM ethanol enhanced PMA-induced hydrolysis of PtdEtn 1.5-2.0-fold during a 2.5-15-min incubation period. Other alcohols, including glycerol, methanol, and 1-propanol, also enhanced PMA-induced PtdEtn hydrolysis. Of the other PLD activators tested, ethanol potentiated the PKC-dependent stimulatory effect of bombesin but failed to alter the apparently PKC-independent stimulatory effect of serum. Pretreatment of [14C]ethanolamine-labeled fibroblasts with 200 mM ethanol for 20 min resulted in increased (approx. 2-fold) hydrolysis of [14C]PtdEtn in isolated membranes. In membranes from ethanol-treated, but not from untreated, cells, PMA further enhanced (approx. 1.5-fold) the production of [14C]ethanolamine. Ethanol exerted none of the above stimulatory effects on phosphatidylcholine hydrolysis. These results suggest that the specific stimulatory action of ethanol on PLD-mediated PtdEtn hydrolysis can occur in vivo and may involve increased binding of a regulatory PKC-isoform to membranes. Introduction Interaction of short-chain alcohols with the cell membrane affects various phospholipase activities which are involved in signal transduction. Thus, in hepatocytes [1], human platelets [2,3], and turkey erythrocyte membranes [4], ethanol was shown to rapidly stimulate a G-protein-dependent inositol phospholipid-specific phospholipase C. This ethanol effect is associated with the formation of two intracellular messengers, inositol-l,4,5-trisphosphate and 1,2-diacylglycerol, which act to release calcium from intracellular stores [5] and to activate protein kinase C (PKC) [6,7], respectively. Phospholipase D (PLD) is another important hydrolytic activity in cell membranes [8,9]. This enzyme cat-

Correspondence to: Z. Kiss, The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA. Abbreviations: PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PtdEtn, phosphatidylethanolamine; PtdCho, phosphatidylcholine; PLD, phospholipase D; PLC, phospholipase C.

alyzes the formation of phosphatidic acid, which appears to be a mitogen in fibroblasts and other cell lines [reviewed in ref. 10]. Importantly, in the presence of ethanol and other short-chain alcohols, PLD also catalyzes the formation of phosphatidylalcohol at the expense of phosphatidic acid. However, it should be stressed that an efficient competition of the transphosphatidylation reaction with the hydrolytic activity requires 50-300 mM concentrations of ethanol. Thus, a significant inhibition of phosphatidic acid formation may occur only at the upper end of physiologically attainable ethanol concentrations (50-100 mM). In fibroblasts, both phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) were found to serve as substrates for PLD, activated by phorbol 12myristate 13-acetate (PMA)[11], nucleotides, or sphingosine [12]. Curiously, in NIH 3T3 cells ethanol not only serves as a substrate for PLD, but it can also stimulate the hydrolysis of PtdEtn [13]. In a most recent study [14], we found that ethanol also potentiated the stimulatory effect of PKC activator PMA on PtdEtn hydrolysis. However, none of these studies [13,14] provided data to suggest the mechanism or the possible occurrence of this ethanol effect in vivo.

89 Here, we report that at 10-50 mM concentrations, ethanol potentiates the stimulatory effects of both PMA and bombesin on the hydrolysis of PtdEtn, suggesting that these actions of ethanol may be physiologically relevant. Pretreatment of fibroblasts with ethanol was also found to sensitize the PtdEtn-hydrolyzing system to the action of PMA in isolated membranes, suggesting that ethanol might act by increasing the membrane content of a regulatory PKC-isoform.

tions were terminated and 14C-labeled water-soluble products of PtdEtn were separated on Dowex-50-WH ÷ resin according to the method of Cook and Wakelam [16], with modifications indicated elsewhere [15]. Measurement of PMA-stimulated hydrolysis of PtdCho was performed in [14C]choline-labeled ceils as described elsewhere [17]; the method of Cook and Wakelam [16] was used for the separation of [14C]choline metabolites. Phospholipids were separated on silica gel H plates as indicated earlier [18].

Materials and Methods

PMA and Dowex-50-WH ÷ were purchased from Sigma; bombesin was from Boehringer Mannheim; fetal calf serum was from Flow Laboratories; [214C]ethanolamine (50 mCi/mmol), [methyl-14C]choline (60 mCi/mmol), and [1-14C]palmitic acid (60 mCi/mmol) were from Amersham; and tissue-culture reagents were bought from GIBCO. Ethanol (analytical purity) was bought from Baker and was redistilled before use.

Cell culture NIH 3T3 clone-7 fibroblasts were continuously cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, penicillin (50 U/ml)/streptomycin (50 /zg/ml) and glutamine (2 mM). Fibroblasts were seeded in 150-mm-diameter plastic dishes, and growing (60-80% confluent) cell populations were harvested after 2 days in culture.

Measurement of PtdEtn hydrolysis Attached NIH 3T3 fibroblasts were incubated with [14C]ethanolamine (0.25 /zCi/ml) for 48 h, washed, incubated for 3 h in the presence of fresh Dulbecco's medium, and then harvested by gentle scraping from the dish. As it was explained elsewhere [15], this procedure ensured that at the beginning of incubation the cells uniformly contained only low amounts of [~4C]ethanolamine (background levels were 800-1000 dpm/106 cells). For the study of bombesin effect, the above labeling procedure was altered in the following way: Ceils were initially labeled with [~4C]ethanolamine in serum-containing medium for 34 h, and then in serum-free medium (containing 1 mg/ml of fatty acid-free albumin) for 14 h; finally, cells were incubated in the above serum-free medium in the absence of [~4C]ethanolamine for 3 h. Aliquots of cell suspension (0.8-1.0-10 6 cells in 1 ml DMEM) were incubated (final volume 0.25 ml) in polypropylene tubes in an incubator at 37°C in the presence of 2 mM unlabeled ethanolamine to prevent further metabolism of newly formed [14C]ethanolamine [11,12,15]. Incuba-

Measurement of PtdEtn hydrolysis in membranes isolated from prelabeled cells NIH 3T3 fibroblasts were labeled with [14C]ethanolamine as indicated above. Washed cells were collected in each experiment from 3 dishes by scraping and disrupted by Dounce homogenization (A pestle; 100 strokes) in 5 ml of ice-cold 20 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 0.2 mM dithiothreitol, and 0.1 mg/ml leupeptin. Remaining intact cells were removed by centrifuging the homogenates at 1000 × g for 5 min. The resulting supernatants were further centrifuged at 16000 x g for 20 min at 4°C. The membrane pellets were resuspended in ice-cold Tris buffer (20 mM; pH 7.5) and used immediately. The assay mixture (0.1 ml) for the determination of phospholipid hydrolysis contained 20 mM Tris-HC1 (pH 7.5), 5 mM MgCI2, 0.1 mM ethanolamine, 0.1 mM ethanolamine phosphate, 0.1 mM CDP-ethanolamine, 64-75 /zg of membrane proteins, and other additions as indicated. The presence of unlabeled ethanolamine derivatives served to prevent further metabolism of newly formed 14Clabeled products of [laC]PtdEtn hydrolysis. Incubations were carried out at 37°C for 20 min in a shaking water bath and were terminated by the addition of 4 ml of chloroform/methanol (1:1, v/v). The water-soluble products of [~4C]PtdEtn hydrolysis were separated as above.

Measurement of phosphatidic acid formation in fibroblasts prelabeled with [14C]palmitic acid Fibroblasts were prelabeled with [1-14C]palmitic acid (0.25 p~Ci/ml) for 24 h. Cells were washed, incubated in fresh Dulbecco's medium for 2 h (to facilitate nearly complete esterification of free labeled palmitic acid; unpublished data), and then harvested as above. Suspended cells were incubated (final volume 0.25 ml) with PMA and/or ethanol for 10 rain as indicated. Phosphatidic acid was separated from other phospholipids and phosphatidylethanol on potassium oxalate (1%) impregnated silica gel H plates (Analtech) by using the solvent system chloroform/methanol/acetone/acetic acid/water (50 : 10 : 15 : 10 : 2, v/v).

90 220

Results

210 200

Time- and concentration-dependent effects of alcohols on PMA-induced PtdEtn hydrolysis Addition of ethanol to []4C]ethanolamine-labeled NIH 3T3 fibroblasts at a concentration as low as 10 mM enhanced PMA-induced PtdEtn hydrolysis almost two-fold at an early period (2.5 min) of treatment (Fig. 1). At later incubation times, there was some decrease in the potentiating effect of ethanol. At each time point examined, ethanol (10 or 50 mM) alone had no significant effects (Fig. 1). It also should be noted that up to 10 min incubation, the potentiating effects of 10 or 50 mM ethanol were not significantly different. Ethanol is not the only, and not even the most effective alcohol, which can enhance PMA-induced PtdEtn hydrolysis. As shown in Fig. 2, methanol and 1-propanol also stimulated PtdEtn hydrolysis, although less effectively than ethanol did, while glycerol was clearly more potent than ethanol in this respect.

Effect of ethanol on growth-factor-stimulated PtdEtn hydrolysis Although PMA is one of the most often used research tools in cell biology, it is not a physiological

12 11 ~

10

~,¢

7

~''6

6

Z ~

5

E -o

,

E

E

3

~

2

2e

I 2.5

I 5.0

I 7.5

10.0

Incubation time [ rain ] Fig. 1. Time-dependent effect of ethanol on PMA-stimulated hydrolysis of PtdEtn in NIH 3T3 cells. Cells were prelabeled with [14C]ethanolamine for 48 h, followed by incubation of suspended cells for 2.5-10 min in the absence ( o ) or presence of 50 mM ethanol (zx), 100 nM PMA (e), PMA plus 10 mM ethanol (A), or PMA plus 50 mM ethanol ( l I), as described in Materials and Methods. The average Inc content of PtdEtn was 1.59.106 dpm/106 cells. Each point represents the mean +_S.E. of two experiments each performed in quadruplicate on the same day. Similar results were obtained in a third experiment.

190

.E f-

~

170

,v;

v

16o

0

',~

130

140

~= ~

12o

LL

110 100

"•1

I

I

I

I

I

20

40

60

80

1O0

/,/

.,

I

200

Alcohol [ mM ] Fig. 2. Concentration-dependent effects of alcohols on PMA-stimulated hydrolysis of PtdEtn in NIH 3T3 cells. Cells were prelabeled with [14C]ethanolamine for 48 h, and then suspended cells were treated with 100 nM PMA for 15 min in the presence of 0-200 mM concentrations of ethanol (e), methanol (A), 1-propanol (11), or glycerol ( 0 ) . Each point represents the mean + S.E. of two experiments each performed in quadruplicate.

agent. Therefore, it was important to determine how ethanol might modify the effects of physiological regulators of phospholipase D. In Swiss 3T3 fibroblasts, bombesin was shown to stimulate PLD-mediated hydrolysis of PtdCho through the PKC system [16,19,20]. In the NIH 3T3 fibroblasts, 50 nM bombesin also stimulated the hydrolysis of PtdCho (not shown) and PtdEtn (Fig. 3) about 1.7- and 1.3-fold, respectively. Interestingly, while ethanol had virtually no effect on bombesin-induced PtdCho hydrolysis (data not shown), the stimulatory effect of bombesin on PtdEtn hydrolysis was potentiated by this agent (Fig. 3). In contrast, the larger stimulatory effect of serum on PtdEtn hydrolysis (which probably reflects the effect(s) of one or more growth factors), was not significantly potentiated by ethanol, except at higher (150-200 mM) concentrations. In order to explain the differential effects of ethanol on the bombesin- and serum-stimulated hydrolysis of PtdEtn, it was important to determine the role of PKC in the mediation of serum effect. This was examined in fibroblasts pretreated with 300 nM PMA for 24 h to down-regulate PMA-dependent PKC activity [21,22]. Such pretreatment abolished the stimulatory effects of newly added PMA (100 nM), bombesin (50 nM), or bombesin plus ethanol (100 mM), but it failed to prevent the stimulatory effect of 10% serum (data not shown). These data suggest that bombesin and serum stimulate PtdEtn hydrolysis by PKC-dependent and

91

.~

forms. These PKC-isoforms are present in NIH 3T3 cells and are only partially down-regulated (in contrast to the a- and /3-isoforms) by prolonged (24 h) TPAtreatment (data not shown).

240 220

~

Comparison of the effects of ethanol on the hydrolysis of PtdEtn and PtdCho

180

~ 16°I~o

"6 1,0-z/"

I

~ 1 2lOO ° - ~r

-

I

I

I

[

I

o

50

100

150

200

Ethanol [ m M ]

Fig. 3. Combined effects of ethanol, bombesin and serum on PtdEtn hydrolysis in NIH 3T3 cells. Cells were prelabeled with [14C]ethanolamine for 48 h, followed by treatment of suspended cells for 15 min with 0-200 mM concentrations of ethanol in the absence (e), or presence of 50 nM bombesin ( • ) , or 10% fetal calf serum ( • ) as described in Materials and Methods. Each point represents the mean + S.E. of three experiments each performed in quadruplicate.

-independent mechanisms, respectively. However, presently we cannot exclude the possibility that the effect of serum involves either the ~- or ~'-PKC-iso-

.~

Recently, Pittner and Fain [23] have shown that addition of ethanol to hepatocytes stimulated PtdCho hydrolysis, and that this effect of ethanol was additive (rather than synergistic) with that of PMA. These authors showed no data on the effect of sphingosine, another stimulator of PLD activity in fibroblasts [12]. Thus, it was of interest to compare the individual and combined effects of ethanol, PMA and sphingosine on the hydrolysis of PtdEtn and PtdCho. To ensure optimal conditions for such comparison, the experiments with [lac]ethanolamine- and [14C]choline-labeled NIH 3T3 cells were always performed with the same cell population on the same day, using the same reagents. In several experiments we invariably found that while ethanol enhanced (although to different degrees) the stimulatory effects of both PMA and sphingosine on PtdEtn hydrolysis (Fig. 4A), it failed to stimulate, both alone or in combination with these agents, the hydrolysis of PtdCho (Fig. 4B).

B

A

65

60 o

D.

55

.~--- O

4s

0

40

0

'""6 "~E m

O-

3s

30

2O •,e @

15

~

10

u_

0

I

I 50

I 100

I 150

I 200

T

I

0

50

I 100

I 150

200

Ethanol [nM ] Fig. 4. Combined effects of ethanol, PMA and sphingosine on the hydrolysis of PtdEtn and PtdCho in NIH 3T3 cells. Cells were prelabeled with [14C]ethanolamine (A) or [14C]choline (B) for 48 h, followed by treatment of suspended cells for 15 min with 0-200 mM concentrations of ethanol in the absence (e) or presence of 20/zM sphingosine ( • ) , 100 nM PMA ( • ) , or PMA plus sphingosine ( • ) , as described in Materials and Methods. Each point represents the mean + S.E. of two experiments each performed in quadruplicate. Similar results were obtained in two other experiments.

92

Hydrolysis of PtdEtn in membranes from untreated and ethanol-treated fibroblasts In membranes isolated from control [14C]ethanolamine-labeled N I H 3T3 fibroblasts, addition of P M A a n d / o r ethanol failed to modify the hydrolysis of PtdEtn (Fig. 5A). Pretreatment of fibroblasts with 200 mM ethanol for 20 min resulted both in increased (approx. 2-fold) formation of [14C]ethanolamine from the prelabeled membrane PtdEtn pool and the sensitization of the PtdEtn-hydrolyzing system to the stimulatory action of PMA.

Effect of ethanol on the formation of phosphatidic acid The interaction of ethanol with PLD can lead to the formation of phosphatidylethanol, instead of phosphatidic acid [9]. This is due to the ability of ethanol to compete with water at the catalytic site of PLD. However, significant formation of phosphatidylethanol, and a corresponding decrease in the formation of phosphatidic acid, requires relatively high (50-300 raM) concentrations of ethanol. Thus, potentiation of PMA-induced hydrolysis by 10 mM ethanol may actually produce phosphatidic acid, instead of phosphatidylethanol. This possibility was tested in [14C]palmitic acid-

TABLE I

Combined effects of etkanol and PMA on the formation of [14C]phospkatidic acid in [14C]palmiticmcid-labeled NIH 3T3 cells Suspended cells (2.2.106/ml), prelabeled with [14C]palmitic acid as described in Materials and Methods, were incubated for 5 min in the absence or presence of PMA (100 nM) and/or ethanol (10 mM). Data are the mean ± S.E. of four independent incubations. The 14C content of PtdEtn and PtdCho was 0.42.106 and 1.28-106 dpm/106 cells, respectively. Similarly low (11%) stimulatory effect of ethanol was obtained in another experiment. Addition

[ 14C]phosphatidicacid (dpm/106 cells)

None PMA Ethanol PMA + ethanol

24350 ± 560 31090 _+420 24190 +_600 34030 _+240

5

labeled fibroblasts. After treatment with PMA or PMA plus 10 mM ethanol for 2.5 min, we could not consistently observe significant effects of 10 mM ethanol on the cellular level of phosphatidic acid. However, after 5-min treatment, P M A and PMA plus 10 mM ethanol enhanced the cellular level of [14C]phosphatidic acid 28% and 40%, respectively (Table I). While the ethanol-induced change in phosphatidic acid formation indicates the involvement of PLD, this change appeared to be too small to play a physiological role. It should be noted here that even this small effect of ethanol on phosphatidic acid formation disappeared (presumably due to rapid metabolism of this phospholipid) when the treatment was extended up to 30 min (data not shown). As expected, in the presence of 100 nM PMA and 10 mM ethanol no significant formation of phosphatidylethanol could be detected (not shown).

4

Discussion

3

Although a previous study from our laboratory [14] established the fact that ethanol can potentiate the stimulatory effect of PMA on PtdEtn hydrolysis, the possible physiological relevance and the mechanism of this ethanol action remained to be characterized. The studies described here indicate that (a) ethanol at a concentration as low as 10 mM can potentiate the effect of PMA on PtdEtn hydrolysis, (b) the effect of ethanol occurs rapidly, and (c) ethanol can also potentiate the stimulatory effects of physiological activators of PKC, such as bombesln. Taken together, these results suggest that the above potentiating effects of ethanol on PtdEtn hydrolysis can occur in vivo. In hepatocytes [1], platelets [2,3] and erythrocytes [4], ethanol was shown to stimulate the hydrolysis of inositol phospholipids, a process which presumably leads to the activation of PKC [24]. Thus, in these cells, in contrast to N I H 3T3 fibroblasts, ethanol might serve

8 ,kCr

7

E 6 ¢-

m~ 0

iN_ A

Fig. 5. Enhanced formation of ethanolamine in membranes isolated from ethanol-treated NIH 3T3 fibroblasts. Membranes isolated from untreated [Iac]ethanolamine-labeled fibroblasts (A) or from fibroblasts treated with 200 mM ethanol for the last 20 min of the labeling period (B), were incubated in the absence ([3), or presence of 100 nM PMA ([]), 200 mM ethanol (l), or PMA plus ethanol (11), as described in Materials and Methods. Results are the mean± S.E. of two experiments each performed in quadruplicate on the same day. Similar results were obtained in a third experiment. * Significantly (P < 0.05) different from untreated control, according to Student's t-test. ** Significantly (P < 0.05) different from ethanol-treated control.

93 both as an activator of PKC and as a potentiator of PKC action. This possibility remains to be examined. Addition of P M A to m e m b r a n e s isolated from untreated fibroblasts failed to enhance PLD-mediated PtdEtn hydrolysis. However, a stimulatory effect of P M A was clearly evident in m e m b r a n e s isolated from ethanol-treated cells. This indicates that only membranes from ethanol-treated cells contained the regulatory PKC-isoform, suggesting that ethanol might stimulate the translocation of this PKC-isoform from the cytosol to membranes. The possible effect of ethanol on the translocation of various PKC-isoforms is presently under investigation in our laboratory. The stimulatory effects of P M A and ethanol in isolated m e m b r a n e s deserve further comments. First, the stimulatory effect of P M A on PtdEtn hydrolysis occurred in the absence of added ATP. In other experiments, addition of 1 m M A T P to m e m b r a n e s failed to potentiate the stimulatory effect of P M A (unpublished data). These data are in agreement with recent findings by Conricode et al. [25] that stimulation of PLD-activity by P M A involves activation of PKC but it does not require an A T P - d e p e n d e n t phosphorylation reaction. Second, isolated m e m b r a n e s from ethanol-treated fibroblasts exhibited enhanced PtdEtn hydrolysis even in the absence of added PKC activators. However, similar to others [26], previously we observed [14] that in fibroblasts scraping causes a transient increase in the cellular content of 1,2-diacylglycerol leading to partial activation of PKC. Consequently, addition of ethanol alone to fibroblasts shortly after scraping caused increased hydrolysis of PtdEtn by a PKC-dependent mechanism [14]. Thus, in the present case, ethanol and 1,2-diacylglycerol (the latter being formed during scraping of cells prior to homogenization) together might have caused alterations in the content a n d / o r activity state of PKC in membranes. This possibility remains to be studied. Recently, ethanol was shown to stimulate PtdCho hydrolysis in hepatocytes by an apparently PKC-independent mechanism [23]. Since ethanol failed to significantly stimulate PtdCho hydrolysis in fibroblasts, it is evident that regulation of phospholipid hydrolysis in these two cellular systems is different. The exact reason for these differences is not known. However, a structural basis for this p h e n o m e n o n may be provided by recent data from Billah's group showing that a variety of cell types contain two different PLD activities exhibiting significant differences in their specificity for PtdCho and PtdEtn [27]. It is possible that PLD activities in hepatocytes and fibroblasts differ in their relative contents, subcellular distribution, and sensitivity to various PKC-isoforms and ethanol. Further experiments are required to determine these possibilities. While the above described effects of ethanol on P L D - m e d i a t e d PtdEtn hydrolysis presumably can oc-

cur in vivo, it remains to be determined whether this mechanism indeed contributes to the pathophysiological effects of ethanol. The very small increases in the cellular contents of phosphatidic acid a n d / o r phosphatidylethanol observed at various concentrations of ethanol are unlikely to have significant effects on signal transduction. It seems more likely that a (perhaps localized) decrease in the cell m e m b r a n e content of PtdEtn might play a role in the mediation of ethanol effects. This latter possibility is supported by observations that important components of the signal transducing system, including the activities of inositol phospholipid-specific phospholipase C [28-31] and PKC [32-34], appear to depend on the cell m e m b r a n e content of PtdEtn. Finally, it is also possible that enhanced PLD-mediated PtdEtn hydrolysis is merely a consequence of the interaction between ethanol and the PKC system, with no relevance to the pathophysiological effects of ethanol. Further studies are required to clarify the role of enhanced PtdEtn hydrolysis in the mediation of in vivo effects of ethanol.

Acknowledgements This work was supported by Grant-In-Aid provided by the University of Minnesota, and by the Hormel Foundation. I am grateful to Mrs. K.S. Crilly for technical assistance and to Mrs. C. Perleberg for secretarial assistance.

References 1 Hoek, J.B., Thomas, A.P., Rubin, R. and Rubin, E. (1987) J. Biol. Chem. 262, 682-691. 2 Rubin, R. and Hoek, J.B. (1988) Biochem. J. 254, 147-153. 3 Rubin, R., Ponnappa, B.C., Thomas, A.P. and Hoek, J.B. (1988) Arch. Biochem. Biophys. 260, 480-492. 4 Rooney, T.A., Hager, R., Rubin, E. and Thomas, A.P. (1989) J. Biol. Chem. 264, 6817-6822. 5 Berridge, M.J. (1987) Ann. Rev. Biochem. 56, 159-193. 6 Nishizuka, Y. (1986) Science 233, 305-312. 7 Kikkawa, V., Kishimoto, A. and Nishizuka, Y. (1989) Ann. Rev. Biochem. 58, 31-44. 8 Exton, J.H. (1990) J. Biol. Chem. 265, 1-4. 9 Billah, M.M. and Anthes, J.C. (1990) Biochem. J. 269, 281-291. 10 Kiss, Z. (1990) Prog. Lipid Res. 29, 141-166. 11 Kiss, Z. and Anderson, W.B. (1989) J. Biol. Chem. 264, 1483-1487. 12 Kiss, Z. and Anderson, W.B. (1990) J. Biol. Chem. 265, 7345-7350. 13 Kiss, Z. and Anderson, W.B. (1989) FEBS Lett. 257, 45-48. 14 Kiss, Z., Crilly, K. and Chattopadhyay, J. (1991) Eur. J. Biochem. 197, 785-790. 15 Kiss, Z. (1991) Lipids 26, 321-323. 16 Cook, S.J. and Wakelam, M.J.O. (1989) Biochem. J. 263, 581-587. 17 Kiss, Z., Chattopadhyay, J. and Pettit, G.R. (1991) Biochem. J. 273, 189-194. 18 Kiss, Z. (1976) Eur. J. Biochem. 67, 557-561. 19 Price, B.D., Morris, J.D.H. and Hall, A. (1989) Biochem. J. 264, 509-515.

94 20 Cook, S.J. and Wakelam, M.J.O. (1991) Biochim. Biophys. Acta 1097, 265-272. 21 Rodriguez-Pena, A. and Rozengurt, E. (1984) Biochem. Biophys. Res. Commun. 120, 1053-1059. 22 Hovis, J.G., Stumpo, D.J., Halsey, D.L. and Blackshear, P.J. (1986) J. Biol. Chem. 261, 10380-10386. 23 Pittner, R.A. and Fain, J.N. (1992) Biochim. Biophys. Acta 1133, 316-320. 24 Hoek, J.B., Thomas, A.P., Rooney, T.A., Higashi, K. and Rubin, E. (1992) FASEB J. 6, 2386-2396. 25 Conricode, K.M., Brewer, K.A. and Exton, J.H. (1992) J. Biol. Chem. 267, 7199-7202. 26 van Veldhoven, P.P. and Bell, R.M. (1988) Biochim. Biophys. Acta 959, 185-196. 27 Wang, P., Anthes, J.C., Siegel, M.I., Egan, R.W. and Billah, M.M. (1991) J. Biol. Chem. 266, 14877-14880.

28 Irvine, R.F., Letcher, A.J. and Dawson, R.M.C. (1984) Biochem. J. 218, 177-185. 29 Wilson, D.B., Bross, T.E., Hoffmann, S.L. and Majerus, P.W. (1984) J. Biol. Chem. 259, 11718-11724. 30 Low, M.G., Carroll, R.C. and Cox, A.C. (1986) Biochem, J. 237, 139-145. 31 Wang, P., Toyoshima, S. and Osawa, T. (1986) J. Biochem. (Tokyo) 100, 1015-1022. 32 Kano-Sueoka, T. and King, D.M. (1988) Cancer Res. 48, 15281532. 33 Kano-Sueoka, T., King, D.M., Fisk, H.A. and Klug, S.J. (1990) J. Cell Physiol, 145, 543-548. 34 Bazzi, M.D., Youakim, M.A. and Nelsestuen, G.L. (1992) Biochemistry 31, 1125-1134.