Phosphatidic acid activation of protein kinase C in LA-N-1 neuroblastoma cells

ELSEVIER

Neuroscience Letters 201 (1995) 199-202

NfUROSCIHCE LETTilIS

Phosphatidic acid activation of protein kinase C in LA-N-1 neuroblastoma cells D o m i n i q u e I , a n g a, A n a n t N. M a l v i y a a, A l p h o n s e H u b s c h a, Julian N. K a n f e r b, L o u i s F r e y s z a,* aLaboratoire de Neurobiologie Mol~culaire des Interactions Cellulaires, Centre de Neurochimie du C.N.R.S., 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France bDepartment of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, Canada Received 19 May 1995; revised version received 25 September 1995; accepted 30 October 1995

Abstract

Phosphatidic acid (PA), a hydrolytic product of phospholipase D activity, stimulated cytosolic protein kinase C (PKC) activity when LA-N-1 neuroblastoma cells in culture were treated with PA, without translocating the enzyme to the membrane. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) translocated and activated PKC in a dogmatic manner. Partially purified PKC activity derived from LA-N-1 neuroblastoma cells was stimulated by PA alone or in the presence of phosphatidylserine or TPA, without affecting [3H]phorbol dibutyrate binding, indicating that the site of action of PA was different from the phorbol ester or diacylglycerol binding site. These resultts suggest an unorthodox pattern of PKC stimulation mediated by PA which appears to be yet another mode of PA signal transduction.

Keywords: Phosphatidic acid; Phorbol ester; Neuroblastoma cells; Protein kinase C; Phospholipases

Protein kinase C (IPKC) activation is a major step in a variety of signalling pathways [21]. PKC constitutes a family of 12 isoenzymes, of which eight members are calcium-independent [9,22]. Diacylglycerol (DAG) was identified as a physiological PKC activator [12]. The DAG is mainly generated by hydrolysis of phosphatidylinositol 4,5-bisphosphate upon phosphoinositide phospholipase C (PLC) activation in response to a variety of growth modulators [24]. DAG is also generated by the activation of phospholipase D (PLD) giving rise to phosphatidic acid (PA). PA is further dephosphorylated by a phosphatidate phosphohydrolase producing DAG. The two-step mechanism of DAG generation in response to growth stimuli has received renewed attention [6]. Furthermore, PA itself serves as a second lipid messenger [15] and is implicated in multiple cellular responses: Ca 2÷ release [23], neutrophil oxidative burst [18], PDGF induced transcriptional event [13], DNA synthesis [19], stimulation of phospho-tyrosine phosphatase [33] and PI-4P kinase [20], inhibition of the p21 Ras GTPaseactivating protein [31], thrombin-incubated actin polym* Corresponding author. Tel.: +33 88 45 66 43; fax: +33 88 61 29 08.

erization [7], PLA 2 activation [26], and PLC-y-1 regulation [11]. The mechanism by which PA is able to modulate these multiple responses remains enigmatic. Since some of these cellular responses triggered by PA are also mediated by PKC, and since a variety of lipids including PA are able to modulate the activity of PKC [16,17,30], we have investigated the mechanism of activation of PKC by PA in the human neuroblastoma cell line LA-N-1 with due comparison to the most accredited PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA). It is well known that diacylglycerol is the physiological analogue of TPA, and the site of DAG binding to PKC is similar but not identical to TPA [3,8]. LA-N-1 cells were cultivated in a Leibovitz's L-15 medium supplemented with 15% fetal calf serum [28]. At confluency (day 8) the medium was replaced by serumfree medium and the cultures treated with TPA (10 -7 M ) or with PA (200/tM). For indicated time, cells were rinsed with NaCI 0.9%, harvested in 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10/~g/ml leupeptin, 25/tg/ml trypsin inhibitor, and homogenized by sonication. The cytosolic and the membrane-bound PKC activity was partially purified

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D. Lang et al. / Neuroscience Letters 201 (1995) 199-202

PKC optimum, added Ca 2+ (1.75 mM) and phosphatidyl-

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effect of TPA in in vitro assay it was necessary to lower the calcium concentration to 1 mM. Thus, 1 mM Ca 2÷, 230/tM PS and TPA (100 n M - 1 / t M ) saved stimulated activity. Over a range of PA concentration studied (100500/~M), it became evident that PA, like PS, activated directly the calcium-dependent PKC activity obtained from LA-N-1 cells homogenate, but to a lesser extent (Fig. 2A). It is interesting to report that no effect of PA on

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TIME (rnin) Fig. 1. In vivo effect of PA and TPA on PKC activity in LA-N-1 neuroblastoma cells. At confluency (day 8) the medium was replaced by serum-free medium and the cultures were treated at the indicated time with TPA (10 -7 M) or with PA (200/~M). Cells were rinsed with NaC1 0.9%, harvested in 20 mM Tris-HC1, pH 7.5, 2 mM EDTA, 2 mM PMSF, 10/zg/ml leupeptin, 25/zg/ml trypsin inhibitor, and homogenized by sonication. The cytosolic and the membrane-bound PKC were purified from the cell homogenates as reported by Block et al. [1]. The standard PKC assay medium contained 20 mM Tris-HC1, pH 7.5, 1 mM EGTA, 1.0 mM CaC12, 5.0 mM MgC12, 2.0 mM PMSF, 230/.tM PS, 20/~g Histone (type III, Sigma), 20/~M ATP, and 3-5 x 105 cpm of 7-[32p]ATP; the final incubation volume was 100/~1. Incubation was performed at 30°C for 10 min. The activity of cytosolic (O) or particulate (e) PKC was evaluated by subtracting the activity measured in the absence of phosphatidylserine from the activity determined in the presence of calcium and PS. Each value represents the mean -+ SEM of two experiments in triplicate.

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by a single step DEAE-cellulose chromatography as reported by Block et al. [1]. Fig. 1A illustrates the time course of translocation/ activation of the Ca2÷-dependent PKC upon treatment of LA-N-1 neuroblastoma cells in culture with TPA (10 -7 M). The action of TPA was apparent by 10 min and maximum translocation of PKC from the cytosol to the particulate compartment was seen by 30 min. In sharp distinction from TPA, the treatment of cells with PA (200/~M) did not elicit any translocation of the PKC to the membrane, but activated specifically the cytosolic enzyme (Fig. 1B). This prompted us to examine the effect of PA on PKC in an in vitro assay system to establish whether PA, or its metabolic products like DAG or free fatty acids, were the stimulating factors. Partially purified PKC was prepared from confluent LA-N-1 cultures [ 1] and assayed in a standard incubation medium in the presence of calcium. For determining the

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Fig. 2. In vitro effect of PA and TPA on LA-N-I PKC activity. Partially purified PKC from LA-N-I homogenate was obtained after harvesting the cells in 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 2 m M PMSF, 10/.tg/ml leupeptin, and 25/.tg/ml trypsin inhibitor, sonicated. After addition of 1% Triton X-100 (final concentration), the solution was mixed for 30 min at 4°C and centrifuged for 20 min at 100 000 x g. Partial purification of the PKC was performed as previously described (Block et al. [1]). The effect of PA on PKC activity was determined in the presence of various combinations of PS (230/.tM), TPA (100 nM), and PA (100-500/.tM) as described in the legend to Fig 1. Results represent the mean _+ SEM of two experiments in triplicate. *P < 0.05, **P < 0.01, compared with the corresponding value obtained in the absence of TPA.

D. Lang et al. /Neuroscience Letters 201 (1995) 199-202

Table 1. Effect of TPA on LA-N-1 PKC activity in the presence of PS or PA Addition

Ca 2+ (1 mM), PS (230/~M) (pmol/ min per mg protein)

Ca 2+ (1 mM), PA (200/~M) (pmol/ min per mg protein)

None TPA (100 nM) TPA (200 nM) TPA (400 nM) TPA (1000 nM)

1957 3298 2656 2887 3391

1153 2862 2845 2922

4_:316 4_:350 4_:320 -+ 260 4:400

_+ 153 ± 300 ± 225 ± 254

Partially purified PKC activity was determined as described in the legend to Fig. 2. Specific PKC activity was obtained by subtracting the value in the presence of calcium alone from the activities obtained in the presence of the various combinations of eofactors as indicated. Results are means .+ SEM of two experiments in triplicate.

PKC activity was observed in the absence of calcium (data not shown). Furthermore, in the presence of PA the addition of TPA increased the PKC activation 1.7-fold (1.4-2.0) as compared with the enzyme activity without the added TPA in the incubation medium (Fig. 2A). Maximal stimulation was obtained with 100 nM TPA. Increasing TPA concentration did not further activate the PKC (Table 1). An unexpected result was obtained when the effect of PA on PKC activity was assayed in the presence of optimal concentration of PS (Fig. 2B). At an optimal PS concentratic,n, PA was able to stimulate PKC activity in vitro. In fact, the PKC activity determined in the presence of PS and PA was comparable to the extent of activity seen with PS and TPA as cofactors. However, in the presence of PS and PA, the addition of TPA did not further increased the activity of the calcium-dependent PKC (Fig. 2B). These results indicate that PA can substitute for TPA directly iln eliciting its effect on PKC stimulation. Taken together, these results suggest also that PA did not interact with the TPA binding site of the enzyme. This found further confirmation by comparing the effect of PA and TPA on the [3H]phorbol dibutyrate ([3H]PDBu) binding. Table 2 shows that PA did not inhibit the [3H]PDBu bound to the partially purified enTable 2 Effect of PA on specific [3H]PDBu binding Addition

Specific [3H]PDBu binding pmol/mg protein (%)

Control PA (200/tM) PA (500pM)

15.0 __. 1.8 (100) 11.0 __.2.2 (80) 14.9 _+ 1.4 (97)

[3H]PDBu binding was detcrmined in the presence of calcium and PS as reported by Sharkey et al. [27]. Specific binding is defined as the total binding subtracting frc,m the non-specific binding (in the presence of 100/.tM TPA). Values are means .+ SEM of two experiments in triplicate. Student's t-test showed no significant variation between PA and control-PDBu binding experiments.

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zyme, whereas phorbol dibutyrate binding was inhibited by TPA. Similar results have been reported for endothelial cells PKC [30], indicating that PA did not interact with the TPA or DAG binding site. These results, and the fact that PA did not interact with the TPA site, indicate that PA activates PKC in vitro by a mechanism distinct from that of PS/TPA, and suggest that PA may have two sites of interaction with PKC. One would be a site interacting with acidic lipids, since PAlike free fatty acids [17,5] mimic the PS requirement and act in synergy with TPA. Whether the PA site of action corresponds to the PS site is not understood at this stage. The observation that PA and PS together were able to activate PKC analogous to the PS/TPA activation, suggests that the enzyme may have a yet unidentified site where PA may intervene. Such a viewpoint is in agreement with a recent finding indicating that PKC has different DAG and phorbol esters binding sites [29]. The active-lipids binding sites remain to be explored and will require some further investigations. Previous studies [16] showed that PA was able to stimulate PKC a in the presence of calcium and PKC ~ in the absence of calcium. It is known that PS levels in the cell remain unchanged, whereas PA levels in vivo are subject to variations upon cellular stimulation involving PLD activation. From this viewpoint, for the regulation of PKC, the cellular changes in PA seem of great importance. Treatment of cultured LA-N-1 cells with PA activated preferentially the cytosolic PKC and did not trigger any translocation of the enzyme to the membrane, thereby indicating that PA may have a physiological role in activating soluble PKC. This is in agreement with the observation of Bocckino et al. [2], who showed that in the liver and the heart homogenates, PA provided markedly different profiles of protein phosphorylation as compared with PS in association with DAG. In conclusion, we may say that in LA-N-1 neuroblastoma cells, PA stimulated the cytosolic calcium-phospholipid-dependent PKC activity without any translocation to membrane. This seems to be yet another potential mechanism for accentuating PA-mediated signal transduction. Precedent exists for PA-specific substantial stimulation of phosphatidylinositol kinases [10] and PLC-7-1 [ 11 ]. It is tempting to suggest that PA may cause a redistribution of PKC from the membrane to the cytosol and activate the cytosolic enzyme as a direct effect of PA. However, it cannot be excluded that in vivo, PA may activate cytosolic phospholipase A [32], which in turn generates free fatty acids and lysophosphatidic acid, thereby stimulating cytosolic PKC [25]. Nevertheless, the activation of native membranous pool of PKC has recently been shown to be regulated by physiological stimuli [4], in sharp contrast to the common belief that one of the prerequisites for PKC activation is the translocation of the enzyme from cytosol to the membrane [14]. In this re-

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