INTRODUCTION

The hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP₂),¹ by a specific phospholipase C (PLC) is one of the earliest key events in the regulation of cellular function by more than 100 different extracellular signaling molecules (reviewed by Noh et al. (1995)). This reaction generates two intracellular messengers: inositol 1,4,5-trisphosphate (IP₃), which induces the release of Ca²⁺ from internal stores, and diacylglycerol, which activates protein kinase C.

Ten mammalian isoforms of PLC have been identified to date, and these can be divided into  type (PLC-1, -2, -3, and -4),  type (PLC-1 and -2), and  type (PLC-1, -2, -3, and -4) enzymes on the basis of amino acid sequence (Noh et al., 1995). The distinct structural features of the different PLC types have been related to specific mechanisms of receptor-mediated enzyme activation. Thus, PLC- isozymes are activated by tyrosine phosphorylation, and PLC- isozymes are activated by heterotrimeric G proteins (Noh et al., 1995); the mechanism of PLC- isozyme activation is not known.

PLC hydrolyzes phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate as well as the physiological substrate, PIP₂, in vitro, with PI-hydrolyzing activity often measured during PLC purification. While purifying PLC-1, the most abundant PLC isoform in brain cytosol, we observed that the PI-hydrolyzing activity of crude brain cytosol decreased more than expected on dilution. Furthermore, addition of crude cytosol to purified PLC-1 markedly enhanced PI-hydrolyzing activity. These observations suggested that brain cytosol contains a component that can enhance the activity of PLC-1 toward PI.

We now describe the purification of this activator and its identification as the microtubule-associated protein tau. We also show that tau enhances the activity of PLC-1 toward PIP₂ to a markedly lesser extent than that apparent with PI and that the effect of tau on PLC- activity toward PIP₂ is greatly increased in the presence of arachidonic acid (AA). Of the three types of PLC, the  type isozymes are most sensitive to activation by tau and AA. These observations suggest that AA, the generation of which is mediated by receptor-activated phospholipase A₂ (PLA₂), can serve as a link between the PLA₂ and PLC pathways and, together with tau, activate PLC- isozymes in the absence of receptor-mediated tyrosine phosphorylation.

EXPERIMENTAL PROCEDURES

Materials

Phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were purchased from Avanti Polar Lipids. Arachidonic acid and cholesterol were purchased from Calbiochem. PI and PIP₂ were purchased from Sigma and Boehringer Mannheim, respectively. [³H]PI and [³H]PIP₂ were purchased from New England Nuclear. Preparative DEAE-5PW (21.5 × 150 mm), analytical phenyl-5PW (7.5 × 75 mm), and analytical heparin-5PW (7.5 × 75 mm) were purchased from Toso Haas Inc.

PLC Isozymes

PLC isozymes (PLC-1, -2, -1, -2, -1, and -2) were purified from HeLa cells that had been transfected with recombinant vaccinia virus containing the entire coding sequence of the respective enzyme as described (Park et al., 1992).

Purification of Activator

All manipulations were performed at 4-6 °C in a refrigerated room or on ice, unless otherwise indicated. During purification, PLC-1-activating activity was measured at 37 °C for 5 min in 200 µl of a reaction mixture containing 20,000 cpm of [³H]PI (DuPont NEN), 150 µM soybean PI (Sigma), PLC-1 (20-50 ng), 3 mM CaCl₂, 2 mM EGTA, 0.1% (w/v) sodium deoxycholate, 50 mM Hepes-NaOH (pH 7.0), and a source of activator. To maintain the stimulated activity in the linear range of the assay, we adjusted the amount of PLC to obtain an unstimulated, basal activity in the range of 500-1200 cpm of inositol 1-phosphate generated. The purification procedure consisted of the following steps.

Fifteen fresh bovine brains (total of 4.5 kg of tissue) were obtained from a local slaughter house and homogenized in 10 liters of a solution containing 10 mM Tris-HCl (pH 7.4), 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol (DTT), leupeptin (2 µg/ml), and aprotinin (2 µg/ml) in a Waring blender. The homogenate was centrifuged at 1000 × g for 10 min, and the resulting supernatant was centrifuged further at 13,000 × g for 1 h. The second supernatant was adjusted to 60% saturation with ammonium sulfate and then centrifuged for 30 min at 13,000 × g. The resulting precipitate was stored at 70 °C.

The frozen ammonium sulfate precipitate was thawed and resuspended by adding 2 volumes of distilled water, heated at 95 °C for 5 min, and centrifuged at 1000 × g for 15 min. The resulting supernatant was filtered through Whatman no. 1 paper, and the filtrate was adjusted to 5% (w/v) trichloroacetic acid and centrifuged at 17,000 × g for 10 min. The resulting pellet was immediately resuspended in 20 ml of 1 M Tris-HCl (pH 8.4) and dialyzed extensively against a solution containing 50 mM Tris-HCl (pH 7.4), 1 mM EGTA, and 0.1 mM DTT. After dialysis, insoluble materials were removed by centrifugation at 75,000 × g for 10 min.

The final supernatant (420 mg of protein) from the previous step was divided into two equal portions, each of which was applied to a preparative TSKgel DEAE-5PW HPLC column (21.5 × 150 mm) that had been equilibrated with 50 mM Tris-HCl (pH 7.4), 1 mM EGTA, and 0.1 mM DTT. Proteins were eluted at a flow rate of 5 ml/min by washing with equilibration buffer for 5 min and then applying a linear gradient of 0-0.3 M NaCl over 35 min and a second linear gradient of 0.3-1 M NaCl over 5 min. Fractions (5 ml) were collected and assayed for PLC-1-activating activity. Peak fractions (25-27) from the two identical runs were combined.

Solid KCl was added to the pooled fractions (40 mg of protein) from the previous step to a final salt concentration of 3 M. Insoluble material was removed by centrifugation, and the resulting supernatant was injected into an analytic TSKgel phenyl-5PW HPLC column (7.5 × 75 mm) that had been equilibrated with 20 mM Hepes-NaOH (pH 7.0), 3 M NaCl, 1 mM EGTA, and 0.1 mM DTT. Proteins were eluted at a flow rate of 1 ml/min by sequential application of equilibration buffer for 5 min and consecutive decreasing linear gradients of 3-1.2 M NaCl over 10 min and 1.2 to 0 M NaCl over 25 min. Fractions (1 ml) were collected and assayed for activator activity. Peak fractions (26 and 27) were pooled and washed with 20 mM Hepes-NaOH (pH 7.0), 1 mM EGTA, and 0.1 mM DTT in a Centriprep-30 (Amicon) concentrator to lower the salt concentration to <0.1 M.

The washed fraction (4 mg of protein) from the previous column was applied to a TSKgel heparin-5PW column (7.5 × 75 mm) that had been equilibrated with 20 mM Hepes-NaOH (pH 7.0), 1 mM EGTA, and 0.1 mM DTT. Proteins were eluted at a flow rate of 1 ml/min by sequential application of equilibration buffer for 15 min and linear NaCl gradients of 0-0.64 M over 40 min and 0.64 to 1 M over 10 min. Fractions (1 ml) were collected and assayed for activator activity. Peak fractions (32 and 33) were pooled, concentrated, divided into portions, and stored at 70 °C.

Purified proteins from the heparin column (700 µg) were separated by preparative SDS-PAGE on an 8% gel (3-mm thickness, single-well comb). The gel was stained lightly with Coomassie Brilliant Blue, and visualized protein bands were excised from the gel with a razor blade. The proteins were subsequently eluted with an Electro-Eluter (C.B.S. Scientific, Del Mar, CA), after which Coomassie Brilliant Blue was extracted with isobutanol and SDS was removed by precipitation with ice-cold acetone.

Cyanogen Bromide Cleavage and Amino Acid Sequencing

Proteins (10 µg each) electroeluted from three different bands (bands 1, 2, and 3 in Fig. 3A) were subjected to chemical cleavage with 100 mM CNBr in the presence of 70% (v/v) formic acid for 16 h. The reaction was quenched by adding excess methionine crystals to the reaction mixture. The cleaved products were dried under vaccum, resuspended in 50 mM Tris-HCl (pH 8.4) and subjected to HPLC analysis on Vydac C₁₈ column (4.6 × 250 mm) that had been equilibrated with 0.05% (w/v) trifluoroacetic acid. Peptides were eluted at a flow rate of 1 ml/min by sequential application of equilibration buffer for 20 min and consecutive linear acetonitrile gradients of 0 to 50% (v/v) in 0.05% trifluoroacetic acid over 50 min and 50-100% over 10 min. Two peptides that eluted at 40.2 and 44.5 min and were common to the three elution profiles obtained with bands 1, 2, and 3 were subjected to sequence analysis.

RESULTS

PLC-1-activating Protein in Bovine Brain Cytosol

The addition of crude bovine brain cytosol to purified PLC-1 increased the PI-hydrolyzing activity of the enzyme 15-fold (Fig. 1), suggesting the presence of an activator in brain extract. This putative activator appeared to be a protein that is stable to treatment with heat or acid. Activator activity in the cytosol was (i) largely unaffected by heating for 5 min at 95 °C; (ii) recovered in the 5% (w/v) trichloroacetic acid precipitate of the extract; (iii) resistant to treatment with DNase or RNase; (iv) susceptible to chymotrypsin treatment; and (v) retained by a membrane with a size cut-off of 30 kDa. (Fig. 1).

Effect of bovine brain cytosol on PLC-1 activity.

The heat and acid stability of the PLC-1-activating protein allowed us to obtain a preparation highly enriched in the activator by submitting a 60% saturated ammonium sulfate fraction of crude brain cytosol to these treatments. This enriched preparation was then subjected to HPLC on successive DEAE, phenyl, and heparin columns (Fig. 2). SDS-PAGE of the peak fractions from the final column revealed five closely spaced protein bands with apparent molecular sizes of 48-62 kDa and two additional bands of <43 kDa (Fig. 3A). Pooling peak fractions more narrowly or further purification of the heparin column fractions on an HPLC gel filtration column (TSKgel G3000-SW) or an HPLC Mono Q column also resulted in multiple protein bands similar to those shown in Fig. 3A on SDS-PAGE analysis (data not shown).

Purification of PLC-1-activating protein.

Each of the five protein bands between 48 and 62 kDa was excised from the polyacrylamide gel and electroeluted. After removal of SDS, each eluted protein was tested for PLC-1-activating activity. All of the eluted proteins activated PLC-1 (Fig. 3B).

Identification of the PLC-1 Activator as Tau

Three of the electroeluted proteins (bands 1, 2, and 3) were individually cleaved with CNBr and analyzed on a C₁₈ HPLC column. The three elution profiles were similar (data not shown), suggesting that the three proteins are related. Two peptides that eluted at 40.2 and 44.5 min and that were common to all three proteins were sequenced. The peptides yielded sequences of EDHAQGDYTLQDQEGD and VSKGKDGTGPDDKKTK, respectively, both of which showed a perfect match to bovine brain tau sequences encoded by exon 1 and 5, respectively (Fig. 4A).

Identification of the PLC-1 activator as tau.

The pooled heparin column peak fraction and electroeluted proteins were subjected to immunoblot analysis with a monoclonal antibody to tau (Fig. 4B). Not only the proteins with molecular sizes between 48 and 62 kDa (bands 1 to 5) but also proteins smaller than 43 kDa were recognized by the antibody, suggesting that nearly all of the proteins in the heparin column fraction are either tau isoforms or their proteolytic fragments. Furthermore, tau proteins purified from bovine brain by the standard procedure (Baudier et al., 1987) yielded multiple bands on SDS-PAGE and activated PLC-1 with a potency and efficacy similar to those of the heparin column fraction (Fig. 4C). The concentration dependence profile showed that half-maximal activation of PLC-1 was achieved at ~0.2 µM tau (data not shown).

All PLC isozymes require Ca²⁺ for catalysis, but the sensitivity to Ca²⁺ varies with specific isozyme and substrate. The effect of tau on PLC-1, PLC-1, and PLC-1, representatives of each type of PLC, was evaluated with PI or PIP₂ as substrates at various Ca²⁺ concentrations (Fig. 5). At Ca²⁺ concentrations of >0.1 µM, tau proteins increased the PI-hydrolyzing activity of PLC-1 by up to 15-20-fold; in contrast, tau had no marked effect on the PI-hydrolyzing activity of PLC-1 and induced only a 3-4-fold increase in that of PLC-1 at Ca²⁺ concentrations above 10 µM. Tau did not have a marked effect on the PIP₂-hydrolyzing activity of any of the three PLC isozymes; only an approximately 2-fold activation was observed for all three enzymes at high Ca²⁺ concentrations.

Effects of Various Fatty Acids on the Activation of PLC-1 by Tau

Irvine et al. (1979) showed that unsaturated fatty acids such as oleic acid and AA stimulated PLC activity in crude brain cytosol with a [³H]inositol-labeled microsomal fraction from rat liver as substrate. However, such unsaturated fatty acids do not directly affect the activities of purified PLC isozymes, including that of PLC-1.² The brain cytosol preparation used by Irvine et al. (1979), likely contained tau proteins in addition to PLC-1, the most abundant PLC isozyme in brain cytosol, and tau may thus have mediated the effect of unsaturated fatty acids on PLC activity.

We therefore investigated the effect of AA on PI and PIP₂ hydrolysis catalyzed by PLC-1. We included PE, PS, and cholesterol in the substrate to mimic the composition of cell membranes. In addition, equal concentrations of both [³H]PI and [³H]PIP₂ were added in the substrate, which thus comprised PI, PIP₂, PS, cholesterol, and PE in a molar ratio of 1:1:1:1:4, and [³H]inositol 1-phosphate and [³H]IP₃ generated by PLC-1 were separated by ion-exchange chromatography in order to compare directly the effects of tau and AA on PI hydrolysis and on PIP₂ hydrolysis. PC was not included in the substrate because it inhibits tau-dependent activation of PLC-1 (see below). AA affected neither the PI- nor the PIP₂-hydrolyzing activity of PLC-1 in the absence of tau, but it increased both activities in a concentration-dependent manner in the presence of tau (Fig. 6). For both activities, the extent of activation was a maximal at 25 µM AA and decreased at higher concentrations; a similar decrease at higher concentrations of unsaturated fatty acid was also observed in the experiment of Irvine et al. (1979).

Effects of AA and tau on the activity of PLC-1 toward a mixed micellar substrate containing PI, PIP

The dependence of the PIP₂-hydrolyzing activity of PLC-1 on tau concentration was examined with the mixed micellar substrate containing AA. Maximal activation was apparent at 0.15 µM tau, in contrast to the tau concentration of 0.5 µM required for maximal activation of PI hydrolysis in the absence of AA and other lipids (data not shown). We also examined the effects of fatty acids other than AA on PIP₂ hydrolysis by PLC-1 in the presence of 0.3 µM tau proteins (Fig. 7). Unsaturated fatty acids, including AA, linolenic acid, linoleic acid, oleic acid, and palmitoleic acid, stimulated PIP₂-hydrolyzing activity in the presence, but not in the absence, of tau. In contrast, the corresponding saturated fatty acids (arachidic acid, stearic acid, and palmitic acid) had no effect on PIP₂-hydrolyzing activity in the absence or the presence of tau.

-hydrolyzing activity of PLC-1.

Isozyme Specificity of Tau- and AA-dependent Activation of PIP₂ Hydrolysis by PLC-1

The combined effects of tau and AA on PIP₂ hydrolysis by PLC-1, -2, -1, -2, -1, and -2 were compared (Fig. 8). At 0.1 µM Ca²⁺, marked effects of tau and AA were apparent only with PLC-1 and PLC-2 (Fig. 8A); however, at 1 µM Ca²⁺, tau and AA stimulated the PLC- isozymes to activity levels 30-50% of those apparent with the PLC- isozymes (Fig. 8B).

Effect of PC on Tau- and AA-dependent PLC-1 Activity

The addition of PC to the mixed micellar substrate containing [³H]PI, [³H]PIP₂, PS, cholesterol, AA, and PE in a molar ratio of 1:1:1:1:1:4 (where 1 corresponds to a concentration of 30 µM) resulted in a concentration-dependent inhibition of both PI and PIP₂ hydrolysis by PLC-1 in the presence of tau (Fig. 9A). Half-maximal inhibition was apparent at 30-40 µM PC. No inhibiting effect of PC was observed in the absence of tau. Lyso-PC (50 µM) inhibited PIP₂ hydrolysis by PLC-1 in the absence or the presence of tau by <10% (data not shown).

Effect of PC on tau- and AA-dependent PLC-1 activity.

Thus the conversion of inhibitory PC to lyso-PC and AA by a PLA₂ enzyme might constitute a signal for the activation of PLC- in the presence of tau. We evaluated this hypothesis with mixed micellar substrates containing 30 µM each of [³H]PIP₂, PS, and cholesterol, 120 µM PE, and various concentrations of PC and AA (Fig. 9B). The initial concentrations of AA and PC in the mixed micelle were 0 and 90 µM, respectively. The concentration of AA was then increased incrementally to 90 µM and that of PC was decreased to 0 µM, with the total concentration of both agents maintained constant at 90 µM. Marked activation of PIP₂ hydrolysis was not apparent until the PC concentration decreased below 70 µM and the AA concentration increased above 20 µM. Maximal activity was apparent when both PC and AA were present at 45 µM; at higher concentrations of AA, the activity decreased.

DISCUSSION

We have purified proteins that enhance the activity of PLC-1 toward a micellar substrate containing PI and deoxycholate and identified them as tau isoforms. Tau comprises a family of microtubule-associated proteins that are generated from alternatively spliced transcripts derived from a single gene with 13 exons (reviewed by Lee (1990)). Tau expression is largely restricted to brain and is developmentally regulated. Six different cDNAs capable of encoding isoforms comprised of between 304 and 448 residues have been isolated for bovine tau; these correspond to mRNA species lacking one or more of exons 3, 6, 8, and 10 (Himmer, 1989).

Tau proteins did not markedly increase PLC-1 activity toward micellar PIP₂. Furthermore, when common lipid components of membranes (PE, PS, and cholesterol) were incorporated into micelles, activation of PLC-1 by tau was not apparent with either the PI or PIP₂ as substrate. The addition of an unsaturated fatty acid to the substrate restored tau-dependent activation of both PI and PIP₂ hydrolysis at low Ca²⁺ concentrations. Of the unsaturated fatty acids tested, AA was the efficacious activator, and efficacy decreased in the rank order palmitoleic acid (16:1) > linolenic acid (18:3) > linoleic acid (18:2) > oleic acid (18:1). The corresponding saturated fatty acids, arachidic acid (20:0), stearic acid (18:0), and palmitic acid (16:0) were ineffective. Maximal (15-20-fold) activation of PLC-1 toward PIP₂ in micelles containing 30 µM each of PIP₂, PS, and cholesterol and 120 µM PE was observed at 0.15 µM tau and 25 µM AA.

Several studies have examined the effects of lipids on PLC activity. Uunsaturated fatty acids were shown to increase PLC activity in rat brain cytosol ~10-fold (Irvine et al., 1979). The effect was probably attributable to the presence of both tau and PLC-1 in the brain cytosol. An approximately 3-4-fold activation by unsaturated fatty acids of a 68-kDa PLC purified from rat liver cytosol was observed when the hydrolysis of micellar PI was measured in the presence of 2 mM Ca²⁺ (Takenawa and Nagai, 1981). It is now thought that the 68-kDa enzyme was a proteolytic fragment of PLC-1 (Taylor et al., 1992). Saturated fatty acids had no effect on brain or liver PLC activity.

Abundunt membrane phospholipids such as PC, PE, and PS were shown to have no marked effect on the activities of PLC- isozymes (James et al., 1995) and PLC-1 (Jones and Carpenter, 1993) in detailed kinetic studies performed with mixed micellar PIP₂ substrates. An approximately 3-fold activation by PS was observed for PLC-1, PLC-1, and PLC-1 with a monolayer substrate containing PIP₂, whereas PC had no effect (Boguslavsky et al., 1994). However, marked inhibition of the PLC-1 activity by PC was observed with PI presented as small unilamellar vesicles (Hofmann and Majerus, 1982). These studies suggest that the activity of PLC, like that of many enzymes that act on lipid substrates, depends on the composition and physical condition of the substrate.

In the present study, PC had no significant effect on basal PLC-1 activity but markedly inhibited activity stimulated by tau and AA. This observation suggested that the activation of PLC-1 by tau might be facilitated by a concomitant decrease in PC concentration and increase in AA concentration, both of which occur in cells on activation of the 85-kDa cytosolic PLA₂ (cPLA₂) that is known to be coupled to various receptors (reviewed by Dennis (1994) and by Kramer (1994); Clark et al. (1991)). This enzyme requires submicromolar concentrations of Ca²⁺ and preferentially hydrolyzes PC with unsaturated fatty acids in the sn-2 position: The rank order of preference for sn-2 acyl chains is 20:4 > 18:3 > 18:2 > 18:1 > 16:1, and the preference order for C₂₀ acyl chains is 20:4 > 20:3 > 20:2 > 20:1 > 20:0 (Hanel et al., 1993). In contrast, secreted PLA₂ enzymes with molecular sizes of 13-18 kDa require millimolar concentrations of Ca²⁺ for catalytic activity, show a preference for PE, and are nonselective with regard to sn-2 fatty acids. A 40-kDa Ca²⁺-independent PLA₂ identified in myocardium preferentially hydrolyzes AA-containing PC (Hazen et al., 1990), whereas an 80-kDa Ca²⁺-independent PLA₂ from macrophages lacks specificity for AA-containing lipids (Dennis, 1994).

Submicromolar concentrations of Ca²⁺ are required for the translocation of cPLA₂ to membranes rather than for catalytic activity and this translocation is a prerequisite for activation (Clark et al., 1991; Sharp et al., 1991). Activation of cPLA₂ may occur secondarily to receptor-mediated activation of a PLC that results in an increase in the cytosolic Ca²⁺ concentration (Kramer, 1994). Initial activation of a PLC- isozyme, for example, in response to ligand occupancy of a G protein-coupled receptor may thus result in an increase in intracellular Ca²⁺, which in turn results in activation of cPLA₂ and subsequent activation of PLC- isozymes. Therefore, activation by the combined action of tau and AA may represent a mechanism by which PLC- isozymes can be activated independently of tyrosine phosphorylation. Jones and Carpenter (1993) observed that incorporation of phosphatidic acid into a micellar substrate containing PIP₂ and Triton X-100 enhanced PLC-1 activity 40-fold; they therefore proposed that PLC-1 can be activated independently of tyrosine phosphorylation if phosphatidic acid is generated by the action of phospholipase D.

Evidence also suggests that the activation of cPLA₂ may occur at basal cytosolic Ca²⁺ concentration; that is, independently of PLC-mediated IP₃ generation (Currie et al., 1992; Kast et al., 1993). The addition of bombesin to Swiss 3T3 cells resulted in the rapid (within 2 s) release of AA and concomitant depletion of PC, without effects on other phospholipids (Currie et al., 1992). The initial AA release was dependent on neither the influx of extracellular Ca²⁺ nor the mobilization of intracelluar Ca²⁺ by IP₃. Furthermore, the increased concentration of AA was sustained over several minutes, whereas the increase in lyso-PC was more transitory. In another study, the association of cPLA₂ with membranes, the increase in cPLA₂ activity, and the liberation of AA in HEL-30 cells treated with tumor necrosis factor- were all independent of PLC activation (Kast et al., 1993). Thus, ligation of receptors that are directly coupled to cPLA₂ but not to PLC may induce PIP₂ breakdown by stimulation of PLC- isozymes indirectly through tau and AA.

Several studies are consistent with the notion that stimulation of PLC by endogenously released AA occurs in cells. Incubation of human trophoblasts with AA stimulates PLC activity (Zeitler and Handwerger, 1985). Further studies with these cells suggested that the stimulation of phosphoinositide metabolism and placental lactogen release are mediated by initial activation of PLA₂ (Zeitler et al., 1991). AA but not other biologically important fatty acids stimulates phosphoinositide metabolism in and catecholamine release from bovine adrenal chromaffin cells (Negishi et al., 1990). AA was also shown to increase phosphoinositide breakdown and glutamate release in rat hippocampal tissue (Lynch and Voss, 1990), to induce phosphoinositide breakdown and diacylglycerol generation in human platelets (Siess et al., 1983), and to increase intracellular Ca²⁺ by mobilizing an IP₃-sensitive Ca²⁺ pool in isolated rat pancreatic islets (Wolf et al., 1986) and a human leukemic T cell line (Chow and Jondal, 1990). The AA-induced Ca²⁺ release was shown to be not due to the metabolites of AA (Wolf et al., 1986)

In addition to serving as a precursor for the biosynthesis of prostaglandins, thromboxanes, leukotrienes, and other eicosanoids, AA has been proposed to act as a modulator or second messenger in signal transduction (reviewed by Sumida et al., 1993). AA and other unsaturated fatty acids activate protein kinase C directly (reviewed by Nishizuka (1992); McPhail et al. (1984); Touny et al. (1990)). Furthermore, AA has been shown to activate guanylate cyclase (Gerzer et al., 1986) and to inhibit both Ca²⁺-calmodulin-dependent protein kinase (Piomelli et al., 1989) and guanine nucleotide binding to the  subunit of G_z (Glick et al., 1996). The effective concentrations of AA in these studies were in the range of 10⁴ M. Although it is difficult to determine the local concentration of released AA at a precise moment in time, intracellular concentrations of 50-100 µM have been measured in activated cells (Wolf et al., 1986; Nishikawa et al., 1988).

Tau proteins are predominantly expressed in neuronal tissue (Lee 1990; Mandelkow and Mandelkow, 1993). However, the above examples of the potential linkage between AA and PLC activation include both neuronal and non-neuronal cells. Furthermore, we have shown that non-neuronal tissues also contain protein components that can activate PLC-1.² Such activating proteins purified from bovine lung are also resistant to heat and acid treatment, exhibit extensive size heterogeneity, and activate PLC- isozymes relatively specifically in the presence but not in the absence of AA.² However, the lung proteins are larger than tau proteins and are not recognized by antibodies to tau. These observations suggest that the putative linkage of cPLA₂ activation to PLC- activation may not be restricted to neuronal cells.

Activation by tau and AA was relatively specific for PLC- isozymes at physiological (submicromolar) concentrations of Ca²⁺. This specificity may be attributable to the unique structural features of PLC- isozymes. Unlike PLC- and PLC- isozymes, PLC- isozymes each contain a Src homology 3 (SH3) domain, which is characterized by the ability to bind proline-rich sequences. Tau proteins possess several sequences rich in proline; two sequences, PTPPTR and RTPPKSP, encoded by exon 9 are similar to the two classes of consensus SH3-binding sequences, PLXR and RXLPX (critical prolines are underlined; X indicates any amino acid; other residues are partially conserved), respectively (Feng et al., 1994). The two consensus sequences were derived for the Src and phosphatidylinositol 3-kinase SH3 domains and may differ from that for the PLC- SH3 domain. However, attempts to co-immunoprecipitate PLC-1 and tau from bovine brain cytosol were not successful (data not shown). It is also of interest that the neurofibrillary tangles typical of the brains of individuals with Alzheimer's disease consist largely of tau proteins that are abnormally phosphorylated, probably by microtubule-associated protein kinase and glycogen synthase kinase 3, at Ser-Pro and Thr-Pro motifs (reviewed by Mandelkow and Mandelkow (1993)). Tau contains 17 Ser-Pro and Thr-Pro motifs, three of which are present in the putative SH3-binding sequences PTPPTR and RTPPKSP. It is possible that phosphorylation of these sites alters interaction of tau with PLC-, thereby undermining the PLA₂-PLC linkage, in brains affected by Alzheimer's disease.

All PLC isozymes have a pleckstrin homology (PH) domain near their amino terminus (Noh et al., 1995). PLC- isozymes, unlike other PLC isoforms, possess another PH domain that is split by the SH domain. Although one function of PH domains appears to be to bind PIP₂ (Harlan et al., 1994), alignment of 92 such domains identified to date revealed marked sequence diversity, and there is neither a conserved surface patch nor a cavity in the known structures that could help identify regions crucial for a common function (Hyvönen et al., 1995). The overall topology of the PH domain has been suggested to be similar to those of fatty acid-binding proteins (Yoon et al., 1994). It is therefore possible that AA interacts with one of the two PH domains of PLC- and cooperates with tau bound to the SH3 domain to enhance enzyme activity.

In conclusion, our observation that tau proteins together with AA activate PLC- activity in vitro suggests that receptor-mediated activation of cPLA₂ might result in the activation of PLC- in neuronal cells. Such a link between the two phospholipase pathways could provide for activation of phosphoinositide metabolism in the absence of or in coordination with direct receptor-mediated stimulation of a PLC enzyme.