Activation of Phospholipase C-γ by the Concerted Action of Tau Proteins and Arachidonic Acid

Abstract

Phospholipase C-γ (PLC-γ) isozymes are thought to be activated by receptor-induced tyrosine phosphorylation. Proteins that activate PLC-γ1 have now been purified from bovine brain and identified as members of the tau family of microtubule-associated proteins. Activation of PLC-γ by tau was enhanced in the presence of unsaturated fatty acids such as arachidonic acid, saturated fatty acids being ineffective. Maximal (15-20-fold) activation was apparent in the presence of 0.15 µM tau and 25 µM arachidonic acid (AA). The effect of tau and AA was specific to PLC-γ isozymes in the presence of submicromolar concentrations of Ca2+ and was markedly inhibited by phosphatidylcholine. These results suggest that in cells that express tau, receptors coupled to cytosolic phospholipase A2 may activate PLC-γ isozymes indirectly in the absence of tyrosine phosphorylation through the hydrolysis of phosphatidylcholine to generate AA. Phospholipase C-γ (PLC-γ) isozymes are thought to be activated by receptor-induced tyrosine phosphorylation. Proteins that activate PLC-γ1 have now been purified from bovine brain and identified as members of the tau family of microtubule-associated proteins. Activation of PLC-γ by tau was enhanced in the presence of unsaturated fatty acids such as arachidonic acid, saturated fatty acids being ineffective. Maximal (15-20-fold) activation was apparent in the presence of 0.15 µM tau and 25 µM arachidonic acid (AA). The effect of tau and AA was specific to PLC-γ isozymes in the presence of submicromolar concentrations of Ca2+ and was markedly inhibited by phosphatidylcholine. These results suggest that in cells that express tau, receptors coupled to cytosolic phospholipase A2 may activate PLC-γ isozymes indirectly in the absence of tyrosine phosphorylation through the hydrolysis of phosphatidylcholine to generate AA. INTRODUCTIONThe hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), 1The abbreviations used are: PIP2phosphatidylinositol 4,5-bisphosphatePLCphospholipase CIP3inositol 1,4,5-trisphosphatePIphosphatidylinositolAAarachidonic acidPLA2phospholipase A2PSphosphatidylserinePEphosphatidylethanolaminePCphosphatidylcholineDTTdithiothreitolHPLChigh pressure liquid chromatographyPAGEpolyacrylamide gel electrophoresiscPLA2cytosolic PLA2PHpleckstrin homology. 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. (28Noh D.-Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-114Crossref PubMed Scopus (254) Google Scholar)). 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 β type (PLC-β1, -β2, -β3, and -β4), γ type (PLC-γ1 and -γ2), and δ type (PLC-δ1, -δ2, -δ3, and -δ4) enzymes on the basis of amino acid sequence (Noh et al., 28Noh D.-Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-114Crossref PubMed Scopus (254) Google Scholar). The distinct structural features of the different PLC types have been related to specific mechanisms of receptor-mediated enzyme activation. Thus, PLC-γ isozymes are activated by tyrosine phosphorylation, and PLC-β isozymes are activated by heterotrimeric G proteins (Noh et al., 28Noh D.-Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-114Crossref PubMed Scopus (254) Google Scholar); the mechanism of PLC-δ 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-γ1, 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-γ1 markedly enhanced PI-hydrolyzing activity. These observations suggested that brain cytosol contains a component that can enhance the activity of PLC-γ1 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-γ1 toward PIP2 to a markedly lesser extent than that apparent with PI and that the effect of tau on PLC-γ activity toward PIP2 is greatly increased in the presence of arachidonic acid (AA). Of the three types of PLC, the γ 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-γ isozymes in the absence of receptor-mediated tyrosine phosphorylation.EXPERIMENTAL PROCEDURESMaterialsPhosphatidylserine (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 IsozymesPLC isozymes (PLC-β1, -β2, -γ1, -γ2, -δ1, and -δ2) 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., 29Park D. Jhon D.-Y. Kriz R. Knopf J. Rhee S.G. J. Biol. Chem. 1992; 267: 16048-16055Abstract Full Text PDF PubMed Google Scholar).Purification of ActivatorAll manipulations were performed at 4-6°C in a refrigerated room or on ice, unless otherwise indicated. During purification, PLC-γ1-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-γ1 (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.(i) Preparation of Bovine Brain Cytosolic ExtractsFifteen 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.(ii) Heat and Acid TreatmentThe 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.(iii) Preparative HPLC on a DEAE-5PW ColumnThe 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-γ1-activating activity. Peak fractions (25-27) from the two identical runs were combined.(iv) Reverse Phase HPLC on a TSKgel Phenyl-5PW ColumnSolid 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.(v) HPLC on a TSKgel Heparin-5PW ColumnThe 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.(vi) Electroelution of Proteins from SDS-Polyacrylamide GelsPurified 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 SequencingProteins (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.DISCUSSIONWe have purified proteins that enhance the activity of PLC-γ1 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 (21Lee G. Cell Motil. Cytoskeleton. 1990; 15: 199-203Crossref PubMed Scopus (77) Google Scholar)). 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, 13Himmer A. Mol. Cell. Biol. 1989; 9: 1389-1396Crossref PubMed Scopus (280) Google Scholar).Tau proteins did not markedly increase PLC-γ1 activity toward micellar PIP2. Furthermore, when common lipid components of membranes (PE, PS, and cholesterol) were incorporated into micelles, activation of PLC-γ1 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-γ1 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 et al., J. PubMed Scopus Google Scholar). The effect was to the presence of both tau and PLC-γ1 in the brain activation by unsaturated fatty acids of a PLC purified from rat cytosol was observed when the hydrolysis of micellar PI was measured in the presence of 2 mM Ca2+ and J. Biol. Chem. Full Text PDF PubMed Google Scholar). is now thought that the enzyme was a of et al., Biophys. 1992; PubMed Scopus Google Scholar). fatty acids had effect on brain or PLC membrane such as and were shown to have effect on the of PLC-β isozymes et al., A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google and PLC-γ1 and G. J. Biol. Chem. Full Text PDF PubMed Google in studies performed with micellar PIP2 activation by was observed for PLC-γ1, and with a substrate containing PIP2, had effect et al., M. Jhon Rhee S.G. PubMed Scopus Google Scholar). of the activity by was observed with PI as and J. Biol. Chem. Full Text PDF PubMed Google Scholar). These studies suggest that the activity of PLC, that of enzymes that on lipid on the and of the the had effect on basal PLC-γ1 activity markedly inhibited activity stimulated by tau and AA. This suggested that the activation of PLC-γ1 by tau be by a in concentration and increase in AA both of which in cells on activation of the cytosolic PLA2 that is to be coupled to receptors (reviewed by J. Biol. Chem. Full Text PDF PubMed Google and by M. R. G. Scholar); et al. Kriz Knopf Cell. Full Text PDF PubMed Scopus Google Scholar)). This enzyme submicromolar concentrations of Ca2+ and hydrolyzes with unsaturated fatty acids in the The rank order of for is > > > > and the order for is > > > > et al., PubMed Scopus Google Scholar). PLA2 enzymes with of concentrations of Ca2+ for show a for and are with to fatty PLA2 identified in hydrolyzes et al., J. Biol. Chem. 1990; Full Text PDF PubMed Google an PLA2 from for lipids J. Biol. Chem. Full Text PDF PubMed Google concentrations of Ca2+ are for the of to membranes than for activity and this is a for activation et al., Kriz Knopf Cell. Full Text PDF PubMed Scopus Google et al., G. D. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Activation of may to receptor-mediated activation of a PLC that results in an increase in the cytosolic Ca2+ concentration M. R. G. Scholar). activation of a PLC-β for in to of a G may in an increase in intracellular which in results in activation of and activation of PLC-γ activation by the of tau and AA may a mechanism by which PLC-γ isozymes can be activated of tyrosine phosphorylation. and G. J. Biol. Chem. Full Text PDF PubMed Google observed that of acid into a micellar substrate containing PIP2 and enhanced PLC-γ1 activity that PLC-γ1 can be activated of tyrosine phosphorylation acid is generated by the of phospholipase also that the activation of may at basal cytosolic Ca2+ that of generation et al., J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google et al., R. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The addition of to cells in the 2 release of AA and of effects on et al., J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). The AA release was on the of extracellular Ca2+ the of Ca2+ by Furthermore, the increased concentration of AA was over the increase in was more the of with the increase in and the of AA in cells with were of PLC activation et al., R. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Thus, of receptors that are coupled to not to PLC may PIP2 by of PLC-γ isozymes indirectly through tau and AA.Several studies are with the that of PLC by AA in of with AA PLC activity and Mol. Google Scholar). studies with these cells suggested that the of and release are mediated by activation of PLA2 et al., PubMed Scopus Google Scholar). AA not fatty acids in and release from bovine cells et al., M. Biophys. 1990; PubMed Scopus Google Scholar). AA was also shown to increase and release in rat and J. 1990; PubMed Scopus Google to and generation in et al., J. Biol. Chem. Full Text PDF PubMed Google and to increase intracellular Ca2+ by an Ca2+ in isolated rat et al., J. M. J. Biol. Chem. Full Text PDF PubMed Google and a and M. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). The Ca2+ release was shown to be not to the of AA et al., J. M. J. Biol. Chem. Full Text PDF PubMed Google addition to as a for the of and AA been to as a or second in (reviewed by et al., R. Full Text PDF PubMed Scopus Google Scholar). AA and unsaturated fatty acids activate protein kinase C (reviewed by 1992; PubMed Scopus Google Scholar); et al. R. PubMed Scopus Google Scholar); et al. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar)). Furthermore, AA been shown to activate et al., R. Biochim. Biophys. Acta. PubMed Scopus Google and to both protein kinase et al., D. A. 1989; PubMed Scopus Google and to the of et al., J. G. J. Biol. Chem. Full Text Full Text PDF Scopus Google Scholar). The concentrations of AA in these studies were in the range of is to the local concentration of AA at a in intracellular concentrations of µM have been measured in activated cells et al., J. M. J. Biol. Chem. Full Text PDF PubMed Google et al., M. PubMed Scopus Google Scholar).Tau proteins are in G. Cell Motil. Cytoskeleton. 1990; 15: 199-203Crossref PubMed Scopus (77) Google and Full Text PDF PubMed Scopus Google Scholar). the of the between AA and PLC activation both and Furthermore, we have shown that also protein components that can activate proteins purified from bovine are also to and acid and activate PLC-γ isozymes in the presence not in the absence of the proteins are than tau proteins and are not by to tau. These observations suggest that the of activation to PLC-γ activation may not be restricted to by tau and AA was specific for PLC-γ isozymes at physiological concentrations of This may be to the structural features of PLC-γ PLC-β and PLC-δ PLC-γ isozymes each a 3 which is by the to Tau proteins in two and by are to the two of and are amino residues are et al., PubMed Scopus Google Scholar). The two were derived for the and phosphatidylinositol and may from that for the PLC-γ to PLC-γ1 and tau from bovine brain cytosol were not not is also of that the of the brains of with largely of tau proteins that are by microtubule-associated protein kinase and kinase 3, at and (reviewed by and Full Text PDF PubMed Scopus Google Scholar)). Tau contains and three of which are in the and is that phosphorylation of these of tau with the in brains by PLC isozymes have a amino (Noh et al., 28Noh D.-Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-114Crossref PubMed Scopus (254) Google Scholar). PLC-γ PLC that is by the one function of to be to PIP2 et al., PubMed Scopus Google of such identified to sequence and is a a in the that for a common function et al., M. M. M. M. M. J. 1995; PubMed Scopus Google Scholar). The of the been suggested to be to of fatty proteins et al., R. PubMed Scopus Google Scholar). is that AA with one of the two of PLC-γ and with tau to the to enhance enzyme that tau proteins together with AA activate PLC-γ activity in that receptor-mediated activation of in the activation of PLC-γ in a link between the two phospholipase pathways for activation of in the absence of or in with receptor-mediated of a PLC INTRODUCTIONThe hydrolysis of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), 1The abbreviations used are: PIP2phosphatidylinositol 4,5-bisphosphatePLCphospholipase CIP3inositol 1,4,5-trisphosphatePIphosphatidylinositolAAarachidonic acidPLA2phospholipase A2PSphosphatidylserinePEphosphatidylethanolaminePCphosphatidylcholineDTTdithiothreitolHPLChigh pressure liquid chromatographyPAGEpolyacrylamide gel electrophoresiscPLA2cytosolic PLA2PHpleckstrin homology. 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. (28Noh D.-Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-114Crossref PubMed Scopus (254) Google Scholar)). 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 β type (PLC-β1, -β2, -β3, and -β4), γ type (PLC-γ1 and -γ2), and δ type (PLC-δ1, -δ2, -δ3, and -δ4) enzymes on the basis of amino acid sequence (Noh et al., 28Noh D.-Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-114Crossref PubMed Scopus (254) Google Scholar). The distinct structural features of the different PLC types have been related to specific mechanisms of receptor-mediated enzyme activation. Thus, PLC-γ isozymes are activated by tyrosine phosphorylation, and PLC-β isozymes are activated by heterotrimeric G proteins (Noh et al., 28Noh D.-Y. Shin S.H. Rhee S.G. Biochim. Biophys. Acta. 1995; 1242: 99-114Crossref PubMed Scopus (254) Google Scholar); the mechanism of PLC-δ 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-γ1, 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-γ1 markedly enhanced PI-hydrolyzing activity. These observations suggested that brain cytosol contains a component that can enhance the activity of PLC-γ1 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-γ1 toward PIP2 to a markedly lesser extent than that apparent with PI and that the effect of tau on PLC-γ activity toward PIP2 is greatly increased in the presence of arachidonic acid (AA). Of the three types of PLC, the γ 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-γ isozymes in the absence of receptor-mediated tyrosine phosphorylation.