Prolonged ischemia differently affects phospholipase C acting against phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in brain subsynaptosomal fraction

The effect of 10 min ischemia on the activity of phospholipase C acting against [3H]inositol-phosphatidylinositol (PI) and [3H]inositol-phosphatidylinositol 4,5-bisphosphate (PIP2) in the brain subsynaptosomal fractions was investigated. In the presence of endogenous CaCl2, specific activity of phospholipase C acting on phosphatidylinositol was as follows: synaptic cytosol (SC) greater than synaptic vesicles (SV) greater than synaptic plasma membrane SPM). Brain ischemia activated phospholipase C acting on PI by about 60% and 40% in SV and SPM, respectively. The enzyme of synaptic cytosol was not affected by ischemic insult. Phospholipase C acting against PIP2 in the presence of endogenous calcium expressed the specific activity in the following order: SV greater than SPM greater than SC. After 10 min of brain ischemia, activity of phospholipase C acting on PIP2 was significantly suppressed in all subsynaptosomal fractions by about 50-60%. These results indicate that prolonged ischemia produced activation exclusively of phospholipase C acting against phosphatidylinositol.     

Stimulation of phosphatidylinositol 4,5-bisphosphate phospholipase C activity by phosphatidic acid

Phosphatidic acid was a potent activator of the phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity associated with human platelet membranes. Lysophosphatidic acid was half as active as phosphatidic acid, and shortening the fatty acid chain reduced the effectiveness of the corresponding phosphatidic acid. Compounds lacking either the phosphate group (diacylglycerol or phorbol ester) or the fatty acid (glycerol phosphate) were not activators. When the negative charge was contributed by a carboxyl group (fatty acid or phosphatidylserine), stimulation of phospholipase C was weak but detectable. Structural analogs of phosphatidic acid (lipopolysaccharide, lipid A, and 2,3-diacylglucosamine 1-phosphate) were less effective but also enhanced PtdIns-P2 hydrolysis. Phosphatidic acid potentiated the activation of phospholipase C by alpha-thrombin, chelators, and guanine nucleotides. Phosphatidylinositol 4-phosphate and PtdIns-P2 were also effective activators of PtdIns-P2 degradation. Other phospholipids were without effect. The production of inositol 1,4,5-trisphosphate and diacylglycerol via the activation of phospholipase C provides a rationale for the cellular responses evoked by phosphatidic acid and the ability of this phospholipid to potentiate and initiate hormonal responses.     

Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate

A soluble phospholipase C from rat liver was purified to homogeneity using phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. After ammonium sulfate fractionation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose CL-6B, hydroxylapatite, Reactive Blue 2 dye-linked agarose, and Mono S cation exchanger. Under the conditions of the assay, the pure enzyme had a specific activity of 407 mumol/mg protein/min. It migrated as a single band with a molecular mass of 87 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The water-soluble product formed during the hydrolysis of PIP2 by the purified enzyme was inositol 1,4,5-trisphosphate. The enzyme shows one-half of maximum velocity at 2 microM Ca2+ with PIP2 as substrate. Between 0 and 100 microM Ca2+, the enzyme shows approximately the same activity with phosphatidylinositol 4-phosphate (PIP) as it does with PIP2, and very low activity with phosphatidylinositol. The enzyme is activated by low concentrations of basic proteins; for example, with PIP2 as substrate, 1 microgram/ml histone activates the enzyme 3.6-fold. The enzyme shows an almost absolute requirement for monovalent salts which can be met by different alkali metal halides. A second, minor peak of PIP2-hydrolyzing phospholipase C activity was resolved during chromatography of the enzyme on hydroxylapatite. The substrate specificity suggests that PIP and PIP2 are normal substrates of this enzyme. Under physiological conditions of activation, the enzyme may therefore generate inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in amounts determined by the ratio of PIP and PIP2 present in the cellular membranes.     

Thrombin- and nucleotide-activated phosphatidylinositol 4,5-bisphosphate phospholipase C in human platelet membranes

Thrombin, nucleotides, and chelators elicited a phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity that was associated with human platelet membranes. Both alpha- and gamma-thrombin enhanced phospholipase C activity, whereas active site-inhibited alpha-thrombin did not stimulate PtdIns-P2 hydrolysis. PtdIns-P2 phospholipase C was also activated by nucleoside triphosphates, citrate, EDTA, and NaF. Magnesium was an inhibitor of PtdIns-P2 hydrolysis stimulated by nucleotides and chelators. Only PtdIns-P2 was degraded by the phospholipase C activated by alpha-thrombin, nucleotides, and chelators. The soluble fraction phospholipase C activity was also stimulated at low protein concentrations by nucleotides; however, soluble fraction phospholipase C activity cleaved both PtdIns-P2 and phosphatidylinositol 4-phosphate and was inhibited by chelators, suggesting the presence of a different enzyme in this compartment. The pH optimum for the membrane-associated phospholipase C in the presence of alpha-thrombin or nucleotides was 6.0, and the PtdIns-P2 phospholipase C was inhibited by neomycin and high detergent concentrations. Guanine nucleotides did not synergistically activate phospholipase C in the presence of alpha-thrombin. The characteristics of the membrane-associated PtdIns-P2 phospholipase C suggest that this enzyme is involved in platelet activation by the low-affinity alpha- or gamma-thrombin-dependent pathway.     

Subcellular localization and kinetic properties of phosphatidylinositol 4,5-bisphosphate phospholipase C and inositol phosphate enzymes from human peripheral blood mononuclear cells

Peripheral blood mononuclear cells from normal donors exhibited phosphatidylinositol 4,5-bisphosphate phospholipase C (PIP2-PLC), inositol 1,4,5-trisphosphate (IP3) and inositol 1-phosphate (IP)-monophosphatase activities which were mostly recovered in the cytosol fraction. In both cytosol and particulate fractions PIP2-PLC displayed the highest activity at pH 6.2, whereas IP3 and IP-monophosphatases showed the same optimal pH at 7.0. While the PIP2-PLC displayed close apparent Km values in cytosol and particulate fractions, both inositol-monophosphatases were found to show substrate affinities for IP and IP3 characteristic of these two fractions, with an higher affinity in the soluble fraction.     

GTP gamma S-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate soluble phospholipase C from human platelets requires soluble GTP-binding protein

GTP-binding activity was fractionated into two peaks (GI and GII) by chromatography on heparin-agarose. GTP-dependent PLC activity eluted as a single peak, which co-chromatographed with GTP-binding peak GII. Rechromatography of peak GII on heparin-agarose, in the presence of 0.5% sodium cholate, resulted in separation of PLC and GTP-binding activities, and loss of GTP-dependent PLC activity. Recombining fractions containing PLC and GTP-binding activities restored GTP-dependent PLC activity. A specific GTP-binding protein of 29,000 daltons was identified in peak GII by Western blotting of column fractions with [alpha-32P]GTP. These results demonstrate that the soluble phospholipase C from human platelets is regulated by GTP S-binding protein (G29).     

Rapid and transient thrombin stimulation of phosphatidylinositol 4,5-bisphosphate synthesis but not of phosphatidylinositol 3,4-bisphosphate independent of phospholipase C activation in platelets

When platelets are stimulated by thrombin they immediately undergo inositol lipid hydrolysis via phospholipase C activation. However, subsequently an increased production of phosphatidylinositol 4,5-bisphosphate is observed. Phospholipases C were inhibited by lowering the cytoplasmic free calcium concentration by preincubation with Quin-2-tetra(acetoxymethyl) ester. Aggregation and secretion were also totally suppressed. Under these conditions we observed an increased labeling of phosphatidylinositol 4,5-bisphosphate, indicating a stimulation of inositol lipid kinases, independent of lipid hydrolysis by phospholipase C. Conversely the production of phosphatidylinositol 3,4-bisphosphate was totally abolished. These results suggest a different regulation of the kinases/phosphatases responsible for the production of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate.     

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl₂, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P₂) resulted in the formation of [³H-]InsP ₃. GTP-gamma-S (125 µM) stimulated the production of [³H-]InsP ₃. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [³H-]Ptdlns(4,5)P ₂ hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca²⁺. Addition of phorbol ester (100 nM PMA) enhanced the ³²P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca²⁺, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P ₂-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate -adrenoceptor mediated Ptdlns(4,5)P ₂ hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.List of abbreviations ATP Adenosine 5-Trphosphate - CSU Catalytic Subunit of cyclic AMP-dependent protein kinase - DG Diacylglycerol - DMSO Dimethylsulfoxide - DTT DL-dithiothreitol - EDTA Ethylenedinitrilotetraacetic Acid - EGTA Ethyleneglycol-0,0-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - GTP-gamma-S Guanosine 5-O-(3-thiotriphosphate) - HPTLC High Performance Thin Layer Chromatography - InsP ₃ Inositol monophosphate - InsP ₂ Inositol bisphosphate - InsP ₃ Inositol trisphosphate - MES 2-Morpholinoethanesulfonic acid - MOPS 3-[N-Morpholino]Propanesulfonic acid - PAGE Polyacrylamide-gel Electrophoresis - PKC Protein Kinase C - PLase C Phospholipase C - PMA Phorbol 12-Myristate 13-Acetate - PMSF Phenylmethylsulfonyl Fluoride - PtdSer Phosphatidylserine - PtdIns Phosphatidyl inositol - PT Pertussis Toxin - Ptdlns(4)P Phosphatidylinositol 4-monophosphate - Ptdlns (4,5)PZ-Phosphatidylinositol4,5-bisphosphate - SDS-Sodium Dodecyl Sulfate Tris-Tris(hydroxymethyl) aminomethane     

Fluorescent phosphatidylinositol 4,5-bisphosphate derivatives with modified 6-hydroxy group as novel substrates for phospholipase C

The capacity to monitor spatiotemporal activity of phospholipase C (PLC) isozymes with a PLC-selective sensor would dramatically enhance understanding of the physiological function and disease relevance of these signaling proteins. Previous structural and biochemical studies defined critical roles for several of the functional groups of the endogenous substrate of PLC isozymes, phosphatidylinositol 4,5-bisphosphate (PIP(2)), indicating that these sites cannot be readily modified without compromising interactions with the lipase active site. However, the role of the 6-hydroxy group of PIP(2) for interaction and hydrolysis by PLC has not been explored, possibly due to challenges in synthesizing 6-hydroxy derivatives. Here, we describe an efficient route for the synthesis of novel, fluorescent PIP(2) derivatives modified at the 6-hydroxy group. Two of these derivatives were used in assays of PLC activity in which the fluorescent PIP(2) substrates were separated from their diacylglycerol products and reaction rates quantified by fluorescence. Both PIP(2) analogues effectively function as substrates of PLC-δ1, and the K(M) and V(max) values obtained with one of these are similar to those observed with native PIP(2) substrate. These results indicate that the 6-hydroxy group can be modified to develop functional substrates for PLC isozymes, thereby serving as the foundation for further development of PLC-selective sensors.     

Effects of phosphatidylinositol 4,5-bisphosphate and neomycin on phospholipase D: Kinetic studies

The kinetics of phosphatidylcholine-specific phospholipase D activated by phosphatidylinositol 4,5-bisphosphate (PIP₂) and inhibition by neomycin were studied in an enzyme preparation partially purified from human hepatocarcinoma cell line. It was found that phospholipase D was marginally activated by phosphatidyl-4-phosphate (PIP) and phosphatidylethanolamine (PE). In contrast, it was considerably activated by PIP₂ in different concentration of phosphatidylcholine (PC). Sphingomyelin (SM), lysophosphatidylcholine (LPC) and phosphatidylserine (PS) were neither substrates nor inhibitors of the phospholipase D. PIP₂ induced an allosteric effect on phospholipase D and a negative cooperative effect with respect to phosphatidylcholine as indicated in the Lineweaver-Burk plot. In the absence of PIP₂, a straight line was obtained, whereas a downward concave curve was observed in the presence of 25 M of PIP₂. The Hill coefficient and the apparent K_m of phosphatidylcholine in the presence of 25 M PIP₂ were calculated to be 0.631 and 10.79 mM, respectively. PIP₂ also increased the maximal velocity (V_max) of the phospholipase D reaction, suggesting that the affinity of substrate to enzyme was decreased, and the turnover number of the enzyme (kcat) was increased by PIP₂. The activation of phospholipase D by PIP₂ was dose dependent up to 50 M of PIP₂. The Ka of PIP₂ was 15.8 mM. Neomycin, a polycationic glycoside, was shown to be an uncompetitive inhibitor of phospholipase D, and revealed the formation of a neomycin-PIP₂ complex. The Ki of neomycin was estimated to be 8.7 mM.     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-alpha

Cytosolic phospholipase A(2)-alpha (cPLA(2)) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA(2) is dependent on a rise in cytosolic Ca(2+) concentration, membrane association via the Ca(2+)-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA(2) via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), including the role of a "pleckstrin homology (PH)-like" region of cPLA(2) (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate+the Ca(2+) ionophore, A23187, or epidermal growth factor+A23187, expression of the PH domain of phospholipase C-delta1 (which sequesters membrane PIP(2)) attenuated cPLA(2) activity. Stimulated cPLA(2) activity was also attenuated by the expression of cPLA(2) 135-366, or cPLA(2) 2-366, and expression of a PIP(2)-specific 5'-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP(2), with high affinity, only cPLA(2) 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA(2)s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA(2) activity can be modulated by sequestration or depletion of cellular PIP(2), although the interaction of cPLA(2) with membrane PIP(2) appears to be indirect, or of weak affinity.     

In vitro synthesis of 32P-labelled phosphatidylinositol 4,5-bisphosphate and its hydrolysis by smooth muscle membrane-bound phospholipase C

For studies of phospholipase C (PLC) activity in cell-free systems, 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2) was prepared enzymatically by phosphorylating phosphatidylinositol 4-phosphate (PIP) in the presence of [gamma-32P]ATP using a PIP kinase partially purified from bovine retinae. PLC activity was determined by incubating membranes of DDT1 MF-2 cells with 32P-PIP2 and measuring remaining non-hydrolyzed substrate as well as accumulation of the hydrolysis product, inositol trisphosphate (IP3). Guanine nucleotides stimulated PIP2 hydrolysis and IP3 release. Additional increase in IP3 accumulation was observed with adrenaline plus guanine nucleotides.     

Partial purification and some properties of a phospholipase C from Pseudomonas sp. strain KS3.2

An extracellular phospholipase C was partially purified from Pseudomonas sp. strain KS3.2. The enzyme was composed of an approximately 18-kDa peptide. Maximal enzyme activity was found at pH 7.2 and 50 degrees C. The enzyme retained activity between pH 8 and 9, and 50% activity at about 52 degrees C for 30 min. The enzyme sample showed the highest activity on phosphatidylcholine and low activity toward other phospholipids.     

Partial purification and characterization of phospholipase C from Yersinia enterocolitica

About 34% of the strains of Yersinia enterocolitica isolated from raw milk were found to produce lecithinase. A selected strain produced phospholipase C at 22°C and 37°C; production was optimum at 37°C in the stationary phase (14–16 h). A decrease in phospholipase C activity at various storage temperatures (—5°C, 4°C, 37°C) was also observed, although the enzyme was active over a wide range of temperature (5–65°C) and pH (3mD5–7mD5). The phospholipase C was partially purified by ammonium sulphate precipitation and Sephadex column chromatography, and characterized.     

Partial purification and characterization of phospholipase C from Yersinia enterocolitica

About 34% of the strains of Yersinia enterocolitica isolated from raw milk were found to produce lecithinase. A selected strain produced phospholipase C at 22 degrees C and 37 degrees C; production was optimum at 37 degrees C in the stationary phase (14-16 h). A decrease in phospholipase C activity at various storage temperatures (-5 degrees C, 4 degrees C, 37 degrees C) was also observed, although the enzyme was active over a wide range of temperature (5-65 degrees C) and pH (3.5-7.5). The phospholipase C was partially purified by ammonium sulphate precipitation and Sephadex column chromatography, and characterized.     

Arf6 promotes autophagosome formation via effects on phosphatidylinositol 4,5-bisphosphate and phospholipase D

Macroautophagy (in this paper referred to as autophagy) and the ubiquitin-proteasome system are the two major catabolic systems in cells. Autophagy involves sequestration of cytosolic contents in double membrane-bounded vesicles called autophagosomes. The membrane source for autophagosomes has received much attention, and diverse sources, such as the plasma membrane, Golgi, endoplasmic reticulum, and mitochondria, have been implicated. These may not be mutually exclusive, but the exact sources and mechanism involved in the formation of autophagosomes are still unclear. In this paper, we identify a positive role for the small G protein Arf6 in autophagosome formation. The effect of Arf6 on autophagy is mediated by its role in the generation of phosphatidylinositol 4,5-bisphosphate (PIP(2)) and in inducing phospholipase D (PLD) activity. PIP(2) and PLD may themselves promote autophagosome biogenesis by influencing endocytic uptake of plasma membrane into autophagosome precursors. However, Arf6 may also influence autophagy by indirect effects, such as either by regulating membrane flow from other compartments or by modulating PLD activity independently of the mammalian target of rapamycin.     

Interaction of cerebral-cortical membranes with exogenously added phosphatidylinositol 4,5-bisphosphate. Effects on measured phospholipase C activity.

Exogenously added phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is rapidly associated with cerebral-cortical membranes. Substrate association with membranes was promoted by Mg2+, but inhibited by bivalent chelators. Once associated with the membrane, the PtdInsP2 was resistant to displacement by EDTA. The apparent phospholipase C activity was dependent on the degree of association of substrate with membranes. After preincubation of membranes with substrate, PtdInsP2 hydrolysis was independent of the incubation volume, indicating that substrate and membrane-associated phospholipase C were not independently diluted. Hydrolysis of the membrane-associated substrate was stimulated by Ca2+, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), guanosine 5'[gamma-thio]triphosphate and carbachol in the presence of p[NH]ppG. Carbachol in the absence of guanine nucleotides, GDP, GTP, ATP and pyrophosphate was ineffective. These results demonstrate that exogenously added PtdInsP2 substrate is rapidly associated with membranes and hydrolysed by a phospholipase C whose activity is regulated by guanine nucleotides and agonist in the presence of guanine nucleotides. Use of exogenously added substrate for studies on the regulation of membrane phospholipase C requires consideration as to possible effects of incubation conditions on the partitioning of substrate into membranes.     

Mechanism of protein kinase C activation by phosphatidylinositol 4,5-bisphosphate.

The mechanism of protein kinase C (PKC) activation by phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI) was investigated by using Triton X-100 mixed micellar methods. The activation of PKC by PIP2, for which maximal activity was 60% of that elicited by sn-1,2-diacyglycerol (DAG), was similar to activation by DAG in several respects: (1) activation by PIP2 and DAG required phosphatidylserine (PS) as a phospholipid cofactor, (2) PIP2 and DAG reduced the concentration of Ca2+ and PS required for activation, (3) the concentration dependences of activation by PIP2 and DAG depended on the concentration of PS, and (4) PIP2 and DAG complemented one another to achieve maximal activation. On the other hand, PIP2 activation of PKC differed from activation by DAG in several respects. With increasing concentrations of PIP2, (1) the optimal concentration of PS required was constant at 12 mol%, (2) the maximal activity at 12 mol% PS increased, and (3) the cooperativity for PS decreased. PIP2 did not inhibit [3H]phorbol 12,13-dibutyrate (PDBu) binding of PKC at saturating levels of PS; however, at subsaturating levels of PS, PIP2 enhanced [3H]PDBu binding by acting as a phospholipid cofactor. PIP did not function as an activator but served as a phospholipid cofactor in the presence of PS. While PIP2, PIP, and PI did not support DAG-dependent PKC activation as phospholipid cofactors, their presence reduced the amount of PS required for maximal activation to as low as 2 mol% from 8 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of protein kinase C isozymes with phosphatidylinositol 4,5-bisphosphate.

Interaction of protein kinase C (PKC) isozymes with phosphatidylinositol 4,5-bisphosphate (PIP2) was investigated by monitoring the changes in the intrinsic fluorescence of the enzyme, the kinase activity, and phorbol ester binding. Incubation of PKC I, II, and III with PIP2 resulted in different rates of quenching of PKC fluorescence and different degrees of inactivation of these enzymes. Other inositol-containing phospholipids such as phosphatidylinositol and phosphatidylinositol 4-phosphate also caused differential rates of quenching of the intrinsic fluorescence of these enzymes. These latter two phospholipids were, however, less potent in the inactivation of PKCs than PIP2. The IC50 of PIP2 were 2, 4, and 11 microM for PKC I, II, and III, respectively. Inactivation of PKCs by PIP2 cannot be reversed by extensive dilution of PIP2 with Nonidet P-40 nor by digestion of PIP2 with phospholipase C. Interaction of PIP2 with the various PKC isozymes was greatly facilitated in the presence of Mg2+ or Ca2+ as evidenced by the accelerated quenching of the PKC fluorescence, however, these divalent metal ions protected PKC from the PIP2-induced inactivation. Binding of PIP2 to PKC in the absence of divalent metal ion also caused a reduction of [3H]phorbol 12,13-dibutyrate binding as a result of reducing the affinity of the enzyme for phorbol ester. Based on gel filtration chromatography, it was estimated that one molecule of PKC interacted with one PIP2 micelle with an aggregation number of 80-90. The PIP2-bound PKC could further interact with phosphatidylserine in the presence of Ca2+ to form a larger complex. Binding of PKC to both PIP2 and phosphatidylserine in the presence of Ca2+ was also evident by changes in the intrinsic fluorescence of PKC. As the interaction of PKC with PIP2, but not with phosphatidylserine, could be enhanced by millimolar concentrations of Mg2+, we propose that PIP2 may be a component of the membrane anchor for PKC under basal physiological conditions when [Ca2+]i is low and Mg2+ is plentiful. Under the in vitro assay conditions, PIP2 could stimulate PKC activity to a level approximately 10-20% of that by diacylglycerol. The stimulatory effect of PIP2 on PKC apparently is not due to binding to the same site recognized by diacylglycerol or phorbol ester, because PIP2 cannot effectively compete with phorbol 12,13-dibutyrate in the binding assay.