Insulin selectively stimulates nuclear phosphoinositide-specific phospholipase C (PI-PLC) beta1 activity through a mitogen-activated protein (MAP) kinase-dependent serine phosphorylation

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen-activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide-specific phospholipase C (PI-PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the beta family of PI-PLC (i.e. beta1, beta2, beta3, and beta4), insulin only activated the beta1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [(32)P]orthophosphate, we ascertained that nuclear PI-PLC-beta1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-beta1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-beta1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.     

Nuclear phospholipase C-beta1b activation during G2/M and late G1 phase in nocodazole-synchronized HL-60 cells

In this study, the activity of nuclear phosphatidylinositol-specific phosholipase C (PI-PLC) was investigated in HL-60 cells blocked at G(2)/M phase by the addition of nocodazole, and released into medium as synchronously progressing cells. Two peaks of an increase in the nuclear PI-PLC activities were detected; an early peak reached a maximum at 1 h after release from the nocodazole block, and a second increase was detected at 8.5 h after the release. Immunoprecipitation studies indicated that the increase in the activity was due to the activation of the nuclear PI-PLC-beta(1). Western blot analysis demonstrated no changes in the level of both a and b splicing variants of PI-PLC-beta(1) in the nuclei of cells isolated at either 1 h or 8.5 h after the block. However, an increase in the serine-phosphorylation of PI-PLC-beta(1b) was detected in the nuclei of HL-60 cells isolated at 1 and 8.5 h after the block, and the presence of MEK-inhibitor PD98059 completely inhibited both the serine phosphorylation and the increase in the PI-PLC activities in vitro. The presence of PI-PLC inhibitor prevented the progression of HL-60 cells through the G(1) into S phase of the cell cycle. These results demonstrate that two peaks of nuclear PI-PLC activities, which are due to a PD98059-sensitive phosphorylation of nuclear PLC-beta(1b) on serine, occur at the G(2)/M and late G(1) phase and are necessary for the progression of the cells through the cell cycle.     

Nuclear phospholipase C: involvement in signal transduction

During the past years, several independent laboratories have highlighted the presence of nuclear signaling pathways based on lipid hydrolysis, which are not a mere duplication of those occurring at the plasma membrane. Among the enzymes of the cycle, nuclear phosphoinositide-specific phospholipase C (PI-PLC) has been analyzed quite extensively. In this context, PI-PLCbeta1 appears to play a key role as a check point in the G1 phase of the cell cycle. It has also been shown that its activation and/or up-regulation is upon the control of type 1 insulin-like growth factor receptor (IGF-R) in both mouse fibroblast and myoblasts, suggesting that its signaling activity is essential for the normal behavior of the cell, at least in culture. The recent discovery of a possible involvement of the deletion of PI-PLCbeta1 gene in the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML) in humans strengthens the contention that nuclear PI-PLC signaling is essential for physiological processes such as cell growth and differentiation. Even though PI-PLCbeta1 is present and does not translocate to eukaryotic nuclei, this organelle, even though only in some conditions contains also PI-PLCgamma1 which acts not only as a PI-PLC but also as guanine nucleotide exchange factor (GEF) for PI 3-kinase enhancer (PIKE) and is somehow linked to PI 3-kinase (PI3K) activity. Also members of PI-PLCdelta family are shuttling from the nucleus to the cytoplasm and return and are possibly involved in the control of cell growth. We must also take into account the presence in the nucleus of other phospholipases such as phospholipase A2 (PLA2) and phospholipase D (PLD), which also exert a signaling activity upon external stimuli. On the whole this review highlights the latest development in the PI-PLC cycle in the nucleus, which in terms of activation, regulation and down-stream targets differs substantially from that located at the plasma membrane.     

Involvement of nuclear phosphatidylinositol-dependent phospholipases C in cell cycle progression during rat liver regeneration

Nuclear lipid metabolism is involved in the regulation of cell proliferation. Modulation of the expression and activity of nuclear PI-phospholipase C (PI-PLC) has been reported during liver regeneration after partial hepatectomy, although it has not been determined whether different PLC isoforms play specific roles in the regulation of cell cycle progression. Here, we report evidence that the increased activity of nuclear PLCs in regenerating rat liver occurs before the peak of DNA replication and involves the enzyme activity associated to the chromatin and not that associated to the nuclear membrane. Immunocytochemical analyses indicate that PI-PLC beta(1) isoform is exclusively localized at the chromatin level, PI-PLC beta(1) co-localizes with DNA replication sites much more than PI-PLC gamma(1), which is also present at the nuclear envelope. These findings and the increased amount of PI-PLC gamma(1) occurring after the peak of DNA replication suggest that PI-PLC beta(1) and gamma(1) play different roles in cell cycle progression during regenerating liver. The increased activity of PI-PLC beta(1) constitutively present within the hepatocyte nucleus, should trigger DNA replication, whereas PI-PLC gamma(1) should be involved in G2/M phase transition through lamin phosphorylation.     

Nuclear inositides: PI-PLC signaling in cell growth, differentiation and pathology

The existence of an inositide-dependent nuclear signaling has been clearly shown. In this review we focused on the nuclear PI-PLC signaling activity and its downstream effects. The main isoform present in the nucleus is PI-PLC β1 and this isoform resides in the nuclear domains called speckles and colocalizes with the splicing factor SC35. PI-PLC β1 is also involved in the physiological control of the cell cycle. Moreover, acting on the cyclin D3 promoter plays a crucial role in the process of C2C12 myoblast differentiation. Finally in hematological malignancies such as high-risk MDS, the deletion of PI-PLC β1 gene has been observed. There is the likelihood that the deletion is a prognostic marker in that 66.7% MDS patients bearing the PI-PLC β1 monoallelic deletion evolved into AML. In addition the expression of nuclear PI-PLC β1 in MDS patients is modulated by the demethylating drug azacytidine. Therefore the analysis of nuclear PI-PLC-β1 appears useful for both MDS prognosis and checking of the epigenetic effect of antileukemic drugs.     

Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes

Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level.     

Inositide signaling in the nucleus: From physiology to pathology

A inositide-dependent signaling exists in the nucleus. In this review we focused to the nuclear PI-PLC signaling activity, its downstream effects and its role in haematological malignancies. PI-PLC β1 is involved in the physiological control of the cell cycle and by acting on the Cylin D3 promoter plays a crucial role in the process of C2C12 myoblast differentiation. In hematological malignancies recent studies showed that PI-PLC β1 mono-allelic deletion correlates with a higher risk of AML evolution. Moreover it has been shown that PI-PLC β1 promoter is hypermethylated in high-risk MDS patients and that the amount of PI-PLC β1 mRNA could predict the clinical response to azacitidine, a well known demethylating drug.All in all the data reviewed here pave the way to a new prognostic and therapeutic strategy in some haematological malignancies.     

Two waves of the nuclear phospholipase C activity in serum-stimulated HL-60 cells during G(1) phase of the cell cycle

Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cell cycle progression. We have previously demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells. In this study, the activity of nuclear PI-PLC was investigated in serum-stimulated HL-60 cells. In serum-starved HL-60 cells, two peaks of the activity of nuclear PI-PLC were detected at 30 min and 11 h after the re-addition of serum with no parallel increase in PLC activity in cytosol, postnuclear membranes or total cell lysates. An increase in the serine phosphorylation of b splicing variant of PI-PLCbeta(1) was detected with no change in the amount of PI-PLCbeta(1b) in nuclei isolated at 30 min and 11 h after the addition of serum. PI-PLC inhibitor ET-18-OCH(3) and MEK inhibitor PD 98059 completely abolished serum-mediated increase at both time-points. The addition of inhibitors either immediately or 6 h after the addition of serum had inhibitory effects on the number of cells entering S phase. These results demonstrate that two waves of nuclear PI-PLCbeta(1b) activity occur in serum-stimulated cells during G(1) phase of the cell cycle and that the later increase in the PLC activity is equally important for the progression into the S phase.     

Regulation of nuclear phospholipase C activity

A body of evidence, linking inositide-specific phospholipase C (PI-PLC) to the nucleus, is quite extensive. The main isoform in the nucleus is PI-PLCbeta1, whose activity is up-regulated in response to insulin-like growth factor-1 (IGF-1) or insulin stimulation. Whilst at the plasma membrane this PI-PLC is activated and regulated by Galphaq/alpha(11) and Gbetagamma subunits, there is yet no evidence that qalpha/alpha(11) is present within the nuclear compartment, neither GTP-gamma-S nor AlF4 can stimulate PI-PLCbeta1 activity in isolated nuclei. Here we review the evidence that upon occupancy of type 1 IGF receptor there is translocation to the nucleus of phosphorylated mitogen-activated protein kinase (MAPK) which phosphorylates nuclear PI-PLCbeta1 and triggers its signalling, hinting at a separate pathway of regulation depending on the subcellular location of PI-PLCbeta1. The difference in the regulation of the activity of PI-PLCbeta1mirrors the evidence that nuclear and cytoplasmatic inositides can differ markedly in their signalling capability. Indeed, we do know that agonists which affect nuclear inositol lipid cycle at the nucleus do not stimulate the one at the plasma membrane.     

Diacylglycerol induces fusion of nuclear envelope membrane precursor vesicles

Purified membrane vesicles isolated from sea urchin eggs form nuclear envelopes around sperm nuclei following GTP hydrolysis in the presence of cytosol. A low density subfraction of these vesicles (MV1), highly enriched in phosphatidylinositol (PtdIns), is required for nuclear envelope formation. Membrane fusion of MV1 with a second fraction that contributes most of the nuclear envelope can be initiated without GTP by an exogenous bacterial PtdIns-specific phospholipase C (PI-PLC) which hydrolyzes PtdIns to form diacylglycerides and inositol 1-phosphate. This PI-PLC hydrolyzes a subset of sea urchin membrane vesicle PtdIns into diglycerides enriched in long chain, polyunsaturated species as revealed by a novel liquid chromatography-mass spectrometry analysis. Large unilammelar vesicles (LUVs) enriched in PtdIns can substitute for MV1 in PI-PLC induced nuclear envelope formation. Moreover, MV1 prehydrolyzed with PI-PLC and washed to remove inositols leads to spontaneous nuclear envelope formation with MV2 without further PI-PLC treatment. LUVs enriched in diacylglycerol mimic prehydrolyzed MV1. These results indicate that production of membrane-destabilizing diglycerides in membranes enriched in PtdIns may facilitate membrane fusion in a natural membrane system and suggest that MV1, which binds only to two places on the sperm nucleus, may initiate fusion locally.     

Physiology and pathology of nuclear phospholipase C β1

The existence and function of inositide signaling in the nucleus is well documented and we know that the existence of the inositide cycle inside the nucleus has a biological role. An autonomous lipid-dependent signaling system, independently regulated from its plasma membrane counterpart, acts in the nucleus and modulates cell cycle progression and differentiation.We and others focused on PLCβ1, which is the most extensively investigated PLC isoform in the nuclear compartment. PLCβ1 is a key player in the regulation of nuclear inositol lipid signaling, and, as discussed above, its function could also be involved in nuclear structure because it hydrolyses PtdIns(4,5)P2, a well accepted regulator of chromatin remodelling. The evidence, in a number of patients with myelodysplastic syndromes, that the mono-allelic deletion of PLCβ1 is associated with an increased risk of developing acute myeloid leukemia paves the way for an entirely new field of investigation. Indeed the genetic defect evidenced, in addition to being a useful prognostic tool, also suggests that altered expression of this enzyme could have a role in the pathogenesis of this disease, by causing an imbalance between proliferation and apoptosis. The epigenetics of PLCβ1 expression in MDS has been reviewed as well.     

The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor

The dimerization properties of the ghrelin receptor (GRLN-R) and its non-signalling, naturally occurring, truncated splice variant (GHS-R1b) have been investigated in human embryonic kidney 293 cells heterologously expressing these proteins. Using the techniques of bioluminescence resonance energy transfer and co-immunoprecipitation, we detected the formation of GRLN-R homodimers and GRLN-R/GHS-R1b heterodimers, but ghrelin-induced conformational changes were only detected in the GRLN-R homodimers. When the expression of GHS-R1b exceeded that of GRLN-R, there was a decrease in the cell surface expression of GRLN-R with a consequent decrease in constitutive activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Furthermore, there was no change in ghrelin affinity, and the efficacy of cell signalling as measured by stimulation of PI-PLC and extracellular signal-regulated kinase 1/2 was unchanged. Cellular localization studies suggest that GRLN-R is normally distributed between the plasma membrane and cytosolic fractions, but in the presence of GHS-R1b, GRLN-R is localized to the nucleus. Therefore, we propose that the decrease in GRLN-R constitutive signalling was due to translocation of GRLN-R to the nucleus due to the formation of GRLN-R/GHS-R1b heterodimers. Therefore, GHS-R1b appears to act as a dominant-negative mutant of the full-length GRLN-R.     

Nuclear sphingomyelin pathway in serum deprivation-induced apoptosis of embryonic hippocampal cells

Sphingomyelin (SM) cycle has been involved in the regulation of proliferation, differentiation, and apoptosis. Increases in ceramide have been found after a larger number of apoptotic stimuli including cytokines, cytotoxic drugs, and environmental stresses. Accumulating evidence suggest that the subcellular localization of ceramide generation is a critical factor in determining the cellular behavior. Since recently enzymes involved in ceramide metabolism such as sphingomyelinase, SM synthase, sphingosine kinase and ceramidase have been found in the nucleus of hepatocyte cells, we have studied first the presence and the physicochemical characteristics of SM metabolism enzymes in nuclei isolated from embryonic hippocampal cells (cell line HN9.10e). The activities of sphingomyelinase and SM-synthase have been assayed and the ceramide production evaluated at different times after serum deprivation in these neurones cultivated in serum-deficient medium. We report that both enzymes are present in the nucleus of embryonic hippocampal cells and differ from those present in the homogenate in optimum pH. After serum deprivation, that induces a time-dependent decrease in cell viability and increase of the cell percentage in G1 phase of the cell cycle, a nuclear sphingomyelinase activation together with SM-synthase inhibition and a consequent increase of nuclear ceramide pool have been demonstrated. No similar enzyme activity modifications in homogenate have been identified. The possible role of nuclear sphingomyelinase/sphingomyelin-synthase balance in serum deprivation-induced apoptosis in the embryonic hippocampal cell is discussed.     

Nuclear responses to MPF activation and inactivation in Xenopus oocytes and early embryos

The cell cycle of most organisms is highlighted by characteristic changes in the appearance and activity of the nucleus. Structural changes in the nucleus are particularly evident when a cell begins to divide. At this time, the nuclear envelope is disassembled, the chromatin condenses into metaphase chromosomes, and the chromosomes associate with a newly formed spindle. Upon completion of cell division the nuclear envelope reassembles around the chromosomes as they form telophase nuclei, and subsequently interphase nuclei, in the daughter cells. The cytoplasmic control of nuclear behavior has been the theme of Yoshio Masui's research for much of his career. His pioneering demonstration that the cytoplasm of maturing amphibian oocytes causes the resumption of the meiotic cell cycle when it is injected into an immature oocyte provided unequivocal evidence that a cytoplasmic factor could initiate the transition from interphase to metaphase (M-phase) in intact cells. As described in several reviews in this and the previous issue of Biology of the Cell (see Beckhelling and Ford; Duesbery and Vande Woude; Maller), Masui initially called this activity maturation promoting factor (MPF), but when it was realized that it was a ubiquitous regulator of both mitotic and meiotic cell cycles, MPF came to stand for M-phase promoting factor. Biochemical evidence indicates that MPF activity is composed of a mitotic B-type cyclins and cyclin-dependent kinase 1. The increase in the protein kinase activity of cdk1 initiates the changes in the nucleus associated with oocyte maturation and with the entry into mitosis. This article will attempt to provide a brief summary of the responses of the nucleus to the activation of MPF. In addition, the effect of MPF inactivation on nuclear envelope assembly at the end of mitosis will be discussed. This article is written as a tribute to Yoshio Masui on his retirement from the University of Toronto, and as an expression of gratitude for his guidance while I was a student in his laboratory. I have felt very privileged to have known him as a mentor and a friend.     

Initiation and organization of events during the first cell cycle in mammals: applications in cloning

The technology of cloning involves transplanting a diploid nucleus into a mature oocyte cytoplast. The cytoplast is then activated to initiate the first cell cycle of development as a nuclear transplant embryo. Initiation and regulation of events during the first cell cycle are, therefore, critical for proper reprogramming of the donor nucleus and development as a cloned embryo. Activation is normally induced by the sperm and is mediated by a series of intracellular free calcium ([Ca(2+)](i)) oscillations that last for several hours. Although it is not known precisely how the sperm induces activation, current evidence favors the delivery, by the sperm, of a soluble protein factor that causes the production of IP3. IP3 acts to open a Ca(2+) channel in the endoplasmic reticulum and release Ca(2+) into the cytosol. A variety of methods have been used to duplicate or replace the sperm-induced [Ca(2+)](i) increase to cause activation in nuclear transplant embryos. It has been found that treatments that cause a single transient [Ca(2+)](i) activate some oocytes with the level of activation increasing as the oocyte ages. Attempts have been made to extend the period of time over which [Ca(2+)](i) oscillations occur. This has been successful in increasing activation rates of less mature oocytes but the techniques are still cumbersome. An alternative method, that has been very successful, is the combination of a treatment that elevates [Ca(2+)](i) and a treatment that maintains low levels of maturation promoting factor for several hours after the initial [Ca(2+)](i) elevation. The sperm also contributes the centrosome that organizes microtubules during the first cell cycle. One current hypothesis for regulation of sperm centrosomal activity consists of a dephosphorylation of sperm connecting piece proteins following sperm entry into the oocyte and activation of the oocyte. Dephosphorylation of these proteins results in the disassembly of the connecting piece and assembly of a functional centrosome. In nuclear transfer, centrosomal components are contributed by the donor cell. If the cell is fused to the cytoplast before centriole replication then a single aster forms. If the cell is fused after centriole replication then two asters form. In either case and even in parthenogenetic oocytes, which do not have centrioles, the first cell cycle progresses to metaphase. However, progress is slow and some defects are observed in the assembly of chromosomes into a metaphase plate.     

Nuclear localization of Chfr is crucial for its checkpoint function

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257–259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.     

Phospholipids chiral at phosphorus. Stereochemical mechanism for the formation of inositol 1-phosphate catalyzed by phosphatidylinositol-specific phospholipase C.

The phosphatidylinositol-specific phospholipase C (PI-PLC) from mammalian sources catalyzes the simultaneous formation of both inositol 1,2-cyclic phosphate (IcP) and inositol 1-phosphate (IP). It has not been established whether the two products are formed in sequential or parallel reactions, even though the latter has been favored in previous reports. This problem was investigated by using a stereochemical approach. Diastereomers of 1,2-dipalmitoyl-sn-glycero-3-(1D- [16O,17O]phosphoinositol) ([16O,17O]DPPI) and 1,2-dipalmitoyl-sn-glycero-3-(1D-thiophosphoinositol) (DPPsI) were synthesized, the latter with known configuration. Desulfurization of the DPPsI isomers of known configurations in H2(18)O gave [16O,18O]DPPI with known configurations, which allowed assignment of the configurations of [16O,17O]DPPI on the basis of 31P NMR analyses of silylated [16O,18O]DPPI and [16O,17O]DPPI (the inositol moiety was fully protected in this operation). (Rp)- and (Sp)-[16O,17O]DPPI were then converted into trans- and cis-[16O,17O]IcP, respectively, by PI-PLC from Bacillus cereus, which had been shown to proceed with inversion of configuration at phosphorus [Lin, G., Bennett, F. C., & Tsai, M.-D. (1990) Biochemistry 29, 2747-2757]. 31P NMR analysis was again used to differentiate the silylated products of the two isomers of IcP, which then permitted assignments of IcP with unknown configuration derived from transesterification of (Rp)- and (Sp)-[16O,17O]DPPI by bovine brain PI-PLC-beta 1. The results indicated inversion of configuration, in agreement with the steric course of the same reaction catalyzed by PI-PLCs from B. cereus and guinea pig uterus reported previously. For the steric course of the formation of inositol 1-phosphate catalyzed by PI-PLC, (Rp)- and (Sp)-[16O,17O]DPPI were hydrolyzed in H2(18)O to afford 1-[16O,17O,18O]IP, which was then converted to IcP chemically and analyzed by 31P NMR. The results indicated that both B. cereus PI-PLC and the PI-PLC-beta 1 from bovine brain catalyze conversion of DPPI to IP with overall retention of configuration at phosphorus. These results suggest that both bacterial and mammalian PI-PLCs catalyze the formation of IcP and IP by a sequential mechanism. However, the conversion of IcP to IP was detectable by 31P NMR only for the bacterial enzyme. Thus an alternative mechanism in which IcP and IP are formed by totally independent pathways, with formation of IP involving a covalent enzyme-phosphoinositol intermediate, cannot be ruled out for the mammalian enzyme. It was also found that both PI-PLCs displayed lack of stereo-specifically toward the 1,2-diacylglycerol moiety, which suggests that the hydrophobic part of phosphatidylinositol is not recognized by PI-PLC.