Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain

Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract of rat brain. The purification procedures consisted of chromatography of both enzymes on Affi-Gel blue and cellulose phosphate, followed by three sequential high performance liquid chromatography steps, which were different for the two enzymes. The resultant preparations each contained homogeneous enzyme with a Mr of 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these enzymes (PLC-II) was found to hydrolyze phosphatidyl-inositol 4,5-bisphosphate (PIP2) at a rate of 15.3 mumol/min/mg of protein and also phosphatidylinositol 4-monophosphate and phosphatidylinositol (PI) at slower rates. For hydrolysis of PI, this enzyme was activated by an acidic pH and a high concentration of Ca2+ and showed a Vmax value of 19.2 mumol/min/mg of protein. The other enzyme (PLC-III) catalyzed hydrolysis of PIP2 preferentially at a Vmax rate of 12.9 mumol/min/mg of protein and catalyzed that of phosphatidylinositol 4-monophosphate slightly. The rate of PIP2 hydrolysis by this enzyme exceeded that of PI under all conditions tested. Neither of these enzymes had any activity on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid. These two enzymes showed not only biochemical but also structural differences. Western blotting showed that antibodies directed against PLC-II did not react with PLC-III. Furthermore, the two enzymes gave different peptide maps after digestion with alpha-chymotrypsin or Staphylococcus aureus V8 protease. These results suggest that these two forms of PLC belong to different families of PLC.     

Tissue- and cell type-specific expression of mRNAs for four types of inositol phospholipid-specific phospholipase C

The mRNA levels for four types of inositol phospholipid-specific phospholipase C (PLC) in various tissues and cell cultures have been studied by Northern analysis using cDNA probes for PLC isozyme I, II, and III [Sue, P.-G., Ryu, S.H., Moon, K.H., Sue, H.W., and Rhee, S.G. (1988) Proc. Natl. Acad. Sci. USA 85, 5419-5423 and Cell 54, 161-169], and the recently identified isozyme IV. All four types are ubiquitously expressed in rat tissues, but the levels of the mRNAs vary among tissues and cell lines. PLC-I mRNA levels are extremely high in brain and rat C6 glioma cells with lower levels in other tissues tested. PLC-II and -III have a more widespread distribution, with relatively high levels in brain, lung, spleen, thymus, and testis in the case of PLC-II, and in skeletal muscle, spleen, and testis for PLC-III. PLC-II and -III mRNAs were also detected in all cell lines examined except human promyelocytic HL60 cells. PLC-IV mRNA levels are extraordinarily high in spleen and HL60 cells. These results indicate that rat C6 glioma cells, together with most rat tissues, contain all four PLC isozymes. Other cultured cell types examined also contain two or three PLC isozymes except for HL60 cells, which contain only PLC-IV. The concomitant expression of PLC isozymes in cultured cells suggests a diverse function for PLC isozymes in single cells.     

A calcium-binding protein with four EF-hand motifs in Streptomyces ambofaciens

A gene (cabA) encoding a calcium-binding protein was cloned from Streptomyces ambofaciens. CabA was 180 amino acid residues long and contained four typical EF-hand motifs bearing high sequence similarity to the calcium-binding sites in calmodulin. Consistent with this, CabA showed distinct calcium-binding activity, comparable to bovine brain calmodulin. cabA was transcribed throughout growth, as found by S1 nuclease mapping. Southern hybridization experiments showed that a single copy of cabA was present in various Streptomyces species. A hypothetical relationship between CabA and aerial mycelium formation in this strain was examined, since S. ambofaciens showed calcium-dependent aerial mycelium formation. However, disruption of cabA or overexpression of cabA in S. ambofaciens caused no detectable phenotypic changes.     

Molecular characterization of an Arabidopsis gene encoding a phospholipid-specific inositol polyphosphate 5-phosphatase

Phosphoinositides are important molecules that serve as second messengers and bind to a complex array of proteins modulating their subcellular location and activity. The enzymes that metabolize phosphoinositides can in some cases serve to terminate the signaling actions of phosphoinositides. The inositol polyphosphate 5-phosphatases (5PTases) comprise a large protein family that hydrolyzes 5-phosphates from a variety of inositol phosphate and phosphoinositide substrates. We previously reported the identification of 15 putative 5PTase genes in Arabidopsis and have shown that overexpression of the At5PTase1 gene can alter abscisic acid signaling. At5PTase1 and At5PTase2 have been shown to hydrolyze the 5-phosphate from inositol phosphate substrates. We have examined the substrate specificity of the At5PTase11 protein, which is one of the smallest predicted 5PTases found in any organism. We report here that the At5PTase11 gene encodes an active 5PTase enzyme that can only dephosphorylate phosphoinositide substrates containing a 5-phosphate. In addition to hydrolyzing known substrates of 5PTase enzymes, At5PTase11 also hydrolyzes the 5-phosphate from phosphatidylinositol (3,5) bisphosphate. We also show that the At5PTase11 gene is regulated by abscisic acid, jasmonic acid, and auxin, suggesting a role for phosphoinositide action in these signal transduction pathways.     

The isolation and characterization of a cDNA encoding phospholipid-specific inositol polyphosphate 5-phosphatase

We report the cDNA cloning and characterization of a novel human inositol polyphosphate 5-phosphatase (5-phosphatase) that has substrate specificity unlike previously described members of this large gene family. All previously described members hydrolyze water soluble inositol phosphates. This enzyme hydrolyzes only lipid substrates, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate. The cDNA isolated comprises 3110 base pairs and predicts a protein product of 644 amino acids and M(r) = 70,023. We designate this 5-phosphatase as type IV. It is a highly basic protein (pI = 8.8) and has the greatest affinity toward phosphatidylinositol 3,4,5-trisphosphate of known 5-phosphatases. The K(m) is 0.65 micrometer, 1/10 that of SHIP (5.95 micrometer), another 5-phosphatase that hydrolyzes phosphatidylinositol 3,4,5-trisphosphate. The activity of 5-phosphatase type IV is sensitive to the presence of detergents in the in vitro assay. Thus the enzyme hydrolyzes lipid substrates in the absence of detergents or in the presence of n-octyl beta-glucopyranoside or Triton X-100, but not in the presence of cetyltriethylammonium bromide, the detergent that has been used in other studies of the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Remarkably SHIP, a 5-phosphatase previously characterized as hydrolyzing only substrates with d-3 phosphates, also readily hydrolyzed phosphatidylinositol 4,5-bisphosphate in the presence of n-octyl beta-glucopyranoside but not cetyltriethylammonium bromide. We used antibodies prepared against a peptide predicted by the cDNA to identify the 5-phosphatase type IV enzyme in human tissues and find that it is highly expressed in the brain as determined by Western blotting. We also performed Western blotting of mouse tissues and found high levels of expression in the brain, testes, and heart with lower levels of expression in other tissues. mRNA was detected in many tissues and cell lines as determined by Northern blotting.     

Functional analysis of calcium-binding EF-hand motifs of visinin-like protein-1

Visinin-like protein-1 (VILIP-1), a myristoylated calcium sensor protein with three EF-hand motifs, modulates adenylyl cyclase activity. It translocates to membranes when a postulated "calcium-myristoyl switch" is triggered by calcium-binding to expose its sequestered myristoyl moiety. We investigated the contributions of the EF-hand motifs to the translocation of VILIP-1 to membranes and to the modulation of adenylyl cyclase activity. Mutation of residues crucial for binding calcium within each one of the EF-hand motifs indicated that they all contributed to binding calcium. Simultaneous mutations of all of the three EF-hand motifs completely abolished VILIP-1's ability to bind calcium, attenuated but did not eliminate its modulation of adenylyl cyclase activity, and abolished its calcium-dependence for association with cellular membranes. These results show that the calcium-binding EF-hand motifs of VILIP-1 do not have an essential role in modulating adenylyl cyclase activity but instead have a structural role in activating the "calcium-myristoyl switch" of VILIP-1.     

Role of phospholipase C in Dictyostelium: formation of inositol 1,4,5-trisphosphate and normal development in cells lacking phospholipase C activity.

The micro-organism Dictyostelium uses extracellular cAMP to induce chemotaxis and cell differentiation. Signals are transduced via surface receptors, which activate G proteins, to effector enzymes. The deduced protein sequence of Dictyostelium discoideum phosphatidylinositol-specific phospholipase C (PLC) shows strong homology with the mammalian PLC-delta isoforms. To study the role of PLC in Dictyostelium, a plc- mutant was constructed by disruption of the PLC gene. No basal or stimulated PLC activity could be measured during the whole developmental programme of the plc- cells. Loss of PLC activity did not result in a visible alteration of growth or development. Further analysis showed that developmental gene regulation, cAMP-mediated chemotaxis and activation of guanylyl and adenylyl cyclase were normal. Although the cells lack PLC activity, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was present at only slightly lower concentrations compared with control cells. Mass analysis of inositol phosphates demonstrated the presence of a broad spectrum of inositol phosphates in Dictyostelium, which was unaltered in the plc- mutant. Cell labelling experiments with [3H]inositol indicated that [3H]Ins(1,4,5)P3 was formed in a different manner in the mutant than in control cells.     

Phosphatidyl inositol 4,5-bisphosphate-specific phospholipase C in bovine rod outer segment membranes.

Polyphosphoinositide-specific phosphodiesterase (phospholipase C) activity against phosphatidylinositol 4,5-bisphosphate has been examined in disrupted bovine retinal rod outer segments. The enzyme was strictly modulated by free calcium ion concentration and maximally activated at 10(-5) M Ca2+ (91 +/- 4 nmoles phosphatidylinositol 4,5-bisphosphate hydrolyzed/min/mg of protein). Guanine nucleotides did not affect in vitro phospholipase C activity either in the presence or absence of light, carbachol or epinephrine. The pH optimum at 10(-5) M Ca2+ in the presence of sodium deoxycholate was 6.5. The enzyme of bovine rod outer segments was concluded to be indirectly regulated by the phototransduction events.     

The role of EF-hand domains and C2 domain in regulation of enzymatic activity of phospholipase Czeta

Sperm-specific phospholipase C-zeta (PLCzeta) induces Ca2+ oscillations and egg activation when injected into mouse eggs. PLCzeta has such a high Ca2+ sensitivity of PLC activity that the enzyme can be active in resting cells at approximately 100 nM Ca2+, suitable for a putative sperm factor to be introduced into the egg at fertilization (Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and Miyazaki, S. (2004) J. Biol. Chem. 279, 10408-10412). In the present structure-function analysis, deletion of EF1 and EF2 of the N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-hydrolyzing activity in vitro and loss of Ca2+ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 or mutation of EF1 or EF2 at the x and z positions of the putative Ca2+-binding loop little affected the Ca2+ sensitivity of the PLC activity, whereas deletion of EF1 to EF3 caused 12-fold elevation of the EC50 of Ca2+ concentration. Thus, EF1 and EF2 are important for the PLCzeta activity, and EF3 is responsible for its high Ca2+ sensitivity. Deletion of four EF-hand domains or the C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisites for PLCzeta activity. Screening of interactions between the C2 domain and phosphoinositides revealed that C2 has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P but not to PI(4,5)P2 or acidic phospholipids. PI(3)P and PI(5)P reduced PLCzeta activity in vitro, suggesting that the interaction could play a role for negative regulation of PLCzeta.     

Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs

Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1–19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, ‘black’ and ‘gray’ clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.     

Cyclic and noncyclic inositol phosphates are formed at different ratios by phospholipase C isozymes

The cyclic inositol phosphate content in the product of PLC-beta, gamma, and delta mediated cleavage of three phosphoinositides, PtdIns, PtdIns(4)p, and PtdIns(4,5)P2, was measured under several different experimental conditions. The ratio of cyclic to noncyclic product generally decreased in the order PLC-beta greater than PLC-delta greater than PLC-gamma. For all three enzymes the ratio decreased in the order PtdIns greater than PtdIns(4)P greater than PtdIns(4,5)P2. For all combinations of the three enzymes and three substrates cyclic product content was always higher at pH 5.5 than at pH 7.0. The effect of Ca2+ on the ratio of cyclic to noncyclic was also measured. The ratio remained constant between 0.5 microM and 2 mM for PtdIns. For PtdIns(4)P and PtdIns(4,5)P2, the ratios were unchanged between 0.5 and 500 microM, but increased abruptly at millimolar Ca2+ concentrations.     

Spectroscopic characterization of the EF-hand domain of phospholipase C delta1: identification of a lipid interacting domain

The interaction of the isolated EF-hand domain of phospholipase C delta1 with arachidonic acid (AA) was characterized using circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectral changes indicate that AA binds to the EF domain. The near-UV CD spectra suggest that the orientations of aromatic residues in the peptide are affected when AA binds to the protein. The fluorescence of the single intrinsic tryptophan located in EF1 was enhanced by the addition of dodecylmaltoside (DDM) and AA suggesting that this region of the protein is involved in hydrophobic interactions. In the presence of a low concentration of DDM it was found that AA induced a change in fluorescence resonance energy transfer, which is indicative of a conformational change. The lipid induced conformational change may play a role in calcium binding because the isolated EF-hand domain did not bind Ca2+ in the absence of lipids, but Ca2+-dependent changes in the intrinsic tryptophan emission were observed when free fatty acids were present. These studies identify specific EF-hand domains as allosteric regulatory domains that require hydrophobic ligands such as lipids.     

Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs

The 'EF-hand' Ca2+-binding motif plays an essential role in eukaryotic cellular signalling, and the proteins containing this motif constitute a large and functionally diverse family. The EF-hand is defined by its helix-loop-helix secondary structure as well as the ligands presented by the loop to bind the Ca2+ ion. The identity of these ligands is semi-conserved in the most common (the 'canonical') EF-hand; however, several non-canonical EF-hands exist that bind Ca2+ by a different co-ordination mechanism. EF-hands tend to occur in pairs, which form a discrete domain so that most family members have two, four or six EF-hands. This pairing also enables communication, and many EF-hands display positive co-operativity, thereby minimizing the Ca2+ signal required to reach protein saturation. The conformational effects of Ca2+ binding are varied, function-dependent and, in some cases, minimal, but can lead to the creation of a protein target interaction site or structure formation from a molten-globule apo state. EF-hand proteins exhibit various sensitivities to Ca2+, reflecting the intrinsic binding ability of the EF-hand as well as the degree of co-operativity in Ca2+ binding to paired EF-hands. Two additional factors can influence the ability of an EF-hand to bind Ca2+: selectivity over Mg2+ (a cation with very similar chemical properties to Ca2+ and with a cytoplasmic concentration several orders of magnitude higher) and interaction with a protein target. A structural approach is used in this review to examine the diversity of family members, and a biophysical perspective provides insight into the ability of the EF-hand motif to bind Ca2+ with a wide range of affinities.     

Two EF-hand motifs in ryanodine receptor calcium release channels contribute to isoform-specific regulation by calmodulin

The mammalian ryanodine receptor Ca²⁺ release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca²⁺. This suggests isoform-specific domains are involved in RyR regulation by CaM. To gain insight into the differential regulation of cardiac and skeletal muscle RyRs by CaM, RyR1/RyR2 chimeras and mutants were expressed in HEK293 cells, and their single channel activities were measured using a lipid bilayer method. All RyR1/RyR2 chimeras and mutants were inhibited by CaM at 2 μM Ca²⁺, consistent with CaM inhibition of RyR1 and RyR2 at micromolar Ca²⁺ concentrations. An RyR1/RyR2 chimera with RyR1 N-terminal amino acid residues (aa) 1–3725 and RyR2 C-terminal aa 3692–4968 were inhibited by CaM at <1 μM Ca²⁺ similar to RyR2. In contrast, RyR1/RyR2 chimera with RyR1 aa 1–4301 and RyR2 4254–4968 was activated at <1 μM Ca²⁺ similar to RyR1. Replacement of RyR1 aa 3726–4298 with corresponding residues from RyR2 conferred CaM inhibition at <1 μM Ca²⁺, which suggests RyR1 aa 3726–4298 are required for activation by CaM. Characterization of additional RyR1/RyR2 chimeras and mutants in two predicted Ca²⁺ binding motifs in RyR1 aa 4081–4092 (EF1) and aa 4116–4127 (EF2) suggests that both EF-hand motifs and additional sequences in the large N-terminal regions are required for isoform-specific RyR1 and RyR2 regulation by CaM at submicromolar Ca²⁺ concentrations.     

Identification of ISC1 (YER019w) as inositol phosphosphingolipid phospholipase C in Saccharomyces cerevisiae

Sphingolipids have emerged as novel bioactive mediators in eukaryotic cells including yeast. It has been proposed that sphingomyelin (SM) hydrolysis and the concomitant generation of ceramide are involved in various stress responses in mammalian cells. The yeast Saccharomyces cerevisiae has inositol phosphosphingolipids (IPS) instead of SM and glycolipids, and synthesis of IPS is indispensable to its growth. Although the genes responsible for the synthesis of IPS have been identified, the gene(s) for the degradation of IPS has not been reported. Here we show that ISC1 (YER019w), which has homology to bacterial neutral sphingomyelinase (SMase), encodes IPS phospholipase C (IPS-PLC). First, we observed that overexpression of ISC1 greatly increased neutral SMase activity, and this activity was dependent on the presence of phosphatidylserine. Cells deleted in ISC1 demonstrated negligible neutral SMase activity. Because yeast cells have IPS instead of SM, we investigated whether IPS are the physiologic substrates of this enzyme. Lysates of ISC1-overexpressing cells demonstrated very high PLC activities on IPS. Deletion of ISC1 eliminated endogenous IPS-PLC activities. Labeling yeast cells with [(3)H]dihydrosphingosine showed that IPS were increased in the deletion mutant cells. This study identifies the first enzyme involved in catabolism of complex sphingolipids in S. cerevisiae.     

Inositol phospholipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C

Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells. They fulfill many important functions through interaction with a wide range of cellular proteins. Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol. The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C. Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development. In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs. The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom.     

Mammalian cells that express Bacillus cereus phosphatidylinositol-specific phospholipase C have increased levels of inositol cyclic 1:2-phosphate, inositol 1-phosphate, and inositol 2-phosphate.

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol. NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated. Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold). The increases correlated with the levels of enzyme expression obtained in each cell type. The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection. The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered. Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.