Biomedical subjects

M Katan

Publications and source records attributed to M Katan.

At least 19 recordsLinked to original sources

PubMed2002

Phospholipase C isoforms in mammalian spermatozoa: potential components of the sperm factor that causes Ca2+ release in eggs.

Injection of a soluble protein factor from mammalian spermatozoa triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilization. This sperm factor also generates inositol 1,4,5-trisphosphate and causes Ca2+ release in sea urchin egg homogenates and frog eggs. Recent studies have indicated that the sperm factor may be an inositol-specific phospholipase C (PLC) activity. This study investigated whether any of the commonly known PLC isoforms are components of the sperm factor. PLCbeta, PLCgamma and PLCdelta isoforms were shown to be present in boar sperm extracts. However, upon column fractionation of sperm extracts, none of the PLC isoforms detected correlated with the ability to cause Ca2+ release in eggs. In addition to our previous work on recombinant PLCs, it was also shown that PLCdelta3, PLCdelta4 and its splice variant PLCdelta4 Alt1 fail to cause Ca2+ release. The recently discovered 255 kDa PLCepsilon isoform also appears unlikely to be a component of the sperm factor, as fractionation of sperm extracts on a gel filtration column demonstrated that the peak of Ca2+-releasing activity was associated with fractions of 30-70 kDa. These findings indicate that the sperm factor that triggers Ca2+ release in eggs does not appear to have a known PLC isoform as one of its components.

Animals↗

PubMed2001

Tyrosine residues in phospholipase Cgamma 2 essential for the enzyme function in B-cell signaling.

Phospholipase Cgamma (PLCgamma) isoforms are regulated through activation of tyrosine kinase-linked receptors. The importance of growth factor-stimulated phosphorylation of specific tyrosine residues has been documented for PLCgamma1; however, despite the critical importance of PLCgamma2 in B-cell signal transduction, neither the tyrosine kinase(s) that directly phosphorylate PLCgamma2 nor the sites in PLCgamma2 that become phosphorylated after stimulation are known. By measuring the ability of human PLCgamma2 to restore calcium responses to the B-cell receptor stimulation or oxidative stress in a B-cell line (DT40) deficient in PLCgamma2, we have demonstrated that two tyrosine residues, Tyr(753) and Tyr(759), were important for the PLCgamma2 signaling function. Furthermore, the double mutation Y753F/Y759F in PLCgamma2 resulted in a loss of tyrosine phosphorylation in stimulated DT40 cells. Of the two kinases that previously have been proposed to phosphorylate PLCgamma2, Btk, and Syk, purified Btk had much greater ability to phosphorylate recombinant PLCgamma2 in vitro, whereas Syk efficiently phosphorylated adapter protein BLNK. Using purified proteins to analyze the formation of complexes, we suggest that function of Syk is to phosphorylate BLNK, providing binding sites for PLCgamma2. Further analysis of PLCgamma2 tyrosine residues phosphorylated by Btk and several kinases from the Src family has suggested multiple sites of phosphorylation and, in the context of a peptide incorporating residues Tyr(753) and Tyr(759), shown preferential phosphorylation of Tyr(753).

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2001

Sequence analysis identifies TTRAP, a protein that associates with CD40 and TNF receptor-associated factors, as a member of a superfamily of divalent cation-dependent phosphodiesterases.

CD40 is a member of the tumor necrosis factor (TNF) receptor family. CD40-mediated signal transduction involves the recruitment of several cytoplasmic proteins and induces expression of a large number of genes. TTRAP, a novel protein that interacts with the cytoplasmic domain of CD40 and with TNF-receptor associated factors (TRAFs), has been cloned and shown to inhibit nuclear factor-kappaB activation (NF-kappaB). By using various bioinformatics-based sequence and structure analyses of proteins involved in signaling by the TNF receptor family, we found that TTRAP is a member of a superfamily of Mg(2+)/Mn(2+)-dependent phosphodiesterases. More precisely, our results suggest that TTRAP is related to the human APE1, a Mg(2+)-dependent endonuclease. This potential novel function of TTRAP raises the intriguing possibility for a role of APE1-like DNA-repair endonucleases in TNF receptor family-mediated signaling and functions.

Amino Acid Motifs↗

PubMed2001

Real time fluorescence imaging of PLC gamma translocation and its interaction with the epidermal growth factor receptor.

The translocation of fluorescently tagged PLC gamma and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor--effector interactions. The translocation of PLC gamma to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC gamma isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC gamma, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P(2)), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P(2) substrate could also be visualized. At later times, internalization of PLC gamma, which could lead to separation from the substrate, was observed. The data support a direct binding of PLC gamma to the receptor as the main site of the plasma membrane recruitment. The presence of PLC gamma in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.

Animals↗

PubMed2001

Consumption of a solid fat rich in lauric acid results in a more favorable serum lipid profile in healthy men and women than consumption of a solid fat rich in trans-fatty acids.

Solid fats are used in food manufacturing to provide texture and firmness to foods. Such fats are rich in either saturated or trans-fatty acids, both of which increase the risk of coronary heart disease. Epidemiological and experimental studies suggest that trans-fatty acids increase risk more than do saturates because they lower serum high density lipoprotein (HDL) cholesterol. However, there appear to be differences between saturates in their effect on HDL cholesterol. We investigated whether the consumption of a solid fat rich in lauric acid (C12:0) would result in a more favorable blood lipid profile than the consumption of a solid fat rich in trans-fatty acids. We fed 32 healthy men and women two controlled diets in a 2 x 4-wk randomized crossover design. The diets consisted of a background diet supplemented with margarines. In the trans-diet, 9.2% of energy was provided by trans-fatty acids and 12.9% by saturated fatty acids. In the Sat-diet, energy intake was 0% from trans-fatty acids and 22.9% from saturated fatty acids. Lauric acid composed one third of all saturates in the Sat-diet. Serum HDL cholesterol was 0.36 mmol/L lower at the end of the trans-diet than at the end of the Sat-diet (95% confidence interval, -0.46 to -0.26), whereas serum low density lipoprotein cholesterol and triglyceride concentrations remained stable. Serum total cholesterol was 0.31 mmol/L (95% confidence interval, -0.48 to -0.14) lower at the end of the trans-diet than at the end of the Sat-diet. Consumption of a solid fat rich in lauric acid gives a more favorable serum lipoprotein pattern than consumption of partially hydrogenated soybean oil rich in trans-fatty acids. Thus, solid fats rich in lauric acids, such as tropical fats, appear to be preferable to trans-fats in food manufacturing, where hard fats are indispensable.

Adolescent↗

PubMed2000

Structural requirements for catalysis and membrane targeting of mammalian enzymes with neutral sphingomyelinase and lysophospholipid phospholipase C activities. Analysis by chemical modification and site-directed mutagenesis.

The sequence similarity with bacterial neutral sphingomyelinase resulted in the isolation of putative mammalian counterparts and, subsequently, identification of similar molecules in a number of other eukaryotic organisms. Based on sequence similarities and previous characterization of the mammalian enzymes, we have chemically modified specific residues and performed site-directed mutagenesis in order to identify critical catalytic residues and determinants for membrane localization. Modification of histidine residues and the substrate protection experiments demonstrated the presence of reactive histidine residues within the active site. Site directed mutagenesis suggested an essential role in catalysis for two histidine residues (His-136 and His-272), which are conserved in all sequences. Mutations of two additional histidines (His-138 and His-151), conserved only in eukaryotes, resulted in reduced neutral sphingomyelinase activity. In addition to sphingomyelin, the enzyme also hydrolyzed lysophosphatidylcholine. Exposure to an oxidizing environment or modification of cysteine residues using several specific compounds also inactivated the enzyme. Site-directed mutagenesis of eight cysteine residues and gel-shift analysis demonstrated that these residues did not participate in the catalytic reaction and suggested the involvement of cysteines in the formation/breakage of disulfide bonds, which could underlie the reversible inactivation by the oxidizing compounds. Cellular localization studies of a series of deletion mutants, expressed as green fluorescent protein fusion proteins, demonstrated that the transmembrane region contains determinants for the endoplasmic reticulum localization.

Amino Acid Sequence↗

PubMed2000

Inhibition of Ca(2+) signalling by p130, a phospholipase-C-related catalytically inactive protein: critical role of the p130 pleckstrin homology domain.

p130 was originally identified as an Ins(1,4,5)P(3)-binding protein similar to phospholipase C-delta but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P(3)-mediated Ca(2+) signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P(3) with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P(3). Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca(2+) signalling. When fura-2-loaded COS-1(p130) cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca(2+) concentration, observed in control cells, was inhibited in COS-1(p130). This inhibition was not accompanied by the decreased production of Ins(1,4,5)P(3); the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P(3) could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P(3)-mediated Ca(2+) signalling.

3T3 Cells↗

PubMed2000

Different Ca2+-releasing abilities of sperm extracts compared with tissue extracts and phospholipase C isoforms in sea urchin egg homogenate and mouse eggs.

A soluble phospholipase C (PLC) from boar sperm generates InsP(3) and hence causes Ca(2+) release when added to sea urchin egg homogenate. This PLC activity is associated with the ability of sperm extracts to cause Ca(2+) oscillations in mammalian eggs following fractionation. A sperm PLC may, therefore, be responsible for causing the observed Ca(2+) oscillations at fertilization. In the present study we have further characterized this boar sperm PLC activity using sea urchin egg homogenate. Consistent with a sperm PLC acting on egg PtdIns(4,5)P(2), the ability of sperm extracts to release Ca(2+) was blocked by preincubation with the PLC inhibitor U73122 or by the addition of neomycin to the homogenate. The Ca(2+)-releasing activity was also detectable in sperm from other species and in whole testis extracts. However, activity was not observed in extracts from other tissues. Moreover recombinant PLCbeta1, -gamma1, -gamma2, -delta1, all of which had higher specific activities than boar sperm extracts, were not able to release Ca(2+) in the sea urchin egg homogenate. In addition these PLCs were not able to cause Ca(2+) oscillations following microinjection into mouse eggs. These results imply that the sperm PLC possesses distinct properties that allow it to hydrolyse PtdIns(4,5)P(2) in eggs.

Animals↗

PubMed2000

A neutral magnesium-dependent sphingomyelinase isoform associated with intracellular membranes and reversibly inhibited by reactive oxygen species.

Activation of neutral sphingomyelinase(s) and subsequent generation of ceramide has been implicated in a wide variety of cellular responses. Although this enzyme(s) has not been purified and cloned from higher organisms, one mammalian cDNA has been previously isolated based on its similarity to the bacterial enzyme. To further elucidate the function of this neutral sphingomyelinase, we studied its relationship with enzymes present in mammalian cells and tissues, its subcellular localization, and properties that could be important for the regulation of its activity. Using specific antibodies, it is suggested that the enzyme could represent one of several forms of neutral sphingomyelinases present in the extract from brain particulate fraction. In PC12 cells, the enzyme is localized in the endoplasmic reticulum and is not present in the plasma membrane. The same result has been obtained in several cell lines transfected or microinjected with plasmids encoding this enzyme. The molecular and enzymatic properties of the cloned neutral magnesium-dependent sphingomyelinase, produced using baculovirus or bacterial expression systems, have been analyzed, demonstrating the expected ion dependence and substrate specificity. The enzyme activity also has a strong requirement for reducing agents and is reversibly inhibited by reactive oxygen species and oxidized glutathione. The studies demonstrate that the cellular localization and some properties of this enzyme are distinct from properties previously associated with neutral magnesium-dependent sphingomyelinases in crude or partially purified preparations.

Animals↗

PubMed2000

Domain organization of p130, PLC-related catalytically inactive protein, and structural basis for the lack of enzyme activity.

The 130-kDa protein (p130) was isolated as a novel inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]-binding protein similar to phospholipase C-delta1 (PLC-delta1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. & Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. & Hirata, M. (1996) Biochem. J. 313, 319-325]. To test experimentally the domain organization of p130 and structural basis for lack of PLC activity, we subjected p130 to limited proteolysis and also constructed a number of chimeras with PLC-delta1. Trypsin treatment of p130 produced four major polypeptides with molecular masses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were calculated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC-delta1 and demonstrating that all domains of p130, including the unique region at the C-terminus, are stable, tightly folded structures. p130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that p130 has intrinsically no PLC activity. A number of chimeric molecules between p130 and PLC-delta1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two proteins, as only some domains of p130 could replace the corresponding structures in PLC-delta1 to form a functional enzyme. These results suggest that p130 and the related proteins could represent a new protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.

Animals↗

PubMed1999

Involvement of EF hand motifs in the Ca(2+)-dependent binding of the pleckstrin homology domain to phosphoinositides.

The pleckstrin homology (PH) domains of phospholipase C (PLC)-delta1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by PLC-delta1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca(2+)-dependent activation of PLC-delta1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC-delta1 and p130 possess the EF-hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to PtdIns(4,5)P2 is regulated by changes in free Ca2+ concentration within the physiological range. A Ca2+ dependent increase in the binding to PtdIns(4,5)P2 was observed with a full-length PLC-delta1, while the isolated PH domain did not show any Ca2+ dependence. However, the connection of the EF-hand motifs to the PH domain restored the Ca2+ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC-delta1, and the EF-hand motifs were again required for the PH domain to exhibit a Ca2+ dependent increase in the binding to PtdIns(4,5)P2. The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P2 showed no Ca2+ dependent enhancement of binding. However, when present within a chimera also containing PLC-delta1 EF-hand motifs, the Ca2+ dependent binding was again observed. These results suggest that the binding of Ca2+ to the EF-hand motifs can modulate binding to PtdIns(4,5)P2 mediated by the PH domain.

Blood Proteins↗

PubMed1999

Modular PH and C2 domains in membrane attachment and other functions.

The pleckstrin homology and C2 domains are modular protein structures involved in mediating intermolecular interactions. Although they represent distinct domains, there are several parallels regarding their function and type of interactions in which they participate. Both domains are stable structural entities that incorporate variable regions which, in different proteins, can be adapted to perform a specific function through binding to membrane phospholipids or specific protein ligands. A number of recent examples illustrate the function of some of these domains in regulated membrane attachment, with an important role in many cellular signalling pathways.

Binding Sites↗

PubMed1998

Families of phosphoinositide-specific phospholipase C: structure and function.

A large number of extracellular signals stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). PI-PLC isozymes have been found in a broad spectrum of organisms and although they have common catalytic properties, their regulation involves different signalling pathways. A number of recent studies provided an insight into domain organisation of PI-PLC isozymes and contributed towards better understanding of the structural basis for catalysis, cellular localisation and molecular changes that could underlie the process of their activation.

Animals↗

PubMed1998

Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1.

Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.

Amino Acid Sequence↗

PubMed1998

Replacements of single basic amino acids in the pleckstrin homology domain of phospholipase C-delta1 alter the ligand binding, phospholipase activity, and interaction with the plasma membrane.

The pleckstrin homology (PH) domain of phosphatidylinositol-specific phospholipase C-delta1 (PLC-delta1) binds to both D-myo-inositol 1,4, 5-trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) with high affinities. We have previously identified a region rich in basic amino acids within the PH domain critical for ligand binding (Yagisawa, H., Hirata, M., Kanematsu, T., Watanabe, Y., Ozaki, S., Sakuma, K., Tanaka, H., Yabuta, N., Kamata, H., Hirata, H., and Nojima, H. (1994) J. Biol. Chem. 269, 20179-20188; Hirata, M., Kanematsu, T., Sakuma, K., Koga, T., Watanabe, Y., Ozaki, S., and Yagisawa, H. (1994) Biochem. Biophys. Res. Commun. 205, 1563-1571). To investigate the role of these basic residues, we have performed site-directed mutagenesis replacing each of the basic amino acid in the N-terminal 60 residues of PLC-delta1 (Lys24, Lys30, Lys32, Arg37, Arg38, Arg40, Lys43, Lys49, Arg56, Lys57, and Arg60) with a neutral or an acidic amino acid. The effects of these mutations on the PH domain ligand binding properties and their consequence for substrate hydrolysis and membrane interactions of PLC-delta1 were analyzed using several assay systems. Analysis of [3H]-Ins(1,4,5)P3 binding, measurement of the binding affinities, and measurements of phospholipase activity using PtdIns(4,5)P2-containing phospholipid vesicles, demonstrated that residues Lys30, Lys32, Arg37, Arg38, Arg40, and Lys57 were required for these PLC-delta1 functions; in comparison, other mutations resulted in a moderate reduction. A subset of selected mutations was further analyzed for the enzyme activity toward substrate present in cellular membranes of permeabilized cells and for interaction with the plasma membrane after microinjection. These experiments demonstrated that mutations affecting ligand binding and PtdIns(4,5)P2 hydrolysis in phospholipid vesicles also resulted in reduction in the hydrolysis of cellular polyphosphoinositides and loss of membrane attachment. All residues (with the exception of the K43E substitution) found to be critical for the analyzed PLC-delta1 functions are present at the surface of the PH domain shown to contain the Ins(1,4,5)P3 binding pocket.

Amino Acid Sequence↗

PubMed1997

Distinct specificity in the binding of inositol phosphates by pleckstrin homology domains of pleckstrin, RAC-protein kinase, diacylglycerol kinase and a new 130 kDa protein.

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130 kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130 kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.

Amino Acid Sequence↗

PubMed1997

Regulation of inositol lipid-specific phospholipase cdelta by changes in Ca2+ ion concentrations.

Studies of inositol lipid-specific phospholipase C (PLC) have elucidated the main regulatory pathways for PLCbeta and PLCgamma but the regulation of PLCdelta isoenzymes still remains obscure. Here we demonstrate that an increase in Ca2+ ion concentration within the physiological range (0.1-10 microM) is sufficient to stimulate PLCdelta1, but not PLCgamma1 and PLCbeta1, to hydrolyse cellular inositol lipids present in permeabilized cells. The activity of PLCdelta1 is further enhanced in the presence of phosphatidylinositol transfer protein (PI-TP). Both full activation by Ca2+ ions and stimulation in the presence of PI-TP require an intact PH domain involved in the membrane attachment of PLCdelta1. The physiological implication of this study is that PLCdelta1 could correspond to a previously uncharacterized PLC responsible for Ca2+ ion-stimulated inositol lipid hydrolysis observed in many cellular systems.

Animals↗

PubMed1997

Phosphoinositide-specific phospholipase C delta1 activity toward micellar substrates, inositol 1,2-cyclic phosphate, and other water-soluble substrates: a sequential mechanism and allosteric activation.

The kinetics of full-length and PH domain truncated cloned PI-PLC delta1 from rat toward soluble substrates [inositol 1, 2-(cyclic)-phosphate (cIP) and glycerophosphoinositol phosphates (GPIPx)] as well as PI in detergent micelles provide the following insights into the mechanism of this enzyme. (i) That cIP is a substrate for the enzyme implies a two-step mechanism for PI hydrolysis [intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)]. The dependence of enzyme activity on cIP is sigmoidal, suggesting a transition between less active and more active forms of the enzyme that is affected by substrate. (ii) Interfaces increase the kcat for cIP (but do not affect the cooperativity), and this allosteric activation requires an intact PH domain. (iii) Phosphorylation of the soluble inositol phosphodiesters GPI, GPIP, and GPIP2 enhances PI-PLC delta1 activity by dramatically increasing kcat and decreasing Km. For these phosphodiesters, the substrate saturation curve is no longer sigmoidal but hyperbolic, indicating the phosphorylated substrate can shift the enzyme to the activated form. (iv) Given the kinetic parameters for cIP hydrolysis and the constant ratio of cIP/I-1-P generated during PI hydrolysis, the cIP produced in situ is either released (and not readily rebound since its concentration is well below Km) or attacked by a water molecule for the generation of the acyclic product.

Allosteric Regulation↗