Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated a cDNA encoding a novel protein, PLC-L(2), with homology to the phospholipase C-like protein PLC-L and delta-type phospholipase C. PLC-L(2) contains a relatively well-conserved PH domain, PLC catalytic region, and X and Y domains. However, it did not have PLC activity. This inactivation was thought to be caused by the replacement of two amino acids that are essential for PLC activity, His356 and Tyr552, with Thr and Phe in the X and Y domain. PLC-L(2) has a wide distribution with strong expression in skeletal muscle and mapped to chromosome 3p24-25. The PH domain of PLC-L(2) bound strongly to PI(4,5)P(2) and Ins(1,4,5)P(3), and moderately to PI(4)P and PI(3,4,5)P(3). PLC-L(2) predominantly localized to perinuclear areas in both myoblast and myotube C2C12 cells. Ectopically expressed GFP-PLC-L(2) also mainly localized in perinuclear areas, including endoplasmic reticulum in COS 7 cells. Furthermore, the expression of GFP-PH showed the same intracellular distribution as the full-length PLC-L(2). All these results suggest that PLC-L(2) plays an important role in the regulation of Ins(1,4, 5)P(3) around the endoplasmic reticulum on which the Ins(1,4,5)P(3) receptor exists.
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PMID:Identification and characterization of a new phospholipase C-like protein, PLC-L(2). 1058 Nov 72

Members of the phospholipase C-beta (PLC-beta) family of proteins are activated either by G alpha or G beta gamma subunits of heterotrimeric G proteins. To define specific regions of PLC-beta 3 that are involved in binding and activation by G beta gamma, a series of fragments of PLC-beta 3 as glutathione-S-transferase (GST) fusion proteins were produced. A fragment encompassing the N-terminal pleckstrin homology (PH) domain and downstream sequence (GST-N) bound to G protein beta 1 gamma 2 in an in vitro binding assay, and binding was inhibited by G protein alpha subunit, G alpha i1. This PLC-beta 3 fragment also inhibited G beta gamma-stimulated PLC-beta activity in a reconstitution system, while having no significant effect on G alpha q-stimulated PLC-beta 3 activity. The N-terminal G beta gamma binding region was delineated further to the first 180 amino acids, and the sequence Asn150-Ser180, just distal to the PH domain, was found to be required for the interaction. Mutation of basic residues 154Arg, 155Lys, 159Lys, and 161Lys to Glu within this region reduced G beta gamma binding affinity and specifically reduced the EC50 for G beta gamma-dependent activation of the mutant enzyme 3-fold. Basal activity and G alpha q-dependent activation of the enzyme were unaffected by the mutations. While these basic residues may not directly mediate the interaction with G beta gamma, the data provide evidence for an N-terminal G beta gamma binding region of PLC-beta 3 that is involved in activation of the enzyme.
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PMID:Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits. 1067 30

Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the betagamma subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-beta(2) (PLC-beta(2)), which is regulated by Gbetagamma, replaces the PH domain of PLC-delta(1) which binds to, but is not regulated by, Gbetagamma. We found that attachment of the PH domain of PLC-beta(2) onto PLC-delta(1) not only causes the membrane-binding properties of PLC-delta(1) to become similar to those of PLC-beta(2), but also results in a Gbetagamma-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.
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PMID:The pleckstrin homology domain of phospholipase C-beta(2) links the binding of gbetagamma to activation of the catalytic core. 1071 48

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is known to regulate a wide range of molecular targets and cellular processes, from ion channels to actin polymerization [1] [2] [3] [4] [5] [6]. Recent studies have used the phospholipase C-delta1 (PLC-delta1) pleckstrin-homology (PH) domain fused to green fluorescent protein (GFP) as a detector for PI(4,5)P(2) in vivo [7] [8] [9] [10]. Although these studies demonstrated that PI(4,5)P(2) is concentrated in the plasma membrane, its association with actin-containing structures was not reported. In the present study, fluorescence imaging of living NIH-3T3 fibroblasts expressing the PLC-delta1 PH domain linked to enhanced green fluorescent protein (PH-EGFP) reveals intense, non-uniform fluorescence in distinct structures at the cell periphery. Corresponding fluorescence and phase-contrast imaging over time shows that these fluorescent structures correlate with dynamic, phase-dense features identified as ruffles and with microvillus-like protrusions from the cell's dorsal surface. Imaging of fixed and permeabilized cells shows co-localization of PH-EGFP with F-actin in ruffles, but not with vinculin in focal adhesions. The selective concentration of the PH-EGFP fusion protein in highly dynamic regions of the plasma membrane that are rich in F-actin supports the hypothesis that localized synthesis and lateral segregation of PI(4,5)P(2) spatially restricts actin polymerization and thereby affects cell spreading and retraction.
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PMID:Dynamics of phosphatidylinositol 4,5-bisphosphate in actin-rich structures. 1087 4

Pleckstrin homology (PH) domains are small protein modules of around 120 amino acids found in many proteins involved in cell signalling, cytoskeletal rearrangement and other processes. Although several different protein ligands have been proposed for PH domains, their only clearly demonstrated physiological function to date is to bind membrane phosphoinositides. The PH domain from phospholipase C-delta(1) binds specifically to PtdIns(4,5)P(2) and its headgroup, and has become a valuable tool for studying cellular PtdIns(4,5)P(2) functions. More recent developments have demonstrated that a subset of PH domains recognizes the products of agonist-stimulated phosphoinositide 3-kinases. Fusion of these PH domains to green fluorescent protein has allowed dramatic demonstrations of their independent ability to drive signal-dependent recruitment of their host proteins to the plasma membrane. We discuss the structural basis for this 3-phosphoinoistide recognition and the role that it plays in cellular signalling. PH domains that bind specifically to phosphoinositides comprise only a minority (perhaps 15%) of those known, raising questions as to the physiological role of the remaining 85% of PH domains. Most (if not all) PH domains bind weakly and non-specifically to phosphoinositides. Studies of dynamin-1 have indicated that oligomerization of its PH domain may be important in driving membrane association. We discuss the possibility that membrane targeting by PH domains with low affinity for phosphoinositides could be driven by alteration of their oligomeric state and thus the avidity of their membrane binding.
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PMID:Signal-dependent membrane targeting by pleckstrin homology (PH) domains. 1092 21

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.
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PMID:Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation. 1095 90

Eleven distinct isoforms of phosphoinositide-specific phospholipase C (PLC), which are grouped into four subfamilies (beta, gamma, delta, and epsilon), have been identified in mammals. These isozymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to inositol 1,4,5-trisphosphate and diacylglycerol in response to the activation of more than 100 different cell surface receptors. All PLC isoforms contain X and Y domains, which form the catalytic core, as well as various combinations of regulatory domains that are common to many other signaling proteins. These regulatory domains serve to target PLC isozymes to the vicinity of their substrate or activators through protein-protein or protein-lipid interactions. These domains (with their binding partners in parentheses or brackets) include the pleckstrin homology (PH) domain [PtdIns(3)P, beta gamma subunits of G proteins] and the COOH-terminal region including the C2 domain (GTP-bound alpha subunit of Gq) of PLC-beta; the PH domain [PtdIns(3,4,5)P3] and Src homology 2 domain [tyrosine-phosphorylated proteins, PtdIns(3,4,5)P3] of PLC-gamma; the PH domain [PtdIns(4,5)P2] and C2 domain (Ca2+) of PLC-delta; and the Ras binding domain (GTP-bound Ras) of PLC-epsilon. The presence of distinct regulatory domains in PLC isoforms renders them susceptible to different modes of activation. Given that the partners that interact with these regulatory domains of PLC isozymes are generated or eliminated in specific regions of the cell in response to changes in receptor status, the activation and deactivation of each PLC isoform are likely highly regulated processes.
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PMID:Regulation of phosphoinositide-specific phospholipase C. 1139 9

FYVE domains are small zinc-finger-like domains found in many proteins that are involved in regulating membrane traffic and have been shown to bind specifically to phosphatidylinositol 3-phosphate (PtdIns-3-P). FYVE domains are thought to recruit PtdIns-3-P effectors to endosomal locations in vivo, where these effectors participate in controlling endosomal maturation and vacuolar protein sorting. We have compared the characteristics of PtdIns-3-P binding by the FYVE domain from Hrs-1 (the hepatocyte growth factor-regulated tyrosine kinase substrate) with those of specific phosphoinositide binding by Pleckstrin homology (PH) domains. Like certain PH domains (such as that from phospholipase C-delta(1)), the Hrs-1 FYVE domain specifically recognizes a single phosphoinositide. However, while phosphoinositide binding by highly specific PH domains is driven almost exclusively by interactions with the lipid headgroup, this is not true for the Hrs-1 FYVE domain. The phospholipase C-delta(1) PH domain shows a 10-fold preference for binding isolated headgroup over its preferred lipid (phosphatidylinositol 4,5-bisphosphate) in a membrane, while the Hrs-1 FYVE domain greatly prefers (more than 50-fold) intact lipid in a bilayer over the isolated headgroup (inositol 1,3-bisphosphate). By contrast with reports for certain PH domains, we find that this preference for membrane binding over interaction with soluble lipid headgroups does not require FYVE domain oligomerization.
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PMID:High-affinity binding of a FYVE domain to phosphatidylinositol 3-phosphate requires intact phospholipid but not FYVE domain oligomerization. 1145 98

Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-delta1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.
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PMID:Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity. 1147 Aug 24

The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C delta1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the beta1-loop1-beta2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 are bona fide PH domain proteins, with broad specificity and moderate affinity for PIPs.
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PMID:Phosphoinositide binding by the pleckstrin homology domains of Ipl and Tih1. 1237 6


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