Action of Phosphatidylinositol-specific Phospholipase Cγ1 on Soluble and Micellar Substrates

Zhou, Chun; Horstman, Debra A.; Carpenter, Graham; Roberts, Mary F.

doi:10.1074/jbc.274.5.2786

Cited by 26 publications

(23 citation statements)

References 32 publications

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“…The exact basis for this effect is uncertain but Nishibe et al (1990) suggested that it mimicked the role of profilin or glucosphingolipids in cells and selectively inhibited the PIP 2 -hydrolyzing activity of unphosphorylated PLC␥1 compared with phosphorylated control. Zhou et al (1999) proposed that Triton and Ca 2ϩ both modified the accessibility of the substrate at the membrane interface.…”

Section: Discussionmentioning

confidence: 99%

Activation of Phospholipase Cγ2 by Tyrosine Phosphorylation

Özdener

Dangelmaier

Ashby

et al. 2002

Molecular Pharmacology

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Phospholipase C␥2 (PLC␥2) has been implicated in collageninduced signal transduction in platelets and antigen-dependent signaling in B-lymphocytes. It has been suggested that tyrosine kinases activate PLC␥2. We expressed the full-length cDNA for human PLC␥2 in bacteria and purified the recombinant enzyme. The recombinant enzyme was Ca 2ϩ -dependent with optimal activity in the range of 1 to 10 M Ca 2ϩ . In vitro phosphorylation experiments with recombinant PLC␥2 and recombinant Lck, Fyn, and Lyn tyrosine kinases showed that phosphorylation of PLC␥2 led to activation of the recombinant enzyme. Using site-directed mutagenesis, we investigated the role of specific tyrosine residues in activation of PLC␥2. A mutant form of PLC␥2, in which all three tyrosines at positions 743, 753, and 759 in the SH2-SH3 linker region were replaced by phenylalanines, exhibited decreased Lckinduced phosphorylation and completely abolished the Lckdependent activation of PLC␥2. Individual mutations of these tyrosine residues demonstrated that tyrosines 753 and 759, but not 743, were responsible for Lck-induced activation of PLC␥2. To confirm these results, we procured a phosphospecific antibody to a peptide containing phosphorylated tyrosines corresponding to residues 753 and 759. This antibody recognized phosphorylated wild-type PLC␥2 on Western blots but did not interact with unphosphorylated PLC␥2 or with PLC␥2 containing mutated tyrosine residues at 753 and 759. Using this antibody, we showed in intact platelets that collagen, a PLC␥2-dependent agonist, induces phosphorylation of PLC␥2 at Y753 and Y759. These studies demonstrate the importance of these two tyrosine residues in regulating the activity of PLC␥2.

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Section: Discussionmentioning

confidence: 99%

Activation of Phospholipase Cγ2 by Tyrosine Phosphorylation

Özdener

Dangelmaier

Ashby

et al. 2002

Molecular Pharmacology

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“…1A). We also tested the ability of PtdIns(3,4,5)P 3 to stimulate PLC␥ activity measured at pH 5.0, where a pH-dependent conformational change "uncaps" the active site of the enzyme (30). Consistent with the model that tyrosine phosphorylation also uncaps the active site, enzymatic activity of basal state PLC␥ is elevated at pH 5.0 and comparable to that of phosphorylated enzyme at either pH 5.0 or pH 6.8.…”

Section: Discussionmentioning

confidence: 99%

p110β and p110δ Phosphatidylinositol 3-Kinases Up-regulate FcεRI-activated Ca2+ Influx by Enhancing Inositol 1,4,5-Trisphosphate Production

Smith

Surviladze

Gaudet

et al. 2001

Journal of Biological Chemistry

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Fc⑀RI-induced Ca2؉ signaling in mast cells is initiated by activation of cytosolic tyrosine kinases. Here, in vitro phospholipase assays establish that the phosphatidylinositol 3-kinase (PI 3-kinase) lipid product, phosphatidylinositol 3,4,5-triphosphate, further stimulates phospholipase C␥2 that has been activated by conformational changes associated with tyrosine phosphorylation or low pH. A microinjection approach is used to directly assess the consequences of inhibiting class IA PI 3-kinases on Ca 2؉ responses after Fc⑀RI cross-linking in RBL-2H3 cells. Injection of antibodies to the p110␤ or p110␦ catalytic isoforms of PI 3-kinase, but not antibodies to p110␣, lengthens the lag time to release of Ca 2؉ stores and blunts the sustained phase of the calcium response. Ca 2؉ responses are also inhibited in cells microinjected with recombinant inositol polyphosphate 5-phosphatase I, which degrades inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ), or heparin, a competitive inhibitor of the Ins(1,4,5)P 3 receptor. This indicates a requirement for Ins(1,4,5)P 3 to initiate and sustain Ca 2؉ responses even when PI 3-kinase is fully active. Antigeninduced cell ruffling, a calcium-independent event, is blocked by injection of p110␤ and p110␦ antibodies, but not by injection of 5-phosphatase I, heparin, or antip110␣ antibodies. These results suggest that the p110␤ and p110␦ isoforms of PI 3-kinase support Fc⑀RI-induced calcium signaling by modulating Ins(1,4,5)P 3 production, not by directly regulating the Ca 2؉ influx channel.Cross-linking of the high affinity receptor for IgE, Fc⑀RI, on mast cells results in the activation of tyrosine kinases Lyn and Syk, followed by tyrosine phosphorylation and activation of PLC␥ 1 isoforms and other downstream effectors (1). Subsequent hydrolysis of phosphatidylinositol 4,5-diphosphate by PLC␥ leads to the formation of Ins(1,4,5)P 3 , the release of Ca 2ϩ from internal stores (2), and the influx of Ca 2ϩ through storeoperated channels (3). Calcium influx, in turn, supports degranulation and the release of inflammatory mediators (4). This scenario was complicated in recent years by observations that PLC␥ activation and Ca 2ϩ responses and secretion are all diminished in mast cells treated with inhibitors of PI 3-kinases (5, 6). It was subsequently discovered that full activation of PLC␥ proteins in mast cells requires both tyrosine phosphorylation, potentially mediated by Tec family kinases (7), and interaction with the lipid products of PI 3-kinase (8). PtdIns(3,4,5)P 3 mediates membrane recruitment and phosphorylation of PLC␥1 in RBL-2H3 cells (8) and increases the activity of both PLC␥1 and PLC␥2 against lipid micelle substrates in vitro (9, 10).Despite demonstrated roles for PI 3-kinase in the activation of PLC␥, its specific contribution to the temporal regulation of Fc⑀RI-mediated Ca 2ϩ signaling is unresolved. Although wortmannin treatment can slightly increase the lag time leading to the initial rise in calcium after antigen stimulation, it does not block the initial response (1...

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“…Around the barrel rim, there are regions where hydrophobic side chains are exposed to solvent. The structural similarity of the bacterial PI-PLC to the mammalian catalytic domain and similar kinetic features (12)(13)(14) suggest that the smaller enzyme can provide a simple model for understanding how different interfaces modulate catalysis.Previous work has shown that the PI-PLC from Bacillus cereus or Bacillus thuringiensis not only prefers micellar to monomeric substrate (15) but is also activated by binding to phosphatidylcholine (PC) interfaces (2,16,17). The binding has an allosteric effect on the enzyme, since it increases k cat and decreases K m for hydrolysis of the soluble substrate cIP.…”

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confidence: 99%

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confidence: 99%

Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

Shao

Shi

Wehbi

et al. 2007

Journal of Biological Chemistry

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The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8 Å resolution. The W47A/ W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp 3 Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membranepartitioning residues.Phosphatidylinositol-specific phospholipase C (PI-PLC) 2 enzymes catalyze the specific cleavage of the sn-3-phosphodiester bond in phosphatidylinositol (PI) and phosphoinositides. PI hydrolysis occurs in two sequential reactions (1-3): (i) an intramolecular phosphotransferase reaction at a phospholipid/ aggregate surface to produce diacylglycerol and water-soluble 1,2-cyclic inositol phosphate (cIP) (or cIP x in the case of a phosphoinositide substrate), followed by (ii) a phosphodiesterase reaction where water-soluble cIP is hydrolyzed to inositol-1-phosphate. Since their substrate is presented in an interface, their activity can often be modulated by the composition of the interface. Eukaryotic PI-PLCs, as critical components of phosphatidylinositol-mediated signaling pathways (4, 5), have multiple domains for regulation of enzyme activity both by regulating partitioning to membranes and by allosteric effects on catalysis, and these are often hard to separate. However, the much smaller bacterial PI-PLC enzymes are secreted, single domain proteins whose crystal structures (6 -8) resemble the catalytic domain of PLC␦ 1 , the only mammalian PI-PLC for which there is a structure (9 -11). The molecular topology is that of a distorted TIM barrel. Around the barrel rim, there are regions where hydrophobic side chains are exposed to solvent. The structural similarity of the bacterial PI-PLC to the mammalian catalytic domain and...

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Action of Phosphatidylinositol-specific Phospholipase Cγ1 on Soluble and Micellar Substrates

Cited by 26 publications

References 32 publications

Activation of Phospholipase Cγ2 by Tyrosine Phosphorylation

Activation of Phospholipase Cγ2 by Tyrosine Phosphorylation

p110β and p110δ Phosphatidylinositol 3-Kinases Up-regulate FcεRI-activated Ca2+ Influx by Enhancing Inositol 1,4,5-Trisphosphate Production

Dimer Structure of an Interfacially Impaired Phosphatidylinositol-specific Phospholipase C

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