Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate.

We studied the binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta) to vesicles containing the negatively charged phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). PLC-delta did not bind significantly to large unilamellar vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but bound strongly to vesicles formed from mixtures of PC and PIP2. The apparent association constant for the putative 1:1 complex formed between PLC-delta and PIP2 was Ka congruent to 10(5) M-1. The binding strength increased further (Ka congruent to 10(6) M-1) when the vesicles also contained 30% PS. High-affinity binding of PLC-delta to PIP2 did not require Ca2+. PLC-delta bound only weakly to vesicles formed from mixtures of PC and either PS or phosphatidylinositol (PI); binding increased as the mole fraction of acidic lipid in the vesicles increased. We also studied the membrane binding of a small basic peptide that corresponds to a conserved region of PLC. Like PLC-delta, the peptide bound weakly to vesicles containing monovalent negatively charged lipids; unlike PLC-delta, it did not bind strongly to vesicles containing PIP2. Our data suggest that a significant fraction of the PLC-delta in a cell could be bound to PIP2 on the cytoplasmic surface of the plasma membrane.     

Stimulation of phosphatidylinositol 4,5-bisphosphate phospholipase C activity by phosphatidic acid

Phosphatidic acid was a potent activator of the phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity associated with human platelet membranes. Lysophosphatidic acid was half as active as phosphatidic acid, and shortening the fatty acid chain reduced the effectiveness of the corresponding phosphatidic acid. Compounds lacking either the phosphate group (diacylglycerol or phorbol ester) or the fatty acid (glycerol phosphate) were not activators. When the negative charge was contributed by a carboxyl group (fatty acid or phosphatidylserine), stimulation of phospholipase C was weak but detectable. Structural analogs of phosphatidic acid (lipopolysaccharide, lipid A, and 2,3-diacylglucosamine 1-phosphate) were less effective but also enhanced PtdIns-P2 hydrolysis. Phosphatidic acid potentiated the activation of phospholipase C by alpha-thrombin, chelators, and guanine nucleotides. Phosphatidylinositol 4-phosphate and PtdIns-P2 were also effective activators of PtdIns-P2 degradation. Other phospholipids were without effect. The production of inositol 1,4,5-trisphosphate and diacylglycerol via the activation of phospholipase C provides a rationale for the cellular responses evoked by phosphatidic acid and the ability of this phospholipid to potentiate and initiate hormonal responses.     

Inhibition of neuronal nitric oxide synthase by phosphatidylinositol 4,5-bisphosphate and phosphatidic acid.

Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) were found to inhibit strongly the citrulline formation activity of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39). Such inhibition was not observed with any other phospholipid examined. A kinetic analysis of purified nNOS showed no significant change in apparent K(m) for L-Arg or NADPH caused by these inhibitory phospholipids. Electron paramagnetic resonance analysis revealed no significant spectral perturbation of the ferriheme or flavin semiquinone upon the addition of PIP2. On the other hand, a lower enhancement of the NADPH diaphorase activity by Ca(2+)-calmodulin was observed in the presence of PIP2 and PA, and the citrulline formation activity was protected from phospholipid inhibition by preincubation with Ca(2+)-calmodulin. Moreover, trypsin digestion analysis showed that the cleavage site within the calmodulin-binding site of nNOS was specifically protected from trypsin by the addition of PIP2 and PA. These results strongly suggest that PIP2 and PA inhibit the citrulline formation activity of nNOS by blocking the interaction of the enzyme with Ca(2+)-calmodulin.     

Neurogranin binds to phosphatidic acid and associates to cellular membranes

Neurogranin (Ng) is a 78-amino-acid-long protein concentrated at dendritic spines of forebrain neurons that is involved in synaptic plasticity through the regulation of CaM (calmodulin)-mediated signalling. Ng features a central IQ motif that mediates binding to CaM and is phosphorylated by PKC (protein kinase C). We have analysed the subcellular distribution of Ng and found that it associates to cellular membranes in rat brain. In vitro binding assays revealed that Ng selectively binds to PA (phosphatidic acid) and that this interaction is prevented by CaM and PKC phosphorylation. Using the peptide Ng-(29-47) and a mutant with an internal deletion (Ng-IQless), we have shown that Ng binding to PA and to cellular membranes is mediated by its IQ motif. Ng expressed in NIH-3T3 cells accumulates at peripheral regions of the plasma membrane and localizes at intracellular vesicles that can be clearly visualized following saponin permeabilization. This distribution was affected by PLD (phospholipase D) and PIP5K (phosphatidylinositol 4-phosphate 5-kinase) overexpression. Based on these results, we propose that Ng binding to PA may be involved in Ng accumulation at dendritic spines and that Ng could modulate PA signalling in the postsynaptic environment.     

Fragmentation and re-assembly of the Golgi apparatus in vitro. A requirement for phosphatidic acid and phosphatidylinositol 4,5-bisphosphate synthesis.

Recent work from our laboratory demonstrated that phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), are required to maintain the structural integrity of the Golgi apparatus. To investigate the role of these lipids in regulating Golgi structure and function, we developed a novel assay to follow the release of post-Golgi vesicles. Isolated rat liver Golgi membranes were incubated with [(3)H]CMP sialic acid to radiolabel endogenous soluble and membrane glycoproteins present in the late Golgi and trans-Golgi network. The release of post-Golgi secretory vesicles was determined by measuring incorporation of (3)H-labeled proteins into a medium speed supernatant. Vesicle budding was dependent on temperature, cytosol, energy and time. Electron microscopy of Golgi fractions prior to and after incubation demonstrated that the stacked Golgi cisternae generated a heterogeneous population of vesicles (50- to 350-nm diameter). Inhibition of phospholipase D-mediated PA synthesis, by incubation with 1-butanol, resulted in the complete fragmentation of the Golgi membranes in vitro into 50- to 100-nm vesicles; this correlated with diminished PtdIns(4,5)P(2) synthesis. Following alcohol washout, PA synthesis resumed and in the presence of cytosol PtdIns(4,5)P(2) synthesis was restored. Most significantly, under these conditions the fragmented Golgi elements reformed into flattened cisternae and the re-assembled Golgi supported vesicle release. These data demonstrate that inositol phospholipid synthesis is essential for the structure and function of the Golgi apparatus.     

Nuclear translocation of phospholipase C-delta1 is linked to the cell cycle and nuclear phosphatidylinositol 4,5-bisphosphate

Nuclear phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, fluctuate throughout the cell cycle and are linked to proliferation and differentiation. Here we report that phospholipase C-delta(1) accumulates in the nucleus at the G(1)/S boundary and in G(0) phases of the cell cycle. Furthermore, as wild-type protein accumulated in the nucleus, nuclear phosphatidylinositol 4,5-bisphosphate levels were elevated 3-5-fold, whereas total levels were decreased compared with asynchronous cultures. To test whether phosphatidylinositol 4,5-bisphosphate binding is important during this process, we introduced a R40D point mutation within the pleckstrin homology domain of phospholipase C-delta(1), which disables high affinity phosphatidylinositol 4,5-bisphosphate binding, and found that nuclear translocation was significantly reduced at G(1)/S and in G(0). These results demonstrate a cell cycle-dependent compartmentalization of phospholipase C-delta(1) and support the idea that relative levels of phosphoinositides modulate the portioning of phosphoinositide-binding proteins between the nucleus and other compartments.     

Casein kinase I is regulated by phosphatidylinositol 4,5-bisphosphate in native membranes.

Casein kinase I activity is present in cells as a cytosolic and a membrane-bound enzyme. Previously, the erythroid membrane-bound casein kinase I was shown to associate with purified integral membrane proteins; this association and protein kinase activity was regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Bazenet, C.E., Brockman, J.L., Lewis, D., Chan, C., and Anderson, R.A. (1990) J. Biol. Chem. 265, 7369-7376). Here we show that both the membrane-bound and the cytosolic casein kinase interact with native membranes and that this interaction is regulated by the membrane content of PIP2. On native membranes, casein kinase I activity is potently inhibited by small increases (10-20%) in the membrane content of either exogenously added or intrinsic PIP2. However, the majority of the intrinsic content of PIP2 in isolated membranes does not inhibit casein kinase, suggesting that this PIP2 is not accessible. Regulation of the casein kinases on membranes is sensitive to detergents and to chymotrypsin treatment of membranes.     

The influence of charge on phosphatidic acid bilayer membranes.

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, 1,2-dihexadecyl-sn-glycerol-3-phosphoric acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of the phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; 1,2-dimyristoyl-sn-glycerol-3-phosphoric acid, 1,3-dimyristoylglycerol-2-phosphoric acid and 1,2-dipalmitoyl-sn-glycerol-3-phosphoric acid. The phase transition temperatures (Tt) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90 degrees light scattering. The Tt values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states. The Tt vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 (pK1). The regions between the first and the second pK of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H+ concentration results in an abrupt change of the transition temperature. The slope of the Tt vs. pH diagram beyond pK2 becomes very steep. This is the result of reduced hydrocarbon interaction energy, which was demonstrated by differential scanning calorimetry (Blume, A. and Eibl, H., unpublished data).     

Thrombin- and nucleotide-activated phosphatidylinositol 4,5-bisphosphate phospholipase C in human platelet membranes

Thrombin, nucleotides, and chelators elicited a phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity that was associated with human platelet membranes. Both alpha- and gamma-thrombin enhanced phospholipase C activity, whereas active site-inhibited alpha-thrombin did not stimulate PtdIns-P2 hydrolysis. PtdIns-P2 phospholipase C was also activated by nucleoside triphosphates, citrate, EDTA, and NaF. Magnesium was an inhibitor of PtdIns-P2 hydrolysis stimulated by nucleotides and chelators. Only PtdIns-P2 was degraded by the phospholipase C activated by alpha-thrombin, nucleotides, and chelators. The soluble fraction phospholipase C activity was also stimulated at low protein concentrations by nucleotides; however, soluble fraction phospholipase C activity cleaved both PtdIns-P2 and phosphatidylinositol 4-phosphate and was inhibited by chelators, suggesting the presence of a different enzyme in this compartment. The pH optimum for the membrane-associated phospholipase C in the presence of alpha-thrombin or nucleotides was 6.0, and the PtdIns-P2 phospholipase C was inhibited by neomycin and high detergent concentrations. Guanine nucleotides did not synergistically activate phospholipase C in the presence of alpha-thrombin. The characteristics of the membrane-associated PtdIns-P2 phospholipase C suggest that this enzyme is involved in platelet activation by the low-affinity alpha- or gamma-thrombin-dependent pathway.     

The effector domain of myristoylated alanine-rich C kinase substrate binds strongly to phosphatidylinositol 4,5-bisphosphate

Both the myristoylated alanine-rich protein kinase C substrate protein (MARCKS) and a peptide corresponding to its basic effector domain, MARCKS-(151-175), inhibit phosphoinositide-specific phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in vesicles (Glaser, M., Wanaski, S., Buser, C. A., Boguslavsky, V., Rashidzada, W., Morris, A., Rebecchi, M., Scarlata, S. F., Runnels, L. W., Prestwich, G. D., Chen, J., Aderem, A., Ahn, J., and McLaughlin, S. (1996) J. Biol. Chem. 271, 26187-26193). We report here that adding 10-100 nm MARCKS-(151-175) to a subphase containing either PLC-delta or -beta inhibits hydrolysis of PIP(2) in a monolayer and that this inhibition is due to the strong binding of the peptide to PIP(2). Two direct binding measurements, based on centrifugation and fluorescence, show that approximately 10 nm PIP(2), in the form of vesicles containing 0.01%, 0.1%, or 1% PIP(2), binds 50% of MARCKS-(151-175). Both electrophoretic mobility measurements and competition experiments suggest that MARCKS-(151-175) forms an electroneutral complex with approximately 4 PIP(2). MARCKS-(151-175) binds equally well to PI(4,5)P(2) and PI(3,4)P(2). Local electrostatic interactions of PIP(2) with MARCKS-(151-175) contribute to the binding energy because increasing the salt concentration from 100 to 500 mm decreases the binding 100-fold. We hypothesize that the effector domain of MARCKS can bind a significant fraction of the PIP(2) in the plasma membrane, and release the bound PIP(2) upon interaction with Ca(2+)/calmodulin or phosphorylation by protein kinase C.     

Erythroid membrane-bound protein kinase binds to a membrane component and is regulated by phosphatidylinositol 4,5-bisphosphate

In the erythrocyte, a membrane-bound serine/threonine protein kinase (a casein kinase) has been shown to phosphorylate a number of membrane proteins, modulating their function. Here we report that the membrane-bound protein kinase binds to membranes by an association with a minor membrane component contained in preparations of glycophorin (possibly a minor glycophorin). The binding of the kinase to glycophorins does not significantly modify kinase activity. However, upon binding, the kinase activity is potently inhibited by phosphatidylinositol 4,5-bisphosphate, and the affinity of the kinase for the glycophorins is increased. Other phospholipids or polyanions such as inositol 1,4,5-trisphosphate or 2,3-diphosphoglycerate do not affect protein kinase activity when the kinase is bound to membranes but do inhibit the solubilized membrane-bound kinase. In the erythrocyte, there is a cytosolic form of the casein kinase which is very similar, having the same molecular weight and substrate specificity as the membrane-bound casein kinase. The cytosolic casein kinase is inhibited by 2,3-diphosphoglycerate but much less so by glycophorin preparations containing phosphoinositol 4,5-bisphosphate. When the sequences of both casein kinases were compared by two-dimensional peptide mapping, it was found that the two kinases were very similar but not identical.     

Phospholipase A1 acting on phosphatidic acid in porcine platelet membranes

1-[14C]Palmitoyl-2-[3H]arachidonoyl-sn-glycerol 3-phosphate was hydrolyzed to form [14C]palmitic acid and 2-[3H]arachidonoyl-glycerophosphate by porcine platelet membranes. This phospholipase A1 activity was relatively specific for phosphatidic acid; the addition of several other phospholipids in equimolar amounts did not have a significant effect on the hydrolysis of radiolabeled phosphatidic acid, and the specific activity for phosphatidic acid hydrolysis was 20-fold higher than that of the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol under the conditions used. This phospholipase A1 acting on phosphatidic acid has properties different from those reported for other phospholipases and lipases present in platelets.     

Molecular dynamics study of a gelsolin-derived peptide binding to a lipid bilayer containing phosphatidylinositol 4,5-bisphosphate

Gelsolin is an actin-severing protein whose action is initiated by Ca(2+) and inhibited by binding to phosphorylated inositol lipid or phosphoinositides. The regions of gelsolin responsible for phosphoinositide binding are comprised of residues 150-169 (G150-169) and 135-142 (G135-142). The corresponding peptides possess similar binding potency as native gelsolin. Their common feature is the presence of arginine and lysine residues that can bind to negatively charged phosphate groups of phosphoinositides. In this work the binding of the G150-169 peptide to a phosphatidylinositol 4,5-bisphosphate (PIP2) cluster in a lipid membrane model was investigated by molecular dynamics calculations (MD) with the AMBER 4.1 force field, taking into account explicit solvent molecules. Initially the structure of G150-169 was simulated by using the electrostatically driven Monte Carlo (EDMC) and MD methods, and the resulting structure agreed within 3.7 A backbone-atom root mean square deviation with the corresponding experimentally derived structure (PDB code: 1SOL). Using this model for the peptide, a subsequent MD simulation of G150-169 in a periodic box containing a model of dimyristoyl-phosphatidylcholine (DMPC) lipids with a cluster of four PIP2 molecules was carried out. During the simulation G150-169 interacted strongly with PIP2 molecules, initially by formation of salt bridges between its N-terminal basic groups and the phosphate groups of PIP2, followed by formation of hydrophobic bonds between the hydrophobic side chains of the peptide and the fatty acid tail of the lipid. As a result of the formation of hydrophobic bonds, the PIP2 molecules were pulled out from the lipid bilayer. This mode of binding differs from those of other PIP2-binding protein motifs such as PH domains that interact solely with the hydrophilic head group of PIP2. These results suggest that dissociation of gelsolin from actin by PIP2 lipids may involve entering of the PIP2 molecules to the gelsolin-actin interface, thereby weakening the interactions between these proteins.     

Interaction of cerebral-cortical membranes with exogenously added phosphatidylinositol 4,5-bisphosphate. Effects on measured phospholipase C activity.

Exogenously added phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is rapidly associated with cerebral-cortical membranes. Substrate association with membranes was promoted by Mg2+, but inhibited by bivalent chelators. Once associated with the membrane, the PtdInsP2 was resistant to displacement by EDTA. The apparent phospholipase C activity was dependent on the degree of association of substrate with membranes. After preincubation of membranes with substrate, PtdInsP2 hydrolysis was independent of the incubation volume, indicating that substrate and membrane-associated phospholipase C were not independently diluted. Hydrolysis of the membrane-associated substrate was stimulated by Ca2+, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), guanosine 5'[gamma-thio]triphosphate and carbachol in the presence of p[NH]ppG. Carbachol in the absence of guanine nucleotides, GDP, GTP, ATP and pyrophosphate was ineffective. These results demonstrate that exogenously added PtdInsP2 substrate is rapidly associated with membranes and hydrolysed by a phospholipase C whose activity is regulated by guanine nucleotides and agonist in the presence of guanine nucleotides. Use of exogenously added substrate for studies on the regulation of membrane phospholipase C requires consideration as to possible effects of incubation conditions on the partitioning of substrate into membranes.     

COMMD1 forms oligomeric complexes targeted to the endocytic membranes via specific interactions with phosphatidylinositol 4,5-bisphosphate

Copper metabolism Murr1 domain 1 (COMMD1) is a 21-kDa protein involved in copper export from the liver, NF-kappaB signaling, HIV infection, and sodium transport. The precise function of COMMD and the mechanism through which COMMD1 performs its multiple roles are not understood. Recombinant COMMD1 is a soluble protein, yet in cells COMMD1 is largely seen as targeted to cellular membranes. Using co-localization with organelle markers and cell fractionation, we determined that COMMD1 is located in the vesicles of the endocytic pathway, whereas little COMMD1 is detected in either the trans-Golgi network or lysosomes. The mechanism of COMMD1 recruitment to cell membranes was investigated using lipid-spotted arrays and liposomes. COMMD1 specifically binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the absence of other proteins and does not bind structural lipids; the phosphorylation of PtdIns at position 4 is essential for COMMD1 binding. Proteolytic sensitivity and molecular modeling experiments identified two distinct domains in the structure of COMMD1. The C-terminal domain appears sufficient for lipid binding, because both the full-length and C-terminal domain proteins bind to PtdIns(4,5)P2. In native conditions, endogenous COMMD1 forms large oligomeric complexes both in the cytosol and at the membrane; interaction with PtdIns(4,5)P2 increases the stability of oligomers. Altogether, our results suggest that COMMD1 is a scaffold protein in a distinct sub-compartment of endocytic pathway and offer first clues to its role as a regulator of structurally unrelated membrane transporters.     

Interaction of protein kinase C isozymes with phosphatidylinositol 4,5-bisphosphate.

Interaction of protein kinase C (PKC) isozymes with phosphatidylinositol 4,5-bisphosphate (PIP2) was investigated by monitoring the changes in the intrinsic fluorescence of the enzyme, the kinase activity, and phorbol ester binding. Incubation of PKC I, II, and III with PIP2 resulted in different rates of quenching of PKC fluorescence and different degrees of inactivation of these enzymes. Other inositol-containing phospholipids such as phosphatidylinositol and phosphatidylinositol 4-phosphate also caused differential rates of quenching of the intrinsic fluorescence of these enzymes. These latter two phospholipids were, however, less potent in the inactivation of PKCs than PIP2. The IC50 of PIP2 were 2, 4, and 11 microM for PKC I, II, and III, respectively. Inactivation of PKCs by PIP2 cannot be reversed by extensive dilution of PIP2 with Nonidet P-40 nor by digestion of PIP2 with phospholipase C. Interaction of PIP2 with the various PKC isozymes was greatly facilitated in the presence of Mg2+ or Ca2+ as evidenced by the accelerated quenching of the PKC fluorescence, however, these divalent metal ions protected PKC from the PIP2-induced inactivation. Binding of PIP2 to PKC in the absence of divalent metal ion also caused a reduction of [3H]phorbol 12,13-dibutyrate binding as a result of reducing the affinity of the enzyme for phorbol ester. Based on gel filtration chromatography, it was estimated that one molecule of PKC interacted with one PIP2 micelle with an aggregation number of 80-90. The PIP2-bound PKC could further interact with phosphatidylserine in the presence of Ca2+ to form a larger complex. Binding of PKC to both PIP2 and phosphatidylserine in the presence of Ca2+ was also evident by changes in the intrinsic fluorescence of PKC. As the interaction of PKC with PIP2, but not with phosphatidylserine, could be enhanced by millimolar concentrations of Mg2+, we propose that PIP2 may be a component of the membrane anchor for PKC under basal physiological conditions when [Ca2+]i is low and Mg2+ is plentiful. Under the in vitro assay conditions, PIP2 could stimulate PKC activity to a level approximately 10-20% of that by diacylglycerol. The stimulatory effect of PIP2 on PKC apparently is not due to binding to the same site recognized by diacylglycerol or phorbol ester, because PIP2 cannot effectively compete with phorbol 12,13-dibutyrate in the binding assay.     

Guanine nucleotide and pyrophosphate activate exogenous phosphatidylinositol 4,5-bisphosphate hydrolysis in rat liver plasma membranes

5'-guanylylimidodiphosphate (GppNHp) in the presence of deoxycholate, stimulated the phospholipase C-mediated hydrolysis of exogenous [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) to myo-[3H]inositol 1,4,5-trisphosphate in rat liver plasma membranes. Activation was not specific for guanine nucleotides as 5'-adenylylimidodiphosphate, imidodiphosphate and pyrophosphate stimulated the enzyme with similar efficacies and potencies. Enzyme activation by GppNHp was most pronounced when [3H]PIP2 was used as substrate. No added Ca++ was required for [3H]PIP2 breakdown but hydrolysis was inhibited by divalent ion chelators. GppNHp stimulation was apparent in the presence of Ca++ or Mg++ as well as chelator concentrations that partially inhibited the enzyme, indicating that this effect was not attributed to changes in affinity of these divalent cations for the enzyme or substrate. These results suggest that guanine nucleotides can stimulate the hydrolysis of exogenous [3H]PIP2 in rat liver membranes by a non-specific effect probably due to the interaction of the diphosphate moiety with the enzyme or substrate.     

Gelsolin in complex with phosphatidylinositol 4,5-bisphosphate inhibits caspase-3 and -9 to retard apoptotic progression

Apoptosis, or programmed cell death, occurs because of the activation of a protease cascade amplification circuit that includes the critical effector caspase-3. Previously, we identified the widely expressed actin modulatory protein gelsolin as a prominent substrate of caspase-3 and demonstrated that the N-terminal gelsolin cleavage product promotes apoptosis. Here we show that phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3, 4-bisphosphate in pure micelles or mixed vesicles prevent caspase-3 cleavage of gelsolin. Moreover, phosphatidylinositol 4, 5-bisphosphate-gelsolin strongly inhibits caspase-3 and -9 activity through the formation of a stable phosphatidylinositol 4, 5-bisphosphate-gelsolin-caspase complex. In addition, phosphatidylinositol 4,5-bisphosphate-gelsolin prevents apoptotic progression mediated by caspase-3 in a cell-free system, and phosphatidylinositol 4,5-bisphosphate-gelsolin-caspase-9 and phosphatidylinositol 4,5-bisphosphate-gelsolin-caspase-3 complexes form in mouse embryonic fibroblasts during apoptosis induction when stimulated with fibronectin, to delay cell death. The results suggest that gelsolin can act as both an effector and an inhibitor of caspase-3, the latter in concert with phosphatidylinositol 4, 5-bisphosphate, and other membrane phospholipids to regulate the onset and progression of apoptosis.     

Structural changes in profilin accompany its binding to phosphatidylinositol, 4,5-bisphosphate.

The effect on the structure of profilin of phosphatidylinositol 4,5-bisphosphate (PIP2) binding was probed by fluorescence and circular dichroism (CD) spectroscopy. Fluorescence of Trp3 and Trp31 of profilin at 292 nm showed a linear decrease in solution emission at 340 nm as PIP2/profilin was increased from 0 to 80:1, apparently due to a static quenching mechanism involving formation of a nonfluorescent PIP2/profilin complex. CD spectra revealed an increase of up to 3.3-fold in the molar ellpticity at 222 nm for profilin as it binds PIP2, as well as changes in the Cotton effect between 250 and 310 nm. These results are consistent with a possible increase in the alpha-helix content of profilin triggered by the binding of PIP2.     

The influence of charge on bilayer membranes. Calorimetric investigations of phosphatidic acid bilayers.

The pH-dependence of the phase transition of dimyristoyl phosphatidic acid and dihexadecyl phosphatidic acid has been investigated using differential scanning calorimetry. Varying the pH induces different degrees of ionization of the polar head group. The changes in transition temperature with pH as observed by calorimetry are in good agreement with those obtained by measuring the changes in light scattering, whereas the transition temperatures reported by the fluorescent probe N-phenylnaphthylamine do not always coincide with those determined from calorimetry [1]. The observed maximum of the transition temperature at pH 3.5 corresponds to a minimum in the transition enthalpy vs. pH diagram. At this pH a particular stable bilayer phase is formed. Full protonation of phosphatidic acids leads to suspensions of mycrocrystals. The transition enthalpy approaches the value of the melting enthalpy of crystalline anhydrous phosphatidic acid. The decrease in the transition enthalpy at high pH values is due to a change in the hydrocarbon chain interactions induced by the doubly charged head groups. The cooperativity of the transition varies with the degree of ionization of the head group, being lower for doubly charged phosphatidic acids.