Alexis Traynor-Kaplan

The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells

Integrin-mediated adhesion is known to stimulate production of phosphatidylinositol 4,5-bisphosphate (4,5-PIP2) and increase 4,5-PIP2 hydrolysis in response to platelet-derived growth factor (PDGF). We now show that treatment of cells with lovastatin, which inhibits modification of small GTP-binding proteins, reduced PIP2 levels and decreased calcium mobilization in response to PDGF and thrombin. In cell lysates, GTP gamma S stimulated PIP 5-kinase activity, and this effect was blocked by botulinum C3 exoenzyme, suggesting that Rho was responsible. GTP-bound recombinant Rho stimulated PIP 5-kinase activity, whereas GDP-Rho was much less potent and GTP-bound Rac was ineffective. Microinjected botulinum C3 exoenzyme caused diminished calcium mobilization in response to PDGF or thrombin. Conversely, microinjection of activated Rho reversed the decrease in calcium mobilization normally seen in nonadherent cells. These data demonstrate that Rho regulates 4,5-PIP2 synthesis and, indirectly, 4,5-PIP2 hydrolysis. They also raise the possibility that PIP2 synthesis could mediate the effects of Rho on the actin cytoskeleton.

An inositol tetrakisphosphate-containing phospholipid in activated neutrophils

Inositol (l,4,5)trisphosphate (InsP3)1 and tetrakisphosphate (InsP4)2 have been observed in a variety of cell types and have been proposed to play roles in the receptor-mediated rise in intracellular Ca2+ (refs 2, 3). Recently, they have been shown to act synergistically in the activation of a Ca2+-dependent K+ channel in lacrimal acinar cells3. InsP3 is the product of phospholipase C (PLC) action on phosphatidylinositol 4,5-bisphosphate (PtdInsP2), whereas InsP4 is believed to arise from phosphorylation of InsP3 by a cytosolic kinase4. Although sought as a source for InsP4, PtdInsP3 has not been identified in any specific cell type2. There were early reports of InsP4-containing phospholipids in crude extract from bovine brain5, but this finding was later withdrawn6. Recently, however, a membrane-bound enzyme (Type 1 PI kinase) which adds phosphate onto the 3 position of inositol phospholipids has been identified7 and the phosphatidylinositol-3-phosphate (PtdIns(3)P) product characterized. This suggests that several forms of phosphoinositides may exist and could be precursors for some of the variety of soluble inositol phosphate products which have been reported in recent years. Here we report the appearance of another novel phosphoinositide containing four phosphates, phosphatidylinositol trisphosphate (PtdInsP3) which we find only in activated but not in unstimulated neutrophils from human donors.

Membrane-permeant Esters of Phosphatidylinositol 3,4,5-Trisphosphate

Phosphoinositide 3-OH kinases and their products, D-3 phosphorylated phosphoinositides, are increasingly recognized as crucial elements in many signaling cascades. A reliable means to introduce these lipids into intact cells would be of great value for showing the physiological roles of this pathway and for testing the specificity of pharmacological inhibitors of the kinases. We have stereospecifically synthesized di-C8-PIP3/AM and di-C12-PIP3/AM, the heptakis(acetoxymethyl) esters of dioctanoyl- and dilauroylphosphatidylinositol 3,4,5-trisphosphate, in 14 steps from myo-inositol. The ability of these uncharged lipophilic derivatives to deliver phosphatidylinositol 3,4,5-trisphosphate across cell membranes was demonstrated on 3T3-L1 adipocytes and T84 colon carcinoma monolayers. Insulin stimulation of hexose uptake into adipocytes was inhibited by the kinase inhibitor wortmannin and was largely restored by di-C8-PIP3/AM, which had no effect in the absence of insulin. Thus phosphatidylinositol 3,4,5-trisphosphate or a metabolite was necessary but not sufficient for stimulation of hexose transport. In T84 epithelial monolayers, di-C12-PIP3/AM mimicked epidermal growth factor in inhibiting chloride secretion and potassium efflux, suggesting that phosphatidylinositol 3,4,5-trisphosphate was sufficient to modulate these fluxes and mediate epidermal growth factor’s action.

A Tyrosine Kinase Signaling Pathway Accounts for the Majority of Phosphatidylinositol 3,4,5-Trisphosphate Formation in Chemoattractant-stimulated Human Neutrophils

The signaling pathway leading from G protein-coupled chemoattractant receptors to the generation of oxidants by NADPH oxidase in human neutrophils requires the formation of the lipid mediator phosphatidylinositol 3,4,5-trisphosphate (PIP3). Two mechanisms through which PIP3 can be generated have been described in human leukocytes. One pathway involves the coupling of the src-related tyrosine kinase Lyn to the “classical” p85/p110 form of phosphatidylinositol 3-kinase. The second paradigm utilizes a novel form of phosphatidylinositol 3-kinase whose activity is directly regulated by G protein βγ subunits. In this paper, we show that formation of PIP3 in chemoattractant-stimulated neutrophils is substantially attenuated by inhibitors that specifically block tyrosine kinase activity. These data suggest that the Lyn activation pathway plays a major role in the formation of this important lipid messenger during chemoattractant stimulation of human neutrophils.

A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START.

In an effort to study further the mechanism of Cdc28 function and cell cycle commitment, we describe here a genetic approach to identify components of pathways downstream of the Cdc28 kinase at START by screening for mutations that decrease the effectiveness of signaling by Cdc28. The first locus to be characterized in detail using this approach was PKC1 which encodes a homolog of the Ca(2+)‐dependent isozymes of the mammalian protein kinase C (PKC) superfamily (Levin et al., 1990). By several genetic criteria, we show a functional interaction between CDC28 and PKC1 with PKC1 apparently functioning with respect to bud emergence downstream of START. Consistent with this, activity of the MAP kinase homolog Mpk1 (a putative Pkc1 effector) is stimulated by activation of Cdc28. Furthermore, we demonstrate a cell cycle‐dependent hydrolysis of phosphatidylcholine to diacylglycerol (a PKC activator) and choline phosphate at START. Diacylglycerol production is stimulated by Cdc28 in cycling cells and is closely associated with Cdc28 activation at START. These results imply that the activation of Pkc1, which is known to be necessary during bud morphogenesis, is mediated via the CDC28‐dependent stimulation of PC‐PLC activity in a novel cell cycle‐regulated signaling pathway.

Cross-talk between calcium and cAMP-dependent intracellular signaling pathways. Implications for synergistic secretion in T84 colonic epithelial cells and rat pancreatic acinar cells.

Treatment of various cells with combinations of agents that increase either cAMP or cytosolic calcium can lead to synergistic responses. This study examined interactions, or cross-talk, between these two intracellular messengers and its implication for signaling in two secretory cell types, T84 human colonic epithelial cells and rat pancreatic acinar cells. T84 cell chloride secretion was measured in Ussing chambers. Acinar cell activation was monitored as amylase secretion. Cytosolic calcium was assessed via fura-2 microfluorimetry. A cell-permeant analogue of cAMP synergistically enhanced secretory responses to calcium-mobilizing hormones in both cell types, but paradoxically reduced overall calcium mobilization. The reduction in calcium mobilization could be attributed to an inhibition of calcium influx in T84 cells, although a different mechanism likely operates in acinar cells. The effects of the cAMP analogue were reproduced by other agents that increase cAMP. Furthermore, econazole, an inhibitor of calcium influx, potentiated secretory responses to calcium-dependent stimulation in T84 cells without itself inducing secretion. We conclude that there is cross-talk between calcium and cAMP-dependent signaling pathways at the level of second messenger generation in two secretory cell types. This cross-talk appears to regulate the extent of secretory responses.

Activation of Human Neutrophil NADPH Oxidase by Phosphatidic Acid or Diacylglycerol in a Cell-free System ACTIVITY OF DIACYLGLYCEROL IS DEPENDENT ON ITS CONVERSION TO PHOSPHATIDIC ACID

The superoxide-generating neutrophil NADPH oxidase can be activated in cell-free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg2+ and agonist concentration. Activation of NADPH oxidase by phosphatidic acids did not appear to require their conversion to corresponding diacylglycerols by phosphatidate phosphohydrolase, since diacylglycerols were much slower than phosphatidic acids to activate the system and required the presence of ATP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a means other than the activation of protein kinase C. Instead, dioctanoylglycerol was converted to dioctanoylphosphatidic acid by an endogenous diacylglycerol kinase present in the cell-free reaction system. This conversion was sensitive to the diacylglycerol kinase inhibitor R59949 and explains the markedly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was suboptimal for NADPH oxidase activation but could synergize with the unmetabolized dioctanoylglycerol to activate superoxide generation.

Inhibition of Ca2+-dependent Cl− secretion in T84 cells: membrane target(s) of inhibition is agonist specific

Previous studies have indicated that Ca2+-dependent Cl- secretion across monolayers of T84 epithelial cells is subject to a variety of negative influences that serve to limit the overall extent of secretion. However, the downstream membrane target(s) of these inhibitory influences had not been elucidated. In this study, nuclide efflux techniques were used to determine whether inhibition of Ca2+-dependent Cl- secretion induced by carbachol, inositol 3,4,5,6-tetrakisphosphate, epidermal growth factor, or insulin reflected actions at an apical Cl- conductance, a basolateral K+ conductance, or both. Pretreatment of T84 cell monolayers with carbachol or a cell-permeant analog of inositol 3,4,5,6-tetrakisphosphate reduced the ability of subsequently added thapsigargin to stimulate apical125I-, but not basolateral86Rb+, efflux. These data suggested an effect on an apical Cl- channel. Conversely, epidermal growth factor reduced Ca2+-stimulated86Rb+but not125I-efflux, suggesting an effect of the growth factor on a K+ channel. Finally, insulin inhibited125I-and86Rb+effluxes. Thus effects of agents that inhibit transepithelial Cl- secretion are also manifest at the level of transmembrane transport pathways. However, the precise nature of the membrane conductances targeted are agonist specific.

Activation by calcium alone of chloride secretion in T84 epithelial cells

1 The goal of this study was to determine if an increase in cytoplasmic calcium concentration ([Ca2+]i), in the absence of additional second messengers derived from membrane phospholipid turnover, is a sufficient signal to induce chloride secretion across monolayers of the human colonic epithelial line, T84. 2 Thapsigargin was used to increase [Ca2+]i by inhibiting the endomembrane Ca2+‐ATPase. [Ca2+]i was monitored in monolayers by fura‐2 fluorescence spectroscopy, chloride secretion by measuring changes in short circuit current (Isc) in modified Ussing chambers, and inositol phosphates were measured by radio‐h.p.l.c. of extracts of cells prelabelled with [3H]‐inositol. 3 Thapsigargin increased [Ca2+]i and Isc in parallel, without increasing any inositol phosphates. The effect of thapsigargin on Isc was abolished by the intracellular calcium chelator, bis‐(o‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). 4 Increasing [Ca2+]i with thapsigargin did not prevent a subsequent calcium response to carbachol or histamine if extracellular calcium was available. In the absence of extracellular calcium, only one such release of calcium to hormonal stimulation occurred when cells were pretreated with thapsigargin, and a second response to either carbachol or histamine was essentially abolished. 5 Addition of carbachol or histamine to thapsigargin‐treated cells mounted in Ussing chambers caused a transient further increase in Isc followed by termination of the response, even though [Ca2+]i continued to rise. 6 We conclude that an elevation in [Ca2+]i is a sufficient signal to induce chloride secretion in T84 cells. Rather than being required to stimulate secretory responses, additional second messengers induced by hormonal secretagogues (such as inositol phosphates) may in fact serve to limit the secretory response.

Membrane-Permeant 3-OH-Phosphorylated Phosphoinositide Derivatives†

A crucial role in the regulation of epithelial chloride secretion is played by the phosphoinositide PtdIns(3,4,5)P3 . Membrane-permeant derivatives of this and other naturally occurring phosphoinositides have been synthesized. These derivatives, which can be bioactivated, were used in investigations on nasal epithelia of patients suffering from cystic fibrosis.