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Biomedical subjects

A Toker

Publications and source records attributed to A Toker.

At least 55 records · Page 3Linked to original sources
PubMed1998

The synthesis and cellular roles of phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) is a membrane phospholipid found in all eukaryotic cells which regulates many important cellular processes, including organization of the cytoskeleton, vesicular trafficking and platelet activation. PtdIns 4,5-P2 can either act as a substrate for lipid kinases and phosphatases, or directly interact with proteins. Recent work has focused on the characterization of the enzymes that regulate PtdIns 4,5-P2 levels in the cell. The discovery of proteins whose activities are modified by interaction with PtdIns 4,5-P2 has helped to redefine the role of this lipid in cell biology.

1-Phosphatidylinositol 4-Kinase↗
PubMed1997

Activation of phosphoinositide 3-OH kinase by the alpha6beta4 integrin promotes carcinoma invasion.

We demonstrate that the alpha6beta4 integrin promotes carcinoma invasion through a preferential and localized targeting of phosphoinositide-3 OH kinase (PI3K) activity. Stable expression of alpha6beta4 increased carcinoma invasion in a PI3K-dependent manner, and transient expression of a constitutively active PI3K increased invasion in the absence of alpha6beta4. Ligation of alpha6beta4 stimulated significantly more PI3K activity than ligation of beta1 integrins, establishing specificity among integrins for PI3K activation. Alpha6beta4-regulated PI3K activity was required for the formation of lamellae, dynamic sites of motility, in carcinoma cells. The small G protein Rac is required downstream of PI3K for invasion. These studies define a mechanism by which the alpha6beta4 integrin promotes carcinoma invasion and invoke a novel function for PI3K signaling.

Androstadienes↗
PubMed1997

Signalling through the lipid products of phosphoinositide-3-OH kinase.

When a stimulatory agonist molecule binds at the exterior of the cell membrane, a second messenger transduces the signal to the interior of the cell. Second messengers can be derived from phospholipids in the membrane by the action of the enzymes phospholipase C or phosphoinositide-3-OH kinase (PI(3)K). PI(3)K is a key player in many cellular responses, including the movement of organelle membranes, shape alteration through rearrangement of cytoskeletal actin, transformation and chemotaxis. But how PI(3)K mediates these responses is only now becoming clear.

Animals↗
PubMed1997

Identification of a novel pathway important for proliferation and differentiation of primary erythroid progenitors.

Homodimerization of the erythropoietin (EPO) receptor (EPO-R) in response to EPO binding transiently activates the receptor-associated protein tyrosine kinase JAK2. Tyrosine phosphorylation of the EPO-R creates "docking sites" for SH2 domain(s) in signaling molecules such as the protein tyrosine phosphatases SH-PTP1 and SH-PTP2, phosphoinositide 3-kinase (PI3 kinase), and STAT5. However, little is known about the specific intracellular signals essential for proliferation and differentiation of erythroid progenitors. Here we show that an EPO-R containing only one cytosolic (phospho)tyrosine residue, Y479, induces a signal transduction pathway sufficient for proliferation and differentiation of fetal liver progenitors of erythroid colony-forming units from EPO-R(-/-) mice as well as for proliferation of cultured hematopoietic cells. This cascade involves sequential EPO-induced recruitment of PI3 kinase to the EPO-R and activation of mitogen-activated protein kinase activity, independent of the Shc/Grb2-adapter pathway and of STAT5. Protein kinase C epsilon may be one of the mediators connecting PI3 kinase with the mitogen-activated protein kinase signaling cascade. Our results identify a signaling cascade important in vivo for erythroid cell proliferation and differentiation.

Animals↗
PubMed1997

The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C.

Phosphoinositide 3-kinase has been implicated as an activator of cell motility in a variety of recent studies, yet the role of its lipid product, phosphatidylinositol 1,4,5-trisphosphate (PtdIns-3,4,5-P3), has yet to be elucidated. In this study, three independent preparations of PtdIns-3,4,5-P3 were found to increase the motility of NIH 3T3 cells when examined utilizing a microchemotaxis chamber. Dipalmitoyl L-alpha-phosphatidyl-D-myo-inositol 3,4,5-triphosphate (Di-C16-PtdIns-3,4,5-P3) also produced actin reorganization and membrane ruffling. Cells pretreated with 12-O-tetradecanoylphorbol-13-acetate to cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility induced by Di-C16-PtdIns-3,4,5-P3. These results are consistent with previous observations that PtdIns-3,4,5-P3 activates Ca2+-independent PKC isoforms in vitro and in vivo and provide the first demonstration of an in vivo role for the lipid products of the phosphoinositide 3-kinase. PtdIns-3,4,5-P3 appears to directly initiate cellular motility via activation of a PKC family member.

3T3 Cells↗
PubMed1997

Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate.

The regulation of the serine-threonine kinase Akt by lipid products of phosphoinositide 3-kinase (PI 3-kinase) was investigated. Akt activity was found to correlate with the amount of phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) in vivo, and synthetic PtdIns-3,4-P2 activated Akt both in vitro and in vivo. Binding of PtdIns-3,4-P2 occurred within the Akt pleckstrin homology (PH) domain and facilitated dimerization of Akt. Akt mutated in the PH domain was not activated by PI 3-kinase in vivo or by PtdIns-3, 4-P2 in vitro, and it was impaired in binding to PtdIns-3,4-P2. Examination of the binding to other phosphoinositides revealed that they bound to the Akt PH domain with much lower affinity than did PtdIns-3,4-P2 and failed to increase Akt activity. Thus, Akt is apparently regulated by the direct interaction of PtdIns-3,4-P2 with the Akt PH domain.

3T3 Cells↗
PubMed1997

Determination of the specific substrate sequence motifs of protein kinase C isozymes.

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (alpha, betaI, betaII, gamma, delta, epsilon, eta, mu, and zeta) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position -3. All isozymes, except PKC mu, selected peptides with basic amino acids at positions -6, -4, and -2. PKC alpha, -betaI, -betaII, -gamma, and -eta selected peptides with basic amino acid at positions +2, +3, and +4, but PKC delta, -epsilon, -zeta, and -mu preferred peptides with hydrophobic amino acid at these positions. At position -5, the selectivity was quite different among the various isozymes; PKC alpha, -gamma, and -delta selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKC mu showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position -3 to +2 for PKC alpha, -betaI, -betaII, -gamma, -delta, and -eta were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKC alpha, -betaI,-delta, -zeta, and -mu were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.

Amino Acid Sequence↗
PubMed1997

Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion.

In the present study, we have synthesized and N-myristoylated peptides derived from the pseudosubstrate sequences of protein kinase C (PKC)-alpha, -delta, and -epsilon [Myr-PKC-alpha-(15-28), Myr-PKC-delta-(142-153), and Myr-PKC-epsilon-(149-164)], three isoforms present in rat lacrimal gland, and a peptide derived from the sequence of the endogenous inhibitor of protein kinase A [Myr-PKI-(17-25)]. Lacrimal gland acini were preincubated for 60 min with the myristoylated peptides (10(-10) to 3 x 10(-7) M), then protein secretion was stimulated with a phorbol ester, phorbol 12,13-dibutyrate (10(-6) M); vasoactive intestinal peptide (10(-8) M); a cholinergic agonist, carbachol (10(-5) M); or an alpha 1-adrenergic agonist, phenylephrine (10(-4) M), for 20 min. In intact lacrimal gland acini, Myr-PKC-alpha-(15-28) inhibited phorbol 12,13-dibutyrate-induced protein secretion. This effect was not reproduced by the acetylated peptide or by the myristoylated PKI, which inhibited vasoactive intestinal peptide-induced protein secretion, a response mediated by protein kinase A. Carbachol-induced protein secretion was inhibited by all three peptides. In contrast, phenylephrine-induced protein secretion was inhibited only by Myr-PKC-epsilon-(149-164), whereas Myr-PKC-alpha-(15-28) and Myr-PKC-delta-(142-153) had a stimulatory effect. None of these myristoylated peptides affected the calcium increase evoked by cholinergic or alpha 1-adrenergic agonists. We concluded that phorbol ester- and receptor-induced protein secretion involve different PKC isoforms in lacrimal gland.

Adrenergic Agents↗
PubMed1996

D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate.

Phorbol 12-myristate 13-acetate (PMA) uncaps a small number of the fast-growing (barbed) ends of actin filaments, thereby eliciting slow actin assembly and extension of filopodia in human blood platelets. These reactions, which also occur in response to immunologic perturbation of the integrin glycoprotein (GP) IIb-IIIa, are sensitive to the phosphoinositide 3-kinase inhibitor wortmannin. Platelets deficient in GPIIb-IIIa integrins or with GPIIb-IIIa function inhibited by calcium chelation or the peptide RGDS have diminished PMA responsiveness. The effects of PMA contrast with thrombin receptor stimulation by >/=5 microM thrombin receptor-activating peptide (TRAP), which causes rapid and massive wortmannin-insensitive actin assembly and lamellar and filopodial extension. However, we show here that wortmannin can inhibit filopod formation if the thrombin receptor is ligated using suboptimal doses (<1 microM) of TRAP. Phosphatidylinositol 3,4-bisphosphate inhibits actin filament severing and capping by human gelsolin in vitro. The findings implicate D3 polyphosphoinositides and integrin signaling in PMA-mediated platelet stimulation and implicate D3 containing phosphoinositides generated in response to protein kinase C activation and GPIIb-IIIa signaling as late-acting intermediates leading to filopodial actin assembly.

Actin Cytoskeleton↗
PubMed1996

Stimulation of an insulin receptor activates and down-regulates the Ca2+-independent protein kinase C, Apl II, through a Wortmannin-sensitive signaling pathway in Aplysia.

Activation of tyrosine kinase-linked receptors has been shown to stimulate Ca2+-independent protein kinase C isoforms in nonneuronal cells. We have examined this signaling pathway in the nervous system. Incubating bag cell neurons from the marine mollusk Aplysia californica with concentrations of insulin known to stimulate a tyrosine kinase-linked receptor in these cells persistently activated and down-regulated the Ca2+-independent protein kinase C (Apl II), whereas insulin only transiently activated and did not down-regulate the Ca2+-activated protein kinase C (Apl I). The effects of insulin may be mediated by activation of phosphoinositide 3-kinase because (a) diC16phosphatidylinositol 3,4,5-trisphosphate, a synthetic phosphoinositide 3-kinase product, stimulated autophosphorylation of baculovirus-expressed Apl II, but not of Apl I, and (b) wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the activation and down-regulation of Apl II by insulin but not the transient activation of Apl I. These results suggest that activators of tyrosine kinase-linked receptors may mediate some of their effects in neurons through activation of Ca2+-independent protein kinase C isoforms.

Androstadienes↗
PubMed1995

Phosphorylation of the platelet p47 phosphoprotein is mediated by the lipid products of phosphoinositide 3-kinase.

Platelet stimulation by thrombin or the thrombin receptor activating peptide (TRAP) results in the activation of phosphoinositide 3-kinase and the production of the novel polyphosphoinositides phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3). We have shown previously that these lipids activate calcium-independent protein kinase C (PKC) isoforms in vitro (Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M. and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PKC in response to TRAP is detected by the phosphorylation of the major PKC substrate in platelets, the p47 phosphoprotein, also known as pleckstrin. Here we provide evidence for two phases of pleckstrin phosphorylation in response to TRAP. A rapid phase of pleckstrin phosphorylation (< 1 min) precedes the peak of PtdIns-3,4-P2 production and is unaffected by concentrations of wortmannin (10-100 nM) that block production of this lipid. However prolonged phosphorylation of pleckstrin (> 2 min) is inhibited by wortmannin concentrations that block PtdIns-3,4-P2 production. Phorbol ester-mediated pleckstrin phosphorylation was not affected by wortmannin and wortmannin had no effect on purified platelet PKC activity. Phosphorylation of pleckstrin could be induced using permeabilized platelets supplied with exogenous gamma-32P[ATP] and synthetic dipalmitoyl PtdIns-3,4,5-P3 and dipalmitoyl PtdIns-3,4-P2 micelles, but not with dipalmitoyl phosphatidylinositol 3-phosphate or phosphatidylinositol 4,5-bisphosphate. These results suggest two modes of stimulating pleckstrin phosphorylation: a rapid activation of PKC (via diacylglycerol and calcium) followed by a slower activation of calcium-independent PKCs via PtdIns-3,4-P2.

Androstadienes↗
PubMed1995

Phosphoinositide 3-kinase binds constitutively to alpha/beta-tubulin and binds to gamma-tubulin in response to insulin.

Recently we reported the localization of phosphoinositide 3-kinase (PI 3-kinase) by immunofluorescence to microtubule bundles and the centrosome (Kapeller, R., Chakrabarti, R., Cantley, L., Fay, F., and Corvera, S. (1993) Mol. Cell. Biol. 13, 6052-6063). In complementary experiments we used the recombinant p85 subunit of PI 3-kinase to identify proteins that associate with phosphoinositide 3-kinase and found that phosphoinositide 3-kinase associates with alpha/beta-tubulin. The association occurs in vivo but was not significantly affected by growth factor stimulation. We localized the region of p85 that interacts with alpha/beta-tubulin to the inter-SH2 domain. These results support the immunofluorescence data and show that p85 directly associates with alpha/beta-tubulin. We then determined whether phosphoinositide 3-kinase associates with gamma-tubulin. We found a dramatic growth factor-dependent association of phosphoinositide 3-kinase with gamma-tubulin. Phosphoinositide 3-kinase associates with gamma-tubulin in response to insulin and, to a lesser extent, in response to platelet-derived growth factor. Neither epidermal growth factor nor nerve growth factor treatment of cells results in association of phosphoinositide 3-kinase and gamma-tubulin. Phosphoinositide 3-kinase is also immunoprecipitated with antibodies to pericentrin in response to insulin, indicating that phosphoinositide 3-kinase is recruited to the centrosome. Neither phosphoinositide 3-kinase activity, nor intact microtubules are necessary for the association. Treatment of cells with 0.5 M NaCl dissociates gamma-tubulin from the centrosome and disrupts the association of phosphoinositide 3-kinase with pericentrin, but not gamma-tubulin. Recombinant p85 binds to gamma-tubulin from both insulin stimulated and quiescent cells. These results suggest that the association of phosphoinositide 3-kinase with gamma-tubulin is direct. These data suggest that phosphoinositide 3-kinase may be involved in regulating microtubule responses to insulin and platelet-derived growth factor.

3T3 Cells↗
PubMed1995

Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets.

Cells respond to diverse external stimuli by polymerizing cytoplasmic actin, and recent evidence indicates that GTPases can specify where this polymerization takes place. Actin assembly in stimulated blood platelets occurs where sequestered monomers add onto the fast-growing (barbed) ends of actin filaments (F-actin), which are capped in the resting cells. We report that D3 and D4 polyphosphoinositides, Pl(4)P, Pl(4,5)P2, Pl(3,4)P2, and Pl(3,4,5)P3, uncap F-actin in resting permeabilized platelets. The thrombin receptor-activating peptide (TRAP), GTP, and GTP gamma S, but not GDP beta S, also uncap F-actin in permeabilized platelets. GDP beta S inhibits TRAP-induced F-actin uncapping, and Pl(4,5)P2 overcomes this inhibition. Constitutively active mutant Rac, but not Rho, activates uncapping of F-actin. Pl(4,5)P2-binding peptides derived from gelsolin inhibit F-actin uncapping by TRAP, Rac, and GTP gamma S. TRAP and Rac induce rapid Pl(4,5)P2 synthesis in permeabilized platelets. The findings establish a signaling pathway for actin assembly involving Rac in which the final message is phosphoinositide-mediated F-actin uncapping.

Actins↗
PubMed1995

Carbachol, substance P, and phorbol ester promote the tyrosine phosphorylation of protein kinase C delta in salivary gland epithelial cells.

The initiation of saliva formation by parotid acinar cells, which comprise the majority of cells in this salivary gland, is initiated by the release of neurotransmitters (acetylcholine, substance P) from parasympathetic nerves. In response to substance P and the muscarinic agonist carbachol, two ligands that activate phospholipase C-linked receptors, which stimulate fluid secretion, PKC delta was phosphorylated on tyrosine residues. The maximal agonist-dependent tyrosine phosphorylation occurred within seconds of the addition of either agonist and then returned rapidly to a smaller increased level. Phorbol ester also caused a rapid increase in tyrosine phosphorylation, which reached a maximal level 5 min after the addition of phorbol 12-myristate 13-acetate. The increase in tyrosine phosphorylation of PKC delta was blocked by tyrosine kinase inhibitors genistein and staurosporine. Ionophore-mediated elevation of [Ca2+]i or activation of the beta-adrenergic receptor, epidermal growth factor receptor, or insulin receptor did not promote the tyrosine phosphorylation of PKC delta. These results indicate that tyrosine phosphorylation plays a role in early signal transduction events promoted by the activation of muscarinic and substance P receptors and suggests that the tyrosine phosphorylation of PKC delta has a role in the activation of fluid secretion by neurotransmitters binding to phospholipase C-linked receptors.

Alkaloids↗
PubMed1995

Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation.

Platelet stimulation by thrombin leads to the activation of phosphoinositide 3-kinase (PI 3K) and to the production of the D3 phosphoinositides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4P2) and 3,4,5-trisphosphate (PdtIns-3,4,5-P3). Because changes in the levels of these phosphoinositides correlate with the kinetics of actin assembly, they have been proposed to mediate actin assembly, causing cell shape changes. Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, were used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of PdtIns-3,4-P2 and PdtIns-3,4,5-P3 in thrombin receptor-activating peptide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation sites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a primary phase of aggregation followed by progressive disaggregation. Flow cytometry analysis with the PAC1 monoclonal antibody or with FITC-labeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin also inhibited, in a dose-dependent manner, platelet aggregation induced by the binding of the monoclonal antibodies P256 and LIBS-6 to GPIIb-IIIa. LIBS Fab-induced aggregation also led to the production of PdtIns-3,4-P2. Platelet secretion, as evidenced by the release of preloaded 14C-5-hydroxy-tryptamine secretion or P-selectin up-regulation, was not affected by PI 3-K inhibition. These results demonstrate that the generation of D3 phosphoinositides is not required for actin assembly in TRAP-activated platelets. However, PI 3-K stimulation is necessary for prolonged GPIIb-IIIa activation and irreversible platelet aggregation. PI 3-K stimulation downstream of GPIIb-IIIa engagement may provide positive feedback required to sustain active GPIIb-IIIa.

Actins↗
PubMed1994

Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3.

The effect of phosphoinositides on the activity of protein kinase C (PKC) isotypes was investigated. PKC alpha, beta I, beta II, gamma, delta, epsilon, eta, and zeta were expressed in baculovirus-infected insect cells and purified by column chromatography. The calcium-activated PKC isotypes alpha, beta I, beta II, and gamma were not significantly activated by any of the phosphoinositides investigated (phosphatidylinositol-4-phosphate (PtdIns-4-P), PtdIns-3-P, PtdIns-4,5-P2, PtdIns-3,4-P2, and PtdIns-3,4,5-P3) when added in the presence of concentrations of phosphatidylserine that give maximal stimulation. The calcium-insensitive PKC isotypes delta, epsilon, and theta also showed little response to PtdIns-3-P, PtdIns-4-P, or PtdIns-4,5-P2 when these lipids were added in the presence of phosphatidylserine. In contrast, PtdIns-3,4-P2 and PtdIns-3,4,5-P3 caused a 5-15-fold stimulation of these enzymes compared with phosphatidylserine alone. 50% maximal stimulation of PKC epsilon by PtdIns-3,4,5-P3 occurred when this lipid was present at about 1% of the carrier PtdIns-4,5-P2 (about 100 nM). These lipids had little effect on baculovirus-expressed PKC zeta, which was constitutively active. A short chain version of PtdIns-3,4,5-P3, dioctanoyl-PtdIns-3,4,5-P3, activated PKC delta, epsilon, and eta in the absence of other lipids, whereas a short chain version of PtdIns-4,5-P2, dihexanoyl-PtdIns-4,5-P2, did not. Since PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are nominally absent in unstimulated cells and appear within seconds to minutes of stimulation by various cell activators, these lipids could act as second messengers to activate PKC delta, epsilon, or eta in vivo.

Animals↗
PubMed1992

Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain. Amino acid sequence of phosphorylated forms.

A potent inhibitor of protein kinase C (PKC), inhibitor protein-1 (KCIP-1), isolated from sheep brain has been shown to consist of eight isoforms by reverse-phase HPLC. Direct protein sequence analysis has revealed these to be the same as those of 14-3-3 protein, described as an activator of tyrosine and tryptophan hydroxylases involved in neurotransmitter biosynthesis. The N-termini of KCIP-1 isoforms were shown to be acetylated, and secondary structure predictions revealed a high degree of alpha-helix with an amphipathic nature. KCIP-1 showed no inhibitory activity towards protein kinase M (the catalytic fragment of PKC) and had no effect on the activities of three other protein kinases, cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase II and casein kinase 2. Four forms of KCIP-1 were shown to be substrates for PKC in vitro, but none were phosphorylated by the other protein kinases mentioned above.

14-3-3 Proteins↗