Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin

The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.     

Purification procedures determine the proteasome activation properties of REG gamma (PA28 gamma)

The proteasome activation properties of recombinant REG gamma molecules depend on purification procedures. Prior to ammonium sulfate precipitation recombinant REG gamma activates the trypsin-like catalytic subunit of the proteasome; afterwards it activates all three catalytic subunits. The expanded activation specificity is accompanied by reduced stability of the REG gamma heptamer providing support for the idea that a "tight" REG gamma heptamer suppresses the proteasome's chymotrypsin-like and postglutamyl-preferring active sites. In an attempt to determine whether REG gamma synthesized in mammalian cells also exhibits restricted activation properties, extracts were prepared from several mammalian organs and cell lines. Surprisingly, endogenous REG gamma was found to be largely monomeric. In an alternate approach, COS7 cells were cotransfected with plasmids expressing FLAG-REG gamma and REG gamma. The expressed FLAG-REG gamma molecules were shown to form oligomers with untagged REG gamma subunits, and the mixed oligomers preferentially activated the proteasome's trypsin-like subunit. Thus, REG gamma molecules synthesized in mammalian cells also exhibit restricted activation properties.     

Role of associated gamma-chain in tyrosine kinase activation via murine Fc gamma RIII.

Type III receptors for the Fc portion of IgG (Fc gamma RIII), initially characterized on macrophages and NK cells, are also expressed on several pre-B cell lines. Surface expression of Fc gamma RIII requires the association of the ligand binding alpha-chain with homodimeric gamma-chains. Type II Fc gamma R is homologous to Fc gamma RIII alpha-chain in the extracellular portion and differs in the transmembrane and cytoplasmic domains. The role of Fc gamma R in cell activation was investigated by expressing Fc gamma RIII and the lymphocyte-specific b1 isoform of Fc gamma RII (Fc gamma RIIb1) in an Fc gamma R-negative, sIgG-positive B-cell line. We found that, in contrast to Fc gamma RIIb1, Fc gamma RIII triggers the same events of cell activation as sIG i.e. Ca2+ mobilization, tyrosine phosphorylation and IL-2 secretion. By expressing cytoplasmic domain-lacking Fc gamma RIII alpha-chain in the absence or in the presence of gamma-chains, we demonstrated that cell activation via Fc gamma RIII requires the co-expression of gamma-chains, and is independent of the cytoplasmic portion of the alpha-chain. Furthermore, the cytoplasmic portion of the gamma-chain, fused to the extracellular and transmembrane domains of Fc gamma RII confers on the chimeric receptor the ability to trigger cell activation. Mutation of one tyrosine residue in the cytoplasmic domain of the gamma-chain prevented triggering of cytoplasmic signals. We therefore demonstrate that a tyrosine-containing motif, present in the cytoplasmic domain of the associated gamma-chain, is necessary and sufficient to trigger cell activation via Fc gamma RIII.     

Differential mitogen-activated protein kinase stimulation by Fc gamma receptor IIa and Fc gamma receptor IIIb determines the activation phenotype of human neutrophils

Fc gamma Rs mediate immune complex-induced tissue injury. The hypothesis that Fc gamma RIIa and Fc gamma RIIIb control neutrophil responses by activating mitogen-activated protein kinases was examined. Homotypic and heterotypic cross-linking of Fc gamma RIIa and/or Fc gamma RIIIb resulted in a rapid, transient increase in ERK and p38 activity, with maximal stimulation between 1 and 3 min. Fc gamma RIIa and Fc gamma RIIIb stimulated distinct patterns of ERK and p38 activity, and heterotypic cross-linking failed to stimulate synergistic activation of either ERK or p38 activity. Both Fc gamma RIIa and Fc gamma RIIIb required activation of a nonreceptor tyrosine kinase and phosphatidylinositol 3-kinase for stimulation of ERK and p38. Inhibition of ERK activation with PD98059 enhanced H2O2 production stimulated by homotypic and heterotypic Fc gamma R cross-linking. Inhibition of p38 with SB203580 attenuated H2O2 production stimulated by Fc gamma RIIIb or heterotypic cross-linking, but had no effect on Fc gamma RIIa-stimulated H2O2 production. On the other hand, PD98059 inhibited actin polymerization stimulated by Fc gamma R cross-linking, while SB203580 had no effect. Inhibition of actin polymerization with cytochalasin D enhanced p38 activity stimulated by either Fc gamma RIIa or Fc gamma RIIIb, but cytochalasin D only enhanced H2O2 production stimulated by Fc gamma RIIIb. Our data indicate that Fc gamma RIIa and Fc gamma RIIIb independently activate ERK and p38. The two receptors demonstrate different efficacies for ERK and p38 activation, and they do not act cooperatively. ERK and p38 provide stimulatory and inhibitory signals for neutrophil responses to immune complexes. In addition, these data indicate that actin reorganization may play a role in mediating p38-dependent activation of respiratory burst upon stimulation of Fc gamma RIIIb in neutrophils.     

Arachidonic acid metabolites alter G protein-mediated signal transduction in heart. Effects on muscarinic K+ channels

The muscarinic acetylcholine receptor (mAChR)-stimulated, inwardly rectifying K+ current (IK [ACh]) was examined in single bullfrog atrial cells using the whole-cell patch clamp technique. IK[ACh] was activated either by bath addition of 1 microM ACh or via activation of the G protein, Gk, with guanosine-gamma-thiotriphosphate (GTP gamma S). Arachidonic acid (AA) modulated IK[ACh] under both conditions. AA decreased mAChR-stimulated IK[ACh] and increased the rate of decay from the peak current (desensitization). In addition, AA affected GTP gamma S-activated IK[ACh] by modulation of Gk. The effects of AA and its metabolites on Gk were assessed by examining their effects on both the basal rate of Gk activation by GTP gamma S, and the mAChR-mediated increase in activation rate produced by nanomolar ACh. AA increased the basal rate of GTP gamma S-mediated IK[ACh] activation, but reduced the ACh-induced augmentation of this rate. All of the effects of AA on GTP gamma S-mediated IK[ACh] activation were produced by metabolites. A lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), decreased the basal and ACh-enhanced rate of IK[ACh] activation in both the presence and absence of exogenous AA. In contrast, indomethacin (INDO), a cyclooxygenase inhibitor, increased the basal rate of IK[ACh] activation by GTP gamma S in both the presence and absence of exogenous AA, and reversed the effects of AA on the ACh-augmented basal rate. AA metabolites produced via lipoxygenase and cyclooxygenase pathways thus have opposing effects on the signal transduction pathway from mAChR to IK[ACh]. We directly tested a lipoxygenase pathway metabolite, LTC4, on GTP gamma S-mediated IK[ACh] activation and found that it not only overcame the inhibitory effects of NDGA, but also increased both the basal and ACh-augmented rate of IK[ACh] activation. From these data, we propose that AA metabolites modulate the function of Gk by altering its kinetic properties.     

Activation of peroxisome proliferator-activated receptor gamma inhibits osteoprotegerin gene expression in human aortic smooth muscle cells

Increasing evidence indicates an important role of PPAR gamma activation in modulating the development and progression of atherosclerosis, however, the mechanisms involved in these effects are not well understood since the PPAR gamma-regulated genes in vascular smooth muscle cells (VSMC) are poorly defined. Here we reported that PPAR gamma ligands, GW7845, ciglitazone and troglitazone had the effect of inhibiting osteoprotegerin (OPG) expression in human aortic smooth muscle cells (HASMC). The effect of GW7845 and ciglitazone on OPG expression was completely abolished by GW9662, a PPAR gamma antagonist. Overexpression of PPAR gamma in HASMC by the infection of a PPAR gamma adenovirus dramatically decreased OPG expression. In addition, PPAR gamma activation inhibited OPG promoter activity. Taken together, our data suggest that OPG expression is a novel PPAR gamma target gene in VSMC and downregulation of OPG expression by PPAR gamma activation provides a new insight into the understanding of the role of PPAR gamma in atheroscelrosis and hypertension.     

Characterization of ATP-stimulated guanylate cyclase activation in rat lung membranes

Many of the effects of ANP are mediated through the elevation of cellular cGMP levels by the activation of particulate guanylate cyclase. While the stimulation of this enzyme is receptor-mediated, the molecular mechanism of activation remains unknown. In this study we present evidence that ATP as well as its analogues adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) and adenylylimidophosphate (AMPPNP) activates guanylate cyclase from rat lung membranes and markedly potentiates the effect of ANP on the enzyme. The order of potency is ATP gamma S greater than ATP greater than AMPPNP. The enzyme activation by adenine nucleotide and ANP together is much more than the sum of the individual activations, suggesting that ATP may be the physiological component essential for the ANP-stimulated guanylate cyclase activation. The ATP gamma S-stimulated guanylate cyclase activity diminishes in the presence of various kinds of detergents, suggesting either that the conformation of an ATP binding site in guanylate cyclase is altered by detergents or that protein-protein interaction may be involved in the activation of guanylate cyclase by ATP. Guanylate cyclase from rat lung membranes is poorly activated by ANP and/or ATP gamma S after removing the cytosolic and weakly membrane-associated proteins or factors by centrifugation. Pre-incubation of the membranes with ATP gamma S retains enzyme activation after membrane washing. These results suggest either that ATP gamma S stabilizes the conformation of nucleotide binding site in guanylate cyclase from denaturation by membrane washing, or that the stimulatory effect of ATP on guanylate cyclase activity may be mediated by accessory proteins or non-protein cofactors which are lost during membrane washing, but remain bound to membranes by ATP gamma S pretreatment.     

Responses of neutrophils to anti-integrin antibodies depends on costimulation through low affinity Fc gamma Rs: full activation requires both integrin and nonintegrin signals

The relative contribution of integrin and nonintegrin signals to neutrophil activation is incompletely understood. Immobilized anti-integrin Abs were previously shown to induce robust activation of neutrophils without any additional stimulus, suggesting that cross-linking of integrins is sufficient for full activation of the cells. However, the possible contribution from other receptors has not been tested in this system. In this study, we show that neutrophil responses to anti-integrin Abs requires costimulation through low-affinity Fc gamma Rs. Murine neutrophils lacking the FcR gamma-chain or Fc gamma RIII failed to respond to immobilized Abs against beta(1), beta(2), or beta(3) integrins and the activation of wild-type cells could be prevented by blocking Abs against Fc gamma RII/III. Plate-bound anti-CD18 Abs initiated a respiratory burst from human neutrophils, but this response was abrogated when the F(ab')(2) of the same Abs were used or the cells were preincubated with Fc gamma RIIA-blocking Abs. Lack of Fc gamma RIII or administration of Fc gamma R-blocking Abs had no effect on responses of TNF-stimulated cells plated on fibrinogen or rICAM-1. TNF restored the respiratory burst of Fc gamma RIII-deficient neutrophils plated on anti-CD18 mAbs. The p38 MAPK inhibitor SB203580 attenuated the responses of neutrophils to anti-CD18 mAbs or TNF stimulation on a fibrinogen surface. Taken together, these results indicate that activation of low-affinity Fc gamma Rs is required for neutrophil responses induced by anti-integrin Abs and suggest that a second coactivation signal (e.g., through TNF or FcR ligation) is indispensable for full integrin-mediated activation of neutrophils. These second signals are interchangeable and they may converge on the p38 MAPK.     

Protein adsorption at solid-liquid interfaces: Part I--Affinities of proteins for alumina surface

Extent of adsorption (gamma pw) of bovine serum albumin, beta-lactoglobulin, gelatin and myosin at the alumina-water interface has been measured as function of protein concentration (Cp) at several temperatures, pH, and ionic strengths of the medium. gamma pw for proteins in most cases increases with increase of protein concentration but it attains maximum value gamma pw(m) when Cp is high. Values of maximum adsorption have been examined in terms of molecular orientation, molecular size and shape and unfolding of the packed proteins at the interface. In few cases, gamma pw increases with increase of Cp without reaching a real state of saturation as a result of aggregation of molecules or extensive unfolding of the protein at the interface. In the case of beta-lactoglobulin at pH 5.2 and ionic strength 0.05, gamma pw in high concentration region decreases to zero value when Cp increases. For myosin at 45 degrees C and pH 6.4, and also at 27 degrees and pH 7.8, the values of gamma pw are all negative and these negative values increase with increase of Cp. All these results have been explained in terms of significant competitions of water and protein for binding to the surface sites of the powdered alumina. Adsorption of myosin has also been found to be affected in the presence of NaCl, KCl, CaCl2, KI, Na2SO4, LiCl and urea. The relative affinities of the adsorption of various proteins for the surface of alumina at different physical conditions of the system have been compared in terms of maximum values of adsorption attained when gamma pw is varied with Cp. The affinities are shown to be compared more precisely in terms of the standard free energy decrease for the saturation of the surface by protein as a result of the change in its concentration from zero to unity in the mole fraction scale.     

Activated alumina as an energy source for peptide bond formation: Consequences for mineral-mediated prebiotic processes

Summary. The catalytic properties of various forms of alumina were tested for alanine dimerization reaction. The catalytic efficiency of alumina depends on the structure, as well as on acid/base properties of the catalyst. The highest yields of Ala₂ were achieved on activated alumina with surface of neutral pH (about 3% conversion after 2 weeks). Thermal analysis of Ala + alumina reaction systems shows that the thermal behavior of amino acid changes substantially in contact with the activated surface of the alumina catalyst. The reaction of Ala is detected as being strongly endothermic by differential thermal analysis of pure amino acid (above 250°C). The alanine endothermic reaction is shifted substantially to lower values (below 200°C) and hardly detectable if activated alumina is present. The reaction mechanism of amino acid activation on alumina surface and its significance for mineral-catalyzed prebiotic peptide bond formation are discussed. Received June 6, 2000 Accepted July 27, 2000     

Activation of phospholipase C gamma by PI 3-kinase-induced PH domain-mediated membrane targeting.

Signaling via growth factor receptors frequently results in the concomitant activation of phospholipase C gamma (PLC gamma) and phosphatidylinositol (PI) 3-kinase. While it is well established that tyrosine phosphorylation of PLC gamma is necessary for its activation, we show here that PLC gamma is regulated additionally by the lipid products of PI 3-kinase. We demonstrate that the pleckstrin homology (PH) domain of PLC gamma binds to phosphatidylinositol 3,4,5-trisphosphate [PdtIns(3,4,5)P3], and is targeted to the membrane in response to growth factor stimulation, while a mutated version of this PH domain that does not bind PdtIns(3,4,5)P3 is not membrane targeted. Consistent with these observations, activation of PI 3-kinase causes PLC gamma PH domain-mediated membrane targeting and PLC gamma activation. By contrast, either the inhibition of PI 3-kinase by overexpression of a dominant-negative mutant or the prevention of PLC gamma membrane targeting by overexpression of the PLC gamma PH domain prevents growth factor-induced PLC gamma activation. These experiments reveal a novel mechanism for cross-talk and mutual regulation of activity between two enzymes that participate in the control of phosphoinositide metabolism.     

MIP-1 gamma promotes receptor-activator-of-NF-kappa-B-ligand-induced osteoclast formation and survival

Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes, and have also been reported to modulate osteoclast differentiation from hemopoietic precursor cells of the monocyte-macrophage lineage. In this study, we examined the effect of MIP-1 gamma, a C-C chemokine family member, on receptor activator of NF-kappa B ligand (RANKL)-stimulated osteoclast differentiation, survival, and activation. RANKL induced osteoclasts to dramatically increase production of MIP-1 gamma and to also express the MIP-1 gamma receptor CCR1, but had only minor effects on the related C-C chemokines MIP-1 alpha and RANTES. Neutralization of MIP-1 gamma with specific Ab reduced RANKL-stimulated osteoclast differentiation by 60-70%. Mature osteoclasts underwent apoptosis within 24 h after removal of RANKL, as shown by increased caspase 3 activity and DNA fragmentation. Apoptosis was reduced by the addition of exogenous MIP-1 gamma or RANKL, both of which increased NF-kappa B activation in osteoclasts. Neutralization studies showed that the prosurvival effect of RANKL was in part dependent on its ability to induce MIP-1 gamma. Finally, osteoclast activation for bone resorption was stimulated by MIP-1 gamma. Taken together, these results demonstrate that MIP-1 gamma plays an important role in the differentiation and survival of osteoclasts, most likely via an autocrine pathway.     

Action of chlortetracycline on the enzymes of the producer itself, Streptomyces aureofaciens]

When preparing a cell-free extract of Streptomyces aureofaciens from the culture to which chlortetracycline (1000 gamma/ml) was added before disintegration with alumina, a considerable decrease in the enzyme activity was reached. The presence of chlortetracycline during disintegration of the cells by means of glass beads did not substantially affect the enzyme activity of the cell-free extract. Addition of chlortetracycline directly to the reaction mixture for the enzyme assay (up to a concentration of 200 gamma/ml) did not induce any inhibition effect. The results suggested that during studying enzyme systems of organisms producing secondary metabolites, the product might distort the results on the use of an inconvenient method.     

The SH2-containing 5'-inositol phosphatase (SHIP) is tyrosine phosphorylated after Fc gamma receptor clustering in monocytes.

Current models of Fc gamma R signal transduction in monocytes describe a molecular cascade that begins upon clustering of Fc gamma R with the phosphorylation of critical tyrosine residues in the cytoplasmic domains of Fc gamma RIIa or the gamma-chain subunit of Fc gamma RI and Fc gamma RIIIa. The cascade engages several other tyrosine-phosphorylated molecules, either enzymes or adapters, to manifest ultimately an array of biological responses, including phagocytosis, cell killing, secretion of a variety of inflammatory mediators, and activation. Continuing to assess systematically the molecules participating in the cascade, we have found that the SH2-containing 5'-inositol phosphatase (SHIP) is phosphorylated on tyrosine early and transiently after Fc gamma R clustering. This molecule in other systems, such as B cells and mast cells, mediates an inhibitory signal. We find that clustering of either Fc gamma RIIa or Fc gamma RI is effective in inducing SHIP phosphorylation, that SHIP binds in vitro to a phosphorylated immunoreceptor tyrosine-based activation motif, peptide from the cytoplasmic domain of Fc gamma RIIa in activation-independent fashion, although SHIP binding increases upon cell activation, and that Fc gamma RIIb and Fc gamma RIIc are not responsible for the observed SHIP phosphorylation. These findings prompt us to propose that SHIP inhibits Fc gamma R-mediated signal transduction by engaging immunoreceptor tyrosine-based activation motif-containing cytoplasmic domains of Fc gamma RIIa and Fc gamma RI-associated gamma-chain.     

Involvement of the MKK6-p38gamma cascade in gamma-radiation-induced cell cycle arrest

The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38gamma (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for gamma-irradiation-induced G(2) arrest. Activation of the MKK6-p38gamma cascade is sufficient to induce G(2) arrest in cells, and expression of dominant negative alleles of MKK6 or p38gamma allows cells to escape the DNA damage-induce G(2) delay. Activation of p38gamma is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by gamma irradiation leads to the activation of MKK6, p38gamma, and Cds1 and that activation of both MKK6 and p38gamma is essential for the proper regulation of the G(2) checkpoint in mammalian cells.     

Membrane stimulation of cGMP phosphodiesterase activation by transducin: comparison of phospholipid bilayers to rod outer segment membranes.

To clarify the role of phospholipids in G protein-effector interactions of vertebrate phototransduction, transducin activation of cGMP phosphodiesterase (PDE) has been reconstituted on the surface of well-defined phosphatidylcholine (PC) vesicles, using purified proteins from bovine rod outer segments (ROS). PC vesicles enhanced PDE stimulation by the GTP-gamma S-bound transducin alpha subunit (T alpha-GTP gamma S) as much as 17-fold over activation in the absence of membranes. In the presence of 3.5 microM accessible PC in the form of large (100 nm) unilamellar vesicles, 500 nM T alpha-GTP gamma S stimulated PDE activity to more than 70% of the maximum activity induced by trypsin. Activation required PC, PDE, and T alpha-GTP gamma S, but did not require prior incubation of any of the components, and occurred within 4 s of mixing. The PC vesicles were somewhat more efficient than urea-washed ROS membranes in enhancing PDE activation. Half-maximal activation occurred at accessible phospholipid concentrations of 3.8 microM for PC vesicles, and 13 microM for ROS membranes. Titrations of PDE with T alpha-GTP gamma S in the presence of membranes indicated a high-affinity (Kact less than 250 pM) activation of PDE by a small fraction (0.5-5%) of active T alpha-GTP gamma S, as did titrations of ROS with GTP gamma S. When activation by PC vesicles was compared to PDE binding to membranes, the results were consistent with activation enhancement resulting from formation of a T alpha-GTP gamma S-dependent PDE-membrane complex with half-maximal binding at phospholipid concentrations in the micromolar range. The value of the apparent dissociation constant, KPL, associated with the activation enhancement was estimated to be in the range of 2.5 nM (assuming an upper limit value of 1600 phospholipids/site) to 80 nM (for a lower limit value of 50 phospholipids/site). Another component of membrane binding was more than 100-fold weaker and was not correlated with activation by T alpha-GTP gamma S. Low ionic strength disrupted the ability of ROS membranes, but not PC vesicles, to bind and activate PDE. Removal of PDE's membrane-binding domain by limited trypsin digestion eliminated both the binding of PDE to vesicles and the ability of PDE to be activated by T alpha-GTP gamma S and membranes. These results suggest that ROS membrane stimulation of PDE activation by T alpha-GTP gamma S is due almost exclusively to the phospholipids in the disk membrane.     

Roles of G beta gamma in membrane recruitment and activation of p110 gamma/p101 phosphoinositide 3-kinase gamma

Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.     

Guanosine 5'-O-(3-thiotriphosphate) and guanosine 5'-O-(2-thiodiphosphate) activate G proteins and potentiate fibroblast growth factor-induced DNA synthesis in hamster fibroblasts

Addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to intact Chinese hamster lung fibroblasts (CCL39) depolarized by high K+ concentrations results in activation of phosphoinositide-specific phospholipase C (PLC) (at GTP gamma S concentrations greater than 0.1 mM), inhibition of adenylate cyclase (between 10 microM and 0.5 mM), and activation of adenylate cyclase (above 0.5 mM). Since GTP gamma S-induced activation of PLC is dramatically enhanced upon receptor-mediated stimulation of PLC by alpha-thrombin, we conclude that in depolarized CCL39 cells GTP gamma S directly activates various guanine nucleotide-binding regulatory proteins (G proteins) coupled to PLC (Gp(s)) and to adenylate cyclase (Gi and Gs). Pretreatment of cells with pertussis toxin strongly inhibits GTP gamma S-induced activation of PLC and inhibition of adenylate cyclase. GTP gamma S cannot be replaced by other nucleotides, except by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which mimics after a lag period of 15-20 min all the effects of GTP gamma S, with the same concentration dependence and the same sensitivity to pertussis toxin. We suggest that GDP beta S is converted in cells into GTP beta S, which acts as GTP gamma S. Since cell viability is not affected by a transient depolarization, these observations provide a simple method to examine long-term effects of G protein activation on DNA synthesis. We show that a transient exposure of G0-arrested CCL39 cells to GTP gamma S or GDP beta S under depolarizing conditions is not sufficient by itself to induce a significant mitogenic response, but markedly potentiates the mitogenic action of fibroblast growth factor, a mitogen known to activate a receptor-tyrosine kinase. The potentiating effect is maximal after 60 min of pretreatment with 2 mM GTP gamma S. GDP beta S is equally efficient but only after a lag period of 15-20 min. Mitogenic effects of both guanine nucleotide analogs are suppressed by pertussis toxin. Since the activation of G proteins by GTP gamma S under these conditions vanishes after a few hours, we conclude that a transient activation of G proteins facilitates the transition G0----G1 in CCL39 cells, whereas tyrosine kinase-induced signals are sufficient to mediate the progression into S phase.     

Protein tyrosine phosphorylation induced via the IgG receptors Fc gamma Ri and Fc gamma RII in the human monocytic cell line THP-1.

We have investigated the role of protein tyrosine phosphorylation in transmembrane signaling via the IgG receptors Fc gamma RI and Fc gamma RII in the human monocytic cell line THP-1. Fc gamma RI and Fc gamma RII were selectively engaged using the anti-Fc gamma RI mAb 197 (IgG2a) and the anti-Fc gamma RII mAb IV.3 (IgG2b). Addition to cells of mAb 197, but not addition of IgG2a mAb of irrelevant specificity, resulted in the rapid induction of cytoplasmic protein tyrosine phosphorylation as assessed by antiphosphotyrosine immunoblotting. A similar pattern of tyrosine phosphorylation was induced by mAb IV.3, but not by control IgG2b mAb. The induction of tyrosine phosphorylation by anti-Fc gamma R mAb was not dependent on antibody Fc region-FcR interactions, because tyrosine phosphorylation was also induced by cross-linked anti-Fc gamma RI F(ab')2 fragments and by cross-linked anti-Fc gamma RII Fab fragments. To investigate the relationship of Fc gamma R-induced tyrosine phosphorylation and activation of phospholipase C, which is known to follow Fc gamma R engagement, we assessed the effect of the tyrosine kinase inhibitor herbimycin A on Fc gamma R-induced Ca2+ flux. Herbimycin A strongly inhibited cellular Ca2+ flux induced by mAb 197, but did not inhibit Ca2+ flux induced by aluminum fluoride, suggesting that tyrosine phosphorylation may be important in regulating Fc gamma R-mediated activation of phospholipase C. Consistent with this, mAb 197 induced rapid phosphorylation of the gamma-1 isoform of phospholipase C. Finally, herbimycin A strongly inhibited the induction of TNF-alpha mRNA accumulation by Fc gamma R cross-linking. These results suggest that protein tyrosine phosphorylation may play an important role in the activation of phospholipase C and in the induction of monokine gene expression that follows engagement of Fc gamma R in human monocytes.