Phosphoinositide 3-kinase γ mediates microglial phagocytosis via lipid kinase-independent control of cAMP.

Microglial phagocytosis plays a key role in neuroprotective and neurodegenerative responses of the innate immune system in the brain. Here we investigated the regulatory function of phosphoinositide 3-kinase γ (PI3Kγ) in phagocytosis of bacteria and Zymosan particles by mouse brain microglia in vitro and in vivo. Using genetic and pharmacological approaches our data revealed PI3Kγ as an essential mediator of microglial phagocytosis. Unexpectedly, microglia expressing lipid kinase deficient mutant PI3Kγ exhibited similar phagocytosis as wild-type cells. These data suggest kinase-independent stimulation of cAMP phosphodiesterase activity by PI3Kγ as a crucial mediator of phagocytosis. In sum our findings indicate PI3Kγ-dependent suppression of cAMP signaling as a critical regulatory element of microglial phagocytosis.

Luminal decoration of blood vessels by activated perivasal mast cells in allergic rhinitis.

BACKGROUND: In allergic diseases, like in rhinitis, antigen challenge induces rapid degranulation of tissue resident mast cells and subsequent recruitment of leukocytes in response to soluble immunmodulators. The fate of mast cell-derived, membrane associated factors in inflamed tissue remained however unresolved. METHODS: Components of the mast cell granular membrane, including the unique marker CD63var, were examined by FACS and by confocal laser scanning microscopy in cell culture and in diseased human tissue. RESULTS: We discovered that selected mast cell membrane components appeared on the surface of distinct bystander cells. Acceptor cells did not acquire these molecules simply by uptake of soluble material or in the form of exosomes. Instead, physically stable cell-to-cell contact was required for transfer, in which a Notch2-Jagged1 interaction played a decisive role. This process is activation-dependent, unidirectional, and involves a unique membrane topology. Endothelial cells were particularly efficient acceptors. In organotypic 3D in vitro cultures we found that transferred mast cell molecules traversed an endothelial monolayer, and reappeared focally compacted on its distal surface, away from the actual contact zone. Moreover, we observed that such mast cell-derived membrane patches decorate microcapillaries in the nasal mucosa of allergic rhinitis patients. CONCLUSION: Direct membrane transfer from perivasal mast cells into nearby blood vessels constitutes a novel mechanism to modulate endothelial surface features with apparent significance in allergic diseases.

Key role of PI3Kγ in monocyte chemotactic protein-1-mediated amplification of PDGF-induced aortic smooth muscle cell migration.

BACKGROUND AND PURPOSE: Vascular smooth muscle cell (SMC) migration within the arterial wall is a crucial event in atherogenesis and restenosis. Monocyte chemotactic protein-1/CC-chemokine receptor 2 (MCP-1/CCR2) signalling is involved in SMC migration processes but the molecular mechanisms have not been well characterized. We investigated the role of PI3Kγ in SMC migration induced by MCP-1. EXPERIMENTAL APPROACHES: A pharmacological PI3Kγ inhibitor, adenovirus encoding inactive forms of PI3Kγ and genetic deletion of PI3Kγ were used to investigate PI3Kγ functions in the MCP-1 and platelet-derived growth factor (PDGF) signalling pathway and migration process in primary aortic SMC. KEY RESULTS: The γ isoform of PI3K was shown to be the major signalling molecule mediating PKB phosphorylation in MCP-1-stimulated SMC. Using a PI3Kγ inhibitor and an adenovirus encoding a dominant negative form of PI3Kγ, we demonstrated that PI3Kγ is essential for SMC migration triggered by MCP-1. PDGF receptor stimulation induced MCP-1 mRNA and protein accumulation in SMCs. Blockade of the MCP-1/CCR2 pathway or pharmacological inhibition of PI3Kγ reduced PDGF-stimulated aortic SMC migration by 50%. Thus PDGF promotes an autocrine loop involving MCP-1/CCR2 signalling which is required for PDGF-mediated SMC migration. Furthermore, SMCs isolated from PI3Kγ-deficient mice (PI3Kγ(-/-)), or mice expressing an inactive PI3Kγ (PI3Kγ(KD/KD)), migrated less than control cells in response to MCP-1 and PDGF. CONCLUSIONS AND IMPLICATIONS: PI3Kγ is essential for MCP-1-stimulated aortic SMC migration and amplifies cell migration induced by PDGF by an autocrine/paracrine loop involving MCP-1 secretion and CCR2 activation. PI3Kγ is a promising target for the treatment of aortic fibroproliferative pathologies.

GABAA receptor-associated phosphoinositide 3-kinase is required for insulin-induced recruitment of postsynaptic GABAA receptors.

Type A gamma-aminobutyric acid (GABAA) receptors mediate most of the fast inhibitory synaptic transmission within the vertebrate brain. The regulation of this inhibition is vital in modulating neural activity. One regulator of GABAA receptor function is insulin, which can serve to enhance GABAA receptor-mediated miniature inhibitory postsynaptic currents, via an increase in the number of receptors at the plasma membrane. We set out to investigate the molecular mechanisms involved in the insulin-induced potentiation of GABAA receptor-mediated responses, by examining the role of phosphoinositide 3-kinase (PI3-K), a key mediator of the insulin response within the brain. We found that PI3-K associates with the GABAA receptor, and this interaction is increased following insulin treatment. Additionally, the beta2 subunit of the GABAA receptor appears to mediate the insulin-stimulated association with the N-terminal SH2 domain of the p85 subunit of PI3-K. Our results imply a mechanism whereby insulin can regulate changes in synaptic transmission through its downstream actions on the GABAA receptor.

Phosphoinositide 3-kinase gamma: a key modulator in inflammation and allergy.

Chronic inflammation and allergy involve the activation of tissue-resident cells and, later on, the invasion of effector cells. We have previously shown that the loss of phosphoinositide 3-kinase (PI3K) gamma impairs chemokine-dependent migration of neutrophils and macrophages both in vitro and in vivo. On the other hand, PI3K gamma is not required either during phagocytic processes or in the activation of bactericidal activities like granule secretion and particle-mediated respiratory burst in neutrophils. Tissue mast cells are key regulators in allergy and inflammation and release histamine upon clustering of their IgE receptors. We have demonstrated that murine mast cell responses are exacerbated in vitro and in vivo by autocrine signals, and require functional PI3K gamma. Adenosine, acting through the A(3) adenosine receptor, as well as other agonists of G(alpha i)-coupled receptors, transiently increased PtdIns(3,4,5) P (3) exclusively via PI3K gamma. PI3K gamma-derived PtdIns(3,4,5) P (3) was instrumental for initiation of a sustained influx of external Ca(2+) and degranulation. Mice that lacked PI3K gamma did not form oedema when challenged by passive systemic anaphylaxis. PI3K gamma thus relays inflammatory signals through various GPCRs, and is thus central to mast cell function. Taken together, this suggests that pharmaceutical targeting of PI3K gamma might alleviate inflammation at both early and late stages of the allergic response.

The ATP-binding site of brain phosphatidylinositol 4-kinase PI4K230 as revealed by 5'-p-fluorosulfonylbenzoyladenosine.

The ATP-binding site of purified bovine brain phosphatidylinositol 4-kinase 230 (PI4K230) was studied by its reaction with 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an ATP-like alkylating reagent. Four hundred to eight hundred micromolar FSBA inactivated PI4K230 specifically with apparently first-order kinetics and resulted in 50% loss of enzyme activity in 36--130 min. The specificity of the reaction with FSBA was demonstrated by the lack of inactivation with 5'-p-fluorosulfonylbenzoyl chloride and by protection with ATP and ATP analogues against inactivation. Most ATP analogues competed with FSBA inactivation in order of their increasing hydrophobicity, parallel to their inhibitory potency in activity measurements. The specific binding of FSBA to PI4K230 was demonstrated also by Western-blot experiments. These results suggest that FSBA-reactive group(s) involved in the enzyme activity are located near to the ATP-binding site in a hydrophobic region of native PI4K230. Experiments with site-directed mutagenesis indicate that the conserved Lys-1792 plays essential role in the enzyme activity and serves as one target of affinity labelling by FSBA. Prevention of both Lys-1792-directed and Lys-1792-independent binding of FSBA by Cibacron Blue 3GA suggest that these sites are located spatially close to each other.

Activation loop sequences confer substrate specificity to phosphoinositide 3-kinase alpha (PI3Kalpha ). Functions of lipid kinase-deficient PI3Kalpha in signaling.

Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3Kalpha (p85/p110alpha). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110alpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110alpha hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70(S6k), retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3Kalpha. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3Kalpha hybrids to dissect PI3Kalpha function as lipid and protein kinase.

Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine.

The specific phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 have been invaluable tools for elucidating the roles of these enzymes in signal transduction pathways. The X-ray crystallographic structures of PI3Kgamma bound to these lipid kinase inhibitors and to the broad-spectrum protein kinase inhibitors quercetin, myricetin, and staurosporine reveal how these compounds fit into the ATP binding pocket. With a nanomolar IC50, wortmannin most closely fits and fills the active site and induces a conformational change in the catalytic domain. Surprisingly, LY294002 and the lead compound on which it was designed, quercetin, as well as the closely related flavonoid myricetin bind PI3K in remarkably different orientations that are related to each other by 180 degrees rotations. Staurosporine/PI3K interactions are reminiscent of low-affinity protein kinase/staurosporine complexes. These results provide a rich basis for development of isoform-specific PI3K inhibitors with therapeutic potential.

Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase-, rho-, and rac-dependent signaling pathways.

Leptin plays a key role regulating food intake, body weight and fat mass. These critical parameters are associated with an increased risk for digestive and mammary gland cancer in the Western population. Here we determined whether leptin contributes to the invasive phenotype of colonic and kidney epithelial cells at various stages of the neoplastic progression. First, leptin potently (EC50 = 10-30 ng/ml) induces invasion of collagen gels by premalignant familial adenomatous colonic cells PC/AA/C1 and nontumorigenic MDCK kidney epithelial cells, their src-transformed counterparts, and the human adenocarcinoma colonic cells LoVo and HCT-8/S11. Leptin and its Ob-Rb receptors were consistently identified by RT-PCR and immunoblotting in these cell lines, as well as in human colonic epithelial crypts, polyps, colonic tumor resections, and adjacent mucosa. Leptin-induced invasion was effectively blocked by pharmacological inhibitors of several downstream signaling pathways involved in cell transformation, namely, JAK2 tyrosine kinase (AG490), phosphoinositide PI3'-kinase (wortmannin and LY294002), mTOR kinase (rapamycin), and protein kinases C (GF109203X, Gö6976). Accordingly, leptin induces transient elevation of the PI3'-kinase lipid products in JAK2 immunoprecipitates prepared from parental MDCK cells. The leptin effect on invasion was potentiated by the activated form of the small GTPase RhoA and was abrogated by dominant negative mutants of RhoA, Rac1, and the p110alpha of PI3'-K. Our data indicate that leptin may exert a local and beneficial effect on migration of normal colonic epithelial cells and reparation of the inflamed or wounded digestive mucosa. We also emphasize a new role for leptin, linking the nutritional and body fat status to digestive cancer susceptibility by stimulating the invasive capacity of colonic epithelial cells at early stages of neoplasia. This finding has potential clinical implications for colon cancer progression and management of obesity.

Analysis of the murine phosphoinositide 3-kinase gamma gene.

Phosphoinositide 3-kinase gamma is preferentially expressed in leukocytes. PI3Kgamma is activated by betagamma subunits of heterotrimeric G-proteins, which thus link seven transmembrane helix receptor activation to phosphatidylinositol (3,4,5)-trisphosphate production. Here we describe the molecular cloning of the murine PI3Kgamma cDNA, the PI3Kgamma gene structure, its chromosomal assignment and the analysis of promoter activity. The mouse cDNA shares 86% identity to its pig and human orthologues at the nucleotide level. The MmPI3Kgamma gene spans approximately 30kb and comprises 11 exons. RACE-PCR indicated the presence of multiple start sites generating 5' UTRs with different lengths, the longest being 874bp. The putative promoter region contains no TATA box but several putative binding sites for hematopoietic specific transcription factors. A 1200bp long sequence upstream the first transcription start site was found to possess tissue specific promoter activity. Deletion constructs revealed two contiguous regions, with activator function, ranging from positions -139 to -557, and with inhibitory function, ranging from positions -557 to -892. FISH analysis revealed that the MmPI3Kgamma is located on chromosome 12 band B and that the human orthologue is positioned on chromosome 7q22.2-22.3. In spite of some differences in the ATP-binding site, recombinant murine PI3Kgamma protein is equally sensitive to wortmannin as its human counterpart. This suggests that mouse models will provide reliable results in the assessments of novel PI3Kgamma inhibitors.

Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation.

Phosphoinositide 3-kinase (PI3K) activity is crucial for leukocyte function, but the roles of the four receptor-activated isoforms are unclear. Mice lacking heterotrimeric guanine nucleotide-binding protein (G protein)-coupled PI3Kgamma were viable and had fully differentiated neutrophils and macrophages. Chemoattractant-stimulated PI3Kgamma-/- neutrophils did not produce phosphatidylinositol 3,4,5-trisphosphate, did not activate protein kinase B, and displayed impaired respiratory burst and motility. Peritoneal PI3Kgamma-null macrophages showed a reduced migration toward a wide range of chemotactic stimuli and a severely defective accumulation in a septic peritonitis model. These results demonstrate that PI3Kgamma is a crucial signaling molecule required for macrophage accumulation in inflammation.

Structure and function of phosphoinositide 3-kinases.

Phosphoinositide kinases (PI3Ks) play an important role in mitogenic signaling and cell survival, cytoskeletal remodeling, metabolic control and vesicular trafficking. Here we summarize the structure-function relationships delineating the activation process of class I PI3Ks involving various domains of adapter subunits, Ras, and interacting proteins. The resulting product, PtdIns(3,4,5)P3, targets Akt/protein kinase B (PKB), Bruton's tyrosine kinase (Btk), phosphoinositide-dependent kinases (PDK), integrin-linked kinase (ILK), atypical protein kinases C (PKC), phospholipase Cgamma and more. Surface receptor-activated PI3Ks function in mammals, insects, nematodes and slime mold, but not yeast. While many members of the class II family have been identified and characterized biochemically, it is presently unknown how these C2-domain containing PI3Ks are activated, and which PI substrate they phosphorylate in vivo. PtdIns 3-P is produced by Vps34p/class III PI3Ks and operates via the PtdIns 3-P-binding proteins early endosomal antigen (EEA1), yeast Vac1p, Vps27p, Pip1p in lysosomal protein targeting. Besides the production of D3 phosphorylated lipids, PI3Ks have an intrinsic protein kinase activity. For trimeric GTP-binding protein-activated PI3Kgamma, protein kinase activity seems to be sufficient to trigger mitogen-activated protein kinase (MAPK). Recent disruption of PI3K genes in slime mold, Caenorhabditis elegans, Drosophila melanogaster and mice further underlines the importance of PI3K signaling systems and elucidates the role of PI3K signaling in multicellular organisms.

Microquantification of cellular and in vitro F-actin by rhodamine phalloidin fluorescence enhancement.

Based on the enhancement of rhodamine phalloidin fluorescence after its binding to actin filaments we have developed a technique to quantify F-actin, drastically (>> 100 times) reducing consumption of the expensive fluorescent dye and sample material in comparison to previous methods. Depolymerization of F-actin is prevented by utilizing short incubation times and stabilization of the filaments by actin-binding proteins or formaldehyde. Equilibrium and kinetic mathematical models relating rhodamine fluorescence with F-actin concentrations were used to predict the optimal assay conditions. The method has been applied to measure relative and absolute F-actin concentrations in cytosolic fractions and stimulus-induced actin polymerization in neutrophils. The cells were lysed with octy1-beta-D-glucopyranoside, which is compatible with the assay due to its high critical micelle concentration. As the assay takes less than 1 h and eliminates all previously required washing or extraction steps, it is faster and much simpler than any other presented up to now for quantification of filamentous actin. Moreover, the method is unique for reliable and easy F-actin measurements in cell-free systems.

Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK.

Phosphoinositide 3-kinases (PI3Ks) activate protein kinase PKB (also termed Akt), and PI3Kgamma activated by heterotrimeric guanosine triphosphate-binding protein can stimulate mitogen-activated protein kinase (MAPK). Exchange of a putative lipid substrate-binding site generated PI3Kgamma proteins with altered or aborted lipid but retained protein kinase activity. Transiently expressed, PI3Kgamma hybrids exhibited wortmannin-sensitive activation of MAPK, whereas a catalytically inactive PI3Kgamma did not. Membrane-targeted PI3Kgamma constitutively produced phosphatidylinositol 3,4, 3,4,5-trisphosphate and activated PKB but not MAPK. Moreover, stimulation of MAPK in response to lysophosphatidic acid was blocked by catalytically inactive PI3Kgamma but not by hybrid PI3Kgammas. Thus, two major signals emerge from PI3Kgamma: phosphoinositides that target PKB and protein phosphorylation that activates MAPK.

The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic.

Bailie et al. [In Vitro Cell Dev. Biol. (1992) 28A, 621-624] reported that primary cultures of rat hepatocytes possess low affinity binding sites for nerve growth factor (NGF). NGF treatment of primary cultures of rat hepatocytes with a maximally effective concentration of NGF (20 ng/ml, 0.8 nM) caused acute phasic activation of Raf-1 and p42(MAPkinase), and a smaller sustained activation of B-Raf. The transient increase in Raf-1 and p42(MAPkinase) activity returned to baseline within approximately 30 min. NGF treatment of hepatocytes did not induce expression of cyclin dependent kinase (cdk) inhibitor proteins, but instead stimulated cdk2 activity and increased [3H]thymidine incorporation into DNA. In contrast to hepatocytes, NGF treatment of PC12 pheochromocytoma cells caused large sustained activations of B-Raf and p42(MAPkinase), and a lower phasic activation of Raf-1. The sustained activations of B-Raf and p42(MAPkinase) were for more than 5 h. Treatment of PC12 cells with NGF increased p21(Cip1/WAF-1) expression, reduced cdk2 activity and inhibited DNA synthesis, the opposite to the effects of NGF treatment of hepatocytes. However when p42(MAPkinase) was chronically activated in hepatocytes, via infection with an inducible oestrogen receptor-Raf-1 fusion protein, expression of p21(Cip-1/WAF1) and p16(INK4a) cdk inhibitor proteins increased, cdk2 activity decreased, and DNA synthesis decreased. Equally, treatment of hepatocytes with 50 mM ethanol elevated the basal activity of p42(MAPkinase) and temporally extended the ability of NGF treatment to activate p42(MAPkinase). Ethanol and NGF co-treatment increased expression of p21(Cip-1/WAF1) and p16(INK4a) cdk inhibitor proteins and decreased hepatocyte DNA synthesis. These data demonstrate that NGF can cause either acute/phasic or sustained activation of the MAP kinase cascade in different cell types. Acute activation of the MAP kinase cascade correlated with increased DNA synthesis. In contrast, sustained activation of the MAP kinase cascade correlated with increased expression of cdk inhibitor proteins, a reduction in cdk activity, and an inhibition of DNA synthesis. These data suggest a general mechanism exists where acute activation of the MAP kinase cascade promotes G1 progression/S phase entry and that chronic activation of the MAP kinase cascade inhibits this process.

GTPgammaS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor.

In a cell-free system from neutrophil cytosol GTP(&ggr ;)S can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTP(&ggr ;)S stimulation was susceptible to an excess of GDP, but not Bordetella pertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPgammaS.Gialpha2 or Gbeta1gamma2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPgammaS. When recombinant, constitutively activated and GTPgammaS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations >100 times the endogenous, however, they were ineffective. Purified Cdc42/Rac-interactive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPgammaS. The action of GTPgammaS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B-sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPgammaS under physiologic conditions. The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPgammaS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPgammaS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.

The Ras/Rac1/Cdc42/SEK/JNK/c-Jun cascade is a key pathway by which agonists stimulate DNA synthesis in primary cultures of rat hepatocytes.

The ability of signaling via the JNK (c-Jun NH2-terminal kinase)/stress-activated protein kinase cascade to stimulate or inhibit DNA synthesis in primary cultures of adult rat hepatocytes was examined. Treatment of hepatocytes with media containing hyperosmotic glucose (75 mM final), tumor necrosis factor alpha (TNFalpha, 1 ng/ml final), and hepatocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Glucose, TNFalpha, or HGF treatments increased phosphorylation of c-Jun at serine 63 in the transactivation domain and stimulated hepatocyte DNA synthesis. Infection of hepatocytes with poly-L-lysine-coated adenoviruses coupled to constructs to express either dominant negatives Ras N17, Rac1 (N17), Cdc42 (N17), SEK1-, or JNK1- blunted the abilities of glucose, TNFalpha, or HGF to increase JNK1 activity, to increase phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesis. Furthermore, infection of hepatocytes by a recombinant adenovirus expressing a dominant-negative c-Jun mutant (TAM67) also blunted the abilities of glucose, TNFalpha, and HGF to stimulate DNA synthesis. These data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pathway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (GSK3) activity and increased c-Jun DNA binding. Co-infection of hepatocytes with recombinant adenoviruses to express dominant- negative forms of PI3 kinase (p110alpha/p110gamma) increased basal GSK3 activity, blocked the abilities of glucose and HGF treatments to inhibit GSK3 activity, and reduced basal c-Jun DNA binding. However, expression of dominant-negative PI3 kinase (p110alpha/p110gamma) neither significantly blunted the abilities of glucose and HGF treatments to increase c-Jun DNA binding, nor inhibited the ability of these agonists to stimulate DNA synthesis. These data suggest that signaling by the JNK/stress-activated protein kinase cascade, rather than by the PI3 kinase cascade, plays the pivotal role in the ability of agonists to stimulate DNA synthesis in primary cultures of rat hepatocytes.