Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P(3), the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gbetagammas) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gbetagammas, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)Kgamma, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P(3), activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)Kgamma to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gbetagammas, p101 and the Ras-RBD interaction all have important roles in the regulation of PI(3)Kgamma in vivo and that they can simultaneously, but differentially, control distinct PI(3)Kgamma effectors.

A variety of genetic and inhibitor studies have shown that phosphoinositide 3-kinase gamma (PI3Kgamma) plays an essential role in a number of physiological responses, including neutrophil chemotaxis, mast cell degranulation, and cardiac function []. PI3Kgamma is currently thought to be composed of a p110gamma catalytic subunit and a single regulatory subunit, p101. The binding of p110gamma to p101 dramatically increases the activation of the complex by Gbetagamma subunits and, hence, is thought to be critical for the coupling of PI3Kgamma to G protein coupled receptors []. Here, we characterize a new regulatory subunit for PI3Kgamma. p84 is present in human, mouse, chicken, frog, and fugu genomes and is located beside the p101 locus. It is broadly expressed in cells of the murine immune system. Both recombinant and endogenous p84 bind p110gamma specifically and with high affinity. Binding of p84 to p110gamma substantially increases the ability of Gbetagamma to stimulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) production both in vitro and in vivo. However, the p84/p110gamma heterodimer is approximately 4-fold less sensitive to Gbetagammas than p101/p110gamma. Endogenous murine p84 expression is substantially reduced in the absence of p110gamma expression. We conclude that p110gamma has two potential regulatory subunits in vivo, p84 and p101.

PI3Ks play important roles in the signaling pathways used by a wide variety of cell surface receptors on neutrophils. Class IB PI3K plays a major role in the initial generation of PtdIns(3,4,5)P₃ by Gi-coupled G-protein coupled receptors (GPCRs) (e.g., receptors for fMLP, C5a, LTB₄). Class IA PI3Ks generate PtdIns(3,4,5)P₃ downstream of receptors which directly or indirectly couple to protein tyrosine kinases such as integrins, FcγRs, cytokine receptors, and GPCRs. The PtdIns(3,4,5)P₃ made by Class I PI3Ks regulates the activity of several different effector proteins, many of which are plasma membrane GEFs or GAPs for small GTPases. Class III PI3K generates PtdIns(3)P in the phagosome membrane and plays an important role in efficient assembly of the NADPH oxidase at this location. Much still remains to be discovered about the molecular details that govern activation of PI3Ks and the mechanisms by which these enzymes regulate complex cellular processes, such as neutrophil spreading, chemotaxis, phagocytosis, and killing of pathogens. However, it is clear from recent use of transgenic mouse models and isoform-selective PI3K inhibitors that these pathways are important in regulating neutrophil recruitment to sites of infection and damage in vivo. Thus, PI3K pathways may present novel opportunities for selective inhibition in some inflammatory pathologies.

Our data suggest that Ras activate PI3Kgamma at the level of the membrane, by allosteric modulation and/or reorientation of the PI3Kgamma, implying that Ras can activate PI3Kgamma without its membrane translocation. This view is supported by structural work that has suggested binding of Ras to PI3Kgamma results in a change in the structure of the catalytic pocket.

The molecular mechanisms by which receptors regulate the Ras Binding Domains of the PIP 3 -generating, class I PI3Ks remain poorly understood, despite their importance in a range of biological settings, including tumorigenesis, activation of neutrophils by pro-inflammatory mediators, chemotaxis of Dictyostelium and cell growth in Drosophila. We provide evidence that G protein-coupled receptors (GPCRs) can stimulate PLCb2/b3 and diacylglycerol-dependent activation of the RasGEF, RasGRP4 in neutrophils. The genetic loss of RasGRP4 phenocopies knock-in of a Ras-insensitive version of PI3Kc in its effects on PI3Kc-dependent PIP 3 accumulation, PKB activation, chemokinesis and reactive oxygen species (ROS) formation. These results establish a new mechanism by which GPCRs can stimulate Ras, and the broadly important principle that PLCs can control activation of class I PI3Ks.

One of the major, progesterone-dependent proteins secreted into the uterine lumen of the mare is a 19-kDa lipocalin (P19). It associates strongly with the embryonic capsule that envelops the young horse conceptus in early gestation, suggesting that it may be involved in sustaining early development. However, it was not known whether the protein was transported through the capsule and/or trophoblast layer and into the yolk sac cavity. To address this question, polyclonal antisera were raised against a C-terminal peptide (based on the deduced amino acid sequence of P19) and a recombinant-derived P19 fusion protein. The antiserum raised against the C-terminal peptide recognized P19 on Western blots of denatured uterine secretions (subjected to SDS-PAGE), but it did not bind to the protein in tissue sections. However, the antiserum raised against the recombinant-derived fusion protein recognized P19 both on Western blots and in histological sections. Western blot analysis of tissues and fluids collected from early-pregnant mares demonstrated significant quantities of P19 in the endometrium and uterine secretions and in the embryonic capsule, the chorion, and the yolk sac fluid, showing that the protein is transferred through to the developing embryo. Concentrations of immunoreactive P19 declined during gestation so that, by Day 30, it had virtually disappeared from both maternal and fetal tissues and fluids. Immunohistochemical staining of endometrial biopsies collected during early pregnancy localized P19 to the glandular and luminal epithelia and to the lumina of the endometrial glands. The capsule and the trophoblast layer of the chorion from early (Days 16-17) horse conceptuses also stained positively with localization of P19 to the apical surface of the trophoblast cells. There was no detectable staining either in or on the embryonic disc. The presence of P19 in both the trophoblast layer and the yolk sac fluid suggests that P19 passes into the yolk sac fluid via trophoblast cells.

Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.