Ras (p21ras) interacts directly with the catalytic subunit of phosphatidylinositol-3-OH kinase in a GTP-dependent manner through the Ras effector site. In vivo, dominant negative Ras mutant N17 inhibits growth factor induced production of 3' phosphorylated phosphoinositides in PC12 cells, and transfection of Ras, but not Raf, into COS cells results in a large elevation in the level of these lipids. Therefore Ras can probably regulate phosphatidylinositol-3-OH kinase, providing a point of divergence in signalling pathways downstream of Ras.

The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.

External signals that control the activity of proteins encoded by the ras proto-oncogenes have not previously been characterized. It is now shown that stimulation of the antigen receptor of T lymphocytes causes a rapid activation of p21ras. The mechanism seems to involve a decrease in the activity of GAP, the GTPase-activating protein, on stimulation of protein kinase C. In lymphocytes, p21ras may therefore be an important mediator of the action of protein kinase C.

Ras proteins signal through direct interaction with a number of effector enzymes, including type I phosphoinositide (PI) 3-kinases. Although the ability of Ras to control PI 3-kinase has been well established in manipulated cell culture models, evidence for a role of the interaction of endogenous Ras with PI 3-kinase in normal and malignant cell growth in vivo has been lacking. Here we generate mice with mutations in the Pi3kca gene encoding the catalytic p110alpha isoform that block its interaction with Ras. Cells from these mice show proliferative defects and selective disruption of signaling from growth factors to PI 3-kinase. The mice display defective development of the lymphatic vasculature, resulting in perinatal appearance of chylous ascites. Most importantly, they are highly resistant to endogenous Ras oncogene-induced tumorigenesis. The interaction of Ras with p110alpha is thus required in vivo for certain normal growth factor signaling and for Ras-driven tumor formation.

We have reported previously that Ras interacts with the catalytic subunit of phosphoinositide 3‐kinase (PI 3‐kinase) in a GTP‐dependent manner. The affinity of the interaction of Ras‐GTP with p85alpha/p110alpha is shown here to be approximately 150 nM. The site of interaction on the p110alpha and beta isoforms of PI 3‐kinase lies between amino acid residues 133 and 314. A point mutation in this region, K227E, blocks the GTP‐dependent interaction of PI 3‐kinase p110alpha with Ras in vitro and the ability of Ras to activate PI 3‐kinase in intact cells. In addition, this mutation elevates the basal activity of PI 3‐kinase in intact cells, suggesting a direct influence of the Ras binding site on the catalytic activity of PI 3‐kinase. Using an in vitro reconstitution assay, it is shown that the interaction of Ras‐GTP, but not Ras‐GDP, with PI 3‐kinase leads to an increase in its enzymatic activity. This stimulation is synergistic with the effect of tyrosine phosphopeptide binding to p85, particularly at suboptimal peptide concentrations. These data show that PI 3‐kinase is regulated by a number of mechanisms, and that Ras contributes to the activation of this lipid kinase synergistically with tyrosine kinases.

The Ras proteins are key regulators of the growth of eukaryotic cells, but their direct target enzymes, or 'effectors', are unknown. The protein encoded by the c-raf-1 proto-oncogene is thought to function downstream of p21ras because disruption of Raf blocks signalling by Ras in a number of systems. Here we report that the amino-terminal cysteine-rich regulatory region of p74c-raf-1 expressed as a glutathione-S-transferase (GST) fusion protein binds directly to Ras with relatively high affinity (50 nM). The binding is strictly dependent on the Ras protein being in the active GTP-bound conformation rather than the inactive GDP-bound state. Raf-GST interacts with wild-type and oncogenic Ras (Val 12) but fails to interact with a biologically inert effector mutant of Ras (Ala 38) and a dominant negative mutant (Asn 17). A peptide based on the effector region of Ras inhibits the interaction. Raf-GST acts as a potent competitive inhibitor of the GTPase-activating proteins p120GAP and neurofibromin. In addition, Raf itself displays weak GTPase-stimulating activity towards Ras. It is therefore likely that Raf is a direct effector of Ras.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been implicated in signal transduction through tyrosine kinase-and heterotrimeric G-proteinlinked receptors. We report herein the cloning and characterization of p110␦, a novel class I PI3K. Like p110␣ and p110␤, other class I PI3Ks, p110␦ displays a broad phosphoinositide lipid substrate specificity and interacts with SH2͞SH3 domaincontaining p85 adaptor proteins and with GTP-bound Ras. In contrast to the widely distributed p110␣ and ␤, p110␦ is exclusively found in leukocytes. In these cells, p110␣ and ␦ both associate with the p85␣ and ␤ adaptor subunits and are similarly recruited to activated signaling complexes after treatment with the cytokines interleukin 3 and 4 and stem cell factor. Thus, these class I PI3Ks appear not to be distinguishable at the level of p85 adaptor selection or recruitment to activated receptor complexes. However, distinct biochemical and structural features of p110␦ suggest divergent functional͞regulatory capacities for this PI3K. Unlike p110␣, p110␦ does not phosphorylate p85 but instead harbors an intrinsic autophosphorylation capacity. In addition, the p110␦ catalytic domain contains unique potential proteinprotein interaction modules such as a Pro-rich region and a basic-region leucine-zipper (bZIP)-like domain. Possible selective functions of p110␦ in white blood cells are discussed.Phosphoinositide 3-kinases (PI3Ks) phosphorylate the 3Ј OH position of the inositol ring of inositol lipids, generating phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. PI3K enzymes have been identified in plants, slime molds, yeast, fruit flies, and mammals (1) and play a role in signal transduction via tyrosine kinase-and G-protein-linked receptors (2-5). In addition, PI3Ks have a function in membrane trafficking events, either constitutive or induced upon receptor stimulation (for review, see ref. 6).

Cytotoxic drug resistance is a major cause of cancer treatment failure. We report an RNA interference screen to identify genes influencing sensitivity of different cancer cell types to chemotherapeutic agents. A set of genes whose targeting leads to resistance to paclitaxel is identified, many of which are involved in the spindle assembly checkpoint. Silencing these genes attenuates paclitaxel-induced mitotic arrest and induces polyploidy in the absence of drug. We also identify a ceramide transport protein, COL4A3BP or CERT, whose downregulation sensitizes cancer cells to multiple cytotoxic agents, potentiating endoplasmic reticulum stress. COL4A3BP expression is increased in drug-resistant cell lines and in residual tumor following paclitaxel treatment of ovarian cancer, suggesting that it could be a target for chemotherapy-resistant cancers.