Chromosomal instability at a mutational hotspot in polyoma middle T-antigen affects its ability to activate the ARF-p53 tumor suppressor pathway.

We have isolated spontaneous mutants of polyoma virus middle T-antigen (PyMT) that do not activate the ARF-p53 pathway based on their inability to block REF52 cell division. The REF52 cells containing these mutants have a flat untransformed morphological phenotype and do not express the ARF protein. The PyMT mutations in the different cell isolates so far analysed occur at a mutational hotspot in the PyMT sequence between nucleotides 1241 and 1249, which contains nine consecutive cytosines. In one set of mutants a single cytosine was deleted, while in another mutant set an additional cytosine was inserted. Both these mutations result in frameshifts, generating altered PyMT proteins containing amino-acid sequences derived from each of the two other alternative reading frames of the polyoma virus early region. Both types of mutations result in the loss of the C-terminal PyMT region containing the membrane-binding hydrophobic region and result is mislocalization of the PyMT mutant proteins. Revertant wild-type PyMT (containing nine cytosines) was easily detected in transformants generated after infection of REF52 cells expressing high amounts of dominant negative p53 with retroviruses containing either mutation. We demonstrate that wild-type PyMT revertants are derived from mutations in the hotspot sequence of the integrated mutant PyMT sequences.

Cancer targets in the Ras pathway.

Ras proteins play a direct causal role in human cancer and in other diseases. Mutant H-Ras, N-Ras, and K-Ras occur in varying frequencies in different tumor types, for reasons that are not known. Other members of the Ras superfamily may also contribute to cancer. Mutations also occur in downstream pathways, notably B-Raf, PTEN, and PI 3' kinase: These pathways interact at multiple points, including cyclin D1, and act synergistically. In some cases mutations in Ras and effectors are mutually exclusive; in other cases, they coexist. Drugs blocking elements of the pathway are in different stages of clinical development. One of these, the Raf kinase/VEGF-R2 inhibitor Sorafenib, has already been approved for treatment of renal cancer and is being tested in other indications. However, therapeutic targets in the Ras pathway have not yet been fully validated as bona fide targets.

Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway.

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf-MEK-extracellular signal-regulated kinase pathway in epithelial oncogenesis.

H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes.

The mechanisms underlying downregulation of the cadherin/catenin complexes and beta-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and beta-catenin distribution in epidermal keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and alpha-catenin and relocalization of beta-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong association of p85alpha and p110alpha subunits of PI3K with beta-catenin is induced in V12Ras-expressing keratinocytes, and in vitro binding assays show a direct interaction between beta-catenin and p85alpha. Overexpression of either V12Ras or constitutively active p110alpha induces metabolic stabilization of beta-catenin and promotes its accumulation in cytoplasmic and nuclear pools. In addition, the interaction of beta-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110alpha transformants though no changes in glycogen synthase kinase 3 beta activity could be detected. Nevertheless, in V12Ras transformants the in vivo phosphorylation of beta-catenin in Ser residues is strongly decreased. These results indicate that H-Ras activation induces the relocalization and cytoplasmic stabilization of beta-catenin by a mechanism involving its interaction with PI3K.

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase.

p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85alpha-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

Interaction of Ras with phosphoinositide 3-kinase gamma.

Phosphoinositide 3-kinase gamma (PI3Kgamma) can be activated in vitro by both alpha and betagamma subunits of heterotrimeric G-proteins and does not interact with p85, the regulatory subunit of PI3Kalpha. Here we demonstrate the binding of Ras to PI3Kgamma in vitro. An N-terminal region of PI3Kgamma was identified as a binding site for Ras. After co-expression with PI3Kgamma in COS-7 cells, Ras induced only a modest increase in PI3K activity compared with the stimulation of PI3Kalpha by Ras in the same cells.

Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by 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.

Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.

Upon detachment from the extracellular matrix, epithelial cells enter into programmed cell death, a phenomenon known as anoikis, ensuring that they are unable to survive in an inappropriate location. Activated ras oncogenes protect cells from this form of apoptosis. The nature of the survival signals activated by integrin engagement and usurped by oncogenic Ras are unknown: here we show that in both cases phosphoinositide 3-OH kinase (PI 3-kinase), but not Raf, mediates this protection, acting through protein kinase B/Akt (PKB/Akt). Constitutively activated PI 3-kinase or PKB/Akt block anoikis, while inhibition of PI 3-kinase abrogates protection by Ras, but not PKB/Akt. Inhibition of either PI 3-kinase or PKB/Akt induces apoptosis in adherent epithelial cells. Attachment of cells to matrix leads to rapid elevation of the levels of PI 3-kinase lipid products and PKB/Akt activity, both of which remain high in Ras-transformed cells even in suspension. PI 3-kinase acting through PKB/Akt is therefore implicated as a key mediator of the aberrant survival of Ras-transformed epithelial cells in the absence of attachment, and mediates matrix-induced survival of normal epithelial cells.

Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB.

The viability of vertebrate cells depends on survival factors which activate signal transduction pathways that suppress apoptosis. Defects in anti-apoptotic signalling pathways are implicated in many pathologies including cancer, in which apoptosis induced by deregulated oncogenes must be forestalled for a tumour to become established. Phosphatidylinositol-3-kinase (PI(3)K) is involved in the intracellular signal transduction of many receptors and has been implicated in the transduction of survival signals in neuronal cells. We therefore examined the role of PI(3)K, its upstream effector Ras, and its putative downstream protein kinase effectors PKB/Akt and p70S6K (ref. 5) in the modulation of apoptosis induced in fibroblasts by the oncoprotein c-Myc. Here we show that Ras activation of PI(3)K suppresses c-Myc-induced apoptosis through the activation of PKB/Akt but not p70S6K. However, we also found that Ras is an effective promoter of apoptosis, through the Raf pathway. Thus Ras activates contradictory intracellular pathways that modulate cell viability. Induction of apoptosis by Ras may be an important factor in limiting the expansion of somatic cells that sustain oncogenic ras mutations.

R-Ras can activate the phosphoinositide 3-kinase but not the MAP kinase arm of the Ras effector pathways.

BACKGROUND: The small GTPase R-Ras displays a less potent transforming activity than the closely related Ras oncogene products. Although R-Ras has been reported to interact with c-Raf1 and Ral-GDS in vitro, the pathways by which it exerts its effects on cellular proliferation are not known. RESULTS: Both Ras and R-Ras interact with phosphoinositide (PI) 3-kinase in vitro, and induce elevation of the levels of PI 3-kinase lipid products in intact cells. Unlike Ras, R-Ras does not activate Raf or mitogen-activated protein (MAP) kinase in cells. In co-transfection assays, the serine/threonine protein kinase PKB (or Akt) is effectively stimulated by R-Ras, Ras, mutants of Ras that activate PI 3-kinase but not other effectors, and activated forms of PI 3-kinase. Ras and R-Ras stimulate PKB/Akt through a non-autocrine mechanism that involves PI 3-kinase. The constitutive activation of PI 3-kinase alone is sufficient to activate PKB/Akt, but not the MAP kinase ERK or the stress-activated protein kinase, Jun N-terminal kinase. Transformation assays in fibroblasts suggest that PKB/Akt and Raf are part of distinct oncogenic signalling pathways. CONCLUSIONS: Both the Raf-MAP kinase and PI 3-kinase-PKB/Akt pathways are activated by Ras, but only the PI 3-kinase-PKB/Akt pathway is activated by R-Ras. PI 3-kinase, and downstream targets such as PKB/Akt, are likely to be essential mediators of transformation induced by R-Ras. PI 3-kinase, as well as Raf, is thus implicated also in Ras transformation.

Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation.

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.

Phosphatidylinositol 3' kinase: one of the effectors of Ras.

Ras proteins are proto-oncogene products that are critical components of signalling pathways leading from cell surface receptors to control of cellular proliferation, morphology and differentiation. the ability of Ras to activate the MAP kinase pathway through interaction with the serine/threonine kinase Raf is now well established. However, recent work has shown that Ras can also interact directly with the catalytic subunit of phosphatidylinositol 3' kinase and is involved in control of the lipid kinase in intact cells. A model is presented in which both tyrosine phosphoprotein interaction with the regulatory p85 subunit and Ras. GTP interaction with the catalytic p110 subunit is required to achieve optimal activation of phosphatidylinositol 3'kinase in response to extracellular stimuli. The ability of Ras to regulate phosphatidylinositol 3' kinase may be important both in Ras control of cellular morphology through the actin cytoskeleton and also in Ras control of DNA synthesis.

The activation of phosphatidylinositol 3-kinase by Ras.

BACKGROUND: Activation of the mammalian phosphatidylinositol 3-kinase complex can play a critical role in transducing growth factor responses. The lipid kinase complex, which is made up of p85 alpha and p110 alpha regulatory and catalytic subunits, becomes associated with a number of activated receptor protein tyrosine kinases, but the mechanism of its activation has not yet been defined. Recent evidence indicates that Ras can bind to the p85 alpha/p110 alpha complex. We describe here the functional regulation of the mammalian phosphatidylinositol 3-kinase complex by Ras. RESULTS: Expression of p110 alpha, the catalytic subunit of phosphatidylinositol 3-kinase, in the fission yeast, Schizosaccharomyces pombe, has been used to demonstrate an inhibitory effect of p85 alpha on p110 alpha activity in intact cells; inhibition did not result from a decrease in p110 alpha expression. In this cellular context, we have investigated the effect of a constitutively active mutant of Ras, v-Ras, either on p85 alpha or p110 alpha-alone, or on the p85 alpha/p110 alpha complex. In the presence of the p85 alpha/p110 alpha complex, v-Ras suppressed cell growth, but an effector-domain mutant of v-Ras did not. The growth-suppressive effect of v-Ras was not seen for any other combination of expressed proteins. The phenotype induced by v-Ras was consistent with activation of the p85 alpha/p110 alpha complex: it was sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin, and the cells accumulated 3-phosphorylated polyphosphoinositides. Activation of purified p85 alpha/p110 alpha by purified recombinant Ras in vitro was also demonstrated. CONCLUSIONS: The phosphatidylinositol 3-kinase complex, p85 alpha/p110 alpha, shows a suppressed catalytic function in vivo when compared with free p110 alpha. This complex can, however, be activated by Ras. We suggest that the phosphatidylinositol 3-kinase p85 alpha/p110 alpha complex is a downstream effector of Ras.

Phosphatidylinositol-3-OH kinase as a direct target of Ras.

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.

A complex of Grb2 adaptor protein, Sos exchange factor, and a 36-kDa membrane-bound tyrosine phosphoprotein is implicated in ras activation in T cells.

T lymphocytes contain both Grb2, an SH2 and SH3 domain containing adaptor protein, and Sos, a guanine nucleotide exchange factor for Ras. Immunoprecipitates of Sos from the lysates of T cells contain a 36-kDa protein which is phosphorylated on tyrosine residues in response to T cell receptor/CD3 cross-linking. In vitro studies using different bacterially synthesized GST-Sos fusion proteins confirm the formation of complexes containing p36 and the proline-rich COOH-terminal domain of Sos. The use of mutant GST-Grb2 proteins in which both SH3 domains have been mutationally inactivated shows that Grb2 binds to tyrosine phosphorylated p36 via its SH2 domain. In Jurkat cells phosphorylated p36 is localized exclusively in the particulate fraction. In addition, another SH2 domain-containing protein, p52Shc is tyrosine phosphorylated upon TCR.CD3 cross-linking and associates with a 150-kDa phosphotyrosine containing protein. Taken together these data suggest that activation of Ras in T cells via the TCR.CD3 complex might be controlled, at least in part, by mechanisms similar to those found in fibroblasts, involving in this case formation of a complex of Grb2, Sos, and a membrane-bound tyrosine phosphoprotein of molecular mass 36-kDa.