Biomedical subjects

Adrienne D Cox

Publications and source records attributed to Adrienne D Cox.

25 records · Page 2Linked to original sources

PubMed2003

Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1.

Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. We have shown recently that Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane but the mechanism of compartmentalized signalling was not determined. Here we show that, in response to Src-dependent activation of phospholipase Cgamma1, the Ras guanine nucleotide exchange factor RasGRP1 translocated to the Golgi where it activated Ras. Whereas Ca(2+) positively regulated Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, was limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induced differentiation of PC12 cells, transformed fibroblasts and mediated radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane.

Animals↗

PubMed2002

Ras mediates radioresistance through both phosphatidylinositol 3-kinase-dependent and Raf-dependent but mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-independent signaling pathways.

Cells transformed by the oncogenic small GTPase, Ras, display a radioresistant phenotype in response to ionizing radiation (IR). To determine the mechanisms by which Ras mediates radioresistance in epithelial cells, we assessed the importance of three major survival pathways that can be activated by Ras [phosphatidylinositol 3-kinase (PI3-K)>Akt, nuclear factor kappaB (NF-kappaB), and Raf>mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)>extracellular signal-regulated kinase] as necessary or sufficient for Ras-mediated radioresistance in matched pairs of RIE-1 rat intestinal epithelial cells expressing oncogenic Ras or empty vector (RIE-Ras and RIE-vector). Inhibiting PI3-K with LY294002 sensitized RIE-1 cells to IR in a dose-dependent manner, indicating that PI3-K is necessary for radioresistance, whereas inhibition of NF-kappaB with the super-repressor IkappaBalpha had little effect on survival. Expression of either the constitutively active catalytic subunit of PI3-K, p110alpha-CAAX, or the Ras effector domain mutant 12V/40C, which retains binding to PI3-K but is impaired in binding to other Ras effectors, was sufficient to confer partial radioresistance. Expression of either a constitutively active form of the serine/threonine kinase Raf-1 or the Ras effector domain mutant 12V/35S, which retains binding to Raf but is impaired in binding to other Ras effectors, was also sufficient to confer partial radioresistance. Surprisingly, however, even complete inhibition of MEK activity by using U0126 resulted in no change in post-IR survival whatsoever. Thus, whereas Raf contributes to Ras-mediated radioresistance, this is accomplished through a MEK-independent pathway. Taken together, these results indicate that multiple pathways, including both PI3-K-dependent and Raf-dependent but MEK-independent signaling, are required for Ras-mediated radioresistance in epithelial cells. Finally, we demonstrate that Ras-mediated radioresistance can be uncoupled from Ras-mediated transformation, in that PI3-K is required for radioresistance but not transformation, whereas MEK and NF-kappaB are required for transformation but not radioresistance in RIE-1 epithelial cells.

Animals↗

PubMed2002

R-Ras C-terminal sequences are sufficient to confer R-Ras specificity to H-Ras.

Activated versions of the similar GTPases, H-Ras and R-Ras, have differing effects on biological phenotypes: Activated H-Ras strongly transforms many fibroblast cell lines causing dramatic changes in cell shape and cytoskeletal organization. In contrast, R-Ras transforms fewer cell lines and the transformed cells display only some of the morphological changes associated with H-Ras transformation. H-Ras cells can survive in the absence of serum whereas R-Ras cells seem to die by an apoptotic-like mechanism in response to removal of serum. H-Ras can suppress integrin activation and R-Ras specifically antagonizes this effect. To map sequences responsible for these differences we have generated and investigated a panel of H-Ras and R-Ras chimeras. We found that the C-terminal 53 amino acids of R-Ras were necessary and sufficient to specify the contrasting biological properties of R-Ras with respect to focus morphology, reactive oxygen species (ROS) production and reversal of H-Ras-induced integrin suppression. Surprisingly, we found chimeras in which the focus formation and integrin-mediated phenotypes were separated, suggesting that different effectors could be involved in mediating these responses. An integrin profile of H-Ras and R-Ras cell pools showed no significant differences; both activated H-Ras and R-Ras expressing cells were found to have reduced beta(1) activity, suggesting that the activity state of the beta(1) subunit is not sufficient to direct an H-Ras transformed cell morphology.

3T3 Cells↗

PubMed2002

A distinct class of dominant negative Ras mutants: cytosolic GTP-bound Ras effector domain mutants that inhibit Ras signaling and transformation and enhance cell adhesion.

Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.

3T3 Cells↗

PubMed2002

Farnesyltransferase inhibitors: promises and realities.

Farnesyltransferase inhibitors have recently shown clinical efficacy against leukemias, gliomas and even non-small-cell lung cancers, especially when administered in combination with taxanes. It is possible that the critical target downstream of farnesyltransferase responsible for these effects is not either Ras or RhoB, the two most cited possibilities - but the hunt is on.

Alkyl and Aryl Transferases↗

PubMed2002

Ras signalling on the endoplasmic reticulum and the Golgi.

Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane.

3T3 Cells↗

PubMedDate not supplied

Ras family signaling: therapeutic targeting.

Mutationally activated and oncogenic versions of the ras genes were first identified in human tumors in 1982. This discovery prompted great interest in the development of anti-Ras strategies as novel, target-based approaches for cancer treatment. The three human ras genes represent the most frequently mutated oncogenes in human cancers. Consequently, a considerable research effort has been made to define the function of Ras in normal and neoplastic cells and to target Ras for cancer treatment. Among the anti-Ras strategies that are under evaluation in the clinic are pharmacologic inhibitors designed to prevent: (1) association with the plasma membrane (farnesyltransferase inhibitors), (2) downstream signaling (Raf and MEK protein kinase inhibitors), (3) autocrine growth factor signaling (EGF receptor inhibitors), or (4) gene expression (H-ras and c-raf-1). Although a number of these inhibitors have demonstrated potent anti-tumor activities in preclinical models, phase l-lll clinical trials have revealed unexpected complexities in Ras function and in the clinical development of target-based therapies. We review the current status of anti-Ras drug development, issues that have complicated their progression to the clinic, and possible future strategies for targeting Ras.

Alkyl and Aryl Transferases↗