Faculty Bibliography

Signal transduction along the Ras/MAPK pathway has been generally thought to take place at the plasma membrane. It is now evident that the plasma membrane is not the only platform capable of Ras/MAPK signal induction. Fusion of Ras with green fluorescent protein and the development of genetically encoded fluorescent probes for Ras activation have revealed signaling events on a variety of intracellular membranes including endosomes, the Golgi apparatus and the endoplasmic reticulum. Thus, the Ras/MAPK pathway is spatially compartmentalized within cells and this may afford greater complexity of signal output

We previously characterized the G60A mutant of Ras and showed that the switch regions of the GTP-bound but not the GDP-bound form of this mutant adopt an 'open conformation' similar to that seen in nucleotide-free Ras. Here, we mutate Lys147 of the conserved (145)SAK(147) motif in the G60A background and characterize the resulting double mutant (DM). We show that RasDM is the first structure of a Ras protein with identical GDP- and GTP-bound structures. Both structures adopt the open conformation of the active form of RasG60A. The increase in the accessible surface area of the nucleotide is consistent with a 4-fold increase in its dissociation rate. Stopped-flow experiments show no major difference in the two-step kinetics of association of GDP or GTP with the wild type, G60A, or RasDM. Addition of Sos fails to accelerate nucleotide exchange. Overexpression of the G60A or double mutant of Ras in COS-1 cells fails to activate Erk and shows a strong dominant negative effect. Our data suggest that flexibility at position 60 is required for proper Sos-catalyzed nucleotide exchange and that structural information is somehow shared among the switch regions and the different nucleotide binding motifs

Hepatic fibrosis occurs during most chronic liver diseases and is driven by inflammatory responses to injured tissue. Because DCs are central to modulating liver immunity, we postulated that altered DC function contributes to immunologic changes in hepatic fibrosis and affects the pathologic inflammatory milieu within the fibrotic liver. Using mouse models, we determined the contribution of DCs to altered hepatic immunity in fibrosis and investigated the role of DCs in modulating the inflammatory environment within the fibrotic liver. We found that DC depletion completely abrogated the elevated levels of many inflammatory mediators that are produced in the fibrotic liver. DCs represented approximately 25% of the fibrotic hepatic leukocytes and showed an elevated CD11b+CD8- fraction, a lower B220+ plasmacytoid fraction, and increased expression of MHC II and CD40. Moreover, after liver injury, DCs gained a marked capacity to induce hepatic stellate cells, NK cells, and T cells to mediate inflammation, proliferation, and production of potent immune responses. The proinflammatory and immunogenic effects of fibrotic DCs were contingent on their production of TNF-alpha. Therefore, modulating DC function may be an attractive approach to experimental therapeutics in fibro-inflammatory liver disease

Ras proteins are small GTPases that play key roles in the regulation of several cellular processes such as growth, differentiation, and transformation. Although Ras signaling was thought to occur uniformly on the inner leaflet of the plasma membrane, a growing body of evidence indicates that Ras activation happens dynamically within defined plasma membrane microdomains and at other specific intracellular compartments, thus ensuring the generation of distinct signal outputs. Yet the mechanisms that control the spatiotemporal segregation of Ras proteins remain poorly characterized. We have recently shown that the differential modification of Ras proteins by ubiquitination is a crucial factor that controls Ras intracellular trafficking and signaling potential. To better understand the process of Ras ubiquitination, it is important to establish assays that not only provide information about the nature of the ubiquitin modification involved, but also enable the monitoring of the dynamics of this process. In this chapter, we will describe biochemical and biophysical methodologies, namely immunoprecipitation, nickel-chelate affinity chromatography, and bioluminescence resonance energy transfer (BRET), for monitoring the ubiquitination of Ras proteins. Although the description focuses on Ras, the assays described can in principle be applied to the study of a range of proteins of interest that may be subject to ubiquitination, and the use of the different methods in parallel should provide new insights into the nature and dynamics of protein ubiquitination

The ubiquitin ligase Siah2 has been shown to regulate prolyl hydroxylase 3 (PHD3) stability with concomitant effect on HIF-1alpha availability. Because HIF-1alpha is implicated in tumorigenesis and metastasis, we used SW1 mouse melanoma cells, which develop primary tumors with a propensity to metastasize, in a syngeneic mouse model to assess a possible role for Siah2 in these processes. Inhibiting Siah2 activity by expressing a peptide designed to outcompete association of Siah2-interacting proteins reduced metastasis through HIF-1alpha without affecting tumorigenesis. Conversely, inhibiting Siah2 activity by means of a dominant-negative Siah2 RING mutant primarily reduced tumorigenesis through the action of Sprouty 2, a negative regulator of Ras signaling. Consistent with our findings, reduced expression of PHD3 and Sprouty2 was observed in more advanced stages of melanoma tumors. Using complementary approaches, our data establish the role of Siah2 in tumorigenesis and metastasis by HIF-dependent and -independent mechanisms

The kinetics of Ras activation by Son of sevenless (SOS) changes profoundly when Ras is tethered to membranes, instead of being in solution. SOS has two binding sites for Ras, one of which is an allosteric site that is distal to the active site. The activity of the SOS catalytic unit (SOS(cat)) is up to 500-fold higher when Ras is on membranes compared to rates in solution, because the allosteric Ras site anchors SOS(cat) to the membrane. This effect is blocked by the N-terminal segment of SOS, which occludes the allosteric site. We show that SOS responds to the membrane density of Ras molecules, to their state of GTP loading and to the membrane concentration of phosphatidylinositol-4,5-bisphosphate (PIP2), and that the integration of these signals potentiates the release of autoinhibition

PURPOSE: Elevated phospholipase D (PLD) activity provides a survival signal in several human cancer cell lines and suppresses apoptosis when cells are subjected to the stress of serum withdrawal. Thus, targeting PLD survival signals has potential to suppress survival in cancer cells that depend on PLD for survival. Honokiol is a compound that suppresses tumor growth in mouse models. The purpose of this study was to investigate the effect of honokiol on PLD survival signals and the Ras dependence of these signals. EXPERIMENTAL DESIGN: The effect of honokiol upon PLD activity was examined in human cancer cell lines where PLD activity provides a survival signal. The dependence of PLD survival signals on Ras was investigated, as was the effect of honokiol on Ras activation. RESULTS: We report here that honokiol suppresses PLD activity in human cancer cells where PLD has been shown to suppress apoptosis. PLD activity is commonly elevated in response to the stress of serum withdrawal, and, importantly, the stress-induced increase in PLD activity is selectively suppressed by honokiol. The stress-induced increase in PLD activity was accompanied by increased Ras activation, and the stress-induced increase in PLD activity in MDA-MB-231 breast cancer cells was dependent on a Ras. The PLD activity was also dependent on the GTPases RalA and ADP ribosylation factor. Importantly, honokiol suppressed Ras activation. CONCLUSION: The data provided here indicate that honokiol may be a valuable therapeutic reagent for targeting a large number of human cancers that depend on Ras and PLD for their survival