Ras-induced cellular events (review)

Ras is a crucial regulator of cell growth in eukaryotic cells. Activated Ras can stimulate signal transduction cascades, leading to cell proliferation, differentiation or apoptosis. It is also one of the most commonly mutated genes in both solid tumours and haematologic neoplasias. In leukaemia and tumours, aberrant Ras signalling can be induced directly by Ras mutation or indirectly by altering genes that associate with Ras or its signalling pathways. A requisite for Ras function is localization to the plasma membrane, which is induced by the post-translational modification farnesylation. Molecules that interfere with this Ras modification have been used as antitumour agents. Ras is emerging as a dual regulator of cell functions, playing either positive or negative roles in the control of proliferation or apoptosis. The diversity of Ras-mediated effects may be related in part to the differential involvement of Ras homologues in distinct cellular processes or to the expanding array of Ras effectors.     

Ras-induced cellular events

Ras is a crucial regulator of cell growth in eukaryotic cells. Activated Ras can stimulate signal transduction cascades, leading to cell proliferation, differentiation or apoptosis. It is also one of the most commonly mutated genes in both solid tumours and haematologic neoplasias. In leukaemia and tumours, aberrant Ras signalling can be induced directly by Ras mutation or indirectly by altering genes that associate with Ras or its signalling pathways. A requisite for Ras function is localization to the plasma membrane, which is induced by the post-translational modification farnesylation. Molecules that interfere with this Ras modification have been used as antitumour agents. Ras is emerging as a dual regulator of cell functions, playing either positive or negative roles in the control of proliferation or apoptosis. The diversity of Ras-mediated effects may be related in part to the differential involvement of Ras homologues in distinct cellular processes or to the expanding array of Ras effectors.     

p66SHC: The apoptotic side of Shc proteins

Initially identified as components of the signaling pathways triggered by receptor tyrosine kinases and leading to Ras activation, Shc proteins have been more recently implicated in the regulation of signals controlling not only cell proliferation, but also cell survival and apoptosis. Here we briefly review the current understanding of Shc proteins as promoters of apoptosis. Specifically, we focus on the 66 kDa isoform of ShcA, whose paramount importance in the regulation of oxidative stress responses leading to cell apoptosis and ageing has been by now firmly established.This revised version was published online in March 2005 with corrections to the title.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

Extracellular signal-regulated activation of Rap1 fails to interfere in Ras effector signalling.

The small GTPase Rap1 has been implicated in both negative and positive control of Ras-mediated signalling events. We have investigated which extracellular signals can activate Rap1 and whether this activation leads to a modulation of Ras effector signalling, i.e. the activation of ERK and the small GTPase Ral. We found that Rap1 is rapidly activated following stimulation of a large variety of growth factor receptors. These receptors include receptor tyrosine kinases for platelet-derived growth factor (PDGF) and epithelial growth factor (EGF), and G protein-coupled receptors for lysophosphatidic acid (LPA), thrombin and endothelin. At least three distinct pathways may transduce a signal towards Rap1 activation: increase in intracellular calcium, release of diacylglycerol and cAMP synthesis. Surprisingly, activation of endogenous Rap1 fails to affect Ras-dependent ERK activation. In addition, we found that although overexpression of active Rap1 is able to activate the Ral pathway, activation of endogenous Rap1 in fibroblasts does not result in Ral activation. Rap1 also does not negatively influence Ras-mediated Ral activation. We conclude that activation of Rap1 is a common event upon growth factor treatment and that the physiological function of Rap1 is likely to be different from modulation of Ras effector signalling.     

Novel aspects of Ras proteins biology: regulation and implications.

The importance of Ras proteins as crucial crossroads in cellular signaling pathways has been well established. In spite of the elucidation of the mechanism of RAS activation by growth factors and the delineation of MAP kinase cascades, the overall framework of Ras interactions is far from being complete. Novel regulators of Ras GDP/GTP exchange have been identified that may mediate the activation of Ras in response to changes in intracellular calcium and diacylglycerol. The direct activation of Ras by free radicals such as nitric oxide also suggests potential regulation of Ras function by the cellular redox state. In addition, the array of Ras effectors continues to expand, uncovering links between Ras and other cellular signaling pathways. Ras is emerging as a dual regulator of cellular functions, playing either positive or negative roles in the regulation of proliferation and apoptosis. The signals transmitted by Ras may be modulated by other pathways triggered in parallel, resulting in the final order for proliferation or apoptosis. The diversity of ras-mediated effects may be related in part to differential involvement of Ras homologues in distinct cellular processes. The study of Ras posttranslational modifications has yielded a broad battery of inhibitors that have been envisaged as anti-cancer agents. Although an irreversible modification, Ras isoprenylation appears to be modulated by growth factors and by the activity of the isoprenoid biosynthetic pathway, which may lead to changes in Ras activity.     

Grb2 associated binder 2 couples B-cell receptor to cell survival

B-cell fate during maturation and the germinal center reaction is regulated through the strength and the duration of the B-cell receptor signal. Signaling pathways discriminating between apoptosis and survival in B cells are keys in understanding adaptive immunity. Gab2 is a member of the Gab/Dos adaptor protein family. It has been shown in several model systems that Gab/Dos family members may regulate both the anti-apoptotic PI3-K/Akt and the mitogenic Ras/MAPK pathways, still their role in B-cells have not been investigated in detail. Here we studied the role of Gab2 in B-cell receptor mediated signaling. We have shown that BCR crosslinking induces the marked phosphorylation of Gab2 through both Lyn and Syk kinases. Subsequently Gab2 recruits p85 regulatory subunit of PI3-K, and SHP-2. Our results revealed that Ig-alpha/Ig-beta, signal transducing unit of the B-cell receptor, may function as scaffold recruiting Gab2 to the signalosome. Overexpression of Gab2 in A20 cells demonstrated that Gab2 is a regulator of the PI3-K/Akt but not that of the Ras/MAPK pathway in B cells. Accordingly to the elevated Akt phosphorylation, overexpression of wild-type Gab2 in A20 cells suppressed Fas-mediated apoptosis, and enhanced BCR-mediated rescue from Fas-induced cell death. Although PH-domain has only a stabilizing effect on membrane recruitment of Gab2, it is indispensable in mediating its anti-apoptotic effect.     

Mathematical model of the role of intercellular signalling in intercellular cooperation during tumorigenesis

Objectives: Intercellular cooperation has been hypothesized to enhance cell proliferation during cancer metastasis through autocrine signalling cascades and mathematical models can provide valuable insights into underlying mechanisms of metastatic tumorigenesis. Here, we present a model that incorporates signal‐stimulated cell proliferation, and investigate influences of diffusion‐driven heterogeneity in signal concentration on proliferation dynamics. Materials and methods: Our model incorporates signal production through both autocrine and paracrine pathways, and signal diffusion and loss for a metastasizing cell population at a host site. We use the signalling pathway of IL‐6 for illustration where this signalling species forms an intermediate complex with its receptor IL‐6R. This in turn forms a heterodimeric complex with transmembrane protein gp130, ultimately resulting in production of downstream signals. Cell population dynamics are taken to follow a modified logistic equation for which the rate term is dependent on local IL‐6 concentration. Results and conclusions: Our spatiotemporal model agrees closely with experimental results. The model is also able to predict two phenomena typical of metastatic tumorigenesis – host tissue preference and long periods of proliferation dormancy. It confirms that diffusivity of the signalling species in a host tissue plays a significant role during the process. Our results show that the proliferation–apoptosis balance is tipped in favour of the former for host sites that have relatively smaller signal diffusivities.     

Restricted-range gradients and travelling fronts in a model of juxtacrine cell relay

Patterning events in development often depend on the transmission over a range of several cell diameters of signals emanating from a localized source. Experimental studies of such long-range signalling by members of the TGF-β family of growth factors suggests that a cell-relay mechanism in which cells signal only with their immediate neighbours (i.e., juxtacrine signalling) may be operating in some tissues. Here, this possibility is investigated through the analysis of a model of juxtacrine signalling. Depending on the strength of the signal relay between cells, a localized signal source can generate either stable gradients or travelling fronts of cell activation. Both of these behaviors could in principle be involved in the long-range transmission of signals and patterning of cell fates by cell relays. There are significant and surprising differences between the gradients generated by the mechanism studied here, and those generated by the diffusion of a morphogen. In particular, there is an upper limit on the distance over which any given level of cell activation can be attained in a relay-mediated gradient, irrespective of the strength of signal source.     

The role of phosphotyrosine phosphatases in haematopoietic cell signal transduction

Phosphotyrosine phosphatases (PTPases) are the enzymes which remove phosphate groups from protein tyrosine residues. An enormous number of phosphatases have been cloned and sequenced during the past decade, many of which are expressed in haematopoietic cells. This review focuses on the biochemistry and cell biology of three phosphatases, the transmembrane CD45 and the cytosolic SH2-domain-containing PTPases SHP-1 and SHP-2, to illustrate the diverse ways in which PTPases regulate receptor signal transduction. The involvement of these and other PTPases has been demonstrated in haematopoietic cell development, apoptosis, activation and non-responsiveness. A common theme in the actions of many haematopoietic cell PTPases is the way in which they modulate the thresholds for receptor signalling, thereby regulating critical events in the positive and negative selection of lymphocytes. There is growing interest in haematopoietic PTPases and their associated regulatory proteins as targets for pharmaceutical intervention and in the involvement of these enzymes in human disease.     

Lipid rafts in T cell receptor signalling

The molecular events and the protein components that are involved in signalling by the T cell receptor (TCR) for antigen have been extensively studied. Activation of signalling cascades following TCR stimulation depends on the phosphorylation of the receptor by the tyrosine kinase Lck, which localizes to the cytoplasmic face of the plasma membrane by virtue of its post-translational modification. However, the precise order of events during TCR phosphorylation at the plasma membrane, remains to be defined. A current theory that describes early signalling events incorporates the function of lipid rafts, microdomains at the plasma membrane with distinct lipid and protein composition. Lipid rafts have been implicated in diverse biological functions in mammalian cells. In T cells, molecules with a key role in TCR signalling, including Lck, localize to these domains. Importantly, mutant versions of these proteins which fail to localise to raft domains were unable to support signalling by the TCR. Biochemical studies using purified detergent-resistant membranes (DRM) and confocal microscopy have suggested that upon stimulation, the TCR and Lck-containing lipid rafts may come into proximity allowing phosphorylation of the receptor. Further, there are data suggesting that phosphorylation of the TCR could depend on a transient increase in Lck activity that takes place within lipid rafts to initiate signalling. Current results and a model of how lipid rafts may regulate TCR signalling are discussed.     

Ras and Phosphoinositide 3-Kinase: Partners in Development and Tumorigenesis

Much progress has been made in understanding the myriad of intracellular signalling pathways responsible for control of cell physiology. Signalling downstream of receptor tyrosine kinases (RTKs) is probably the most studied signalling mechanism to date and many of the molecular components and corresponding interactions involved have been delineated. Importantly, deregulation of RTK signalling has been implicated in the formation and maintenance of many human tumours. Two of the pivotal molecular components in RTK signalling, Ras and phosphoinositide 3-kinase (PI 3-kinase), have been shown to bind to each other, leading to the activation of PI 3-kinase. However, in addition to this Ras - PI 3-kinase interaction, first described over a decade ago, several other molecular interactions have more recently been described that appear to mediate the same signal. This has brought into question the physiological relevance of the Ras – PI 3-kinase interaction during RTK signalling. Through disruption of the interaction in a mouse model, we have now confirmed that the interaction is highly functional in vivo both during mammalian development and during Ras-induced tumorigenesis. Many questions still remain: in this Perspective, we explore the remaining uncertainties surrounding the role of this signalling mechanism, as well as the future directions that will likely shed further light on its role within cells.     

Recent advances in Ca(2+)-dependent Ras regulation and cell proliferation

Our understanding of the mechanisms whereby growth factors stimulate cell proliferation through the Ras pathway stems largely from studies of the canonical pathway involving recruitment of Ras activators and inhibitors to the vicinity of receptor tyrosine kinases via phosphotyrosine-binding adaptor proteins. Ca(2+) has seldom joined the party, despite the identification of phospholipase Cgamma and Ca(2+) entry as receptor tyrosine kinase-dependent signals. Mechanisms by which Ca(2+) can directly influence Ras activity have remained relatively elusive. Similarly, the mechanisms whereby Ca(2+) modulates the cell cycle have been equally murky, and yet there are some interesting parallels in the role of Ras and Ca(2+) in cell cycle re-entry. This review focuses on a number of novel mechanisms that link Ca(2+) with the regulation of Ras activity and signaling output. Their collective discovery adds to the complexities of Ras regulation and raises further questions about the role of Ca(2+) signals in Ras-dependent cell proliferation.     

Ras signaling in tumor necrosis factor-induced apoptosis.

Tumor necrosis factor (TNF) exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. Our previous studies have shown that enforced expression of an activated H-ras oncogene converted non-tumorigenic, TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells that also became very sensitive to TNF-induced apoptosis. This finding suggested that Ras activation may play a role in TNF-induced apoptosis. In this study we investigated whether Ras activation is an obligatory step in TNF-induced apoptosis. Introduction of two different molecular antagonists of Ras, the rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras-transformed 10TEJ cells inhibited TNF-induced apoptosis. Similar results were obtained with L929 cells, a fibroblast cell line sensitive to TNF-induced apoptosis, which does not have a ras mutation. While Ras is constitutively activated in TNF-sensitive 10TEJ cells, TNF treatment increased Ras-bound GTP in TNF-sensitive L929 cells but not in TNF-resistant 10T1/2 cells. Moreover, RasN17 expression blocked TNF-induced Ras-GTP formation in L929 cells. These results demonstrate that Ras activation is required for TNF-induced apoptosis in mouse fibroblasts.     

Non-genomic oestrogen receptor signal in B lymphocytes: An approach towards therapeutic interventions for infection,autoimmunity and cancer

The non-genomic membrane bound oestrogen receptor (mER) regulates intracellular signals through receptor-ligand interactions. The mER, along with G-protein coupled oestrogen receptor GPR 30 (GPER), induces diverse cell signalling pathways in murine lymphocytes. The mER isoform ER-alpha46 has recently been demonstrated in human B and T lymphocytes as an analogue receptor for chemokine CCL18, the signalling events of which are not clearly understood. Ligand-induced mER and GPER signalling events are shared with BCR, CD19 mediated intracellular signalling through phospholipase C, PIP2/IP3/PI3 mediated activation of Akt, MAP kinase, and mTOR. Oestrogen has the ability to induce CD40-mediated activation of B cells. The complete signalling pathways of mER, GPR30 and their interaction with other signals are targeted areas for novel drug development in B cells during infection, autoimmunity and cancer. Therefore, an in depth investigation is critical for determining shared signal outputs during B cell activation. Here, we focus on the mode of action of membrane bound ER in B cells as therapeutic checkpoints.     

Apoptotic signal transduction: emerging pathways.

Apoptosis is a counterbalance to mechanisms of cell proliferation and is critically important in regulation of the immune system, development, and normal tissue homeostasis. Mammalian signal transduction pathways affecting apoptosis are more complex than their counterparts in the nematode Caenorhabditis elegans, a valuable model system that has provided powerful initial insights into key molecules regulating apoptosis. Despite this complexity, substantial progress has been made in recent years towards defining the nature and detail of signalling pathways bringing about apoptosis in mammalian cells. In particular, the identity and precise substrate specificities of a large family of caspase enzymes, implicated as critical components of the apoptotic machinery, have been defined. In addition, the mechanism by which the cell surface Fas receptor mediates induction of apoptosis, via activation of caspases, has recently been elucidated. A prominent role for mitochondria in cell death pathways has also recently emerged, a clear theme being that mitochondria can trigger degradative events by the release of apoptogenic proteins (e.g., cytochrome c) from the intermembrane space to the cytosol. This review focuses on recent progress in these areas and discusses integration of this knowledge in our overall understanding of the processes that control apoptosis.     

Negative receptor signalling

Binding of external factors to cell membrane receptors triggers intracellular signalling pathways that ultimately determine if the cell proliferates, differentiates or undergoes apoptosis. Activated receptors also initiate a cascade of events, called negative receptor signalling, that decreases the amplitude of positive signals and modulates the level of cell stimulation. Recent studies have revealed that negative signalling by receptor tyrosine kinases involves coordinated action of ubiquitin ligases (i.e. Cbl), adaptor proteins (i.e. Grb2 and CIN85), inhibitory molecules (i.e. Sprouty), cytoplasmic kinases (i.e. activated Cdc42-associated kinase) and phosphoinositol metabolites. These inhibitory signals are essential for normal cell functioning, and their deregulation often results in human diseases.