The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

Recent discoveries have suggested the concept that intracellular signals are the sum of multiple, site‐specified subsignals, rather than single, homogeneous entities. In the context of cancer, searching for compounds that selectively block subsignals essential for tumor progression, but not those regulating “house‐keeping” functions, could help in producing drugs with reduced side effects compared to compounds that block signaling completely. The Ras‐ERK pathway has become a paradigm of how space can differentially shape signaling. Today, we know that Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site‐specific regulatory mechanisms, distinctively engaging effector pathways and switching‐on diverse genetic programs to generate different biological responses. The Ras effector pathway leading to ERKs activation is also under strict, space‐related regulatory processes. These findings may open a gate for aiming at the Ras‐ERK pathway in a spatially restricted fashion, in our quest for new anti‐tumor therapies.     

Distinct utilization of effectors and biological outcomes resulting from site-specific Ras activation: Ras functions in lipid rafts and Golgi complex are dispensable for proliferation and transformation

Ras proteins are distributed in different types of plasma membrane microdomains and endomembranes. However, how microlocalization affects the signals generated by Ras and its subsequent biological outputs is largely unknown. We have approached this question by selectively targeting RasV12 to different cellular sublocalizations. We show here that compartmentalization dictates Ras utilization of effectors and the intensity of its signals. Activated Ras can evoke enhanced proliferation and transformation from most of its platforms, with the exception of the Golgi complex. Furthermore, signals that promote survival emanate primarily from the endoplasmic reticulum pool. In addition, we have investigated the need for the different pools of endogenous Ras in the conveyance of upstream mitogenic and transforming signals. Using targeted RasN17 inhibitory mutants and in physiological contexts such as H-Ras/N-Ras double knockout fibroblasts, we demonstrate that Ras functions at lipid rafts and at the Golgi complex are fully dispensable for proliferation and transformation.     

Coassembly of flotillins induces formation of membrane microdomains, membrane curvature, and vesicle budding

Endocytosis has a crucial role in many cellular processes. The best-characterized mechanism for endocytosis involves clathrin-coated pits [1], but evidence has accumulated for additional endocytic pathways in mammalian cells [2]. One such pathway involves caveolae, plasma-membrane invaginations defined by caveolin proteins. Plasma-membrane microdomains referred to as lipid rafts have also been associated with clathrin-independent endocytosis by biochemical and pharmacological criteria [3]. The mechanisms, however, of nonclathrin, noncaveolin endocytosis are not clear [4, 5]. Here we show that coassembly of two similar membrane proteins, flotillin1 and flotillin2 [6-8], is sufficient to generate de novo membrane microdomains with some of the predicted properties of lipid rafts [9]. These microdomains are distinct from caveolin1-positive caveolae, are dynamic, and bud into the cell. Coassembly of flotillin1 and flotillin2 into microdomains induces membrane curvature, the formation of plasma-membrane invaginations morphologically similar to caveolae, and the accumulation of intracellular vesicles. We propose that flotillin proteins are defining structural components of the machinery that mediates a clathrin-independent endocytic pathway. Key attributes of this machinery are the dependence on coassembly of both flotillins and the inference that flotillin microdomains can exist in either flat or invaginated states.     

Transformation of cells overexpressing a tyrosine kinase by phospholipase D1 and D2.

Phospholipase D (PLD) activity is elevated in response to most mitogenic signals. Two mammalian PLD genes (PLD1 and PLD2) have been cloned and their gene products have been characterized. PLD1 is a downstream target of the Ras/RalA GTPase cascade implicated in mitogenic and oncogenic signaling. Consistent with a role in mitogenic signaling, elevated expression of PLD1 transforms cells overexpressing the epidermal growth factor (EGF) receptor (EGFR). However, PLD2 colocalizes with the EGFR in caveolin-enriched light membrane microdomains. We therefore investigated whether PLD2 could also contribute to the transformation of cells overexpressing a tyrosine kinase. We report here that elevated expression of PLD2 transforms rat fibroblasts overexpressing either the EGFR or c-Src. Since overexpression of a tyrosine kinase is a common genetic alteration in several human cancers, these data suggest that elevation of either PLD1 or PLD2 may contribute to the progression to a malignant phenotype in cells with elevated tyrosine kinase activity.     

Differential Ras activation between caveolae/raft and non-raft microdomains

Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu-Ras, was fused to the C-terminal hypervariable regions of H-Ras and K-Ras to create indicators for Ras activation within caveolae/rafts (Raichu-tH) and non-raft domains (Raichu-tK) of the plasma membrane, respectively. Raichu-tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device (DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer (FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.     

Cloning oncogenic ras-regulated genes by differential display

The coordinated regulation of gene expression is a key cellular function that specifies cell characteristics as well as controls normal physiological processes of the organism. Deregulation of this gene expression leads to a variety of abnormal conditions such as cancer. The ras oncogene is one of the most frequently found mutations in various types of human cancer. The mutated Ras protein constitutively elicits multiple mitogenic signals to the nucleus to alter gene expression of target genes that are involved in a broad range of normal cellular functions. Thus the identification of these genes may provide an important tool toward the understanding of these pathogenic processes. As a first step to reveal these processes at the molecular level and to dissect the key pathway employed by oncogenic Ras protein, we have looked for its target genes in rodent model cell lines using the differential display method. Our initial screening has isolated a number of genes either up- or downregulated by oncogenic ras activation. Although the functional analyses of these genes in terms of ras-mediated cell transformation will be the major challenge, differential display has come to be a very efficient tool that helped us move to the next step. In this short report, we focus primarily on the technical aspects of differential display and experimental designs used in this study.     

Abolition of cyclin-dependent kinase inhibitor p16Ink4a and p21Cip1/Waf1 functions permits Ras-induced anchorage-independent growth in telomerase-immortalized human fibroblasts

Human cells are more resistant to both immortalization and malignant transformation than rodent cells. Recent studies have established the basic genetic requirements for the transformation of human cells, but much of this work relied on the expression of transforming proteins derived from DNA tumor viruses. We constructed an isogenic panel of human fibroblast cell lines using a combination of gene targeting and ectopic expression of dominantly acting mutants of cellular genes. Abolition of p21(Cip1/Waf1) and p16(Ink4a) functions prevented oncogenically activated Ras from inducing growth arrest and was sufficient for limited anchorage-independent growth but not tumorigenesis. Deletion of the tumor suppressor p53 combined with abolition of p16(Ink4a) function failed to mimic the introduction of simian virus 40 large T antigen, indicating that large T antigen may target additional cellular functions. Ha-Ras and Myc cooperated only to a limited extent, but in the absence of Ras, Myc cooperated strongly with the simian virus 40 small t antigen to elicit aggressive anchorage-independent growth. The experiments reported here further define specific components of human transformation pathways.     

Electron microscopic imaging of Ras signaling domains

Ras isoform-specific signaling from the plasma membrane appears to be regulated by interactions with distinct functional microdomains. We have developed protocols allowing the generation of 2-D spatial maps describing cell surface microdomain distributions. The combined electron microscopic (EM)-statistics approach provides nanometer scale resolution allowing both inner and outer leaflet domains to be visualized and cross-correlated with each other or with a protein of interest. In particular, the technique has allowed the interaction of Ras isoforms with signaling microdomains and proteins regulating these compartments to be screened. By allowing detailed monitoring of cell surface organization and compartmentalization, the approach has widespread potential for studies of plasma membrane-dependent cell biology, including regulated signaling and membrane trafficking.     

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.     

Wild-type NRas and KRas perform distinct functions during transformation

The ras proto-oncogenes, of which there are four isoforms, are molecular switches that function in signal transduction pathways to control cell differentiation, proliferation, and survival. How the Ras isoforms orchestrate cellular processes that affect behavior is poorly understood. Further, why cells express two or more Ras isoforms is unknown. Here, using a genetically defined system, we show that the presence of both wild-type KRas and NRas isoforms is required for transformation because they perform distinct nonoverlapping functions: wild-type NRas regulates adhesion, and KRas coordinates motility. Remarkably, we find that Ras isoforms achieve functional specificity by engaging different signaling pathways to affect the same cellular processes, thereby coordinating cellular outcome. Although we find that signaling from both isoforms intersects in actin and microtubule cytoskeletons, our results suggest that KRas signals through Akt and Cdc42 while NRas signals through Raf and RhoA. Our analyses suggest a previously unappreciated convergence of different Ras isoforms on the dynamics of the processes involved in transformation.     

Starvation-associated mutagenesis in yeast Saccharomyces cerevisiae is affected by Ras2/cAMP signaling pathway

The number of revertants with restored ability to form colony increases in a time-dependent manner during long-term selective starvation of dense mutant microbial cultures. This is due to starvation-associated (also called adaptive) mutations that arise in a replication independent manner. Here we report that in Saccharomyces cerevisiae the frequency of starvation-associated reversions of mutant genes whose products are necessary for amino acids biosynthesis are influenced by Ras2/cAMP signaling pathway. This signaling pathway is a yeast general regulatory pathway involved in nutritional sensing, UV response, sporulation control and life span control and its changes are manifested in both, cell cycle and life cycle. Inactivation of the RAS2 gene causes an increase in number of starvation-associated revertants in comparison to an isogenic wild type strain and a strain with constitutively activated Ras2/cAMP signaling pathway. Therefore, we suggest that starvation-associated mutagenesis is different from spontaneous mutagenesis and is related to the cellular capacity to adopt distinct physiological states in response to environmental signals.     

Nonconventional trafficking of Ras associated with Ras signal organization

Ras signaling to its downstream effectors appears to include combinations of extracellular-signal-regulated Ras activation at the plasma membrane (PM) and endomembranes, dynamic lateral segregation in the PM, and translocation of Ras from the PM to intracellular compartments. These processes are governed by the C-terminal polybasic farnesyl domain in K-Ras 4B and by the cysteine-palmitoylated C-terminal farnesyl domains in H-Ras and N-Ras. K-Ras 4B has no palmitoylated cysteines. Depalmitoylation/repalmitoylation of H-/N-Ras proteins promotes their cellular redistribution and signaling by mechanisms as yet unknown, possibly involving chaperones. Palmitoylation of H-/N-Ras also promotes their association with 'rasosomes', randomly diffusing nanoparticles that apparently provide a means by which multiple copies of activated Ras and its signal can spread rapidly. Ubiquitination of H-Ras evidently targets it to the endosomes. The polybasic farnesyl domain of K-Ras 4B was shown to act as a target for Ca++/calmodulin, which sequesters the active protein from the PM, thereby facilitating its trafficking to Golgi apparatus and early endosomes. Protein kinase C-dependent phosphorylation of S181 in K-Ras 4B was shown to provide a regulated farnesyl-electrostatic switch on K-Ras 4B, which promotes its translocation to the mitochondria. All these translocation events are characterized by nonconventional trafficking of the farnesyl-modified Ras proteins and seem to govern the selectivity and probably also the robustness of the Ras signal. In this review, we discuss the various modifications and interactions of the farnesylated C-terminus, the trafficking of Ras proteins in the PM and between the PM and the endomembranes, and the relevance of the subcellular localization of Ras for Ras function.     

Mitochondrial DNA copy number is regulated by cellular proliferation: a role for Ras and p66(Shc)

The abundance of mitochondria is regulated by biogenesis and division. These processes are controlled by cellular factors, given that, for example, mitochondria have to replicate their DNA prior to cell division. However, the mechanisms that allow a synchronization of cell proliferation with mitochondrial genome replication are still obscure. We report here our investigations on the role of proliferation and the contribution of Ras and p66Shc in the regulation of mitochondrial DNA copy number. Ras proteins mediate a variety of receptor-transduced mitogenic signals and appear to play an essential role in the cellular response to growth factors. P66Shc is a genetic determinant of life span in mammals and has been implicated in the regulation of receptor signaling and various mitochondrial functions. First, we confirmed previous reports showing that mitochondrial DNA is replicated during a specific phase of the cell cycle (the pre-S phase) and provided novel evidences that this process is regulated by mitogenic growth factors. Second, we showed that mitochondrial DNA replication is activated following Ras-induced cellular hyper-proliferation. Finally, we showed that p66Shc expression induces mitochondrial DNA replication, both in vitro and in vivo. We suggest that mitochondria are target of intracellular signaling pathways leading to proliferation, involving Ras and p66Shc, which might function to integrate cellular bio-energetic requirements and the inheritance of mitochondrial DNA in a cell cycle-dependent manner.     

Germline Mutations in Components of the Ras Signaling Pathway in Noonan Syndrome and Related Disorders

Ras proteins control a variety of critical cellular processes, and somatic mutations in RAS genes (and other members of signaling networks regulated by Ras) are common in human malignancies. Ras proteins are guanosine triphosphate (GTP)-binding proteins that cycle between active GTP-bound and inactive guanosine diphosphate (GDP) bound conformations. Cancer-associated Ras mutations typically alter amino acids G12, G13 or Q61. These mutant Ras proteins display impaired GTPase activity and are resistant to GTPase activating proteins (GAPs). We and others recently discovered novel germline KRAS mutations in individuals diagnosed with Noonan or cardio-facio-cutanous (CFC) syndrome, two clinically overlapping disorders characterized by short stature, distinct facial anomalies, heart defects, and other developmental abnormalities. We found that the mutant K-Ras proteins encoded by NS-associated alleles have less pronounced biochemical defects than known Ras oncoproteins, which likely explains why these mutations are tolerated in the germline. Together with the recent findings of mutations in other members of the Ras signaling cascade in CFC syndrome and in Costello syndrome, another clinically related disorder, it is now clear that Noonan-like features are common phenotypic consequences of systemic deregulation of the Ras pathway. The discovery of germline mutations in this group of related genetic disorders underscores the pivotal role of the degree and duration of Ras activation in cell fate decisions during embryonic development and morphogenesis.     

Mechanisms regulating Raf-1 activity in signal transduction pathways

Raf-1 is a key protein involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Biochemical and genetic studies have demonstrated that Raf-1 functions downstream of activated tyrosine kinases and Ras and upstream of mitogen-activated protein kinase (MAPK) and MAPK kinase (MKK or MEK) in many signaling pathways. A major objective of our laboratory has been to determine how Raf-1 becomes activated in response to signaling events. Using mammalian, baculovirus, and Xenopus systems, we have examined the roles that phosphorylation and protein-protein interactions play in regulating the biological and biochemical activity of Raf-1. Our studies have provided evidence that the activity of Raf-1 can be modulated by both Ras-dependent and Ras-independent pathways. Recently, we reported that Arg89 of Raf-1 is a residue required for the association of Raf-1 and Ras. Mutation of this residue disrupted interaction with Ras and prevented Ras-mediated, but not protein kinase C-or tyrosine kinase-mediated, enzymatic activation of Raf-1 in the baculovirus expression system. Further analysis of this mutant demonstrated that kinase-defective Raf-1 proteins interfere with the propagation of proliferative and developmental signals by binding to Ras and blocking Ras function. Our findings have also shown that phosphorylation events play a role in regulating Raf-1. We have identified sites of in vivo phosphorylation that positively and negatively alter the biological and enzymatic activity of Raf-1. In addition, we have found that some of these phosphorylation sites are involved in mediating the interaction of Raf-1 with potential activators (Fyn and Src) and with other cellular proteins (14-3-3). Results from our work suggest that Raf-1 is regulated at multiple levels by several distinct mechanisms. © 1995 wiley-Liss, Inc.     

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.     

Mitochondrial DNA copy number is regulated by cellular proliferation: A role for Ras and p66Shc

The abundance of mitochondria is regulated by biogenesis and division. These processes are controlled by cellular factors, given that, for example, mitochondria have to replicate their DNA prior to cell division. However, the mechanisms that allow a synchronization of cell proliferation with mitochondrial genome replication are still obscure. We report here our investigations on the role of proliferation and the contribution of Ras and p66^Shc in the regulation of mitochondrial DNA copy number. Ras proteins mediate a variety of receptor-transduced mitogenic signals and appear to play an essential role in the cellular response to growth factors. P66^Shc is a genetic determinant of life span in mammals and has been implicated in the regulation of receptor signaling and various mitochondrial functions. First, we confirmed previous reports showing that mitochondrial DNA is replicated during a specific phase of the cell cycle (the pre-S phase) and provided novel evidences that this process is regulated by mitogenic growth factors. Second, we showed that mitochondrial DNA replication is activated following Ras-induced cellular hyper-proliferation. Finally, we showed that p66^Shc expression induces mitochondrial DNA replication, both in vitro and in vivo. We suggest that mitochondria are target of intracellular signaling pathways leading to proliferation, involving Ras and p66^Shc, which might function to integrate cellular bio-energetic requirements and the inheritance of mitochondrial DNA in a cell cycle-dependent manner.