Ras GTPases-H-Ras, N-Ras, and K-Ras 4B/4A-operate as key molecular switches that convey extracellular signals from surface receptors to the interior of the cell, thereby regulating essential processes including proliferation, differentiation, and survival (15,34). It is well known that Ras must be attached to the inner leaflet of the plasma membrane (PM) to be functional (50). This is accomplished by lipidic additions to the protein C terminus (33), which contains the essential signal for localizing Ras to membranes: the CAAX box (where C is cysteine, A is alyphatic amino acid, and X is serine/methionine). This motif undergoes posttranslational modifications that make it more hydrophobic. The cysteine is farnesylated, the AAX sequence is proteolyzed, and the newly C-terminal cysteine is carboxymethylated (50). However, a second signal is necessary for efficiently positioning Ras in the membrane. This is accomplished by palmitoylation of cysteine 181 in N-Ras, and cysteines 181 and 184 in H-Ras. In the case of K-Ras 4B the second signal is attained by a polybasic motif of six lysines (175 to 180) that interacts electrostatically with the negatively charged membrane (24-26).Recently, a new twist has been provided by findings indicating that Ras isoforms are distinctively segregated in different PM microdomains with unique biochemical and physicochemical characteristics, H-Ras can be found in bulk membrane and in lipid rafts, both caveolar and noncaveolar. K-Ras is exclusively located in bulk membrane, whereas N-Ras can only be detected in noncaveolar lipid rafts (35,(38)(39)(40). Furthermore, recent reports indicate that Ras is also present in endomembranes such as endosomes, endoplasmic reticulum (ER), and the Golgi complex (10,37,45). The significance of this distribution seems to go beyond that of a transient event associated to transport and/or recycling. Instead, a pool of Ras appears to reside in these organelles, and therein Ras can productively engage downstream effectors (10,11,37,45). Moreover, at these endomembranes Ras regulation is undertaken by proteins that operate in a location-specific fashion. As such, the guanine nucleotide exchange factor RasGRP specifically regulates Ras activation at the Golgi complex (7, 9), whereas SOS and RasGRF undertake Ras regulation at the ER. Likewise, stimuli such as lysophosphatidic acid preferentially activate the Ras pool at the ER, whereas calcium ionophores are more effective in activating PM Ras (4).The fact that exogenous stimuli activate Ras distinctively depending on its localization and that Ras regulation at different sites requires the participation of specific intermediaries hints at the necessity for a location-specific control. This, in term, may imply that Ras functions at its different sites may not be totally redundant. Thus, a selective activation of Ras at each of its locations could be intended to generate variability in its biochemical and biological outputs. It is known that Ras regulates numerous cellular functions through the activation of an...

Nearly 50% of human malignancies exhibit unregulated RAS-ERK signaling; inhibiting it is a valid strategy for antineoplastic intervention. Upon activation, ERK dimerize, which is essential for ERK extranuclear, but not for nuclear, signaling. Here, we describe a small molecule inhibitor for ERK dimerization that, without affecting ERK phosphorylation, forestalls tumorigenesis driven by RAS-ERK pathway oncogenes. This compound is unaffected by resistance mechanisms that hamper classical RAS-ERK pathway inhibitors. Thus, ERK dimerization inhibitors provide the proof of principle for two understudied concepts in cancer therapy: (1) the blockade of sub-localization-specific sub-signals, rather than total signals, as a means of impeding oncogenic RAS-ERK signaling and (2) targeting regulatory protein-protein interactions, rather than catalytic activities, as an approach for producing effective antitumor agents.

Individual tumour cells move in three-dimensional environments with either a rounded or an elongated 'mesenchymal' morphology. These two modes of movement are tightly regulated by Rho family GTPases: elongated movement requires activation of Rac1, whereas rounded/amoeboid movement engages specific Cdc42 and Rho signalling pathways. In siRNA screens targeting the genes encoding guanine nucleotide exchange factors (GEFs), we found that the Ras GEF RasGRF2 regulates conversion between elongated- and rounded-type movement. RasGRF2 suppresses rounded movement by inhibiting the activation of Cdc42 independently of its capacity to activate Ras. RasGRF2 and RasGRF1 directly bind to Cdc42, outcompeting Cdc42 GEFs, thereby preventing Cdc42 activation. By this mechanism, RasGRFs regulate other Cdc42-mediated cellular processes such as the formation of actin spikes, transformation and invasion in vitro and in vivo. These results demonstrate a role for RasGRF GEFs as negative regulators of Cdc42 activation.

Subcellular localization influences the nature of Ras/extracellular signal-regulated kinase (ERK) signals by unknown mechanisms. Herein, we demonstrate that the microenvironment from which Ras signals emanate determines which substrates will be preferentially phosphorylated by the activated ERK1/2. We show that the phosphorylation of epidermal growth factor receptor (EGFr) and cytosolic phospholipase A 2 (cPLA 2 ) is most prominent when ERK1/2 are activated from lipid rafts, whereas RSK1 is mainly activated by Ras signals from the disordered membrane. We present evidence indicating that the underlying mechanism of this substrate selectivity is governed by the participation of different scaffold proteins that distinctively couple ERK1/2, activated at defined microlocalizations, to specific substrates. As such, we show that for cPLA 2 activation, ERK1/2 activated at lipid rafts interact with KSR1, whereas ERK1/2 activated at the endoplasmic reticulum utilize Sef-1. To phosphorylate the EGFr, ERK1/2 activated at lipid rafts require the participation of IQGAP1. Furthermore, we demonstrate that scaffold usage markedly influences the biological outcome of Ras site-specific signals. These results disclose an unprecedented spatial regulation of ERK1/2 substrate specificity, dictated by the microlocalization from which Ras signals originate and by the selection of specific scaffold proteins.

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.

Highlights d MiNETi (Mixed Network Integration) enables the integration of multi-omics datasets d MiNETi provides an integrated view of HRAS signaling from different subcellular sites d HRAS controls its interactome, phosphoproteome, and transcriptome site specifically d HRAS regulates cell migration and p53-mediated apoptosis from endomembranes

H-Ras must adhere to the plasma membrane to be functional. This is accomplished by posttranslational modifications, including palmitoylation, a reversible process whereby H-Ras traffics between the plasma membrane and the Golgi complex. At the plasma membrane, H-Ras has been proposed to occupy distinct sublocations, depending on its activation status: lipid rafts/detergent-resistant membrane fractions when bound to GDP, diffusing to disordered membrane/soluble fractions in response to GTP loading. Herein, we demonstrate that H-Ras sublocalization is dictated by its degree of palmitoylation in a cell type-specific manner. Whereas H-Ras localizes to detergent-resistant membrane fractions in cells with low palmitoylation activity, it locates to soluble membrane fractions in lineages where it is highly palmitoylated. Interestingly, in both cases GTP loading results in H-Ras diffusing away from its original sublocalization. Moreover, tilting the equilibrium between palmitoylation and depalmitoylation processes can substantially alter H-Ras segregation and, subsequently, its biochemical and biological functions. Thus, the palmitoylation/depalmitoylation balance not only regulates H-Ras cycling between endomembranes and the plasma membrane but also serves as a key orchestrator of H-Ras lateral diffusion between different types of plasma membrane and thereby of H-Ras signaling.

The expression of MIC genes on enterocytes under stressful conditions and their function as ligands of intraepithelial gammadelta and CD8 T cells, together with the data presented here suggest a potential role of MIC genes in the pathogenesis of CD.