Ras GTPases mediate a wide variety of cellular processes by converting a multitude of extracellular stimuli into specific biological responses including proliferation, differentiation and survival. In mammalian cells, three ras genes encode four Ras isoforms (H-Ras, K-Ras4A, KRas4B and N-Ras) that are highly homologous but functionally distinct. Differences between the isoforms, including their post-translational modifications and intracellular sorting, mean that Ras has emerged as an important model system of compartmentalised signalling and membrane biology. Ras isoforms in different subcellular locations are proposed to recruit distinct upstream and downstream accessory proteins and activate multiple signalling pathways. Here, we summarise data relating to isoform specific signalling, its role in disease and the mechanisms promoting compartmentalised signalling. Further understanding of this field will reveal the role of Ras signalling in development, cellular homeostasis and cancers and may suggest new therapeutic approaches.

The ubiquitously expressed major Ras isoforms: H-, Kand N-Ras, are highly conserved, yet exhibit different biological outputs. We have compared the relative efficiencies with which epidermal or hepatocyte growth factor activates Ras isoforms and the requirement for specific isoforms in the activation of downstream pathways. We find that the relative coupling efficiencies to each Ras isoform are conserved between stimuli. Furthermore, in both cases, inhibition of receptor endocytosis led to reduced N-and H-Ras activation, but K-Ras was unaffected. Acute knockdown of each isoform with siRNA allows endogenous Ras isoform function and abundance to be probed. This revealed that there is significant variation in the contribution of individual isoforms to total Ras across a panel of cancer cell lines although typically KXNbH. Intriguingly, cancer cell lines where a significant fraction of endogenous Ras is oncogenically mutated showed attenuated activation of canonical Ras effector pathways. We profiled the contribution of each Ras isoform to the total Ras pool allowing interpretation of the effect of isoform-specific knockdown on signalling outcomes. In contrast to previous studies indicating preferential coupling of isoforms to Raf and PtdIns-3-kinase pathways, we find that endogenous Ras isoforms show no specific coupling to these major Ras pathways.

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The ErbB-4 receptors are unique in the EGFR/ErbB family for the ability to associate with WW domain-containing proteins. To identify new ligands of the cytoplasmic tail of ErbB-4, we panned a brain cDNA phage library with ErbB-4 peptides containing sequence motifs corresponding to putative docking sites for class-I WW domains. This approach led to identification of AIP4/Itch, a member of the Nedd4-like family of E3 ubiquitin protein ligases, as a protein that specifically interacts with and ubiquitinates ErbB-4 in vivo. Interaction with the ErbB-4 receptors occurs via the WW domains of AIP4/Itch. Functional analyses demonstrate that AIP4/Itch is recruited to the ErbB-4 receptor to promote its polyubiquitination and degradation, thereby regulating stability of the receptor and access of receptor intracellular domains to the nuclear compartment. These findings expand our understanding of the mechanisms contributing to the integrity of the ErbB signaling network and mechanistically link the cellular ubiquitination pathway of AIP4/Itch to the ErbB-4 receptor.

Differential subcellular compartmentalization of the three main Ras isoforms (H‐Ras, N‐Ras and K‐Ras) is believed to underlie their biological differences. Modulatable interactions between cellular membranes and Ras C‐terminal hypervariable region motifs determine differences in trafficking and the relative proportions of each isoform in cell‐surface signalling nanoclusters and intracellular endoplasmic reticulum/Golgi, endosomal and mitochondrial compartments. Ras regulators, effectors and scaffolds are also differentially distributed, potentially enabling preferential coupling to specific signalling pathways in each subcellular location. Here we summarize the mechanisms underlying compartment‐specific Ras signalling and the outputs generated.

Oncogenic Ras mutations render the protein constitutively active and promote tumorigenesis via chronic stimulation of effector pathways. In A549 lung adenocarcinoma approx. 50% of the total Ras population is constitutively active, yet these cells display only weak activation of the effectors: ERK1/2 (extracellular-signal-regulated kinase 1/2) and Akt. In order to identify key negative regulators of oncogenic Ras signalling we performed a phosphatome RNAi (RNA interference) screen in A549 cells and ranked their effects on phosphorylation of Ser473 of Akt. As expected, the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) emerged as a leading hit: knockdown elevated Akt activation to 70% of maximal generated by acute EGF (epidermal growth factor) stimulation. Importantly, we identified other phosphatases with similar potencies including PTPN2 (T-cell protein tyrosine phosphatase; also known as TC-PTP) and PTPRJ (protein tyrosine phosphatase receptor type J; also known as DEP-1/CD148). Potentiation of Akt phosphorylation by knockdown of PTEN or PTPRJ was contingent on the presence of oncogenic K-Ras. Our data reveal a synergy between oncogene function and the loss of a tumour suppressor within the same pathway that was necessary for full effector activation since each alone failed to elicit significant Akt phosphorylation. Taken together, these data reveal potent regulators of Akt signalling which contribute to ameliorating the consequences of oncogenic K-Ras activity.

Aberrant expression of the epidermal growth factor receptor family has been implicated in the pathogenesis and progression of breast cancer and associated with poor prognosis. To evaluate the prognostic impact of the ErbB receptors expression profile, we analyzed a well-characterized series of 145 primary breast carcinomas for the simultaneous expression of epidermal growth factor receptor (EGFR/HER-1), ErbB-2 (HER-2), ErbB-3 (HER-3), and ErbB-4 (HER-4), using immunohistochemistry. Tumors were considered negative or positive for each marker when less than or more than 25% of the cancer cells were immunopositive. Expression of EGFR, ErbB-2, ErbB-3, and ErbB-4 was observed in 31 (21.4%), 65 (44.8%), 72 (49.7%), and 81 (55.9%) of the cases, respectively. There were significant associations between EGFR expression and pT status (P = 0.01), and between ErbB-3 expression and pN (P = 0.003), menopausal (P = 0.01) and PR (P < 0.001) status. The majority of the cases co-expressed two or more receptors. ErbB-3 resulted positive in 51/81 (63.0%) of the ErbB-4 positive cases and ErbB-3/ErbB-4 co-expression was statistically significant (P = 0.0003). As expected, ErbB-2 expression was associated with reduced overall survival at 15 years of follow-up (P = 0.04), even after adjusting for a series of other prognostic factors (P = 0.05). Moreover, cumulative analysis of ErbB-2/3/4 expression showed a strong positive association between higher total ErbB-2/3/4 expression score and worse prognosis (P = 0.002). The simultaneous expression in cancer cells of more than one ErbB receptor identifies a subset of breast cancer patients at high risk for poor survival.

The research and treatment of non-small cell lung cancer (NSCLC) have achieved some important advances in recent years. Nonetheless, the overall survival rates for NSCLC remain low, indicating the importance to effectively develop new therapies and improve current approaches. The understanding of the function of different biomarkers involved in NSCLC progression, survival and response to therapy are important for the development of early detection tools and treatment options. Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (K-RAS) are two of the main significant biomarkers for the management of NSCLC. Mutations in these genes were associated with development and response to therapies. For example, the use of small molecule tyrosine kinase (TK) inhibitors and immunotherapy has led to benefits in some, but not all patients with altered EGFR. In contrast, there is still no effective approved drug to act upon patients harbouring K-RAS mutations. In addition, K-RAS mutations have been associated with lack of activity of TK inhibitors. However, promising approaches aimed to inhibit mutant K-RAS are currently under study. Therefore, this review will discuss these approaches and also EGFR therapies, and hopefully, it will draw attention to the need of continued research in the field in order to improve the outcomes in NSCLC patients.