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.     

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.     

Oncogenic Ras in tumour progression and metastasis

The ras genes give rise to a family of related GTP-binding proteins that exhibit potent transforming potential. Mutational activation of Ras proteins promotes oncogenesis by disturbing a multitude of cellular processes, such as gene expression, cell cycle progression and cell proliferation, as well as cell survival, and cell migration. Ras signalling pathways are well known for their involvement in tumour initiation, but less is known about their contribution to invasion and metastasis. This review summarises the role and mechanisms of Ras signalling, especially the role of the Ras effector cascade Raf/MEK/ERK, as well as the phosphatidylinositol 3-kinase/Akt pathway in Ras-mediated transformation and tumour progression. In addition, it discusses the impact of Rho GTPases on Ras-mediated transformation and metastasis.     

RalGDS family members couple Ras to Ral signalling and that's not all

Ras proteins function as molecular switches that are activated in response to signalling pathways initiated by various extracellular stimuli and subsequently bind to numerous effector proteins leading to the activation of several signalling cascades within the cell. Ras and Ras-related proteins belong to a large superfamily of small GTPases characterized by significant sequence and function similarities. Several evidence indicate the existence of complex signalling networks that link Ras with its relatives in the family. A key role in this cross-talk is played by guanine nucleotide exchange factors (GEFs) that serve both as regulators and as effectors of Ras family proteins. The members of the RalGDS family, RalGDS, RGL, RGL2/Rlf and RGL3, can interact with activated Ras through their Ras Binding Domain (RBD), but may function as effectors for other Ras family members. They possess a REM-CDC25 homology region like RasGEFs, but specifically activate only RalA and RalB and not Ras or other Ras-related small GTPases. In this review we provide an update on this recently discovered family of GEFs, highlighting their crucial role in coupling activated Ras to activation of Ral, thus regulating several fundamental cell processes, and also discussing some evidence supporting Ras-independent additional functions of RalGDS proteins.     

SH3‐class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth

Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras‐like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras‐subfamily‐specific guanine nucleotide exchange factors (RasGEFs). Although Ras‐protein networks are essential for virulence in all pathogenic fungi, the importance of RasGEF proteins is largely unexplored. A. fumigatus encodes four putative RasGEFs that represent three separate classes of RasGEF proteins (SH3‐, Ras guanyl nucleotide‐releasing protein [RasGRP]–, and LTE‐class), each with fungus‐specific attributes. Here, we show that the SH3‐class and RasGRP‐class RasGEFs are required for properly timed polarity establishment during early growth and branch emergence as well as for cell wall stability. Further, we show that SH3‐class RasGEF activity is essential for polarity establishment and maintenance, a phenotype that is, at least, partially independent of the major A. fumigatus Ras proteins, RasA and RasB. Finally, loss of both SH3‐class RasGEFs resulted in avirulence in multiple models of invasive aspergillosis. Together, our findings suggest that RasGEF activity is essential for the integration of multiple signalling networks to drive invasive growth in A. fumigatus.     

The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.     

TRADD contributes to tumour suppression by regulating ULF-dependent p19Arf ubiquitylation

Tumour necrosis factor receptor (TNFR)-associated death domain (TRADD) protein is a central adaptor in the TNFR1 signalling complex that mediates both cell death and inflammatory signals. Here, we report that Tradd deficiency in mice accelerated tumour formation in a chemical-induced carcinogenesis model independently of TNFR1 signalling. In vitro, primary cells lacking TRADD were less susceptible to HRas-induced senescence and showed a reduced level of accumulation of the p19(Arf) tumour suppressor protein. Our data indicate that TRADD shuttles dynamically from the cytoplasm into the nucleus to modulate the interaction between p19(Arf) and its E3 ubiquitin ligase ULF, thereby promoting p19(Arf) protein stability and tumour suppression. These results reveal a previously unknown tumour-suppressive role for nuclear TRADD, augmenting its long-established cytoplasmic functions in inflammatory and immune signalling cascades. Our findings also make an important contribution to the rapidly expanding field of p19(Arf) post-translational regulation.     

Rheb is an essential regulator of S6K in controlling cell growth in Drosophila

Understanding the mechanisms through which multicellular organisms regulate cell, organ and body growth is of relevance to developmental biology and to research on growth-related diseases such as cancer. Here we describe a new effector in growth control, the small GTPase Rheb (Ras homologue enriched in brain). Mutations in the Drosophila melanogaster Rheb gene were isolated as growth-inhibitors, whereas overexpression of Rheb promoted cell growth. Our genetic and biochemical analyses suggest that Rheb functions downstream of the tumour suppressors Tsc1 (tuberous sclerosis 1)-Tsc2 in the TOR (target of rapamycin) signalling pathway to control growth, and that a major effector of Rheb function is ribosomal S6 kinase (S6K).     

A Ras1-Cdc24 signal transduction pathway mediates thermotolerance in the fungal pathogen Cryptococcus neoformans

Pathogenic microorganisms must precisely regulate morphogenesis to survive and proliferate within an infected host. This regulation is often controlled by conserved signal transduction pathways that direct morphological changes in varied species. One such pathway, whose components include Ras proteins and the PAK kinase Ste20, allows the human fungal pathogen Cryptococcus neoformans to grow at high temperature. Previously, we found that Ras1 signalling is required for differentiation, thermotolerance and pathogenesis in C. neoformans. We show here that the guanine nucleotide exchange factor Cdc24 is a Ras1 effector in C. neoformans to mediate the ability of this fungus to grow at high temperature and to cause disease. In addition, we provide evidence that the Ras1-Cdc24 signalling cascade functions specifically through one of the three Cdc42/Rac1 homologues in C. neoformans. In conclusion, our studies illustrate how components of conserved signalling cascades can be specialized for different downstream functions, such as pathogenesis.     

Rheb promotes cell growth as a component of the insulin/TOR signalling network

Insulin signalling is a potent stimulator of cell growth and has been proposed to function, at least in part, through the conserved protein kinase TOR (target of rapamycin) [corrected]. Recent studies suggest that the tuberous sclerosis complex Tsc1-Tsc2 may couple insulin signalling to Tor activity [corrected]. However, the regulatory mechanism involved remains unclear, and additional components are most probably involved. In a screen for novel regulators of growth, we identified Rheb (Ras homologue enriched in brain), a member of the Ras superfamily of GTP-binding proteins. Increased levels of Rheb in Drosophila melanogaster promote cell growth and alter cell cycle kinetics in multiple tissues. In mitotic tissues, overexpression of Rheb accelerates passage through G1-S phase without affecting rates of cell division, whereas in endoreplicating tissues, Rheb increases DNA ploidy. Mutation of Rheb suspends larval growth and prevents progression from first to second instar. Genetic and biochemical tests indicate that Rheb functions in the insulin signalling pathway downstream of Tsc1-Tsc2 and upstream of TOR. Levels of rheb mRNA are rapidly induced in response to protein starvation, and overexpressed Rheb can drive cell growth in starved animals, suggesting a role for Rheb in the nutritional control of cell growth.     

Ras oncogenes: split personalities

Extensive research on the Ras proteins and their functions in cell physiology over the past 30 years has led to numerous insights that have revealed the involvement of Ras not only in tumorigenesis but also in many developmental disorders. Despite great strides in our understanding of the molecular and cellular mechanisms of action of the Ras proteins, the expanding roster of their downstream effectors and the complexity of the signalling cascades that they regulate indicate that much remains to be learnt.     

AKAP-Lbc enhances cyclic AMP control of the ERK1/2 cascade

Mitogen-activated protein kinase (MAPK) cascades propagate a variety of cellular activities. Processive relay of signals through RAF-MEK-ERK modulates cell growth and proliferation. Signalling through this ERK cascade is frequently amplified in cancers, and drugs such as sorafenib (which is prescribed to treat renal and hepatic carcinomas) and PLX4720 (which targets melanomas) inhibit RAF kinases. Natural factors that influence ERK1/2 signalling include the second messenger cyclic AMP. However, the mechanisms underlying this cascade have been difficult to elucidate. We demonstrate that the A-kinase-anchoring protein AKAP-Lbc and the scaffolding protein kinase suppressor of Ras (KSR-1) form the core of a signalling network that efficiently relay signals from RAF, through MEK, and on to ERK1/2. AKAP-Lbc functions as an enhancer of ERK signalling by securing RAF in the vicinity of MEK1 and synchronizing protein kinase A (PKA)-mediated phosphorylation of Ser 838 on KSR-1. This offers mechanistic insight into cAMP-responsive control of ERK signalling events.     

Ras mutation cooperates with ��-catenin activation to drive bladder tumourigenesis

Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-Ras^Q61L or K-Ras^G12D) with an activating β-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor β-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo.     

Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane

Ras-GTP imaging studies using the Ras-binding domain (RBD) of the Ras effector c-Raf as a reporter for overexpressed Ras have produced discrepant results about the possible activation of Ras at the Golgi apparatus. We report that RBD oligomerization provides probes for visualization of endogenous Ras-GTP, obviating Ras overexpression and the side effects derived thereof. RBD oligomerization results in tenacious binding to Ras-GTP and interruption of Ras signalling. Trimeric RBD probes fused to green fluorescent protein report agonist-induced endogenous Ras activation at the plasma membrane (PM) of COS-7, PC12 and Jurkat cells, but do not accumulate at the Golgi. PM illumination is exacerbated by Ras overexpression and its sensitivity to dominant-negative RasS17N and pharmacological manipulations matches Ras-GTP formation assessed biochemically. Our data illustrate that endogenous Golgi-located Ras is not under the control of growth factors and argue for the PM as the predominant site of agonist-induced Ras activation.     

Bradykinin signalling to MAP kinase: cell-specific connections versus principle mitogenic pathways

Mitogenic signalling pathways from G protein-coupled receptors (GPCRs) to the mitogen-activated protein kinase (MAPK) cascade may involve alpha- or betagamma-subunits of heterotrimeric G proteins, receptor or non-receptor tyrosine kinases, adaptor molecules, phosphoinositide 3-kinases, protein kinase C, and probably other proteins. The majority of models describing the connection of different signalling proteins within a mitogenic pathway are based on experimental data obtained by co- and overexpression of epitope-tagged MAPK together with the respective GPCR and other signalling proteins of interest in transfectable cell lines. Here the link of the bradykinin B2 receptor (B2R) to MAPK in the COS-7 cell expression system is compared with mitogenic signalling pathways of bradykinin in various tumour cell lines. It becomes evident that in natural or tumour cells expressing individual amounts and different isoforms of signalling proteins completely other relations between B2R and MAPK may exist than in COS-7 cells, suggesting a high degree of cellular specificity in mitogenic signalling.     

Bcl-2 promotes premature senescence induced by oncogenic Ras

The expression of the apoptosis inhibitory protein, Bcl-2, is increased in naturally senescing human fibroblasts and upon induction of their senescence-like growth arrest by oxidative stress, implying its role in maintaining their extended viability. Oncogenic Ras(V12) protein induces signaling cascades that result in the premature senescence of primary fibroblast cells, which are insensitive to oncogene-dependent apoptosis. Here we show that constitutive expression of Bcl-2 accelerates selected features of the Ras-induced senescence program in primary human fibroblasts. Yet, Bcl-2 also inhibits fibroblast apoptosis induced by exogenous H(2)O(2), while both signals induce an increased endogenous Bcl-2 expression in these cells. Together, these data suggest a context-dependent phenotypic function of Bcl-2 in the regulation of overlapping cell fate specification programs, with potential implications for both physiology and multistep tumorigenesis.