Ras85D: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Effects of Mutation | Ras as Oncogene | References

Gene name - Ras oncogene at 85D

Synonyms - Ras1

Cytological map position - 85D11--85D27

Function - signal transduction protein

Keywords - RAS pathway and EGF receptor-ligand complex, eye, wing, terminal group, FGF signaling, oncogene

Symbol - Ras85D

FlyBase ID: FBgn0003205

Genetic map position - 3-[49]

Classification - GTPase

Cellular location - cytoplasmic

NCBI link: Entrez Gene
Ras85D orthologs: Biolitmine
Recent literature
Vega-Cuesta, P., Ruiz-Gomez, A., Molnar, C., Organista, M. F., Resnik-Docampo, M., Falo-Sanjuan, J., Lopez-Varea, A. and de Celis, J. F. (2020). Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability. Dev Biol. PubMed ID: 32061885
Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. This study finds that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.
Rambur, A., Lours-Calet, C., Beaudoin, C., Bunay, J., Vialat, M., Mirouse, V., Trousson, A., Renaud, Y., Lobaccaro, J. A., Baron, S., Morel, L. and de Joussineau, C. (2020). Sequential Ras/MAPK and PI3K/AKT/mTOR pathways recruitment drives basal extrusion in the prostate-like gland of Drosophila. Nat Commun 11(1): 2300. PubMed ID: 32385236
One of the most important but less understood step of epithelial tumourigenesis occurs when cells acquire the ability to leave their epithelial compartment. This phenomenon, described as basal epithelial cell extrusion (basal extrusion), represents the first step of tumour invasion. Implication of emblematic signalling pathways such as Ras/MAP Kinase and PI3K/protein kinase B (AKT)/mTOR signalling pathways, is scarcely described in this phenomenon. This paper reports a unique model of basal extrusion in the Drosophila accessory gland. There, it was demonstrated that both Ras/MAPK and PI3K/AKT/mTOR pathways are necessary for basal extrusion. Furthermore, as in prostate cancer, this study shows that these pathways are co-activated. This occurs through set up of Epidermal Growth Factor Receptor (EGFR) and Insulin Receptor (InR) dependent autocrine loops, a phenomenon that, considering human data, could be relevant for prostate cancer.
Sawyer, J. K., Kabiri, Z., Montague, R. A., Allen, S. R., Stewart, R., Paramore, S. V., Cohen, E., Zaribafzadeh, H., Counter, C. M. and Fox, D. T. (2020). Exploiting codon usage identifies intensity-specific modifiers of Ras/MAPK signaling in vivo. PLoS Genet 16(12): e1009228. PubMed ID: 33296356
Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. By altering codon usage as a novel platform to control signaling output, the Drosophila genome was screened for modifiers specific to either weak or strong Ras-driven eye phenotypes. This screen enriched for regions of the genome not previously connected with Ras phenotypic modification. The underlying gene from one modifier mapped to the ribosomal gene RpS21. In multiple contexts, it was shown that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.
Morris, O., Deng, H., Tam, C. and Jasper, H. (2020). Warburg-like metabolic reprogramming in aging intestinal stem cells contributes to tissue hyperplasia. Cell Rep 33(8): 108423. PubMed ID: 33238124
In many tissues, stem cell (SC) proliferation is dynamically adjusted to regenerative needs. How SCs adapt their metabolism to meet the demands of proliferation and how changes in such adaptive mechanisms contribute to age-related dysfunction remain poorly understood. This study identified mitochondrial Ca(2+) uptake as a central coordinator of SC metabolism. Live imaging of genetically encodasensors in intestinal SCs (ISCs) of Drosophila reveals that mitochondrial Ca(2+) uptake transiently adapts electron transport chain flux to match energetic demand upon proliferative activation. This tight metabolic adaptation is lost in ISCs of old flies, as declines in mitochondrial Ca(2+) uptake promote a "Warburg-like" metabolic reprogramming toward aerobic glycolysis. This switch mimics metabolic reprogramming by the oncogene Ras(V12) and enhances ISC hyperplasia. These data identify a critical mechanism for metabolic adaptation of tissue SCs and reveal how its decline sets aging SCs on a metabolic trajectory reminiscent of that seen upon oncogenic transformation.
Krautz, R., Khalili, D. and Theopold, U. (2020). Tissue-autonomous immune response regulates stress signalling during hypertrophy. Elife 9. PubMed ID: 33377870
Postmitotic tissues are incapable of replacing damaged cells through proliferation, but need to rely on buffering mechanisms to prevent tissue disintegration. By constitutively activating the Ras/MAPK-pathway via Ras(V12)-overexpression in the postmitotic salivary glands of Drosophila larvae, the glands adaptability to growth signals and induced hypertrophy was overridden. The accompanied loss of tissue integrity, recognition by cellular immunity and cell death are all buffered by blocking stress signalling through a genuine tissue-autonomous immune response. This novel, spatio-temporally tightly regulated mechanism relies on the inhibition of a feedback-loop in the JNK-pathway by the immune effector and antimicrobial peptide Drosomycin. While this interaction might allow growing salivary glands to cope with temporary stress, continuous Drosomycin expression in Ras(V12)-glands favors unrestricted hypertrophy. These findings indicate the necessity to refine therapeutic approaches that stimulate immune responses by acknowledging their possible, detrimental effects in damaged or stressed tissues.
Dong, Y. L., Vadla, G. P., Lu, J. J., Ahmad, V., Klein, T. J., Liu, L. F., Glazer, P. M., Xu, T. and Chabu, C. Y. (2021). Cooperation between oncogenic Ras and wild-type p53 stimulates STAT non-cell autonomously to promote tumor radioresistance. Commun Biol 4(1): 374. PubMed ID: 33742110
Oncogenic RAS mutations are associated with tumor resistance to radiation therapy. Cell-cell interactions in the tumor microenvironment (TME) profoundly influence therapy outcomes. However, the nature of these interactions and their role in Ras tumor radioresistance remain unclear. This study used Drosophila oncogenic Ras tissues and human Ras cancer cell radiation models to address these questions. It was discovered that cellular response to genotoxic stress cooperates with oncogenic Ras to activate JAK/STAT non-cell autonomously in the TME. Specifically, p53 is heterogeneously activated in Ras tumor tissues in response to irradiation. This mosaicism allows high p53-expressing Ras clones to stimulate JAK/STAT cytokines, which activate JAK/STAT in the nearby low p53-expressing surviving Ras clones, leading to robust tumor re-establishment. Blocking any part of this cell-cell communication loop re-sensitizes Ras tumor cells to irradiation. These findings suggest that coupling STAT inhibitors to radiotherapy might improve clinical outcomes for Ras cancer patients.
Rackley, B., Seong, C. S., Kiely, E., Parker, R. E., Rupji, M., Dwivedi, B., Heddleston, J. M., Giang, W., Anthony, N., Chew, T. L. and Gilbert-Ross, M. (2021). The level of oncogenic Ras determines the malignant transformation of Lkb1 mutant tissue in vivo. Commun Biol 4(1): 142. PubMed ID: 33514834
The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. This study generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. Low-level expression of oncogenic Ras (RasLow) was shown to promote the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHigh) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, this study has characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. This molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS were shown to exhibit worse overall survival and increased AMPK activation. These results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.
Cheng, K. C., Chen, Y. H., Wu, C. L., Lee, W. P., Cheung, C. H. A. and Chiang, H. C. (2021). Rac1 and Akt Exhibit Distinct Roles in Mediating Abeta-Induced Memory Damage and Learning Impairment. Mol Neurobiol. PubMed ID: 34273104
Accumulated β-amyloid (Aβ) in the brain is the hallmark of Alzheimer's disease (AD). Despite Aβ accumulation is known to trigger cellular dysfunctions and learning and memory damage, the detailed molecular mechanism remains elusive. Recent studies have shown that the onset of memory impairment and learning damage in the AD animal is different, suggesting that the underlying mechanism of the development of memory impairment and learning damage may not be the same. In the current study, with the use of Aβ42 transgenic flies as models, this study found that Aβ induces memory damage and learning impairment via differential molecular signaling pathways. In early stage, Aβ activates both Ras and PI3K to regulate Rac1 activity, which affects mostly on memory performance. In later stage, PI3K-Akt is strongly activated by Aβ, which leads to learning damage. Moreover, reduced Akt, but not Rac1, activity promotes cell viability in the Aβ42 transgenic flies, indicating that Akt and Rac1 exhibit differential roles in Aβ regulating toxicity. Taken together, different molecular and cellular mechanisms are involved in Aβ-induced learning damage and memory decline; thus, caution should be taken during the development of therapeutic intervention in the future.
Chen, D., Roychowdhury-Sinha, A., Prakash, P., Lan, X., Fan, W., Goto, A. and Hoffmann, J. A. (2021). A time course transcriptomic analysis of host and injected oncogenic cells reveals new aspects of Drosophila immune defenses. Proc Natl Acad Sci U S A 118(12). PubMed ID: 33737397
Oncogenic RasV12 cells injected into adult males proliferated massively after a lag period of several days, and led to the demise of the flies after 2 to 3 wk. The injection induced an early massive transcriptomic response that, unexpectedly, included more than 100 genes encoding chemoreceptors of various families. The kinetics of induction and the identities of the induced genes differed markedly from the responses generated by injections of microbes. Subsequently, hundreds of genes were up-regulated, attesting to intense catabolic activities in the flies, active tracheogenesis, and cuticulogenesis, as well as stress and inflammation-type responses. At 11 d after the injections, GFP-positive oncogenic cells isolated from the host flies exhibited a markedly different transcriptomic profile from that of the host and distinct from that at the time of their injection, including in particular up-regulated expression of genes typical for cells engaged in the classical antimicrobial response of Drosophila.
Beatty, J. S., Molnar, C., Luque, C. M., de Celis, J. F. and Martin-Bermudo, M. D. (2021). EGFRAP encodes a new negative regulator of the EGFR acting in both normal and oncogenic EGFR/Ras-driven tissue morphogenesis. PLoS Genet 17(8): e1009738. PubMed ID: 34411095
Activation of Ras signaling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, it is imperative to identify those genes cooperating with activated Ras in driving tumoral growth. This work identified a novel EGFR inhibitor, which was named EGFRAP, for EGFR adaptor protein. Elimination of EGFRAP potentiates activated Ras-induced overgrowth in the Drosophila wing imaginal disc. EGFRAP interacts physically with the phosphorylated form of EGFR via its SH2 domain. EGFRAP is expressed at high levels in regions of maximal EGFR/Ras pathway activity, such as at the presumptive wing margin. In addition, EGFRAP expression is up-regulated in conditions of oncogenic EGFR/Ras activation. Normal and oncogenic EGFR/Ras-mediated upregulation of EGRAP levels depend on the Notch pathway. Elimination of EGFRAP does not affect overall organogenesis or viability. However, simultaneous downregulation of EGFRAP and its ortholog PVRAP results in defects associated with increased EGFR function. Based on these results, it is proposed that EGFRAP is a new negative regulator of the EGFR/Ras pathway, which, while being required redundantly for normal morphogenesis, behaves as an important modulator of EGFR/Ras-driven tissue hyperplasia. It is suggested that the ability of EGFRAP to functionally inhibit the EGFR pathway in oncogenic cells results from the activation of a feedback loop leading to increase EGFRAP expression. This could act as a surveillance mechanism to prevent excessive EGFR activity and uncontrolled cell growth.
Kohashi, K., Mori, Y., Narumi, R., Kozawa, K., Kamasaki, T., Ishikawa, S., Kajita, M., Kobayashi, R., Tamori, Y. and Fujita, Y. (2021). Sequential oncogenic mutations influence cell competition. Curr Biol. PubMed ID: 34314674 At the initial stage of carcinogenesis, newly emerging transformed cells are often eliminated from epithelial layers via cell competition with the surrounding normal cells. For instance, when surrounded by normal cells, oncoprotein RasV12-transformed cells are extruded into the apical lumen of epithelia. During cancer development, multiple oncogenic mutations accumulate within epithelial tissues. However, it remains elusive whether and how cell competition is also involved in this process. Using a mammalian cell culture model system, this study investigated what happens upon the consecutive mutations of Ras and tumor suppressor protein Scribble. When Ras mutation occurs under the Scribble-knockdown background, apical extrusion of Scribble/Ras double-mutant cells is strongly diminished. In addition, at the boundary with Scribble/Ras cells, Scribble-knockdown cells frequently undergo apoptosis and are actively engulfed by the neighboring Scribble/Ras cells. The comparable apoptosis and engulfment phenotypes are also observed in Drosophila epithelial tissues between Scribble/Ras double-mutant and Scribble single-mutant cells. Furthermore, mitochondrial membrane potential is enhanced in Scribble/Ras cells, causing the increased mitochondrial reactive oxygen species (ROS). Suppression of mitochondrial membrane potential or ROS production diminishes apoptosis and engulfment of the surrounding Scribble-knockdown cells, indicating that mitochondrial metabolism plays a key role in the competitive interaction between double- and single-mutant cells. Moreover, mTOR (mechanistic target of rapamycin kinase) acts downstream of these processes. These results imply that sequential oncogenic mutations can profoundly influence cell competition, a transition from loser to winner. Further studies would open new avenues for cell competition-based cancer treatment, thereby blocking clonal expansion of more malignant populations within tumors (Kohashi, 2021).
Enomoto, M., Takemoto, D. and Igaki, T. (2021). Interaction between Ras and Src clones causes interdependent tumor malignancy via Notch signaling in Drosophila. Dev Cell 56(15): 2223-2236.e2225. PubMed ID: 34324859
Cancer tissue often comprises multiple tumor clones with distinct oncogenic alterations such as Ras or Src activation, yet the mechanism by which tumor heterogeneity drives cancer progression remains elusive. This study shows in Drosophila imaginal epithelium that clones of Ras- or Src-activated benign tumors interact with each other to mutually promote tumor malignancy. Mechanistically, Ras-activated cells upregulate the cell-surface ligand Delta while Src-activated cells upregulate its receptor Notch, leading to Notch activation in Src cells. Elevated Notch signaling induces the transcriptional repressor Zfh1/ZEB1, which downregulates E-cadherin and cell death gene hid, leading to Src-activated invasive tumors. Simultaneously, Notch activation in Src cells upregulates the cytokine Unpaired/IL-6, which activates JAK-STAT signaling in neighboring Ras cells. Elevated JAK-STAT signaling upregulates the BTB-zinc-finger protein Chinmo, which downregulates E-cadherin and thus generates Ras-activated invasive tumors. These findings provide a mechanistic explanation for how tumor heterogeneity triggers tumor progression via cell-cell interactions.
Zhu, J. Y., Huang, X., Fu, Y., Wang, Y., Zheng, P., Liu, Y. and Han, Z. (2021). Pharmacological or genetic inhibition of hypoxia signaling attenuates oncogenic RAS-induced cancer phenotypes. Dis Model Mech. PubMed ID: 34580712
Oncogenic Ras mutations are highly prevalent in hematopoietic malignancies. However, it is difficult to directly target oncogenic RAS proteins for therapeutic intervention. This study has developed a Drosophila Acute Myeloid Leukemia (AML) model induced by human KRASG12V, which exhibits a dramatic increase in myeloid-like leukemia cells. Both genetic and drug screens were performed using this model. The genetic screen identified 24 candidate genes able to attenuate the oncogenic RAS-induced phenotype, including two key hypoxia pathway genes HIF1A and ARNT (HIF1B). The drug screen revealed echinomycin, an inhibitor of HIF1A, could effectively attenuate the leukemia phenotype caused by KRASG12V. Furthermore, this study showed that echinomycin treatment could effectively suppress oncogenic RAS-driven leukemia cell proliferation using both human leukemia cell lines and a mouse xenograft model. These data suggest that inhibiting the hypoxia pathway could be an effective treatment approach for oncogenic RAS-induced cancer phenotype, and that echinomycin is a promising targeted drug to attenuate oncogenic RAS-induced cancer phenotypes.
Cong, B., Nakamura, M., Sando, Y., Kondo, T., Ohsawa, S. and Igaki, T. (2021). JNK and Yorkie drive tumor malignancy by inducing L-amino acid transporter 1 in Drosophila. PLoS Genet 17(11): e1009893. PubMed ID: 34780467.
Identifying a common oncogenesis pathway among tumors with different oncogenic mutations is critical for developing anti-cancer strategies. This study performed transcriptome analyses on two different models of Drosophila malignant tumors caused by Ras activation with cell polarity defects (RasV12/scrib-/-) or by microRNA bantam overexpression with endocytic defects (bantam/rab5-/-), followed by an RNAi screen for genes commonly essential for tumor growth and malignancy. Juvenile hormone Inducible-21 (JhI-21), a Drosophila homolog of the L-amino acid transporter 1 (LAT1), was identified is upregulated in these malignant tumors with different oncogenic mutations and knocking down of JhI-21 strongly blocked their growth and invasion. JhI-21 expression was induced by simultaneous activation of c-Jun N-terminal kinase (JNK) and Yorkie (Yki) in these tumors and thereby contributed to tumor growth and progression by activating the mTOR-S6 pathway. Pharmacological inhibition of LAT1 activity in Drosophila larvae significantly suppressed growth of RasV12/scrib-/- tumors. Intriguingly, LAT1 inhibitory drugs did not suppress growth of bantam/rab5-/- tumors and overexpression of bantam rendered RasV12/scrib-/- tumors unresponsive to LAT1 inhibitors. Further analyses with RNA sequencing of bantam-expressing clones followed by an RNAi screen suggested that bantam induces drug resistance against LAT1 inhibitors via downregulation of the TMEM135-like gene CG31157. These observations unveil an evolutionarily conserved role of LAT1 induction in driving Drosophila tumor malignancy and provide a powerful genetic model for studying cancer progression and drug resistance.
Manzanero-Ortiz, S., de Torres-Jurado, A., Hernandez-Rojas, R. and Carmena, A. (2021). Pilot RNAi Screen in Drosophila Neural Stem Cell Lineages to Identify Novel Tumor Suppressor Genes Involved in Asymmetric Cell Division. Int J Mol Sci 22(21). PubMed ID: 34768763
A connection between compromised asymmetric cell division (ACD) and tumorigenesis was proven some years ago using Drosophila larval brain neural stem cells, called neuroblasts (NBs), as a model system. Since then, it has been learned that compromised ACD does not always promote tumorigenesis, as ACD is an extremely well-regulated process in which redundancy substantially overcomes potential ACD failures. Considering this, a pilot RNAi screen was performed in Drosophila larval brain NB lineages using Ras(V)(12) scribble (scrib) mutant clones as a sensitized genetic background, in which ACD is affected but does not cause tumoral growth. First, as a proof of concept, this study has tested known ACD regulators in this sensitized background, such as lethal (2) giant larvae and warts. Although the downregulation of these ACD modulators in NB clones does not induce tumorigenesis, their downregulation along with Ras(V)(12) scrib does cause tumor-like overgrowth. Based on these results, 79 RNAi lines randomly screened detecting 15 potential novel ACD regulators/tumor suppressor genes. It is concluded that Ras(V)(12) scrib is a good sensitized genetic background in which to identify tumor suppressor genes involved in NB ACD, whose function could otherwise be masked by the high redundancy of the ACD process.
Zhou, Y., Liu, J. and Liu, J. L. (2022). Connecting Ras and CTP synthase in Drosophila. Exp Cell Res: 113155. PubMed ID: 35427600
CTP synthase (CTPS), the enzyme responsible for the last step of de novo synthesis of CTP, forms filamentous structures termed cytoophidia in all three domains of life. This study reports that oncogenic Ras regulates cytoophidium formation in Drosophila intestines. Overexpressing active Ras induces elongate and abundant cytoophidia in intestinal stem cells (ISCs) and enteroblasts (EBs). Knocking-down CTPS in ISCs/EBs suppresses the over proliferation phenotype induced by ectopic expression of active Ras. Moreover, disrupting cytoophidium formation increases the number of proliferating cells in the background of overexpressing active Ras. Therefore, these results demonstrate a link between Ras and CTPS (Zhou, 2022).
Chen, D., Lan, X., Huang, X., Huang, J., Zhou, X., Miao, Z., Ma, Y., Goto, A., Ji, S. and Hoffmann, J. A. (2023). Single Cell Analysis of the Fate of Injected Oncogenic RasV12 Cells in Adult Wild Type Drosophila. J Innate Immun 15(1): 442-467. PubMed ID: 36996781
=Dish-cultured oncogenic RasV12 cells into adult male flies and single cell transcriptomics was used to examine their destiny within the host after 11 days. The preinjection samples were identified in the 11-day postinjection samples in all 16 clusters of cells, of which 5 disappeared during the experiment in the host. The other cell clusters expanded and expressed genes involved in the regulation of cell cycle, metabolism, and development. In addition, three clusters expressed genes related to inflammation and defense. Predominant among these were genes coding for phagocytosis and/or characteristic for a plasmatocytes (the fly equivalent of macrophages). A pilot experiment indicated that the injection into flies of oncogenic cells, in which two of most strongly expressed genes had been previously silenced by RNA interference, into flies resulted in a dramatic reduction of their proliferation in the host flies as compared to controls. As has been shown earlier, the proliferation of the injected oncogenic cells in the adult flies is a hallmark of the disease and induces a wave of transcriptions in the experimental flies. We hypothesize that this results from a bitter dialogue between the injected cells and the host, while the experiments presented here should contribute to deciphering this dialogue.
Cappucci, U., Casale, A. M., Proietti, M., Marinelli, F., Giuliani, L. and Piacentini, L. (2022). WiFi Related Radiofrequency Electromagnetic Fields Promote Transposable Element Dysregulation and Genomic Instability in Drosophila melanogaster. Cells 11(24). PubMed ID: 36552798
Exposure to artificial radio frequency electromagnetic fields (RF-EMFs) has greatly increased in recent years, thus promoting a growing scientific and social interest in deepening the biological impact of EMFs on living organisms. The current legislation governing the exposure to RF-EMFs is based exclusively on their thermal effects, without considering the possible non-thermal adverse health effects from long term exposure to EMFs. This study investigated the biological non-thermal effects of low-level indoor exposure to RF-EMFs produced by WiFi wireless technologies, using Drosophila melanogaster as the model system. Flies were exposed to 2.4 GHz radiofrequency in a Transverse Electromagnetic (TEM) cell device to ensure homogenous controlled fields. Signals were continuously monitored during the experiments and regulated at non thermal levels. The results of this study demonstrate that WiFi electromagnetic radiation causes extensive heterochromatin decondensation and thus a general loss of transposable elements epigenetic silencing in both germinal and neural tissues. Moreover, the findings provide evidence that WiFi related radiofrequency electromagnetic fields can induce reactive oxygen species (ROS) accumulation, genomic instability, and behavioural abnormalities. Finally, this study demonstrated that WiFi radiation can synergize with Ras(V12) to drive tumor progression and invasion. All together, these data indicate that radiofrequency radiation emitted from WiFi devices could exert genotoxic effects in Drosophila and set the stage to further explore the biological effects of WiFi electromagnetic radiation on living organisms.
Cabrera, A. J. H., Gumbiner, B. M. and Kwon, Y. V. (2023). Remodeling of E-cadherin subcellular localization during cell dissemination. Mol Biol Cell 34(5): ar46. PubMed ID: 36989029
Given the role of E-cadherin (E-cad) in holding epithelial cells together, an inverse relationship between E-cad levels and cell invasion during the epithelial-mesenchymal transition and cancer metastasis has been well recognized. This study reports that E-cad is necessary for the invasiveness of Ras(V12)-transformed intestinal epithelial cells in Drosophila. E-cad/β-catenin disassembles at adherens junctions and assembles at invasive protrusions--the actin- and cortactin-rich invadopodium-like protrusions associated with the breach of the extracellular matrix (ECM)--during dissemination of Ras(V12)-transformed intestinal epithelial cells. Loss of E-cad impairs the elongation of invasive protrusions and attenuates the ability of Ras(V12)-transformed cells to compromise the ECM. Notably, E-cad and cortactin affect each other's localization to invasive protrusions. Given the essential roles of cortactin in cell invasion, these observations indicate that E-cad plays a role in the invasiveness of Ras(V12)-transformed intestinal epithelial cells by controlling cortactin localization to invasive protrusions. Thus this study demonstrates that E-cad is a component of invasive protrusions and provides molecular insights into the unconventional role of E-cad in cell dissemination in vivo.
Martinez-Abarca Millan, A., Soler Beatty, J., Valencia Exposito, A. and Martin-Bermudo, M. D. (2023). Drosophila as Model System to Study Ras-Mediated Oncogenesis. The Case of the Tensin Family of Proteins. Genes (Basel) 14(7). PubMed ID: 37510408
Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, tissue growth induced by oncogenic Ras is restrained by the induction of cellular senescence, and additional mutations are required to induce tumor progression. Therefore, identifying cooperating cancer genes is of paramount importance. Recently, the tensin family of focal adhesion proteins, TNS1-4, have emerged as regulators of carcinogenesis, yet their role in cancer appears somewhat controversial. Around 90% of human cancers are of epithelial origin. This study used the Drosophila wing imaginal disc epithelium as a model system to gain insight into the roles of two orthologs of human TNS2 and 4, blistery (by) and PVRAP, in epithelial cancer progression. This study has generated null mutations in PVRAP and found that, as is the case for by and mammalian tensins, PVRAP mutants are viable. This study also found that elimination of either PVRAP or by potentiates Ras(V12)-mediated wing disc hyperplasia. Furthermore, the results have unraveled a mechanism by which tensins may limit Ras oncogenic capacity, the regulation of cell shape and growth. These results demonstrate that Drosophila tensins behave as supressors of Ras-driven tissue hyperplasia, suggesting that the roles of tensins as modulators of cancer progression might be evolutionarily conserved.
Singh, J., Karunaraj, P., Luf, M., Pfleger, C. M. (2023). Lysines K117 and K147 play conserved roles in Ras activation from Drosophila to mammals. G3 (Bethesda), 13(11) PubMed ID: 37665961
Ras signaling plays an important role in growth, proliferation, and developmental patterning. Maintaining appropriate levels of Ras signaling is important to establish patterning in development and to prevent diseases such as cancer in mature organisms. The Ras protein is represented by Ras85D in Drosophila and by HRas, NRas, and KRas in mammals. In the past dozen years, multiple reports have characterized both inhibitory and activating ubiquitination events regulating Ras proteins. Inhibitory Ras ubiquitination mediated by Rabex-5 or Lztr1 is highly conserved between flies and mammals. Activating ubiquitination events at K117 and K147 have been reported in mammalian HRas, NRas, and KRas, but it is unclear if these activating roles of K117 and K147 are conserved in flies. Addressing a potential conserved role for these lysines in Drosophila Ras activation requires phenotypes strong enough to assess suppression. Therefore, oncogenic Ras, RasG12V, which biases Ras to the GTP-loaded active conformation, was used. Double mutants RasG12V,K117R and RasG12V,K147R and triple mutant RasG12V,K117R,K147R were created to prevent lysine-specific post-translational modification of K117, K147, or both, respectively. Their phenotypes were compared to RasG12V in the wing to reveal the roles of these lysines. Although RasG12V,K147R did not show compelling or quantifiable differences from RasG12V, RasG12V,K117R showed visible and quantifiable suppression compared to RasG12V, and triple mutant RasG12V,K117R,K147R showed dramatic suppression compared to RasG12V and increased suppression compared to RasG12V,K117R. These data are consistent with highly conserved roles for K117 and K147 in Ras activation from flies to mammals.

There are three homologs to vertebrate ras in Drosophila: Ras1 (more precisely termed Ras oncogene at 85D), Ras2 (or Ras oncogene at 64D) and Ras3, also referred to as Rap1. The three Ras proteins are functionally distinct. This overview will focus on Ras1, but before doing that, some information on Ras64D is desireable. Ras64D is implicated in vesicular trafficking in garland cells, a ring of cells around the esophagus implicated in the removal of toxic materials from the hemolymph by endocytosis. Ras64D is also expressed in the antennal-maxillary complex and a specific set of cells in the central nervous system (Salzberg, 1993). Ras64D and Ras opposite (Rop), are a jointly regulated gene pair. Drosophila Rop is homologous to C. elegans UNC-18 and rat munc-18/n-Sec1/rbSec1 proteins, implicated in the final steps of neurotransmitter exocytosis in nerve terminals, and the bovine mSec1 protein implicated in the secretion of catecholamines in chromaffin cells. The distribution of Ras64D in the cortex of the garland cell is identical to that of Rop, suggesting mutual regulation, and suggesting that Ras2 might be a component of the exocytic/endocytic cycle in this cell (Halachmi, 1995).

In contrast to the restricted expression of Ras64D, Ras85D, the subject of this report, is expressed ubiquitously and is involved in what is arguably the most widely used biochemical pathway in differentiation, the Ras pathway. What is it about Ras85D that makes it so important, and what are its functions in differentiation?

When Ras85D was isolated and sequence in 1984, mammalian Ras oncogenes had already been sequenced. Ha-ras and Ki-ras were isolated from their respective murine sarcoma viruses, Harvey murine sarcoma and Kirsten murine sarcoma. A third gene, N-ras had been isolated from a neuroblastoma cell line. These three genes are closely related; they share about 70% amino acid sequence homology and each has three introns in identical positions. Ras85D is closest in sequence to these three genes, the first 79 amino acids being identical, with an overall homology of 75%. In comparison, the 50% homology of these mammalian oncogenes to RAS64B is definitely less pronounced (Neuman-Silberberg, 1984 and Brock, 1987).

Ras and the Ras pathway are involved in a number of patterning and cellular signaling events in Drosophila development:

  1. Ras functions downstream of the EGF-receptor to establish follicular cell fate during oogenesis (Schnorr, 1996).

  2. The Ras pathway functions downstream of the receptor tyrosine kinase Torso to activate terminal cell fate immediately after fertilization, although Ras itself may not be required in this process (Hou, 1995). Another study shows that in Torso signalling, binding to 14-3-3 by Raf is necessary but not sufficient for activation of Raf and overexpressed 14-3-3 requires Ras to activate Raf (Li, 1997).

  3. The Ras pathway functions downstream of the receptor tyrosine kinase EGF-R in the establishment of ventral ectoderm fate (Golembo, 1996).

  4. The Ras pathway functions downstream of the receptor tyrosine kinase Breathless in the regulation of tracheal cell migration and migration of midline glia (Reichman-Fried, 1994).

  5. The Ras pathway functions downstream of the receptor tyrosine kinase Fibroblast growth factor receptor 1 in muscle precursors and in the central nervous system; it is involved in determining the fate of heart cell precursors (Beiman, 1996).

  6. The Ras pathway functions downstream of the EGF-R in the determination of wing cell fate (Schnepp, 1996).

  7. The Ras pathway converges with the Rutabaga-adenylyl cyclase pathway to modulate potassium ion-channel activity at the larval neuromuscular junction (Zhong, 1996).

  8. The Ras pathway functions downstream of the EGF-R and the receptor tyrosine kinase Sevenless in mediation involving the decision between neuronal and non-neuronal differentiation in photoreceptor precursors (Wassarman, 1995).

How does Ras function in signaling and cell fate determination? Ras is a molecular switch, cycling between an inactive GDP-bound and active GTP-bound form. Two other proteins interact with Ras85D in its activation and inactivation. Son of sevenless (Sos) is a guanine nucleotide-releasing factor that activates Ras by promoting the exchange of GDP for GTP. Gap1, a GTPase-activating protein inactives Ras85D by stimulating Ras's intrinsic GTPase activity, and as a result, creating the inactive form of Ras with bound GDP (Wassarman, 1995).

In Drosophila, the Ras pathway is best understood as the downstream signal transduction pathway of the Sevenless receptor tyrosine kinase, involved in the determination of R7 photoreceptor cell fate. A physical link between Ras85D and the Sevenless receptor tyrosine kinase is provided by Downstream of receptor kinase (Drk), known in vertebrates as Grb-2; this protein binds Sos and autophosphorylated Sevenless through its Src homology domains (Simon, 1993 and Oliver, 1993). The sole function of Ras, with respect to the serine/threonine protein kinase Raf, directly downstream of Ras, may be to recruit Raf to the plasma membrane, where it is activated. It appears that four proteins (Drk, Sos, Ras, and Raf) are recruited to the autophosphorylated Sevenless receptor tyrosine kinase, forming an interacting protein complex during activation of the Ras pathway (reviewed by Wassarman, 1995).

Ras is targeted to membranes where it adheres by the posttranslational attachment of isoprenoid lipids. Prenylation is a process involving covalent linkage of lipid groups near the C-terminal end of Ras. Vertebrate proteins involved in prenylation and involved immediately with Ras in the Ras pathway are well documented. Multiple Rases, guanine nucleotide exchange factors, GTPase-activating proteins, prenyltransferases, and Grb-2s each consist of families of proteins that function in different contexts (Boguski, 1993).

What accounts for the specificity of the Ras signal? This is not a trivial question. How does the cell know that the signal comes from EGF-R, Sevenless, FGF-R1, Breathless, or Torso? The most probable answer is that Ras signaling is context dependent. Using an analogy as an example, there are different contexts and consequences for yelling "fire," whether one is in a theater or on a rifle range. Torso signaling takes place in the context of a recently fertilized embryo; Breathless signaling takes place in tracheal precursors; FGF-R1 signaling takes place in the context of mesodermal cell fate, and Sevenless signaling takes place in the context of R7 photoreceptor determination. Ras signaling is not the only determiner of cell fate in each of these contexts, but each context is adorned with other signaling events that reinforce and restrict the signals received through the ras pathway. One indicator of the preexisting cell fate accompaning Ras signaling is the fact that different ligands and cofactors are made available by surrounding cells to each of the receptor tyrosine kinases involved in Ras signaling: Spitz, Gurken, and Vein for the EGF-R, Branchless for Breathless, Argos for Sevenless, and Trunk for Torso. Cofactors to receptor signaling such as Brainiac, Rhomboid and Star provide additional context dependent information to Ras pathway functioning. In addition there are other inputs to the Ras pathway than include Abl, Shc, Src, KSR (Kinase suppressor of ras), Cbl, Neurofibromin, Map kinase (Rolled in Drosophila), Protein kinase A, and RasGRF (responsive to Ca++ levels), each of which are documented in the various Ras Evolutionary Homologs sections. Thus context is likely to be the main determiner of specificity in Ras pathway signaling.

The role of Ras1 in promoting growth of the Drosophila wing

The Ras GTPase links extracellular mitogens to intracellular mechanisms that control cell proliferation. To understand how Ras regulates proliferation in vivo, Ras was either activated or inactivated in cell clones in the developing Drosophila wing. Cells lacking Ras are smaller, have reduced growth rates, accumulate in G1, and undergo apoptosis due to cell competition. Conversely, activation of Ras increases cell size and growth rates and promotes G1/S transitions. Ras upregulates the growth driver dMyc, and both Ras and dMyc increase levels of cyclin E posttranscriptionally. It is proposed that Ras primarily promotes growth and that growth is coupled to G1/S progression via cyclin E. Interestingly, upregulation of growth by Ras does not deregulate G2/M progression or a developmentally regulated cell cycle exit (Prober, 2000).

Cellular growth, defined as accumulation of mass, accompanies most cell divisions and allows cells to maintain a consistent cell size. Despite the growing knowledge of how cell division is regulated, little is known about how cells monitor their size to coordinate growth with the cell cycle. Ras, a major effector of extracellular signals, is shown here to function in vivo in the Drosophila wing to regulate cellular growth. When Ras activity is reduced, using either a null allele or by expressing a dominant-negative allele of Ras, growth slows, cell size decreases, and cell death due to cell competition increases. Conversely, activation of Ras increases growth rates and cell size. Since the Ras/cAMP pathway regulates cellular growth in response to nutrient levels in budding yeast, the results presented here indicate that growth regulation by Ras is conserved in both single and multicelled organisms. However, it was unexpectedly found that activation of Ras is not sufficient to accelerate cell division. This contrasts with Ras's ability to increase cell cycle rates in isolated cell culture systems. The data presented here indicate that the difference between results in cell culture and whole organisms is due to Ras-independent control of G2/M progression in vivo. In addition, the work presented here implies that, contrary to expectation, increased growth is not sufficient to accelerate cell proliferation (Prober, 2000).

In addition to promoting growth, Ras activity also controls the length of G1. This has also been observed in mammalian cells, and it has been suggested that Ras promotes cell proliferation by acting on components of the cell cycle machinery that regulate G1/S progression. Although Drosophila Ras might promote G1/S progression and cellular growth independently, the observations presented here suggest that in the developing wing, Ras's primary effect is to promote growth, and its cell cycle effects are secondary. Ras continues to promote growth when coexpressed with Stg, which reverses the cell cycle effects of Ras, and when coexpressed with RBF, which has a dominant effect in relationship to Ras in slowing the cell cycle. This contrasts with the proposal that the role of Ras in cell cycle progression is to inhibit Rb by directing its phosphorylation (Prober, 2000).

It is proposed that there is parallel and independent control of G1/S and G2/M transitions in Drosophila wing disc cells. Cellular growth due to Ras or dMyc drives G1/S transitions by promoting translation of cyclin E. Ras may also drive growth via proteins other than dMyc; this could feed back to upregulate translation of dmyc mRNA. Alternatively, Ras may regulate cellular growth and the G1/S cell cycle machinery in parallel. Stg/Cdc25, which is regulated primarily at the transcriptional level, drives G2/M transitions. Signaling molecules capable of regulating coordinated growth and patterning such as Vein may regulate G1/S transitions via Ras, dMyc, or other growth-promoting proteins and regulate G2/M transitions via transcription factors that modulate transcription of Stg/Cdc25 (Prober, 2000).

Other proteins that promote growth, such as the Drosophila homologs of dMyc and Phosphoinositide 3-Kinase (dPI3K), have effects on cell cycle progression similar to Ras. Upregulating these proteins in the developing wing truncates G1, elongates G2, and increases growth rates, while downregulating them cause the opposite effects. Furthermore, the resulting growth rates are inversely proportional to the length of G1. Given these similarities, it is proposed that cellular growth is rate limiting for G1/S progression in wing imaginal cells (Prober, 2000).

How might cellular growth drive G1/S transitions? Coupling of cellular growth to G1/S progression might be explained by a mechanism in which unstable, translationally regulated proteins are rate limiting for G1/S transitions. Cyclin E, a short-lived protein, is rate limiting for G1/S progression in wing discs. Both RasV12 and dMyc posttranscriptionally increase levels of cyclin E. As with the yeast G1 cyclin Cln3, the 5' untranslated region of Drosophila cyclin E contains several open reading frames (uORFs). It has been proposed that the Cln3 uORF reduces initiation of translation at the downstream Cln3 translation start site. As a result, more ribosomes are needed to achieve efficient translation of Cln3. Since the abundance of ribosomes correlates with growth rate, the uORF renders translation of Cln3 sensitive to the rate of cellular growth. The data presented here suggest that a similar mechanism may regulate production of cyclin E in the developing wing. Cyclin E would thus act as a 'growth sensor' to couple growth rates to G1/S progression. This hypothesis could be tested by mutating the cyclin E uORFs and assaying how cyclin E protein levels and G1/S progression respond to ectopic Ras or dMyc (Prober, 2000).

The data suggest that the effects of Ras on cellular growth and the cell cycle are at least partially mediated by dMyc. Mammalian Myc transcription factors activate expression of many genes involved in cellular growth and metabolism, and Drosophila dMyc is a potent growth driver in vivo. Upregulation of dMyc by Ras appears to be posttranscriptional. Ras might act by inhibiting degradation of dMyc protein, as has been demonstrated in mammalian cell culture. Alternatively, Ras might stimulate growth via other proteins, such as components of the dPI3K/dAkt/dS6 Kinase pathway, which promote cellular growth in Drosophila. Increased growth due to these proteins could then feed back to promote translation of extant dmyc mRNA. However, dMyc and dPI3K cannot be mediating all of Ras's effects, since unlike Ras they do not affect cell fate or cell adhesion. These additional functions of Ras, along with the ability to increase Myc protein levels, likely contribute to the strong synergistic action of Ras and Myc in oncogenesis (Prober, 2000).

RasV12 accelerates G1/S transitions but fails to accelerate rates of cell division. This is similar to findings with overexpressed dMyc. However, coexpressing either RasV12 or dMyc with String (Stg), the G2/M rate limitor, does accelerate cell division. This suggests that regulation of Stg is independent of both Ras and dMyc. It is therefore proposed that there is parallel and independent control of G1/S and G2/M transitions during wing development. Signaling molecules capable of regulating coordinated growth and patterning, such as Vein, Decapentaplegic, and Wingless might control G1/S transitions by regulating growth via Ras, dMyc, or other growth-promoting proteins. These signaling molecules might also, unlike Ras and dMyc, control G2/M transitions by modulating transcription of stg. Analysis of more than 40 kb of the stg promoter has revealed an extensive array of regulatory modules that respond to different patterning signals and thus integrate complex patterning information. A model in which cyclin E acts as a growth sensor and Stg acts as a 'pattern sensor' is attractive, as it allows coordination of independent growth and patterning signals by the cell cycle machinery (Prober, 2000).

This model is supported not only by experiments but also by characteristics of normal development. Early in wing development, rapid growth and cell proliferation take place, and as the disc prepares to differentiate into an adult wing, growth and proliferation slow. During the rapid growth phase, cells express stg RNA uniformly and at high levels and have a very short G2. This suggests that cell cycle length is primarily regulated at G1/S and that growth may thus be rate limiting for cell proliferation at this stage. As growth slows near the end of wing development, disc cells express stg periodically and in patterns and acquire a much longer G2. Thus, at a time when many detailed patterning decisions are being made, cell cycle length may become primarily regulated by Stg at G2/M (Prober, 2000).

Studies of the role of Ras in cancer have focused on its role in driving quiescent cells into the cell cycle; its ability to promote G1/S transitions in cycling cells, and its effects on cell adhesion and the cytoskeleton. In contrast, little is known regarding its role in promoting cellular growth. Drosophila Ras, like vertebrate Ras, promotes G1/S transitions and alters the adhesive properties and identities of cells. Significantly, Ras promotes growth and its effects on the cell cycle are secondary. This suggests that activation of Ras, as well as other oncogenes such as Myc, may promote cancer by driving cellular growth. In light of this, it is interesting to note that Drosophila disc cells increase their mass 6-fold prior to exiting a developmentally regulated G1 cell cycle arrest early in larval development. The increase in cellular mass suggests that cellular growth may be promoting exit from this arrest. By analogy, the ability of Ras to promote exit from quiescence in cancer may also be a consequence of growth promotion. Consistent with this idea, cellular hypertrophy is commonly observed during neoplastic progression in mice and humans, suggesting that the findings presented in this paper are relevant to mammalian pathology (Prober, 2000).

The Ras-Erk-ETS-signaling pathway is a drug target for longevity

Identifying the molecular mechanisms that underlie aging and their pharmacological manipulation are key aims for improving lifelong human health. This study has identified a critical role for Ras-Erk-ETS signaling in aging in Drosophila. Inhibition of Ras was shown to be sufficient for lifespan extension downstream of reduced insulin/IGF-1 (IIS) signaling. Moreover, direct reduction of Ras or Erk activity leads to increased lifespan. ETS transcriptional repressor Anterior open (Aop) was identified as central to lifespan extension caused by reduced IIS or Ras attenuation. Importantly, it was demonstrates that adult-onset administration of the drug trametinib, a highly specific inhibitor of Ras-Erk-ETS signaling, can extend lifespan. This discovery of the Ras-Erk-ETS pathway as a pharmacological target for animal aging, together with the high degree of evolutionary conservation of the pathway, suggests that inhibition of Ras-Erk-ETS signaling may provide an effective target for anti-aging interventions in mammals (Slack, 2015).

The key role of IIS in determining animal lifespan has been well appreciated for more than two decades and shows strong evolutionary conservation. Alleles of genes encoding components of this pathway have also been linked to longevity in humans. Multiple studies have demonstrated the importance of the PI3K-Akt-Foxo branch of IIS, while this study has identified an equally important role for Ras-Erk-ETS signaling in IIS-dependent lifespan extension (Slack, 2015).

Downstream of chico, preventing the activation of either Ras or PI3K is sufficient to extend lifespan. Ras can interact directly with the catalytic subunit of PI3K, which is required for maximal PI3K activation during growth. Thus, inhibition of Ras could increase lifespan via inactivation of PI3K. However, several lines of evidence indicate that the Erk-ETS pathway must also, if not solely, be involved. In this study and elsewhere, it has been demonstrated that direct inhibition of the Ras-dependent kinase, Erk, or activation of the Aop transcription factor, a negative effector of the Ras-Erk pathway, is sufficient to extend lifespan. Importantly, this study shows that Ras-Erk-ETS signaling is genetically linked to chico because activation of Aop is required for lifespan extension due to chico loss of function. Furthermore, altering the ability of Chico to activate Ras or PI3K does not result in equivalent phenotypes: it has been shown that mutation of the Grb2/Drk docking site in Chico is dispensable for multiple developmental phenotypes associated with chico mutation, while disruption of the Chico-PI3K interaction is not. Overall, the observations strongly suggest that lifespan extension downstream of chicomutation involves inhibition of the Ras-Erk-ETS-signaling pathway (Slack, 2015).

A simple model integrates the role of Ras-Erk-ETS signaling with the PI3K-Akt-Foxo branch in extension of lifespan by reduced IIS. It is proposed that, downstream of Chico, the IIS pathway bifurcates into branches delineated by Erk and Akt, with inhibition of either sufficient to extend lifespan, as is activation of either responsive TF, Aop or Foxo. The two branches are not redundant, because mutation of chico or the loss of its ability to activate either branch results in the same magnitude of lifespan extension. Furthermore, Aop and Foxo are each individually required downstream of chico mutation for lifespan extension. At the same time, the effects of the two branches are not additive, as simultaneous activation of Aop and Foxo does not extend lifespan more than activation of either TF alone. Taken together, these data suggest that the two pathways re-join for transcriptional regulation, where Aop and Foxo co-operatively regulate genes required for lifespan extension. The model is corroborated by a previous finding that, in the adult gut and fat body, some 60% of genomic locations bound by Foxo overlap with regions of activated-Aop binding (Alic, 2014; Slack, 2015).

It is proposed that functional interactions of Aop and Foxo at these sites may be such that each factor is both necessary and sufficient to achieve the beneficial changes in target gene expression upon reduced IIS. It remains to be determined how promoter-based Foxo and Aop interactions produce such physiologically relevant, transcriptional changes. It is, however, curious that activation of either TF alone promotes longevity when one is known as a transcriptional activator (Foxo) and the other as a transcriptional repressor (Aop). A subset of Foxo-bound genes, albeit a minority, has been consistently observed that are transcriptionally repressed when Foxo is activated (Alic, 2014). Furthermore, the Foxo target gene myc is downregulated in larval muscle when Foxo is active under low insulin conditions, while deletion of foxo or its binding site within the myc promoter results in de-repression of myc expression in adipose of fed larvae (Teleman, 2008). Thus, on some promoters under certain conditions, Drosophila Foxo appears to act as a transcriptional repressor. Mammalian Foxo3a may also directly repress some genes. It will therefore be important to test whether the lifespan-relevant interactions between Foxo and Aop occur on promoters where Foxo acts as a repressor with Foxo possibly acting as a cofactor for Aop or vice versa (Slack, 2015).

In mediating the effects of IIS on lifespan, the Ras-Erk-ETS- and PI3K-Akt-Foxo-signaling pathways both appear to inhibit Aop/Foxo. To understand why signaling might be so wired, it is important to consider that the two pathways are also regulated by other stimuli, such as other growth factors, stress signals, and nutritional cues. The re-joining of the two branches at the transcriptional level would therefore allow for their outputs to be integrated, producing a concerted transcriptional response, a feature that is also seen in other contexts. For example, stability of the Myc transcription factor is differentially regulated in response to Erk and PI3K signals, allowing it to integrate signals from the two kinases. Transcriptional integration in response to RTK signaling also confers specificity during cell differentiation, with combinatorial effects of multiple transcriptional modulators inducing tissue-specific responses to inductive Ras signals. Similar integrated responses of lifespan could be orchestrated by transcriptional coordination of Aop and Foxo (Slack, 2015).

Direct inhibition of Ras in Drosophila can extend lifespan, suggesting that the role of Ras in aging is evolutionarily conserved. In budding yeast, deletion of RAS1 extends replicative lifespan, and deletion of RAS2 increases chronological lifespan by altering signaling through cyclic-AMP/protein kinase A (cAMP/PKA), downregulation of which is sufficient to extend both replicative and chronological lifespan. This role of cAMP/PKA in aging may be conserved in mammals, as disruption of adenylyl cyclase 5' and PKA function extend murine lifespan. However, cAMP/PKA are not generally considered mediators of Ras function in metazoa. Instead, the data suggest that signaling through Erk and the ETS TFs mediates the longevity response to Ras. Interestingly, fibroblasts isolated from long-lived mutant strains of mice and long-lived species of mammals and birds show altered dynamics of Erk phosphorylation in response to stress, further suggesting a link between Erk activity and longevity. Importantly, the ETS TFs are conserved mediators of Ras-Erk signaling in mammals. Investigation of the effects of Ras inhibition on mammalian lifespan and the role of the mammalian Aop ortholog Etv6 are now warranted (Slack, 2015).

A role for Ras-Erk-ETS signaling in lifespan offers multiple potential targets for small-molecule inhibitors that could function as anti-aging interventions. Importantly, due to the key role of this pathway in cancer, multiple such inhibitors exist or are in development (Slack, 2015).

This study has shown that trametinib, a highly specific allosteric inhibitor of the Mek kinase, prolongs Drosophila lifespan, thus validating the Ras-Erk-ETS pathway as a pharmacological target for anti-aging therapeutics. Trametinib joins a very exclusive list of FDA-approved drugs that promote longevity in animals, the most convincing other example being rapamycin (Slack, 2015).

Rapamycin not only increases lifespan in multiple organisms, including mammals, but also improves several indices of function during aging (Ehninger, 2014; Lamming, 2013). While rapamycin can protect against tumor growth, the effects on longevity appear to be independent of cancer prevention, as rapamcyin-treated animals still develop tumors and rapamycin can increase lifespan in tumor-free species. Furthermore, increased activity of certain tumor suppressors such as lnk4a/Arf and PTEN as well as the RasGrf1 deficiency all increase lifespan independently of anti-tumor activity. The findings that trametinib can increase lifespan inDrosophila, which are mainly post-mitotic in adulthood, and that doses of trametinib that increase lifespan do not alter proliferation rates of ISCs inDrosophila suggest that the anti-aging effects of trametinib are separable from its anti-cancer activity (Slack, 2015).

Finally, due to the high degree of evolutionary conservation in the Ras-Erk-ETS pathway, this study suggests the intriguing possibility that pharmacological inhibition of Ras-Erk-ETS may also increase lifespan in mammal (Slack, 2015).

The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila

Using the Drosophila larval neuromuscular junction, this study shows a PI3K-dependent pathway for synaptogenesis (a pro-syaptogenesis pathway) which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, the functional hierarchy within the pathway was determined. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, an antagonistic pathway (an anti-synaptogenesis pathway) was identified that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways (Jordan-Alvarez, 2017).

The epistasis assays have determined the in vivo functional links between PI3K and other previously known pro-synaptogenesis factors. Epistasis assays are based on the combined expression of two or more UAS constructs. Several double combinations in this study have produced a phenotype in spite of the apparent ineffectiveness of the single constructs. This type of results underscores the necessity to use epistasis assays in order to reveal functional interactions in vivo, hence, biologically relevant. In addition to the pro-synaptogenesis signaling, the study has revealed an anti-synaptogenesis pathway that composes a signaling equilibrium to determine the actual number of synapses. The magnitude of the synapse number changes elicited by the factors tested here are mostly within the range of 20%-50%. Are these values significant to cause behavioral changes? Reductions in the order of 30% of excitatory or inhibitory synapses in adult Drosophila local olfactory interneurons transform perception of certain odorants from attraction to repulsion and vice versa. In schizophrenia patients, a 16% loss of inhibitory synapses in the brain cortex has been reported. In Rhesus monkeys, the pyramidal neurons in layer III of area 46 in dorsolateral prefrontal cortex show a 33% spine loss, and a significant reduction in learning task performance during normal aging. Thus, it seems that behavior is rather sensitive to small changes in synapse number irrespective of the total brain mass (Jordan-Alvarez, 2017).

The signaling interactions analyzed here were chosen because they were reported in other cellular systems and species previously. Some of these interactions have been confirmed (e.g., Gbb/Wit), while others have proven ineffective in the context of synaptogenesis (e.g., Ras85D/PI3K binding). Likely, the two signaling pathways, pro- and anti-synaptogenesis, are not the only ones relevant for synapse formation. For example, in spite of the null condition of the gbb and wit mutant alleles used here, the resulting synaptic phenotypes are far less extreme than expected if these two factors would be the only source of signaling for synaptogenesis. Although it could be argued that the incomplete absence of synapses in the mutant phenotypes could result from maternal perdurance, Wit is not part of the oocyte endowment while Gbb is. Three alternative possibilities may be considered, additional ligands for Wit, additional receptors for Gbb, and a combination of the previous two. Beyond the identity of these putative additional ligands and receptors, the stoichiometry between ligands and receptors may certainly be relevant. Actually, Gbb levels are titrated by Crimpy. An equivalent quantitative regulation could operate on Wit. The reported data on Wit illustrate already the diversity of the functional repertoire of this receptor. Wit can form heteromeric complexes with Thick veins (Tkv) or Saxophone (Sax) receptors to receive Dpp/BMP4 or Gbb/BMP7 as ligands. However, the same study also showed that Wit could dimerize with another receptor, Baboon, upon binding of Myoglianin to activate a different and antagonistic signaling pathway, TGFβ/activin-like (Jordan-Alvarez, 2017).

The Gbb/Wit/PI3K signaling analyzed in this study is likely not the only pro-synaptogenesis pathway in flies and vertebrates. The ligand Wingless (Wg), member of the Wnt family, and the receptors Frizzled have been widely documented as relevant in neuromuscular junction development, albeit data on synapse number are scant. Interestingly, however, the downstream intermediaries can be as diverse as those mentioned above for Wit. Although generally depicted as linear pathways, a more realistic image would be a network of cross-interacting signaling events whose in situ regulation and cellular compartmentalization remains fully unexplored (Jordan-Alvarez, 2017).

The quantitative regulation of receptors is most relevant to understand their biological effects. In that context, is worth noting that Tkv levels are distinctly regulated from those of Wit and Sax through ubiquitination in the context of neurite growth. On the other hand, although the receptor Wit is considered a RSTK type, the functional link with PI3K is a feature usually associated to the RTK type instead. The link of Wit with a kinase has a precedent with LIMK1 that binds to, and is functionally downstream from, Wit in the context of synapse stabilization. Thus, Wit should be considered a wide spectrum receptor in terms of its ligands, co-receptor partners and, consequently, signaling pathways elicited. Actually, the Wit amino acid sequence shows both, Tyr and Ser/Thr motifs justifying its initial classification as a 'dual' type of receptor. In this report this study did not determine if Wit heterodimerizes with other receptors, as canonical RSTKs do, or if it forms homodimers, as canonical RTKs do. However, the lack of synaptogenesis effects by the putative co-receptors, Tkv and Sax, and the phenotypic similarity with the manipulation of the standard RTK signaling effector Cic, leaves open the possibility that Wit could play RTK-like functions, at least in the context of synaptogenesis (Jordan-Alvarez, 2017).

Consistent with the proposal of a dual mechanism for Wit, its activation seems to be a requirement to elicit two independent signaling steps, PI3K and Ras85D, that could reflect RTK and RSTK mechanisms, respectively. Both steps are independent because the mutated form of PI3K unable to bind Ras85D, PI3KΔRBD, is as effective as the normal PI3K to elicit synaptogenesis. PI3K and Ras85D signaling, however, seem to converge on Bsk revealing a novel feature of this crossroad point. The activity level of Bsk is known to be critical in many signaling processes. The peculiarities of Bsk/JNK activity include its coordinated regulation by p38a and Slpr in the context of stress heat response without interference on the developmental context. Another modulator, Puc, was described as a negative feed-back loop in the context of oxidative stress. The Puc mediated loop is operative also for synaptogenesis, while that of p38a/Slpr is relevant for p38a only, as shown here. Further, Ras85D represents an additional regulator in the neural scenario. The triple regulation of Bsk/JNK by Ras85D, Puc and the MAPKs seems to stablish a narrow range of activity thresholds within which normal number of synapses is determined (Jordan-Alvarez, 2017).

The concept of signaling thresholds is also unveiled in this study by the identification of another signaling pathway that opposes synapse formation. The pro- and anti-synaptogenesis pathways have similar constituents, including small GTPases, MAPKs and transcriptional effectors, Mad/Smad, which are canonical for RSTK receptors. The RSTK type II receptor Put, which can mediate diverse signaling pathways according to the co-receptor bound can be discarded in either the pro- or the anti-synaptogenesis pathways. Thus, the main receptor for the anti-synaptogenesis pathway remains to be identified (Jordan-Alvarez, 2017).

Concerning small GTPases, the pro-synaptogenesis pathway uses Ras85D while its counterpart uses the poorly studied Ras64B. The anti-synaptogenesis pathway includes an additional member of this family of enzymes, Rala. This small GTPase plays a role in the exocyst-mediated growth of the muscle membrane specialization that surrounds the synaptic bouton as a consequence of synapse activity. That is, Rala can influence synapse physiology acting from the postsynaptic side. The experimental expression of a constitutively active form of Rala in the neuron does not seem to affect the overall synaptic terminal branching. However, the null ral mutant shows reduced synapse branching and its vertebrate homolog is expressed in the central nervous system. This study found that Rala under-expression in neurons yields an elevated number of synapses. Thus, it is likely that this small GTPase acts as a break to synaptogenesis, hence its inclusion in the antagonistic pathway (Jordan-Alvarez, 2017).

Synaptogenesis and neuritogenesis are distinct processes since each one can be differentially affected by the same mutant (e.g.: Hiw). Both features, however, share some signals (e.g., Wnd, Hep). This signaling overlap is akin to the case of axon specification versus spine formation for constituents of the apico/basal polarity complex Par3-6/aPKC [127]. These and other examples illustrated in this study underscore the need to discriminate between synapses and boutons. This study is focused on the cell autonomous signaling that takes place in the neuron. Non-cell autonomous signals (e.g., originated in the glia or hemolymph circulating) have not been considered. The active role of glia in axon pruning and bouton number has been the subject of other studies. Considering the reported role of Hiw through the midline glia in the remodeling of the giant fiber interneuron it is not unlikely that the glia-to-neuron signaling may share components with the neuron autonomous signaling addressed here (Jordan-Alvarez, 2017).

The summary scheme (see Summary diagram of antagonistic signaling pathways for synaptogenesis and their interactions) describes the scenario where two signaling pathways mutually regulate each other. Epistasis assays are the only experimental approach for in vivo studies of more than one signaling component, albeit this type of assay is only feasible in Drosophila Thus, it is plausible that vertebrate synaptogenesis will be regulated by a similar antagonistic signaling (Jordan-Alvarez, 2017).

The regulatory equilibrium as a mechanism to determine a biological parameter is the most relevant feature in this scenario for several reasons. First, because this type of mechanism can respond very fast to changes in the physiological status of the cell, and, second because it provides remarkable precision to the trait to be regulated, synapse number in this case. Although bi-stable regulatory mechanisms are known in other contexts, the case of synapse number may seem unexpected because the highly dynamic nature of synapse number has been recognized only recently. Consequently, a molecular signaling mechanism endowed with proper precision and time resolution must sustain this dynamic process. The balanced equilibrium uncovered in this study, although most likely still incomplete in terms of its components, offers such a mechanism (Jordan-Alvarez, 2017).

Distinct roles and requirements for Ras pathway signaling in visceral versus somatic muscle founder specification

Pleiotropic signaling pathways must somehow engender specific cellular responses. In the Drosophila mesoderm, Ras pathway signaling specifies muscle founder cells from among the broader population of myoblasts. For somatic muscles, this is an inductive process mediated by the ETS-domain downstream Ras effectors Pointed and Aop (Yan). For the circular visceral muscles, despite superficial similarities, a significantly different specification mechanism is at work. Not only is visceral founder cell specification not dependent on Pointed or Aop, but Ras pathway signaling in its entirety can be bypassed. These results show that de-repression, not activation, is the predominant role of Ras signaling in the visceral mesoderm and that, accordingly, Ras signaling is not required in the absence of repression. The key repressor acts downstream of the transcription factor Lame duck and is likely a member of the ETS transcription factor family. These findings fit with a growing body of data that point to a complex interplay between the Ras pathway, ETS transcription factors, and enhancer binding as a crucial mechanism for determining unique responses to Ras signaling (Zhou, 2019).

Ras acts as a molecular switch between two forms of consolidated memory in Drosophila

Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. This study demonstrates that the Ras-->Raf-->rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. The initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. These effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. It is concluded that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM (Noyes, 2020).

Consolidation is a process required for the formation of long-lasting memories. This process of converting memories that are initially sensitive to disruption from a variety of insults to more resilient ones is well conserved and many of its characteristics are shared across species. For example, memory in invertebrates and vertebrates lasts longer following multiple spaced training sessions, undergoes both molecular/cellular and systems consolidation, and can be disrupted by inhibition of protein synthesis (Noyes, 2020).

The fruit fly Drosophila melanogaster forms two distinguishable types of consolidated aversive olfactory memory: 1) anesthesia-resistant memory (ARM), which reportedly decays to negligible levels by 4 d after conditioning, can be generated by a single training session; 2) protein synthesis-dependent long-term memory (PSD-LTM), which shows limited decay, requires spaced training. These two types of consolidated memory are not independent from one another. The formation of ARM impairs either the formation or expression of PSD-LTM. Although circuit mechanisms possibly responsible for this relationship are beginning to be dissected, the molecular requirements in the mushroom body (MB), a brain region critical for the storage and retrieval of PSD-LTM and ARM remain unknown (Noyes, 2020).

The small GTPase Ras85D (Ras) is a Drosophila homolog of the mammalian Ras family genes KRAS, NRAS, and HRAS. Activated Ras proteins act as signaling switches, initiating signaling cascades through multiple downstream effector proteins. Precise induction and regulation of Ras activity is essential for mammalian synaptic plasticity and memory. Although upstream regulators of Ras, like NF1 and DRK, have been explored for their roles in Drosophila learning and memory Ras itself has not been thoroughly examined. A large RNA interference (RNAi) screen identified Ras85D as a memory suppressor gene but did not detail its specific role in memory suppression (Noyes, 2020).

This study reports that Ras activity in the MB acts as a switch between the two forms of consolidated memory, required both for PSD-LTM and inhibition of ARM. Increasing Ras activity dramatically reduced memory expression. This effect was determined to be due to Ras regulation of ARM. Knockdown of Ras enhanced the consolidation of ARM, leading to an overall increase in memory, while Ras knockdown eliminated PSD-LTM following spaced training. Although the effect of Ras on both ARM and PSD-LTM was found to be mediated by Raf, it is independent from the canonical downstream extracellular signal-regulated kinase (ERK). Instead, Ras/Raf-mediated inhibition of rho kinase (ROCK) suppresses ARM and is required for PSD-LTM (Noyes, 2020).

Based on the results, a model in which ARM consolidation is suppressed by a training-induced increase in Ras activity. Raf activity is increased in γ MBn following training, presumably through Ras, but the receptor(s) initiating this signaling are not known. Ras can be regulated through G-coupled protein receptors. It is possible that dopamine or an unknown coneurotransmitter released from dopaminergic neurons (DAn) during training initiates Ras signaling. This would provide a link between MP1 DAn, which are proposed to gate LTM, and Ras. The participation of ROCK in consolidation suggests that PSD-LTM and ARM are modulated by changes in the actin cytoskeleton (36) but does not directly indicate whether these changes occur in the pre- or post-synaptic compartments. Of the several genes known to be required for ARM, Bruchpilot (Brp) is the only one with a well-established, specific subcellular compartmentalization. Brp is localized to presynaptic active zones and is required for normal presynaptic morphology and synaptic transmission, indicating that ARM may result from a form of presynaptic plasticity in the MB. Additionally, the DAn that are required for memory formation innervate MB axons and modulate synaptic strength between MBn and downstream MB output neurons. The results demonstrating that artificial activation of Ras increases axonal pERK in γ MBn is evidence that Ras/Raf signaling participates in axonal signal transduction and is consistent with a previous report highlighting a role for presynaptic Raf activity in γ MBn. ROCK activity in mammalian axons is critical for a number of processes; however, it has not been tested whether ROCK signaling occurs in γ MBn axons (Noyes, 2020).

The hypothesis that ARM inhibits the formation PSD-LTM was based on the observation that spaced training, which generates PSD-LTM, eliminates or precludes ARM. Subsequent research at the systems neuroscience level revealed that two sets of neurons, MP1 DAn and serotonergic projection neurons (SPn), appear to be responsible for the promotion of PSD-LTM through the suppression of ARM. The activity of these neurons is increased during spaced training. This activity reduces ARM, while inhibiting their activity enhances ARM. Blocking the activity of either set of neurons during spaced training does not prevent memory formation but prevents the formation of PSD-LTM. This suggests that without SPn and MP1 DAn activity, ARM occurs by default and is preferentially expressed at the expense of PSD-LTM. Ras fulfills the requirements as the intracellular and molecular switch regulating the inverse relationship between ARM and PSD-LTM. The suppression of ARM and formation of PSD-LTM both require Ras in γ MBn, which are downstream in the circuit from the ARM/PSD-LTM-gating MP1 DAn that synapse directly on to γ MBn (Noyes, 2020).

The mammalian counterpart for ARM, if one exists, is unknown. Protein synthesis-independent ARM has been reported to be measurable up to 4 d after conditioning, while mammalian protein synthesis-independent memory lasts only hours. Despite the lack of a clear and direct mammalian counterpart to ARM, it is becoming apparent that many of the same genes that are involved in ARM also play a role in mammalian memory and plasticity. Ras, Raf, and CDC42 negatively regulate ARM but in mammals are positive regulators of LTM. Conversely, reduced ROCK or dunce, the latter purported to function through the SPn, impair ARM. In mammals, inhibition of ROCK or a mammalian ortholog of dnc (48), PDE4, enhances memory. It seems likely that discovering more genetic regulators of ARM will reveal previously unknown genetic regulators of mammalian memory. Based on the genes and their functions discussed in this paper, it is possible that factors that promote ARM in Drosophila function in memory suppression in mammals (Noyes, 2020).

The effect of ROCK on memory is not restricted to γ MBn. ROCK is also required in α/β MBn for ARM. In this neuron type, the effects of ROCK are not mediated by Ras but through Drk, the Drosophila homolog of Grb2. It is interesting to consider whether the ROCK substrate(s) mediating enhanced ARM in α/β and γ MBn are the same even though the upstream signaling components are distinct. Several ROCK targets have been established as important for normal memory, including cofilin and nonmuscle myosin II (Noyes, 2020).

A recent report indicates that ERK activity in γ MBn slows forgetting. The current results revealing that ERK knockdown reduces memory support this conclusion. However, the former report finds that Raf RNAi expression in γ MBn reduces memory, which is at odds with the finding that Raf RNAi enhances memory. The most likely explanation for this discrepancy is the use of different gal4/UAS-RNAi combinations that produce different levels of gene knockdown. It is interesting to consider that Raf signaling in γ MBn might regulate three forms of memory: consolidated ARM and PSD-LTM through ROCK and labile memory through ERK (Noyes, 2020).


cDNA clone length - 916

Exons - 3

Bases in 3' UTR - 345


Amino Acids - 190

Structural Domains

Three Drosophila genes homologous to the Ha-ras probe were isolated and mapped to positions 85D, 64B, and 62B on chromosome 3. Two of these genes (termed Dras 1 and Dras 2) were sequenced. In the case of Dras 1, which contains multiple introns, a cDNA clone was isolated and sequenced. In the case of Dras2, the nucleotide sequence of the genomic clone was determined. Each gene codes for a protein with a predicted molecular weight of 21.6 kd. Alignment of the amino acid sequence of Dras 1 with the vertebrate Ha-ras protein shows that at the amino terminus and central portion (residues 1-121 and 137-164) the two proteins are remarkably similar, and have an overall homology of 75%. The Dras 2 gene lacks significant homology to the vertebrate counterpart at the extreme amino terminus and is homologous only between positions 28-120 and 139-161 (overall homology of 50%). This result suggests that the N terminus of p21 forms a distinct regulatory or functional domain. At the carboxy terminus, the major region of variability among the vertebrate Ras proteins, the two Drosophila sequences also display considerable variability. However, both appear to be similar to exon 4B of the Ki-ras gene (Neuman-Silberberg, 1984).

The Ras homologs of Drosophila melanogaster located at 85D and 64B on the polytene chromosome map were cloned using the Ha-ras gene of Harvey murine sarcoma virus as a probe. The genomic sequences of Dmras85D and Dmras64B were determined and shown to differ from previously published sequences. Dmras85D is much more similar to the Ha-ras, Ki-ras, and N-ras genes than it is to either the Dmras64B gene or to the Ras genes of Saccharomyces cerevisiae. Comparison of the Dmras85D genomic sequences with the previously published nucleotide sequence (Neuman-Silberberg, 1984) shows that the positions of the two introns are not conserved relative to the positions of the introns in Dmras64B or in vertebrate Ras genes. Analysis by blot hybridization shows Dmras64B and Dmras85D transcripts are dissimilar. The data suggest that the divergence of the Dmras genes was ancient, and that Dmras85D and Dmras64B have different functions (Brock, 1987).

Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, an F156L mutation was introduced into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activates the transforming potential of wild-type Ras, it does not impair that of oncogenic Ras. Ras (156L) exhibits an extremely rapid off rate for bound GDP/GTP in vitro and shows increased levels of GTP bound Ras in vivo. The F156L mutation causes loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity; instead, these structural changes of the F156L mutation weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch (Quilliam, 1995).

Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. The uncatalyzed hydrolysis of GTP was analyzed, and the understanding derived applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from a chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, the chemical analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge, going from the ground state to the transition state. The existence of a transition state leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl transfer, including kinases and phosphatases (Maegley, 1996).

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change was determined by simulation molecular dynamics. A holonomic constraint directs the system from the known GTP-bound structure (with the gamma-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD-Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of alpha2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of alpha2 and facilitates the unwinding of a helical turn of alpha2 (residues 66-69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition (Ma, 1997).

For more information on the Ras pathway, see the Corkscrew and Rolled/MAPK sites. Targets of the Ras pathway include Pointed, Yan, Phyllopod, DJun, and Dorsal. See these individual sites for more infomation about the consequences of Ras signaling.

Ras85D: Evolutionary Homologs | Regulation | Protein Interactions | Effects of Mutation | Ras as Oncogene | References
date revised:  25 April 2024  

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