BIOLOGICAL OVERVIEW

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 Ras^V12 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).

Ras^V12 accelerates G1/S transitions but fails to accelerate rates of cell division. This is similar to findings with overexpressed dMyc. However, coexpressing either Ras^V12 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).

GENE STRUCTURE

Exons - 3

Bases in 3' UTR - 345

PROTEIN STRUCTURE

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.

date revised:  10 June 2024  

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