Gene name - Ras oncogene at 85D
Synonyms - Ras1
Cytological map position - 85D11--85D27
Function - signal transduction protein
Symbol - Ras85D
FlyBase ID: FBgn0003205
Genetic map position - 3-
Classification - GTPase
Cellular location - cytoplasmic
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:
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 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, andundergo apoptosis due to cell competition. Conversely, activation of Ras increases cell size and growth rates and promotes G1/S transitions. Ras upregulates thegrowth 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).
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.
Exons - 3
Bases in 3' UTR - 345
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. Inthe case of Dras 1, which contains multiple introns, a cDNA clone was isolated and sequenced. In thecase of Dras2, the nucleotide sequence of the genomic clone was determined. Each gene codes for aprotein with a predicted molecular weight of 21.6 kd. Alignment of the amino acid sequence of Dras 1with the vertebrate Ha-ras protein shows that at the amino terminus and central portion (residues 1-121and 137-164) the two proteins are remarkably similar, and have an overall homology of 75%. The Dras2 gene lacks significant homology to the vertebrate counterpart at the extreme amino terminus and ishomologous only between positions 28-120 and 139-161 (overall homology of 50%). This resultsuggests that the N terminus of p21 forms a distinct regulatory or functional domain. At the carboxyterminus, the major region of variability among the vertebrate Ras proteins, the two Drosophilasequences also display considerable variability. However, both appear to be similar to exon 4B ofthe Ki-ras gene (Neuman-Silberberg, 1984).
The Ras homologs of Drosophila melanogaster located at 85D and 64B on the polytene chromosomemap were cloned using the Ha-ras gene of Harvey murine sarcoma virus as a probe. The genomicsequences of Dmras85D and Dmras64B were determined and shown to differ from previouslypublished sequences. Dmras85D is much more similar to the Ha-ras, Ki-ras, and N-ras genes than it isto either the Dmras64B gene or to the Ras genes of Saccharomyces cerevisiae. Comparison of theDmras85D genomic sequences with the previously published nucleotide sequence (Neuman-Silberberg, 1984) shows that the positions of the two introns are not conserved relativeto the positions of the introns in Dmras64B or in vertebrate Ras genes. Analysis by blot hybridization shows Dmras64B and Dmras85Dtranscripts are dissimilar. The data suggest that thedivergence of the Dmras genes was ancient, and that Dmras85D and Dmras64B have differentfunctions (Brock, 1987).
Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Rassuperfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its locationwithin the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role instructural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-relatedprotein 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 Rasstructure and function. Whereas introduction of this mutation activates the transforming potential ofwild-type Ras, it does not impair that of oncogenic Ras. Ras (156L) exhibits an extremely rapidoff 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 indisruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structuralchanges contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) thatperturb GTPase activity; instead, these structural changes of the F156L mutation weaken Ras contacts with Mg2+ and its guanine nucleotidesubstrate and result in increased rates of GDP/GTP dissociation. Altogether, these observationsdemonstrate the essential role of this conserved residue in Ras structure and its function as a regulatedGDP/GTP switch (Quilliam, 1995).
Despite the biological and medical importance of signal transduction via Ras proteinsand despite considerable kinetic and structural studies of wild-type and mutant Rasproteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. The uncatalyzed hydrolysis of GTP wasanalyzed, and the understanding derived applied to the Ras-catalyzed reaction.Evaluation of previous mechanistic proposals from a chemical perspective suggeststhat proton abstraction from the attacking water by a general base and stabilization ofcharge development on the gamma-phosphoryl oxygen atoms would not be catalytic.Rather, the chemical analysis focuses attention on the GDP leaving group, including thebeta-gamma bridge oxygen of GTP, the atom that undergoes the largest change incharge, going from the ground state to the transition state. The existence of a transition state leads to a newcatalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 tothis 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 thebeta-nonbridging phosphoryl oxygens and a network of interactions that positions thenucleophilic water molecule and gamma-phosphoryl group with respect to one anothermay also contribute to catalysis. It is speculated that a significant fraction of theGAP-activated GTPase activity of Ras arises from an additional interaction of thebeta-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 toother enzymes that catalyze phosphoryl transfer, including kinases andphosphatases (Maegley, 1996).
Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in thesignal transduction pathway. The path for the conformational change was determined by simulation moleculardynamics. A holonomic constraint directs the system from the known GTP-boundstructure (with the gamma-phosphate removed) to the GDP-bound structure. The simulation is donewith 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 theprotein, 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 thetransition. In the switch II region, the conformational changes of alpha2 and loop 4 are stronglycoupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region andGlu-37 in switch I region stabilizes the intermediate conformation of alpha2 and facilitates theunwinding of a helical turn of alpha2 (residues 66-69), which in turn permits the larger scale motion ofloop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important inthe transition (Ma, 1997).
date revised: 18 December 97
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