Ras oncogene at 85D


Table of contents

Raf, a serine/threonine kinase (part 1/2)

Formation of the tail region of the Drosophila larva requires the activities of the terminal class genes. Genetic and molecular analyses of these genes suggests that localized activation of the receptor tyrosine kinase Torso at the posterior egg pole triggers a signal transduction pathway. This pathway, mediated through the serine/threonine protein kinase D-raf and the protein tyrosine phosphatase Corkscrew, controls the domains of expression of the transcription factors Tailless and Huckebein. In this paper, the molecular and developmental characterization of mutations in the D-raf gene are characterized. Mutations that alter conserved residues known to be necessary for kinase activity are associated with a null phenotype, demonstrating that D-raf kinase activity is required for its role in torso signaling. Another mutation, D-rafPB26, which prematurely truncates the kinase domain shows a weaker maternal effect phenotype that is strikingly similar to the corkscrew maternal effect phenotype, suggesting that a lower amount of kinase activity decreases the terminal signaling pathway. Finally, molecular and developmental characterization of two mutations that affect the late D-raf zygotic function(s) implies a novel role for D-raf in cell fate establishment in the eye. One of these mutations, D-rafC110, is associated with a single amino acid change within the putative D-raf regulatory region, while the other, D-rafHM-7, most likely reduces the wild-type amount of D-raf protein (Melnick, 1993).

Somatic control over the germline stem cell lineage during Drosophila spermatogenesis

Stem cells divide both to produce new stem cells and to generate daughter cells that can differentiate. The underlying mechanisms are not well understood, but conceptually are of two kinds. Intrinsic mechanisms may control the unequal partitioning of determinants leading to asymmetric cell divisions that yield one stem cell and one differentiated daughter cell. Alternatively, extrinsic mechanisms, involving stromal cell signals, could cause daughter cells that remain in their proper niche to stay stem cells, whereas daughter cells that leave this niche differentiate. Drosophila spermatogenesis has been used as a model stem cell system to show that there are excess stem cells and gonialblasts in testes that are deficient for Raf activity. In addition, the germline stem cell population remains active for a longer fraction of lifespan than in wild type. Finally, raf is required in somatic cells that surround germ cells. It is concluded that a cell-extrinsic mechanism regulates germline stem cell behaviour (Tran, 2000).

The testis proliferation center in Drosophila melanogaster includes the germline and somatic stem cells that maintain spermatogenesis. As a germline stem cell divides, one daughter becomes a gonialblast, while the other remains a stem cell. To amplify the germline population, each gonialblast executes four divisions as 2° spermatogonia, which exit the mitotic cycle and enter a meiotic and differentiation program as a clone of 16 spermatocytes. The gonialblast and its progeny are encysted by somatic cells derived from cyst progenitor stem cells. Mutants affecting 2° spermatogonia have been characterized: signal transduction pathways were tested for possible involvement at earlier decision points in this germline stem cell lineage. Null mutations in raf, encoding a serine/threonine kinase involved in receptor tyrosine kinase pathways, are lethal in larvae, but such flies carrying a heat shock (HS)-Raf transgene can survive to fertile adults by using daily heatshocks. By withdrawing heat shock when adults eclose, animals become progressively raf deficient as the protein decays. Testes from such hypomorphic raf-deficient males 5 days after eclosion were indistinguishable from controls. By day 7, however, 43 of 44 raf mutant testes show great expansion of the proliferation center. The increase in cell number is due to excess, early stage germ cells, and continues such that on day 15, raf -deficient testes are filled with these cells at the expense of post-mitotic cells (Tran, 2000).

To identify the earliest defect in germ-cell progression, the fusome, a membrane and cytoskeletal organelle specific to the germline that is spheroid throughout stem cells and gonialblasts, but branches extensively throughout interconnected 2° spermatogonia, was tested. In raf-deficient testes, fusome structure is not normal. Unbranched fusomes are found in many cells, even those located some distance from the hub, where, in wild-type testes, only branched fusomes interconnecting 2° spermatogonia are found. Although branched fusomes do appear in raf-deficient testes, unlike wild-type testes, these fusomes usually appear only after the appearance of an intervening region containing many excess germ cells that have only spheroid fusomes. These excess germ cells do not result from an increased frequency of germline stem cell divisions, because the M-phase index for the tier of cells adjacent to the hub in raf-deficient testes is almost identical to that in the wild type (Tran, 2000).

Cytoplasmic Bag-of-marbles protein (Bam-C), which first accumulates in 2° spermatogonial cells was tested and was found to be required for their progression into spermatocytes. In the wild type, the total population of germline stem cells and gonialblasts comprises the few non-staining cells between the hub and the first rows of 2° spermatogonia. In raf-deficient testes, the first Bam-C-expressing germ cells are located much further from the hub. Also, most of the intervening non-staining cells contain unbranched fusomes, consistent with these cells representing excess gonialblasts and/or germline stem cells (Tran, 2000).

To verify this, M5-4 and S1-33, markers expressed in hub cells, germline stem cells and gonialblasts, but not in 2° spermatogonia, were examined. In raf-deficient testes, while hub cells appear normal, the number of germ cells expressing these markers is greatly increased. Furthermore, marker expression persists for a significantly longer fraction of adult lifespan. For instance, by day 19, all control testes have M5-4 marker expression in fewer than three germ cells, rather than the average 5 to 9 germline stem cells plus associated gonialblasts contained in testes from young adults. In contrast, only 17% of raf-deficient testes on day 19 show loss of germ cell expression, whereas 48% maintain expression at least equivalent to that of day 1, and a further 35% still show increased expression. These data suggest that germline stem cells and gonialblasts remain active for a significantly longer period than in the wild type. This was further tested by directly counting the number of cycling germline stem cells, which were judged to be cells that are located adjacent to the hub and contain a spherical fusome and nuclear anillin, a late interphase marker. Whereas wild-type testes averaged 3.1 late interphase germline stem cells per testis on day 1, on day 19 aged cohorts average only 0.8. This suggests an age-induced quiescence of the germline stem cell population. In contrast to this, in raf-deficient testes, late interphase germline stem cell numbers are maintained over a 19 day period suggesting that germline stem cells as a population remain active for a longer period than in wild-type (Tran, 2000).

To determine whether raf function is required in the germ line, which exhibits the phenotypes described above, or in the surrounding somatic cells, raf null mutant clones were generated. Persistent germline clones indicate the existence of a raf null germline stem cell. In all cases, progression through spermatogenesis is normal as judged by groups of 16 mutant germ cells that are morphologically indistinguishable from surrounding wild-type spermatocytes. Thus, raf function is dispensable in germ cells. In contrast, no cyst cell clones were recovered, suggesting that raf is necessary for viability or proliferation of cyst progenitor cells. Since the previous analysis was under hypomorphic conditions for raf, the mosaic analysis was repeated, introducing a HS-Raf transgene to provide a basal level of raf function so that raf-deficient cyst cells might survive. Persistent raf-deficient cyst cell clones indicate the existence of a raf-deficient cyst progenitor cell. Such testes have excess early stage, raf+ germ cells. Thus, raf is required in the cyst cell lineage. In addition, in raf-deficient testes two cyst cell markers normally expressed in later-stage cyst cells, LacZ600 and Eyes absent, are now expressed prematurely in somatic cells adjacent to the hub, probably the cyst progenitor cells. This shows that raf function is required in somatic cells surrounding germline stem cells (Tran, 2000).

Germline stem cell divisions lead to a distinction between a self-renewing daughter cell and a sister cell committed to differentiation as a gonialblast. These data suggest that a somatic signal influences this decision by limiting the self-renewing potential. It cannot yet be said how this potential is encoded. It is hypothesized is that in raf-deficient testes, where cyst progenitor cell identity is disturbed, the signal is lost, and excess stem cell potential is produced. Upon division, both daughters of the germline stem cell inherit some stem cell character. At steady state, this increases the number of cells that become gonialblasts, and somehow prolongs the active state of the stem cell population. Thus, a somatic cell defect leads to a tumor in the germline stem cell lineage, which suggests that some tumors of progenitor cell populations could be initiated by genetic lesions in support cells, rather than in the tumorous cells themselves. In the testis, the Raf-dependent signal may be delivered by cyst progenitor cells, or their cyst-cell daughters. Mosaic analysis shows that depletion of Raf from just one of the two cyst progenitor cells surrounding a germline stem cell causes a defect. This may indicate a dose effect, where one heterozygous somatic cell is not sufficient to allow normal signaling. It is likely that the signal transducer Raf is engaged, owing to activation of the Epidermal growth factor receptor pathway in somatic cells. It is noted that in raf-deficient testes, differentiation of 2° spermatogonia is blocked because they do not transit to the spermatocyte stage. It is believed that this is a secondary effect owing to the defective cyst lineage, since a cyst cell signal governs this later transition from 2° spermatogonia to spermatocytes (Tran, 2000).

Somatic signals have been postulated to affect germline stem cell behaviour in the Drosophila ovary. However, as has been found here, the characterized signals are necessary to maintain germline stem cells, rather than restrict their self-renewing potential. Additionally, raf-deficient ovaries exhibit no increases in early stage germ cells. Thus, despite the superficial similarities of early germ cell development in ovary and testis, oogenesis and spermatogenesis are emerging as complementary systems from which different principles of stem cell regulation will emerge (Tran, 2000).

Dual function of Ras in Raf activation

The small guanine nucleotide binding protein p21Ras plays an important role in the activation of the Raf kinase. However, the precise mechanism by which Raf is activated remains unclear. It has been proposed that the sole function of p21Ras in Raf activation is to recruit Raf to the plasma membrane. Two basic observations constitute the basis of this model: (1) purified Raf-1 cannot be activated by Ras-GTP in vitro, and (2) in cultured cells, Raf-1 can be activated by being targeted to the membrane without co-transfection of activated Ras. This activation of membrane-targeted Raf cannot be inhibited by a dominant negative form of Ras (Li, 1998 and references).

All Raf proteins share three conserved regions: CR1, CR2, and CR3. CR1 and CR2 are located in the N-terminal regulatory region, which acts to suppress the catalytic activity of the C-terminal CR3 kinase domain. Removal of the N terminus (including CR1 and CR2) results in constitutive activation of Raf-1 in mammalian cells. The activation of Raf requires its direct interaction with Ras in its activated GTP-bound form. Two regions within CR1 have been identified that bind Ras: the Ras-binding domain (RBD) and a cysteine-rich domain (CRD). The RBD associates with the Ras effector domain with high affinity, and Arg89, located in the RBD of Raf-1, is a key residue that mediates this interaction. A mutation in this residue, R89L, abolishes the association between Ras and Raf-1, as well as Ras-dependent activation of Raf-1. The CRD also associates with Ras through residues different from those that contact the RBD. Interestingly, it was demonstrated that the CRD:Ras interaction only occurs once the association of RBD with Ras has occurred. The Drosophila Draf protein is structurally and functionally homologous to mammalian Raf-1. Human RAF-1 is 46% identical in amino acid sequence to Draf, and is able to substitute in Drosophila for Draf for viability and signal transduction. A Drosophila mutation, DrafC110, associated with a reduced level of Draf activity, has been isolated that cannot support the survival of the animal. This mutation is an amino acid change in Arg217, equivalent to Arg89 of Raf-1. The DrafC110 R217L mutation prevents the Draf:Ras1 interaction, suggesting that like Arg89 in Raf-1, Arg217 is essential for Ras1:Draf interaction. Dominant intragenic suppressors of DrafC110 have been identified that restore viability to DrafC110 flies. Two of these suppressors, Su2 and Su3, identify mutations P308L and F290I, respectively, in the CRD of Draf. When the analogous DrafC110 mutation along with either of its intragenic suppressors are introduced into mammalian Raf-1, the suppressors do not function to restore the lost Ras:Raf association. Rather, they increase the basal level activity of Raf-1 (Li, 1998 and references).

To further understand the mechanism of Raf activation by Ras, the activity of a number of Raf mutations were examined in the complete absence of Ras activity. These experiments are feasible in Drosophila because it is possible to generate eggs completely devoid of Ras1 or Draf activity using the mosaic 'FLP-DFS' technique. This is achieved following generation of germline mosaics that allow production of eggs derived from germline cells homozygous for either Ras1 or Draf protein null mutations. The embryos derived from females that lack maternal Draf or Ras1 activities are referred to as Draf or Ras1 embryos, respectively. Draf plays a central role in Tor signal transduction because embryos lacking maternal Draf gene activity have phenotypes identical to those of tor null embryos, resulting in the complete absence of posterior tll expression. Weaker Draf alleles exhibit reduced levels of tll expression. Therefore, visualization of tll expression allows one to determine the level of activity of Tor, Draf, or its downstream signal transducer. Previously, it had been proposed that N-terminal truncation or membrane targeting of Raf results in Ras-independent 'constitutive activation' of Raf. The signaling activity of an N-terminally truncated Draf (DrafdeltaN) mutation expressed in embryos completely devoid of Ras1 activity has been reexamined. Contrary to the current model, it is demonstrated that Ras1 is necessary for the activation of DrafdeltaN. Further, it is shown that the activity of membrane-targeted DrafdeltaN is still sensitive to the presence of Ras1. Finally, the role of Ras1 in Draf activation was examined using point mutations in Draf that disrupt its association with Ras1. DrafC110 in combination with one of its intragenic suppressors, Su3, is still regulated by Ras1 and Tor, although Su3 does not restore the Draf C110:Ras1 molecular interaction. The unregulated enzymatic activity of Raf-1, due to the Su3 mutation, is not sufficient for the mutant Draf to transduce Tor signals, suggesting that Ras1, which is not physically interacting with the mutant Draf protein, is required for the activation of the mutant Draf. Taken together, these results suggest that Ras has an essential function in Raf activation beyond translocating Raf to the plasma membrane (Li, 1998).

Currently, the exact mechanism by which Raf is regulated is still unknown. In addition to Ras, other Raf-interacting proteins have been isolated as potential Raf activators. These include 14-3-3 and KSR. Studies with the Drosophila 14-3-3 genes indicate that they cannot encode the 'Raf activator' since these proteins are necessary, but not sufficient for Draf signaling. The situation with KSR is less clear. KSR associates with 14-3-3 and also with Raf-1 at the membrane in a Ras-dependent manner. It has been suggested that KSR is a ceramide-activated protein (CAP) kinase that phosphorylates and activates Raf-1 in vitro. However, it has been found that KSR plays a structural role in modulating Raf/MEK/MAPK signal propagation and it does not appear to phosphorylate Raf-1. A 'two-step' mechanism by which Ras activates Raf is proposed. It is suggested that first activated Ras binds to Raf at the membrane and brings Raf into close proximity to its activator. The conformational change that results from the binding of Ras to the N-terminal region of Raf relieves the inhibitory effects of the N terminus, exposing the CR3 kinase domain to the 'Raf activator'. Since it has been shown that Ras-GTP does not directly activate Raf, the existence of a 'Raf activator' has to be postulated. As a second step, it is proposed that, independent of the binding of Ras to Raf, activated Ras activates the unknown 'Raf activator'. A test of this model obviously awaits the identification of the 'Raf activator' (Li, 1998 and references).

Drosophila Pin1 prolyl isomerase Dodo is a MAP kinase signal responder during oogenesis

Transcription factor CF2 is required for the expression of rhomboid gene. Dodo is an enzyme that facilitates the degradation of CF2, which regulates expression of the rhomboid gene in follicle cells. This chain of events is required to establish the dorsal/ventral polarity of the developing oocyte. The epidermal growth factor receptor (Egfr) signal transduction system that functions in follicle cells, specifies the dorsal follicle cell fates in the Drosophila egg chamber (Hsu, 2001).

CF2 degradation in the anterodorsal follicle cells requires phosphorylation at the single MAPK site (PAT40P). Replacement of Thr 40 with alanine by site-directed mutagenesis renders the mutant protein (CF2A40) resistant to proteolysis (Mantrova, 1998). This MAPK recognition site also constitutes the potential Dodo target. If Dodo is a responder to the MAPK signal, as is proposed, the phosphorylation status at this site should directly affect the binding affinity of Dodo for CF2. Binding assays in vitro comparing phosphorylated and unphosphorylated CF2 as well as the wild type and the A40 mutant demonstrate that Dodo binding affinity for the CF2 protein is indeed markedly decreased when the MAPK/Dodo recognition site is unphosphorylated, either owing to the absence of MAPK or as a result of the mutation (Hsu, 2001).

Interestingly, although the hs-dodo transgene can rescue the ventralized dodo-null phenotype, expression of the transgene by itself does not cause a significant level of reciprocal (dorsalized) phenotype. This finding is consistent with the hypothesis that the function of Dodo requires, a priori, an activated Egfr-MAPK signaling pathway. In other words, Dodo acts as a responder to the Egfr-MAPK signaling although it is not part of the signaling cascade itself. However, this hypothesis also predicts that increased levels of Dodo should exacerbate the deleterious effect of an ectopically expressed and constitutively activated MAPK signaling pathway. This prediction was tested with a transgenic fly strain containing a constitutively active D-raf mutant complementary DNA (cDNA) under the control of heat-shock promoter. D-Raf acts upstream of MAPK in this pathway and the hs-D-raf gain-of-function (hs-D-rafgof) transgene has been shown to induce the dorsalized phenotype such as expanded dorsel appendages. One copy of this transgene is weakly penetrant, inducing 16.6% phenocopies. This low penetrance can be enhanced more than threefold by adding only one copy of the hs-dodo transgene, to a degree comparable to two copies of D-rafgof. The enhancing effect of dodo on D-rafgof is also reflected in the CF2 expression pattern. In most stage-10 eggs derived from hs-dodo/+;hs-D-rafgof/+ females, CF2 is degraded throughout the follicle cell population, similar to that observed in homozygous hs-D-rafgof egg chambers (Hsu, 2001).

Raf-1 is involved in the regulation of the interaction between guanine nucleotide exchange factor and Ha-ras. Evidences for a function of Raf-1 and phosphatidylinositol 3-kinase upstream to Ras

The role in patterning of quantitative variations of MAPK activity in signaling from the Drosophila Torso (Tor) receptor tyrosine kinase (RTK) has been examined. Activation of Tor at the embryonic termini leads to differential expression of the genes tailless and huckebein. Using a series of mutations in the signal transducers Corkscrew/SHP-2 and D-Raf, it has been demonstrated that quantitative variations in the magnitude of MAPK activity trigger both qualitatively and quantitatively distinct transcriptional responses. When terminal activity is progressively removed, there is a corresponding progressive malformation and eventual loss of terminal cuticular structures. The first terminal cuticular elements that are malformed or lost require the highest terminal activation (e.g., the anal tuft and posterior spiracles visualized by the presence of Filzkorper material). The next elements that are malformed or lost require intermediate levels of terminal signal (e.g., the abdominal 8 (A8) denticle belt and the posterior spiracles). Finally, the last elements that are malformed or lost require the lowest levels of terminal activity (e.g., posterior A7). While in the absence of D-raf activity, no activated MAPK (dp-ERK) is observed at the posterior pole. In csw null mutant embryos, where the tll and Hb expression domains are present though mispositioned, reduced levels of dp-ERK reactivity are observed. Collectively, these results reveal that a precise transcriptional response translates into a specific cell identity (Ghiglione, 1999).

Two chimeric receptors, Torextracellular-Egfrcytoplasmic and Torextracellular-Sevcytoplasmic, cannot fully functionally replace the wild-type Tor receptor, revealing that the precise activation of MAPK involves not only the number of activated RTK molecules but also the magnitude of the signal generated by the RTK cytoplasmic domain. For example, analysis of Torextracellular-Egfrcytoplasmic reveals that the posterior domain of Hunchback does not retract from the posterior pole, but rather remains as a terminal cap. Further, the anterior border of this posterior Hb domain is shifted posteriorly. Altogether, these results illustrate how a gradient of MAPK activity controls differential gene expression and thus, the establishment of various cell fates. The roles of quantitative mechanisms in defining RTK specificity are discussed. It is possible that in some instances, the generation of differing magnitudes of activity from the cytoplasmic domains of specific RTKs might be dependent on the specific affinities of the downstream signal transducers to the receptor. Csw binds through one of its SH2 domains to only one phosphotyrosine on Tor. Perhaps a higher or lower affinity of Csw to this site, or addition of another site that would also engage the second SH2 domain of Csw, would increase or decrease signal output. Presumably, in each individual cell there exists a mechanism built into the enhancer elements of the promoters of both tll and hkb that acts to read directly the magnitude of Tor signaling. In the tll promoter, a Tor-response element that mediates the repression of tll has been identified, indicating that the Tor signal activates tll by a mechanism of derepression. A putative candidate for this repressor activity is encoded by the transcription factor Grainyhead. Grainyhead binds to the Tor-response element and can be directly phosphorylated by MAPK in vitro: a decrease in Gh activity has been shown to cause tll expansion in early embryos. Further, the transcriptional corepressor Groucho is required for terminal patterning. Further characterization of how Gh and/or Gro activities are regulated by activated MAPK should clairify how differing levels of phosphorylation translate into derepression of terminal target genes (Ghiglione, 1999).

Progression of the morphogenetic furrow in the Drosophila eye: the roles of Hedgehog, Decapentaplegic and the Raf pathway

During Drosophila eye development, Hedgehog (Hh) protein secreted by maturing photoreceptors directs a wave of differentiation that sweeps anteriorly across the retinal primordium. The crest of this wave is marked by the morphogenetic furrow, a visible indentation that demarcates the boundary between developing photoreceptors located posteriorly and undifferentiated cells located anteriorly. Evidence is presented that Hh controls progression of the furrow by inducing the expression of two downstream signals. The first signal, Decapentaplegic (Dpp), acts at long range on undifferentiated cells anterior to the furrow, causing them to enter a 'pre-proneural' state marked by upregulated expression of the transcription factor Hairy. Acquisition of the pre-proneural state appears essential for all prospective retinal cells to enter the proneural pathway and differentiate as photoreceptors. The second signal, presently unknown, acts at short range and is transduced via activation of the Serine-Threonine kinase Raf. Activation of Raf is both necessary and sufficient to cause pre-proneural cells to become proneural, a transition marked by downregulation of Hairy and upregulation of the proneural activator, Atonal (Ato), which initiates differentiation of the R8 photoreceptor. The R8 photoreceptor then organizes the recruitment of the remaining photoreceptors (R1-R7) through additional rounds of Raf activation in neighboring pre-proneural cells. Dpp signaling is not essential for establishing either the pre-proneural or proneural states, or for progression of the furrow. Instead, Dpp signaling appears to increase the rate of furrow progression by accelerating the transition to the pre-proneural state. In the abnormal situation in which Dpp signaling is blocked, Hh signaling can induce undifferentiated cells to become pre-proneural but does so less efficiently than Dpp, resulting in a retarded rate of furrow progression and the formation of a rudimentary eye (Greenwood, 1999).

The signal transduction pathway downstream of receptor tyrosine kinases involving Ras and Raf has been shown to have multiple roles during the formation of the Drosophila eye, most notably in mediating signals by the Sevenless and Epidermal Growth Factor receptors. Further, misexpression of activated forms of Ras, or the EGF receptor, anterior to the furrow, can initiate photoreceptor differentiation. It was therefore asked whether this signaling pathway might also be utilized in the induction of Ato. To constitutively activate the Raf transduction pathway in the eye disc, a myristylated and truncated form of human Raf, which is called Raf*, was expressed in clones of cells using the Flp-out technique. Under conditions that cause expression of constitutively active Raf* in most cells, the anterior stripe of peak Ato expression is observed to expand several cell diameter lengths forward, to fill the region just anterior to the position of the furrow where Hairy is normally expressed at peak level. However, the anterior limit of peak Ato expression does not extend past that of Hairy expression. Examination of Hairy under the same conditions reveals that the normally high levels of expression just anterior to the furrow are diminished. This reduction in Hairy expression, alone, is not likely to account for the expansion of Ato expression, because Ato expression is not altered in eye discs lacking hairy activity. However the combined loss of both Hairy and the related repressor protein Emc causes ectopic Ato expression anterior to the furrow. In the presence of indiscriminate Raf* activity, Emc expression, like that of Hairy, appears to be diminished anterior to the furrow. Hence, the expansion of Ato expression can be attributed to the concomitant reduction in both Hairy and Emc expression (Greenwood, 1999).

Constitutive expression of Raf* also induces ectopic photoreceptor differentiation anterior to the furrow, as assayed by the expression of the neuronal antigen 22C10. Similar to the expansion of Ato induced by Raf*, ectopic photoreceptor differentiation is restricted to a dorso-ventral stripe of cells just anterior to the normal position of the furrow. Not all the cells within this column differentiate as photoreceptors. Rather, there is a saltatory pattern of differentiation that can be attributed to the process of lateral inhibition, which normally restricts the number of proneural cells that differentiate as photoreceptors. Similar results are obtained under conditions that create small, rare clones of cells expressing Raf*. In this case, small, isolated clusters of photoreceptors, located anterior to the endogenous furrow and surrounded by what appears to be an ectopic furrow (marked in this experiment by the expression of a dpp-lacZ reporter gene) are observed. As is observed for neural differentiation induced by widespread expression of Raf*, small clones of Raf* expressing cells initiate ectopic photoreceptor development only when they are close to the furrow. These findings indicate that during normal development, Dpp signaling anterior to the furrow creates a pool of cells; these cells are primed to enter the proneural pathway, but blocked from doing so by the expression of high levels of Hairy and Emc. These cells are referred to as being 'pre-proneural'. Release from this block requires an additional signal, which is induced at short range by Hh signaling and transduced by activation of the Raf pathway. Raf, but not the Drosophila EGF receptor, is required for Ato expression and photoreceptor differentiation. The results seen with activated Raf suggest that endogenous Raf may normally regulate Ato expression. Therefore, Ato expression was examined in clones of cells that lack Raf function. Clones of raf minus cells marked by the expression of a nuclear-localized form of beta-galactosidase were generated using the Flp-out technique. Raf minus cells do not express Ato. Moreover, the absence of Ato expression is cell-autonomous, indicating that Raf is normally required to transduce a signal that is essential for Ato expression (Greenwood, 1999).

Raf normally mediates signals from receptor tyrosine kinases. Two candidate receptor tyrosine kinases, which could activate Raf ahead of the furrow, are the Drosophila EGF receptor (Egfr) and one of the Drosophila FGF receptor homologs, Heartless (Htl). Egfr is expressed at high levels ahead of the furrow, and is required for photoreceptor differentiation. Similarly, two ligands for Egfr, Spitz and Vein, are active within the furrow and regulate photoreceptor differentiation. However, Ato is expressed in Egfr minus clones, indicating that Egfr is not essential for the Raf-dependent activation of Ato expression. Htl is also expressed in the morphogenetic furrow. Similar to Egfr, however, removal of Htl has no effect on Ato expression. It is possible that both Egfr and Htl can receive the Ato inducing signal, accounting for the ability of cells lacking one or the other function to initiate Ato expression. Alternatively, Raf may transduce a signal that is received by another receptor (Greenwood, 1999).

It is envisaged that pre-proneural cells are metastable, having a latent proneural capacity that is actively held in check by proneural repressors such as Hairy and Emc. How does activation of Raf precipitate the transition to the proneural state? Because the simultaneous loss of both Hairy and Emc activities causes an expansion of Ato expression similar to that resulting from the expression of activated Raf, it has been suggested that Raf activation may normally induce transition to the proneural state by blocking the expression or activity of these repressors. Consistent with this possibility, Hairy contains potential phosphorylation sites for MAPK, a kinase downstream of Raf in the signaling pathway. Daughterless expression is also upregulated in the furrow and is necessary to maintain Ato expression. Moreover, Daughterless, like Hairy, contains phosphorylation sites for MAPK, raising the possibility that Raf activity may directly potentiate proneural activators at the same time that it downregulates the activities of their repressors. Similar events may also occur in mammalian neural differentiation, as NGF-induced differentiation of the mammalian neuronal cell line PC12 is mediated by the phosphorylation of HES-1, a Hairy related protein (Greenwood, 1999).

Identification of autosomal regions involved in Drosophila Raf function

Raf is an essential downstream effector of activated Ras in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified 'Raf activator.' In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, a genetic screen was conducted to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). A collection of chromosomal deficiencies representing ~70% of the autosomal euchromatic genomic regions was examined for the abilities of these regions to enhance the lethality associated with a hypomorphic viable allele of Draf, DrafSu2. Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of DrafSu2, causing lethality in DrafSu2 hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3epsilon, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. Src64B interacts genetically with Draf and an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, a genetic interaction was discovered between DrafSu2 and Df(3L)vin5 that reveals a novel role for Draf in limb development. Loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs (Li, 2000).

Src64B and Tec29A are removed by two deficiencies that each dominantly enhance the lethality of DrafSu2. They were selected as candidate genes for these two deficiencies because a survey of FlyBase for genes in the regions removed by the deficiencies did not yield other genes more likely to be involved in Draf function. The Src64BDelta17 allele in homozygotes enhances DrafSu2, confirming that Src64B genetically interacts with DrafSu2. Overexpression of an activated form of Src64B in early embryos can cause activation of the Tor target gene tll and cuticular defects similar to those caused by gain-of-function mutations in tor. These results are consistent with a role for Src64B in Tor signaling and/or Draf activation. It was not possible to demonstrate that Tec29A could enhance Draf using an available mutant allele of Tec29A. However, indirect evidence has been obtained suggesting a requirement of Tec29A in Tor signaling. (1) Tec29A206 homozygous mutant embryos exhibit defects in the terminal structures that are specified by the Tor pathway. Specifically, they show defective mouth parts and shortened Filzkörper, phenotypes consistent with disruption of Tor signaling. (2) Reducing the activity of Tec29A suppresses a gain-of-function tor allele. Most strikingly, embryos zygotically homozygous for Tec29A206 that are derived from torY9 mothers exhibit mouth parts and Filzkörper indistinguishable from those of Tec29A206 embryos. (3) Mutation of Tec29A restores most of the ventral denticle bands that would have been deleted due to torY9, suggesting that Tec29A is genetically epistatic to tor. However, many of the embryos still exhibit minor disruptions in the ventral denticle bands, a defect reminiscent of weak tor gain-of-function mutations. This suggests that homozygosity for Tec29A206 cannot completely suppress torY9. (4) Possibly, while Tec29A may be required for Tor signaling, Tec29A206 may not be a null allele and therefore cannot completely suppress torY9. This would be consistent with the inability of this allele to enhance DrafSu2. Alternatively, the maternally contributed Tec29A may be able to partially mediate signaling by the mutant TorY9 protein. (5) Tec29A may not be an absolute requirement for Tor signaling, but rather it may function in a separate pathway that in conjunction with Tor is required for the differentiation of terminal structures (Li, 2000).

The likelihood that Src64B and Tec29A are involved in Draf activation is based upon data from in vitro studies of mammalian c-Src function. Src kinases can phosphorylate and activate Raf-1 in vitro, and the tyrosine residues phosphorylated by Src are important for Raf-1 activation. Tec kinases are very similar to Src kinases in the kinase domain, but lack the C-terminal regulatory tyrosine and the N-terminal myristylation site that are specific for Src family members. Tec kinases interact with and are activated by Src through phosphorylation. It has been shown in Drosophila that Tec29A is regulated by Src64B and that both are required for the growth of ring canals of the egg chamber. Although it has not been documented that Tec can phosphorylate Raf in vivo, given the similarities in the kinase domain, it is not unreasonable to propose that Tec could do so. Finally, consistent with these results, the two genomic regions containing Src64B and Tec29A have also been identified as required for the function of Corkscrew (Csw) in a similar screen for modifiers of a partial loss-of-function csw allele (Li, 2000).

The proper expression of en in the posterior compartment of imaginal discs is essential for maintaining compartmental boundaries and patterning of Drosophila limbs. Despite much insight into the events required for Hh signaling, little is known about the mechanism(s) by which en expression is controlled in the posterior compartment. Two instances have been identified where a further reduction in Draf function, due to the presence of a deficiency, results in defects in posterior pattern elements in the limbs. DrafSu2/Y; Df(3L)vin5/+ male survivors exhibit notching only in the posterior region of the wing, and partial pattern duplications in the posterior compartment. Since no specific role for Draf has been described in the limbs, the requirements for Draf in the imaginal discs were examined. Since clonal analysis with null alleles is uninformative, because Draf mutant clones do not develop, Draf was conditionally provided to the developing animals in a Draf null background (Li, 2000).

As a result of withholding Draf during the second and early third larval instars, animals with anterior pattern element duplications in the posterior compartment were frequently observed. By examining the imaginal discs of these animals, it could be determined that when there are insufficient levels of Draf, en expression is no longer restricted to the normal posterior compartment, which suggests that Draf may act to repress/restrict En expression. Along with ectopic expression of En in the anterior compartment and increased levels of En in the posterior compartment, a new mirror image anterior compartment devoid of en expression was induced. This observation is consistent with the observation that when En is ectopically expressed, ectopic anterior pattern elements are induced. Also, ectopic expression of En in the anterior compartment induces expression of high levels of Hh and Dpp, which are responsible for overgrowth and the duplication of anterior pattern elements. Indeed, when Hh was examined in the partially rescued Draf null males, it was found to be widely ectopically expressed. The posteriorly restricted wing notching observed in DrafSu2/Y; Df(3L)vin5/+ male survivors is also consistent with a requirement for Draf in negatively regulating en, since elevated levels of En expression in the posterior compartment partially inactivate both en and inv, which are necessary for the development and terminal differentiation of posterior fates. Taken together, these observations suggest that the Df(3L)vin5 deficiency contains a gene that participates with Draf in patterning of the limbs (Li, 2000).

Drosophila Raf's N terminus contains a novel conserved region and can contribute to torso RTK signaling

Drosophila Raf (DRaf) contains an extended N terminus, in addition to three conserved regions (CR1-CR3); however, the function(s) of this N-terminal segment remains elusive. In this study, a novel region within Draf's N terminus that is conserved in BRaf proteins of vertebrates was identified and termed conserved region N-terminal (CRN). The N-terminal segment can play a positive role(s) in the Torso receptor tyrosine kinase pathway in vivo, and its contribution to signaling appears to be dependent on the activity of Torso receptor, suggesting this N-terminal segment can function in signal transmission. Circular dichroism analysis indicates that DRaf's N terminus (amino acids 1-117) including CRN (amino acids 19-77) is folded in vitro and has a high content of helical secondary structure as predicted by proteomics tools. In yeast two-hybrid assays, stronger interactions between DRaf's Ras binding domain (RBD) and the small GTPase Ras1, as well as Rap1, were observed when CRN and RBD sequences were linked. Together, these studies suggest that DRaf's extended N terminus may assist in its association with the upstream activators (Ras1 and Rap1) through a CRN-mediated mechanism(s) in vivo (Ding, 2010).

Amino acids 19-77) within Draf's N terminus, conserved for Raf genes of most invertebrates and BRaf genes of vertebrates, was identified and termed CRN. This conserved region has not been described by others, but potential roles for the extended N terminus have been proposed in two reports. One found that in HeLa cells, the N terminus of BRaf may mediate Raf dimerization to generate BRaf-BRaf or BRaf-CRaf complexes, and play an important regulatory role in calcium-induced BRaf activation. Another study reported that deletion of BRaf's N terminus did not affect BRaf-CRaf dimer formation. Instead, it was found that N-terminal residues appeared to facilitate interaction with HRas in vitro. In accordance with the previous study, stronger interactions between DRaf's RBD (Ras binding domain) and the small GTPase Ras1δCAAX were observed when N-terminal and RBD sequences were linked in a yeast two-hybrid analysis. This suggested that the N terminus might assist in Ras1 binding. Furthermore, the identity of specific residues in the N terminus that might participate in Ras1 binding were mapped to the CRN region (amino acids 19-77). Two known Raf motifs, RBD and CRD, are involved in Raf's interaction with Ras. This studies, and previous results using BRaf, suggest that the N-terminal residues of DRaf and BRaf proteins, particularly the CRN region, might be another element that plays a role(s) in Ras-Raf coupling (Ding, 2010).

The small GTPase Rap shares with Ras nearly identical Raf binding regions that comprise switch 1 and the lipid moiety. Rap functions as an antagonist of Ras in regulating CRaf activity, but can activate BRaf in a parallel way with Ras. Isoform-specific features of different Raf family members may explain their distinct responses to Rap. In flies, both Ras1 and Rap1 can interact with and activate DRaf. Thus, it was reasonable to test whether DRaf's N terminus including CRN might also assist in Rap1 binding. In agreement with this idea, stronger interaction between RBD and Rap1δCAAX was observed when DRaf's CRN and RBD sequences were linked in vitro, further suggesting that the N terminus may contribute to both Ras1 and Rap1 binding potentially through a CRN-mediated mechanism(s) in vivo (Ding, 2010).

No direct interaction between Ras1 or Rap1 and the isolated DRaf N-terminal segment (amino acids 1-117) was detected, or when the N terminus was linked with the Ras1/Rap1 binding-deficient RBDR174L. Thus, the contribution of DRaf's N-terminal residues to Ras1 and Rap1 binding requires the presence of RBD. It is possible that the CRN-containing N terminus may assist in Raf-Ras interaction by making RBD more accessible to Ras1 and/or in a sequential manner, subsequent to RBD-Ras1 interaction, by stabilizing the RBD-Ras1 complex. Deletion of CRN may result in conformational or structural changes that reduce Ras1 binding affinity. Structural analysis of these complexes may provide important clues and help to understand the molecular mechanism(s) by which CRN assists in Ras-Raf interaction. The computational analysis suggested conserved CRN has the propensity to form two α-helical structures (α1 and α2) and contains a putative phosphorylation motif T-S-K located in α2. In agreement, DRaf's N terminus (amino acids 1-117) was folded in vitro and had a high content of helical secondary structure. These findings may help to establish a basis for future determination of molecular structure (Ding, 2010).

Although no verified binding partner(s) for DRaf or BRaf's N terminus has been identified, it is still possible that CRN may interact with other regulatory factors in vivo, that may affect Ras or Rap binding and/or function in activation of DRaf and BRaf. If so, the conserved structural features of CRN most likely relate to these regulatory events in vivo. Site-directed mutagenesis of conserved sites/motifs could provide useful information regarding the molecular mechanism(s) of CRN's role in the activation of DRaf and BRaf (Ding, 2010).

This in vitro study of DRaf's N terminus was initiated on the basis of in vivo findings using both loss- and gain-of-function genetic assays that deletion of N-terminal residues consistently reduces DRaf's signal potential in the Torso pathway. When expressed at high levels, FL DRaf enhanced the gain-of-function effects of the torRL3 allele much more significantly than DRafδN114. In embryos from trk-/- mothers, addition of FL DRaf, but not DRAFδN114, partially restored the A8 denticle belt structure. These findings indicate that the N terminus can play a positive role(s) in Torso RTK signaling. Interestingly, the contribution of DRaf's N terminus in the Torso pathway appeared to be dependent on upstream receptor activity, suggesting its role in transmission of the signal. Together with yeast two-hybrid data it is proposed that the presence of N-terminal residues may facilitate the association of DRaf with the upstream regulators Ras1 and Rap1, thereby assisting in transmission of the RTK signal in vivo (Ding, 2010).

For instance, in the trk- background, a small amount of active GTP-Ras1 and GTP-Rap1 are likely present, mostly due to activation by residual upstream Trunk activity, the presence of Torso-like ligand, and/or the intrinsic activity of the Torso receptor. The trk1 mutation used in this analysis results in protein truncation at the last 16 amino acids. It is possible that overexpression of FL DRaf proteins in this background increases the likelihood of interaction between abundant DRaf proteins and membrane bound GTP-Ras1 or GTP-Rap1. This in turn, could elevate the RTK signal and partially restore development of the A8 denticle belt structure in some embryos. In contrast, deletion of the N terminus could destabilize Ras1-DRaf (or Rap1-DRaf) coupling or decrease the duration of interaction, resulting in reduced DRaf signal transmission. This may explain why expression of DRafδN114 failed to rescue the A8 denticle belt in embryos from trk-/- mothers (Ding, 2010).

Previously, an auto-inhibitory role had been assigned to residues compromising the first half of the DRaf protein, in addition to their functions in promoting its activity. Deletion of the N-terminal amino acids 1-272 (including the N terminus and CR1) or 1-402 (including the N terminus, CR1, and CR2) of DRaf at least partially relieved these negative effects. In this study, although removal of the N-terminal 1-114 residues did not result in constitutive DRafδN114 activity in embryos lacking the maternal Torso receptor, it is still possible that the N terminus may contribute to auto-inhibitory effects. Together with CR1 and CR2, these N-terminal residues (1-114) may help maintain DRaf's inactive conformation. If so, the N terminus might play dual roles, both positively and negatively regulating DRaf. Therefore, its contribution to signaling may be neutralized by this auto-inhibition and consequently result in a subtle in vivo effect. If so, selective mutagenesis of the 'inhibitory' motifs/sites in the N-terminal region or removal of other cofactors involved in its negative regulation may amplify signaling differences between FL DRaf and DRafδN114. Ras binding has been thought crucial to recruit Raf to the membrane and promote its RTK signaling activity. However, the Drosophila Torso pathway appears tolerant of alterations in Ras1-DRaf coupling. Draf C110 has a R174L point mutation in the RBD domain and likely comprised for Ras1 binding. The RBDR174L is Ras binding deficient in the yeast two-hybrid assay. However, tll expression patterns and cuticles of the embryos derived from mothers with Draf C110/Draf C110 germ cells were indistinguishable from those of wild-type embryos, suggesting a mechanism(s) independent of RBD-Ras1 interaction might function in recruiting DRaf to the membrane. In agreement with this model, it has been found that membrane translocation of CRaf could be mediated by its interaction with phosphatidic acid (PA) and independent of Ras binding. This PA binding site is also conserved in ARaf, BRaf, and DRaf. Thus, DrafC110 could be recruited to the cell membranes by associating with PA. Moreover, it is known that Raf's CRD participates in Ras binding through its interaction with the lipid moiety of Ras. Once at the membrane, it is also possible that the interaction between DrafC110's CRD and Ras1 could further promote its membrane attachment and result in relatively normal Torso signal production. In this study, the presence of RBD, CRD, and the potential PA binding site may be sufficient to promote DRaf's activation in Torso signaling. This may explain why at approximately endogenous wild-type protein level maternally expressed DRafδN114 is able to rescue the embryonic terminal defects of Draf11-29 mutants. Together, considering the Torso pathway's tolerance of alterations in Ras1-DRaf coupling and the minor role DRaf's N terminus plays in Ras1 binding, it is reasonable that the phenotypic consequences of removing these N-terminal residues (DRafδN114) are not great in Torso signaling. The subtle phenotypic effects of DRaf's N terminus could also be due to compensation provided by potential autoregulatory feedback or alternative redundant processes in the in vivo system. In this study, the expression of DRaf proteins at a low level appeared to sensitize the assay system. It was found that deletion of the N terminus seemed to increase the threshold of DRaf protein levels required for normal signaling. Furthermore, by adding one copy of the ectopic torRL3 allele or removing wild-type maternal Trunk activity the sensitivity of the Torso pathway was apparently increased. These allowed the embryonic terminal system to display enhanced differences between FL DRaf and DRafδN114 proteins (Ding, 2010).

Why is this N terminus with its 'subtle' functional effects conserved during evolution, and what is its biological relevance? There are numerous RTK pathways functioning in Drosophila cellular and developmental processes. In spite of the identical Ras-Raf-MEK signal cassette they share, these RTK pathways can lead to different biological responses. Previous studies indicated that such specificity might be due to the difference in the intensity and/or duration of the signal. This suggested that the magnitude of Raf signal could function as a critical determinant of biological responses. Participation of multiple DRaf elements in Ras1 or Rap1 binding could be a good strategy to modulate its activity. Normally, tight association with Ras1 or Rap1 through RBD and CRD regions is required and sufficient to initiate the activation of DRaf, while minor adjustments/regulation of interaction by the CRN region could optimize signaling potential and reduce variability. Thus, the extended N terminus including CRN may play a role(s) as one element in a multidomain effort to promote DRaf's interaction with Ras1 and Rap1, participating and assisting in regulation to reliably attain maximal signal output (Ding, 2010).

Table of contents

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

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