Ras oncogene at 85D
In Drosophila, the Ras1 gene is required downstream of receptor tyrosine kinases for correct eye development, embryonic patterning, wing vein formation, and border cell migration. A P-element allele of Ras1, Ras1(5703), affects viability, eye morphogenesis, and early and late stages of oogenesis. Flies transheterozgyous for Ras1(5703) and existing EMS-induced Ras1 alleles are viable and exhibit a range of eye and eggshell defects. Differences in the severity of these phenotypes in different tissues suggest that there are allele-specific effects of Ras1 in development. Analysis of rescue constructs demonstrates that these differential phenotypes are due to loss of function in Ras1 alone and not due to effects on neighboring genes. Females mutant at the Ras1 locus lay eggs with reduced or missing dorsal eggshell structures. Dominant interactions are observed between Ras1 mutants and other dorsoventral pathway mutants, including Egfr(Torpedo) and gurken. Ras1 is also epistatic to K10. Unlike Egfr and gurken mutants, Ras1 females are moderately fertile, laying eggs with ventralized eggshells that can hatch normal larvae. These results suggest that Ras1 may have a different requirement in the patterning of the eggshell axis than in the patterning of the embryonic axis during oogenesis (Schnorr, 1996).
In Drosophila, Drk (an SH2 adaptor protein), Sos (a putative activator of Ras1), Ras1, raf and rolled/MAP kinase have been shown to be required for signaling from Sevenless and the torso receptor tyrosine kinase. From these studies, it was unclear whether these components act in a single linear pathway as suggested by the genetic analysis or whether different components serve to
integrate different signals. Removing each of these components during the development of the adult epidermal structures produces a very similar set of phenotypes. These phenotypes resemble those caused by loss-of-function mutations in the Drosophila EGF receptor homolog. It appears that these components form a signaling cassette, which mediates all aspects of DER signaling but that is not required for other signaling processes during epidermal development (Diaz-Benjumea, 1994).
Mutations of the Drosophila homeotic proboscipedia gene (pb, the Hox-A2/B2 homolog) provoke dose-sensitive defects. These effects were used to search for dose-sensitive dominant modifiers of pb function. Two identified interacting genes are the proto-oncogene Ras1 and its functional antagonist Gap1, prominent intermediaries in known signal transduction pathways. Ras1+ is a positive modifier of pb activity both in normal and ectopic cell contexts, while Gap1, the Ras1-antagonist, has an opposite effect. Ras1-modulated changes were observed in homeotic effects on cell identity (bristle to distal sex combs, wing trichomes to veins, veins to trichomes or veins to bristles). Only a small number of cell identities in precise contexts are changed by HSPB activity. This suggests that most cells are aware of their positions and their correctly associated fates, perhaps as a consequence of cell-cell communication. Ras1-dependent modifications of segmental identity are also observed. These occur in a concerted fashion on groups of adjacent cells, again suggesting cell communication. A general role for Ras1 in homeotic function is likely, since Ras1+ activity also modulates functions of the homeotic loci Sex combs reduced and Ultrabithorax. These data suggest that the modulation occurs by an independent mechanism for the transcriptional control of the homeotic loci
themselves, or of the Ras1/Gap1 genes. Taken together the data support a role for Ras1-mediated cell signaling in the homeotic control of segmental differentiation (Boube, 1997).
The Src family of nonreceptor tyrosine kinases has been implicated in many signal transduction
pathways. However, due to a possible functional redundancy in vertebrates, there is no genetic
loss-of-function evidence that any individual Src family member has a crucial role for receptor tyrosine
kinase (RTK) signaling. An extragenic suppressor of Raf, Su(Raf)1, has been isolated that encodes a
Drosophila Src family gene (Src42A) identical to the previously cloned DSrc41. Characterization of Src42A mutations shows that Src42A acts
independent of Ras1 and that it is, unexpectedly, a negative regulator of RTK signaling. Src42A negatively regulates Egfr signaling during oogenesis and negatively regulates receptor tyrosine kinase signaling in the eye. For example Src42A suppresses the rough eye phenotype caused by expression of hyperactive Ras or Raf. Src42A mutation also leads to defects in head and tail morphology, tracheal development and wing morphogenesis. This study
provides the first evidence that Src42A defines a negative regulatory pathway parallel to Ras1 in the
RTK signaling cascade. A possible model for Src42A function is discussed (Lu, 1999).
The functional status of Ras, Raf, Mek, or Mapk proteins does not appear to alter the ability of wild-type Src42A to repress receptor tyrosine kinase signaling: this would favor a model in which Src42A defines a branch pathway parallel to the main Ras/Mapk cascade with an integration point downstream of Mapk. This model is consistent with the reduction of maternal Src42A activity, which can still enhance Torso receptor tyrosine kinase signaling in the absence of Ras1 protein. The manner in which Src42A acts to modulate receptor tyrosine kinase signaling is similar in two ways to another branch pathway component, Kinase suppress of Ras-1 (ksr-1), of C. elegans. (1) Src42A does not appear to dramatically alter RTK-mediated processes when mutated alone. For example, mitotic clones of Src42A mutant cells in the eye do not produce extra photoreceptor cells. (2) The negative role of Src42A is only revealed when the Ras/Mapk cascade is compromised or hyperactived. Recently, Therrien (1998) reported the isolation of Src42A as a suppressor of a dominant negative form of fly ksr in the eye. In attempts to understand more about Src42A, a genetic screen was performed that isolated loss-of-function mutation in Egfr, rolled, and a new gene, semang, as suppressors of Src42A mutants (Zhang, 1999). It is suggested that Src42A works together with other branch pathway modulators such as Ksr to regulate signal transduction downstream of Egfr and other RTKs. If each branch pathway modulator takes over only a part of the total regulatory power, it would explain why Src42A or ksr-1 shows mild phenotypes when mutated alone. However, the phenotypes of Src42A do not overlap with two other Drosophila Src family members, Src64 and Tec29, both of which are involved in ring canal development during oogenesis. It is tempting to speculate that Src42A is activated following the activation of Egfr RTK via a Ras-1 independent mechanism. The signal from the activated Src42A would then integrate, in a negative fashion, with that from the main Ras/MAPK pathway to determine the final readout of an RTK pathway (Lu, 1999).
Connector enhancer of KSR (CNK) is a multidomain protein required
for RAS signaling. Its C-terminal portion (CNKC-term)
directly binds to RAF. The N-terminal portion of
CNK (CNKN-term) strongly cooperates with RAS, whereas
CNKC-term efficiently blocks RAS- and RAF-dependent
signaling when overexpressed in the Drosophila eye. Two
effector loop mutants of RASV12, S35 and C40, which
selectively activate the mitogen-activated protein kinase (MAPK) and
phosphatidylinositol-3-kinase pathways, respectively, do not cooperate
with CNK. However, a strong cooperation is observed between CNK and
RASV12G37, an effector loop mutant known in mammals to
activate specifically the RAL pathway. Two domains
in CNKN-term that are critical for cooperation with RAS have been identified.
These results suggest that CNK functions in more than one pathway
downstream of RAS. CNKc-term seems to regulate RAF, a
component of the MAPK pathway, whereas CNKN-term seems to
be involved in a MAPK-independent pathway (Therrien, 1999).
The ability of CNK or CNKN-term to enhance
activated RAS but not activated RAF suggests that this effect occurs
upstream of RAF or in a pathway parallel to RAF. Two independent sets
of results presented in this paper are consistent with the idea that
the CNK/RAS cooperation is mediated by a
RAF/MAPK-independent pathway. The first is the fact that the
coexpression of CNKN-term and
RAS1V12 does not produce higher levels of
activated MAPK. The second set of evidence is the striking
observation that CNK strongly cooperates with
RAS1V12G37, a RAS effector loop mutant known to
stimulate the RAL pathway in mammals, but not with two other RAS
effector loop mutants known to stimulate the MAPK and PI3-K pathways. The data cannot rule out the alternative hypothesis that the
enhancement of RAS1V12G37 by CNK results from
CNK-mediated stimulation of the MAPK pathway acting synergistically
with the RAS1V12G37-stimulated pathway. This
possibility would be analogous to the strong cooperation observed in
mammalian cells between the MAPK pathway and the RAL pathway to
transform cells. Consistent with this possibility, it has been found that a
loss-of-function mutation in the
rolled/mapk locus does not suppress the
RAS1V12G37 mild rough-eye phenotype but
suppresses the CNK/RAS1V12G37 cooperation. Moreover, the
RAS1V12G37 mutant is leaky and stimulates MAPK
when expressed in S2 cells to approximately 10% of the levels obtained
with RAS1V12 or RAS1V12S35. It is thus formally possible that CNK functions in
the MAPK pathway, which collaborates efficiently with another pathway
also stimulated by RAS1V12G37. A strong argument
against this model is the observation that CNK strongly cooperates with
RAS1V12 but not with
RAS1V12S35, a RAS effector loop mutant known to
stimulate the RAF/MAPK pathway. It would be difficult to
reconcile this observation with a model in which CNK is merely
enhancing signaling through the MAPK pathway. More likely, the
phenotypic effect of suppressing MAPK signaling might be due to the
fact that MAPK is involved in secondary developmental defects resulting from the strong stimulation of the
RAS1V12G37-specific pathway. Alternatively, it
might reflect the fact that the
CNK/RAS1V12G37-stimulated pathway also
requires a basal level of MAPK signaling to mediate its effects. A
similar dependency in basal MAPK activity has been suggested recently
for the RAL pathway to induce the differentiation of F9 embryonal
carcinoma cells (Therrien, 1999).
It is expect that RAS1, like its mammalian homologues, controls the RAL
pathway in Drosophila. Because the effector loop regions of Drosophila RAS1 and mammalian RAS proteins are identical in sequence and because the mammalian RALA and Drosophila RAL
are nearly identical, it is
likely that RAS1V12G37 also stimulates the RAL
pathway in Drosophila. Although little is known regarding
the RAL pathway in Drosophila, it has been reported recently
that overexpression of activated RAL in the Drosophila eye
disrupts the normal actin cytoskeleton assembly but does not interfere
with cell differentiation. This result is consistent with studies
conducted in mammalian cells that suggested that RAL controls the
organization of the actin cytoskeleton. Based on these findings,
it will be interesting to determine whether
RAS1V12G37 has a similar effect on the actin
cytoskeleton and whether CNK enhances this effect (Therrien, 1999).
The CNK/RAS cooperation clearly depends on the integrity of the
SAM and the CRIC domains. SAM domains have been found in various types
of proteins and seem to mediate homodimerization and/or heterodimerization with other SAM domain-containing proteins. The
CRIC domain is a unique region shared by all CNK homologs identified
thus far. The
boundaries of this domain (~ 80 amino acids) have been arbitrarily defined based on sequence homology. Its functional relevance was initially suggested by a cnk loss-of-function allele,
cnkXE-726, which has a 3-amino acid
in-frame deletion within this region. The elucidation of the
functions of the SAM and CRIC domains of CNK awaits the molecular
characterization of their effect on RAS signaling and the
identification of the proteins that interact with them (Therrien, 1999).
Mutations have been characterized in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both
Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions (Tapon, 2001).
The enhanced growth observed in the Tsc1 or Tsc2 mutants most resembles the results of inactivating PTEN or increasing Ras1 or dmyc activity. In each of these situations, there is a reduction in the length of the G1 phase. In contrast, increased growth driven by Cyclin D/cdk4 does not alter the distribution of cells in different phases of the cell cycle. The effects of the combined overexpression of Tsc1 and Tsc2 displays genetic interactions with multiple pathways. The phenotype is influenced by alterations in the levels of dS6K, PTEN, Ras1, dmyc, cyclin D, and cdk4. Thus, Tsc1 and Tsc2 may function downstream of the point of convergence of these pathways. Alternatively, Tsc1 and Tsc2 may primarily antagonize one of these pathways, but this effect could be overcome by increasing the activity of one of the others (Tapon, 2001).
Calcineurin is a Ca2+-calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca2+ signals into changes in cell function and development. A dominant modifier screen was carried out in the Drosophila eye using an activated form of Calcineurin A1 (FlyBase name: Protein phosphatase 2B at 14D), the catalytic subunit, to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. A second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways have suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of these results, it is proposed that calcineurin, upon activation by Ca2+-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of Sprouty and the GTPase-activating protein Gap1 (Sullivan, 2002).
To examine the interaction between calcineurin and individual components of the Egfr pathway, the ability of mutations in these components to modify the activated calcineurin phenotype was tested. Hypomorphic mutations in Egfr, Ras, pnt, sty, Gap1, and small wing modified activated calcineurin, although this was not the case for most downstream components of the Egfr pathway. TCAGB (ectopically expressed activated calcineurin) is enhanced by removing one copy of Egfr, Ras, or pnt and was suppressed by Gap1 and small wing. Both TCAGB and TCAG (another form of ectopically expressed activated calcineurin) suppress the rough eye caused by hypermorphic Egfr alleles: flies that have one copy of EgfrE1 and TCAGB have a rough eye that closely resembles that of TCAGB alone. TCAG is not detectably modified by hypomorphic Egfr, Ras, or pnt alleles. Aside from CS3-3, none of the modifier groups corresponded to Egf receptor/Ras signaling components that genetically interact with TCAG. However, it is possible that these genes are present among the 61 single hits, which have not been characterized (Sullivan, 2002).
The Drosophila larval hematopoietic system has been used as an in vivo model for the genetic and functional genomic analysis of oncogenic cell overproliferation. Ras regulates cell proliferation and differentiation in multicellular eukaryotes. To further elucidate the role of activated Ras in cell overproliferation, a collagen promoter-Gal4 strain was generated to overexpress RasV12 (Ras-act) in Drosophila hemocytes. Activated Ras causes a dramatic increase in the number of circulating larval hemocytes (blood cells); this increase is caused by cellular overproliferation. This phenotype is mediated by the Raf/MAPK pathway. The mutant hemocytes retain the ability to phagocytose bacteria as well as to differentiate into lamellocytes. Microarray analysis of hemocytes overexpressing RasV12 vs. Ras+ identified 279 transcripts that are differentially expressed threefold or more in hemocytes expressing activated Ras. This work demonstrates that it will be feasible to combine genetic and functional genomic approaches in the Drosophila hematopoietic system to systematically identify oncogene-specific downstream targets (Asha, 2003).
One overall finding is that many of the genes that are upregulated in Ras-act cells include genes that function in cell cycle regulation and DNA replication. These genes include both positive and negative regulators of cell proliferation. The cyclin-dependent kinase inhibitor dacapo (which antagonizes the function of cyclin E/cdk2 complexes), as well as the wee1 kinase (which inactivates cdc2), are both induced. There is currently no known function for either gene in promoting cell cycle progression. Thus the induction of these genes may represent a negative feedback mechanism that attempts to reduce cell proliferation under conditions of excessive cell proliferation. Another possibility is that these two genes have currently unknown roles in promoting cell cycle progression. The microarray data also show that regulators that promote all stages of cell cycle progression are induced, not only those that promote the G1/S transition. These data therefore suggest that both the G1/S and G2/M cell cycle transitions may be influenced by an increase in Ras activity (Asha, 2003).
A second finding is that many of the transcriptional targets known to be induced by Ras1 in other tissues are not induced in Ras-act hemocytes. Therefore, although the RTK/Ras pathway induces the expression of phyllopod in the eye disc, mirror in the ovary, and blistered and ribbon in the tracheal cells, none of these genes are obviously induced in Ras-act hemocytes. This is consistent with tissue-specific factors acting together with Ras to determine which target genes are expressed in each cell type. Another gene whose expression is modulated by Ras activity is the pro-apoptotic gene, hid (also known as Wrinkled). It is believed that the anti-apoptotic effect of Ras in embryos is mediated in part by a reduction in hid transcription. The hid RNA level does not decrease in Ras-act cells, indicating that this mechanism may not be of importance in hemocytes. Ras may still inactivate hid in these cells via MAPK-mediated phosphorylation of the Hid protein. Other pro-apoptotic genes like reaper or grim are not expressed in either Ras-wt or Ras-act hemocytes (Asha, 2003).
Finally, the data indicate that the large overproliferation of hemocytes in response to activated Ras does not lead to a general activation of the immune response. Among the 134 Drosophila immune-regulated genes induced by septic injury and fungal infection, only 6 genes are upregulated and 4 genes are downregulated by a factor of 3 or more in Ras-act hemocytes. The 6 upregulated genes in Ras-act are Tep2 (complement like), alpha-2M receptor like (complement binding), a trypsin-like serine protease (phenol oxidase cascade), a serpin (serine protease inhibitor), spz (antifungal response), and Tl (antifungal response). The 4 downregulated genes include Tep1 (complement like), Rel (transcription factor), Metchnikowin (antimicrobial response), and a lipase (Asha, 2003).
It is concluded that activated versions of both Ras and the Hop Jak kinase induce leukemia-like phenotypes in Drosophila larvae. Further, it is possible to isolate sufficient quantities of larval hemocytes to conduct microarray expression studies. By comparing the expression profiles from different oncogene-induced leukemia cells, coupled with mutational analysis of the newly identified targets, it should be possible to systematically characterize the critical, oncogene-specific target genes. This approach could prove beneficial to the treatment of human cancers (Asha, 2003).
The Drosophila PDGF/VEGF receptor (PVR) has known functions in the guidance of cell migration. It has been demonstrated that during embryonic hematopoiesis, PVR has a role in the control of antiapoptotic cell survival. In Pvr mutants, a large fraction of the embryonic hemocyte population undergoes apoptosis, and the remaining blood cells cannibalistically phagocytose their dying peers. Consequently, total hemocyte numbers drop dramatically during embryogenesis, and large aggregates of engorged macrophages carrying multiple apoptotic corpses form. Hemocyte-specific expression of the pan-caspase inhibitor p35 in Pvr mutants eliminates hemocyte aggregates and restores blood cell counts and morphology. Additional rescue experiments suggest involvement of the Ras pathway in PVR-mediated blood cell survival. In cell culture, PVR has been demonstrated to directly control survival of a hemocyte cell line. This function of PVR shows striking conservation with mammalian hematopoiesis and establishes Drosophila as a model to study hematopoietic cell survival in development and disease (Brückner, 2004).
Pvr mutant rescue experiments demonstrate that activated Ras is sufficient to restore hemocyte survival. This result resembles findings from survival signaling by DER, which was shown to inhibit action of the proapoptotic protein HID by phosphorylation through Ras-activated MAPK. Since PVR signaling triggers MAPK activation in Schneider cells and may have the same effect in embryonic hemocytes, it is likely that the Ras/MAPK pathway is a route of antiapoptotic PVR signaling. Expression of dominant-negative RasN17 did not lead to large hemocyte aggregates but induced mild enlargement of hemocytes at a low penetrance. This mild phenotype points to weak defects in blood cell survival. The incomplete effect of RasN17 may be due to a number of reasons. RasN17may be too weak to fully block endogenous Ras signaling, or Ras signaling may be redundant with other signaling pathways that are active in PVR-dependent cell survival. In Pvr1 rescue experiments, activated RasV12 was used, which was shown to ectopically activate other signaling pathways such as the PI3K pathway. Therefore, rescue by RasV12 may involve a number of downstream pathways, but the Ras/MAPK pathway itself may still be central to the observed effect, consistent with a current model for apoptosis in Drosophila. Regardless of the upstream pathways involved, inhibition of caspases by the baculovirus inhibitor of apoptosis p35 was sufficient to rescue the Pvr mutant hemocyte death and aggregation phenotype. In these rescue experiments, p35 appeared slightly less potent than RasV12, which may be due to the inability of p35 to inhibit the upstream caspase Dronc, or partially insufficient expression levels, since p35 inhibits caspases by stoichiometric binding (Brückner, 2004).
The role of Drosophila PVR in trophic cell survival emphasizes the high degree of conservation between Drosophila and vertebrate PDGF/VEGF family receptor function. In vivo and cell culture work now provides the basis to study cell survival in a simple but highly conserved hematopoietic system. In vertebrates, control of cell survival is an important aspect of hematopoiesis and stem cell maintenance. Antiapoptotic cell survival and its aberrant prolongation are a major mechanism in the formation of human neoplasias. In many cases, connections to deregulated upstream signaling pathways remain unclear. Interestingly, in Acute Myeloid Leukemias (AML), more than one-third of cases are associated with specific activating mutations in the PDGF/VEGF receptors Flt3 and c-Kit, and activating fusions of PDGFßR replace the more common oncogenic BCR-ABL fusions in some cases of Chronic Myeloid Leukemia (CML). The contribution of these and other disease-associated genes to aspects of cell survival versus proliferation is still difficult to assess in vivo, yet their mechanism of action is important for the selection of molecularly targeted therapies. Drosophila embryonic hematopoiesis allows the in vivo study of blood cell survival independent of cell proliferation, and this work has demonstrated the antiapoptotic potential of the Drosophila PDGF/VEGF receptor and activated signaling components such as RasV12. It will be interesting to exploit the system further by testing disease-related genes of the same and particularly other families for their in vivo potential to rescue blood cell survival in Drosophila. Complementary to these in vivo findings, a Drosophila cell culture system was established for the study of PVR-dependent blood cell survival. Genome-wide RNAi screens will allow identification of modifiers of PVR-dependent blood cell survival (Brückner, 2004).
Determination of cell fate at the posterior termini of the Drosophila embryo is specified by the activation of the Torso receptor tyrosine kinase. This signaling pathway is mediated by the serine/threonine kinase D-raf and a protein tyrosine phosphatase corkscrew. Expression of an activated form of Ras1 during oogenesis resulted in embryos with tor gain-of-function phenotypes. Mammalian p21ras variants were injected into early Drosophila embryos. The injection of activated mammalian p21v-ras rescues the maternal-effect phenotypes of both tor and csw null mutations. These rescuing effects of p21v-ras are dependent on the presence of maternally derived D-raf activity. Wild-type embryos show a terminal-class phenotype resembling csw mutation when injected with p21rasN17, a dominant-negative form of p21ras. The maternal-effect phenotype embryos lacking Son of sevenless (Sos) exhibit a terminal-class phenotype. The Drosophila p21ras, encoded by Ras1, is an intrinsic component of the tor signaling pathway, where it is both necessary and sufficient in specifying posterior terminal cell fates. p21ras/Ras1 operates upstream of the D-raf kinase in this signaling pathway (Lu, 1993).
Activation of the receptor tyrosine kinase (RTK) Torso defines the spatial domains of expression of the transcription factors Tailless and Huckebein. Previous analyses have demonstrated that Ras1 (p21ras) operates upstream of the D-Raf (Raf1) serine/threonine kinase in this signaling pathway. D-Raf can be activated by Torso in the complete absence of Ras1. This result is supported by analysis of D-Raf activation in the absence of either the exchange factor Son of sevenless (Sos) or the adaptor protein drk (Grb2), as well as by the phenotype of a D-Raf mutation that abolishes binding of Ras1 to D-Raf. This study provides in vivo evidence that Raf can be activated by an RTK in a Ras-independent pathway (Hou, 1995).
The terminal portions of the Drosophila body pattern are specified by the localized activity of the receptor tyrosine kinase Torso (Tor) at each pole of the early embryo. Tor activity elicits the transcription of two 'gap' genes, tailless (tll) and huckebein (hkb), in overlapping but distinct domains by stimulating the Ras signal transduction pathway. Quantitative variations in the level of Ras activity can specify qualitatively distinct transcriptional and morphological responses. Low levels of Ras activity at the posterior pole direct tll but not hkb transcription; higher levels drive transcription of both genes. Correspondingly, low levels of Ras activity specify a limited subset of posterior terminal structures, whereas higher levels specify a larger subset. When a constitutively active 1X RasV12 gene is expressed in torso mutant embryos, brachyenteron (byn) is expressed in a small terminal cap, whereas the domain of expression in 2X RasV12 is much broader. Because both the activation and repression of terminal byn expression is known to depend, respectively, on tll and hkb, it is surmised that higher levels of Ras activity are required at the posterior of wild-type (ras+) embryos to drive sufficiently high levels of Hkb expression to repress byn expression (a phenomenon not observed with even 2X RasV12 ectopic expression). 1X RasV12 forms the least terminal of the posterior terminal structures: the eighth abdominal dentical band and the posterior spiracles. The extent of restoration is considerably greater in 2X RasV12 embryos: these form additional terminal structures such as the anal tuft and anal pads.The response to Ras activity is not uniform along the body. Instead, levels of Ras activity that suffice to drive tll and hkb transcription at the posterior pole fail to drive their expression in more central portions of the body, apparently due to repression by other gap gene products. The levels of Huckebein and/or Kruppel through the embryo might be responsible for a failure to express hkb in response to moderate RasV12 activity. It is concluded that tll and hkb transcription, as well as the terminal structures, are specified by two inputs: a gradient of Ras activity, which emanates from the pole, and the opposing influence of more centrally deployed gap genes, which repress the response to Ras (Greenwood, 1997).
14-3-3 proteins have been shown to interact with Raf-1 and cause its activation when overexpressed. However, their precise role in Raf-1 activation is still enigmatic, as they are ubiquitously present in cells and found to associate with Raf-1 in vivo regardless of Raf's activation state. The function of the Drosophila 14-3-3 gene leonardo (leo) has been analyzed in the Torso (Tor) receptor tyrosine kinase (RTK) pathway. In the syncytial blastoderm embryo, activation of Tor triggers the Ras/Raf/MEK pathway that controls the transcription of tailless (tll). In the absence of Tor, overexpression of leo is sufficient to activate tll expression. The effect of leo requires D-Raf and Ras1 activities but not KSR or DOS, two recently identified essential components of Drosophila RTK signaling pathways. Tor signaling is impaired in embryos derived from females lacking maternal expression of leo. It is proposed that binding to 14-3-3 by Raf is necessary but not sufficient for the activation of Raf and that overexpressed Drosophila 14-3-3 requires Ras1 to activate D-Raf (Li, 1997).
Primordial germ cells (PGCs) undergo proliferation, invasion, guided migration, and aggregation to form the gonad. In Drosophila, the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development, and coactivation of STAT and Ras is required for PGC proliferation and invasive migration. Embryos mutant for stat92E or Ras1 have fewer PGCs, and these cells migrate slowly, errantly, and fail to coalesce. Conversely, overactivation of these molecules causes supernumerary PGCs, their premature transit through the gut epithelium, and ectopic colonization. A requirement for RTK in Drosophila PGC development is analogous to the mouse, in which the RTK c-kit is required, suggesting a conserved molecular mechanism governing PGC behavior in flies and mammals (Li, 2003).
STAT92E plays an essential role in mediating the phenotypic effects of gain-of-function mutations of Torso, TorGOF, but is only minimally required for wild-type Tor function in patterning the terminal structures of the Drosophila embryo. To investigate whether wild-type Tor nevertheless activates STAT92E, an antibody was used that recognizes the phosphorylated, or active form of STAT92E (pSTAT92E) to examine the activation status of STAT92E in different genetic backgrounds. In early embryos, pSTAT92E is detected in the anterior and posterior terminal regions in a pattern reminiscent of Tor activation. By analyzing embryos mutant for loss- or gain-of-function mutations of tor as well as those lacking JAK, encoded by hopscotch (hop), it was concluded that the early STAT92E activation is dependent on Tor but not Hop, suggesting that Tor may activate STAT92E independent of Hop. Because STAT92E contributes only marginally to the expression of the Tor target gene tailless (tll), it was of interest to find whether the early activation of STAT92E by Tor had any other biological functions. It was evident that Tor activation correlates temporally and spatially with the formation of PGCs, which are localized at the posterior pole of the early embryo. Tor-dependent activation of STAT92E as well as that of the Ras-MAPK signaling cassette, as detected by an antibody against activated ERK/MAPK (diphospho-ERK), persists in pole cells at this stage. STAT92E activation was detected in PGCs during their migration and in the gonads of late embryos, that are formed following the migration of pole cells through a complex route. These observations indicate that STAT92E and Ras1/Draf activation may play a role in PGC development (Li, 2003).
So far the only known function of Tor has been in pattern formation, since Tor protein is present only transiently in early embryos. Therefore, the finding that Tor is involved in germ cell migration was initially unexpected. However, there is a precedent for the requirement of an RTK in germ cell migration in the mouse. Mutations in the mouse genes dominant white-spotting (W) cause migration and proliferation defects in germ cells as well as a few other cell types. W encodes the protooncoprotein c-kit, an RTK that is expressed on the membrane of mouse PGCs. Sl encodes the c-kit ligand termed stem cell factor (SCF), which is localized on the membrane of somatic cells associated with PGC migratory pathways. Interestingly, c-kit and Tor share structural similarities and both are structurally similar to the platelet derived growth factor (PDGF) receptor, in which an insert region separates the intracellular kinase domain. Moreover, similar to Tor and the PDGF receptor, c-kit is able to activate STAT molecules as well as the Ras-MAPK cascade. Although true molecular homologs of c-kit and SCF are not yet found in the Drosophila genome, the functional and structural similarities between Tor and c-kit suggest that flies and mice share molecular mechanisms for regulating primordial germ cell proliferation and migration (Li, 2003).
In addition to germ cells, the ovarian border cells of Drosophila are also capable of invasive and guided migration. Border cells of the Drosophila ovary are follicle cells that, during oogenesis, delaminate as a cluster six to ten cells from the anterior follicle epithelium, invade the nurse cells, and migrate toward the oocyte. Interestingly, it has been shown that the detachment and guided migration of these cells require STAT92E activation. Mutations in components of the Hop/STAT92E pathway cause border cell migration defects. In addition, border cell migration also requires RTK signaling. An RTK related to mammalian PDGF and VEGF receptors, PVR, is required in border cells for their guided migration toward the oocyte. PVR appears functionally redundant with another fly RTK, EGFR, in guiding border cells. Taken together, these results indicate that the invasive behavior and guided migration of Drosophila ovarian border cells require both STAT92E and RTK activation. In light of the results from analyzing PGC migration, it is proposed that activation of both STAT and components downstream of RTK signaling may serve as a general mechanism for invasive and guided cell migration (Li, 2003).
It has been shown that actin-based cytoskeletal reorganization plays a crucial role in cell shape changes and movements. The identification of STAT and Ras coactivation as an essential requirement for germ cell migration raises an interesting question of how activated STAT and Ras coordinate the cytoskeletal reorganization required for germ cell migration. STAT92E has been shown to be involved in the transcriptional activation of many signaling molecules as well as key transcription factors. A recent systematic search for STAT92E target genes has revealed a plethora of genes that might be directly activated by STAT92E, among which are those involved in the regulation of cytoskeletal movements and actin reorganization. Upregulation of such genes in response to spatial cues should facilitate cell movements. In addition, Ras and other small GTP proteins have been implicated in multiple cellular processes that require cytoskeletal reorganization. It remains to be determined how these two signaling pathways coordinate germ cell movements in response to guidance cues from surrounding somatic tissues (Li, 2003).
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