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Previous Article | Next Article 
Molecular and Cellular Biology, November 2002, p. 7658-7666, Vol. 22, No. 21
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.21.7658-7666.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
National Creative Research Initiatives Center for Cell Growth Regulation and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea
Received 23 January 2002/ Returned for modification 29 March 2002/ Accepted 23 July 2002
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the GTPases, Ras has been the most thoroughly characterized. It plays the role of a master switch in mitogenic signaling pathways including the mitogen-activated protein kinase (MAPK) and phosphatidylinositide-3 kinase pathways. Rap1 (also known as Roughened, Ras3, and Dras62B in Drosophila melanogaster) is another well-studied GTPase with a structure highly homologous to that of Ras. Rap1 was originally identified by its ability to reverse cellular transformation induced by oncogenic Ras (18). Rap1 is more than 50% identical to Ras and is thought to block the mitogenic activity of the cell by trapping the Ras effector C-Raf into an inactive complex, silencing MAPK activity (9, 14). However, many recent findings suggest that Rap1 could act as an activator of the MAPK pathway in other cellular contexts. For example, Rap1 was found to interact with and activate B-Raf (27), resulting in sustained MAPK activation (35, 36). These inconsistent observations obscure the relationship between Rap1 and MAPK, and thus, a deeper probing into the function of Rap1 is required in order to explain its seemingly contradictory functions within the MAPK signaling pathway.
Recently, the regulation of Rap1 activity has been extensively studied, and consequently, multiple Rap1-GEFs have been identified. They include smgGDS (23), C3G (12), Epac/cyclic AMP-GEF (11, 16), CalDAGGEF1 (17), and PDZ-GEF/RA-GEF/CNrasGEF/nRapGEP1 (10, 20, 26, 28). Among them, PDZ-GEF has attracted considerable attention because of its unique structural characteristics. PDZ-GEF contains multiple functional domains including cyclic nucleotide monophosphate-binding, Ras-exchange motif, PDZ, and Ras/Rap1-associating (RA) domains, as well as GEF catalytic domains.
Although several insightful findings pertaining to the functions of PDZ-GEF and other Rap1 GEFs have been made, they were based either on biochemical methods or on cell line-based overexpression studies, which have a limited in vivo relevance. Thus, we decided to define the physiological role of PDZ-GEF in the Drosophila system, which allows highly convenient genetic and histological studies. Using the dPDZ-GEF mutant fly, the first genetic model for a Rap1 GEF, we have elucidated dPDZ-GEF/Rap1-specific signaling in context of the MAPK signaling pathway.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Northern blot analysis. Total RNAs, extracted by the easy-Blue system (Intron, Seoul, Korea), were separated by electrophoresis on denaturing formaldehyde agarose gels in MOPS (morpholinepropanesulfonic acid) buffer, transferred onto a nylon membrane, and successively hybridized with nick-translated 32P-labeled cDNA probes for dPDZ-GEF. Hybridized probes were visualized by autoradiography.
Analysis of eye phenotypes. To obtain the eye-specific mitotic clone, the dPDZ-GEF1 (l(2)K13720) allele was recombined onto the FRT40A y+ chromosome, to generate the y w; dPDZ-GEF1 FRT40A/CyO line. Females from this line were crossed to males of either y w ey-flp; FRT40A or y w; gmr-hid FRT40A/CyO; ey-GAL4 UAS-flp line.
The genotypes of the control eyes shown in Fig. 5 are as follows: UAS-Raswt/+ and gmr-GAL4/UAS-Raswt for Fig. 5B, Rase1b/+ and gmr-GAL4/+;Rase1b/+ for Fig. 5C, Rrv(R)B1/+ and gmr-GAL4/+;Rrv(R)B1/+ for Fig. 5D, phl11/+ and phl11/+;gmr-GAL4/+ for Fig. 5E, dSorLH110/+ and dSorLH110/+;gmr-GAL4/+ for Fig. 5F, rl1/+ and gmr-GAL4/rl1 for Fig. 5G. All of these were analyzed by scanning electron microscopy (SEM) and tangential section, and each eye was shown to be normal with intact photoreceptor numbers.
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Analysis of ovaries. For phalloidin and Hoechst staining, ovaries were fixed for 20 min in 9.4% formaldehyde in phosphate-buffered saline and stained with phalloidin-tetramethyl rhodamine isocyanate (Sigma) overnight at 4°C. After being washed, the same samples were stained with Hoechst 33258 for 4 min. The stained ovaries were examined with an LSM510 laser confocal microscope (Carl Zeiss). Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analyses were performed as described previously (8). In situ hybridization experiments were performed with a digoxigenin-labeled RNA probe of the same fragment used for the Northern blot analysis (Fig. 1B).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To understand the physiological roles of dPDZ-GEF, we have isolated flies containing mutations in the dPDZ-GEF locus for genetic analyses. While searching the Berkeley Drosophila Genome Project P-element database, we found two P-element insertion mutants, l(2)k13720 and EP(2)388, with insertion at the 5' upstream region of the dPDZ-GEF gene (designated here dPDZ-GEF1 and dPDZ-GEF2, respectively, Fig. 1E). While dPDZ-GEF2 showed no visible phenotype, dPDZ-GEF1 homozygous flies showed lethality, with a reduced hatching rate below 50% (data not shown). We expected that P-element insertion at the 5' flanking region of dPDZ-GEF in dPDZ-GEF1 would prevent dPDZ-GEF expression. As predicted, we found that the expression of dPDZ-GEF was almost completely abolished in the dPDZ-GEF1 homozygous larvae (Fig. 1C) while the nearby genes were unaffected (Fig. 1D).
To further prove that the dPDZ-GEF1 lethality is specifically due to the P-element insertion and to exclude the possibility that this lethality is due to a second-site mutation in the same chromosome arm bearing the dPDZ-GEF1 mutation, we attempted to rescue the phenotype of dPDZ-GEF1 by precisely excising out the existing P{lacW} element from the insertion locus. By mobilizing the P-element by use of the delta2-3 line, we obtained a considerable number of revertants, which displayed no abnormal effects on viability or fertility (data not shown). These revertants could also fully complement the dPDZ-GEF1 allele (data not shown), and we named this allele dPDZ-GEFrv-1. Thus, we could conclude that the dPDZ-GEF1 is a reliable loss-of-function allele of the dPDZ-GEF gene.
dPDZ-GEF is required for normal eye development. To study the physiological role of dPDZ-GEF, we attempted to use transallelic conditions to obtain a hypomorphic phenotype of the dPDZ-GEF gene. dPDZ-GEF1/dPDZ-GEF2 flies, which have decreased dPDZ-GEF expression, showed a slight rough eye phenotype (Fig. 2C and E), with some missing photoreceptor cells (Fig. 2D). Because the precisely excised allele dPDZ-GEFrv-1 did not show any phenotype in the eye morphology and photoreceptor cell number (Fig. 2F and G), we hypothesized that dPDZ-GEF is involved in the eye development process. To verify this hypothesis, dPDZ-GEF loss-of-function clones were induced in the Drosophila eye by ey-flp mitotic recombination technique (25) as briefly described in Materials and Methods. The dPDZ-GEF1 homozygotic ommatidia, indicated as red eye pigment by mini-white P-element marker, displayed a sunken and disarrayed morphology (Fig. 2H and J). Further tangential sections revealed the loss of many photoreceptor cells in this area (Fig. 2I, -/- area).
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From the observations shown in Fig. 2 and 3, we concluded that dPDZ-GEF plays important roles in eye development and photoreceptor cell specification in Drosophila.
dPDZ-GEF1 homozygous escapees display a rolled phenotype.
While conducting experiments for determining the lethal phase in dPDZ-GEF1 homozygotes, we found that there is some fluctuation in lethality, possibly due to temperature variations, implying that this mutant is temperature sensitive. Indeed, we were able to obtain the escapees of the lethal phenotype of dPDZ-GEF1 homozygotes by large-scale culture of the flies at a low temperature (18°C). We were able to find more than 40 escapees at a low frequency (
0.01%) between dPDZ-GEF1/CyO parents, distinguished by the Cy dominant marker.
These dPDZ-GEF1 homozygous mutants displayed striking features. The wing edges were rolled downward with frayed margins (Fig. 4C; compare with Fig. 4A), and the eyes became small and rough compared to those of the wild-type flies (Fig. 4D, left panels), a phenotype identical to the loss-of-function eye made through mitotic clone analyses (Fig. 2H through J and 3B and D). We sectioned each eye and could observe the loss of photoreceptor cells in the dPDZ-GEF1 homozygous mutant (Fig. 4D, right panels). These phenotypes are highly similar to the one already described in the rolled mutant, the Drosophila MAPK mutant (Fig. 4B and middle panels of Fig. 4D; see also reference 4 for more-severe phenotypes). This phenotypical relationship suggests that dPDZ-GEF and Drosophila MAPK are involved in the same signaling pathway.
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Ras is not a functional target of dPDZ-GEF. As dPDZ-GEF was required for photoreceptor cell fate determination and as the dPDZ-GEF loss-of-function mutants displayed a phenotype similar to that of the rolled mutant, it appeared highly likely that dPDZ-GEF lies upstream of the conventional Ras-MAPK pathway. Therefore, we decided to examine the relationship between Ras and dPDZ-GEF. We speculated that dPDZ-GEF may enhance the activity of wild-type Ras by stimulating its GDP-GTP exchange activity, resulting in a more severely disrupted eye.
However, surprisingly, coexpression of dPDZ-GEF and wild-type Ras resulted in a rather normal eye, with a normal ommatidia array and photoreceptor structure (Fig. 5B). To confirm this, we used the loss-of-function mutant of Ras (Rase1b) to reduce the dosage of the endogenous Ras gene. At one-half dosage background of Ras, the dPDZ-GEF-induced rough eye phenotype and increase of photoreceptor cells are not reduced (Fig. 5C). This is in stark contrast to similar experiments in which Ras dosage reduction resulted in a phenotype rescue in Son of sevenless (Sos) (5) or dC3G (15) gain-of-function mutants. Thus, we came to the unexpected conclusion that Ras is not a functional target of dPDZ-GEF but rather an inhibitor of dPDZ-GEF signaling.
Rap1 is a functional target of dPDZ-GEF. Next, we examined whether Rap1 is a downstream target of dPDZ-GEF. As described, Rap1 is a Ras-family GTPase of considerable importance in photoreceptor cell development. We used the Raprv(R)B1 allele, a loss-of-function mutant of Rap1, to examine whether there is a functional interaction between PDZ-GEF and Rap1 in Drosophila (13). The number of increased photoreceptors due to dPDZ-GEF overexpression is reduced compared to that of the wild type when one copy of Rap1 is removed (Fig. 5D). Thus, we were able to conclude that the rough eye phenotype caused by dPDZ-GEF is accomplished by Rap1 activation. This is consistent with numerous recent reports (10, 20, 21, 26, 30) that mammalian PDZ-GEF is a Rap1-specific GEF.
MAPK is a downstream target of the dPDZ-GEF signaling pathway. The phenotype similarity between the dPDZ-GEF mutants and the rolled mutant (Fig. 4) prompted us to further investigate whether the dPDZ-GEF mutant eye phenotype is mediated by MAPK signaling components. Interestingly, the rough eye phenotype and irregular ommatidia array, as well as the increased photoreceptor number caused by dPDZ-GEF overexpression, were strongly suppressed by D-Raf (Fig. 5E), dMEK (Fig. 5F), or dMAPK (Fig. 5G) dosage reduction. We quantified this genetic interaction by counting and calculating the percentage of ommatidia having extra photoreceptor cells for each eye (Fig. 5, lower right panel). These results strongly support that dPDZ-GEF modulates eye development through the MAPK signaling cascade.
rhomboid expression is strongly induced by dPDZ-GEF. The transcriptional regulation of rhomboid is important in the establishment of EGF receptor-dependent MAPK activation (24). Furthermore, rhomboid is a transcriptional target of the MAPK pathway (22). To further confirm whether dPDZ-GEF activates MAPK, we examined rhomboid expression in the eye under a gmr-GAL4-driven dPDZ-GEF overexpression background. As expected, we were able to detect a much stronger expression of rhomboid in the eye disk overexpressing dPDZ-GEF (Fig. 6B) than in the control eye disk (Fig. 6A) by analyzing the activities of the rhoAA69 enhancer trap reporter.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rap1 was originally thought to inhibit the Ras-MAPK signaling pathway (18), and subsequent studies mainly focused on how Rap1 inhibits the Ras-MAPK pathway (9, 14). However, recent studies on Rap1 action have hinted that Rap1 may have other roles besides inhibiting Ras. Both Ras and Rap1 exhibited mitogenic effects in the Swiss 3T3 cell line (37), and most signals that activate Ras also stimulate Rap1 activation (39). These results suggested that Rap1 plays a Ras-like role in cell growth regulation. In support of this, B-Raf has been suggested to be a specific effector for Rap1. B-Raf can bind to and is activated by Rap1 in vitro (27), and Rap1 was shown to play an important role in the cyclic AMP- or NGF-stimulated activation of B-Raf, which leads to the activation of MAPK (35, 36).
In concert with these findings, the view on the in vivo function of Drosophila Rap1 has also been changed. The Roughened mutation, which causes a reduction in photoreceptor cell number, was originally regarded as a hyperactivated Rap1 allele that inhibits the Ras-dependent MAPK pathway (13). However, genetic analyses revealed that this allele cannot be regarded as a simple hypermorph (19), and Raprv(R)B1, a loss-of-function allele of Rap1, showed no phenotype related to the expected hyperactivation of the Ras-MAPK pathway (3). Furthermore, Rap1 overexpression by gmr-GAL4 caused increases in photoreceptor cell number with defects in eye morphogenesis (15), implicating that Rap1 is rather a positive regulator of photoreceptor cell differentiation. This conclusion is also consistent with the original observation that the Rap1rv(R)B1/Rap1Roughened genotype reduces the photoreceptor cell number more severely than does +/Rap1Roughened (13).
As shown in Fig. 3F, overexpression of dPDZ-GEF also displayed a rough eye phenotype, in a manner similar to that of Rap1 overexpression, with an increase in photoreceptor cell number (Fig. 3H), and this rough eye phenotype was suppressed by reduction of Rap1 activity (Fig. 5D). These results suggest that dPDZ-GEF plays a crucial role in Rap1-mediated ommatidium formation and eye development. The phenotypic similarity between loss-of-function mutants of dPDZ-GEF and Rap1 in ovariole development (Fig. 7) further confirms this connection in the other cellular processes besides photoreceptor cell fate determination.
Although dPDZ-GEF-deficient ommatidia survived with only partial defects in photoreceptor structure (Fig. 3B and D and Fig. 4D, right panels), Rap1-deficient ommatidia did not survive to adult stage, leaving only a scar in their location (3). This implies that another activator, possibly dC3G or other unidentified GEFs, could maintain the minimal activity of Rap1 in the dPDZ-GEF1 homozygotic eye. In addition, suppression of the dPDZ-GEF-induced rough eye phenotype by Rap1 dosage reduction was weaker than those induced by the dosage reductions of downstream components such as D-Raf, dMEK, and dMAPK (Fig. 5). This suggests that dPDZ-GEF could activate another target, possibly another GTPase, to affect the activity of D-Raf and the MAPK signaling pathway. It has been reported that in the mammalian system PDZ-GEF was also able to activate another Rap-subfamily GTPase, Rap2 (10, 30). Indeed, a homologue of Rap2 exists in Drosophila and is involved in eye development (33), although its loss-of-function mutation has not yet been reported. However, we must be aware of the possibility that the Drosophila version of PDZ-GEF may not behave exactly in the same way as mammalian PDZ-GEF. Nonetheless, Rap2 should be investigated as a possible downstream target of dPDZ-GEF.
dPDZ-GEF, a novel MAPK regulator. Our findings that dPDZ-GEF is phenotypically related to rolled and is also involved in photoreceptor cell differentiation implicate that dPDZ-GEF plays an important role in the MAPK signaling pathway. As shown in Fig. 5, dPDZ-GEF involves the activities of the well-known downstream components of the MAPK pathway D-Raf, the product of pole hole, dMEK, the product of Drosophila Suppressor of Ras1, and dMAPK, the product of rolled. Thus, in addition to the traditional Ras-MAPK signaling pathway for photoreceptor cell determination, we now propose the presence of a Ras-independent and dPDZ-GEF/Rap1-dependent MAPK signaling pathway, where dPDZ-GEF/Rap1 signaling molecules functionally substitute Sos/Ras1. The idea that Rap1 and Ras1 share and compete for the same effector molecule(s) has been proposed since the discovery of Rap1 (15, 18, 38); however, no further studies have addressed this issue. In this report, we convincingly demonstrated that the dPDZ-GEF/Rap1 signaling pathway and the Sos/Ras signaling pathway converge at D-Raf, the only orthologue of mammalian Rafs in Drosophila (Fig. 8). While a recent review has cautiously suggested that Rap1-dependent signaling may be involved in MAPK signaling in Drosophila (7), our results clearly demonstrate that dPDZ-GEF and Rap1 are involved in the MAPK signaling pathway, at least in photoreceptor cell fate determination.
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Another novel finding is that Ras1 can inversely inhibit Rap1-dependent signaling (Fig. 5C). Considering our findings demonstrating that Rap1 and Ras share effector molecule(s), it could be expected that overexpression of either Ras or Rap1 inhibits the other by competing for effector molecule(s). Previous reports have shown the inhibition of Ras-mediated MAPK activation by Rap1 (9, 14); we have demonstrated for the first time that Ras1 is also able to suppress the Rap1-dependent signaling pathway.
In summary, our findings demonstrate that dPDZ-GEF is highly involved in the MAPK signaling pathway, specifically in photoreceptor cell development, while the analysis of wing vein patterns and ovary developments also showed that dPDZ-GEF has other roles that are independent of the MAPK signaling pathway. Further research is required on dPDZ-GEF as an important upstream regulator of the MAPK pathway and modulator of morphogenesis.
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ACKNOWLEDGMENTS |
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This research was supported by the Ministry of Science and Technology of Korea.
J.H.L. and K.S.C. contributed equally to this work.
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FOOTNOTES |
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