Correspondence to: Hideyuki Okano, Division of Neuroanatomy (D12), Department of Neuroscience, Biomedical Research Center, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan., okano{at}nana.med.osaka-u.ac.jp (E-mail), 81-6-6879-3581 (phone), 81-6-6879-3589 (fax)
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Abstract |
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The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. We have identified a Drosophila homologue of Ral that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRalG20V and DRalS25N, were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRalG20V and DRalS25N act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal did not cause any visible phenotype, whereas DRalG20V and DRalS25N mutants caused defects in the development of various tissues including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein caused defects in the development of hairs and bristles. These phenotypes were genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH2-terminal kinase kinase (JNKK) and Jun NH2-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein caused defects in the process of dorsal closure during embryogenesis and inhibited the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway.
Key Words: bristle, dorsal closure, hair, Jun NH2-terminal kinase, Ral
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Introduction |
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RAL is a member of the small GTPase superfamily and is found in two forms, RalA and RalB (reviewed by
Recently, putative downstream targets for Ral have been identified (reviewed by
During the development of multicellular organisms, a variety of morphologically differentiated cells are generated. Proper regulation of the cytoskeleton is essential for the precise changes in their shape. A well studied example of cell shape change is the development of hairs and bristles in Drosophila, in which the epithelial cells that secrete cuticle form hairs and bristles that point posteriorly or distally. A number of studies have shown that regulation of the cytoskeleton is required to regulate the development of these structures (
In this paper, we report the identification and characterization of a Ral GTPase in Drosophila. Constitutively active and dominant negative mutants of Drosophila Ral (DRal) were generated and used for functional characterization, both in vitro and in vivo. Our results indicate that Ral regulates developmental cell shape changes through inhibition of the JNK pathway.
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Materials and Methods |
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Cloning and Sequencing of the DRal cDNA
Degenerate primers were designed to amplify Ras-like genes by PCR. The sequences were: GGIGTIGGIAA(A/G)(A/T)(C/G)(A/C/G/T)GC(A/C/G/T)(C/T)T(A/C/G/T)AC and A(C/T)TC(C/T)TGICC(A/C/G/T)GC(A/C/G/T)GT(A/G)TC. PolyA+ mRNA was prepared from S2 cells using a Micro Fast Track kit (Invitrogen Corp.) and used as the template for synthesizing cDNAs using a first strand cDNA kit (Pharmacia Biotech, Inc.). The PCR procedure was: five cycles at 94°C for 1 min, 46°C for 1 min, and 72°C for 1 min, followed by 45 cycles at 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min. PCR products were subcloned into the pT7 blue T vector (Novagen, Inc.), transformed into JM109 cells, and subjected to DNA sequencing according to standard protocols. The PCR product of DRal was 32P-labeled and used as a probe to screen an eye imaginal disc cDNA library. Five 1.2-kb cDNAs were identified that contained the entire open reading frame encoding DRal, a 261-bp 5' untranslated region and a 354-bp 3' untranslated region. The nucleotide sequence encompassing the open reading frame was determined by sequencing the cDNAs from both directions.
Northern Blotting
RNA samples were prepared from eye imaginal discs of third-instar larvae according to the method previously described by
In situ Hybridization to Polytene Chromosomes
The DRal cDNA was labeled with digoxigenin using a random-primer kit (Boehringer Mannheim Corp.) and hybridized with squashed Polytene chromosomes, as described previously (
Site-directed Mutagenesis and Plasmid Constructions
The DRal cDNA in pBluescript was used as the template for site-directed mutagenesis with QuickChangeTM and Chameleon Kits (Stratagene). The constitutively active DRalG20V mutation was created using an oligonucleotide with a base change from GGC to GTC, converting amino acid 20 from Gly to Val. The dominant negative DRalS25N mutation was created using an oligonucleotide with a base change from TCC to AAC, converting amino acid 25 from Ser to Asn. Mutations were confirmed by DNA sequencing. The cDNA inserts with or without mutations were excised from pBluescript and then ligated into either pGEX (Pharmacia Biotech, Inc.), for the expression of glutathione S-transferase (GST)-fusion proteins in Escherichia coli, or into pUAST (
Purification of GST Fusion Proteins
To purify GST fusion proteins (GST-DRal, GST-DRalG20V, GST-DRalS25N, and GST-RalGDS) from E. coli, transformed E. coli were initially grown in Luria-Bertani's broth at 37°C to an absorbance of 0.8 (OD = 600 nm), and subsequently transferred to 25°C. Isopropyl-1-ß-D-thiogalactopyranoside was added to a final concentration of 100 µM and further incubation was carried out for 10 h at 25°C. The GST fusion proteins were purified from E. coli by glutathione Sepharose 4B, in accordance with the manufacturer's instructions.
RalGDS Assay
GST-DRal and GST-DRal mutants (8 pmol each) were preincubated for 10 min at 30°C in 20 µl of reaction mixture (50 mM Tris/HCl, pH 7.5, 2 µM [3H]GDP [1,5003,000 dpm/pmol], 5 mM MgCl2, 10 mM EDTA, 1 mM DTT, and 1 mg/ml BSA). After preincubation, 1 µl of 400 mM MgCl2 was added. To this preincubation mixture, 29 µl of reaction mixture (50 mM Tris/HCl, pH 7.5, 170 µM GTP, and 1 mg/ml BSA) containing GST-RalGDS (10 pmol) was added, and the mixture was further incubated for 530 min at 30°C. Assays were quantified by rapid filtration on nitrocellulose filters (
RalGAP Assay
RalGAP was partially purified from bovine brain cytosol as described previously ([32P]GTP [8,00012,000 cpm/pmol], 5 mM MgCl2, 10 mM EDTA, 1 mM DTT, and 1 µg/ml BSA). After preincubation, 1 µl of 340 mM MgCl2 was added. To this preincubation mixture, 30 µl of reaction mixture (50 mM Tris/HCl, pH 7.5, 1.3 mM GTP, 0.3 mM MgCl2, and 1 mg/ml BSA) containing RalGAP (7 µg of protein) was added, and the second incubation was performed for 15 min at 30°C. Assays were quantified by rapid filtration on nitrocellulose filters. The actual catalytic rates (Kcat) were calculated from the decrease in radioactive
[32P]GTP in the presence or absence of RalGAP (
Other Biochemical Assays of DRal
The Kd values for GDP or GTPS of, dissociation rate of GDP (K-1) from, and the steady-state rate (Kss) of GTP hydrolysis of the mutant forms of DRal were determined as described previously (
Genetics
Plasmids were injected into the embryos of w1118; Dr/TMS, Sb P[ry+, 2-3] (from the Bloomington stock center) to generate transgenic lines, as described previously (
Histological Analyses
In situ hybridization to embryonic and larval tissues was performed as described by
For scanning EM, adult flies or isolated wings were dehydrated in a graded ethanol series and dried using a critical point drier. The mounted samples were ion-coated and observed with a scanning electron microscope (Hitachi Instruments, Inc.).
For phalloidin staining, pupal wings were dissected away from the surrounding cuticle and fixed in 8% paraformaldehyde/PBS at room temperature for 20 min. The wing samples were washed in 0.1% Triton X-100/PBS (PBT) three times, then incubated in rhodamine-phalloidin/PBT (0.5 mg/ml; Sigma Chemical Co.) overnight at 4°C. After rinsing in three changes of PBT, the wings were mounted and examined with a confocal laser microscope (Olympus).
Pupal nota were dissected and fixed in 4% paraformaldehyde/PBS as described previously (
Preparation and analysis of embryonic cuticle were performed as described previously (
Cell Culture and Transfection Assay
pWAGAL4 was a kind gift from Dr. Yasushi Hiromi (National Institute of Genetics, Japan). S2 cells were grown on 24-well plates to 6080% confluence in Schneider's medium (Sigma Chemical Co.) supplemented with 10% FBS and 0.5% peptone (Difco Laboratories Inc.). The cells were transfected with pWAGAL4 (200 ng) alone, pWAGAL4 (200 ng) plus pUAST-DRal (1 µg), or pWAGAL4 (200 ng) plus pUAST-DRalG20V (1 µg) using Cell Fectin reagent (GIBCO BRL) according to the manufacturer's instructions. After 24 h, the cells were incubated in Drosophila serum-free medium (GIBCO BRL) for 30 min, then treated with 500 mM D-sorbitol for 5 min. Cells were lysed in 40 µl of SDS-PAGE sample buffer containing phosphatase inhibitors (100 nM okadic acid, 200 µM sodium orthovanadate, and 50 mM sodium fluoride), heated at 100°C for 3 min and spun at 10,000 g for 10 min. The resulting supernatant fractions were subjected to SDS-PAGE (12.5% gel) and transferred to a nylon membrane. After blocking in 5% dry milk in PBS + 0.1% Tween-20 overnight at 4°C, the membranes were incubated with anti-ACTIVE JNK (Promega Corp.) or anti-JNK1 (Santa Cruz Biotechnology) antibodies for 1 h at room temperature, and then with HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Signals were detected by ECL reagents (Nycomed Amersham Inc.).
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Results |
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Cloning the cDNA that Encodes the DRal Protein
The known GTPase genes of the Ras family share significant homology in several structurally and functionally important regions. To search for novel Ras-like GTPases in Drosophila, we designed degenerative PCR primers that recognize the nucleotide binding and effector regions of the known GTPases of the Ras family and that were also likely to amplify novel Ras-like GTPases. Using these primers to perform reverse transcriptase PCR, we isolated a number of cDNAs encoding Ras-like GTPases. Some were known genes, such as Ras1 (
The sequence of the cDNA indicated a single open reading frame encoding a protein of 201 amino acids with a predicted molecular mass of 21 kD. The deduced amino acid sequence shared high homology with all of the mammalian Ral proteins (Figure 1). The amino acid sequence in the putative effector domain was identical to that of the mammalian Ral proteins. The CAAX motif at the COOH terminus required for geranylgeranylation (
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To determine the cytological map position of the DRal gene, we performed in situ hybridization with chromosomes from Drosophila salivary glands using the DRal cDNA as the probe. A single signal was detected in the 3E region on the X chromosome (data not shown).
DRal Expression Pattern during Development
To examine the spatiotemporal expression pattern of the DRal mRNA during development, in situ hybridization analysis was performed at various stages of development using a DRal antisense RNA probe. Widespread expression of the DRal transcripts was detected throughout embryogenesis (Figure 2, AC). In the third-instar larval stages, DRal mRNA was also broadly expressed in the brain hemispheres and ventral nerve cords (Figure 2 D), leg discs (Figure 2 E), eye discs (Figure 2 F), and wing discs (Figure 2 G). The sense control probe did not hybridize to these tissues (data not shown).
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Biochemical Characterization of the Constitutively Active and Dominant Negative Mutants of the DRal Protein
Since no mutants of the DRal gene were available, we designed constitutively active and dominant negative DRal mutants based on structurefunction studies of human Ral (
Previously, we characterized the biochemical activities of human wild-type Ral and its mutants (S were similar (~14 and 31 nM, respectively). DRalG20V also showed similar Kd values for both GDP and GTP
S. The Kd values of DRalS25N for GDP and GTP
S were larger than those of wild type, and its affinity for GDP was four- to fivefold higher than for GTP
S. The GDP dissociation constants (K-1) of wild-type DRal, DRalG20V, and DRalS25N were 0.009, 0.006, and 0.09, respectively. RalGDS stimulated the dissociation of GDP from DRal four- to fivefold. RalGDS stimulated the dissociation of GDP from DRalG20V threefold, but did not affect that from DRalS25N. The steady-state rates (Kss) of the GTPase activity of DRal, DRalG20V, and DRalS25N were 0.007, 0.003, and 0.004, respectively. RalGAP stimulated the actual GTPase Kcat of wild-type DRal eightfold, but not that of DRalG20V. The biochemical characteristics of DRalG20V and DRalS25N were almost identical to those of human RalG23V and RalS28N, respectively. These results indicate that Ser-25 of DRal is important for the action of RalGDS, that Gly-20 is important for the action of RalGAP, and that DRalG20V and DRalS25N are constitutively active and dominant negative forms of DRal, respectively.
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Functional Analysis of DRal by Ectopic Expression
To gain insight into the function of DRal in Drosophila development, we examined the effects of overexpressing the dominant mutants described in a specific tissue using the GAL4/UAS (upstream activation sequence) system (
Overexpression of the wild-type DRal protein did not cause any visible phenotype. On the other hand, overexpression of DRalG20V and DRalS25N resulted in a variety of phenotypes that depended on the GAL4 line used. In this study, we focused on the effect of DRalS25N on the development of two cell types that have highly specialized structures, hair and bristles, since the phenotypes were obvious and easy to analyze. The development of these structures is dependent on the proper regulation of the cytoskeleton (
Effects on Wing Hair
Each epithelial cell of the Drosophila wing forms a hair by extending a single process from its apical membrane during pupal development (
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To label the F-actin, the developing wings were dissected from pupae at 3036 h APF and stained with rhodamine-conjugated phalloidin. In the wild-type pupal wings, a single large bundle of F-actin, termed the prehair, is formed in each wing cell (
Effects on Sensory Bristles
The development of sensory bristles provides another excellent model system to study how the cytoskeleton controls cell shape changes. Each external sense organ consists of four cells: the neuron, the sheath, the tormogen (socket forming cell), and the trichogen (shaft forming cell;
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The absence of bristles on the nota expressing DRalS25N could be due to failure in the process of shaft initiation from the trichogen cells. Alternatively, overexpression of the dominant negative DRal protein could disrupt the formation of the trichogen cells. To distinguish between these two possibilities, developing nota from pupae at 2632 h APF were stained with the antibody mAb 22C10 (
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To visualize the F-actin in the developing bristles, pupal nota at 2632 h APF were dissected and stained with rhodamine-conjugated phalloidin. Figure 6 A shows developing microchaetes in wild-type nota. Developing shafts containing F-actin were observed at this stage. On the nota expressing DRalS25N, initiation of shafts was often inhibited (Figure 6 B). Figure 6 C shows a wild-type macrochaete. The developing shaft was filled with well-organized actin bundles that ran parallel to the long axis of the bristle. At the tip, patches of F-actin were observed. On the nota expressing DRalS25N, the development of actin structures in the macrochaetes appeared to be interrupted at the initiation of extension (Figure 6 D).
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Next, we used the GAL4-69B line to examine the effects of expressing wild-type DRal and DRalS25N in the trichogen and hair cells on the nota. The phenotype of the hairs on the nota was similar to that of the wing hairs (Figure 3). They were often shortened, forked, twisted, duplicated, or triplicated (Figure 7A and Figure C). As for the bristles, the GAL4-69B line expressing DRalS25N resulted in a similar phenotype to that caused by sca-GAL4 (i.e., some of the bristles were lost; Figure 7A and Figure C). We expected that these phenotypes were caused by a dominant negative effect on the endogenous DRal protein. To address whether these phenotypes could be caused by decreased function of DRal, wild-type DRal protein was expressed with the dominant negative DRalS25N protein. The loss of bristles and morphological defects resulting from DRalS25N expression were largely rescued by coexpression of the wild-type DRal protein (Figure 7B and Figure D). Therefore, these phenotypes are likely to have resulted from decreased DRal function.
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Effects of Mutations in the JNK Pathway on the DRalS25N-induced Phenotype
To explore other genes associated with the DRal-induced defects described above, flies carrying both sca-GAL4 and UAS-DRalS25N were crossed to a number of mutants for genes known to be involved in the Ras pathway and cytoskeletal regulation. The resulting F1 progenies were scored for modification of the bristle-loss phenotype caused by DRalS25N (Table 2). No effect was seen as a result of halving the dosages of the genes coding for the proteins of the Ras/Raf/ERK pathway or the Rho family of small GTPases, i.e., Ras1 (
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Effects of DRalG20V on Dorsal Closure
It has been shown that both the bsk and hep mutations disrupt the process of dorsal closure during embryonic development (
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DRal Inhibits the Phosphorylation of JNK in Cultured Cells
The genetic data suggested that DRal could act as a negative regulator of the JNK pathway in vivo. We next examined the ability of the constitutively active DRal protein to inhibit JNK activation when overexpressed in tissue culture cells. JNK is activated by phosphorylation on both threonine and tyrosine residues in the Thr-X-Tyr sequence within the catalytic core of the enzyme. Therefore, the level of Bsk/JNK activation in cells was evaluated on Western blots using an antibody that specifically recognizes phosphorylated JNK. S2 cells were transfected with pUAST-DRal or pUAST-DRalG20V together with a plasmid that expresses GAL4 under control of the actin5C promoter, pWAGAL4 (Hiromi, Y., unpublished observations). DRal did not affect the basal level of Bsk phosphorylation in untreated S2 cells (data not shown). It has been shown that JNK is activated by osmotic shock (
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Discussion |
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We have identified a Drosophila gene, DRal, that encodes a protein with strong homology to mammalian Ral GTPases. The Ral proteins identified in mammals so far are easily classified into two types, RalA and RalB, based on their amino acid sequences. Although the amino acid sequence of DRal is more homologous to that of RalA, some residues of DRal, e.g., Glu-103 and Pro-135, are identical to RalB, but not to RalA. Therefore, we could not classify DRal as a homologue of either RalA or RalB. The COOH-terminal region of DRal contains a basic amino acid repeat and a CAAX motif, which are important for post-translational modifications and membrane localization. DRal may be localized to the membrane with Ras and activated by RalGDS, as shown in mammals (
Much of our knowledge about the functions of small GTPases has been obtained from studies using dominant active and dominant negative mutants. In Drosophila, ectopic expression of wild-type or mutant proteins has been successfully used to study the roles of small GTPases in development (see
Development of wing hairs is controlled by both actin and microtubules (
Another structure examined in this work is the external sensory bristle. The development of bristles is also an excellent model system for studying the role of the cytoskeleton in cell shape changes. The trichogen cell extends and forms a bristle shaft during early pupal development (
Our genetic and biochemical data suggest that DRal regulates cell shape changes through the inhibition of the JNK pathway (Figure 8, Figure 9, and Figure 10; Table 2). The JNK pathway has been implicated in cell shape changes and in the regulation of tissue polarity (for review see
Ras mediates its diverse biological functions by activating multiple downstream targets including GEFs for Ral (
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Acknowledgements |
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We would like to thank Norbert Perrimon, Kozo Kaibuchi, and Beth Stronach for comments on the manuscript and valuable discussions. We are grateful to Richard G. Fehon, Mathew Freeman, Toshihiko Hosoya, Fumio Matsuzaki, Marek Mlodzik, Makoto Nakamura, Yasuyoshi Nishida, Masataka Okabe, Norbert Perrimon, Thomas Raabe, Gerald M. Rubin, Kuniaki Takahashi, Yoshihiro Takatsu, Minoru Tateno, and Ryu Ueda. The Bloomington Stock Center for fly stocks, Shinobu C. Fujita for the mAb 22C10, Yasushi Hiromi for the pWAGAL4 plasmid, Hiroko Kouike for sequencing, Ritsuko Shimamura for making the fly medium, and Ken-ichi Kimura, Ryusuke Niwa, Masataka Okabe, Kuniaki Takahashi, Minoru Tateno, and Tadashi Uemura for technical instructions.
This work was supported by grants from the Japanese Ministry of Science, Education, Sports, and Culture to K. Sawamoto, A. Kikuchi, and H. Okano. H. Okano was also supported by the Human Frontier Science Program Organization and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation.
Submitted: January 25, 1999; Revised: May 19, 1999; Accepted: June 10, 1999.
1.used in this paper: APF, after puparium formation; bsk, basket gene; DRal, Drosophila Ral; GAP, GTPase-activating protein; GDP, guanosine 5'-diphosphate; GDS, guanine nucleotide dissociation stimulator; GEFs, guanine nucleotide exchange factors; GST, glutathione S-transferase; hep, hemipterous gene; JNK, Jun NH2-terminal kinase; JNKK, Jun NH2-terminal kinase kinase; PAK, p21 activated kinase; PLD, phospholipase D; UAS, upstream activation sequence
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