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1 UMR 7622-Biologie du Développement; CNRS-Université Pierre et Marie Curie Paris 6 (UPMC), 9, quai Saint-Bernard, 75005 Paris, France
2 Centre de Génétique Moléculaire, CNRS, UPR 2167, 91190 Gif-sur-Yvette, France
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Among MAPK cascades, the Raf (MAPKKK)/MEK (MAPKK)/ERK (MAPK) module can be activated by growth factors like EGF or PDGF that bind RTKs. This initial activation step takes place at the cytoplasmic membrane. In mammals, there are several isoforms for each kinase (reviewed in McKay & Morrison 2007). A two-hybrid screen has isolated a mouse scaffold protein named MP1 (MEK Partner 1) that joins MEK1 to ERK1, thereby enhancing ERK1 activation (Schaeffer et al. 1998). MP1 is a 15-kD protein that belongs to a novel super family of Profilin-Like Adaptors named ProflAP (Kurzbauer et al. 2004; Lunin et al. 2004). MP1 forms a tight heterodimer with p14, a 14-kD protein that also belongs to the ProflAP family and is present on the cytoplasmic face of late endosomes (Wunderlich et al. 2001; Kurzbauer et al. 2004; Lunin et al. 2004). The p14–MP1 interaction is required to recruit the MP1–MEK1–ERK1 module to late endosomes, and this recruitment is crucial for sustained ERK1 activation (Teis et al. 2002).
Like MAPKs, most scaffold proteins are evolutionarily conserved in metazoans, both in sequence and in function. This is the case for KSR and CNK, two proteins regulating Ras-mediated Raf activation, that were initially identified in Drosophila and later shown to play overall similar roles in mammals (reviewed in Claperon & Therrien 2007). The genetically tractable organism Drosophila melanogaster has been very useful in elucidating the role of these two scaffold proteins in ERK signaling, in particular as the Drosophila EGF Receptor (DER)-mediated ERK transduction pathway is involved in many processes during fly development (reviewed in Shilo 2003). A powerful working system is the developing Drosophila wing. Indeed, DER–ERK signaling in wing imaginal discs of larvae and pupae is critical for patterning, control of proliferation, cellular growth and differentiation of wing tissues (Karim & Rubin 1998; Bier 2000; Prober & Edgar 2000; Zecca & Struhl 2002). While initial wing disc proliferation is induced by general activation of DER–ERK signaling, specification and differentiation of veins and interveins, occurring between the third larval and the pupal stages, are based on fine-tuned ERK activation. Longitudinal veins are first determined as broad regions named proveins, each one expressing a specific combination of transcription factors that defines a positional information. From late third-instar larva to pupal stage, localized expression of rhomboïd (rho) in provein cells directs them to differentiate as veins (Sturtevant et al. 1993). rho encodes a protein required for the proteolytic processing of DER ligands (Lee et al. 2001; Urban et al. 2001), which is necessary and sufficient for ERK activation in future vein cells (Guichard et al. 1999). In addition, DER signaling can activate rho expression, which leads to a positive feedback loop of the pathway (Martin-Blanco et al. 1999). Whereas rho is required for the specification of vein cells, blistered (bs), that encodes a homologue of the mammalian Serum Response Factor (SRF), is expressed in the future intervein cells of third instar discs and pupal wings, and controls the formation of intervein tissue (Fristrom et al. 1994; Montagne et al. 1996). bs is repressed by DER–ERK signaling in provein territories and is involved in rho repression in future intervein cells (Roch et al. 1998). The differentiation of vein and intervein cells thus depends of the outcome of a fine-tuned balance between rho and bs expression patterns, that are both regulated by the DER–ERK signaling pathway.
In this paper, we focus on the role of the MP1 scaffold protein in D. melanogaster. Genome databases showed the presence of MP1-like proteins in several metazoans. In Drosophila, the MP1 orthologue is encoded by CG5110, named thereafter dMP1, located on the left arm of chromosome 2. The dMP1 protein presents 45% identity with human and mouse sequences (Kurzbauer et al. 2004; Lunin et al. 2004). Such a high level of sequence conservation suggests that its function in ERK signaling may also be conserved. However, previous work using the Drosophila Schneider S2 cell line showed no role for dMP1 in insulin-mediated ERK activation (Anselmo et al. 2002). To further address the implication of dMP1 in ERK signaling, we used biochemical and genetic approaches. We describe the dMP1 expression pattern during Drosophila development and show that dMP1 interacts with MEK and ERK. By using transgenic Drosophila lines allowing either dMP1 down-regulation by RNA interference or dMP1 over-expression, we demonstrate that dMP1 is involved in the control of cell differentiation during development of the Drosophila wing by regulating ERK vein-promoting function.
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Results |
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The D. melanogaster genome contains one sequence orthologous to mammalian MP1, CG5110, that we named dMP1 (Fig. 1A). This gene, located on chromosome 2 L at 36C10, comprises two exons and a small 78 bp intron. Its annotated transcript (EST RE09173, 610 bp) encodes a 124 amino-acid protein with 45% identity to human and mouse MP1 (Kurzbauer et al. 2004; Lunin et al. 2004). Other ESTs extend beyond RE09173 either in 3' or in 5' (Fig. 1A), suggesting that dMP1 may have more than one promoter and/or terminator.
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As no antibody against mouse MP1 was available, we produced polyclonal antibodies raised against a GST–dMP1 fusion protein. We first checked their specificity by Western blot analysis on a dMP1–6His fusion protein produced in bacteria (Fig. 3A). By using these antibodies on total S2 cell extracts (Fig. 3B), we detected very low amounts of a protein of the expected size (approximately 15 kDa), but only after lysis plus sonication (lane 2) or after lysis in denaturing urea buffer (lane 3). A similar result was obtained with total embryonic extracts (data not shown). These results show that dMP1 is predominantly insoluble and may not be very abundant. We then analyzed dMP1 cellular distribution by producing tagged forms of this protein, either dMP1–FLAG or dMP1–RFP, in S2 cells. Western blots (Fig. 3C) showed that dMP1–FLAG was found preferentially in the insoluble pellet. Unlike the endogenous protein, dMP1–FLAG was also detected in the cytoplasmic and the nuclear soluble fractions. The presence of dMP1–FLAG in the soluble fractions is probably because of the fact that dMP1–FLAG is expressed at a much higher level than the endogenous gene. A minor endogenous dMP1 soluble fraction might thus exist but would not be abundant enough to be detected. Similar cytoplasmic and nuclear localizations were observed for a dMP1–RFP fusion protein produced in S2 cells (Fig. 4A). We also constructed a UAS::mRFP-MP1 transgenic Drosophila line to follow dMP1 localization in flies. When induced with the daughterless::Gal4 (da::Gal4) ubiquitous driver, mRFP-dMP1 localized in the cytoplasm and the nuclei of salivary gland cells (Fig. 4B).
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In mouse, MP1 was identified as a scaffold protein of the ERK signaling pathway that specifically binds the MAPKK–MEK1 and the MAPK–ERK1 (Schaeffer et al. 1998; Wunderlich et al. 2001). In order to see whether these interactions were conserved in Drosophila, we carried out GST pull-down assays and co-immunoprecipitation experiments. The Drosophila genome contains only one MEK and one ERK encoding gene, Dsor1 (CG15793) and rolled (CG12559), respectively. As shown in Fig. 5A, the GST–dMP1 fusion protein directly interacted with in vitro translated MEK and ERK. To detect in vivo interactions, we expressed dMP1–Myc and ERK–FLAG proteins in S2 cells and carried out co-immunoprecipitation experiments on total cell extracts using an anti-Myc antibody. As shown on Fig. 5B, a low amount of ERK–FLAG co-immunoprecipitated with dMP1–Myc. This might indicate that the association between dMP1 and ERK is very transient. The same conclusion arose from co-immunoprecipitation experiments between mouse MP1 and ERK1 (Teis et al. 2002). This is probably because of the intrinsic nature of MEK to ERK signaling, because in mouse the interaction between ERK1 and its activator MEK1 has to be transient to allow efficient signaling (Fukuda et al. 1997; Teis et al. 2002). Taken together, co-immunoprecipitations and GST pull-down assays show that dMP1 can interact with ERK and MEK, and suggest that, as in mouse, it could form a complex with both ERK and MEK.
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As dMP1 interacts with ERK and MEK, we addressed the physiological function of this interaction. As no dMP1 loss-of-function mutant was available, we generated transgenic lines allowing dMP1 inactivation by RNA interference (RNAi). To detect possible ‘Off-Target Effects’ (OTEs), the 499 bp dMP1 sequence used to generate these lines was screened for short sequences that perfectly match unintended targets. Several 16–18 nt sequences were found, but none of them contained more than two consecutive CAN trinucleotides, while sequences containing more than 13 consecutive CAN repeats have been shown to cause OTEs (Ma et al. 2006; Dietzl et al. 2007). Moreover, no homologous sequence above 18 nt was found. Thus, with the criteria defined in several studies, the sequence used to generate our lines is unlikely to induce OTEs (Kulkarni et al. 2006; Ma et al. 2006; Dietzl et al. 2007). Four independent transgenic lines were obtained. When crossed with different Gal4 drivers, they all presented similar phenotypes, which shows that these were not because of transgene position effects. Detailed results for two of these lines, UAS::dsMP1II and UAS::dsMP1III, in which the RNAi transgene is located on the second and third chromosome, respectively, are presented thereafter.
Inactivation of dMP1 was checked in da::Gal4>UAS ::dsMP1 flies obtained by crossing UAS::dsMP1 flies with the ubiquitous da::Gal4 driver line. Control flies were obtained from crosses of UAS::dsMP1 flies with a wild-type strain. Quantitative RT-PCR analysis showed a 1.2 and 1.4-fold reduction of dMP1 transcripts in da::Gal4>UAS::dsMP1II and da::Gal4>UAS::dsMP1III flies, respectively, as regards to UAS::dsMP1/+ flies (Fig. 6A). The amount of dMP1 detected in larval extracts by Western blot analysis was strongly reduced in da::Gal4>UAS::dsMP1II and da::Gal4>UAS::dsMP1III flies in comparison to control flies (Fig. 6B). Thus, UAS::dsMP1II and UAS::dsMP1III lines allow efficient dMP1 down-regulation and can be used as partial loss-of-function alleles.
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To analyze dMP1 requirement during wing development, we used several driver lines allowing Gal4 expression in the wing imaginal disc: engrailed::Gal4 (en::Gal4) drives Gal4 expression in the posterior compartment of all imaginal discs, apterous::Gal4 (ap::Gal4) drives Gal4 expression in the dorsal compartment of the wing disc, and scalloped::Gal4 (sd::Gal4) induces Gal4 expression in regions of the wing disc that will give rise, among others, to the adult wing blade. These driver lines showed very weak ectopic vein phenotypes in a fraction of the flies (Table 1 and Fig. 7B). Flies from crosses between these driver strains and the UAS::dsMP1II and UAS::dsMP1III lines exhibited a high percentage of ectopic veins (Table 1). Furthermore, with the en::Gal4 driver, these ectopic veins were located in the posterior compartment where engrailed is expressed. The same ectopic vein phenotype was observed using UAS::dsMP1–34 935, a third dMP1 RNAi line generated at the Vienna Drosophila RNAi Center (VDRC) with a shorter (307 bp) dMP1 sequence (Dietzl et al. 2007) (Table 1). As expected for the Gal4/UAS system, strength of this phenotype was modulated by temperature. Indeed, all flies from crosses carried out at 25 °C between the ap::Gal4 driver strain and the UAS::dsMP1II line presented fully ballooned wings (data not shown), a phenotype initially described for blistered loss-of-function alleles and resulting from formation of a very high number of ectopic vein cells within intervein tissue. Blisters are formed by ectopic vein cells which do not express integrin, resulting in loss of adhesion between the two cell layers that constitute the wing (Fristrom et al. 1994; Montagne et al. 1996). At 23 °C, only 12.9% of females presented ballooned wings (data not shown), whereas at 20 °C, ap::Gal4>UAS::dsMP1II flies presented no blistered wings but only discrete ectopic veins (Table 1). Thus, the expressivity of the ectopic vein phenotype seemed to be directly correlated with the level of dMP1 inactivation.
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In order to confirm that this ectopic vein phenotype was because of dMP1 down-regulation and not to unspecific off-target effects (OTE), we generated flies heterozygous for a dMP1 RNAi transgene (either UAS::dsMP1II or UAS::dsMP1–34 935), the sd::Gal4 driver and deficiency Df(ED)1175, which deletes the whole dMP1 locus. As shown in Table 1, deleting a copy of dMP1 significantly enhanced the penetrance of the ectopic vein phenotype induced by both dMP1 RNAi transgenes.
One genetic characteristic of scaffold proteins, initially reported for KSR, is that inactivation and over-expression lead to similar phenotypes. Indeed, scaffold complexes are very sensitive to the relative amounts of their components, and can be disrupted either by an excess or a shortage of one of these (Cacace et al. 1999). To see whether this property applies to dMP1, we constructed transgenic lines allowing over-expression of wild-type or RFP-tagged dMP1 (UAS::MP1 and UAS::mRFP-MP1 lines). As shown in Table 1, over-expressing dMP1 with either UAS::MP1 or UAS::mRFP-MP1 also led to ectopic veins. In addition, combining the Df(ED)1175 deficiency or the UAS::dsMP1III transgene with the UAS::MP1 transgene significantly lowered the percentage of ectopic veins (Table 1). These results suggest that dMP1 indeed acts as a scaffold protein.
dMP1 regulates ERK vein-promoting function
In order to assess the temporal requirement for dMP1 function in vein cell differentiation, we crossed the UAS::dsMP1II line with the hs::Gal4 driver strain allowing staged Gal4 expression. We applied 20 min heat-shocks at various times during larval and pupal development, from 39 to 141 h after egg laying. As shown on Fig. 7H and Table 2, ectopic veins arose in 69.3% of females when heat-shock was applied 129 h after egg laying, which corresponds to the early pupal stage. Interestingly, Egfr, that encodes DER, and rho, are required during the same time interval to control vein differentiation (Guichard et al. 1999), suggesting that dMP1 is involved in vein differentiation together with ERK signaling.
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Discussion |
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In this study, we have characterized the D. melanogaster dMP1 gene that encodes the Drosophila homologue of mouse MP1 scaffold protein. This gene is ubiquitously expressed during development. dMP1 is a highly insoluble protein located both in the cytoplasm and the nucleus of cells. Its poor solubility is consistent with reports showing that production of a recombinant mouse MP1 in E. coli yielded very low amounts of a rather insoluble protein (Kurzbauer et al. 2004; Lunin et al. 2004) and that mouse MP1 has been detected in lipid rafts (Foster et al. 2003). Moreover, a mouse MP1–Myc recombinant protein expressed in mammalian cells was also shown to be located both in the cytoplasm and the nucleus (Kurzbauer et al. 2004). We show that dMP1 interacts physically with MEK and ERK and co-immunoprecipitates with ERK, suggesting that the dMP1–MEK–ERK complex is conserved in Drosophila. As dMP1 is also nuclear, this suggests that at least a fraction of the dMP1–ERK complex may be translocated to the nucleus.
Moreover, our data show that the amount of dMP1 has to be precisely regulated to ensure correct wing development, as both dMP1 down-regulation and over-expression lead to similar ectopic wing vein phenotypes. This feature, which was also reported for the KSR scaffold protein (reviewed in Claperon & Therrien 2007), seems to be a general property of scaffold proteins. Indeed, scaffold complexes have been shown to be very sensitive to the relative amounts of their components. The murine MP1–MEK1–ERK1 complex was either favored or disrupted depending on the relative concentration of MP1 (Schaeffer et al. 1998). Furthermore, a quantitative computational model of the MAP kinase cascades suggested that correct signaling required an optimal concentration of scaffold proteins (Levchenko et al. 2000). Altogether, our results indicate that, like mouse MP1, dMP1 would be a scaffold protein involved in the regulation of ERK signaling via formation of a dMP1–MEK–ERK complex. This complex could be disrupted either by dMP1 shortage or by dMP1 excess that could result in sequestrating MEK, ERK, or other participating proteins. Lowering the amount of the dMP1–MEK–ERK complex during wing development might induce ERK signaling deregulation.
dMP1 scaffold protein is required to repress vein differentiation during wing development
Our results point out a role of dMP1 in wing cell differentiation, as dMP1 down-regulation as well as over-expression induces ectopic veins. The same phenotype arises when over-expressing rolled, whereas rolled loss-of-function alleles induce interruption of longitudinal veins (Brunner et al. 1994). Moreover, dMP1 down-regulation enhances the rolled over-expression vein phenotype. These results suggest that dMP1 controls ERK vein-promoting function. Using staged dMP1 inactivation, we show that dMP1 is required to limit vein cell differentiation essentially in young pupae, thus when ERK signaling is required to control vein formation (Guichard et al. 1999). Martin-Blanco et al. (1999) have shown that loss of DER–ERK signaling in third instar larvae results in loss of veins, whereas it induces ectopic veins when applied during pupal development. This suggests that early DER–ERK signaling is involved in vein specification, while it participates in intervein cell differentiation during the pupal stage. Interestingly, we never observed interruption of longitudinal veins when down-regulating dMP1 at any time during development with the hs::Gal4 driver. This result suggests that dMP1 and the dMP1–ERK scaffold complex are primarily required during the pupal stage in future intervein cells to repress vein fate and/or to promote intervein fate. Hence, ERK signaling via the dMP1–ERK complex may be dispensable in future vein cells, and other modules of ERK activation may function in these cells to specify vein identity.
dMP1 could regulate ERK subcellular localization
It has been shown that ERK subcellular localization controls the balance between proliferation and differentiation in Drosophila wings (Marenda et al. 2006). In late larval wing discs and pupal wings, once activated by phosphorylation, ERK is maintained in the cytoplasm. This cytoplasmic retention directs cell differentiation, whereas nuclear translocation of activated ERK induces cell proliferation. Nuclear import of activated ERK involves the Drosophila orthologue of Importin 7, encoded by the moleskin gene (msk) (Lorenzen et al. 2001), but proteins involved in ERK cytoplasmic hold have not been identified so far. In mammals, ERK subcellular localization is also involved in the control of the balance between proliferation and differentiation, and two proteins, PEA-15 and Sef, have been shown to maintain activated ERK in the cytoplasm (Formstecher et al. 2001; Torii et al. 2004). However, no Sef and PEA-15 Drosophila orthologies have been identified (Marenda et al. 2006). dMP1, which is located both in the cytoplasm and in the nucleus, might participate in the regulation of ERK shuttling between the cytoplasm and the nucleus. This regulation could also involve dMP1–MEK interaction. Indeed, MEK, which is mostly cytoplasmic because of its nuclear export sequence, can constantly enter the nucleus by passive diffusion. Cytoplasmic MEK acts as an anchor of inactivated ERK (Fukuda et al. 1997), and nuclear MEK is involved in nuclear export of ERK (Adachi et al. 2000). This process allows restoring the cytoplasmic pool of unphosphorylated ERK, thus ensuring its sustained and continuous activation. dMP1, which interacts with MEK and ERK and may also shuttle between cytoplasm and nucleus, might thus similarly participate with MEK in the nuclear export of ERK.
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Experimental procedures |
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Flies were raised on standard yeast-cornmeal medium at 25 °C. w1118 was used as control strain. Lines allowing dMP1 (CG5110) inactivation or over-expression was generated by standard P-element mediated transformation. A 499-bp dMP1 cDNA fragment (bp 24–523 of EST RE09173, corresponding to bp 17.481.270–17.481.847 of chromosome 2 L NT_033 779 sequence) that contains the whole dMP1 coding sequence was cloned into the pCasper-UASp transformation vector (Rorth 1998). This fragment was also cloned into the pUASp::mRFP GatewayTM vector (Campbell et al. 2002) to generate the mRFP-MP1 transgenic line. For RNA interference inactivation, a head-to-head inverted-repeat of this 499 bp fragment was cloned into pCasper-UASp as described elsewhere (Roignant et al. 2003). The UAS::dsMP1–34 935 line allowing dMP1 RNA interference inactivation was from the Vienna Drosophila RNAi Center (VDRC) (Dietzl et al. 2007), and the Df(2 L)ED1175 line harboring a 383-kb deletion uncovering the dMP1 genomic region was from the Szeged collection center. Transgenic lines were crossed with different driver strains producing Gal4: da::Gal4 (Gal4daG32, Roignant et al. 2003), sd::Gal4 (sdETX4, Campbell et al. 1992), ap::Gal4 (apmd544, a gift of J. Montagne), en::Gal4 (a gift of A. Zider) and hs::Gal4 (Roignant et al. 2003). The UAS::rolled line allowed ERK over-expression (Kumar & Moses 2001).
RNA expression analysis
Total RNA was extracted from embryos, third instar larvae and adults with the RNeasy mini-kit (Qiagen) according to the manufacturer's instructions, and subsequently submitted to RNAse-free DNAse I treatment (Fermentas). Northern blots were carried out with standard protocols, with radiolabeled dMP1 and 18S RNA probes synthesized by in vitro transcription (Riboprobe transcription system, Promega). Eight micrograms of total RNA were loaded on each lane. In situ hybridization on embryos and larvae with DIG-UTP labeled RNA probes followed protocols described elsewhere (Tautz & Pfeifle 1989; Masucci et al. 1990). For quantitative real-time RT-PCR, 2 µg of total RNA were used to synthesize cDNA with SuperScriptTM II reverse transcriptase (Invitrogen). Real-time PCR was carried out with an ICycler iQTM system (BIO-RAD) and the Absolute TM QPCR SYBR® Green Fluorescein Mix (Thermoscientific) under conditions recommended by the manufacturer. The primers used were dMP1 forward (5'-GTTCATACCCACATTCACCAC-3') and reverse (5'-CACAAAGGTCAGGATGAGCG-3') and rp49 forward (5'-CCCAAGATCGTG AAGAAGCG-3') and reverse (5'-AGATACTGTCCCTTGAAGCG-3'). A standard curve of amplification efficiency for each set of primers was generated with a serial dilution of cDNA. Melting curve analysis was carried out to eliminate non-specific products from the reaction. rp49 levels were used for normalization with the standard curve method.
Antibodies
Polyclonal anti-dMP1 antibodies were raised in guinea pig against a GST–dMP1 fusion protein produced by cloning the dMP1 full-length coding-sequence into pGEX-4T1 vector (Pharmacia Biotech). After depletion of anti-GST antibodies on acetonic powder from bacteria expressing GST, their specificity was checked against a dMP1–6His fusion protein produced by cloning the dMP1 full-length coding sequence into pET31b vector (Novagen). Monoclonal anti-FLAG M2 antibody (F3165, Sigma) and anti-Myc 9E10 antibody (sc-40, Santa Cruz Biotechnology) were used to detect fusion proteins expressed in S2 cells. Anti-lamin (ADL67.10) and anti-β-tubulin (E7) antibodies were from the Developmental Studies Hybridoma Bank.
Protein extracts
Embryos or S2 cells were homogenized in RIPA buffer (50 mM Tris pH7.5, 150 mM NaCl, 0.1% SDS, 0.5% NP40, protease inhibitors). dMP1 was solubilized either by sonication in RIPA buffer or by homogenization in urea buffer (50 mM Tris pH7.5, 7 M urea, 0.1% SDS, 0.5 mM EDTA, protease inhibitors). Larval extracts were carried out by formic acid extraction as previously described (Blard et al. 2007). Briefly, 10 third instar larvae were homogenized in RIPA buffer. After centrifugation, the pellet containing insoluble proteins was incubated for 1 h at 4 °C in formic acid 70%, followed by centrifugation, collection of the supernatant, evaporation of formic acid and resuspension in 50 µL of Laemmli buffer. Cytoplasmic and nuclear extracts were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce) according to the manufacturer's instructions.
S2 cell transfection
To transiently express fusion proteins (dMP1–RFP, dMP1–Myc, ERK–FLAG) in Drosophila S2 cells, dMP1 (EST RE09173) and rolled (EST RE08694) full-length coding sequences were cloned into pENTR/D-TOPO vector (Invitrogen). LR-recombination reactions were carried out with these vectors according to the manufacturer's instructions to transfer coding sequences into GatewayTM Drosophila vectors allowing expression of the fusion proteins under control of the actin5C promoter (Huynh & Zieler 1999). S2 cells were cultivated at 25 °C in Schneider medium supplemented with 10% fetal calf serum. 2.5.106 cells were transfected with 1 µg of each plasmid using Effecten® transfection reagent (Qiagen) according to the manufacturer's instructions (1/25 DNA-Effecten® ratio). Cells were collected 48 h after transfection. To study dMP1–RFP localization, 106 transfected cells were plated on a poly-lysine coated slide. After fixation in 4% paraformaldehyde, cells were stained with DAPI, mounted in antifading PVP1 medium (CitifluorTM) and visualized by confocal microscopy. The same protocol was applied to salivary glands of the pUASp::mRFP-dMP1 transgenic line.
Protein–protein interactions
In vitro transcription/translation and GST pull-down assays were carried out as previously described (Salvaing et al. 2003), by cloning dMP1 (EST RE09173), Dsor1 (EST LD4120) and rolled (EST RE08694) full-length coding sequences into expression vectors. For co-immunoprecipitation experiments, S2 cells were co-transfected with plasmids allowing expression of ERK–FLAG and dMP1–Myc. These cells were homogenized in RIPA buffer 48 h after transfection. Approximately 2 mg of protein extracts were immunoprecipitated either with anti-Myc agarose beads (A7470, Sigma) or with protein G-agarose beads plus rabbit serum as a control.
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Acknowledgements |
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Footnotes |
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aPresent address: UMR 1198 INRA-ENVA-Biologie du Développement et Reproduction; FRE 2857 CNRS; F-78 350 Jouy-en-Josas, France.
* Correspondence: Emmanuele.Mouchel-Vielh{at}snv.jussieu.fr |
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Accepted: 29 July 2008
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0.001 < P < 0.05)
