20E-regulated USP expression and phosphorylation in Drosophila melanogaster

Insect Biochemistry and Molecular Biology 33 (2003) 1211–1218 www.elsevier.com/locate/ibmb

20E-regulated USP expression and phosphorylation in Drosophila melanogaster Q. Song ∗, X. Sun, X.-Y. Jin Department of Entomology, University of Missouri, 1-87 Agriculture Building, Columbia, MO 65211, USA Received 1 February 2003; received in revised form 12 May 2003; accepted 28 June 2003

Abstract The developmental profiles of ultraspiracle protein (USP) in the tissues of Drosophila melanogaster were investigated using a USP specific monoclonal antibody (mAb) as a probe. Western blot analysis revealed four USP mAb reactive bands (p46, p48, p54 and p56), each with tissue- and stage-specific expression patterns. The p54 and p56 were expressed in nearly all larval and prepupal tissues tested with fluctuations in abundance. However, the p46 and p48 were detected exclusively in the midgut of prepupae and shown to be the proteolytic products of p54 and p56. A lambda protein phosphatase assay demonstrated that the p56 is the phosphorylated form of p54. The expression and phosphorylation of the p54 USP is regulated by 20E. Protein kinase consensus recognition sequence analysis revealed 10 putative phosphorylation sites in Drosophila USP, with seven sites for protein kinase C (PKC) and three sites for casein kinase II (CKII). The fact that seven out of 10 putative phosphorylation sites reside in the ligand- and DNA-binding domains suggests that phosphorylation may play important role in regulating USP function. Identification of the in vivo USP phosphorylation sites and signal transduction pathways that regulate the specific USP phosphorylation is currently underway.  2003 Elsevier Ltd. All rights reserved. Keywords: Ecdysteroids; EcR; USP; Protein phosphorylation

1. Introduction Insect molting and metamorphosis are regulated by the interaction of 20-hydroxyecdysone (20E) with ecdysone receptor (EcR) and its heterodimer partner ultraspiracle protein (USP) (Riddiford, 1995). Upon activation by ligand, the EcR/USP complex recognizes specific DNA response elements and triggers a cascade of transcription factors that direct the molting process (Henrich et al., 1999; Thummel, 1995). Genes for both EcR and USP have been cloned and sequenced from Drosophila melanogaster (Koelle et al., 1991; Henrich et al., 1990; Oro et al., 1990; Shea et al., 1990), and subsequently from several other dipteran and lepidopteran species (Cherbas and Cherbas, 1996; Henrich et al., 1999). In Drosophila, EcR encodes three ecdysone receptor isoforms, EcR-A, EcR-B1 and EcR-B2 (Talbot et al.,

Corresponding author. Tel.: +1-573-882-9798; fax: +1-573-8821469. E-mail address: [email protected] (Q. Song). ∗

0965-1748/$ - see front matter  2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2003.06.005

1993), whereas usp encodes a single ultraspiracle protein that is the Drosophila homolog of the vertebrate retinoid X receptors (RXRs) (Henrich et al., 1990; Oro et al., 1990; Shea et al., 1990). Heterodimerization of USP with EcR is required for the binding of 20E, sequencespecific interactions with DNA and reporter gene transcription in transfected tissue culture cells (Koelle, 1992; Yao et al., 1992; Thomas et al., 1993; Yao et al., 1993). In vivo studies have shown that USP is co-localized with EcR to ecdysone-regulated puffs in the salivary gland polytene chromosomes, suggesting that USP functions together with EcR in vivo (Talbot, 1993; Yao et al., 1993). USP mutation studies revealed that loss of maternal usp function leads to embryos with a defective chorion and lethality during late embryogenesis with cuticular scarring in posterior abdominal segments (Perrimon et al., 1985; Oro et al., 1992). In contrast, loss of zygotic usp function leads to early larval lethality with some surviving second instar larvae carrying an extra set of posterior spiracles, suggesting a defect in molting of the first instar cuticle (Perrimon et al., 1985; Oro et al., 1992). USP is also required for morphogenetic furrow

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movement during adult eye development (Zelhof et al., 1995) and plays a role in fusion of the wing imaginal disc during metamorphosis (Henrich et al., 1994). Most recently, Hall and Thummel (1998) have shown that the usp is required in late third instar larvae for appropriate developmental and transcriptional responses to the ecdysone pulse that triggers puparium formation. In addition to forming a heterodimer with EcR, USP also forms heterodimers with Drosophila hormone receptor (DHR38) (Yao et al., 1992; Sutherland et al., 1995). Like its vertebrate homolog, USP can also heterodimerize with mammalian nuclear receptors, including retinoid acid receptor (RAR), thyroid receptor (TR) and vitamin D receptor (VDR), to mediate the hormonal responses (Khoury Christianson et al., 1992; Yao et al., 1993). USP was also reported to act as a putative juvenile hormone receptor based on its ability to mediate a transcriptional response to methyl epoxyfarnesoate (JHIII) via a direct repeat (DR12) response element in insect cells (Jones and Sharp, 1997; Jones et al., 2001; Xu et al., 2002). Both EcR and USP are ligand-activated transcriptional factors. Many transcriptional factors are phosphoproteins and their functions are regulated by phosphorylation (Weigel, 1996). Phosphorylation serves as an important mechanism for modulating the structure, activity and lifetime of many proteins. Selective protein phosphorylation underlies regulation of cellular metabolism by a wide variety of agents, ranging from hormones and growth factors to tumor promoters and oncogenes. Studies of other transcriptional factors in vertebrates have shown that phosphorylation of steroid hormone receptors can play important roles in nuclear translocation, ligand and DNA binding, protein–protein interactions and transactivation (Weigel, 1996). In insects, both EcR and USP have recently been demonstrated to be phosphoproteins in Manduca sexta (Song and Gilbert, 1998), in a Chironomus tentans epidermis cell line (Rauch et al., 1998) and in Tenebrio molitor (Nicolaı¨ et al., 2000). Phosphorylation of EcR and USP are regulated by 20E in all three species tested. For example, when the EcR/USP complex from Manduca prothoracic glands was treated with λ protein phosphatase, an enzyme that removes phosphate groups from protein, the ligand-binding activity of the dephosphorylated EcR/USP complex was significantly increased (Song and Gilbert, 1998), suggesting that phosphorylation play important roles in regulating the function of the EcR/USP complex. Despite extensive functional studies of the Drosophila USP at the molecular level, the effect of post-translational modifications, such as phosphorylation, has not yet been studied in Drosophila. In this paper, we reported that Drosophila USP is a phosphoprotein and USP phosphorylation is regulated by 20E.

2. Materials and methods 2.1. Insects D. melanogaster [wild typeore and ecdysone-deficient mutant without childrenrgl (wocrgl)] was reared on an artificial blue diet (FisherSci, catalog # S22315C) at 24 °C under constant darkness. The wocrgl mutant was kindly provided by Dr. L. Gilbert of University of North Carolina and Dr. J. Wismar of Johannes Gutenberg Universita¨ t, Germany. 2.2. Chemicals and reagents SuperSignal West Pico Chemiluminescent substrate for western blot analysis was ordered from Pierce (Rockford, IL). Lambda protein phosphatase (λPP) was obtained from New England Biolabs (Beverly, MA). AB11 USP monoclonal antibody (mAb) was provided by Dr. F.C. Kafatos of Harvard University. Horseradish peroxidase-conjugated goat anti-mouse secondary antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA). Other chemical reagents used for buffers, sample preparation, tissue culture, electrophoresis, and western blot were obtained from Sigma (St Louis, MO), FisherSci (Houston, TX) or BioRad (Hercules, CA). 2.3. Polyacrylamide gel electrophoresis and western blot analysis One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in this study for protein identification by western blotting. The slab gels were prepared following Takacs’ gel formulation (1979) using a 105 × 100 × 1.5 mm Hoefer Mighty Small Gel Caster. Acrylamide separating gel solution (12.5%) was prepared, mixed well and immediately poured into the mini-gel glass plate to a level about 2/3 of the plate height. The gel was gently covered with water-saturated butanol and allowed to polymerize for 30 min to 1 h at room temperature. After polymerization of the separating gel, the butanol was poured off and a stacking gel with 3.0% acrylamide was overlayed on top of the separating gel. Protein samples were prepared according to a protocol modified from Song and Gilbert (1997). In brief, Drosophila tissues were dissected from the indicated stages at room temperature under the Ringers’ solution and homogenized on ice in phosphate-buffered saline (PBS) (136 mM NaCl, 1.1 mM K2HPO4, 2.7 mM KCl, 8.0 mM Na2HPO4, pH 7.4) using a disposable pestle in an Eppendorf tube. The homogenate was centrifuged at 16,000 × g for 10 min at 4 °C to remove debris. Protein concentration was determined using the Bio-Rad protein bioas-

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say kit (Reagent A: catalog # 500-0113. Reagent B: catalog # 500-0114). Protein samples were denatured by adding 3X sample buffer (0.2 M Trish, pH 6.8, 30% glycerol, 6.9% SDS, 15% 2-mercaptoethonol, 0.4% bromophenol blue) and boiled for 10 min prior to sample loading. Molecular weight (MW) standards were used to monitor electrophoresis and for MW calibration. Electrophoresis was carried out in a running buffer (25 mM Tris–HCl, 192 mM glycine, 0.1% SDS) at 50 V for the first 30 min, and then at 100–120 V until completion. Western blotting was employed to monitor the expression of USP in protein samples from different tissues. Proteins (20 µg/lane) separated in gels were transferred onto PVDF membranes in a transferring buffer (gel running buffer containing 15% methanol) for 40–50 min at 20 V with the BioRad Trans-blot SD SemiDry Transfer Cell. After protein transfer, the PVDF membranes were blocked with 5% nonfat dried milk in Tris-buffered saline (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, pH 7.5) (TBST) for 1 h, and then incubated overnight at 4 °C in AB11 USP mAb (1:2000) diluted with blocking solution. After incubation with the primary mAb, the membranes were washed three times with TBST for 10 min each, and then incubated with horseradish peroxidase-conjugated goat antimouse secondary antibody (diluted at 1:2000 with blocking solution) for 90 min at room temperature. After washing with TBST for 30 min, the membranes were treated for 1 min with chemiluminescent substrate (PIERCE, Rockford IL) and the immunoreactive proteins were visualized by exposing an X-ray film to the membrane. 2.4. Tissue and developmental profiles The salivary glands, brain/ring gland complex, midgut, epidermis and fat body were dissected from early wandering larvae and 4 h old prepupae at room temperature in Ringer’s solution. Third instar larvae were staged according to the methods of Andres and Thummel (1994) based on the amount of blue food remaining in their gut following the cessation of feeding. Prepupae were synchronized at 0 h immediately following the inversion of mouthpart. The tissue samples were washed with the Ringer’s, homogenized in the PBS and centrifuged. The protein concentration was determined using BioRad protein assay kit and verified with silverstained gels. The calibrated protein samples were then subjected to SDS-PAGE (12.5%) and western blot analysis as described above. For USP developmental profile studies, both salivary glands and midgut were dissected from early wandering larvae, late wandering larvae, and 0, 2, 4, 6 h old prepupae, respectively. The dissected samples were immediately placed in ice-cold Grace’s medium, homogenized

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in PBS and quantified for protein concentration before being subjected SDS-PAGE and western blot analysis. 2.5. Protease inhibitor treatment To reveal whether the specific USP forms displaying a MW of 46 and 48 kDa identified in the midgut of prepupae were the products of protease activity, the salivary gland sample (10 µl, equivalent to a pair) from early wandering larvae, which contains only two large USP bands with a MW of 54 and 56 kDa, was mixed with the midgut sample (10 µl, equivalent to one mid gut) from 0 h old prepupae and incubated at room temperature for the indicated time periods. The resulting sample was subjected to SDS-PAGE and western blot analysis. To determine which stage of prepupal midgut contains a high level of protease activity, the salivary gland sample from early wandering larvae was incubated with the midgut sample from 0, 2, 4 and 6 h old prepupae, respectively. To investigate whether the protease activity in midgut was involved in converting large USP bands to the small bands, the salivary gland sample (10 µl, equivalent to a pair) from early wandering larvae was mixed with the indicated equivalents of midgut sample from 0 h old prepupae in the presence of the protease inhibitor cocktail (Sigma, catalog # P2714) at the manufacturer’s suggested concentration. 2.6. Lambda protein phosphatase treatment The procedure for λPP treatment of salivary gland sample was identical to that described previously (Song and Gilbert, 1998). In brief, a salivary gland sample (5 µl, one pair equivalent) from an early wandering third instar larva was incubated for 30 min with 1000 unit λPP in 50 µl of reaction buffer according to the manufacturer’s specifications. At the end of the incubation period, the sample was boiled for 10 min following the addition of SDS sample buffer, and subjected to SDSPAGE and western blot analysis. 2.7. Dose and time-course response of USP to 20E incubation The salivary glands from the early wondering stage of third instar larvae homozygous for the ecdysonedeficient wocrgl mutation of D. melanogaster were dissected in Ringer’s solution and immediately placed in a 24-well tissue culture plate containing 200 µl of Grace’s medium. After dissection, the medium was removed from the well and replaced with 500 µl of a fresh Grace’s medium containing the indicated concentrations of 20E dissolved in 100% ethanol. The glands were then incubated for 12 h at room temperature. A control group was incubated in medium containing a comparable volume

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of ethanol. For temporal response, the glands were incubated for the indicated time periods in the presence of 0.01 µM 20E. At the end of the 20E incubation, the salivary glands (20 pairs/concentration or time period) were collected, solublized in 20 µl of 3X SDS sample buffer and boiled for 10 min before being subjected to SDS-PAGE and western blot analysis.

3. Results 3.1. Tissue-specific USP profiles To investigate whether multiple forms of USP exist in Drosophila, five different tissues were prepared from early wandering third instar larvae and 4 h old prepupae. USP expression profiles in these tissues were analyzed by western blot using AB11 USP mAb as a probe. Western blot analysis revealed three major AB11 mAb reactive bands with a MW of 48, 54, and 56 kDa, respectively (subsequently referred to as p48, p54, p56) (Fig. 1). The expression of p48, p54 and p56 was tissuespecific and stage-dependent. The p54 and p56 bands were detected mainly in the salivary glands (Fig. 1, lane 1) and brain/ring gland complex (Fig. 1, lane 2). The p54 and p56 bands were barely detectable in epidermis (Fig. 1, lane 4) and fat body (Fig. 1, lane 5), and were missing in the midgut (Fig. 1, lane 3) of early wandering larvae. At early prepupal development, the p54 and p56 were abundant in all tissue samples except midgut in which only a trace amount of p54 was evident. Surprisingly, the p48 was observed only in the midgut of 4 h old prepupae (Fig. 1, lane 8). In order to reveal the developmental profile of the p48, midgut from different developmental stages of the third instar larvae and early prepupal stage was prepared and subjected to SDS-PAGE and western blot analysis. The data indicated that the p48 was detectable in the midgut of the prepupae (Fig. 2, lanes 3–6), but not in the midgut of early or late wandering larvae (Fig. 2, lanes 1 and 2). The p48 appeared at the time of mouthpart inversion

Fig. 2. Developmental profiles of USP in the midgut of early wandering larvae (EW), late wandering larvae (LW), and 0, 2, 4 and 6 h old prepupae (PP0–6) of D. melanogaster. Arrows indicate the positions of different forms of USP detected by AB11 USP mAb.

(Fig. 2, lane 3), peaked 2 h later (Fig. 2, lane 4), then rapidly declined at 4 h (Fig. 2, lanes 5 and 6). A minor band with a MW of 46 kDa (p46) was detected in the midgut sample from 2 h old prepupae (Fig. 2, lane 4). Although the p54 and p56 were also detected in the midgut of 2 and 4 h old prepupae (Fig. 2, lanes 4 and 5), but they were less abundant compared to the p48. The data suggest that the prepupal midgut specific p46 and p48 might be the breakdown products of the p54 and p56 by midgut proteases. 3.2. Protease inhibitor treatment To test the above hypothesis, the salivary gland samples from early wandering larvae containing only p54 and p56 were incubated for the indicated time periods with the midgut sample from 0 h old prepupae mainly containing the p46 and p48. The data revealed that incubation of the salivary gland sample with the midgut sample resulted in the disappearance of the p54 and p56 within 1 min after incubation started (Fig. 3a). Incubation of salivary gland sample with the midgut sample from 0, 2, 4 and 6 h old prepupae for 30 min resulted in the disappearance of the p54 and p56 in all cases (Fig. 3b). Interestingly, the p46 and p48 USP from midgut were stable. The data suggest that the disappear-

Fig. 1. Western blot analysis of USP expression in the tissues of early wandering third instar larvae (L3) and prepupae 4 h (PP4) after mouthpart inversion of D. melanogaster. Samples (20 µg/lane) were loaded and subjected to 12% SDS-PAGE and western blot analysis. SG, salivary gland; B/R, brain/ring gland complex; Mg, midgut; Ep, epidermis; FB, fat body. Arrows indicate the position of different forms of USP protein detected by AB11 USP mAb.

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3.3. Lambda protein phosphatase treatment To investigate whether Drosophila USP is a phosphoprotein, the salivary gland sample containing the p54 and p56 was treated with λPP. Fig. 4 revealed that the p56 disappeared with a simultaneous increase in the p54 immunoreactivity in the λPP-treated sample (Fig. 4a, lane 2), demonstrating that the p56 is indeed the phosphorylated form of the p54. Based on kinase consensus recognition sequences, 10 putative phosphorylation sites of Drosophila USP were identified, with seven sites for protein kinase C (PKC) and three sites for casein kinase II (CKII) (Fig. 4b). Interestingly, out of 10 putative phosphorylation sites, three are located at the variable domain (A/B), two at the DNA-binding domain (C) and five at the ligand-binding domain (E). None of the putative phosphorylation sites was located at the linker domain (D). All putative phosphorylation sites are at serine residues except for one at a threonine residue. 3.4. Developmental profiles

Fig. 3. Western blot analysis of USP in salivary gland samples in the presence or absence of midgut sample. (a) The salivary gland supernatant from early wandering third instar larvae (equivalent to a pair of salivary glands) was mixed with the midgut supernatant [equivalent to one midgut of 0 h old prepupa (PP0)] and incubated at room temperature for the indicated time periods. The resulting samples were then subjected to 12.5% SDS-PAGE and western blot analysis. (b) The salivary gland supernatant (equivalent to a pair of salivary glands) was incubated with the midgut supernatant (one midgut equivalent) from the indicated stages of prepupae. Arrows indicate the position of different forms of USP protein detected by AB11 USP mAb. (c) The salivary gland supernatant (equivalent to a pair of salivary glands) from the early wandering larva was incubated at room temperature for 30 min in the presence or absence of the PP0 midgut supernatant (one PP0 midgut equivalent) and protease inhibitor (PI) cocktail.

ance of the p54 and p56 might be due to the protease activity of the midgut sample and that the p46 and p48 are the protease resistant remnants of the p54 and p56. When the salivary gland samples from early wandering larvae were incubated for the 30 min with the indicated equivalents of midgut sample from 0 h old prepupae in the presence of protease inhibitor cocktail, the p54 and p56 started to disappear when incubated with as little as 0.01 equivalent of midgut sample. Addition of the protease inhibitor cocktail had no effect on the disappearance of the p54 and p56 (Fig. 3c). The results indicate that a specific, protease inhibitor cocktail resistant protease exists in prepupal midgut.

The developmental profiles of the p54 and p56 in the salivary glands showed that the p54 and p56 were most abundant at the time of mouthpart inversion (PP0) and 2 h thereafter. Interestingly, approximate 60% of USP was in the phosphorylated form at the early wandering larvae, a period following a small peak of hemolymph ecdysteroids that initiates wandering behavior. At the

Fig. 4. Western blot analysis of USP in lambda protein phosphatase (λPP)-treated salivary gland sample of D. melanogaster. (a) Forty micrograms of salivary gland protein from early wandering larvae were incubated for 30 min at 30 °C in the presence (lane 2) or absence (lane 1) of 4000 units of λPP in 200 µl of reaction buffer, respectively. Onefourth of the aliquot was subjected to SDS-PAGE and western blot analysis. (b) Scheme of Drosophila USP indicating putative phosphorylation sites. Sequence of USP cDNA was analyzed with PROSITE. Letters in the boxes denote USP domains (A/B, variable domain; C, DNA-binding domain; D, linker domain; E, hormone binding domain). Numbers indicate the positions of putative phosphorylation sites for casein kinase II (CKII) (above the box) and protein kinase C (PKC) (below the box). The highlighted letter following the number indicates the phosphoamino acid (S, serine; or T, threonine).

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late wandering stage, the phosphorylated form declined to about 50% of total USP content. Despite the significant increase in total USP content at PP0 and PP2, the phosphorylated form remained at about 40–50% of total USP content. At PP4 and PP6, only about 40% USP was in the phosphorylated form. The data suggest that 20E may be responsible for eliciting USP phosphorylation in early wandering larvae, a stage when the glands were initially exposed to a high titer of ecdysteroids that induces wandering behavior. 3.5. Dose- and time-course response of USP to 20E incubation To test this possibility, salivary glands from early wandering third instar ecdysone-deficient wocrgl mutant were prepared and incubated with the indicated concentrations of 20E. Glands from wocrgl mutant have not been exposed to a high titer of hemolymph ecdysteroids and are therefore sensitive to 20E incubation. The data showed that 20E induced USP phosphorylation at concentration as low as at 0.001 µM, with ⬎70% of USP in its phosphorylated form (Fig. 6a, lane 2). 20E at 0.01 µM increased both the nonphosphorylated and phosphorylated USP content, with ~80% in the phosphorylated form (Fig. 6, lane 3). The maximum increase in the nonphosphorylated and phosphorylated USP level was observed at 0.1 µM 20E. At 1 µM or higher, 20E decreased both the nonphosphorylated and phosphorylated USP. Temporal response data revealed that 20E increased the intensity of the nonphosphorylated and phosphorylated USP at 6 h (Fig. 6b, lane 3) and the maximum increase in the phosphorylated and phosphorylated USP form was at 12 h with 80% of USP in the phosphorylated form (Fig. 6b, lane 4). Decrease in the nonphosphorylated and phosphorylated USP was observed with incubations of 24 h or longer (Fig. 6b, lanes 5 and 6).

4. Discussion Multiple isoforms of USP have been identified in all insect species so far studied except in D. melanogaster where only a single copy of USP gene has been identified. However, a recent study revealed that two USP proteins (p54 and p48) were present in the whole body homogenate of D. melanogaster, each with a different developmental expression profile (Henrich et al., 1994). The p54 was most abundant during the larval stage and disappeared in the late wandering larvae and pupal stage while the p48 appeared only in the pupal stage and was not detectable during the larval stages (Henrich et al., 1994). The different expression patterns of p54 and p48 were similar to that from Manduca prothoracic glands in which a small USP and its phosphorylated form were

induced by a high titer of hemolymph ecdysteroids that elicited wandering behavior. It was the small USP and its phosphorylated form that formed a heterodimer partner with EcR (Song and Gilbert, 1998). To reveal the identities of these two USP forms in Drosophila, both developmental and tissue-specific USP expression profiles were investigated with western blot analysis using an USP specific mAb as a probe. The results of the present study revealed four AB11 USP mAb reactive bands (p46, p48, p54, p56) in Drosophila tissues (Figs. 2 and 3), each with tissue-specific and stage-dependent expression profiles. With the exception of the midgut of early wandering larvae, the p54 and p56 were detected in all the larval and prepupal tissues (Fig. 1). However, the p46 and p48 were detected exclusively in the midgut of prepupae (Figs. 1 and 2) and were likely the proteolytic products of the p54 and p56 (Fig. 3). The data suggest that the p54 and p56 might be the native USP proteins since they are close to the predicted size of Drosophila USP from usp transcript (Henrich et al., 1990; Oro et al., 1990; Shea et al., 1990). The treatment of the p54 and p56 Drosophila USP sample with λ protein phosphatase clearly demonstrated that the p56 is indeed the phosphorylated form of the p54 (Fig. 4), a result similar to that described in M. sexta (Song and Gilbert, 1998), C. tentans epidermis cell line (Rauch et al., 1998) and T. molitor (Nicolaı¨ et al., 2000). This is the first demonstration of phosphorylated form of USP in D. melanogaster. Whether the p48 is the phosphorylated form of the p46 remains unclear. The role of the protease resistant p46 and p48 in prepupal midgut remains unknown. The developmental profile of the p54 and p56 expression in the salivary glands of wild type Drosophila revealed that the maximum phosphorylation of USP (60% of total USP in the phosphorylated form) was observed only at the early wandering stage and 40–50% of USP was phosphorylated at other stages (Fig. 5). Perhaps, it is this small but significant increase in USP phosphorylation at the early wandering stage that elicits signaling cascades. For example, in Manduca prothoracic gland, approximate 5–10% of total S6 protein was in the phosphorylated form under nonstimulated experimental conditions (Song and Gilbert, 1995). An increase in the phosphorylated S6 to approximately 20% of total

Fig. 5. Western blot analysis of USP in the salivary glands of early wandering (EW) and late wandering (LW) third instar larvae and 0– 6 h old prepupae (PP0–6) of D. melanogaster. Arrows indicate the position of different forms of USP protein detected by AB11 USP mAb.

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Fig. 6. Western blot analysis of USP expression in the 20E-incubated salivary glands of ecdysone-deffecient wocrgl mutant of D. melanogaster. (a) Dose response of USP expression in the salivary glands incubated for 12 h in vitro with the indicated concentrations of 20E. (b) Temporal response of USP expression to 20E incubation (0.01 µM).

S6 protein in the first 15 min after PTTH stimulation is sufficient to stimulate ecdysteroidogenesis. The maximum ecdysteroid synthesis rate occurred when about 20–30% of S6 was in the phosphorylated form. The small shift from the nonphosphorylated to phosphorylated USP in response to 20E stimulation may be sufficient to activate the EcR/USP complex, resulting in subsequent transcriptional events that lead to molting and metamorphosis. The response of USP expression and phosphorylation to 20E challenge in early wandering third instar glands of wild type larvae was not sensitive since the glands from this stage have already been exposed to a high titer of ecdysteroids (data not shown). Therefore, the ecdysone-deficient wocrgl mutant larvae were used for the dose response study since the glands from the wocrgl mutant have not been exposed to a high titer of hemolymph ecdysteroids (Wismar et al., 2000), and thus are sensitive to a 20E challenge. Functional studies utilizing other steroid hormone and retinoid receptors have revealed that the thyroid hormone, retinoid, vitamin D, and other orphan receptors appear to be specifically and tightly bound to the chromatin at specific sites in the DNA in the absence of ligand (Henrich et al., 1999). These proteins typically form heterodimers with RXRs. Ligand and DNA binding may cause dissociation of repressor molecules, allowing association with general transcription factors, other DNA-binding proteins and/or co-activitors, thus resulting in transcription of the target genes. Since the EcR/USP complex is the insect homolog of the vertebrate RAR/RXR, the same might be true for the EcR/USP complex, it is possible that phosphorylation of USP or EcR elicited by ligand removes the suppressor(s) from the receptor complex, and thus activates the transcriptional cascade.

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Even in the ecdysone-deficient wocrgl mutant, about 40–50% of USP was phosphorylated, indicating that USP phosphorylation is also regulated independently by ligand, perhaps by other unknown signaling pathways. Multiple putative phosphorylation sites for both CKII and PKC suggest multiple levels of regulation by independent pathways. The present study has shown that a single USP protein exists in both the phosphorylated (p56) and nonphosphorylated (p54) forms in Drosophila tissues and that the expression and phosphorylation of the p54 is regulated by 20E. Thus, USP phosphorylation, in addition to USP expression, provides another layer of regulation for USP function. Although 20E has been shown to regulate the in vitro expression of the nonphosphorylated USP in the present study, we do not know whether the increased expression of USP by 20E is due to increase in USP translation or to inhibition in USP degradation or to the combination of both. For the phosphorylated USP, it is possible that the 20E-elicited increase in USP phosphorylation may be due to the direct activation of protein kinases such as PKC and CKII or to the inhibition of protein phosphatase(s). Nevertheless, to understand the role of USP phosphorylation in mediating the function of the EcR/USP complex or the putative function of USP as a JH receptor (Jones and Sharp, 1997), it is critical to identify the in vivo phosphorylation sites of USP and signal transduction pathways that regulate USP phosphorylation. Such investigations are currently underway.

Acknowledgement This research was supported by the grant (C1533026) from the University of Missouri Research Board.

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