A locust DNA-binding protein involved in gene regulation by juvenile hormone

Molecular and Cellular Endocrinology 190 (2002) 177– 185 www.elsevier.com/locate/mce

A locust DNA-binding protein involved in gene regulation by juvenile hormone S. Zhou a, J. Zhang a, M. Hirai a,b, Y. Chinzei b, H. Kayser c, G.R. Wyatt a, V.K. Walker a,* a b

Department of Biology, Queen’s Uni6ersity, Kingston, Ont., Canada K7L 3N6 Department of Medical Zoology, Mie Uni6ersity School of Medicine, Tsu, Japan c No6artis Crop Protection AG, Basle, Switzerland Received 28 June 2001; accepted 12 July 2001

Abstract Although juvenile hormone (JH) has essential roles in insect development and reproduction, the molecular mechanisms of gene regulation by JH remain an enigma. In Locusta migratoria, the partially palindromic 15-nt sequence, GAGGTTCGAGA/TCCTT/C, found upstream of a JH-induced gene, jhp21, was designated as a putative juvenile hormone response element (JHRE). When JH-deprived adult female locusts were treated with the active JH analog, methoprene, a fat body nuclear factor that bound specifically to JHRE appeared after 24 h. Binding exhibited a preference for an inverted repeat with GAGGTTC in the left half-site, a single nucleotide spacer, and a right half-site in which some variation is acceptable. Binding to JHRE was abolished by phosphorylation catalyzed by a C-type protein kinase present in the nuclear extracts. The DNA-binding protein is thus believed to be a transcription factor, which is brought to an active state through the action of JH and then participates in the regulation of certain JH-dependent genes. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: DNA-binding protein; Locust; Juvenile hormone; Hormone-response element; Nuclear factor; Protein kinase C

1. Introduction Despite the elucidation of molecular mechanisms of action of many hormones during recent years, the primary modes of action of the juvenile hormone (JH) of insects remain obscure (Jones, 1995; Davey, 2000). In premetamorphic insects, where JH maintains the larval state by modifying the cellular responses to the moulting hormone, 20-hydroxyecdysone, some effects of JH on the ecdysone-induced gene expression cascade have been identified (Zhou et al., 1998; Zhou and Riddiford, 2001). In adult insects, where JH acts in many species independently of ecdysone to stimulate various processes required for reproduction, two distinct modes of action appear to be used (Wyatt and Davey, 1996). One mode of JH action, seen in the ovarian follicular epithelium, utilizes a membrane re* Corresponding author. Tel.: +1-613-533-6123; fax: + 1-613-5336617. E-mail address: [email protected] (V.K. Walker).

ceptor and protein kinase C (PKC) to bring about enzyme activation without the need for new transcription or protein synthesis (Sevala and Davey, 1989). In the fat body and several other tissues, however, JH acts via alteration of gene expression (Wyatt, 1997). The gene-level responses to JH in both larval and adult insects likely involve a nuclear hormone receptor acting directly on the genome, as has been established for ecdysteroids in insects, as well as for steroidal hormones, thyroid hormones and retinoids in vertebrates. Despite extensive efforts, however, no nuclear receptor for JH has yet been firmly identified (Engelmann, 1995; Charles et al., 1996; Jones and Sharp, 1997; Feyereisen, 1998; Davey, 2000; Belle´s, 2002). In the fat body of the African migratory locust, Locusta migratoria, JH and JH analogs (JHA) induce the transcription of two vitellogenin genes (Vg) with a lag time of 12–24 h (Glinka and Wyatt, 1996). The lag can be lengthened by temporary inhibition of protein synthesis with cycloheximide (Edwards et al., 1993) or shortened by ‘priming’ by the prior application of a

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dose of JH or JHA that is, by itself, too low to induce vitellogenin production (Wyatt et al., 1996). Similar observations have been reported in the cockroach, Blattella germanica, where Vg mRNA is detected as early as 2 h after application of JHA: Vg transcription is blocked by cycloheximide and accelerated by priming with a subthreshold dose of JHA (Comas et al., 1999, 2001). These observations show that JH initially activates a gene or genes whose protein product(s) are required for transcription of the eventual target genes such as Vg. The priming experiments, however, indicate that stimulation by JH is required at least twice in the sequence of events, and the second JH action could be directly upon Vg. The Vgs of amphibia and birds, which are homologous with those of insects, are directly activated by estrogen-receptor complexes (Slater et al., 1989; Nardulli et al., 1996). In the mosquito, Aedes aegypti, the regulation of Vg transcription by 20-hydroxyecdysone has recently been shown to involve both the direct binding of the receptor heterodimer EcR-USP to a DNA response element and synergistic action of other hormone-induced transcription factors (TFs) (Martin et al., 2001). In different taxonomic groups, conservation of a Vg activation mechanism, with substitution of group-specific hormones, might be expected. The priming effect in Locusta, then, would involve the synthesis of one or more proteins needed as cofactors for the hormonal activation of the target genes. The locust hemolymph protein gene, jhp21, is induced by JH in adult female fat body coordinately with the Vgs, though to a lower level of expression. Studies with jhp21 have detected a nuclear protein, whose production depends on JH, which appears to be involved in transcriptional activation (Zhang et al., 1993, 1996). Faithful transcription from the jhp21 promoter was obtained in nuclear extracts from JH-exposed, but not from JH-deprived, fat body, whereas both extracts transcribed from a control, non-selective promoter. Upstream from jhp21 there are three copies of the partially palindromic 15-nucleotide sequence, GAGGTTCGAGA/TCCTT/C. As imperfect palindromes containing the motif AGGT, these resemble known hormone response elements, and particularly the canonical sequence, IR-1 (inverted repeat with one spacer; AGGTCAnTGACCT), known as a response element for ecdysteroids (Antoniewski et al., 1996; Wang et al., 1998). In the JH-activated nuclear extracts, specific transcription was greatly diminished by deletion of the region containing this sequence and restored by addition of two tandem copies of the 15-nucleotide element. This sequence is therefore regarded as a putative juvenile hormone response element (JHRE). Electrophoretic band-shift studies with fat body nuclear extracts revealed a protein that binds to the putative JHRE, and this is regarded as a specific TF, possibly a

JH receptor (Zhang et al., 1996; Zhang and Wyatt, 1996).

2. Materials and methods

2.1. Animals Locusts were reared in the gregarious phase as previously described (Chinzei et al., 1982). Besides the standard regimen of dry diet plus wheat seedlings, sliced carrots were continuously supplemented in the cages to increase the size of fat body and the production of active nuclear extracts (Wyatt et al., 1996). JH-deprived female adult locusts were obtained by inactivation of the corpora allata with 500 mg ethoxyprecocene (Sigma, St. Louis, MO) per locust within 12 h after eclosion. To replace JH activity, the JHA, methoprene (Zoecon Corp., Palo Alto, CA) was topically applied to the locusts (150 mg per locust in acetone) 10 d after precocene treatment.

2.2. Fat-body nuclear extracts Nuclei were isolated from locust fat body (precocene treated as well as 2, 6, 12, 24 and 48 h after methoprene application) and proteins were extracted and precipitated by the procedure developed for cell-free transcription (Zhang et al., 1996). Fat body from about 100 locusts was disrupted in homogenization buffer (10 mM HEPES, pH 7.6, 12 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2 M sucrose, 5% glycerol, 1% skim milk, 0.5 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, and 1 mg/ml aprotinin), layered over cushion buffer (homogenization buffer without skim milk) and centrifuged at 10000×g for 60 min at 4 °C. The pellets were suspended in lysis buffer (10 mM HEPES, pH 7.6, 100 mM KCl, 0.1 mM EDTA, 10% glycerol, 3 mM MgCl2, 1 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, and 1 mg/ml aprotinin) with 0.4 M (NH4)2SO4, pH 8.0. After incubation for 30 min on ice, the lysate was centrifuged for 60 min at 14000×g, 4 °C. The crude nuclear proteins were precipitated by dissolving (NH4)2SO4 (0.3 g/ml) and incubated for 30 min on ice. After centrifugation at 14000× g for 30 min at 4 °C, the precipitate was dissolved in dialysis buffer (25 mM HEPES, pH 7.6, 0.1 mM EDTA, 40 mM KCl, 10% glycerol, and 1 mM DTT) and dialyzed 2×2 h through a Slide-ALyzer cassette (Pierce Inc., Rockford, IL). To test the effect of added JH or JHA, nuclear protein preparations were preincubated at 4 °C for 10 min with JH III, the active analog methoprene or methyl farnesoate (a hormonally-inactive analog) at concentrations of 0.1, 1.0 and 10 mM in 0.2% ethanol,

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or with 0.2% ethanol alone, before addition of the DNA probe.

2.3. Electrophoretic mobility shift assay Synthesized oligonucleotide probes (Table 1) were end-labeled with g-32P-ATP or a-32P-dCTP (Amersham Pharmacia Biotech) with the T4 polynucleotide kinase or Klenow fragment of DNA polymerase I (MBI Fermentas) to a specific activity of 1– 2 ×104 cpm/fmol. Additional oligonucleotides (designated as M6 in Table 1) were designed to test the possibility that binding differences among the initial set might be due to variations in the terminal overhang sequences. For each reaction, 5–10 mg (5 mg in all the shown figures) of nuclear proteins and about 0.1 pmol of labeled probe were incubated for 30 min at room temperature in the electrophoretic mobility shift assay (EMSA) buffer, containing 50 ng/ml poly(dI-dC), 25 mM HEPES (pH 7.6), 5 mM MgCl2, 34 mM KCl and 10% (v/v) glycerol. For the competition experiments, unlabeled competitor oligonucleotides at 2-, 5-, 10-, and 20-fold molar excess were added to the binding reactions at the same time as the labeled JHRE. In the supershift assays, antiserum to Drosophila USP (mAB11; Khoury Christianson et Table 1 Coding strand sequences of synthetic oligonucleotide duplexes used as probes in the gel mobility shift assays

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al., 1992) was preincubated with the nuclear extracts at 4 °C for 30 min. The complexes were resolved using 5% native polyacrylamide gels in 1× TBE buffer at 35 mA, 4 °C. Gels were dried, and bands were visualized either by autoradiography or by PhosphorImaging for quantitative analysis using ImageQuant™ software (Molecular Dynamics).

2.4. Protein phosphorylation and dephosphorylation For phosphorylation, nuclear extracts were supplemented with ATP to a final concentration of 1 mM and incubated at 4 or 22 °C for 5–20 min. For phosphatase treatment, the mixtures were further incubated with 0.5, 1.0 or 2.0 U calf intestinal alkaline phosphatase (CIAP, MBI Fermentas) at 4 °C for 10 min. To determine the involvement of the specific kinases, a serine/threonine kinase inhibitor set (Calbiochem, San Diego, CA) was used. Fat body nuclear extracts were incubated with the kinase inhibitors (10 or 500 mM depending on the inhibitor) or 1% DMSO for 30 min at 4 °C, and then with added 1 mM ATP at 22 °C for 15 min. In the PKC assay (Kikkawa et al., 1982), the nuclear extracts were preincubated at 30 °C for 5–15 min with 1 mM ATP plus 40 mg/ml phosphatidylserine, 8 mg/ml diolein, 20 mM Tris (pH 7.5), 0.5 mM CaCl2, and 5 mM magnesium acetate before carrying out the binding reaction and analysis by EMSA as described above.

3. Results

3.1. Binding specificity of the nuclear protein

The putative JHRE and mutants (M1–M8) are shown; M8 corresponds to IR-1. Underlined letters indicate mutated positions, boxes indicate deletions and inverted repeats are in italics. The central (‘spacer’) nucleotide is shown in bold. Small letters indicate extensions used in labeling reactions. The second set of oligonucleotides (M4– M8) was synthesized to ensure that differences in binding did not result from different extension sequences.

The specificity of the nucleotide sequence to which TF binds was studied with synthetic oligonucleotides containing the native JHRE and a set of mutants (Table 1). Since, in the native JHRE, five out of seven nucleotides each side of the central G are palindromic, the sequences are regarded as modified inverted repeats consisting of two half-sites with a single nucleotide spacer. Each probe was labeled with 32P and tested in the binding assay (Fig. 1(a)); the identity of TF was confirmed by competition with unlabelled probe. An experiment using mutant oligonucleotides with terminal overhang sequences the same as in the JHRE construct (M4 –M8 in Table 1) gave identical results. In another set of experiments, unlabeled probes (JHRE and the mutant set) were tested at 2, 5, 10 and 20-fold molar excess for competition with 32P-JHRE for binding. A 20-fold molar excess of unlabeled JHRE was needed to eliminate the specific TF band completely, and the results using this level of mutant competitors are shown in Fig. 1(b). Mutants M1 and M2, in which two bases in each half-site were altered, did not bind to TF and showed

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competition, and M5, with the left site switched to the canonical AGGTCA and the right site unaltered, showed reduced binding but strong competition. M8, an inverted repeat of the canonical element AGGTCA, showed no binding and partial competition. These results indicated that the intact AGGTTC left half-site is required for optimal binding but modification in the left half-site, including substitution by the canonical sequence AGGTCA, may produce binding with reduced affinity. M4, in which the left half-site was unaltered while the right site was replaced with a direct repeat of the left half-site sequence, showed moderate binding and competition, while M6, with an intact left site and a single base change in the right site bound and competed strongly. This shows that the right site, while tolerating more variation than the left, does influence binding affinity. The weak binding and competition by M7, with both half-sites intact but the central spacer deleted, shows the importance of the spacer. A set of mutants all having identical terminal overhang sequences (Table 1, M6 ) showed the same specificity in binding and competition (not shown) as did the original set, confirming that the observed differences were due to the internal changes and not the terminal sequences. In summary, the data indicate that the protein binding site exhibits preference for an inverted repeat with GAGGTTC in the left half-site, a single nucleotide spacer, and a right half-site that contributes to binding affinity but tolerates some variation.

3.2. Time-course of induction of the DNA-binding protein by a ju6enile hormone analog

Fig. 1. Oligonucleotide sequence specificity for binding of TF. Gel mobility shift assays (shown in gels representative of seven separate experiments) were performed as described in Section 2 using the putative JHRE (J) or mutant oligonucleotides (M1 –M8) listed in Table 1 with nuclear extracts from locusts deprived of JH and treated with methoprene for 48 h. (a) Interactions of 32P-labeled JHRE and mutant oligonucleotides with proteins in the nuclear extracts; the specific TF band, identified by competition with unlabelled JHRE (J+ J), is indicated by an arrow. (b) Competition experiments using 32 P-labeled JHRE together with increasing amounts (2-, 5-, 10- and 20-fold molar excess) of unlabeled JHRE (J) and mutant oligonucleotides (M1 – M8) in the binding reactions were performed. Results using 20-fold molar excess of competitor are shown.

no significant competition with JHRE. M3, where two nucleotides were deleted from the left half-site and central position while the right half-site was unaltered, showed no direct binding but some activity in

The time-course for the appearance of TF was examined in fat body extracts prepared at different times after administration of the JHA, methoprene, to groups of precocene-treated locusts. Several experiments all gave consistent results with respect to TF, which was identified by absence of binding to the mutant probe M1 (Fig. 2(a)). TF was not seen at 2 or 6 h, but appeared faintly at 24 h and strongly at 48 h after methoprene treatment (Fig. 2(a) and (b)). Before the appearance of TF, beginning as early as 2 h, there was a reduction in intensity of a faster-moving non-specific band seen in the precocene-treated locusts, and transient intensification of at least one band of intermediate mobility. While these data do not establish precursor-product relationships, they suggest that the production of TF may involve modification, possibly including subunit association, from pre-existing protein(s), resulting in formation of a specific binding site.

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3.3. Relation of transcription factor to hormone receptors Since USP is a heterodimer partner of EcR (Yao et al., 1993) and has been proposed as a JH receptor (Jones and Sharp, 1997), we tested for the presence of USP in TF by the use of USP antibody. Addition of USP antibody to the nuclear extract did not diminish the TF band and produced only an extremely faint, slow-moving extra band (not shown). In control experiments, when IR-1 was used as the probe, however, anti-USP produced a distinct, supershifted band while a diffuse band with a faster migration than TF was eliminated. These results indicate that, although the nuclear extract contained another protein complex that includes USP and binds to IR-1, USP is not a component of TF. It was possible that TF is a JH receptor, and thus it was of interest to determine whether JH or an active analog, preincubated with the nuclear protein preparations, would influence the binding to the JHRE. In a series of experiments, however, neither JH III nor methoprene significantly increased the intensity of the TF band using nuclear protein extracts

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from either normal mature female locusts (P\0.05, n= 6) or JH-deprived locusts induced with methoprene (P\ 0.05, n= 6).

3.4. Phosphorylation of transcription factor Protein phosphorylation is important in regulating the functions of many hormone receptors (Weigel, 1996). To investigate this possibility for TF, nuclear protein preparations were incubated in EMSA buffer with ATP at 1 mM or less before addition of the labeled JHRE for band-shift analysis. This treatment was found to abolish the specific TF band (Fig. 3(a)), while there appeared to be a transient intensification of faster-moving bands that may correspond to those observed in the time-course experiment described above (Fig. 2). This indicated that phosphorylation by a kinase present in the nuclear extract blocked the DNA-binding activity. We therefore tested the effect of subsequent dephosphorylation by the addition of calf intestinal alkaline phosphatase to the ATPtreated preparations. This treatment restored the specific TF band (Fig. 3(b)). The same conditions, however, did not result in the appearance of the TF

Fig. 2. Time-course of changes in proteins that bind to the putative JHRE in fat body nuclear extracts after application of a JHA to locusts. Extracts were from locusts deprived of JH by precocene (P) application, then treated with methoprene and taken for analysis after 2 – 48 h. Gel mobility shift assays were performed as described in Section 2. (a) Probes were 32P-labeled JHRE or the M1 mutant oligonucleotide (Table 1) which did not bind to the specific TF. (b) The probe was JHRE, and a control (C) using bovine serum albumin instead of nuclear extract was included. These gels are representative of six experiments; while there was some variation in the faster-migrating bands, the appearance of the specific TF complex after methoprene treatment was consistent.

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Fig. 3. The phosphorylation state affects the appearance of the TF band in gel mobility assays with the putative JHRE. Fat body nuclear extracts were prepared from locusts treated with methoprene for 48 h. (a) Protein extracts were incubated with 1 mM ATP at 4 or 22 °C for 5 –20 min (as shown) prior to use in gel mobility shift assays. Controls were incubated without added ATP (-ATP). The TF band is indicated with an arrow. (b) Protein extracts incubated with 1 mM ATP at 22 °C for 15 min (as above) were further incubated at 4 °C with 0.5 U calf intestinal alkaline phosphatase (CIAP) for 10 min before analysis (lane 4). Control extracts were not treated with either ATP or phosphatase (lane 1), treated with ATP only (lane 2), or treated with ATP and subjected to a further 4 °C incubation for 10 min (lane 3). TF is indicated with an arrow. Both (a) and (b) gels were repeated.

band in extracts from precocene-treated controls, nor in enhancement of the band from females that had been exposed to methoprene for 24 h (not shown). To determine the class of kinase responsible for the phosphorylation of TF, several selective inhibitors, including inhibitors of PKC, protein kinase A, Ca2 + / CaM kinase II, myosin light chain kinase and protein G kinase were incubated with the nuclear protein preparations before the addition of ATP. Of these, only staurosporine (a broad-range serine/threonine kinase inhibitor with a preference for PKC) and bisindolylmaleimide I (a specific PKC inhibitor) reduced the loss of the specific band (Fig. 4(a)). Since these results indicate the involvement of a C-type protein kinase, we tested incubation conditions favorable for activating this class of kinase, including the presence of Ca2 + , diolein and phosphatidylserine. Under these conditions the reaction was clearly accelerated (Fig. 4(b)). We conclude that a PKC in the locust nuclear extracts is involved in phosphorylation of TF, which binds to the specific DNA sequence in a dephosphorylated but not in a phosphorylated state.

4. Discussion Our electrophoretic band-shift experiments using protein extract from locust fat body nuclei have examined a specific DNA-binding protein that is present in the tissue activated for synthesis of yolk proteins by JH or an analog but is lacking in JH-deprived tissue. A DNA-binding protein associated with the capacity to transcribe the JH-responsive genes VgA and jhp21 is likely to be a TF and it has been provisionally designated as such (Zhang et al., 1996). Since the sequence element GAGGTTCGAGA/TCCTT/C, which is reiterated three times upstream from the jhp21 coding region, enhanced transcription of the gene in cell-free extracts, it is regarded as a regulatory element, a putative JHRE (Zhang and Wyatt, 1996). Using a set of mutant oligonucleotide probes, we have now examined the target sequence specificity of TF. We found that a partially palindromic element with GAGGTTC in the left half-site and a single nucleotide spacer, corresponding to the native JHRE, showed optimal binding. Replacing the left half-site with the

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sequence AGGTCA, found in many hormone response elements (reviews: Beato et al., 1987; Qiu et al., 1996), reduced binding affinity. There was some tolerance for variation in the right half-site. Certainly, TF does not appear to share the binding characteristics of Aedes aegypti USP, which shows a broad specificity and forms complexes with both direct and inverted repeats of AGGTCA, separated with spacers of varying lengths (Wang et al., 1998). The canonical element IR-1, which has been identified as a response element for ecdysteroids and the mammalian farnesoid receptor FXR (Laffitte et al., 2000), bound the locust TF only weakly. Use of USP antibody, however, showed that other proteins in the same locust nuclear extract, including locust USP (Hayward et al., 1999), bound to IR-1. These results clearly distinguish JHRE from the response elements important for ecdysteroid-regulated gene expression. The tetranucleotide, GTTC, makes up 4/7 of the sequence in the JHRE left half-site but is not part of IR-1. It is also present in putative regulatory elements of the locust Vgs and a cockroach (Periplaneta) oothecin gene

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(Wyatt, 1991; Pau et al., 1987), and might be proposed as selective for the response to JH. The mutant oligonucleotides M4 and M6, which include this motif, bound to JHRE more strongly than M5, which does not, suggesting that it contributes to binding affinity. This motif, however, is also present in the hsp27 ecdysteroid response element (Riddihough and Pelham, 1987) and is not found in putative JH response elements upstream from a second oothecin gene or a cockroach (Blattella) Vg (Belle´ s, 2002), so that it cannot be exclusively related to JH responsiveness. In some systems, the addition of ligand enhances the binding of nuclear receptor proteins to their response elements (Thompson et al., 1998; Wang et al., 2000), although this effect can be modest and time-sensitive (Elke et al., 1999). Since it has been proposed that TF could be a JH receptor (Zhang et al., 1996), it was of interest to determine the effect of added hormone on the interaction of TF with its DNA binding site. When JH or the analogue was added to locust extracts there was neither a significant enhancement nor a diminution of

Fig. 4. The phosphorylation of TF appears to be a function of PKC. (a) Extracts from JHA-exposed locusts were treated with selected protein kinase inhibitors at 4 °C for 30 min prior to addition of 1 mM ATP and incubation at 22 °C for 15 min (lanes 3 – 8). Control extracts were not treated with ATP or kinase inhibitors (lane 1), treated with ATP alone (lane 2), or treated with ATP and the inhibitor vector, 1% dimethylsulfoxide (lane 9). Kinase inhibitors used were: 10 mM staurosporine, a broad-range kinase inhibitor with a preference for PKC (lane 3), 10 mM bisindolylmaleimide, a PKC inhibitor (lane 4), 10 mM H-89, a protein kinase A inhibitor (lane 5), 10 mM KN-93, a calmodulin kinase II inhibitor (lane 6), 10 mM ML-7, a myosin light chain kinase inhibitor (lane 7) and 500 mM protein kinase G inhibitor (lane 8). The specific TF band is indicated with an arrow. (b) Stimulation of endogenous PKC is shown by the addition of activators to nuclear extracts. After incubating extracts with 1 mM ATP at 30 °C in the presence or absence of a PKC activation cocktail (PKCA) containing phosphatidylserine, diolein and Ca + 2 (Section 2) for 5, 10 or 15 min, gel mobility shift assays were done in duplicate. Controls were either not treated with ATP or were treated with cocktail alone. TF is shown with an arrow.

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band intensity. The presence of hormone, therefore, either does not increase the affinity of TF with JHRE or, perhaps less likely, the putative JH receptor extracted from JH-stimulated locusts was saturated and therefore failed to show response to added ligand. TF was detected by band-shift assay in fat body nuclear extracts about 24 h after administration of an active JHA to JH-deprived locusts, and gave a strong band at 48 h. Before the appearance of TF, there was reduction in a higher-mobility protein band and transient changes in intermediate bands that bound to a mutant probe as well as to the JHRE. Perhaps then, after hormone application, pre-existing proteins associate to form the active TF with a specific DNA-binding site. Incubation of the active extracts with ATP resulted in the disappearance of TF and produced an electrophoretic picture similar to that seen with JH-deprived extracts. Subsequent treatment with phosphatase restored the TF band, suggesting that the intracellular changes set in motion by JH may include the dephosphorylation of precursor proteins. This would contrast with the observations that 20-hydroxyecdysone initiates the phosphorylation of a USP isoform during tobacco hornworm (Manduca sexta) moulting and metamorphosis (Song and Gilbert, 1998) and rapidly increases the phosphorylation of USP in the beetle, Tenebrio molitor (Nicolai et al., 2000), and would accord with the distinct and sometimes opposing roles of the two hormones. In the locust, however, dephosphorylation of pre-existing nuclear binding protein is not the sole requirement for JHRE binding, since phosphatase addition to nuclear extracts obtained from JH-deficient females did not result in the appearance or enhancement of the TF band. Indeed, our previous experiments with cycloheximide and priming with low doses of JH showed that protein synthesis is a prerequisite for JH activation of Vg transcription. It is of interest that the capacity of TF to bind to JHRE is abolished by action of PKC, since this class of kinase has central roles in signal transfer for cellular regulation by many membrane-acting hormones and other stimuli (review: Jaken and Parker, 2000). There is also evidence for interactions between the membrane signal transduction and nuclear transcriptional pathways of cellular regulation. For example, in vitro phosphorylation of a retinoic acid receptor by PKC was shown to diminish its ability to bind to its DNA response element, even though in intact cells PKC activity was required for retinoic acid-dependent transcription from an inducible promoter (Tahayato et al., 1993). Among insects, PKC is involved in the action of JH on putative membrane receptors on kissing bug (Rhodnius prolixus) ovarian follicle cells (Sevala and Davey, 1989). In locust fat body, it is possible that PKC-catalyzed inactivation of TF has a role in the repression of Vg (and presumably jhp21 ) transcription that accompanies the end of the vitellogenic

cycle (Glinka and Wyatt, 1996). This might be an aspect of the action of the decapeptide adipokinetic hormone, which has been shown to repress Vg synthesis at this stage (Moshitzky and Applebaum, 1990; Glinka et al., 1994). It is likely that the adipokinetic hormone affects gene transcription since the closely related hypertrehalosemic hormone of Blattella has recently been found to repress the fat body level of Vg mRNA (Comas et al., 2001). We suggest here that JH stimulates the production of a fat body TF possibly through both transcriptional and post-transcriptional regulation. The activity of this factor could then be negatively regulated by PKC, so that when phosphorylated after a transduction signal, it disassociates from a protein complex and loses affinity for JHRE. The phosphorylation state of TF may be critical to reproductive maturation and the subsequent oviposition rhythm. This model proposes too that JH regulation may involve ‘cross-talk’ between regulation mediated by cell surface and nuclear receptors, contributing to the complexity and challenge of understanding the molecular basis of JH action.

Acknowledgements We thank Dr T.S. Dhadialla (Rohm & Haas Inc.) for bacterially expressed USP. S.Z. is partially supported by a Queen’s Graduate Fellowship and M.H. was supported by a Canada–Japan exchange grant to Y.C. This work was supported by an NSERC of Canada Research Partnership Program grant with Novartis Crop Protection AG and Novartis Crop Protection Canada Inc. to GRW and VKW, and by an NSERC grant to VKW.

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