PKC-mediated USP phosphorylation is required for 20E-induced gene expression in the salivary glands of Drosophila melanogaster.код для вставкиСкачать
116 Sun and Song Archives of Insect Biochemistry and Physiology 62:116�7 (2006) PKC-Mediated USP Phosphorylation Is Required for 20E-Induced Gene Expression in the Salivary Glands of Drosophila melanogaster 1 Xiaoping Sun 2 and Qisheng Song * Ecdysone receptor (EcR) and its heterodimer, ultraspiracle protein (USP), are ligand-dependent transcriptional factors that mediate the action of molting hormone 20-hydroxyecdysone. The activities of transcriptional factors are subjected to regulation not only by transcriptional/translational mechanisms, but also by posttranslational mechanisms such as phosphorylation. Protein kinase consensus recognition sequence analysis of Drosophila EcR and USP reveals multiple phosphorylation sites for protein kinase C (PKC) and casein kinase II (CKII) on EcR and USP sequence. By using specific protein kinase inhibitors, we have shown that PKC, not CKII, is responsible for USP phosphorylation. Inhibition of PKC activity by protein kinase inhibitors blocked USP phosphorylation, resulting in inhibition of 20E-induced gene expression at both transcriptional and translational levels. The composite data suggest that PKC-mediated USP phosphorylation is required for 20E-induced gene expression in the salivary glands of Drosophila melanogaster. Arch Insect Biochem Physiol 62:116�7, 2006. � 2006 Wiley-Liss, Inc. KEYWORDS : nuclear receptor; transcriptional and translational expression; protein kinase; inhibitor INTRODUCTION The action of 20E is mediated by ecdysone receptor (EcR). Biochemical and molecular studies Insect steroid hormones, mainly 20-hydroxy- have demonstrated that heterodimerization be- ecdysone (20E), trigger and coordinate the molting tween EcR and its heterodimer partner ultraspiracle and metamorphosis of insects at specific times dur- protein (USP) is required for ligand- and DNA- ing the insect life cycle (Riddiford, 1993). 20E di- binding and for gene transcription (Koelle, 1992; rectly induces a small set of early genes including Yao et al., 1992, 1993; Thomas et al., 1993). Genes the Broad-Complex (BR-C), E74 and E75 (Burtis et for both EcR and USP have been cloned and se- al., 1990; Segraves and Hogness, 1990; DiBello et quenced from Drosophila (Koelle et al., 1991; al., 1991). These genes encode transcription factors Henrich et al., 1990; Oro et al., 1990; Shea et al., that transduce and amplify the hormonal signal by 1990), and subsequently from several other dipteran regulating a large set of late secondary-response and lepidopteran species (Cherbas and Cherbas, genes (Ashburner et al., 1974; Urness and Thummel, 1996; Henrich et al., 1999) as well as from other 1995; Crossgrove et al., 1996). It is the stage- and arthropods such as the ixodid tick Amblyomma tissue-specific activation of these target genes that americanum (Guo et al., 1997) and the fiddler crab directs insect molting and metamorphosis. Uca pugilator (Durica et al., 2002). 1 2 Division of Biological Sciences, University of Missouri, Columbia, Missouri Division of Plant Sciences, University of Missouri, Columbia, Missouri Presented at the XXII International Congress of Entomology in a Symposium entitled 揑nsect Signal Transduction Systems: Current Knowledge and Future Directions,� Brisbane, Australia, 2004. *Correspondence to: Dr. Qisheng Song, Division of Plant Sciences, 1-31 Agriculture Building, University of Missouri, Columbia, MO 65211. E-mail: SongQ@missouri.edu � 2006 Wiley-Liss, Inc. DOI: 10.1002/arch.20130 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology July 2006 doi: 10.1002/arch. USP Phosphorylation and 20E-Induced Gene Expression In Drosophila, EcR encodes three ecdysone re- progesterone relative to its 117 unphosphorylated ceptor isoforms, EcR-A, EcR-B1, and EcR-B2, through counterpart. In addition, PR activity is highest in alternative splicing (Talbot et al., 1993). These three the S phase and lower in the G0/G1 phases of the isoforms differ only in their N-terminal A/B do- cell cycle, but impaired during the G0/M concomi- main, implying that these sequences are respon- tant with lowered PR phosphorylation. Thus, post- sible for directing specific physiological responses translational modification serves as an important to receptor mechanism for modulating the structure, activity, isoforms may explain partially how a single hor- and lifetime of many proteins including receptors 20E. Existence of three ecdysone mone drives such a variety of tissue- and stage-spe- and enzymes. It is the phosphorylation resulting cific responses. Indeed, it has been shown that from hormonal signal that activates the function expression of these three EcR isoforms is differen- of nuclear receptors. tially regulated according to the developmental fate In insects, both EcR and USP have been dem- of larval and adult structures (Talbot et al., 1993; onstrated to be phospho-proteins (Song and Gil- Robinow et al., 1993; Truman et al., 1994). Ge- bert, 1998; Song et al., 2003; Rauch et al., 1998; netic analyses using isoform-specific mutations Nicola� et al., 2000) and EcR and USP phosphory- have provided direct evidence that EcR isoforms lation is regulated by 20E. High levels of EcR and are functionally distinct (Bender et al., 1997; USP phosphorylation in the absence of 20E in both Schubiger et al., 1998). By comparison with EcR抯 Manduca sexta and Drosophila melanogaster (Song complexity, usp encodes a single USP protein that and Gilbert, 1998; Song et al., 2003) suggest that is the Drosophila homolog of the vertebrate retin- their phosphorylation is perhaps also regulated by oid X receptors (RXRs) (Henrich et al., 1990; Oro ligand-independent signaling pathways as reported et al., 1990; Shea et al., 1990). It is the hetero- in vertebrate systems (Weigel, 1996; Lange 2004). dimerization between USP and each of the three isoforms of EcR that provides a mechanism for achieving transcriptional diversity and tissue- and stage-specific response to 20E stimulation. Both EcR and USP are members of the nuclear receptor superfamily and are ligand-activated transcriptional factors. The activities of nuclear receptors are subjected to regulation not only by transcriptional/translational mechanisms, but also by posttranslational mechanisms such as phosphorylation. Studies of other transcription factors in vertebrates have shown that phosphorylation can play roles in nuclear translocation, DNA binding, interactions with other proteins, and transactivation. Transcription factors are frequently phosphorylated at multiple sites and phosphorylation is generally accepted as a positive regulator of steroid receptor Phosphorylation of EcR and USP plays roles in mediating the ligand- and perhaps also DNA-binding activity in the prothoracic glands of M. sexta. 20E-induced expression and phosphorylation of a specific isoform of USP in the Manduca prothoracic glands have been associated with changes in ecdysteroidogenic activity both in vivo and in vitro (Song and Gilbert, 1998), suggesting a feedback mechanism by which ecdysteroid synthesis is downregulated. Thus, phosphorylation provides another layer of mechanisms to regulate the function of the EcR/USP complex. Although EcR and USP are phosphoproteins (Song et al., 2003; Song and Gilbert, 1998; Rauch et al., 1998; Nicola� et al., 2000) and phosphorylation has been implicated in mediating the ligandand DNA-binding activities of the EcR/USP complex (Song and Gilbert, 1998), the effect of EcR function (Weigel, 1996; Lange, 2004). Phosphory- and USP phosphorylation on 20E-induced gene lation of steroid receptors may serve to integrate expression has not yet been explored. In the present signals initiated by growth factors with responses study, we investigated the effect of USP phospho- to steroid hormones in endocrine tissues. For ex- rylation on the expression of 20E-induced genes ample, phosphorylated progesteron receptors (PR) and proteins. Our results indicate that protein ki- are ultrasensitive to subphysiological levels of nase C (PKC)-mediated phosphorylation of USP Archives of Insect Biochemistry and Physiology July 2006 doi: 10.1002/arch. 118 Sun and Song mM 100 at both transcriptional and translational levels in arin, respectively, for the indicated time periods. the salivary glands of D. melanogaster. chelerythrine chloride or 10 mg/ml is required for the expression of 20E-induced genes hep- After incubation, salivary glands were collected in 1.5-ml Eppendorf tubes, homogenized on ice MATERIALS AND METHODS in phosphate-buffered saline (PBS) (136 mM NaCl, 1.1 mM K2HPO4, 2.7 mM KCl, 8.0 mM Na2HPO4, Insects pH 7.4) using a disposable pestle and centrifuged D. melanogaster (wild type ore ) was reared on ar- tificial blue diet (Fisher Scientific, Cat. No. 22315) � at 24 C under constant darkness. The early wandering third instar larvae was synchronized as described by Andres and Thummel (1994) and used � at 16,000g for 10 min at 4 C to remove debris. The protein concentration was determined using the BioRad protein bioassay kit (Reagent A: Catalog No.500-0113; Reagent B: catalog No.500-0114). To monitor the expression and phosphorylation of USP in the kinase inhibitor-treated salivary for tissue dissection and RNA extraction. glands, protein samples were subjected to SDSPAGE and Western blot analysis using AB11 USP- Reagents specific mAb as a probe as described previously Chemical reagents used for buffers, sample (Song et al., 2003). preparation, tissue culture, electrophoresis, and Western blot analysis were obtained from Sigma (St. Louis, MO), Fishers (Houston, TX), BioRad (Her- RT-PCR Analysis of 20E-Induced Gene Transcription in the PKC Inhibitor-Treated Glands cules, CA), and Pierce (Rockford, IL). The kits for RNA extraction, genomic DNA elimination, firststrand cDNA synthesis for RT-PCR, and some substrates were indicated specifically in the text. AB11 USP-specific monoclonal antibody (mAb) was provided by Dr. F. C. Kafatos (Harvard University). (a predetermined effective concentration) for 30 min and then stimulated with 0.5 mM 20E for the indicated time periods of 0, 6, 12, and 24 h, respectively. The control received no 20E stimulation. each treatment using Qiagen Rneasy� Mini Kit Protein Kinase Inhibitors (Catalog No. 74104) and RNA concentration was Salivary glands were dissected from early wandering stage under Ringer抯 solution (3.6 mM NaCl, 54.3 mM KCl, 8.0 mM CaCl2, and 28.3 mM MgCl2) and immediately placed in a 24-well tissue culture ml of Grace抯 insect tissue cul- ture medium. After dissection, the medium was carefully removed from the well, and replaced with 500 mM PKC inhibitor chelerythrine chloride After incubation, total RNA was insolated from Dose and Temporal Response of USP to plate containing 500 Salivary glands were dissected and incubated with100 ml of fresh Grace抯 medium containing the in- dicated concentrations of protein kinase inhibitors determined by measuring the absorbance at 260 nm in a spectrophotometer. RT-PCR was performed for estimation of gene expression levels in salivary glands. Prior to RT-PCR, the extracted total RNA was treated with RNase-free DNase I (Promega, Catalog No. M6101) to eliminate any potential genomic DNA contamination. The first-strand cDNA synthesis was primed using oligo (dT) based on the SupperScript� First-Strand synthesis kit (Invitrogen, Catalog No. 11904-018). About 1.5 mg (general protein kinase inhibitor H89 or PKC-spe- total RNA from each sample was used for the first- cific inhibitor chelerythrine chloride or CKII-spe- strand synthesis. The rp49 gene was used for nor- cific inhibitor heparin) and incubated for 6 h at malization of the compared templates. The PCR room temperature. Control group received Grace抯 cycle numbers for each gene were chosen empiri- medium only. For temporal response study, sali- cally to avoid saturation. The PCR cycle number vary glands were incubated with 200 mM H89 or was constant for a particular sequence in the mul- Archives of Insect Biochemistry and Physiology July 2006 doi: 10.1002/arch. USP Phosphorylation and 20E-Induced Gene Expression TABLE 1. Selected Primers for Ecdysone Response Genes RESULTS Forward primer Reverse primer cccgtcaggttctacatgct cggtttcttggtggtgttct E74A ccctttatcgacgatgcact tgtccattcgcttctcaatg E74B atgtgtccagctccagctct ccgttttggtactgctccag E75B cctcaagcagcgcgagtt gaggcgtggctggtcatc agcagcgtcacttcgaaact cagcgtcttgatcagcacat E78A caggaggacctcatcaagga ctgctaggccaaaagattgc E78B gcagcaacaccaccaactg ttcagcgtgttggcaaagt cgacaatgatgcacacgttc gcattctatccgctcgaaca tacaggcccaagatcgtgaa gacaatctccttgcgcttct Dose and Temporal Response of USP to Intermolt puffs Sgs4 119 Protein Kinase Inhibitors Early-puffs Early-late puffs DHR3 Protein kinase consensus sequence analysis revealed that USP has 10 potential phosphorylation sites, with 5 sites for PKC and 5 for CKII (Song et al., 2003; Rauch et al., 1998). To investigate which protein kinase is responsible for USP phosphory- Stage-specific puffs FTZ-F1A Control Dros RP49 lation, salivary glands from early wandering larvae were incubated for 6 h with the indicated doses of protein kinase inhibitor H89 or PKC-specific inhibitor chelerythrine chloride or CKII-specific in- tiple samples analyzed. The amplification product was analyzed in1.5% EtBr agar gel and visualized using the AlphaImagerTM digital system (Alpha Innotech Corporation, San Leandro, CA). The 20E responsive genes and the corresponding primers used in RT-PCR were listed in Table 1. Profiles in the PKC Inhibitor-Treated Glands using AB11 USP mAb as a probe to monitor the changes in USP phosphorylation patterns. As shown in Figure 1, the common protein kinase inhibitor H89 inhibited USP phosphorylation in a phorylated USP band (p56) started to disappear when the glands were incubated with 100 mM H89 and diminished completely when treated with 200 Salivary glands from early wandering larvae were dissected in Ringer抯 solution and preincubated for mM PKC inhibitor chelerythrine chloride and then stimulated with 0.5 subjected to SDS-PAGE and Western blot analysis dose- and temporal-dependent manner. The phos- 2D Gel Analysis of 20E-Inducted Protein Expression 30 min in Grace抯 medium containing 100 hibitor heparin, respectively. The glands were mM 20E (a predetermined effective dose) for 6 h (the predetermined time period). The control received no 20E stimulation. At end of the incubation, the glands were collected, homogenized, and centrifuged and protein concentration quantified as described above. The resulting protein sample from each treatment was subjected to 2D gel electrophoresis and silver staining as described previously (Sun et al., 2003). In brief, the first dimensional isoelectric focusing (IEF) was performed using BIORAD PROTEIN IEF Cell and ReadyStrip� IPG Strips (PH 3� 11cm, Catalog mM or higher. Meanwhile, the intensity of the un- phosphorylated USP band (p54) was increased accordingly (Fig. 1a). Temporal response study revealed that the phosphorylated USP band started to decline 1 h after incubation with 200 mM H89 and disap- peared completely at 6-h incubation (Fig. 1b). When the salivary glands were incubated with the PKC-specific inhibitor chelerythrine chloride, chelerythrine chloride inhibited USP phosphory- mM and when the lation at the concentration as low as 50 maximum inhibition was glands were treated with 100 observed mM. Meanwhile, the intensity of the unphosphorylated USP band was nearly doubled (Fig. 2a). Temporal response of USP to the PKC inhibitor indicated that the PKC inhibitor was able to block USP phosphorylation in the glands incubated with chelerythrine chlo- number 165-4000) according to the manufacturer抯 ride for as short as 1 h. USP phosphorylation was instructions. Each IPG strip was soaked in rehy- completely inhibited by chelerythrine chloride at dration buffer containing 100 3 h incubation (Fig. 2b). The results suggest that mg protein per treat- ment. After isoelectric focusing, the IPG strip was PKC, which phosphorylates the USP, was inhib- equilibrated and subjected to SDS-PAGE (12.5% ited by chelerythrine chloride, resulting in the dis- gel) and silver staining. appearance of the p56 phosphorylated USP band. Archives of Insect Biochemistry and Physiology July 2006 doi: 10.1002/arch. 120 Fig. 1. Sun and Song Effect of the general protein kinase inhibitor H89 or for the indicated period of time in the presence or ab- mM H89 (b). Protein samples were prepared on USP phosphorylation in the salivary glands of D. sence of 200 melanogaster . The salivary glands from early wandering from the glands and subjected to SDS-PAGE and Western third instar larvae were incubated for 6 h in Grace抯 me- blot analysis using AB11 USP specific mAb as a probe. dium containing the indicated concentrations of H89 (a) Figure 3 reveals that the phosphorylated and expected, the PKC inhibitor also blocked 20E-in- unphosphorylated USP bands in the glands treated duced USP phosphorylation (Fig. 4) in a similar with the CKII inhibitor heparin decreased at a simi- manner as shown in the basal level inhibition (Fig. lar rate in the dose and temporal response studies, 2). All data suggest clearly that PKC, not CKII, is suggesting that CKII has no effect on USP phos- responsible for USP phosphorylation. phorylation, but inhibits its expression. It is not clear that whether the decline in the phosphory- RT-PCR Analysis of 20E-Induced Gene Transcription in lated and unphosphorylated USP was due to the the PKC Inhibitor-Treated Glands inhibition of USP expression or the increase in USP breakdown by proteases. To investigate whether PKC-mediated USP phos- To investigate whether the PKC inhibitor was phorylation affects 20E-induced gene transcription, mM able to block USP phosphorylation in the presence salivary glands were preincubated with 100 of 20E, the glands were preincubated with 100 PKC inhibitor chelerythrine chloride for 30 min mM mM PKC inhibitor chelerythrine chloride for 30 min and then challenged with 0.5 and then challenged with 0.5 dicated time periods. After incubation, the glands Fig. 2. mM 20E for 6 h. As Effects of the PKC-specific inhibitor chelerythrine 20E for the in- chelerythrine chloride (a) or for the indicated period of mM chloride on USP phosphorylation in the salivary glands time in the presence or absence of 100 of D. melanogaster. The salivary glands from early wander- chloride ( b). Protein samples were prepared from the ing third instar larvae were incubated for 6 h in Grace抯 glands and subjected to SDS-PAGE and Western blot analy- medium sis using AB11 USP specific mAb as a probe. containing the indicated concentrations of Archives of Insect Biochemistry and Physiology July 2006 chelerythrine doi: 10.1002/arch. USP Phosphorylation and 20E-Induced Gene Expression Fig. 3. Effects of the CKII-specific inhibitor heparin on 121 for the indicated period of time in the presence or ab- mg/ml heparin (b). Protein samples were pre- USP phosphorylation in the salivary glands of D. melano- sence of 10 gaster. The salivary glands from early wandering third in- pared from the glands and subjected to SDS-PAGE and star larvae were incubated for 6 h in Grace抯 medium Western blot analysis using AB11 USP specific mAb as a a containing the indicated concentrations of heparin ( ) or probe. were collected and analyzed using RT-PCR for tran- completely blocked the expression of these 20E- scriptional expression of 8 randomly selected 20E induced genes. Similar response patterns were ob- response genes (Table 1). These genes represent 4 served for the above-mentioned genes in the glands different response types, i.e., intermolt puff, early- incubated with 20E for 12 and 24 h although larger puff, late-puff, and stage-dependent puff genes variation did occur. However, 20E had no effect (Table 1). Figure 5 reveals that 20E induced the on the expression of the intermolt puff gene Sgs4, expression of the early-puff genes E74A and E75B neither did the PKC inhibitor. For the stage-spe- and the late-puff genes DHR3, E78A, and E78B cific puff FTZ-F1A gene, it expressed only in con- when the glands were incubated with 20E for 6 h trol glands 6 h after incubation with medium and and that the PKC inhibitor completely or nearly was inhibited by 20E. Fig. 4. dicated concentrations of chelerythrine chloride before Effects of the PKC-specific inhibitor chelerythrine mM b). chloride on 20E-stimulated USP phosphorylation in the being challenged with 0.5 salivary glands of D. melanogaster. The salivary glands from samples were prepared from the glands and subjected to early wandering third instar larvae were incubated for 6 h SDS-PAGE and Western blot analysis using AB11 USP spe- in Grace抯 medium containing the indicated concentra- cific mAb as a probe. CC: chelerythrine chloride. a tions of 20E ( ) or preincubated for 30 min with the in- Archives of Insect Biochemistry and Physiology July 2006 doi: 10.1002/arch. 20E for 6 h ( Protein 122 Fig. 5. Sun and Song Effects of the PKC inhibitor chelerythrine chlo- expression of 8 selected genes. The rp49 gene was used ride on the expression of 20E-induced genes (RT-PCR). for normalization of the compared templates. The tran- The salivary glands from early wandering third instar lar- scripts were analyzed in 1.5% EtBr agar gel and visual- vae of D. melanogaster were preincubated for 30 min with ized using the AlphaImagerTM digital system (Alpha mM) and then challenged with Innotech Corporation, San Leandro, CA). CC: chelerythrine chelerythrine chloride (100 20E (0.5 mM ) for the indicated periods of time. Total chloride. CK: control. RNA was insolated for RT-PCR analysis of transcriptional As expected, the PKC inhibitor had no influence 20E or both. When compared to the control, which on FTZ-F1A gene expression. 20E also failed to in- was incubated with medium only (Fig. 6a), 20E duce the expression of E74B transcript, but the PKC induced or increased the expression of a set of pro- inhibitor was able to induce E74B expression, sug- teins (Fig. 6b, protein spots 1�. Meanwhile, 20E gesting that E74B may be negatively regulated by also inhibited or decrease the expression of sev- receptor phosphorylation. Neither 20E nor PKC in- eral proteins (Fig. 6b, protein spots 7�). The PKC hibitor had any effect on the expression of rp49 inhibitor alone had no effect on protein expres- control transcript. These results suggest that PKC- sion profiles except protein spots 12 and 13, which mediated phosphorylation is required for the ex- were induced only by the PKC inhibitor (Fig. 6c). pression of 20E-induced genes and inhibition of PKC As expected, the PKC inhibitor inhibited or reduced activity results in inhibition of USP phosphorylation the expression of 20E-induced proteins (Fig. 6d, and subsequent 20E-induced gene transcription. protein spots 1�. It must be noted that only the major protein spots affected by PKC, 20E, or both 2D Gel Analysis of 20E Induced-Protein Expression in were labeled and discussed. Although the identi- the PKC Inhibitor-Treated Glands ties of these proteins are not known, the data clearly indicate that PKC-mediated USP phospho- Figure 6 shows the protein expression profiles in the glands incubated with the PKC inhibitor or rylation is also required for 20E-induced protein expression. Archives of Insect Biochemistry and Physiology July 2006 doi: 10.1002/arch. USP Phosphorylation and 20E-Induced Gene Expression Fig. 6. Effects of the PKC inhibitor chelerythrine chlo- glands incubated with 0.5 123 mM 20E. c: Protein profile from mM) ride on the expression of 20E-induced proteins. The sali- the glands incubated with chelerythrine chloride (100 vary glands from early wandering third instar larvae of D. only. d: Protein profile from the glands preincubated with melanogaster were preincubated for 30 min with chel- chelerythrine chloride (100 erythrine chloride (100 mM) and then challenged with 20E lenged with 20E (0.5 mM) for 30 min and then chalmM) for 6 h. 20E-induced (spots mM) for 6 h. The resulting protein sample from each #1�, 20E-inhibited (spots #7�), the PKC inhibitor in- treatment was subjected to 2D gel electrophoresis and sil- duced proteins (spots #12 and #13), and 20E-induced but ver staining. a: Protein profile from the control glands blocked by the PKC inhibitor are indicated by arrows. CC: incubated with medium only. b: Protein profile from the chelerythrine chloride. (0.5 Archives of Insect Biochemistry and Physiology July 2006 doi: 10.1002/arch. 124 Sun and Song DISCUSSION in the glands incubated with the PKC inhibitor chelerythrine chloride or the common inhibitor H89. USP is a phosphoprotein in several insect spe- After PKC was demonstrated to be responsible cies including D. melanogaster (Song et al., 2003), for USP phosphorylation, it is critical to know M. sexta (Song and Gilbert, 1998), C. tantans whether the PKC-mediated USP phosphorylation (Rauch et al., 1998), and T. molitor (Nicola� et al., plays any roles in regulating the expression of 20E- 2000). Phosphorylation of USP as well as EcR induced genes and proteins. To investigate that, a modulates the ligand- and perhaps also the DNA- set of randomly selected 20E response genes, rep- binding activity of the EcR/USP complex in Man- resenting 4 different response groups (Table 1), duca (Song and Gilbert, 1998). Although the were used as tools to monitor the transcriptional putative phosphorylation sites of USP for PKC and activities of these genes in the glands that were CKII have been reported in Drosophila and Chiro- preincubated with the PKC inhibitor chelerythrine nomus, based on kinase consensus recognition se- chloride followed by 20E challenge. By using RT- quence analysis (Song et al., 2003; Rauch et al., PCR to measure the transcriptional activities of 1998), the kinase responsible for its phosphoryla- these selected genes, we found that inhibition by tion and the role of USP phosphorylation in 20E- chelerythrine chloride of the PKC-mediated USP induced gene transcription have not yet been phosphorylation blocked the transcriptional ex- explored. The present study demonstrated for the pression of 20E-induced genes, i.e., the early-puff first time in Drosophila that PKC is responsible gene E74A and E75B and the late-puff gene DHR3, for USP phosphorylation and that inhibition of E78A, and E78B (Fig. 5), but had no effect on the PKC activity results in inhibition of USP phospho- genes that were not induced by 20E under the ex- rylation and 20E-induced gene expression at both transcriptional and translational levels, suggesting that PKC-mediated USP phosphorylation is required for 20E-induced gene expression. Protein phosphorylation is regulated by protein kinase, which phosphorylates proteins and by protein phosphatase, which dephosphorylates proteins. When the salivary glands from early wondering larvae were incubated with the common protein kinase inhibitor H89 that inhibits both PKC and CKII at different concentrations (Fig. 1) and the PKC specific inhibitor chelerythrine chloride in the absence (Fig. 2) or presence (Fig. 4) of 20E, the phosphorylated USP band declined or disappeared in a dose- and time-dependent manner while the unphosphorylated USP band increased accordingly. These results indicate that the PKC inhibitors blocked PKC perimental conditions, i.e., the intermolt puff gene Sgs4, the stage-dependent puff gene FTZ-F1A, and the early-puff gene E74B. Similarly, the PKC inhibitor also blocked the expression of 20E-induced proteins (Fig. 6, spots 1� although the identities of these proteins are not known. These results suggest that the PKC-mediated phosphorylation of USP is required for the expression of 20E-induced genes and proteins. Although the PKC inhibitor chelerythrine chloride inhibited PKC activity, thus blocking USP phosphorylation and 20E-induced gene transcription, chelerythrine chloride might also inhibit EcR phosphorylation. Protein consensus recognition sequence analysis of Drosophila EcR reveals multiple phosphorylation sites on EcR sequence, with 10 sites for PKC, 11 sites for CKII, and a single site for PKA (Rauch et al., 1998). It is likely that PKC activities and USP phosphorylation was stopped. On is also the key kinase responsible for EcR phos- the other hand, protein phosphatase activity was not phorylation. Unfortunately, we do not have a good affected by the PKC inhibitor. Protein phosphatase antibody to track the PKC-mediated EcR phospho- continuously dephosphorylated the existing phos- rylation patterns as we did for USP. We could not phorylated USP by removing phosphate group(s) exclude the possibility that other chaperon proteins from USP protein, resulting in the decline or com- associated with the EcR/USP complex (Arbeitman plete disappearance of the phosphorylated USP band and Hogness, 2000; Song et al., 1997) are also Archives of Insect Biochemistry and Physiology July 2006 doi: 10.1002/arch. USP Phosphorylation and 20E-Induced Gene Expression 125 regulated by PKC. Arbeitman and Hogness (2000) lation could also be regulated by ligand-indepen- have shown that chaperone proteins are essential dent pathway(s) as reported in vertebrates (Weigel, for the DNA-binding of the EcR/USP complex. 1996). It is possible that 20E stimulates USP phos- Elimination of any one of six chaperon proteins phorylation at specific sites different from the ones will proportionally reduce ecdysone response element enhanced by ligand-independent pathway(s). We (EcRE) recognition while none of these chaperones could not exclude the possibility that 20E stimu- is necessary for ligand-binding. In vertebrates, lates USP phosphorylation at the same sites as phosphorylation of chaperon proteins can cause ligand-independent mechanisms do, but at a dif- conformational change of these proteins and dis- ferent level. sociate them from steroid receptor, thus activating In Drosophila, six PKC isoforms have been the receptor (Weigel, 1996; Lange, 2004). For ex- identified based on the available genomic sequence ample, in the absence of hormone, PRs are com- (Shieh et al., 2002), including two classical PKCs plexed Upon (PKC53E and eye-PKC), two novel PKCs (PKC98E exposure to progesterone, the ligand-activated re- with several chaperon proteins. and PKCdelta), an atypical PKC (DaPKC), and a ceptor undergoes a conformational change, disso- PKC-related kinase. DaPKC is essential for early ciates from chaperon proteins, dimerizes, and embryonic development and Eye-PKC plays a role interacts with specific progesterone response ele- in the regulation of visual signaling, a G-protein ments in the promoter regions of target genes. It coupled phospholipase Cbeta-mediated cascade. is unclear whether the EcR/USP complex is acti- No information is available on which PKC isoform vated via phosphorylation in a similar manner as is responsible for USP or EcR phosphorylation. in the vertebrate system. Nevertheless, the fact that Identification and characterization of the PKC chelerythrine chloride blocked the transcriptional isoform responsible for EcR and USP phosphory- and translational expression of 20E-induced genes suggests that the PKC-mediated phosphorylation of USP, presumably EcR as well, is required for 20E- lation are currently underway. The data from our previous and present studies suggest that phosphorylation plays important roles in regulating the ligand- and DNA-binding activities of the EcR/USP induced gene expression. Our previous data from Manduca (Song and complex and 20E-induced gene expression. Gilbert, 1998) and Drosophila (Song et al., 2003) show clearly that 20E is responsible for enhancing LITERATURE CITED USP expression and phosphorylation in both species. These results are consistent with the recent Andres AJ, Thummel CS. 1994. Methods for quantitative analysis reports from two independent labs that USP is a phosphoprotein and its phosphorylation is regulated by 20E (Rauch et al., 1998; Nicola� et al., of transcription in larvae and prepupae. In: Goldstein LSB, Fyrberg EA, editors. Drosophila melanogaster: practical uses in cell and molecular biology. New York: Academic Press. p. 565�3. 2000). Although the in vitro enhancement of USP phosphorylation by 20E in Drosophila salivary glands is small (about 20% increase compared to Arbeitman MN, Hogness DS. 2000 Molecular chaperones activate the Drosophila ecdysone receptor, an RXR heterodimer. Cell 101:67�. the control) (Fig. 4), it is sufficient to induce the expression of 20E-induced genes that lead to molt- Ashburner M, Chihara C, Meltzer P, Richards G. 1974. Tem- ing and metamorphosis. Blocking 20E-induced poral control of puffing activity in polytene chromosomes. USP phosphorylation inhibits 20E-induced gene expression as shown in Figure 5. In the unstim- Cold Spring Harbor Symp Quant Biol 38:655�2. Bender M, Imam FB, Talbot WS, Ganetzky BS, Hogness燚S. ulated glands from early wandering larvae, approxi- 1997. Drosophila ecdysone receptor mutations reveal func- mate 40% of the total USP is in phosphorylation tional differences among receptor isoforms. Cell 62:413� form (Figs. 1�, suggesting that USP phosphory- 423. Archives of Insect Biochemistry and Physiology July 2006 doi: 10.1002/arch. 126 Sun and Song Burtis KC, Thummel CS, Jones CW, Karim FD, Hogness DS. ultraspiracle during metamorphosis and in vivo induction 1990. The Drosophila 74EF early puff contains E74, a com- of its phosphorylation by 20-hydroxyecdysone. Insect Mol plex ecdysone-inducible gene that encodes two eto-related Biol 9:242�9. proteins. Cell 61:85�. Oro AE, McKeown M, Evans RM. 1990. Relationship between Cherbas P, Cherbas L. 1996. Moleculor aspects of ecdysteroid hormone action. In: Gilbert LI, Tata JR, Atkinson BG, edi- the product of the Drosophila ultraspiracle locus and the vertebrate retinoid X receptor. Nature 347:298�1. tors. Metamorphosis: postembryonic reprogramming of gene expression in amphibian and insect cells, 3rd ed. San Riddiford LM. 1993. Hormones and Drosophila development. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Diego: Academic Press. p 175�1. Press. 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