Expression and activity of glycogen synthase kinase during vitellogenesis and embryogenesis of Rhipicephalus (Boophilus) microplus

Veterinary Parasitology 161 (2009) 261–269

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Expression and activity of glycogen synthase kinase during vitellogenesis and embryogenesis of Rhipicephalus (Boophilus) microplus Carlos Logullo a,b,*, William H. Witola a,d, Caroline Andrade c, Leonardo Abreu b, Josiana Gomes b, Itabajara da Silva Vaz Jr.c, Saiki Imamura a, Satoru Konnai a, Kazuhiko Ohashi a, Misao Onuma a a Laboratory of Infectious Diseases, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, 060-0818, Sapporo, Hokkaido, Japan b Laborato´rio de Quı´mica e Func¸a˜o de Proteı´nas e Peptı´deos–CBB–UENF, Avenida Alberto Lamego, 2000, Horto, Campos dos Goytacazes, CEP 28015-620, RJ, Brazil c Centro de Biotecnologia and Faculdade de Veterina´ria, UFRGS, Avenida Bento Gonc¸alves, 9500, Porto Alegre, C.P. 15005, CEP 91501-970, RS, Brazil d Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT-06030-3301, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 October 2008 Received in revised form 12 January 2009 Accepted 26 January 2009

Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism in mammals. GSK-3 belongs to a highly conserved family of serine/ threonine protein kinases, whose members are involved in hormonal regulation, nuclear signaling, and cell fate determination in higher eukaryotes. We have cloned and characterized the RmGSK-3 gene from Rhipicephalus (Boophilus) microplus tick embryos. DNA and protein sequence analysis depicted high similarity to the corresponding enzyme, from both vertebrate and invertebrate animals. In addition, the mRNA transcription profile identified during embryogenesis was analyzed. We observed that the RmGSK-3 mRNA rapidly decreases from the 1st to 3rd day of development, and increases from the 3rd to 15th day. After the 15th day of development, we observed a near 50% reduction in RmGSK3 mRNA transcription in comparison to the 1st day. We detected the GSK-3b isoform in egg homogenates throughout embryogenesis using Western blot analysis. RmGSK-3 mRNA was present in fat body, midgut and ovary from partially and fully engorged adult female ticks. The highest mRNA level was observed in ovaries from both developmental stages and in first-day eggs. Furthermore, RmGSK-3 activity correlated with glycogen content variation. Finally, kinase activity in egg homogenates was inhibited by the specific inhibitor, SB-216763. These data suggest that RmGSK-3b may be involved in glycogen metabolism regulation during R. microplus embryogenesis. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Tick egg Embryogenesis Glycogen synthase kinase Metabolism Rhipicephalus (Boophilus) microplus

1. Introduction Rhipicephalus (Boophius) microplus is the most economically damaging tick worldwide (Johnston, 1985). Tick

* Corresponding author at: Laborato´rio de Quı´mica e Func¸a˜o de Proteı´nas e Peptı´deos–CBB–UENF, Avenida Alberto Lamego, 2000, Horto, Campos dos Goytacazes, CEP 28015-620, RJ, Brazil. Tel.: +55 22 27261467; fax: +55 22 27261520. E-mail address: [email protected] (C. Logullo). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.01.029

embryo metabolism has been investigated by our group in order to understand the physiology of this stage of tick development, and also to identify possible targets for the development of control strategies (Logullo et al., 2002; Campos et al., 2006; Moraes et al., 2007). Conventional tick control methods have been based mainly on the use of acaricides; however, the rapid appearance of resistant tick populations, and the presence of chemical residues in meat and milk are aspects that emphasize the need for novel control methods, such as vaccination (Willadsen et al., 1996; Willadsen, 2006) and biological control (Samish

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et al., 2001). Although an anti-tick vaccine may be the most promising control method, its development still depends on the identification and characterization of one or more protective tick antigens (Imamura et al., 2005). One strategy to block disease transmission by ticks would be the identification of new molecular targets for the interruption of its life cycle. In this context, proteins involved in egg formation and development are very attractive targets, since interference in oogenesis and embryogenesis has a pronounced negative effect on the number of larvae that hatch. Moreover, an immune response against diverse antigens can be simultaneously achieved and can assist in the reduction of tick number in successive generations in the field. Glycogen synthase kinase-3 (GSK-3) is a widely conserved and ubiquitously expressed serine/threonine kinase (Fisher et al., 1999; Bax et al., 2001). It was originally described as a key enzyme involved in glycogen metabolism due to its inhibitory action over glycogen synthase (GS) through phosphorylation (Embi et al., 1980). Since GSK-3 is constitutively active in unstimulated cells, GS is maintained in a phosphorylated and inactive state (Cross et al., 1995). Furthermore, GSK-3 has been described as associated with diseases, inflammation and cellular processes (Jope and Johnson, 2004; Jope and Roh, 2006). GSK-3 activity modulation is also being studied as novel targets for therapeutic use in the treatment of diabetes and neurodegenerative diseases (Frame and Cohen, 2001). GSK-3 can be regulated by the phosphatidylinositol 3OH kinase/Akt (PI3K/Akt) pathway. In response to stimulation of cells by insulin or growth factors GSK-3 activity is inhibited by phosphorylation at a specific serine residue in the NH2-terminus (Cross et al., 1995). Phosphorylation at a different regulatory tyrosine residue at the activation loop of the catalytic domain is required for functional activity (Hughes et al., 1993). GSK-3 is expressed in mammals as two highly homologous isoforms, and both possess serine 9 for GSK-3a, serine 21 for GSK-3b, tyrosine 279 for GSK3a, and 216 for GSK-3b, as regulatory residues (Cross et al., 1995). GSK-3 is also associated with the Wnt signaling pathway during Drosophila, Xenopus and sea urchin embryogenesis (Kim and Kimmel, 2000). This pathway promotes specification of cell fates in early embryos, which appears to be conserved in all metazoa, and leads to GSK-3 inhibition by phosphorylation (Ferkey and Kimelman, 2000; Frame and Cohen, 2001). The use of non-conserved regions as targets for selective inhibition of homologous proteins from different species has been explored with triosephosphate isomerase (Tellez-Valencia et al., 2002), glycerol-3-phosphate dehydrogenase (Hannaert et al., 1995; Verlinde et al., 2002) and hsp70 proteins (Kanamura et al., 2002). This inhibition could be obtained by chemical (Zomosa-Signoret et al., 2003) or immunological (Jimenez et al., 2000) methods. These proteins are immunogenic and induce humoral and cellular immunity to immunizations and infections from various pathogens (Kakeya et al., 1999; Kanamura et al., 2002), despite their conserved sequences. The GSK proteins possess a highly conserved sequence at the Nterminal region, however they possess a non-conserved Cterminal region, so a non-conserved region could be a

useful target for the development of immunological or chemical control methods. Embryogenesis of oviparous organism relies on the utilization of yolk components that were stored in the egg during oogenesis (Sappington and Raikhel, 1998). After maturation, oocytes increase and accumulate large amounts of RNA, carbohydrates, lipids and proteins that serve as substrates for functional metabolic pathways of the developing embryo (Oliveira and Machado, 2006). R. microplus embryos accumulate glucose and glycogen during late embryogenesis by activation of gluconeogenesis after cellular blastoderm formation (Campos et al., 2006; Moraes et al., 2007). Fluctuations in glycogen levels during embryogenesis have also been observed for Drosophila (Yamazaki and Nusse, 2002). Glucose-glycogen metabolism is influenced by Wnt pathway components in fly embryos (Yamazaki and Yanagawa, 2003). This work presents the first evidence of GSK-3 in R. microplus tick and may offer novel strategies for the identification of new molecular targets for its control. In this work we identified and cloned a cDNA sequence highly similar to GSK-3b, and characterized its expression pattern during embryogenesis and in adult females’ organs of R. microplus tick. Furthermore, our results point to a possible role of RmGSK-3 in glycogen metabolism during tick embryogenesis. We also suggest RmGSK-3 as a possible target in order to interfere on insulin pathway and embryo development.

2. Materials and methods 2.1. Chemicals Molecular weight standards, glycine, acrylamide, bisacrylamide, TEMED, DMSO, DTT, TRIS, bovine serum albumin, rabbit polyclonal anti-GSK-3b, rabbit antiphospho-GSK-3a/b (pTyr279/216) and protease inhibitors cocktail (aprotinin, leupeptin, pepstatin A and E-64) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Pre-stained molecular weight standards Kaleidoscope1 (208–Myosin (blue), 144–b-galactosidase (magenta), 87–bovine serum albumin (green), 44.1– carbonic anhydrase (violet), 32.7–soybean trypsin inhibitor (orange), 17.7–lysosyme (red) and 7.1–aprotinin (blue) kDa) were purchased from Bio-Rad. DMSO was purchased from Merck (Gibbstown, NJ, USA). All other chemicals were of analytical grade. 2.2. Tick maintenance and egg collection Ticks were obtained from a colony maintained at the Faculdade de Veterina´ria, Universidade Federal do Rio Grande do Sul, Brazil. R. microplus ticks from the Porto Alegre strain, free of Babesia spp., were reared on calves obtained from a tick-free area. Engorged adult females were kept in Petri dishes at 28 8C and 80% relative humidity until completion of oviposition. After oviposition, eggs were separated according to age and maintained under the same conditions. Under these conditions, embryogenesis lasts 18–20 days, with over 90% successful eclosion rates.

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Eggs were collected on different days during embryogenesis and stored at 80 8C until use. 2.3. RNA isolation and first strand cDNA synthesis RNA was isolated from ticks at different developmental stages (1-, 3-, 6-, 9-, 12-, 15-, 18- and 21-day-old eggs and 1-day-old larvae) and from different tissues (fat bodies, ovaries and midguts) of fully engorged females. Fully engorged female ticks were washed with PBS. The dorsal surface was dissected with a scalped blade. Midguts, ovaries and fat bodies were separated with fine-tipped forceps and washed in PBS and tissues rapidly removed. Eggs, larvae and tissues were frozen in liquid N2. Total RNAs were extracted from using the TRIZOL reagent according to the manufacturer’s instructions (Invitrogen, CA, USA). First-strand cDNA was synthesized from 5 mg of total RNA. The reactions were performed at 42 8C for 60 min and at 72 8C for 15 min in the presence of oligo-dT (Hokkaido System Science, Japan) and RAV2 (Takara, Japan) as recommended by the manufacturer. 2.4. Cloning of RmGSK-3 cDNA Degenerate oligonucleotides for the kinase domain conserved region of GSK-3b were used (50 -GTIGCIATHAARAARGTIYTICARGAY-30 , and 50 -YTTRWRYTCIRTRTARTTIGGRTTCAT-30 ). These primers were previously designed based on the sea urchin GSK-3b cDNA sequence (Emily-Fenouil et al., 1998) and utilized in PCR reactions to amplify a partial GSK-3b sequence from the R. microplus egg cDNA. Conditions for amplification with Taq polymerase were: 94 8C for 5 min; 40 cycles at 94 8C for 1 min, 45 8C for 1 min and 72 8C for 1 min; 72 8C for 10 min. The PCR products were resolved on 1.5% agarose electrophoresis gels, and an expected fragment of approximately 600-bp, corresponding to the partial GSK3b cDNA, was excised and purified using the Gene Clean DNA purification kit (BIO 101 Systems). The fragment was cloned in the pGEM1T-Easy vector (Promega) and propagated in Escherichia coli (DH5a strain). The recombinant plasmid was extracted by Qiagen miniprep kit and sequenced using an automated sequencer (Beckman CEQ2000). The predicted GSK-3b sequence for R. microplus was subsequently confirmed by comparative analysis with the GSK-3b sequence data obtained from GenBank. To clone the full-length cDNAs encoding GSK-3b, 50 and 30 rapid amplifications of cDNA Ends (RACE) were performed using the nucleotide sequence of the 600-bp amplified fragment. 30 RACE was performed using the 30 RACE System for rapid amplification of cDNA Ends kit (Invitrogen) and an oligo-(dT) containing adaptor primer according to the manufacturer’s instructions. The oligonucleotide, 50 GCTCGCGCTACTACCGGGCCCCAGAACT-30 was used as a forward primer. A second nested specific primer 50 GACGTGTGGTCGGCGGGTTGTGTGCTGG-30 was used to perform a second PCR reaction. 50 RACE was performed using the 50 RACE System for rapid amplification of cDNA Ends, Version 2.0 (Invitrogen), as specified by the manufacturer. First-strand cDNA was synthesized from

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total RNA of R. microplus using the oligonucleotide 50 CAATGCGGTCCACAAA-30 and reverse transcriptase enzyme (M-MLV RT; Takara). The PCR-amplified dC-tailed cDNA was made using the abridged anchor primer AUAP and a nested gene-specific primer 50 -CCAGGGAGTGGATGTAGGCCAGGCTCCG-30 . Two second nested specific primers (50 -TGGACTTGCTGTAGTGGCGCGCCACGCG-30 and 50 GCCCATGCGAGTCATGATGCGCATGCTG-30 ) were used to perform a second PCR reaction. The product was cloned into pGEMT-Easy and sequenced. Subsequently, to confirm that the 30 -RACE and 50 -RACE products corresponded to the authentic transcript, the full-length cDNA was amplified by PCR with a pair of primers corresponding to the 30 and 50 Ends. The amplified fragment was cloned into pGEMT-Easy and sequenced. 2.5. Sequence analyses Nucleotide analysis and deduced amino acid sequences were carried out using a software package, GENETYX-Win version 4.04 (Software development Co. LTD., Tokyo, Japan) and the BioEdit version 5.0.6 software program (Hall, 1999). Sequence alignment was performed using the Clustal W program. Sequence fragments were compared with those of known proteins in Genbank database. Signal sequence prediction analysis was performed using the SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/). The presence of conserved patterns was determined using InterProScan (Zdobnov and Apweiler, 2001). Similarity was determined from optimized sequence alignments using the CLUSTAL W program (Thompson et al., 1994). The accession numbers for GSK-3 sequences used in the phylogenetic analysis are as follows: Drosophila melanogaster AAM52705, Xenopus laevis AAC42224, Apis mellifera XP 392504, Mus musculus AAH60743, Danio rerio NP_571456 and Bos taurus XP878051. 2.6. Real-time PCR analysis of the GSK-3 gene expression in tick embryogenesis and female organs Total RNA was isolated from R. microplus eggs at different developmental stages (1, 3, 6, 9, 12, 15, 18, 21day-old eggs), ovaries, gut, and fat body of fully and partially engorged females were isolated using the TRIzol Reagent (Sigma) following the manufacturer’s instructions. After reverse transcription of equal amounts (5 mg) of total RNA for all samples, 1 mL of each sample’s product was used as template in the real-time PCR reaction using gene specific primers (validated by RT-PCR) with Master SYBR Green I kit (Roche, Japan). Amplification was performed using a LightCycler1 DX 400 (Roche). Serial dilutions of the cDNA were used for curve calibration. Reaction efficiencies between 85 and 100% were determined from calibration curves for each set of primers. The primer sets were 50 -CGAGGTGTACCTGAACCTGGT-30 (forward) and 50 -CGATGGCAGATGCCCAGAGAC-30 (reverse) for GSK-3 and 50 -GGACGACCGATGGCTACCT-30 (forward) and 50 -TGAGTTGATTGGCGCACTTCT30 (reverse) for 40-S ribosomal protein (GenBank accession no. EW679928) (Pohl et al., 2008). The relative expression ratio of the GSK

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gene was measured using the housekeeping gene, 40S ribosomal protein as endogenous control. The mathematical model (Pfaffl, 2001) and Relative Expression Software Tool (REST-MCSß, version 2) (Pfaffl et al., 2002) were used in the analysis. 2.7. Western blotting Eggs laid were collected on days 1, 6, 12, and 18 after oviposition and homogenized in a Potter-Elvehjem tissue grinder in 20 mm Tris–HCl buffer, pH 7.4 with 0.05 mg/ mL soybean trypsin inhibitor, leupeptin, antipain, and 1 mM benzamidine (approximately 1 g of eggs/10 mL). Egg homogenates were centrifuged at 11000  g for 10 min at 4 8C. The floating lipids and the pellet were discarded, and the supernatant was used for protein analysis. Proteins (40 mg) were first separated by SDSPAGE (10%) and then transferred at 190 mA for 90 min to a PVDF membrane (Towbin et al., 1979). PVDF sheets were then blocked by incubation with 1% (w/v) BSA, 0.05% (v/v) Tween 20 in TBS for 1 h at room temperature. After that the membranes were incubated with polyclonal antibody anti-pTyr279/216 GSK-3a/b (1:200) or GSK-3b (1:400) in blocking buffer 1% (w/v) BSA, 0.05% (v/v) Tween 20 in PBS for 1 h. Goat anti-rabbit IgG conjugated to peroxidase, diluted 1:2000 (v/v), was used as secondary antibody and the signal was generated using 0.013% (w/v) DAB, 0.015% (w/v) CoCl2 in 50 mL of PBS, with 50 mL of 30% H2O2. 2.8. GSK-3 activity in egg homogenate Eggs laid were collected on days 1, 5, 6, 9, 12, and 15 after oviposition and homogenized in a Potter-Elvehjem tissue grinder in 20 mM Tris–HCl buffer, pH 7.4 with 0.05 mg/mL soybean trypsin inhibitor, leupeptin, antipain, and 1 mM benzamidine (approximately 1 g of eggs/10 mL). Egg homogenates were centrifuged at 11000  g for 10 min at 4 8C. The floating lipids and the pellet were discarded, and the supernatant was used for GSK-3 activity. GSK-3b was immunoprecipitated from 100 mg of protein using 2 mL of anti-GSK-3b commercial antibody (Sigma Co.). The immuno-complex was captured with 10 mL of protein A-agarose suspension by incubating the mixture at room temperature under gentle agitation for 20 min. The resin was collected by centrifugation, washed three times and resuspended in reaction buffer (50 mL) [20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 5 mM dithiothreitol, 1 mM ammonium molybdate, 1 mg/mL heparin and 50 mM CREB phosphopeptide (Calbiochem)]. GSK-3 b activity was determined in the absence or presence of the inhibitor SB216763, by incubating the suspension with 100 mM g-[P32]-ATP (500–3000 CPM/pmol) at 37 8C for 30 min (Ryves et al., 1998). After incubation, supernatant aliquots (20 mL) of the supernatant were spotted onto pieces of Whatman P81 phosphocellulose paper strips. The papers were washed three times with phosphoric acid solution (75 mM), dried and immersed in scintillation liquid for radioactivity count determination on a 1600TR TRICARB-Packard. The activity was determined as the amount of GSK-3b required to catalyze the transfer of

1 pmol of phosphate to CREB Phosphopeptide in 1 min at 30 8C in 20 mL. 2.9. Glycogen content Eggs were homogenized by grinding with 1 mL of extraction buffer containing 200 mM sodium acetate, pH 4.8 and centrifuged at 10000  g for 10 min. Aliquots of 100 mL (20 mg/mL) of the supernatant were incubated with 1 unit a-amyloglucosidase (Sigma Chemicals) for 4 h at 40 8C. Liberated glucose was detected with a commercial kit for glucose dosage (Glucox1, Doles, Inc.) at 510 nm. Endogenous glucose was subtracted from control conditions (without a-amyloglucosidase addition). Glycogen content was determined using a standard curve submitted to the same conditions (Moraes et al., 2007). 3. Results GSK-3 is an important enzyme involved in phosphorylation of more than forty different enzymes in eukaryotic cells (Jope and Johnson, 2004). In this work, approximately 600-bp-long cDNA fragment was amplified from R. microplus 6-day-old eggs by RT-PCR. We used a pair of degenerate primers based on the conserved region of GSK3b kinase domain. The fragment was cloned into pGEMTEasy vector and sequenced. The deduced amino acid sequence from the nucleotide sequence of the RT-PCR product showed high identity (above 70%, at amino acid level) with other GSK-3b (Fig. 1). The 50 - and 30 -RACE PCRs were then performed to obtain the sequence containing the entire GSK-3b mRNA encoding region (Fig. 1). Fulllength cDNA corresponded to a 1,230-bp open reading frame (ORF), and 50 and 30 -untranslated regions (GenBank accession number EF142066). The ORF encodes a protein of 410 amino acids with a calculated molecular mass of 45745.03 Da and an estimated pI of 8.97. The amino acid sequence was analyzed using the ScanProsite algorithm for predicted intra-domain features and was found to have one protein kinase domain between residues 55 and 339. Protein kinase ATP-binding region signature (between amino acids 61 and 85) and serine/threonine protein kinases active site signature (between amino acids 176 and 188) were also identified. We also identified two candidate regulatory residues at positions 9 (serine) and 215 (tyrosine). These amino acids correlate with phosphorylation sites responsible for inactivation and activation, respectively, found only in GSK-3b isoform. Amino acid residues R95, R179 and K204 of a positively charged binding pocket were present in R. microplus GSK-3b. Sequence comparison of R. microplus GSK-3b amino acids showed identities of 76, 76, 75, 75, 75 and 62% to that of D. rerio, A. mellifera, M. musculus, B. taurus, X. laevis and D. melanogaster, respectively (Fig. 1). In order to confirm which RmGSK-3 isoform was present in R. microplus eggs, we performed Western blot analysis using egg homogenates from different days of embryo development, as shown in Fig. 2. We used a specific antibody against b constitutive isoform (GSK-3b) and another against phosphorylated a and b isoforms (anti-pTyr279/216 GSK3a/b) as primary antibodies. Only one band was recognized

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Fig. 1. Amino acid sequence alignment of the GSK-3 in R. microplus, D. melanogaster, X. laevis, A. mellifera, M. musculus, D. rerio and B. taurus. The percentage of identical residues between R. microplus and the other organisms is indicated on the left side. The Protein kinases ATP-binding region signature is shown in bold letters with bold underline. The serine/threonine protein kinases active-site signature is shown in underlined italics. Two predicted and conserved phosphorylation sites for GSK3-Beta are shown in black boxes. The positively charged binding pockets are shown in background shading.

with both antibodies in all tested samples (Fig. 2). Additionally, the absence of both the glycine-rich region at NH2-terminus and a serine residue at position 21 in the predicted amino acid sequence (Fig. 1) may suggest the presence of only GSK3-b isoform in R. microplus eggs.

We also investigated RmGSK-3 relative transcription in fat body, midgut and ovary from partially and fully engorged adult female ticks, and first day eggs. The results depicted a differential expression among organs and between developmental stages (Fig. 3). Partially engorged

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Fig. 2. Western blot analysis using antibodies against anti-pTyr279/216 GSK-3a/b and GSK-3b. Newly oviposited R. microplus eggs were maintained at 27 8C with 80% relative humidity. After oviposition, at the days indicated in the figure, eggs (oviposited on days 1, 6, 12, and 18) were homogenized in the sample buffer, resolved by SDS-PAGE (10%), transferred to PVDF membrane and probed with anti-GSK-3b and antipTyr279/216 GSK-3a/b antibodies. Fig. 4. Real-time PCR analysis of mRNA expression profiles for GSK-3 during R. microplus embryogenesis. For Real-time PCR, the analysis was performed on cDNA synthesized from RNA isolated from eggs collected on days 1, 4, 5, 6, 8, 10, 13, and 16 after the beginning of oviposition and from larvae obtained 21 days after the commencement of oviposition. Respective cDNA quantitative values are shown relative to the concentration of the 40S cDNA. The data mean values + SD, normalized relative to 40S transcript levels. Asterisk (*) denotes the difference between the days during embryogenesis and the significance was determined by two way ANOVA test (Kruskal-Wallis).

Fig. 3. mRNA transcription analysis of GSK-3 in tissues at two stages of development. The mRNA transcription (%) is the relative amount of GSK-3 against each unit of tick 40S mRNA. Each column represents the mean and standard deviation of three analyses. For Real-time PCR, the analysis was performed on cDNA synthesized from RNA isolated from ovary, midgut and fat body from partially engorged adult females (PT) and engorged adult females (TL). Eggs were collected on day 1 after the beginning of engorged female oviposition. Respective cDNA quantitative values are shown relative to the concentration of the 40S cDNA. The data mean values + SD, normalized relative to 40S transcript levels. Asterisk (*) denotes the difference between the tissues and the significance was determined by two way ANOVA test (Kruskal–Wallis).

female organs presented high RmGSK-3 relative transcription when compared with corresponding fully engorged female ticks. In both cases the ovaries presented elevated transcript levels. For eggs, relative expression level was near that observed for ovaries from fully engorged females. Moreover, RmGSK-3 appeared to be differentially expressed during tick embryogenesis (Fig. 4). RmGSK-3 transcripts were present throughout embryo development, but varied considerably when related to the ribosomal protein 40S. We observed that RmGSK-3 mRNA rapidly decreases from the 1st to the 3rd day of development, and increases from the 3rd to the 15th day. After the 15th day of development we detected a nearly 50% reduction in RmGSK-3 mRNA relative transcription. Kinase activity was assayed in egg homogenates using specific peptide substrate for GSK-3b (Wang et al., 1994a,b). We observed that RmGSK-3 activity oscillates during embryogenesis with a peak on the 6th day of

development (Fig. 5). Due to the role of RmGSK-3 in carbohydrate metabolism in vertebrates, we also determined glycogen content in eggs during embryo development. Glycogen amounts were coincidently scarce on day 6. Furthermore, when glycogen content peaked on day 9, RmGSK-3 activity was low. Additionally, RmGSK-3 activity from 6th-day egg homogenates was strongly inhibited by a specific inhibitor, SB-216763 (Fig. 6). 4. Discussion Although first identified through its ability to phosphorylate and inhibit glycogen synthase, GSK-3 has been demonstrated to be involved in many normal and pathological cellular processes, including differentiation, transcription, translation, cytoskeletal organization, tumor genesis, cell cycle progression and the development of

Fig. 5. Glycogen metabolism in R. microplus embryogenesis. The specific activity of GSK-3 was measured as a percentage of the maximum activity (100%) obtained from a standard control reaction. After immunoprecipitation using the anti-GSK-3 antibody, the activity was determined by CREB peptide phosphorylation with respect to the consumption of 32ATP. The glycogen amount in egg homogenates was determined by digestion whit a-amyloglucosidase and glucose released was measured as described in Section 2. Data shown are mean  SEM (n = 4).

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Fig. 6. Effect of SB-216463 on GSK-3 activity in 6-day old egg homogenates. The specific activity of GSK-3 was measured in egg homogenates in the presence or absence of SB216763 inhibitor. After immunoprecipitation using the anti-GSK-3 antibody, the activity was determined by CREB peptide phosphorylation with respect to the consumption of [P32]-ATP. Data shown are mean + SEM (n = 6).

neurorogical diseases (Doble and Woodgett, 2003). Moreover, studies from both vertebrate and invertebrate organisms positioned GSK-3 as a kinase essential for the specification of cell fate in early embryos, within the canonical Wnt signaling pathway (Ferkey and Kimelman, 2000; Siegfried and Kimble, 2002). This pathway determines segment polarity in Drosophila and axis formation in Xenopus. Additionally, it promotes cellular growth and differentiation during mammal embryogenesis. The Wnt signaling also plays essential roles in adult tissues, especially in maintaining stem cells in their pluripotent state (Frame and Cohen, 2001). Due to its fundamental functions and conservation, we investigated GSK-3 in R. microplus ticks. To our knowledge, this work presents the first evidence for the presence of this serine/threonine kinase in tick eggs. The deduced amino acid sequence of RmGSK-3 gene showed high similarity with proteins belonging to families of serine/threonine protein kinases and the presence of the catalytic triad characteristic of GSK-3. Also, the comparison of RmGSK-3 to six other GSK-3 from other species revealed conserved amino acids known to be involved in the activation and inactivation of the enzyme. Furthermore, GSK-3 amino acid sequence possesses important functional domains, characteristic of the beta isoform for GSK3. The Western blot probed with antibody against GSK-3b isoform detected the protein in tick eggs. Large amounts of yolk protein are required for embryogenesis in arthropods; most of these are synthesized outside the oocyte. Fat body, ovaries and midgut are the main organs involved in this process (Telfer, 1965; Atella et al., 2005; Moraes et al., 2007). In this sense, we analyzed RmGSK-3 relative transcription in organs from vitellogenic and non-vitellogenic female ticks. Ovaries from partially engorged (non-vitellogenic) female exhibited the higher levels of RmGSK-3 mRNA transcription. Interestingly, all organs from vitellogenic females presented lower RmGSK-3 transcripition levels when compared with non-vitellogenic ticks. Additionally, RmGSK-3 relative expression in one-day-old eggs is closely related

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to the levels presented by ovaries from vitellogenic females. These results suggest that RmGSK-3 transcripts are present in fat body, midgut and especially in ovary. RmGSK-3 relative expression in one-day-old eggs may suggest an RNA uptake process from vitellogenic ovaries. During oocyte formation, large amounts of RNA, carbohydrates, lipids and proteins accumulate in the organelles called yolk granules (Saito et al., 2005). After oocyte maturation and fertilization, the embryogenesis process starts and yolk proteins, RNA, lipids and carbohydrates are utilized for embryo development (Campos et al., 2006; Moraes et al., 2007). Initially, RmGSK-3 mRNA transcription is decreased from day 1 to day 3, during syncitial blastoderm formation (Campos et al., 2006). After cellular blastoderm formation RmGSK-3 mRNA transcription increases up to day 15, accompanying many morphological events as segmentation and differentiation (Campos et al., 2006). Kinase activity in egg homogenates using CREB-tide peptide as substrate (Fiol et al., 1994) and glycogen determination were performed during embryogenesis. RmGSK-3 activity during embryogenesis was elevated during blastoderm transition between days 4 and 6. Interestingly, glycogen levels were elevated when RmGSK-3 activity was low, between the 12th and the 15th days of development. R. microplus embryos accumulate glycogen and exhibit high PEPCK activity levels at this same period of embryogenesis (Moraes et al., 2007). RmGSK-3 transcription pattern did not correlate with its enzymatic activity during embryogenesis, and coincided with the knowledge that this enzyme is post-transcriptionally regulated by phosphorylation (Fiol et al., 1987). Differences due to energy metabolism requirements from distinct microcompartimentalization within the egg should be considered to explain gluconeogenesis and glycogen synthesis at the same time. However, in order for glucose uptake to succeed, the existence of insulin-like machinery cannot be discarded. One of the cellular roles played by insulin includes glycogen synthesis. Studies of the insulin-dependent inactivation of GSK-3 led to the identification of a negatively acting phosphorylated site controlled by phosphatidylinositol 3-kinase (PI3K) signaling, involving direct phosphorylation of GSK-3 at an Nterminal serine (Ser9 in GSK-3b and Ser21 in GSK-3a) by protein kinase B (PKB) (PKB/Akt) (Cross et al., 1995; Cohen, 1999). Modulation of GSK-3 activity by selective inhibitors has emerged as a strategy to reduce hyperglycemia associated with diabetes (Lochhead et al., 2001). Some of these inhibitors mimic insulin action and provoke antidiabetic effects in cells and animal models (Cline et al., 2002; Nikoulina et al., 2002). Furthermore, RmGSK-3 activity was strongly inhibited in egg homogenates from the 6th day by the specific inhibitor, SB-216463. Therefore, we suggest that RmGSK-3 may participate in glycogen metabolism by means of the influence of an insulin-like responsive machinery in R. microplus eggs. Insulin signaling is also conserved in and associated with the development of different organisms (Brogiolo et al., 2001; Wu and Brown, 2006). The results in this study also emphasize that although RmGSK-3 is an effective enzyme involved in glycogen

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