RESEARCH PAPER

Cloning and expression pattern of 14-3-3e fromCulex pipiensDong Hyun KIM1, Yong Hun JO1, Yeon Soo HAN1, Sang Sun KANG2 and Jaesun CHUN3

1 Department of Agricultural Biology, College of Agriculture and Life Science, Chonnam National University, Gwangju, Korea2 School of Science Education and Bio-Science Institute, Chungbuk National University, Chongju, Korea3 Department of Biology Education, Korea National University of Education, Chungbuk, Korea

Correspondence

Jaesun Chun, Department of BiologyEducation, Korea National University ofEducation, Chungbuk 363-791, Korea.Email: chunjs@knue.ac.kr

Received 25 November 2008;accepted 27 November 2008.

doi: 10.1111/j.1748-5967.2009.00223.x

Abstract

14-3-3 proteins play various roles in eukaryotes, such as signal transduction, cellcycle regulation, apoptosis, stress response and cytoskeleton organization. We areinterested in apoptosis induced by mosquito–virus interactions using Culex pipiensas an experimental model. To this end, we cloned a partial fragment (702 bp) of14-3-3e, one of the key proteins known to be involved in apoptosis, from Cx.pipiens. The deduced amino acid sequence (234 residues) had high homology to14-3-3e homologs from various organisms, such as Culex quinquefasciatus (99%),Anopheles gambiae (98%), Aedes aegypti (96%), Drosophila melanogaster (93%),Apis mellifera (91%) and Bombyx mori (89%). Developmental reverse transcrip-tion (RT)-polymerase chain reaction (PCR) analysis showed that Cp14-3-3emRNA is expressed at very high levels in the egg, early pupa, and adult male andfemale, although it was expressed in other developmental stages, such as first andfourth instar larvae. Analysis of RT-PCR for tissue specificity showed that Cp14-3-3e is abundant in the abdomen and ovary, and it was also detectable in all othertissues, such as thorax, midgut and Malpighian tubule. Actinomycin-D, anapoptosis-inducing chemical, induces Cp14-3-3e mRNA expression. Furthermore,poly I : C (RNA virus analog) induces Cp14-3-3e mRNA 3 h after injection. Thus,although further investigation is necessary for elucidating the biological signifi-cance of the induced 14-3-3e, our data suggest that Cp14-3-3e is involved in cellsurvival and signal transduction in Cx. pipiens.

Key words: 14-3-3e, apoptosis, Culex pipiens, Korea, virus.

Introduction

Mosquitoes play a critical role in transmitting human dis-eases. A group of the Culex complex plays a critical role intransmitting viral diseases such as lymphatic filariasis, StLouis encephalitis, equine encephalitis and West Nile virus(WNV) (Kasai et al. 2008). These mosquito-borne viral dis-eases are a major cause of morbidity and mortality in humanpopulations in the world. Vaidyanathan and Scott (2006)reported that midgut cell apoptosis in response to WNVinfection is an active immune response to limit the numberof WNV-infected midgut cells. In doing so, mosquitoesseem to inhibit viral spreading from the midgut to the rest of

the body. In this context, it is intriguing to study virus-induced midgut apoptosis in Culex pipiens.

14-3-3 proteins play a critical role in apoptosis. 14-3-3proteins are intracellular, dimeric, phosphoserine-bindingmolecules that have been identified in eukaryotic organisms,and they are found primarily in the cytoplasmic compart-ment of the cells (Yaffe et al. 2001; van Hemert et al. 2001;Tzivion & Avruch 2002). There are eight mammalianmembers of the 14-3-3 family encoded by separate genes: b,g, z, e, h, q/t and s. Mammalian 14-3-3 proteins bind toprotein kinase C (PKC), phosphotidylinositol 3-kinase 1(PDK1) and apoptosis signal kinase 1 (Ask1) to inhibit theiractivities. 14-3-3 interacts with Raf-1 for Ras-dependent

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© 2009 The AuthorsJournal compilation © 2009 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd

activation of Raf-1 and cell cycle protein phosphatase, celldivision cycle 2 homolog C (cdc25c) for promoting cytoplas-mic translocation of cdc25c (Zha et al. 1996). By binding toapoptosis-promoting protein Bcl associated death promoter(BAD), 14-3-3 prevents BAD from binding to basal celllymphoma-extra large (Bcl-XL). Binding of 14-3-3 to itspartners depends on phosphorylation of the Ser or Thr residuein the specific binding motif RX1–2S/T*X2–3S/T or RX2–3S/T*XP (where * indicates the residue to be phosphorylated)(Yaffe et al. 2001; van Hemert et al. 2001). Thus, it is alsoimportant to characterize protein kinases that phosphorylatethe Ser or Thr residue in these motifs and thereby create14-3-3-binding sites (Yaffe et al. 2001; Chun et al., 2004).

We speculated that the 14-3-3e gene might play a role inapoptosis or survival induced by mosquito pathogens such asPlasmodium species (Han et al. 2000) or virus. To investi-gate our speculation, we cloned the 14-3-3e gene from Cx.pipiens. Then, using reverse transcription (RT)-polymerasechain reaction (PCR) analysis, we found that Cp14-3-3e isabundant in the abdomen and ovary, but also detectable in allother tissues, such as the thorax, midgut and Malpighiantubule. After injection of Actinomycin-D into the adult bodysegment of Cx. pipiens, Cp14-3-3e mRNA was clearlyupregulated from 1 to 6 h. Furthermore, RT-PCR data analy-sis with poly I : C (RNA virus analog) treatment showed thatCp14-3-3e was upregulated at 3 h. Thus, although furtherinvestigation is necessary for elucidating the biological sig-nificance of the induced 14-3-3e, our data suggest that thisnew Cp14-3-3e is involved in cell survival/apoptosis orsignal transduction in Cx. pipiens after viral infection.

Materials and methods

Mosquito model

All experiments were carried out with the Cx. pipiens strains.Mosquitoes were maintained in insectaries at 26–28°C and70% relative humidity, with constant access to 10% sucrose.Mosquitoes were maintained under a photoperiod of 16 hlight : 8 h dark (Jo et al. 2009; Kim et al. 2009).

Cloning of the full length cDNA using RACE PCR

To obtain the full length cDNA sequence of Cp14-3-3e, totalRNA was isolated from adult females using an RNA isolationkit (SV Total RNA Isolation System; Promega, Madison, WI,USA). 5′/3′-RACE PCR was conducted with a RACE kit (5′RACE System for Rapid Amplification of cDNA Ends,Version 2.0 and 3′ RACE System for Rapid Amplification ofcDNA Ends; Invitrogen, Carlsbad, CA, USA). Specificprimers used for 5′-RACE PCR were 5′-GGTAGTCGCCCTTCATTTTG-3′ for gene specific primer (GSP) 1,5′-ACGCCCTTG TTCTCTTCCTT-3′ for GSP 2, and5′-CTCAACCATTTCCGCCGTAT-3′ for GSP 3. In

addition, gene specific primers for 3′-RACE PCR were 5′-CCTGAGCGAGGAAAGCTACAA-3′ (GSP 4) and5′-AGGACGTGAAGACCAAGACG-3′ (GSP 5). PCRproducts were purified using a Gel DNAextraction kit (ExpinGel SV; GeneAll, Seoul, Korea). These products were thencloned into a TA vector (TOPO 2.1 TA Cloning Kit; Invitro-gen). Plasmid was isolated using the plasmid DNA quick-prep kit (Exprep Quick; Geneall), and was sequenced. Thenucleotide sequence of Cx. pipiens 14-3-3e has been assignedthe GenBank Accession Number FJ848979.

Injection

After blood-feeding, 10 ng of poly I : C (RNA virus analog)was injected into the thorax of adult female mosquitoesusing NANOJECT II (Drummond Scientific, Broomall, PA,USA). In addition, 1 ng of Actinomycin-D (an apoptosis-inducing chemical) was also injected into the adult mos-quito. RNA from 1, 3 and 6 h post-injection was analyzed.

Total RNA isolation and RT-PCR

Total RNA was isolated using an RNA isolation kit (SVTotal RNA Isolation System; Promega). For analysis ofdevelopmental patterns of Cp14-3-3e, RNA was isolatedfrom first instar larva, fourth instar larva, early pupa, latepupa, and adult female and male mosquitoes. For tissuespecificity, RNA was isolated from the thorax, abdomen,midgut, Malpighian tubule and ovary. First-strand cDNAwas synthesized from 2 mg total RNA using the AccuPowerRT PreMix (Bioneer) and an oligo (dT)12–18 primer (Invitro-gen). Actin primers were used as a control using 2¥ fluidpremix (Bioneer, Daejeon, Korea) under the same condi-tions. Specific primers used for RT-PCR were as follows:Cp14-3-3e forward primer (5′-GGAACGCGAAAATAACATCTA CAA-3′) and Cp14-3-3e reverse primer (5′-GAATTCTCGGCGGCATCCTTAC-3′).

Results and discussion

Cloning and sequence homology of Cp14-3-3e

To study the role of 14-3-3 in Cx. pipiens, we cloned a partialfragment of Cx. pipiens 14-3-3e using a PCR-based genecloning method. We cloned a partial fragment (702 bp) of14-3-3e, one of the key proteins known to be involved inapoptosis, from Cx. pipiens. Figure 1A shows multiple align-ment of deduced amino acid sequence (234 residues) ofthe Cp14-3-3e with six insect 14-3-3e proteins. Directcomparison between the sequence of amino acids of Cp14-3-3e and Cq14-3-3e shows that there are nine additionalamino acids at the N-terminal end of Cp14-3-3e. In contrast,if the comparison was made between Cp14-3-3e andother insects’ 14-3-3e (Anopheles gambiae, Drosophila

Induction of Culex pipiens 14-3-3e mRNA

193Entomological Research 39 (2009) 192–195© 2009 The Authors. Journal compilation © 2009 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd

melanogaster, Aedes aegypti, Apis mellifera and Bombyxmori), there should be at least 22 amino acid residues at theN-terminal end of Cp14-3-3e. Therefore, to obtain the fulllength cDNA and deduced amino acids of Cp14-3-3e,5′-RACE PCR should be conducted. Figure 1B shows thatCp14-3-3e has high homology to 14-3-3e homolog fromvarious organisms, such as Culex quinquefasciatus (99%), A.gambiae (98%), Ae. aegypti (96%), D. melanogaster (93%),A. mellifera (91%) and B. mori (89%). Phylogenetic treeanalysis shows that Cp14-3-3e belongs to the dipteran14-3-3e family, as expected (Fig. 1C).

Expression pattern

To examine expression patterns of Cp14-3-3e during devel-opmental stages of Cx. pipiens, we conducted RT-PCR usingCp14-3-3e specific primers. As expected, Cp14-3-3e mRNAlevels changed during the life cycle of the Cx. pipiens mos-quito. Cp14-3-3e was expressed at very high levels in theegg, early pupa and adult male, although it was alsoexpressed in other developmental stages, such as first andfourth instar larvae (Fig. 2).

To determine which part of the Culex mosquito is theprimary source of Cp14-3-3e, RT-PCR was performed withcDNA originating from the thorax, abdomen, midgut, Mal-

pighian tubule and ovary (Fig. 3). Cp14-3-3e mRNA wasabundant in the abdomen and ovary, and it was also detect-able in all other tissues, such as the thorax, midgut andMalpighian tubule.

In an attempt to evaluate the potential involvement ofCp14-3-3e in Cx. pipiens apoptosis, RT-PCR was conducted

Figure 1 Sequence homology of Culex pipiens 14-3-3e. (A) Multiple alignment of Cp14-3-3e with other species’ 14-3-3e. Amino acid sequencesused for 14-3-3e alignment are as follows: Culex quinquefasciatus (Cq; GenBank Accession No. XP_001863567), Anopheles gambiae (Ag;Accession No. XP_322009), Aedes aegypti (Aa; Accession No. XP_001655109), Drosophila melanogaster (Dm; Accession No. NP_732312), Apismellifera (Am; Accession No. XP_392479) and Bombyx mori (Bm; Accession No. NP_001091764). The sequences used for this alignment wereretrieved from an NCBI BLAST search. The CLUSTAL X program was used for multiple sequence alignment. “*” indicates completely conservedresidues in all seven sequences. Both “:” and “.” show highly conserved residues. “-” denotes gaps. (B) A matrix comparison of Cp14-3-3e withseveral known 14-3-3e is presented. Values indicate the percentage identity. (C) Phylogenetic tree analysis of Cp14-3-3e and other species’ 14-3-3e.

Figure 2 Temporal expression patterns of Cp14-3-3e during devel-opmental stages. L1, first instar larvae; L4, fourth instar larvae; EP,early pupae; LP, late pupae; AF, 4 day old adult female mosquitoes;AM, 4 day old adult male mosquitoes.

Figure 3 Spatial expression patterns of Cp14-3-3e in adult female ofCulex pipiens. Thx, thorax; Ab, abdomen; Mg, midgut; Mt, Malpighiantubule; Ov, ovary.

D. H. Kim et al.

194 Entomological Research 39 (2009) 192–195© 2009 The Authors. Journal compilation © 2009 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd

after injection of Actinomycin-D to the adult thorax of Cx.pipiens. This experiment showed that Cp14-3-3e mRNA isupregulated from 1 to 6 h (Fig. 4). We examined whether theexpression level of Cp14-3-3e is induced by Actinomycin-D,an apoptisis-inducing chemical. Kumar et al. (2004) previ-ously reported that peroxidase is highly induced during apo-ptosis in midgut cells by either invasion of Plasmodiumberghei or treatment with Actinomycin-D. In this context,Cp14-3-3e might be involved in midgut cell apoptosis,although the precise biological significance of the Cp14-3-3e induction remains to be further studied. In addition,RT-PCR was conducted to determine whether poly I : C(RNA virus analog) influences the expression level of Cp14-3-3e. Data analysis shows that Cp14-3-3e was upregulated3 h after injection (Fig. 5). However, the biological signifi-cance of the induced 14-3-3e remains to be elucidated.

Conclusion

In conclusion, we have cloned Cp14-3-3e and partially char-acterized it at the level of mRNA. This investigation wasperformed to determine whether the adaptor molecule14-3-3 is induced as a stress protein after treatment withActinomycin-D or poly I : C (RNA virus analog). Todevelop a deeper biological understanding of a 14-3-3protein as a molecular chaperone, further study remains tobe performed under other stress conditions with Cx. pipiens.In the present study, we first found that the expression ofCp14-3-3e is significantly upregulated with treatment ofActinomycin-D or poly I : C. Our observations suggest thatCp14-3-3e is an inducible molecular chaperone to overcome

the stress condition. However, the chaperone activity ofCp14-3-3e should be observed together with another func-tional protein, such as mitochondrial protein, citrate syn-thase (CS) or BCL2. These in vivo and in vitro observationsestablish the role of Cp14-3-3e protein as a molecular chap-erone, particularly in apoptosis.

Acknowledgments

This work was supported by a Korea Research FoundationGrant funded by the Korea Government (MOEHRD, BasicResearch Promotion Fund) (KRF-2005-003-C00142).

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Figure 4 Upregulation of Cp14-3-3e by Actinomycin-D.Actinomycin-D was injected into the thorax, and 14-3-3e graduallyincreased in a time-dependent manner. C, control.

Figure 5 Induction patterns of 14-3-3e from Poly I : C (RNA virusanalog) injected mosquitoes. Poly I : C was injected into the thorax.Cp14-3-3e was overexpressed 3 h after poly I : C injection. C, control.

Induction of Culex pipiens 14-3-3e mRNA

195Entomological Research 39 (2009) 192–195© 2009 The Authors. Journal compilation © 2009 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd