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1 Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan
2 ERATO, Japan Science and Technology, Honcho 4-1-8, Kawaguchi, Saitama 332-0012, Japan
3 Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Ubiquitin-mediated proteasomal degradation regulates protein stability in many cellular processes. The E3 ubiquitin ligases of the cullin-RING type ubiquitin ligase family include five members, Cul1–5 (Petroski & Deshaies 2005). Recently, 11 BTB proteins were identified as adaptors for substrate recognition by Cul3 ubiquitin ligase in Caenorhabditis elegans (Xu et al. 2003). The substrate selectivity of Cul3 is thus defined by its specific adaptor proteins, the BTB domain proteins (Furukawa et al. 2003). In Saccharomyces pombe, only three kinds of BTB proteins, Btb1, 2 and 3, have been identified, and all function as adaptors of the S. pombe Cul3 homolog pcu3 (Geyer et al. 2003). In Drosophila, Cul3 is involved in the development of mushroom body neurons (Zhu et al. 2005), the wing, sensory organs (Mistry et al. 2004) and the eye (Ou et al. 2003). One of the specific substrates for Drosophila Cul3 (dCul3) is Cubitus Interruptus (Ci), a Hedgehog signaling transcription factor (Zhang et al. 2006). Furthermore, the human BTB-Kelch protein KLHL12 (kelch-like 12) in the Cul3 ubiquitin ligase complex functions as an adaptor for Dishevelled (Dsv), an essential node of the Wnt-signaling branch point between β-catenin-dependent and -independent pathways (Angers et al. 2006). KLHL12 induces Dsv degradation by the Cul3 ubiquitin ligase complex in a Wnt-dependent manner and negatively regulates the Wnt/β-catenin pathway in vivo (Angers et al. 2006). Another domain of the KLHL12 protein, the Kelch repeat, is essential for the recognition of its specific substrate (Angers et al. 2006). Thus, the combination of the BTB domain and the Kelch repeat is likely important for the recruitment of specific substrates to the Cul3 ubiquitin ligase complex. However, the target substrates mostly remain to be identified.
In the present study, 167 modifiers of thorax development were isolated in Drosophila using a misexpression screen with enhancer promoter (EP) lines and thoracic Gal4 drivers (Pena-Rangel et al. 2002). To screen another set of modifiers, we genetically screened gene search (GS) fly lines (Toba et al. 1999) by means of a GAL4/UAS system (Gustafson & Boulianne 1996). As a result, we isolated a BTB-kelch protein, dKLHL18 (Drosophila kelch-like 18)/CG3571. Biochemical purification of dKLHL18 identified a complex composed of five subunits including dCul3. Physical and genetic association among the complex components was seen, and an E3 ubiquitination through dKLHL18/dCul3 was also detected. Thus, our study suggests that dKLHL18 is a substrate adaptor for cullin 3 ubiquitin ligases.
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Results |
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Candidate genes involved in many cellular functions have been successfully isolated by misexpression screens by crossing thoracic Gal4 drivers with EP lines in which controlled misexpression of a given gene was induced by insertion of a transposable element (EP element) containing UAS sites in the gene locus (Pena-Rangel et al. 2002). To isolate a set of modifiers involved in thoracic development of Drosophila melanogaster, we carried out a modified overexpression/misexpression screen using a GS system, which is similar to the EP line, but with an additional UAS/promoter site, allowing induced misexpression of a given gene transcribed from both sides of the insertions (Fig. 1a) (Toba et al. 1999). The GS lines were crossed with a thoracic Gal4 driver, a pannier (pnr) Gal4 (pnrGal4) (Fig. 1c) (Pena-Rangel et al. 2002), and then the adult thorax phenotype in the F1 progeny was assessed (Fig. 1a). Among the identified fly lines, GS15802 and GS10310, were found to target the same gene, CG3571 (Fig. 1b). Both lines exhibited similar phenotypes: slight dorsal thoracic clefts and disorganization of bristles at the centre of the thorax just after adult emergence (Fig. 1c–e, shown by white arrow). However, both GS15802 and GS10310 fly lines were inserted the P-factor at the promoter-proximal region of the CG3572 gene locus (Fig. 1b), we could not exclude a possible contribution of the CG3572 gene to the observed phenotypes. To address this issue, the CG3571 gene was overexpressed by generation of UAS-CG3571 transgenic fly strains. Overexpression of CG3571 by pnrGal4 and UAS-CG3571 also caused a slight cleft in the adult thorax and loss of bristles at the scutellum (Fig. 1e, shown by white arrowhead). This phenotype was similar to those caused by both GS lines (GS10310 and GS15802), suggesting that misexpression of CG3571 induced the observed thoracic phenotypes.
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The CG3571 gene encodes a BTB domain protein which has not been functionally characterized. The protein product of the CG3571 gene contains a BTB domain, a BACK (BTB and C-terminal Kelch) domain of unknown function, and a Kelch repeat motif (Stogios et al. 2005) (Fig. 2a). The CG3571 protein is 53% identical to the human protein KLHL18 (Fig. 2a), thus we refer to the CG3571 gene product as Drosophila KLHL18 (dKLHL18). To explore the possible functions of dKLHL18, we biochemically purified the dKLHL18-associated complex. For affinity purification (Fukuda et al. 2007; Ohtake et al. 2007; Fujiki et al. 2009), a Drosophila S2 cell line stably expressing the FLAG-tagged-dKLHL18 protein was established. As shown in Fig 2b, expression of FLAG-dKLHL18 protein was detected in all of the subcellular fractions. To test whether dKLHL18 forms a nuclear complex, dKLHL18 was purified by anti-FLAG antibody immunoprecipitation followed by size fractionation by glycerol density gradient centrifugation (Fig. 2c). dKLHL18 was detected in the sequential fractions and appeared to form multisubunit complexes (Fig. 2d). To identify the components of the purified dKLHL18-complex, proteins found in fractions 4 to 6 of the glycerol gradient were directly digested with trypsin protease for protein identification using nanoflow liquid chromatography tandem-mass spectrometry (nanoLC-MS/MS). Five proteins were identified: Drosophila Cullin-based E3 ubiquitin ligase, dCul3/guftagu, CG10324, CG5808 and l(2)37Cb (proteins of unknown function) as well as dKLHL18 (Table 1). We also confirmed that dKLHL18 was co-immunoprecipitated with these four factors (Fig. 2e). These results suggest that dKLHL18 forms a complex with an E3 ligase (dCul3) with three factors of unknown function, at least, in S2 cells.
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To address whether all of the identified factors are integrated into one functional complex in intact flies, we examined genetic interactions of dKLHL18 with the identified proteins using the pnrGal4 driver system. First, we examined the genetic relationship between dKLHL18 and dCul3. A fly with thorax-specific overexpression of dKLHL18, was crossed with gft2, which is a mutant fly line with loss of function of dCul3, generated by ethyl methanesulfonate (EMS) mutagenesis (Ou et al. 2003). This line’s phenotype included a decreased number of microbristles at the thorax (Fig. 3b, white arrowhead), whereas the heterozygote without UAS-dKLHL18 (gft2/+; pnrGal4/+) lacked that characteristic (Fig. 3b), suggesting that dKLHL18 genetically interacts with dCul3.
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dKLHL18 is a component of the Cul3-based E3 ubiquitin ligase complex
Among the complex components, only dCul3 has been functionally characterized as an E3 ubiquitin ligase. Therefore, we asked whether the dKLHL18 complex was associated with the dCul3-mediated ubiquitin ligase. S2 cells were treated with MG132, a proteasome inhibitor, and protein expression levels were monitored by Western blotting with a specific antibody for Myc-tag. We found that the expression levels of CG5808 and CG10324 [but not l(2)37Cb] proteins were increased by MG132 treatment (Fig. 4a). Moreover, transient overexpression of dKLHL18 in the presence of MG132 induced accumulation of CG5808 and CG10324 proteins with higher molecular weights when compared with those without dKLHL18 transfection (Fig. 4a). These results suggest that CG10324 and CG5808 proteins are the targets of dCul3/dKLHL18-mediated ubiquitination. To verify whether these proteins are indeed targeted in vivo, we then analyzed the genetic relationship between dCul3 and CG5808, CG10324 and l(2)37Cb. Mutant flies carrying these genes enhanced the phenotype caused by thorax-specific misexpression of dCul3, the pnrGal4/gftEY11031 phenotype (Fig. 4b). This result suggests that these proteins interact with dCul3 in vivo. Moreover, when CG5808 and l(2)37Cb were knocked down in the thorax using pnrGal4 and UAS-transgenes having an inverted repeat sequence (IR2), thorax formation was severely impaired (Fig. 4c). The effects of the knockdown of CG5808 and l(2)37Cb were consistent with the dKLHL18-mediated ubiquitination and degradation observed in the cells (Fig. 4a). Thus, the data indicate that proper levels of CG5808 and CG10324 proteins (controlled by dKLHL18 and dCul3) are required for normal thorax development.
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Discussion |
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Although the biological functions of CG5808, CG10324 and l(2)37Cb remain uncertain, we can speculate about their roles based on the functional domains (Table 1). CG10324 has a KOW motif, found in RNA-binding proteins (Kyrpides et al. 1996) as well as a G-patch domain, which serves as a single RNA-binding domain in splicing factors (Sampson & Hewitt 2003). CG5808 has a RRM (RNA recognition-motif) and a cyclophilin-type prolyl isomerase catalytic domain, and might therefore regulate the assembly of RNA–protein complexes through alteration to protein structures (Zeng et al. 2001). l(2)37Cb has a DEAD-box type RNA helicase domain and its yeast homolog, Prp2p, was revealed to act in a splicing pathway (Teigelkamp et al. 1994). Thus, all of the other components in the nuclear dKLHL18-dCul3 complex bear RNA-related motifs and are implicated in RNA splicing events. Supporting our speculation that the RNA binding complex regulates RNA splicing through dCul3-mediated ubiquitination, it was recently reported that the dynamics of the spliceosome, pre-mRNA splicing complex requires a ubiquitin-mediated process (Bellare et al. 2008). In this regard, it is notable that the yeast homolog of l(2)37Cb, Prp2p, controls one step of RNA splicing (Teigelkamp et al. 1994). Thus, the dKLHL18/dCul3 E3 ligase complex may directly modulate function of the spliceosome through ubiquitination. Better understanding of the functional linkage between RNA splicing factors and ubiquitination may be gained by further analysis of the dKLHL18–dCul3 complex.
BTB domain-containing proteins are evolutionarily conserved from yeast to humans. In fact, 357 BTB-domain containing proteins have been identified in the human genome and 136 in the Drosophila genome. A subset of BTB proteins bear a similar motif organization composed of MATH (Meprin and TRAF homology), kelch and ankyrin repeat or Zn finger. Among these BTB proteins, KCTD5, one of the potassium channel tetramerization domain proteins (Bayon et al. 2008), as well as RhoBTB2, an atypical RhoGTPase (Wilkins et al. 2004), were reported to function as adaptors for Cul3-mediated ubiquitination. A BTB domain containing transcription factor, PLZF, was shown to interact with Cul3 for ubiquitination of some target substrates for degradation (Furukawa et al. 2003). Moreover, the known Cul3 adaptor protein, SPOP, targets ubiquitination of the histone variant macroH2A, the polycomb group protein BMI-1 (Hernandez-Munoz et al. 2005), death domain-associated protein (Daxx) (Kwon et al. 2006) and the Jun kinase phosphatase, Puckered (Liu et al. 2009). Thus, SPOP appears to target a large variety of proteins involved in different intracellular pathways. Taken together, it is conceivable that uncharacterized BTB domain proteins may function as substrate-specific adaptors to target diverse substrates to Cul3 ubiquitin ligase.
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Experimental procedures |
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To generate the FLAG-tagged-dKLHL18 expression plasmid, the entire coding region of dKLHL18 cDNA was fused to FLAG-tag at the N-terminus using forward primer and ligated into the pAct vector (Promega, Madison, WI; GenBank accession no. AF264723). FLAG-dCul3 expression vector was generated in the same way. dCul3, CG10324, CG5808 and l(2)37Cb were fused to Myc-tag at the C-terminus using reverse primer and subcloned into the pAct vector. Deletion constructs of dKLHL18 were amplified by PCR using the FLAG-dKLHL18 expression vector as the template. For generation of the UAS-dKLHL18 transgenic fly, cDNA encoding dKLHL18 was inserted into pUAST vector (Brand & Perrimon 1993).
Antibodies
Antibodies used in this study were rabbit anti-FLAG (F7425; Sigma, St Louis, MO), Myc tag clone 4A6 (05-724; Millipore, Billerica, MA), beta-tubulin dN-17 (sc-20852; Santa Cruz Biotechnology, Santa Cruz, CA), RNA polymerase II CTD clone 8WG16 (ab817-100; Abcam, Cambridge, MA), histone H2B (07-317; Millipore). ANTI-FLAG M2 Affinity Gel and anti-Myc-Tag (agarose) were purchased from Sigma and MBL (Aichi, Japan) respectively.
Cell culture and transfection
Drosophila Schneider (S2) cells were cultured in Schneider’s Drosophila Medium (1X) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum at 25 °C. Transfections were performed following the manufacturer’s protocol using Cellfectin Reagent (Invitrogen) for both transient and stable expression.
Drosophila melanogaster strains and genetic crosses
All flies were maintained at 25 °C on standard cornmeal/agar medium. GS strains were purchased from DGRC (Kyoto Stock Center) and maintained as described previously (Toba et al. 1999). All of the other mutant flies were obtained from Bloomington Drosophila Stock Center. Inverted repeat transgenic flies were obtained from Fly Stocks of the National Institute of Genetics. The UAS-dKLHL18 line was generated by injection of pUAST-dKLHL18 constructs at the BestGene Inc. All crosses were carried out at 25 °C, and 3 days after larvae developed at 28 °C (Takeyama et al. 2002; Ito et al. 2004).
Complex purification
Schneider (S2) cells stably expressing FLAG-tagged dKLHL18 were generated by co-transfection of pCoBlast vector (Invitrogen) and selected in 25 µg/mL Blastcidin (Takara, Shiga, Japan). Nuclear extracts were prepared as described previously (Fukuda et al. 2007) and incubated at 4 °C with 40 µL of anti-FLAG M2 agarose (Sigma) for 6 h. After five washes with wash buffer [20 mM Tris-HCl (pH 7.5), 0.5 M KCl, 0.2 mM EDTA, 10% glycerol, 0.5% NP-40, 0.2 mM PMSF, 2 µg/mL pepstatin, 2 µg/mL leupeptin, 2 µg/mL aprotinin], the protein complex was eluted in glycerol density gradient buffer [20 mM Tris-HCl (pH 7.5), 0.1 M KCl, 0.2 mM EDTA, 0.05% NP-40, 10% glycerol, 0.2 mM PMSF] supplemented with 400 µg/mL FLAG peptide (Sigma). The eluted complex was fractionated by 10–50% glycerol density gradient centrifugation at 4 °C, 182 000 g using a SW-40 rotor (Beckman Coulter, Fullerton, CA) for 16 h. Gradients were fractionated into 1 mL and fractions were analyzed by Western blotting.
Identification by mass spectrometry
The fractions containing the dKLHL18 complex were concentrated by precipitation with 10% trichloroacetic acid. Derived precipitates were dissolved in 7 M guanidine hydrochloride, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA solution, with 5 mM DTT at 37 °C for 30 min, and cysteine SH groups were alkylated with 10 mM iodoacetamide at 37 °C for 1 h. After alkylation, the solution was desalted by methanol/chloroform precipitation, and precipitates were dissolved in 2 M urea, 50 mM Tris-HCl buffer and subjected to trypsin gold (Promega) digestion overnight at 37 °C. The resulting mixture of peptides was directly subjected to the LC-MS/MS analysis system (Zaplous, AMR, Tokyo, Japan) using Finnigan LTQ mass spectrometry (Thermo Scientific, Waltham, MA) and reverse phase C18 ESI column (0.2 x 50 mm, LC assist). The protein annotation data were verified in the Drosophila NCBI sequences (downloaded April, 2007) using Bioworks software (Ver. 3.3; Thermo Scientific) with quantitation featuring the SEQUEST search algorithm.
Immunoprecipitation and in vivo ubiquitination assay
Immunoprecipitation and in vivo ubiquitination assay were performed as described previously (Ohtake et al. 2007). S2 cells were transfected with cellfectin reagent and plasmid vectors, and after 24 h, the cells were treated with 10 µM MG132 for 6 h. Whole cell lysates were derived by extraction with TNE buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40) and immunoprecipitated with anti-Myc tag agarose. The ubiquitination status was analyzed using Western blot.
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Acknowledgements |
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Footnotes |
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* uskato{at}mail.ecc.u-tokyo.ac.jp |
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Received: 16 April 2009
Accepted: 11 May 2009
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