hDKIR, a human homologue of the Drosophila kelch protein, involved in a ring-like structure

Experimental Cell Research 300 (2004) 72 – 83 www.elsevier.com/locate/yexcr

hDKIR, a human homologue of the Drosophila kelch protein, involved in a ring-like structure Angela Maia,b, Sang-Kee Junga,b,*, Shin Yoneharab a

b

M, F, L Science Center, Tensei-suisan Co., Saga 847-0193, Japan Institute for Virus Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Received 30 September 2003, revised version received 14 June 2004 Availalable online 31 July 2004

Abstract We have previously purified and cloned an apoptosis-inducing protein (AIP) derived from fish infected with the anisakis simplex. Recently, we identified a series of AIP-responsive genes in the HL-60 cell line using a subtractive hybridization method. Here we report the molecular cloning and characterization of one of these genes, which encodes a novel human kelch protein containing 568 amino acid residues, termed hDKIR. The Drosophila Kelch protein localizes to a ring canal structure, which is required for oocyte development. When hDKIR was expressed in cultured-mammalian cells, hDKIR localized to a ring-like structure. Furthermore, when coexpressed with Mayven or Keap1, hDKIR bound to Mayven and recruited Mayven into ring-like structures perfectly. This indicates that kelch homologues can interact with each other in a specific manner and such interaction can affect the subcellular localization of kelch proteins. Finally, domain analysis revealed that both the N-terminal POZ (poxviruses and zinc fingers) and intervening region (IVR) domains of hDKIR are essential for ring-like structure activity, suggesting that the development of the ring-like structure is independent of the ability to bind actin. D 2004 Elsevier Inc. All rights reserved. Keywords: hDKIR; Kelch; Ring canal

Introduction Several organisms use stable intercellular bridges as cytoplasmic connections, probably to allow rapid transfer of information and organelles among cells [1–3]. In Drosophila, ring canals connect the developing oocyte to supporting nurse cells [4]. The Drosophila Kelch protein is a structural component of the ring canals and loss of the Kelch protein causes disorganization of ring canal formation resulting in infertility [5]. A major class of the kelch family contains N-terminal BTB/POZ domain and C-terminal kelch repeats, and is found in a diverse set of organisms [1,6], including virus [7,8], yeast [9,10], Caenorhabditis elegans [11], Drosophila [12], and mammals. The BTB (bric-a-brac, * Corresponding author. M, F, L Science Center, Tensei-suisan, Co., 1-25 Nakase-dori, Karatsu, Saga 847-0193, Japan. Fax: +81 955 75 4441. E-mail address: [email protected] (S.-K. Jung). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.06.023

tramtrack, broad complex)/POZ (poxvirus and zinc fingers) domain was identified in a group of transcription factors such as Bcl6 [13–15], PLZF [16,17], Drosophila Tramtrack [18], and bric-a-brac proteins [19,20]. It has been reported that the BTB/POZ domain is implicated in the regulation of subcellular localization and gene expression through formation of multimeric complexes [21–24]. The kelch-repeats motif was originally discovered as a 6-fold tandem element in the sequence of the Drosophila Kelch protein [4]. The predicted h-sheet repeats structure of kelch-repeat motifs may have functional significance in actin binding, protein folding, or protein–protein interactions [1,6,25]. In mammals, the kelch family proteins containing Nterminal BTB/POZ domain have diverse functions. The human Mayven protein has been characterized as an actinbinding protein that is involved in the dynamic organization of the actin cytoskeleton in brain cells [26]. Keap1 sequesters the Nrf2 transcription factor (NF-E2-related

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factor 2) in the cytoplasm [27,28]. This interaction is downregulated in the presence of electrophilic agents that stimulate translocation of Nrf2 to the nucleus, initiating a cytoprotective electrophilic counterattack response. It has also been reported recently that Keap1 negatively regulates Nrf2 by enhancing its rate of proteasomal degradation [29,30]. Gigaxonin is mutated in a human autosomal recessive neurodegenerative disorder named giant axonal neuropathy [31–33]. The kelch family also includes murine ENC-1 (required for cell differentiation) [34,35], human and bovine Calicin (a component of the sperm head) [36], human NS-1BP (implicated in pre-mRNA splicing) [37], actinfilin (brain-specific actin-binding protein) [38], Nd1 (involved in stabilization of actin filaments) [39], and NRP/ B (involved in neuronal differentiation) [40]. Recently, we cloned a novel kelch protein, hDKIR, identified due to its differential expression following apoptosis-inducing protein (AIP) treatment. AIP derived from parasite-infected fish induces apoptosis in mammalian cells through H2O2 production or L-amino acid depletion [41,42]. In addition, at low concentrations, AIP also inhibits cell growth. We have attempted to investigate differentially expressed genes in HL-60 cells using a subtractive hybridization technique under the condition of cell growth inhibition induced by AIP treatment. Several cDNA fragments were isolated, and we cloned the full-length hDKIR gene from one of them. In this paper, we report the cloning, molecular analysis, and cellular distribution of the novel kelch protein hDKIR.

Methods Cloning of hDKIR cDNA The subtractive hybridization was performed with mRNA purified from AIP-treated HL-60 cells as a tester using the PCR-select cDNAs subtraction kit (Clontech). Clones isolated using the subtraction method encoded partial genes approximately 500 bp in length. To obtain the full-length hDKIR cDNA, a human fetal brain library (Gibco Life Technologies) was screened with the radiolabeled subtracted probe. The cDNA clones selected were sequenced and subjected to homology search using the NCBI databases. Plasmid construction The full-length hDKIR was prepared by PCR amplification and subcloned into the mammalian expression vector pME18S with the c-terminal Myc epitope (AEEQKLISEEDLN) tag or the FLAG epitope (DYKDDDDK) tag [43]. Primers containing EcoRI site in the 5V primer (5VGGAATTCCACCATGGGAGGCATTATGGCCCC-3V) and XhoI site in the 3V primer (5V-CCGCTCGAGCTTCTCGCGGAGAACACAAAC-3V) were used. The resultant PCR

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products were digested with EcoRI and XhoI, and ligated into pME18S-C-FLAG or pME18S-C-Myc. cDNA encoding hDKIR-FLAG was excised from pME18S-hDKIR-FLAG and introduced to EcoRI–XbaI sites of the pIND vector of the ecdysone-inducible expression system (Invitrogen). Keap1 was a kind gift from Dr. M. Yamamoto (Tsukuba University, Tsukuba, Japan). Mayven was cloned from a human fetal brain library (Gibco Life Technologies) using reported sequences [26]. cDNAs encoding various deletion mutants of hDKIR, Keap1, and Mayven were also prepared and subcloned into pME18S-C-FLAG or pME18S-CMyc using the same methods described for the full-length hDKIR. All subcloned cDNAs were analyzed by dideoxynucleotide sequencing to make sure no unwanted mutations had been introduced. The following hDKIR variants were prepared: POZ, M1-L130; intervening region (IVR), D131-E281; KELCH, E282-K568; POZ/IVR, M1-E281; IVR/KELCH, D131-K568; POZ/KELCH, M1-L130 connected to E282– K568. The numbers correspond to amino acid positions in hDKIR. PCR analysis PCR was performed using a panel of 16 different human tissue cDNAs (human MTC panels I and II, Clontech) as templates and the Advantage 2 Polymerase Mix (Clontech). The cDNA fragments encoding hDKIR (698–1017 bp) were produced using the 5V primer 5VTCCTGAAGTGGTACAGCATGAAG-3Vand the 3Vprimer 5V-TGGGTCCCTGCATCTGACTC-3V. The PCR cycle started at 96.58C for 1 min followed by a three-step cycling for 28 cycles: denaturation at 968C for 25 s, annealing at 648C for 25 s, and extension at 728C for 1 min. This was followed by a final extension step at 728C for 5 min. PCR for G3PDH was performed to ensure that an equal quality and quantity of cDNA was used for each reaction. The primers for G3PDH were 5V-TGAAGGTCGGAGTCAACGGATTTGGT-3V and 5V-CATGTGGGCCATGAGGTCCACCAC-3V. The PCR products were subjected to electrophoresis on a 1.5% agarose gel. All of the PCR reactions were performed under conditions in which amplification did not reach saturation levels. Cell culture and transfection HeLa and 293 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), penicillin–streptomycin (100 U/ml–100 Ag/ml), and 2 mM glutamine at 378C in a humid atmosphere (5% CO2–95% air). All culture reagents were purchased from Gibco Life Technologies. Expression vectors containing Mayven, Keap1, hDKIR, or various deletion mutants were transfected into HeLa or 293 cells by the Lipofectamine-Plus method (Gibco Life Technologies). The cells were examined 24–48 h after transfection.

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Fig. 1. Alignment of the amino acid sequences of hDKIR, Mayven, and Drosophila Kelch. The amino acid sequence of hDKIR is predicted from the nucleotide sequence of hDKIR cDNA containing an open reading frame that codes for a protein of 568 amino acids with the Kozak consensus at the initiation codon. Sequences were aligned by clustal method. Identical and similar residues are shaded in black and light, respectively. The BTB/POZ domain is boxed and six kelch-repeats are indicated by arrows.

hDKIR stable transfectant was established as following procedure. hDKIR-FLAG/pIND ecdysone-inducible vector and pVgRXR ecdysone receptor were transfected into HeLa cells sequentially according to the manufacturer’s protocol (Invitrogen). The hDKIR-FLAG-positive clones were selected by G418 and Zeocin resistance, and cells were stained with anti-FLAG antibody before and after induction to confirm expression of hDKIR-FLAG. The expression of hDKIR-FLAG was induced by addition of the inducer Muristerone A (1 mM). Immunofluorescence The transfected cells were washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.2% Triton-X 100 in PBS for 5 min, and blocked in 3% BSA in PBS for 1 h. Cells were incubated for overnight with antiMyc (9E10) or anti-FLAG (M2, Sigma) antibodies in PBS containing 0.1% BSA and 3% normal goat serum, washed, and then incubated with a 1:2000 dilution of Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes), Alexa Fluor 568-conjugated goat anti-mouse IgG (Molecular Probes), or Cy-3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Lab). For double staining of hDKIR and intracellular membrane, cells were first stained for hDKIR as indicated above and then incubated for 20 min with cation lipophilic dye 3,3V-dihexyloxacarbocyanine iodide, DiOC6(3), (Molecular Probes) at 1 Ag/ml; for double staining

of hDKIR and cytoskeleton, cells were then incubated for 40 min with Alexa Fluor 568 phalloidin (Molecular Probes) at 0.2 U/ml; for double staining of hDKIR and Golgi, cells were then incubated for 20 min with Alexa-488-conjugated wheat germ agglutinin (Molecular Probes). Lysosome staining was performed on cells during the last 1 h in culture using 50 nM LysoTracker Red DND-99 reagent (Molecular Probes, L7528). Following incubation with LysoTracker reagent, cells were stained for hDKIR as described above. Immunostained cells were washed, mounted with PermaFluor antifade reagent (Shandon Immunon), and examined and photographed using a Zeiss Axioplan epifluorescence microscope equipped with 63  1.4 NA object lens (Carl Zeiss Co., Ltd.)

Fig. 2. Tissue expression of hDKIR. PCR was performed using MTC panels (Clontech) as templates. PCR amplification was carried out through 28 cycles under conditions in which PCR amplification did not reach saturation. G3PDH was used as a control.

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or a BioRad Radiance confocal laser scanning microscope (BioRad). Images were prepared and analyzed using Adobe Photoshop software. Immunoprecipitation and Western blot analysis The transfected HeLa cells were lysed in lysis buffer (50 mM Tris–HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1

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mM phenylmethylsulfonyl fluoride, 2 Ag/ml aprotinin, leupeptin, and pepstatin). Lysates were clarified by centrifugation at 20,000  g for 30 min at 48C and the supernatants were gently agitated at 48C for 3 h with antiMyc or anti-FLAG antibodies following pre-clear with beads only, then followed by incubation with protein G-Sepharose beads (Amersham-Pharmacia). Immunoprecipitates were then collected by centrifugation, washed five times, and

Fig. 3. The intracellular distribution of hDKIR. (A) HeLa cells transfected with FLAG-tagged hDKIR, FLAG-tagged Mayven, or FLAG-tagged Keap1 cDNAs were fixed and permeabilized as described in Methods section. Each of these proteins was detected by immunofluorescence using mouse anti-FLAG antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. Scale bar, 10 Am. (B) To obtain higher magnification, the staining cells were also viewed using a BioRad Radiance confocal laser scanning microscope. Serial z sections (0.25 Am) were collected and analyzed using LaserSharp image processing software (BioRad). The images shown from left to right indicate from the bottom to the top of the cell. (C) HeLa cells transfected only with FLAG-tagged hDKIR (a, b, d, and e) or cotransfected with pDsRed2-Mito (c) or cotransfected with Bip-GFP (f) were fixed and stained as described in Methods section. Cells were labeled only with anti-FLAG antibody (c and f) or double labeled with a maker for intracellular membrane (DiOC6(3)) (a), lysosome (LysoTracker Red) (b), mitochondria (pDsRed2-Mito) (c), cytoskeleton (Alexa Fluor 568 phalloidin) (d), Golgi (Alexa-488-conjugated wheat germ agglutinin) (e), or endoplasmic reticulum (Bip-GFP) (f). The left-column panels show the fluorescence staining of the expressed hDKIR proteins. Middle-column panels show the fluorescence staining of organelle makers. The images were merged for analysis of colocalization (the right column panels). No overlap between the distribution of hDKIR and that of any of the organelle makers examined was detected. The images were analyzed using Adobe Photoshop software. Scale bar, 10 Am. (D) Muristerone-induced FLAG-tagged hDKIR expression HeLa cells were prepared for immunofluorescence staining. hDKIR was visualized with anti-FLAG antibody. The image was analyzed using Adobe Photoshop. Scale bar, 10 Am.

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Fig. 3 (continued).

A. Mai et al. / Experimental Cell Research 300 (2004) 72–83

Fig. 3 (continued).

denatured in SDS loading buffer in preparation for electrophoresis. Samples were separated by SDS-PAGE (10%) and then transferred to PVDF membranes. The membranes were blocked with 5% skim milk and probed with the primary antibody for 1 h at room temperature. Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibodies and chemiluminescence detection reagents (Renaissance; NEN Life Science).

Results hDKIR encodes a novel human kelch protein A probe corresponding to a hDKIR cDNA fragment, one of about fifty genes differentially expressed during growth arrest upon AIP treatment of HL-60 cells, was used to screen a human fetal brain cDNA library to obtain a full-length 3344-bp cDNA. The cDNA contained an open reading frame for a protein of 568 aa with a predicted relative molecular mass of 63 kDa (Fig. 1). The coding region of hDKIR is exactly the same as the C3IP1, a human clone with unknown function isolated by the NCBI annotation project (NP_067646). The sequence of hDKIR consists of a BTB/ POZ domain at its N-terminus and six kelch-repeats motif at its C-terminus. The overall domain of hDKIR shares a significant homology (41% identity and 59% similarity) with the 76.5-kDa Drosophila Kelch protein and a high degree of homology with several mammalian kelch family proteins including human Mayven or murin Keap1 [4,26,27]. The Drosophila Kelch protein is the largest with 689 aa, and it contains a unique N-terminus of approximately 110 aa prior to the BTB/POZ domain, characterized by two stretches of glutamine residues. However, hDKIR is the smallest protein with 568 aa and lacks the N-terminal extension of Drosophila Kelch. The homology among the three proteins spans the length of the entire protein, including the intervening region between the POZ domain and the kelchrepeats as shown in Fig. 1, suggesting that Drosophila Kelch is the ancestral gene of both Mayven and hDKIR. The POZ domain also has 39–42% identity to the POZ domain of zinc

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finger transcriptional factor (Bcl6, PLZF) [14,17,20]. The signature bGG. . .Y-X6-WQ motif is well conserved in kelchrepeats of hDKIR, indicating that they may form the same hturn propeller structure as seen in the crystal structure of the kelch-related protein galactose oxidase from Hypomyces rosellus [1,44,45]. To examine gene expression patterns of hDKIR in human tissues, PCR screening was performed using human multiple tissue cDNA panels I and II (Clontech) as described in Methods. The hDKIR transcript was ubiquitously expressed at various levels in tissues examined. hDKIR was found at high level in lung, pancreas, prostate, spleen, and testis (Fig. 2), suggesting that hDKIR might play a significant role in these particular organs. Characterization of hDKIR Members of the growing kelch family are present throughout the cell and have diverse functions [3,6]. In Drosophila, Kelch localizes to the rim of canals that are responsible for intercellular cytoplasm transport. We were thus initially interested in the cellular distribution of hDKIR. The FLAG-tagged hDKIR was expressed in HeLa cells and detected by immunofluorescent analysis using the antiFLAG antibody. As shown in Fig. 3A, the intracellular distribution of hDKIR shows a striking ring-like architectural pattern similar to the Drosophila ring canal and is particularly intense in the perinuclear area. Simultaneously, we also examined the subcellular localization of Mayven and Keap1, the two other mammalian kelch proteins. Both Mayven and Keap1 were found to localize in the cytoplasm of transfected cells as shown previously [26,27]. We also investigated whether hDKIR was targeted to any known compartment or membrane core using general subcellular makers (intracellular membrane, DiOC6(3); lysosome, LysoTracker; mitochondria, pDsRed2-Mito vector; Golgi, Alexa-488-conjugated wheat germ agglutinin; endoplasmic reticulum, Bip-GFP; cytoskeleton, Alexa Fluor 568 phalloidin). However, neither of these makers was detected in the hDKIR structures and the ring-like structures did not display morphological similarity to the other known subcellular organelles (Fig. 3C). It indicates that the ring-like structures do not contain any component of the known organelles or membranes. These results are consistent with the finding that hDKIR does not contain a signal sequence or a transmembrane domain. To gain a high magnification image of the ring-like structures, we obtained 26 Z sections of 0.25-Am thickness from the bottom to the top of the cell by using confocal laser scanning microscope; however, we have presented only six representative sections in Fig. 3B. As shown in Fig. 3B, the pore structures containing hDKIR are never occluded, indicating that the hDKIR structures are really ring-like, not dots or puncta. Additionally, we confirmed the existence of the hDKIR ring-like structure in other transfected cells such as 293, NIH3T3, and CHO (data not shown). Moreover, we established ecdysone-

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inducible stable expression transfectants of hDKIR, and the ring-like structures were also observed under low protein expression condition following ecdysone induction (Fig.

3D). These results suggest that hDKIR is the first kelch homologue in mammals that can assemble into ring-like structures.

Fig. 4. hDKIR associates with Mayven and recruits the Mayven protein to ring-like structures. (A) HeLa cells were transfected with the combinations of plasmids as indicated at the top. After 48 h, total cell lysates were prepared, immunoprecipitated with anti-Myc antibody (lane 1 was immunoprecipitated with anti-Myc mixed anti-FLAG antibody), separated by SDS-PAGE, and transferred to PVDF membranes. The membranes were probed with antibodies as indicated on the left, and the reacted proteins were detected using a chemiluminescence reagent. The negative control for immunoprecipitation was performed without anti-Myc antibody and no band was detected. The bottom panel shows the immunoblots analysis of whole cell lysates. (B) hDKIR–Myc with Mayven– FLAG, hDKIR–Myc with Keap1–FLAG, or Mayven–Myc with Keap1–FLAG cDNAs were transfected into HeLa cells. The expression of each protein was detected using mouse anti-FLAG antibody or rabbit anti-Myc antibody, followed by Alexa Fluor 488-conjugated goat anti-mouse Ig or Cy-3-conjugated goat anti-rabbit Ig antibodies, respectively. Photographs in the same rows were taken from the same fields using a filter for rhodamine (left) or fluorescein (middle). The images were analyzed and merged (right) using Adobe Photoshop software. Scale bar, 10 Am.

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Fig. 4 (continued).

hDKIR associates with Mayven and recruits the Mayven protein into ring-like structures Mayven is a cytoplasmic protein that is thought to play a role in the dynamic organization of the cytoskeleton of neurons [26]. Keap1 suppresses the transcriptional activity of Nrf2 and works as a sensor for oxidative stress [27,28]. Because the BTB/POZ domain has been proposed to mediate protein–protein interactions for either homomeric or heteromeric dimerization [22– 24], we supposed that hDKIR might interact with other mammalian kelch homologues and regulate their functions. To examine this possibility, we cotransfected Myctagged hDKIR with either FLAG-tagged Mayven or FLAG-tagged Keap1 cDNAs into HeLa cells and examined their ability to form stable complexes using immunoprecipitation assay. The complexes from cotransfected HeLa cells were immunoprecipitated with the antiMyc antibody and then analyzed by immunoblotting with the anti-FLAG antibody. As shown in Fig. 4A, MayvenFLAG was detected in the hDKIR–Myc immunoprecipitates and Keap1–FLAG was detected in the Mayven-Myc immunoprecipitates, indicating that Mayven formed a complex with hDKIR or Keap1. In contrast, Keap1– FLAG was not detected in the hDKIR–Myc immunoprecipitates. Furthermore, we examined whether Mayven was involved in the hDKIR ring structures. HeLa cells transfected with hDKIR–Myc in combination with Mayven-FLAG or Keap1–FLAG were processed for immunofluorescence analysis. As shown in Fig. 4B, Mayven expressed together with hDKIR localized to the ring structures of hDKIR completely. We also observed

that Keap1 was colocalized with Mayven in the cytoplasm. While Keap1 coexpressed with hDKIR was shown to remain in the cytoplasm mostly with several puncta occasionally, colocalization staining was not observed in the merged image of Keap1 dots and hDKIR ring-like structures. When hDKIR, Mayven and Keap1 were coexpressed, only Mayven was detected in the hDKIR immunoprecipitates (Fig. 4A, lane 5) and was recruited to hDKIR ring (data not shown), resulting in destruction of the Mayven–Keap1 colocalization. It possibly suggests that the hDKIR–Mayven interaction is predominant over the Mayven–Keap1 interaction or hDKIR–Mayven interaction competes with the Mayven– Keap1 interaction for their complex formation. These results indicate that hDKIR associates with Mayven in the same complex and Mayven is recruited to ring-like structures on hDKIR-dependent manner. The POZ/IVR domain of hDKIR participates in the development of ring-like structures Next, we examined which domain of hDKIR is required for ring assembly activity. First, the deletion variants illustrated in Fig. 5A were transfected into HeLa cells and detected by immunofluorescent procedures. As shown in Fig. 5B, the POZ domain had a diffuse distribution with some local concentrations in membrane ruffles; the IVR domain was present throughout cell body; the KELCH domain was located in the nucleus; the POZ/KELCH and IVR/KELCH domains were cytoplasmic; while, importantly, the POZ/IVR domain showed the ring structure distribution more prominent than the full-length intact

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protein. Further, when cotransfected in HeLa, the POZ/IVR and full length of hDKIR colocalized to ring structures completely (data not shown). Next, HeLa cells were transfected with the indicated combination of plasmids

Fig. 5 (continued).

Fig. 5. Domain analysis of hDKIR required for ring-like structure formation. (A) Schematic diagrams of full-length hDKIR and its deletion variants. POZ domain includes approximately 130 amino acids. The IVR is the approximately 150 amino acid region between the POZ and the six kelch-repeats (KELCH). The details were described in Methods. (B) HeLa cells transfected with the deletion variants indicated in A were fixed and permeabilized as described in Methods. Each of these proteins was detected by immunofluorescence staining using mouse anti-FLAG antibody followed by fluorescein isothiocyanate-conjugated goat anti-mouse Ig. (C) HeLa cells were transfected with the combinations of plasmids as indicated at the top. Cell lysates were prepared, immunoprecipitated with anti-Myc antibody, separated by SDS-PAGE, and transferred to PVDF membranes. The membranes were probed with antibodies as indicated on the left, and the reacted proteins were detected using a chemiluminescence reagent (upper and middle panels). The bottom panel shows the immunoblots analysis of whole cell lysates.

and the lysates from transfected cells were subjected to immunoprecipitation and immunoblot analyses. In addition to the intact form, also the POZ/IVR domain, but not the KELCH domain, was detected in the immunoprecipitated complex of hDKIR (Fig. 5C). In conclusion, these results suggest that the POZ/IVR domain of hDKIR is required for ring-like structure localization and that the KELCH domain does not contribute to the ring-like structure assembly. Additionally, we notice a substantial increase in hDKIR levels in cells cotransfected with POZ/IVR, but the mechanisms remain to be elucidated (Fig. 5C).

Discussion In this paper, we cloned a novel gene encoding a kelch protein with two major elements, the N-terminal POZ domain and the C-terminal kelch-repeats motif, termed hDKIR. When hDKIR was expressed in cells, the subcellular distribution of hDKIR showed a striking ring-like structure pattern. While a growing number of kelch family member proteins have been identified, the ring-like structure

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has never been investigated in other mammalian kelch homologues. hDKIR was isolated and cloned as differentially expressed cDNA during growth arrest upon AIP treatment of HL-60 cells. Keap1 was identified to be a negative regulator of Nrf2, and the Keap1–Nrf2 complex was reported to constitute a sensor system for oxidative stress [27,28]. Electrophiles and reactive oxygen species liberate Nrf2 from its cytoplasmic repressor Keap1 and provoke the translocation of Nrf2 from the cytoplasm to the nucleus. Because AIP is a H2O2producing flavoprotein [41,42], we expected that hDKIR could collaborate with Nrf2 or Keap1 in sensing oxidative stress, whereas hDKIR did not affect the subcellular distribution of Nrf2 in the presence of electrophilic agents (data not shown). In addition, the immunoprecipitation and immunofluorescence analyses showed that hDKIR did not interact with Keap1 (Fig. 4) or Nrf2 (data not shown). It seems that hDKIR does not regulate the activity of Nrf-2 or Keap1 at this point. hDKIR associated with Mayven and recruited Mayven into ring-like structures (Fig. 4). Mayven and hDKIR both showed ubiquitous gene expression pattern. Mayven was predominantly expressed in the brain [26]. In contrast, hDKIR was found at high levels in lung and pancreas (Fig. 2). The physiological function for the association of hDKIR with Mayven is currently not understood. Because coexpression of hDKIR with Mayven caused obvious change in cellular distribution of Mayven, it is possible that hDKIR has a role on modulating Mayven function in some tissues. It has been reported that the POZ domain of several kelch proteins forms homomeric oligomer [21,22]. To examine the relationship between oligomerization and the ring-like structure assembly, we performed localization and precipitation studies using various deletion mutants of hDKIR and chimeric proteins of hDKIR with Mayven. As shown in Fig. 5B, the POZ/IVR domain of hDKIR is sufficient to localize to ring-like structures. We also observed that hDKIR or Mayven was colocalized and coprecipitated with the POZ/IVR domain of hDKIR (Fig. 5C and data not shown). Furthermore, the transfected chimeric proteins POZr/IVRm (containing POZ domain of hDKIR and IVR domain of Mayven) and POZm/IVRr (containing POZ domain of Mayven and IVR domain of hDKIR) showed a diffuse cytoplasmic staining of the fusion protein in HeLa cells (data not shown). When coexpressed with each one of the chimeric proteins, hDKIR was co-immunoprecipitated with both the chimeric proteins, but only POZr/IVRm showed partial colocalization with hDKIR to ring-like structures (data not shown). Overall, these results suggest that the POZ/IVR domain participates in the complex formation between the selected kelch families in vivo and that the protein–protein interactions between the kelch families are not sufficient to form or localize to the ringlike structure. Further, both the POZ and IVR domains of hDKIR are essential for localization to ring-like structure. The kelch-repeats motifs found in several actin-associated proteins, Mayven, Nd1, and ENC1, have been shown

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to co-immunoprecipitate with actin through the C-terminal KELCH region, and Drosophila Kelch has been reported to colocalize with actin in ring canals through its C-terminal KELCH domain as well [12,26,35,39]. However, some other kelch-repeats proteins do not bind directly to or colocalize with actin. For example, Calicin is located within an actin-negative structure of spermatozoa termed the calyx, which is involved in the morphogenesis of the spermatocyte in mammal [36]. GFPKeap1 did not colocalize to actin stress fiber or cortical F-actin [27]. As shown by immunofluorescence analysis (Fig. 3C), actin did not show apparent colocalization to the ring-like structure of hDKIR in HeLa cells. We also could not detect actin in the immunoprecipitates of hDKIR or Mayven in HeLa cells (data not shown). From these results and the data of hDKIR deletion mutants, we conclude that the ring assembly activity is independent of actin binding by hDKIR under the conditions studied here. A notable difference between the transfected hDKIR in HeLa cells and Drosophila Kelch is the lack of actin binding activity in hDKIR, suggesting that hDKIR in mammals is not functionally equivalent to Drosophila Kelch. Although our data concerning the localization of hDKIR ring-like structures were obtained using the overexpressing transfected cells, there are some evidences supporting that the phenomenon is not due to simple overexpression of hDKIR. First, hDKIR localized to ring-like structures as early as 6 h after transfection, when the protein just started to be detected (data not shown). Second, the hDKIR structures were also observed under low protein expression condition following ecdysone induction (Fig. 3D). Third, the ring-like structures as shown in the cells expressing the full length or the POZ/IVR domain of hDKIR could not be observed in transfected cells with other mammalian kelch proteins, deletion mutants, or chimeric proteins, indicating that the ring-like structures are not attributed to an abnormal protein accumulation. Further work will be required to determine whether hDKIR localizes to real rings and what role it might play under physiological conditions. It is interesting to note that these ring-like structures shown by hDKIR are amorphous. One possible explanation for the structures is that the hDKIR ring’s maturation is incomplete. We do not exclude a possibility that complete ring assembly may require additional factors regulated by cell or tissue specificity. Finally, it has been reported recently that BTB/POZ proteins, including the KELCHcontaining BTB/POZ proteins, are required for proteasomedependent degradation of some protein [46,47]. This raises an intriguing possibility that hDKIR might function in a protein degradation pathway too. References [1] J. Adams, R. Kelso, L. Cooly, The kelch repeats superfamily of proteins: propellers of cell function, Trends Cell Biol. 10 (2000) 17 – 24.

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