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COMMD6 from amphioxus Branchiostoma belcheri (BbCOMMD6) interacts with creatine kinase and inhibits its activity
The International Journal of Biochemistry & Cell Biology 41 (2009) 2459–2465
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COMMD6 from amphioxus Branchiostoma belcheri (BbCOMMD6) interacts with creatine kinase and inhibits its activity Peipei Li, Shicui Zhang ∗ , Chunxin Fan Department of Marine Biology, Ocean University of China, Qingdao 266003, China
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Article history: Received 27 April 2009 Received in revised form 12 June 2009 Accepted 19 July 2009 Available online 26 July 2009 Keywords: Amphioxus Lancelet Branchiostoma COMMD Creatine kinase
a b s t r a c t COMM domain-containing proteins are a group of recently discovered proteins; their biochemical characterization remains much limited. Here we demonstrate that a cDNA encoding Branchiostoma belcheri COMMD6, designated BbCOMMD6, codes for a protein of 203 amino acids, with a COMM domain at its C-terminal region and an extended N-terminal portion. BbCOMMD6 is mainly present in the cytosol. In contrast to COMMD1, the presence of Cu(II) cannot enhance recombinant BbCOMMD6 dimer formation. Both the pull-down and reverse pull-down assays reveal that BbCOMMD6 interacts with the creatine kinase (CK), an essential enzyme involved in energy metabolism, forming a heterodimer BbCOMMD6-CK. The enzymatic activity assays show that CK activities are inhibited by BbCOMMD6 in a dose-dependent manner. All these data suggest that BbCOMMD6 is involved in energy transduction, via binding to CK and inhibiting activities of CK, and offer first clues to its role as a regulator of CK activities. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction COMM domain-containing or COMMD proteins are a family of recently discovered proteins, which are characterized by the presence of a unique copper metabolism gene MURR1 domain (COMMD) of 70–85 amino acids in their extreme carboxy termini (Burstein et al., 2005; Maine and Burstein, 2007). The founding member of this family, COMMD1 (previously known as MURR1), was originally identified as a gene in close proximity to the imprinted murine gene U2af1-rs1 (Nabetani et al., 1997). Additional members of the family were later identified in a biochemical screen for interacting proteins with homology to MURR1 (Burstein et al., 2005). Ten family members have now been documented in all the vertebrates examined, with significant conservation observed among mammals and even fish. Also, all 10 COMMD genes are present in the slime mold Dictyostelium discoideum, and several homologs are identifiable in the lower organisms including insects, worms as well as several unicellular protozoa (Burstein et al., 2005; Maine and Burstein, 2007). The majority of these genes are only known as open reading frames and their functions remain unexplored. The COMMD domain not only defines this protein family but also serves as an interface for protein–protein interactions. It has been demonstrated that the COMMD domain mediates COMMD1–COMMD1 dimer formation as well as binding to other COMMD proteins such as Cul2, Elongin C and SOCS1 (Burstein et
∗ Corresponding author. Tel.: +86 532 82032787; fax: +86 532 82032787. E-mail address: [email protected] (S. Zhang). 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.07.007
al., 2005; de Bie et al., 2006; Maine et al., 2007; Narindrasorasak et al., 2007). Of the 10 COMMD members, COMMD1 is the best characterized. Through identification of its molecular partners, COMMD1 has been shown to be associated with several biological processes. For example, COMMD1 was involved in copper homeostasis, presumably by regulating the stability of the copper transporter ATP7B (Burstein et al., 2004; de Bie et al., 2007; Spee et al., 2007; Tao et al., 2003); COMMD1 was known to inhibit HIV-1 replication and expression of several pro-inflammatory mediators via its suppression of NF-B activity (Ganesh et al., 2003; Maine et al., 2007), and to be implicated in intracellular sodium regulation through its inhibitory effects on the function of the epithelial sodium channels (Biasio et al., 2004); and COMMD1 was also found to regulate the adaption to hypoxia, by functioning as an inhibitor of the HIF1␣ transcription factor (van de Sluis et al., 2007). Recently, COMMD1 was revealed to form oligomeric complexes targeted the endocytic membranes via specific interactions with phosphatilinositol-4,5bisphosphate, PtdIns(4,5)P2 (Burkhead et al., 2009). However, the exact biological mechanisms are still lacking and further research is required. Now little information is available regarding the biochemical characteristics and functions of the other members of COMMD family except COMMD1. It was only known that COMMD5 was able to affect cell proliferation and gene expression although the precise targets responsible for these effects were not known (Devlin et al., 2003; El Hader et al., 2005; Solban et al., 2000), and COMMD6 was to be able to inhibit NF-B activity as COMMD1 (Burstein et al., 2005; de Bie et al., 2006). In a large scale sequencing of the gut cDNA library of adult amphioxus Branchiostoma belcheri, we
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isolated a cDNA clone exhibiting high identity to COMMD6, designated BbCOMMD6 (GenBank accession number: AAT45381). As B. belcheri occupies a nodal position transient from invertebrates to vertebrates, the study of BbCOMMD6 will provide important implications for understanding the origin and evolution of the functions and molecular interaction systems in which COMMD6 is involved. As an initial step, here we report the identification and expression of BbCOMMD6, and demonstrate that the recombinant BbCOMMD6 interacts with creatine kinase (CK), an essential enzyme for energy metabolism, and inhibits its activity. 2. Materials and methods 2.1. Cloning and sequence analysis of cDNA The gut cDNA library of adult B. belcheri was constructed with SMART cDNA Library Construction Kit (CLONTECH, CA, USA), and more than 5000 clones were sequenced and analyzed for coding probability with the DNATools program. Sequence comparison against the GenBank protein database was performed using the BLAST network server at the NCBI. Multiple protein sequences were aligned using the MegAlign program by CLUSTAL W method in DNASTAR software package. The phylogenetic tree was constructed by the neighbor-joining method within the PHYLIP 3.6 c software package using 1000 bootstrap replicates. 2.2. Expression and purification of recombinant BbCOMMD6 The complete coding region of BbCOMMD6 was amplified by polymerase chain reaction (PCR) with the upstream primer 5 -CGCGATATCATGGCGACGACGCAGTTC-3 (EcoRV site is underlined) and the downstream primer 5 -CCGCTCGAGCTATACTGTTTCTAACAT-3 (XhoI site is underlined), and subcloned into the plasmid expression vector pET30a (Novagen). The identity of the insert was verified by sequencing, and the plasmid was designated pET30a/BbCOMMD6. The cells of Escherichia coli BL21 were transformed with the plasmid pET30a-BbCOMMD6, and cultured overnight in LB broth containing kanamycin (30 g/ml). The culture was diluted 1:100 with LB broth and subjected to further incubation at 37 ◦ C for 3 h. The expression of BbCOMMD6 was induced by addition of isopropyl -d-thiogalactoside (IPTG) to the culture at a final concentration of 0.1 mM. The recombinant protein was purified as described by Fan et al. (2007). Protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard. The total cellular extracts and purified BbCOMMD6 were applied for SDS-PAGE in the absence and presence of -mercaptoethanol in the loading buffer. 2.3. Assay for effect of copper on dimerization of BbCOMMD6 To test if Cu(II) can facilitate the dimerization of BbCOMMD6, aliquots of 10 g BbCOMMD6 in 100 l PBS were incubated with 0, 0.5 and 5 mM CuCl2 at 4 ◦ C overnight, and separated by 12% nativePAGE and SDS-PAGE without -mercaptoethanol (Narindrasorasak et al., 2007). 2.4. Preparation of polyclonal antibody Approximately 4 mg of the purified BbCOMMD6 was emulsified with Freund’s complete adjuvant and injected subcutaneously at multiple sites of two rabbits. Three booster injections of 2 mg BbCOMMD6 mixed with Freund’s incomplete adjuvant were administered subcutaneously at intervals of 2 weeks. Eight days after the final booster, blood was collected from the rabbits by carotid puncture and serum was prepared. The antisera were aliquoted and stored at −70 ◦ C.
2.5. Western blotting The homogenates of B. belcheri were prepared as described by Jiang et al. (2008). The homogenates, cell lysates of IPTG-induced E. coli BL21, control cell lysates and purified protein were run on 12% SDS-PAGE, and immunostained by the method of Fan et al. (2007). 2.6. His-tag pull-down assay and mass spectrometry The His-tag pull-down experiments were performed by the methods of Jiang et al. (2008) and Boutell et al. (1999). The cell lysates of IPTG-induced E. coli BL21 were mixed with 50 l of MagExtractor His-tag particles (TOYOBO, Japan) at 4 ◦ C with agitation overnight. The particles were washed with the binding buffer (50 mM PBS, pH 8.0, containing 0.3 M NaCl and 10 mM imidazole), wash buffer I (50 mM PBS, pH 8.0, containing 0.3 M NaCl and 20 mM imidazole) and wash buffer II (50 mM PBS, pH 8.0, containing 0.3 M NaCl and 40 mM imidazole) for four times each. Subsequently, the particles were incubated with 1 ml of B. belcheri homogenates prepared as above at 4 ◦ C overnight with gentle shaking, and washed three times with the homogenization buffer (50 mM Tris–HCl buffer with 50 mM NaCl, pH 7.2) and three times with the binding buffer. The controls consisted of the particles incubated with both BbCOMMD6 and homogenization buffer, and the particles incubated with pET30a-expressed soluble protein and B. belcheri homogenates. The interacted complexes were eluted by 50–100 l of the elution buffer (50 mM pH 8.0 PBS, containing 0.3 M NaCl and 500 mM imidazole) and separated on 12% SDS-PAGE. To identify the pulled down protein, the Coomassie blue-stained band was cut out, and subjected to trypsin digestion for further process as described by Gevaert and Vandekerckhove (2000) and to peptide mass fingerprinting (PMF) analysis. The data obtained by mass spectrometry were submitted to NCBI database for protein identification using Mascot Explorer Software (http://www. matrixscience.com/, version: 9 April 2007) with ±0.2 Da peptide mass tolerance. Protein scores greater than 79 were considered to be significant (p < 0.05). The mass calibration was done externally on the target using sequazyme mass STDs Kit (ABI, USA). 2.7. Reverse pull-down assay To verify the pull-down result, the reverse pull-down experiment was performed according to the method of Jiang et al. (2008) with slight modification. B. belcheri creatine kinase, BbCK, was expressed and purified. An equal amount (about 0.2 mg) of BbCK and BbCOMMD6 was mixed and incubated on ice for 1 h with gentle shaking, followed by immunoprecipitation with about 2 g of mouse anti-human CK monoclonal antibody (MM-type, Sigma) at 4 ◦ C for 2 h. 20 l of the protein A/G PLUS-Agarose (Santa Cruz Biotechnology) was added to the reaction mixture and incubated at 4 ◦ C overnight. After centrifugation, the agaroses were washed three times with PBS (pH 7.8), and the precipitated proteins were eluted from the agaroses with 50 l of SDS-PAGE loading buffer and separated by 12% SDS-PAGE. For control, the total homogenate of B. belcheri was also run on the same gel. The immunoblotting analysis was carried out as above. Two parallel reactions, incubations of the monoclonal antibody with BbCK alone or with BbCOMMD6 alone followed by interaction with the protein A/G PLUS-Agarose, were the negative controls, and incubation of the monoclonal antibody with the homogenates of B. belcheri, was positive control. 2.8. Crude subcellular distribution assay The cytosolic and mitochondrial fractions prepared from B. belcheri as described by Jiang et al. (2008) were separated by 12% SDS-PAGE and immunostained.
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2.9. Assay for effect of BbCOMMD6 on creatine kinase activity Aliquots of 6 g creatine kinase from rabbit muscle (MM-type; Sigma) or rabbit brain (BB-type; Sigma) solved in 30 l of the elution buffer (PBS, pH 8.0, containing 0.3 M NaCl and 500 mM imidazole) were incubated with 30 l of the elution buffer containing different amounts (0, 1.5, 3, 4.5, 6, 7.5, 9 and 10.5 g, respectively) of recombinant BbCOMMD6 at room temperature for 1 h. The activities of creatine kinase were detected following the instructions of CK test kit (Jiancheng Biotechnology Institute, Nanjing, China). For control, the creatine kinase was incubated with BSA instead of BbCOMMD6 and processed similarly. In parallel, the mixtures of 6 g BbCOMMD6 in the elution buffer and 6 g BB-type creatine kinase or BSA were also subjected to 12% native-PAGE. 3. Results 3.1. Sequence and phylogeny of BbCOMMD6 The full length of BbCOMMD6 cDNA (GenBank accession number: AAT45381) was 1073 bp in length, and its longest ORF encoded a protein of 203 amino acids with a calculated molecular weight (MW) of about 23 kDa (Fig. 1). The deduced protein had a COMM domain at the residues 116–201 at its C-terminal region. Alignment analysis showed that the COMM domain of BbCOMMD6 shared 47.0, 45.3, 44.7, 44.2, 45.8, 43.5, 54.7, 50.0 and 44.4% identity to that of human, chimpanzee, house mouse, dog, cattle, frog, zebrafish, sea urchin and slime mold COMMD6 proteins, respectively. However, its N-terminal region consisted of 115 amino acids, was close to that of frog, zebrafish and sea urchin COMMD6 proteins in length, and exhibited 28.2, 27.9 and 40.9% identity in sequence, individually (Fig. 2A), contrasting to the significantly shorter N-terminal portion of mammalian COMMD6 proteins. The phylogenetic tree constructed using the sequences of representative COMM
Fig. 1. The nucleotide and deduced amino acid sequences of amphioxus (Branchiostoma belcheri) COMMD6 cDNA. The start codon is underlined. The asterisk represents the stop codon. The polyadenylation signal is boxed. The numbering of the nucleotide and amino acid sequences is shown to the left.
Fig. 2. (A) Alignment of the N-terminal portion of COMMD6 proteins including BbCOMMD6. Shaded (with solid black) residues are the amino acids that match the consensus. Gaps introduced into sequences to optimize alignment are represented by (−). The sequences are: Xt (Xenopus tropicalis, NP 001016464), Dr (Danio rerio, XP 001332088), Bb (B. belcheri tsingtaunes, AAT45381), Sp (Strongylocentrotus purpuratus, XP 794885). (B) Phylogenetic tree of COMM domains of COMMD proteins including BbCOMMD6. Bootstrap majority consensus values on 1000 replicates are indicated at each branch point in percent. The sequences are: Dr1 (D. rerio, AAI08024), Dr2 (D. rerio, NP 001002421), Dr3 (D. rerio, NP 001008734), Dr4 (D. rerio, NP 001070206), Dr5 (D. rerio, NP 001003553), Dr6 (D. rerio, XP 001332088), Dr8 (D. rerio, NP 001071186), Dr9 (D. rerio, NP 001082867), Mm1 (Mus musculus, AAH51210), Mm2 (M. musculus, NP 780304), Mm3 (M. musculus, EDL08102), Mm4 (M. musculus, NP 079693), Mm5 (M. musculus, NP 079812), Mm6 (M. musculus, NP 001028304), Mm7 (M. musculus, NP 598611), Mm8 (M. musculus, NP 848714), Mm9 (M. musculus, NP 083911), Mm10 (M. musculus, NP 848464), Hs1 (Homo sapiens, AAH09266), Hs2 (H. sapiens, NP 057178), Hs3 (H. sapiens, NP 036203), Hs4 (H. sapiens, NP 060298), Hs5 (H. sapiens, NP 001074472), Hs6 (H. sapiens, CAQ09795), Hs7 (H. sapiens, AAH47440), Hs8 (H. sapiens, NP 060315), Hs9 (H. sapiens, AAH10892), Hs10 (H. sapiens, NP 057228), Xt1 (X. tropicalis, AAI60794), Xt2 (X. tropicalis, AAI54926), Xt4 (X. tropicalis, NP 001011118), Xb5 (Xenopus borealis, ACC54703), Xt6 (X. tropicalis, AAI71118), Xt7 (X. tropicalis, NP 989169), Xt10 (X. tropicalis, NP 001039088), Cf1 (Canis familiaris, NP 001003055), Cf6 (C. familiaris, XP 534146), Cf9 (C. familiaris, XP 850727), Bb6 (B. belcheri tsingtaunes, AAT45381), Bf6 (Branchiostoma floridae, XP 002247470), Bf8 (B. floridae, XP 002223266), Sp3 (S. purpuratus, XP 001185060), Sp6 (S. purpuratus, XP 794885), Sp7 (S. purpuratus, XP 001179231), Sp8 (S. purpuratus, XP 789228), Sp9 (S. purpuratus, XP 794857), Dd1 (Dictyostelium discoideum, XP 629812), Dd2 (D. discoideum, XP 639903), Dd3 (D. discoideum, XP 638867), Dd4 (D. discoideum, XP 642944), Dd5 (D. discoideum, XP 643905), Dd6 (D. discoideum, XP 637929), Dd7 (D. discoideum, XP 643770), Dd8 (D. discoideum, XP 629572), Dd9 (D. discoideum, XP 635194), Dd10 (D. discoideum, XP 643748), Ci10 (Ciona intestinalis, XP 0021221790).
domain-containing proteins including BbCOMMD6 demonstrated that BbCOMMD6 was clustered together with COMMD6 proteins, separating from all the other nine family members (Fig. 2B). A search of the recently completed draft assembly and automated annotation of Branchiostoma floridae genome was carried out. It revealed the presence of a Florida amphioxus COMMD6
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Fig. 3. SDS-PAGE and Western blotting of BbCOMMD6. (A) SDS-PAGE. Lane M, markers; lane 1, total cellular extracts from E. coli BL21 containing pET30a/BbCOMMD6 before induction; lane 2, total cellular extracts from IPTG-induced E. coli BL21 containing pET30a/BbCOMMD6; lanes 3 and 4, purified recombinant BbCOMMD6 in the presence and absence of -mercaptoethanol. The arrow (→) indicates the localization and size of monomeric and dimeric BbCOMMD6. (B) Western blotting. Totally, ∼6 g of purified protein and ∼85 g of cell lysate proteins were loaded onto the gel. Lane 1, extracts from E. coli BL21 containing pET30a/BbCOMMD6 before induction; lane 2, extracts from IPTG-induced E. coli BL21 containing pET30a/BbCOMMD6; lane 3, recombinant BbCOMMD6 protein; lane 4, extracts of amphioxus B. belcheri. The arrow (→) indicates the localization of the recombinant BbCOMMD6 and the triangle (I) the distribution of native BbCOMMD6 in the amphioxus extracts.
cDNA and its genomic DNA sequence (estExt gwp.C 820054, scaffold 82: 1065728–1071678, http://genome.Jgi-psf.org/cgibin/dispTranscript?db=Brafl1&id=280511&useCoords=1). Sequence comparison showed that BbCOMMD6 shared 96% identity to the deduced protein encoded by the Florida amphioxus gene at the amino acid level (Fig. 2B), suggesting that BbCOMMD6 is highly conserved in inter-species. 3.2. Biochemical properties of recombinant BbCOMMD6 An expression vector including the entire ORF of BbCOMMD6 and a 5 additional His6 tag (this resulted in the increase in the size of the recombinant protein by approximately 5 kDa) of pET30a was constructed and successfully transformed into E. coli. The recombinant BbCOMMD6 was purified and separated on SDS-PAGE in the presence of -mercaptoethanol, which yielded a single band of ∼28 kDa, coinciding with its theoretical size (Fig. 3A). In contrast, the BbCOMMD6 occurred as two bands under SDS-PAGE in the absence of -mercaptoethanol, with one band being ∼28 kDa and the other being ∼55 kDa (Fig. 3A). These indicated that BbCOMMD6 existed as a mixture of monomers and dimers generated via disulfide bond formation. Rabbit antiserum against BbCOMMD6 with a titer of 1:1000 was obtained. Western blotting demonstrated that the antiserum reacted with the cell lysates of IPTG-induced E. coli BL21, forming a band of ∼28 kDa, but not with the lysate of the same E. coli cells before induction. The antiserum also reacted with the homogenates of B. belcheri, forming a band of ∼23 kDa (Fig. 3B), matching the molecular mass predicted by BbCOMMD6 cDNA. These showed that the rabbit antiserum had a conspicuous antigen-specific reactivity. To test if Cu(II) was able to stimulate the dimer formation, BbCOMMD6 was incubated with CuCl2 and separated by nativePAGE. Unexpectedly, little change in the intensity of the fast migrating band (*) was found (Fig. 4A). Similarly, SDS-PAGE in the absence of -mercaptoethanol revealed that the presence of Cu(II) was not able to enhance dimer formation (Fig. 4B). To identify the proteins that might interact with BbCOMMD6, the pull-down technique was applied (Kaelin et al., 1991; Soutoglou et al., 2000). It was found that a protein (A in lane 4 of Fig. 5) from B. belcheri homogenates bound to BbCOMMD6 (B in lane 4 of Fig. 5). The molecular weight of protein A was approximately 42 kDa. A total of 14 peptide fragments of the protein were measured by MALDI-TOF MS5. The measured peptides were matched
against the creatine kinase from B. belcheri, and the MOWSE score obtained was 414, which was 5.2-fold greater than the threshold for identification. The matched peptide fragments were located at the residues 8–20, 21–27, 103–111, 119–124, 133–142, 146–166, 147–166, 204–209, 210–217, 218–230, 302–309, 303–311, 315–336 and 316–336 of the CK (Fig. 6), respectively. The control experiments revealed no protein bound to the particles without BbCOMMD6 (Fig. 5). These suggested that BbCOMMD6 was able to bind to CK in B. belcheri. To verify that BbCOMMD6 can bind to CK, the reverse pulldown assay was carried out. BbCOMMD6 and BbCK were incubated together with the mouse anti-human CK monoclonal antibody, and then with the protein A/G PLUS-Agarose. After washing stringently, the complexes were analyzed by Western blotting. Recombinant BbCOMMD6 was detected in the complexes (Fig. 7, lanes 1–3). Similarly, BbCOMMD6 was also detected in the positive controls
Fig. 4. Effects of copper on the dimerization of BbCOMMD6. (A) Native-PAGE of BbCOMMD6 in the presence of Cu(II). Ten microgram aliquots of BbCOMMD6 were incubated with 0, 0.5 and 5.0 mM CuCl2 before being applied to 12% native-PAGE. (B) SDS-PAGE of BbCOMMD6 in the presence of Cu(II). The same amount of protein in the absence and presence of Cu(II) were applied to 12% SDS-PAGE without mercaptoethanol. The protein bands were stained with Commassie blue dye.
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Fig. 5. SDS-PAGE (12%) analysis of the pulled down proteins. Lane M, markers; lane 1, extracts from E. coli BL21 containing pET30a/BbCOMMD6 after induction; lane 2, the control particles incubated with both recombinant BbCOMMD6 protein and the homogenate buffer; lane 3, the control particles incubated with the pET30a/expressed soluble protein and the tissue homogenates; lane 4, recombinant BbCOMMD6 protein and the interaction protein; lane 5, total protein of the amphioxus homogenate. A indicates the pulled down protein and B the recombinant BbCOMMD6 protein.
including the homogenates of B. belcheri (Fig. 7, lane 7) and the homogenates incubated with the monoclonal antibody and the protein A/G PLUS-Agarose (Fig. 7, lane 6). In contrast, no signals were observed in the negative controls including the incubation of the monoclonal antibody with BbCK alone or with BbCOMMD6 alone, followed by interaction with the protein A/G PLUS-Agarose (Fig. 7, lanes 4 and 5). These showed that BbCOMMD6 was pulled down by forming the BbCOMMD6–BbCK–antibody–agarose complex, indicating that BbCOMMD6 was able to interact with BbCK specifically. 3.3. Subcellular localization of BbCOMMD6 To investigate the subcellular localization of BbCOMMD6, the cytosolic and mitochondrial fractions were prepared from
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Fig. 7. Western blotting of reverse pulled down proteins. Lanes 1–3 show the presence of recombinant BbCOMMD6 in the complexes eluted; lanes 4 and 5 are the two negative controls; lane 6 shows the presence of natural BbCOMMD6 in the complexes eluted; lane 7 indicates the presence of natural BbCOMMD6 in the total homogenates. The arrow (→) indicates the localization of recombinant BbCOMMD6 and the triangle (I) the distribution of native BbCOMMD6 in the amphioxus homogenates.
B. belcheri and analyzed by Western blotting. It showed that BbCOMMD6 was present in the cytosolic fraction; no signal was observed for the mitochondrial fraction (Fig. 8). This indicated that BbCOMMD6 was localized in the cytosol, agreeing with the primary cytosolic localization of COMMD1 in mammalian species (Klomp et al., 2003). 3.4. Inhibition of CK activity by BbCOMMD6 Next we sought to examine if the recombinant BbCOMMD6 has any effect on CK activity. It was found that BbCOMMD6 was capable of inhibiting the activities of both the MM- and BB-type CKs in a concentration-dependent manner, whereas BSA had no influence (Fig. 9). Calculation revealed that on average three BbCOMMD6
Fig. 6. MALDI spectra of trypsin digestion of the pulled down protein. (A) is the PMF data of creatine kinase protein. The 14 peaks of identified peptide fragments are marked with asterisks. (B) shows the identified peptide number, the observed mass and the sequence of the creatine kinase protein, respectively.
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Fig. 8. Western blotting analysis of the BbCOMMD6 subcellular distribution. Lane 1, purified recombinant BbCOMMD6; lane 2, the extracts from E. coli BL21 containing pET30a/BbCOMMD6 before induction; lane 3, the cytosolic fraction of the primitive gut; lane 4, the mitochondrial fraction of the gut. The arrow (→) indicates the localization of recombinant BbCOMMD6 and the triangle (I) the distribution of native BbCOMMD6 in the different cell fractions of the gut.
molecules per one CK was necessary to cause the CK inhibition. This was the first evidence that BbCOMMD6 could specifically suppress CK activities. Of note, native-PAGE demonstrated that the preincubation of BbCOMMD6 with CK resulted in BbCOMMD6-CK dimer formation, while no degraded peptides were observed (Fig. 10), suggesting that BbCOMMD6 inhibited CK activities via directly binding with CK.
Fig. 10. Native-PAGE showing the binding of recombinant BbCOMMD6 to BB-type creatine kinase. The same amount of BB-type creatine kinase was incubated with recombinant BbCOMMD6 or with BSA (as negative control), and then subjected to 12% native-PAGE. The arrow (→) indicates the presence and localization of BbCOMMD6-CK dimer in the mixture of BbCOMMD6 and CK after incubation. The molecular weights of monomeric and dimeric BbCOMMD6 are shown to the left.
4. Discussion
Fig. 9. Inhibition of CK activity by BbCOMMD6. (A) Inhibition of the creatine kinases from rabbit muscle; (B) inhibition of the creatine kinases from rabbit brain. Different concentrations of recombinant BbCOMMD6 and BSA (as negative control) were added to the creatine kinases, and then CK activities were detected following the instructions of CK test kit. Data are mean values ±S.D. from three experiments.
With the exception of COMMD1, the functions and molecular partners of COMMD family members remain essentially unknown. Here we report the identification, expression and functional characterization of a member of COMMD family, COMMD6, in B. belcheri. The deduced 203 amino acids long protein, BbCOMMD6, has a COMM domain at its C-terminus and an extended N-terminal portion, resembling that of frog, zebrafish and sea urchin COMMD6 proteins both in sequence and in length. This is the first member of COMMD family reported in the protochordates. Various protein–protein interaction experiments have shown that COMMD1 is able to dimerize with several members of COMMD family (Burstein et al., 2005; de Bie et al., 2006) and to form complexes with a growing number of other structurally and functionally unrelated proteins (Burkhead et al., 2009; Klomp et al., 2003). Here we demonstrate that BbCOMMD6 is able to interact with CK, forming a heterodimer BbCOMMD6-CK. Moreover, BbCOMMD6 is able to inhibit CK activities. BbCOMMD6 appears to inhibit CK activities by its binding to CK, thereby leading to the conformational change in CK as BbCOMMD6 does not apparently cause the degradation of CK. CK, also known as creatine phosphokinase, is an enzyme catalyzing the reversible transfer of the phosphate group of phosphocreatine to adenosine diphosphate (ADP), generating adenosine triphosphate (ATP) and creatine. Therefore, our results suggest that BbCOMMD6 may be involved in energy transduction, via binding to CK and inhibiting its activities. This is the first report showing the inhibitory effects of COMMD6 on CK activities; it will be of particular interest to determine if this is also true for COMMD6 in other organisms. Interestingly, recent studies have shown that muscle
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RING-finger protein-1 (MuRF1) and ycaC-related protein (ycaCR) are also able to interact with CK (Jiang et al., 2008; Koyama et al., 2007). These results and ours together suggest that CK activity may be regulated by multiple factors. COMMD1 has been shown to bind Cu(II), increasing the COMMD1–COMMD1 homodimer formation (Narindrasorasak et al., 2007). In contrast, the presence of Cu(II) fails enhancing the dimerization of BbCOMMD6. We speculate that the presence of Cu(II) may not facilitate the dimerization of BbCOMMD6; however, further study is necessary to determine if BbCOMMD6 binds with Cu(II). It is also interesting to note that BbCOMMD6 is mainly present in the cytosol although its detailed cellular distribution is still to be determined. Previous studies have shown that COMMD1 is distributed throughout the cell including the nucleus, with main localization in the cytoplasm (Klomp et al., 2003; Maine and Burstein, 2007), and COMMD5 is a primarily nuclear protein (Solban et al., 2000). It appears that different COMMD proteins are localized in different cellular compartments. It is nuclear if there is a relationship between the different cellular distribution of these proteins and their biological significance. In summary, this study highlights for the first time a novel function of COMMD6 in energy transduction through its interacting with CK and inhibiting CK activities. It will be of particular interest to examine if COMMD6 in other organisms also plays a role as a regulator of CK activities. Acknowledgement This work was supported by the Ministry of Science and Technology (MOST) of China (2008AA09Z409). References Biasio W, Chang T, McIntosh CJ, McDonald FJ. Identification of Murr1 as a regulator of the human epithelial sodium channel. J Biol Chem 2004;279:5429–34. Boutell JM, Thomas P, Neal JW, Weston VJ, Duce J, Harper PS, et al. Aberrant interactions of transcriptional repressor proteins with the Huntington’s disease gene product, huntingtin. Hum Mol Genet 1999;8:1647–55. Burkhead JL, Morgan CT, Shinde U, Haddock G, Lutsenko S. COMMD1 forms oligomeric complexes targeted to the endocytic membranes via specific interactions with phosphatidylinositol 4,5-bisphosphate. J Biol Chem 2009;284(1):696–707. Burstein E, Ganesh L, Dick RD, van de Sluis B, Wilkinson JC, Klomp LW, et al. A novel role for XIAP in copper homeostasis through regulation of MURR1. EMBO J 2004;23:244–54. Burstein E, Hoberg JE, Wilkinson AS, Rumble JM, Csomos RA, Komarck CM, et al. COMMD proteins: a novel family of structural and functional homologs of MURR1. J Biol Chem 2005;280:22222–32.
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de Bie P, van de Sluis B, Burstein E, Duran KJ, Berger R, Duckett CS, et al. Characterization of COMMD protein–protein interactions in NF-kB signalling. Biochem J 2006;398:63–71. de Bie P, van de Sluis B, Burstein E, Peter VE, van de Berqhe PV, Muller P, et al. Distinct Wilson’s disease mutations in ATP7B are associated with enhanced binding to COMMD1 and reduced stability of ATP7B. Gastroenterology 2007;133(4):1316–26. Devlin AM, Solban N, Tremblay S, Gutkowska J, Schurch W, Orlov SN, et al. HCaRG is a novel regulator of renal epithelial cell growth and differentiation causing G2M arrest. Am J Physiol Renal Physiol 2003;284:F753–62. El Hader C, Tremblay S, Solban N, Gigras D, Beliveau R, Orlov SN, et al. HCaRG increases renal cell migration by a TGF-a autocrine loop mechanism. Am J Physiol Renal Physiol 2005;289:F1273–80. Fan C, Zhang S, Liu Z, Li L, Luan J, Saren G. Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma belcheri with implications to the origin of vertebrate liver. Int J Biochem Cell Biol 2007;39: 450–61. Ganesh L, Burstein E, Guha-Niyogi A, Louder MK, Mascola JR, Klomp LW, et al. The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes. Nature 2003;426:853–7. Gevaert K, Vandekerckhove J. Protein identification methods in proteomics. Electrophoresis 2000;21:1145–54. Jiang S, Sun X, Zhang S. The ycaC-related protein from the amphioxus Branchiostoma belcheri (BbycaCR) interacts with creatine kinase. FEBS J 2008;275(18):4597–605. Kaelin Jr WG, Pallas DD, DeCaprio JA, Kaye FJ, Livingston DM. Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell 1991;64:521–32. Klomp AE, van de Sluis B, Klomp LW, Wijmenga C. The ubiquitously expressed MURR1 protein is absent in canine copper toxicosis. J Hepatol 2003;39:703–9. Koyama S, Hata S, Witt CC, Ono Y, Lerche S, Koichi O, et al. Muscle RING-finger protein1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis. J Mol Biol 2007;376:1224–36. Maine GN, Burstein E. COMMD proteins: COMMing to the scene. Cell Mol Life Sci 2007;64:1997–2005. Maine GN, Mao X, Komarck CM, Burstein E. COMMD1 promotes the ubiquitination of NF-kB subunits through a Cullin-containing ubiquitin ligase. EMBO J 2007;26:436–47. Nabetani A, Hatada I, Morisaki H, Oshimura M, Mukai T. Mouse U2af1-rs1 is a neomorphic imprinted gene. Mol Cell Biol 1997;17:789–98. Narindrasorasak S, Kulkarni P, Deschamps P, She YM, Sarkar B. Characterization and copper binding properties of human COMMD1 (MURR1). Biochemistry 2007;46:3116–28. Solban N, Jia HP, Richard S, Tremblay S, Devlin AM, Peng J, et al. HCaRG, a novel calcium-regulated gene coding for a nuclear protein, is potentially involved in the regulation of cell proliferation. J Biol Chem 2000;275: 32234–43. Soutoglou E, Katrakili N, Talianidis I. Acetylation regulates transcription factor activity at multiple levels. Mol Cell 2000;5:745–51. Spee B, Arends B, van Wees AM, Bode P, Penning LC, Rothuizen J. Functional consequences of RNA interference targeting COMMD1 in a canine hepatic cell line in relation to copper toxicosis. Anim Genet 2007;38(2):168–70. Tao TY, Liu F, Klomp L, Wijmenga C, Gitlin JD. The copper toxicosis gene product Murr1 directly interacts with the Wilson disease protein. J Biol Chem 2003;278:41593–6. van de Sluis B, Muller P, Duran K, Chen A, Groot AJ, Klomp LW, et al. Increased activity of hypoxia-inducible factor 1 is associated with early embryonic lethality in Commd1 null mice. Mol Cell Biol 2007;27(11):4142–56.
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COMMD6 from amphioxus Branchiostoma belcheri (BbCOMMD6) interacts with creatine kinase and inhibits its activity Peipei Li, Shicui Zhang ∗ , Chunxin Fan Department of Marine Biology, Ocean University of China, Qingdao 266003, China
a r t i c l e
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Article history: Received 27 April 2009 Received in revised form 12 June 2009 Accepted 19 July 2009 Available online 26 July 2009 Keywords: Amphioxus Lancelet Branchiostoma COMMD Creatine kinase
a b s t r a c t COMM domain-containing proteins are a group of recently discovered proteins; their biochemical characterization remains much limited. Here we demonstrate that a cDNA encoding Branchiostoma belcheri COMMD6, designated BbCOMMD6, codes for a protein of 203 amino acids, with a COMM domain at its C-terminal region and an extended N-terminal portion. BbCOMMD6 is mainly present in the cytosol. In contrast to COMMD1, the presence of Cu(II) cannot enhance recombinant BbCOMMD6 dimer formation. Both the pull-down and reverse pull-down assays reveal that BbCOMMD6 interacts with the creatine kinase (CK), an essential enzyme involved in energy metabolism, forming a heterodimer BbCOMMD6-CK. The enzymatic activity assays show that CK activities are inhibited by BbCOMMD6 in a dose-dependent manner. All these data suggest that BbCOMMD6 is involved in energy transduction, via binding to CK and inhibiting activities of CK, and offer first clues to its role as a regulator of CK activities. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction COMM domain-containing or COMMD proteins are a family of recently discovered proteins, which are characterized by the presence of a unique copper metabolism gene MURR1 domain (COMMD) of 70–85 amino acids in their extreme carboxy termini (Burstein et al., 2005; Maine and Burstein, 2007). The founding member of this family, COMMD1 (previously known as MURR1), was originally identified as a gene in close proximity to the imprinted murine gene U2af1-rs1 (Nabetani et al., 1997). Additional members of the family were later identified in a biochemical screen for interacting proteins with homology to MURR1 (Burstein et al., 2005). Ten family members have now been documented in all the vertebrates examined, with significant conservation observed among mammals and even fish. Also, all 10 COMMD genes are present in the slime mold Dictyostelium discoideum, and several homologs are identifiable in the lower organisms including insects, worms as well as several unicellular protozoa (Burstein et al., 2005; Maine and Burstein, 2007). The majority of these genes are only known as open reading frames and their functions remain unexplored. The COMMD domain not only defines this protein family but also serves as an interface for protein–protein interactions. It has been demonstrated that the COMMD domain mediates COMMD1–COMMD1 dimer formation as well as binding to other COMMD proteins such as Cul2, Elongin C and SOCS1 (Burstein et
∗ Corresponding author. Tel.: +86 532 82032787; fax: +86 532 82032787. E-mail address: [email protected] (S. Zhang). 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.07.007
al., 2005; de Bie et al., 2006; Maine et al., 2007; Narindrasorasak et al., 2007). Of the 10 COMMD members, COMMD1 is the best characterized. Through identification of its molecular partners, COMMD1 has been shown to be associated with several biological processes. For example, COMMD1 was involved in copper homeostasis, presumably by regulating the stability of the copper transporter ATP7B (Burstein et al., 2004; de Bie et al., 2007; Spee et al., 2007; Tao et al., 2003); COMMD1 was known to inhibit HIV-1 replication and expression of several pro-inflammatory mediators via its suppression of NF-B activity (Ganesh et al., 2003; Maine et al., 2007), and to be implicated in intracellular sodium regulation through its inhibitory effects on the function of the epithelial sodium channels (Biasio et al., 2004); and COMMD1 was also found to regulate the adaption to hypoxia, by functioning as an inhibitor of the HIF1␣ transcription factor (van de Sluis et al., 2007). Recently, COMMD1 was revealed to form oligomeric complexes targeted the endocytic membranes via specific interactions with phosphatilinositol-4,5bisphosphate, PtdIns(4,5)P2 (Burkhead et al., 2009). However, the exact biological mechanisms are still lacking and further research is required. Now little information is available regarding the biochemical characteristics and functions of the other members of COMMD family except COMMD1. It was only known that COMMD5 was able to affect cell proliferation and gene expression although the precise targets responsible for these effects were not known (Devlin et al., 2003; El Hader et al., 2005; Solban et al., 2000), and COMMD6 was to be able to inhibit NF-B activity as COMMD1 (Burstein et al., 2005; de Bie et al., 2006). In a large scale sequencing of the gut cDNA library of adult amphioxus Branchiostoma belcheri, we
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isolated a cDNA clone exhibiting high identity to COMMD6, designated BbCOMMD6 (GenBank accession number: AAT45381). As B. belcheri occupies a nodal position transient from invertebrates to vertebrates, the study of BbCOMMD6 will provide important implications for understanding the origin and evolution of the functions and molecular interaction systems in which COMMD6 is involved. As an initial step, here we report the identification and expression of BbCOMMD6, and demonstrate that the recombinant BbCOMMD6 interacts with creatine kinase (CK), an essential enzyme for energy metabolism, and inhibits its activity. 2. Materials and methods 2.1. Cloning and sequence analysis of cDNA The gut cDNA library of adult B. belcheri was constructed with SMART cDNA Library Construction Kit (CLONTECH, CA, USA), and more than 5000 clones were sequenced and analyzed for coding probability with the DNATools program. Sequence comparison against the GenBank protein database was performed using the BLAST network server at the NCBI. Multiple protein sequences were aligned using the MegAlign program by CLUSTAL W method in DNASTAR software package. The phylogenetic tree was constructed by the neighbor-joining method within the PHYLIP 3.6 c software package using 1000 bootstrap replicates. 2.2. Expression and purification of recombinant BbCOMMD6 The complete coding region of BbCOMMD6 was amplified by polymerase chain reaction (PCR) with the upstream primer 5 -CGCGATATCATGGCGACGACGCAGTTC-3 (EcoRV site is underlined) and the downstream primer 5 -CCGCTCGAGCTATACTGTTTCTAACAT-3 (XhoI site is underlined), and subcloned into the plasmid expression vector pET30a (Novagen). The identity of the insert was verified by sequencing, and the plasmid was designated pET30a/BbCOMMD6. The cells of Escherichia coli BL21 were transformed with the plasmid pET30a-BbCOMMD6, and cultured overnight in LB broth containing kanamycin (30 g/ml). The culture was diluted 1:100 with LB broth and subjected to further incubation at 37 ◦ C for 3 h. The expression of BbCOMMD6 was induced by addition of isopropyl -d-thiogalactoside (IPTG) to the culture at a final concentration of 0.1 mM. The recombinant protein was purified as described by Fan et al. (2007). Protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard. The total cellular extracts and purified BbCOMMD6 were applied for SDS-PAGE in the absence and presence of -mercaptoethanol in the loading buffer. 2.3. Assay for effect of copper on dimerization of BbCOMMD6 To test if Cu(II) can facilitate the dimerization of BbCOMMD6, aliquots of 10 g BbCOMMD6 in 100 l PBS were incubated with 0, 0.5 and 5 mM CuCl2 at 4 ◦ C overnight, and separated by 12% nativePAGE and SDS-PAGE without -mercaptoethanol (Narindrasorasak et al., 2007). 2.4. Preparation of polyclonal antibody Approximately 4 mg of the purified BbCOMMD6 was emulsified with Freund’s complete adjuvant and injected subcutaneously at multiple sites of two rabbits. Three booster injections of 2 mg BbCOMMD6 mixed with Freund’s incomplete adjuvant were administered subcutaneously at intervals of 2 weeks. Eight days after the final booster, blood was collected from the rabbits by carotid puncture and serum was prepared. The antisera were aliquoted and stored at −70 ◦ C.
2.5. Western blotting The homogenates of B. belcheri were prepared as described by Jiang et al. (2008). The homogenates, cell lysates of IPTG-induced E. coli BL21, control cell lysates and purified protein were run on 12% SDS-PAGE, and immunostained by the method of Fan et al. (2007). 2.6. His-tag pull-down assay and mass spectrometry The His-tag pull-down experiments were performed by the methods of Jiang et al. (2008) and Boutell et al. (1999). The cell lysates of IPTG-induced E. coli BL21 were mixed with 50 l of MagExtractor His-tag particles (TOYOBO, Japan) at 4 ◦ C with agitation overnight. The particles were washed with the binding buffer (50 mM PBS, pH 8.0, containing 0.3 M NaCl and 10 mM imidazole), wash buffer I (50 mM PBS, pH 8.0, containing 0.3 M NaCl and 20 mM imidazole) and wash buffer II (50 mM PBS, pH 8.0, containing 0.3 M NaCl and 40 mM imidazole) for four times each. Subsequently, the particles were incubated with 1 ml of B. belcheri homogenates prepared as above at 4 ◦ C overnight with gentle shaking, and washed three times with the homogenization buffer (50 mM Tris–HCl buffer with 50 mM NaCl, pH 7.2) and three times with the binding buffer. The controls consisted of the particles incubated with both BbCOMMD6 and homogenization buffer, and the particles incubated with pET30a-expressed soluble protein and B. belcheri homogenates. The interacted complexes were eluted by 50–100 l of the elution buffer (50 mM pH 8.0 PBS, containing 0.3 M NaCl and 500 mM imidazole) and separated on 12% SDS-PAGE. To identify the pulled down protein, the Coomassie blue-stained band was cut out, and subjected to trypsin digestion for further process as described by Gevaert and Vandekerckhove (2000) and to peptide mass fingerprinting (PMF) analysis. The data obtained by mass spectrometry were submitted to NCBI database for protein identification using Mascot Explorer Software (http://www. matrixscience.com/, version: 9 April 2007) with ±0.2 Da peptide mass tolerance. Protein scores greater than 79 were considered to be significant (p < 0.05). The mass calibration was done externally on the target using sequazyme mass STDs Kit (ABI, USA). 2.7. Reverse pull-down assay To verify the pull-down result, the reverse pull-down experiment was performed according to the method of Jiang et al. (2008) with slight modification. B. belcheri creatine kinase, BbCK, was expressed and purified. An equal amount (about 0.2 mg) of BbCK and BbCOMMD6 was mixed and incubated on ice for 1 h with gentle shaking, followed by immunoprecipitation with about 2 g of mouse anti-human CK monoclonal antibody (MM-type, Sigma) at 4 ◦ C for 2 h. 20 l of the protein A/G PLUS-Agarose (Santa Cruz Biotechnology) was added to the reaction mixture and incubated at 4 ◦ C overnight. After centrifugation, the agaroses were washed three times with PBS (pH 7.8), and the precipitated proteins were eluted from the agaroses with 50 l of SDS-PAGE loading buffer and separated by 12% SDS-PAGE. For control, the total homogenate of B. belcheri was also run on the same gel. The immunoblotting analysis was carried out as above. Two parallel reactions, incubations of the monoclonal antibody with BbCK alone or with BbCOMMD6 alone followed by interaction with the protein A/G PLUS-Agarose, were the negative controls, and incubation of the monoclonal antibody with the homogenates of B. belcheri, was positive control. 2.8. Crude subcellular distribution assay The cytosolic and mitochondrial fractions prepared from B. belcheri as described by Jiang et al. (2008) were separated by 12% SDS-PAGE and immunostained.
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2.9. Assay for effect of BbCOMMD6 on creatine kinase activity Aliquots of 6 g creatine kinase from rabbit muscle (MM-type; Sigma) or rabbit brain (BB-type; Sigma) solved in 30 l of the elution buffer (PBS, pH 8.0, containing 0.3 M NaCl and 500 mM imidazole) were incubated with 30 l of the elution buffer containing different amounts (0, 1.5, 3, 4.5, 6, 7.5, 9 and 10.5 g, respectively) of recombinant BbCOMMD6 at room temperature for 1 h. The activities of creatine kinase were detected following the instructions of CK test kit (Jiancheng Biotechnology Institute, Nanjing, China). For control, the creatine kinase was incubated with BSA instead of BbCOMMD6 and processed similarly. In parallel, the mixtures of 6 g BbCOMMD6 in the elution buffer and 6 g BB-type creatine kinase or BSA were also subjected to 12% native-PAGE. 3. Results 3.1. Sequence and phylogeny of BbCOMMD6 The full length of BbCOMMD6 cDNA (GenBank accession number: AAT45381) was 1073 bp in length, and its longest ORF encoded a protein of 203 amino acids with a calculated molecular weight (MW) of about 23 kDa (Fig. 1). The deduced protein had a COMM domain at the residues 116–201 at its C-terminal region. Alignment analysis showed that the COMM domain of BbCOMMD6 shared 47.0, 45.3, 44.7, 44.2, 45.8, 43.5, 54.7, 50.0 and 44.4% identity to that of human, chimpanzee, house mouse, dog, cattle, frog, zebrafish, sea urchin and slime mold COMMD6 proteins, respectively. However, its N-terminal region consisted of 115 amino acids, was close to that of frog, zebrafish and sea urchin COMMD6 proteins in length, and exhibited 28.2, 27.9 and 40.9% identity in sequence, individually (Fig. 2A), contrasting to the significantly shorter N-terminal portion of mammalian COMMD6 proteins. The phylogenetic tree constructed using the sequences of representative COMM
Fig. 1. The nucleotide and deduced amino acid sequences of amphioxus (Branchiostoma belcheri) COMMD6 cDNA. The start codon is underlined. The asterisk represents the stop codon. The polyadenylation signal is boxed. The numbering of the nucleotide and amino acid sequences is shown to the left.
Fig. 2. (A) Alignment of the N-terminal portion of COMMD6 proteins including BbCOMMD6. Shaded (with solid black) residues are the amino acids that match the consensus. Gaps introduced into sequences to optimize alignment are represented by (−). The sequences are: Xt (Xenopus tropicalis, NP 001016464), Dr (Danio rerio, XP 001332088), Bb (B. belcheri tsingtaunes, AAT45381), Sp (Strongylocentrotus purpuratus, XP 794885). (B) Phylogenetic tree of COMM domains of COMMD proteins including BbCOMMD6. Bootstrap majority consensus values on 1000 replicates are indicated at each branch point in percent. The sequences are: Dr1 (D. rerio, AAI08024), Dr2 (D. rerio, NP 001002421), Dr3 (D. rerio, NP 001008734), Dr4 (D. rerio, NP 001070206), Dr5 (D. rerio, NP 001003553), Dr6 (D. rerio, XP 001332088), Dr8 (D. rerio, NP 001071186), Dr9 (D. rerio, NP 001082867), Mm1 (Mus musculus, AAH51210), Mm2 (M. musculus, NP 780304), Mm3 (M. musculus, EDL08102), Mm4 (M. musculus, NP 079693), Mm5 (M. musculus, NP 079812), Mm6 (M. musculus, NP 001028304), Mm7 (M. musculus, NP 598611), Mm8 (M. musculus, NP 848714), Mm9 (M. musculus, NP 083911), Mm10 (M. musculus, NP 848464), Hs1 (Homo sapiens, AAH09266), Hs2 (H. sapiens, NP 057178), Hs3 (H. sapiens, NP 036203), Hs4 (H. sapiens, NP 060298), Hs5 (H. sapiens, NP 001074472), Hs6 (H. sapiens, CAQ09795), Hs7 (H. sapiens, AAH47440), Hs8 (H. sapiens, NP 060315), Hs9 (H. sapiens, AAH10892), Hs10 (H. sapiens, NP 057228), Xt1 (X. tropicalis, AAI60794), Xt2 (X. tropicalis, AAI54926), Xt4 (X. tropicalis, NP 001011118), Xb5 (Xenopus borealis, ACC54703), Xt6 (X. tropicalis, AAI71118), Xt7 (X. tropicalis, NP 989169), Xt10 (X. tropicalis, NP 001039088), Cf1 (Canis familiaris, NP 001003055), Cf6 (C. familiaris, XP 534146), Cf9 (C. familiaris, XP 850727), Bb6 (B. belcheri tsingtaunes, AAT45381), Bf6 (Branchiostoma floridae, XP 002247470), Bf8 (B. floridae, XP 002223266), Sp3 (S. purpuratus, XP 001185060), Sp6 (S. purpuratus, XP 794885), Sp7 (S. purpuratus, XP 001179231), Sp8 (S. purpuratus, XP 789228), Sp9 (S. purpuratus, XP 794857), Dd1 (Dictyostelium discoideum, XP 629812), Dd2 (D. discoideum, XP 639903), Dd3 (D. discoideum, XP 638867), Dd4 (D. discoideum, XP 642944), Dd5 (D. discoideum, XP 643905), Dd6 (D. discoideum, XP 637929), Dd7 (D. discoideum, XP 643770), Dd8 (D. discoideum, XP 629572), Dd9 (D. discoideum, XP 635194), Dd10 (D. discoideum, XP 643748), Ci10 (Ciona intestinalis, XP 0021221790).
domain-containing proteins including BbCOMMD6 demonstrated that BbCOMMD6 was clustered together with COMMD6 proteins, separating from all the other nine family members (Fig. 2B). A search of the recently completed draft assembly and automated annotation of Branchiostoma floridae genome was carried out. It revealed the presence of a Florida amphioxus COMMD6
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Fig. 3. SDS-PAGE and Western blotting of BbCOMMD6. (A) SDS-PAGE. Lane M, markers; lane 1, total cellular extracts from E. coli BL21 containing pET30a/BbCOMMD6 before induction; lane 2, total cellular extracts from IPTG-induced E. coli BL21 containing pET30a/BbCOMMD6; lanes 3 and 4, purified recombinant BbCOMMD6 in the presence and absence of -mercaptoethanol. The arrow (→) indicates the localization and size of monomeric and dimeric BbCOMMD6. (B) Western blotting. Totally, ∼6 g of purified protein and ∼85 g of cell lysate proteins were loaded onto the gel. Lane 1, extracts from E. coli BL21 containing pET30a/BbCOMMD6 before induction; lane 2, extracts from IPTG-induced E. coli BL21 containing pET30a/BbCOMMD6; lane 3, recombinant BbCOMMD6 protein; lane 4, extracts of amphioxus B. belcheri. The arrow (→) indicates the localization of the recombinant BbCOMMD6 and the triangle (I) the distribution of native BbCOMMD6 in the amphioxus extracts.
cDNA and its genomic DNA sequence (estExt gwp.C 820054, scaffold 82: 1065728–1071678, http://genome.Jgi-psf.org/cgibin/dispTranscript?db=Brafl1&id=280511&useCoords=1). Sequence comparison showed that BbCOMMD6 shared 96% identity to the deduced protein encoded by the Florida amphioxus gene at the amino acid level (Fig. 2B), suggesting that BbCOMMD6 is highly conserved in inter-species. 3.2. Biochemical properties of recombinant BbCOMMD6 An expression vector including the entire ORF of BbCOMMD6 and a 5 additional His6 tag (this resulted in the increase in the size of the recombinant protein by approximately 5 kDa) of pET30a was constructed and successfully transformed into E. coli. The recombinant BbCOMMD6 was purified and separated on SDS-PAGE in the presence of -mercaptoethanol, which yielded a single band of ∼28 kDa, coinciding with its theoretical size (Fig. 3A). In contrast, the BbCOMMD6 occurred as two bands under SDS-PAGE in the absence of -mercaptoethanol, with one band being ∼28 kDa and the other being ∼55 kDa (Fig. 3A). These indicated that BbCOMMD6 existed as a mixture of monomers and dimers generated via disulfide bond formation. Rabbit antiserum against BbCOMMD6 with a titer of 1:1000 was obtained. Western blotting demonstrated that the antiserum reacted with the cell lysates of IPTG-induced E. coli BL21, forming a band of ∼28 kDa, but not with the lysate of the same E. coli cells before induction. The antiserum also reacted with the homogenates of B. belcheri, forming a band of ∼23 kDa (Fig. 3B), matching the molecular mass predicted by BbCOMMD6 cDNA. These showed that the rabbit antiserum had a conspicuous antigen-specific reactivity. To test if Cu(II) was able to stimulate the dimer formation, BbCOMMD6 was incubated with CuCl2 and separated by nativePAGE. Unexpectedly, little change in the intensity of the fast migrating band (*) was found (Fig. 4A). Similarly, SDS-PAGE in the absence of -mercaptoethanol revealed that the presence of Cu(II) was not able to enhance dimer formation (Fig. 4B). To identify the proteins that might interact with BbCOMMD6, the pull-down technique was applied (Kaelin et al., 1991; Soutoglou et al., 2000). It was found that a protein (A in lane 4 of Fig. 5) from B. belcheri homogenates bound to BbCOMMD6 (B in lane 4 of Fig. 5). The molecular weight of protein A was approximately 42 kDa. A total of 14 peptide fragments of the protein were measured by MALDI-TOF MS5. The measured peptides were matched
against the creatine kinase from B. belcheri, and the MOWSE score obtained was 414, which was 5.2-fold greater than the threshold for identification. The matched peptide fragments were located at the residues 8–20, 21–27, 103–111, 119–124, 133–142, 146–166, 147–166, 204–209, 210–217, 218–230, 302–309, 303–311, 315–336 and 316–336 of the CK (Fig. 6), respectively. The control experiments revealed no protein bound to the particles without BbCOMMD6 (Fig. 5). These suggested that BbCOMMD6 was able to bind to CK in B. belcheri. To verify that BbCOMMD6 can bind to CK, the reverse pulldown assay was carried out. BbCOMMD6 and BbCK were incubated together with the mouse anti-human CK monoclonal antibody, and then with the protein A/G PLUS-Agarose. After washing stringently, the complexes were analyzed by Western blotting. Recombinant BbCOMMD6 was detected in the complexes (Fig. 7, lanes 1–3). Similarly, BbCOMMD6 was also detected in the positive controls
Fig. 4. Effects of copper on the dimerization of BbCOMMD6. (A) Native-PAGE of BbCOMMD6 in the presence of Cu(II). Ten microgram aliquots of BbCOMMD6 were incubated with 0, 0.5 and 5.0 mM CuCl2 before being applied to 12% native-PAGE. (B) SDS-PAGE of BbCOMMD6 in the presence of Cu(II). The same amount of protein in the absence and presence of Cu(II) were applied to 12% SDS-PAGE without mercaptoethanol. The protein bands were stained with Commassie blue dye.
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Fig. 5. SDS-PAGE (12%) analysis of the pulled down proteins. Lane M, markers; lane 1, extracts from E. coli BL21 containing pET30a/BbCOMMD6 after induction; lane 2, the control particles incubated with both recombinant BbCOMMD6 protein and the homogenate buffer; lane 3, the control particles incubated with the pET30a/expressed soluble protein and the tissue homogenates; lane 4, recombinant BbCOMMD6 protein and the interaction protein; lane 5, total protein of the amphioxus homogenate. A indicates the pulled down protein and B the recombinant BbCOMMD6 protein.
including the homogenates of B. belcheri (Fig. 7, lane 7) and the homogenates incubated with the monoclonal antibody and the protein A/G PLUS-Agarose (Fig. 7, lane 6). In contrast, no signals were observed in the negative controls including the incubation of the monoclonal antibody with BbCK alone or with BbCOMMD6 alone, followed by interaction with the protein A/G PLUS-Agarose (Fig. 7, lanes 4 and 5). These showed that BbCOMMD6 was pulled down by forming the BbCOMMD6–BbCK–antibody–agarose complex, indicating that BbCOMMD6 was able to interact with BbCK specifically. 3.3. Subcellular localization of BbCOMMD6 To investigate the subcellular localization of BbCOMMD6, the cytosolic and mitochondrial fractions were prepared from
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Fig. 7. Western blotting of reverse pulled down proteins. Lanes 1–3 show the presence of recombinant BbCOMMD6 in the complexes eluted; lanes 4 and 5 are the two negative controls; lane 6 shows the presence of natural BbCOMMD6 in the complexes eluted; lane 7 indicates the presence of natural BbCOMMD6 in the total homogenates. The arrow (→) indicates the localization of recombinant BbCOMMD6 and the triangle (I) the distribution of native BbCOMMD6 in the amphioxus homogenates.
B. belcheri and analyzed by Western blotting. It showed that BbCOMMD6 was present in the cytosolic fraction; no signal was observed for the mitochondrial fraction (Fig. 8). This indicated that BbCOMMD6 was localized in the cytosol, agreeing with the primary cytosolic localization of COMMD1 in mammalian species (Klomp et al., 2003). 3.4. Inhibition of CK activity by BbCOMMD6 Next we sought to examine if the recombinant BbCOMMD6 has any effect on CK activity. It was found that BbCOMMD6 was capable of inhibiting the activities of both the MM- and BB-type CKs in a concentration-dependent manner, whereas BSA had no influence (Fig. 9). Calculation revealed that on average three BbCOMMD6
Fig. 6. MALDI spectra of trypsin digestion of the pulled down protein. (A) is the PMF data of creatine kinase protein. The 14 peaks of identified peptide fragments are marked with asterisks. (B) shows the identified peptide number, the observed mass and the sequence of the creatine kinase protein, respectively.
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Fig. 8. Western blotting analysis of the BbCOMMD6 subcellular distribution. Lane 1, purified recombinant BbCOMMD6; lane 2, the extracts from E. coli BL21 containing pET30a/BbCOMMD6 before induction; lane 3, the cytosolic fraction of the primitive gut; lane 4, the mitochondrial fraction of the gut. The arrow (→) indicates the localization of recombinant BbCOMMD6 and the triangle (I) the distribution of native BbCOMMD6 in the different cell fractions of the gut.
molecules per one CK was necessary to cause the CK inhibition. This was the first evidence that BbCOMMD6 could specifically suppress CK activities. Of note, native-PAGE demonstrated that the preincubation of BbCOMMD6 with CK resulted in BbCOMMD6-CK dimer formation, while no degraded peptides were observed (Fig. 10), suggesting that BbCOMMD6 inhibited CK activities via directly binding with CK.
Fig. 10. Native-PAGE showing the binding of recombinant BbCOMMD6 to BB-type creatine kinase. The same amount of BB-type creatine kinase was incubated with recombinant BbCOMMD6 or with BSA (as negative control), and then subjected to 12% native-PAGE. The arrow (→) indicates the presence and localization of BbCOMMD6-CK dimer in the mixture of BbCOMMD6 and CK after incubation. The molecular weights of monomeric and dimeric BbCOMMD6 are shown to the left.
4. Discussion
Fig. 9. Inhibition of CK activity by BbCOMMD6. (A) Inhibition of the creatine kinases from rabbit muscle; (B) inhibition of the creatine kinases from rabbit brain. Different concentrations of recombinant BbCOMMD6 and BSA (as negative control) were added to the creatine kinases, and then CK activities were detected following the instructions of CK test kit. Data are mean values ±S.D. from three experiments.
With the exception of COMMD1, the functions and molecular partners of COMMD family members remain essentially unknown. Here we report the identification, expression and functional characterization of a member of COMMD family, COMMD6, in B. belcheri. The deduced 203 amino acids long protein, BbCOMMD6, has a COMM domain at its C-terminus and an extended N-terminal portion, resembling that of frog, zebrafish and sea urchin COMMD6 proteins both in sequence and in length. This is the first member of COMMD family reported in the protochordates. Various protein–protein interaction experiments have shown that COMMD1 is able to dimerize with several members of COMMD family (Burstein et al., 2005; de Bie et al., 2006) and to form complexes with a growing number of other structurally and functionally unrelated proteins (Burkhead et al., 2009; Klomp et al., 2003). Here we demonstrate that BbCOMMD6 is able to interact with CK, forming a heterodimer BbCOMMD6-CK. Moreover, BbCOMMD6 is able to inhibit CK activities. BbCOMMD6 appears to inhibit CK activities by its binding to CK, thereby leading to the conformational change in CK as BbCOMMD6 does not apparently cause the degradation of CK. CK, also known as creatine phosphokinase, is an enzyme catalyzing the reversible transfer of the phosphate group of phosphocreatine to adenosine diphosphate (ADP), generating adenosine triphosphate (ATP) and creatine. Therefore, our results suggest that BbCOMMD6 may be involved in energy transduction, via binding to CK and inhibiting its activities. This is the first report showing the inhibitory effects of COMMD6 on CK activities; it will be of particular interest to determine if this is also true for COMMD6 in other organisms. Interestingly, recent studies have shown that muscle
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RING-finger protein-1 (MuRF1) and ycaC-related protein (ycaCR) are also able to interact with CK (Jiang et al., 2008; Koyama et al., 2007). These results and ours together suggest that CK activity may be regulated by multiple factors. COMMD1 has been shown to bind Cu(II), increasing the COMMD1–COMMD1 homodimer formation (Narindrasorasak et al., 2007). In contrast, the presence of Cu(II) fails enhancing the dimerization of BbCOMMD6. We speculate that the presence of Cu(II) may not facilitate the dimerization of BbCOMMD6; however, further study is necessary to determine if BbCOMMD6 binds with Cu(II). It is also interesting to note that BbCOMMD6 is mainly present in the cytosol although its detailed cellular distribution is still to be determined. Previous studies have shown that COMMD1 is distributed throughout the cell including the nucleus, with main localization in the cytoplasm (Klomp et al., 2003; Maine and Burstein, 2007), and COMMD5 is a primarily nuclear protein (Solban et al., 2000). It appears that different COMMD proteins are localized in different cellular compartments. It is nuclear if there is a relationship between the different cellular distribution of these proteins and their biological significance. In summary, this study highlights for the first time a novel function of COMMD6 in energy transduction through its interacting with CK and inhibiting CK activities. It will be of particular interest to examine if COMMD6 in other organisms also plays a role as a regulator of CK activities. Acknowledgement This work was supported by the Ministry of Science and Technology (MOST) of China (2008AA09Z409). References Biasio W, Chang T, McIntosh CJ, McDonald FJ. Identification of Murr1 as a regulator of the human epithelial sodium channel. J Biol Chem 2004;279:5429–34. Boutell JM, Thomas P, Neal JW, Weston VJ, Duce J, Harper PS, et al. Aberrant interactions of transcriptional repressor proteins with the Huntington’s disease gene product, huntingtin. Hum Mol Genet 1999;8:1647–55. Burkhead JL, Morgan CT, Shinde U, Haddock G, Lutsenko S. COMMD1 forms oligomeric complexes targeted to the endocytic membranes via specific interactions with phosphatidylinositol 4,5-bisphosphate. J Biol Chem 2009;284(1):696–707. Burstein E, Ganesh L, Dick RD, van de Sluis B, Wilkinson JC, Klomp LW, et al. A novel role for XIAP in copper homeostasis through regulation of MURR1. EMBO J 2004;23:244–54. Burstein E, Hoberg JE, Wilkinson AS, Rumble JM, Csomos RA, Komarck CM, et al. COMMD proteins: a novel family of structural and functional homologs of MURR1. J Biol Chem 2005;280:22222–32.
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