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Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; and CREST, Japan Science and Technology Corporation, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
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Introduction |
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Despite the large number of protein substrates for the ubiquitin proteolytic pathway, relatively few E3s have been characterized at the molecular level. E3s whose amino acid sequences are known include members of the HECT (homologous to E6-AP carboxyl terminus) family (Huibregtse et al. 1995) and members of the RING finger–containing protein family (Joazeiro et al. 1999; Lorick et al. 1999; Freemont 2000). HECT-family E3s include E6-AP, which targets p53 for ubiquitylation in the presence of human papilloma virus E6 (Scheffner et al. 1993), and Nedd4, which ubiquitylates epithelial Na+ channel subunits (Staub et al. 1997). E3s that contain a RING finger, a motif defined by a total of eight cysteine and histidine residues that coordinate two zinc ions, include those that mediate protein degradation according to the N-end rule (Xie & Varshavsky 1999); Mdm2, which catalyses both its own ubiquitylation and that of p53 (Fang et al. 2000; Honda & Yasuda 2000); the anti-apoptotic proteins c-IAP1 and XIAP (Yang et al. 2000); Cbl, which targets the receptor for epidermal growth factor (Joazeiro et al. 1999; Waterman et al. 1999); the anaphase-promoting complex or cyclosome (APC/C), which ubiquitylates mitotic cyclins and anaphase inhibitors (securins) (Gmachl et al. 2000; Leverson et al. 2000); and SCF complexes, whose substrates include G1 cyclins, cyclin-dependent kinase inhibitors, I
B, and ß-catenin (Yaron et al. 1998; Hatakeyama et al. 1999; Kamura et al. 1999; Kitagawa et al. 1999; Nakayama et al. 2000). Furthermore, RING fingers of otherwise unrelated proteins, such as BRCA1, Siah-1, TRC-8, Praja1 and AO7, support E2-dependent ubiquitylation (Lorick et al. 1999), suggesting that this domain plays a general role in the ubiquitin system.
A new class of ubiquitylation enzyme, termed E4, the prototype of which is yeast Ufd2, was recently identified (Koegl et al. 1999). Together with E1 (Uba1), E2 (Ubc4), and a HECT-type E3 (Ufd4), E4 (Ufd2) is required for the assembly of a polyubiquitin chain on artificial substrates that comprise proteins with a fused ubiquitin moiety at their NH2-termini and which are preferentially targeted for degradation. Ufd2 and its homologs in other eukaryotes share a conserved domain designated the U box (Koegl et al. 1999). The U box of Ufd2 mediates the interaction of this protein with ubiquitin-conjugated targets and is therefore likely an essential functional unit of E4 enzymes. The predicted three-dimensional structure of the U box is similar to that of the RING finger, despite the lack in the former of the hallmark metal-chelating residues of the latter (Aravind & Koonin 2000).
We have recently shown that all six mammalian U-box proteins tested mediate ubiquitylation by functioning as E3s (Hatakeyama et al. 2001). Deletion of the U-box domain or mutation of its most conserved amino acids resulted in the loss of E3 activity, suggesting that such activity requires an intact U box. One of these proteins, UFD2a, attaches ubiquitin to lysine residues of ubiquitin other than those at positions 29, 48, or 63, a property that is not usually exhibited by HECT-type or RING finger–containing E3s. Taken together, these observations suggest that U-box proteins constitute a new family of E3s, some of which may also function as E4s to mediate the assembly of polyubiquitin chains on proteins ubiquitylated by another E3 enzyme.
Through its interaction with the AAA-type ATPase Cdc48, which possesses chaperone activity, Ufd2 is thought to contribute to cell survival under stressful conditions in yeast (Koegl et al. 1999). We have also recently shown that, in mammals, UFD2a binds to VCP, an ortholog of yeast Cdc48 (Kaneko et al. 2003). Coordination of the chaperone activity of VCP (or Cdc48) with the ability of UFD2a (or Ufd2) to bind ubiquitin chains is implicated in endoplasmic reticulum-associated degradation (ERAD). These proteins are thus thought to act at the cytosolic face of the endoplasmic reticulum to promote the translocation of ERAD substrates across the membrane of this organelle and to present them to the proteasome.
The U-box protein CHIP interacts with the molecular chaperones Hsc70 and Hsp90 through its tetratricopeptide repeat (TPR) domain and is thereby thought to participate in the cellular stress response to the accumulation of unfolded or misfolded proteins. The combination of CHIP and Hsp90 mediates ubiquitylation of the glucocorticoid receptor, and that of CHIP and Hsc70 targets the immature cystic fibrosis transmembrane conductance regulator (CFTR) for proteasomal degradation (Connell et al. 2001; Meacham et al. 2001). CHIP has been proposed to act as a cochaperone that regulates the balance between the folding of these two proteins and their degradation by the ubiquitin-proteasome pathway. We and others have shown that CHIP itself possesses ubiquitin ligase activity (Hatakeyama et al. 2001; Jiang et al. 2001; Murata et al. 2001). CHIP is thus thought to act as an E3 in the quality control of protein folding, selectively ubiquitylating unfolded proteins associated with molecular chaperones. We have also recently shown that CHIP and Hsp70 are associated both with Parkin, a RING finger–containing E3 that when mutated contributes to autosomal recessive juvenile parkinsonism, and with the Pael receptor, a substrate targeted by Parkin for degradation (Imai et al. 2002). These interactions are thought to be related to the ERAD pathway. Some U-box proteins thus appear to interact with molecular chaperones to mediate the ubiquitylation of misfolded proteins.
We now show that mammalian U-box proteins in general physically interact with molecular chaperones or cochaperones: UFD2a binds to VCP; UFD2b to DnaJc7; CHIP to Hsp90, Hsp70, and EKN1; UIP5 (KIAA0860) to VCP; and CYC4 to CRN and Hsp90. Through their association with molecular chaperones, most U box–type E3s may function as ‘quality control E3s’ in the clearance of abnormal proteins from the cell.
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Results |
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Proteins that associate with mammalian U-box proteins other than UFD2a and CHIP have not been previously identified. We therefore set out to identify the binding partners of other U-box proteins in order to examine further the relation between the function of these proteins and that of molecular chaperones. UFD2a and UFD2b are mammalian homologs of yeast Ufd2, which binds to Cdc48. Given that UFD2a interacts with VCP, a mammalian homolog of Cdc48, we examined the possible interaction between mouse UFD2b and VCP in human HEK293T cells. Immunoprecipitation and immunoblot analysis revealed that recombinant UFD2a, but not UFD2b, interacted with VCP in the transfected cells (Fig. 1A). To identify potential binding partners of UFD2b, we performed yeast two-hybrid screening with a cDNA fragment encoding amino acids 1–539 of mouse UFD2b as the bait. From 1.7 x 106 transformants obtained with a mouse T cell lymphoma cDNA library that were able to grow on Leu- and Trp-deficient medium, 23 positive clones were isolated after two rounds of growth in the absence of His and screening for ß-galactosidase activity. The nucleotide sequences of six of the 23 clones were highly homologous to that of human DnaJc7 cDNA, and the longest of these clones encoded all but the five NH2-terminal amino acids (residues 6–494) of mouse DnaJc7 (Ohtsuka & Hata 2000). The interaction between mouse UFD2b and DnaJc7 was verified in yeast (Fig. 1B).
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2.0 kb in size and is abundant in testis, kidney, heart, and brain as well as present in smaller amounts in liver (data not shown). Recombinant mouse UFD2b, but not UFD2a, also interacted with recombinant mouse DnaJc7 in HEK293T cells (Fig. 1D). We also showed that mouse DnaJc7 interacted with mouse Hsp90 in a yeast two-hybrid assay (data not shown) and that recombinant mouse DnaJc7 interacted with endogenous Hsp90 in HEK293T cells (Fig. 1E). These findings demonstrated that UFD2a and UFD2b, two mammalian homologs of yeast Ufd2, associate with the chaperone VCP and the cochaperone DnaJc7, respectively, suggesting that UFD2a and UFD2b might play diverse roles in collaboration with different molecular chaperone systems. CHIP interacts with Hsp90 or Hsp70 through its TPR domain and charged region
The molecular chaperones Hsp90 and Hsc70 are thought to function as substrate receptors for the ubiquitin ligase activity of CHIP (Connell et al. 2001; Meacham et al. 2001; Murata et al. 2001). However, the regions of CHIP responsible for the interactions with Hsp90 and Hsc70 have not been previously determined. To examine whether the TPR region of CHIP is sufficient for its binding to Hsp90 or Hsp70, we constructed truncation mutants of mouse CHIP lacking the U box (
U), the three TPRs (TPR), or both the charged region and U box (CU) (Fig. 2A). An in vivo binding assay in transfected HEK293T cells revealed that full-length (FL) CHIP and the U mutant bound to both Hsp90 and Hsp70, whereas the TPR and CU mutants did not (Fig. 2B), indicating that the interaction between CHIP and Hsp90 or Hsp70 requires both the TPR and the charged region of CHIP in vivo.
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In certain instances, CHIP does not appear to rely on Hsp70 or Hsp90 for recognition of target substrates (Imai et al. 2002). We therefore investigated whether regions of CHIP other than the TPR domain either contribute to substrate recognition directly or interact with other chaperones or cochaperones. To identify potential binding partners for CHIP, we performed yeast two-hybrid screening with a cDNA fragment encoding the TPR mutant, which contains amino acids 129–304 of the mouse protein, as the bait. From 8 x 105 transformants obtained with a HeLa cell cDNA library, four positive clones were isolated. The nucleotide sequence of one of these clones was highly similar to that of mouse EKN1 cDNA, and the interaction between CHIP and EKN1 was verified in yeast (Fig. 2C). Although the function of EKN1 has been unknown, the protein contains a domain implicated in movement within the nucleus and three TPRs (Fig. 2D). Mouse EKN1 mRNA is abundant in testis but is also present in smaller amounts in kidney (data not shown). An in vivo binding assay revealed that recombinant mouse CHIP interacted specifically with recombinant human EKN1 in transfected HEK293T cells (Fig. 2E). To determine whether the TPRs of EKN1 might interact with molecular chaperones, we immunoprecipitated HA-EKN1 or HA-VCP (negative control) from transfected HEK293T cells and subjected the resulting precipitates to immunoblot analysis with antibodies to Hsp70. The HA-EKN1 precipitate contained endogenous Hsp70 whereas the HA-VCP precipitate did not (Fig. 2F). These results thus demonstrate that EKN1 binds to Hsp70. They further suggest that CHIP relies on at least two molecular chaperone–dependent mechanisms for substrate recognition: one mediated by the direct interaction with Hsp90 or Hsp70, and another mediated by the EKN1–dependent interaction with Hsp70.
Interaction of UIP5 with VCP
We next attempted to identify binding partners of mouse UIP5 (also known as KIAA0860), which contains both a U box and a RING finger (Fig. 3A), only the former of which is responsible for ubiquitin ligase activity (Hatakeyama et al. 2001). We performed yeast two-hybrid screening with a cDNA encoding full-length mouse UIP5 as the bait. From 1.6 x 106 transformants obtained with a mouse T cell lymphoma cDNA library, six positive clones were isolated. The nucleotide sequence of one of these clones was almost identical to that of mouse VCP cDNA. To determine which regions of UIP5 are sufficient for binding to VCP, we constructed two deletion mutants lacking either the U box (U) or the RING finger (R) (Fig. 3A). Whereas full-length (FL) UIP5 and the U mutant bound to VCP in the yeast two-hybrid assay, the R mutant did not (Fig. 3B), indicating that the interaction between UIP5 and VCP is dependent on the RING finger of UIP5. FLAG-UIP5 was also shown to interact specifically with HA-VCP in HEK293T cells (Fig. 3C).
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Identification of CRN as a binding protein for CYC4
To identify binding partners for the U-box protein CYC4, we performed yeast two-hybrid screening with a cDNA for full-length human CYC4 as the bait. From a total of 1.3 x 105 transformants obtained with a mouse T cell lymphoma cDNA library, nine positive clones were isolated. The nucleotide sequences of three of these nine clones were highly homologous to that of the Drosophila melanogaster crooked neck (CRN) gene, which is thought to play an important role in development of the central and peripheral nervous systems in the fly (Zhang et al. 1991). The interaction between human CYC4 and mouse CRN was verified in yeast (Fig. 4A) The mouse CRN cDNA encodes a protein of 690 amino acid residues that shares 65% sequence identity with Drosophila CRN (Fig. 4B) and which contains 16 TPRs, two W-F/Y motifs, and a nuclear localization signal. The mRNA for mouse CRN is abundant in heart, liver, and testis but is also present in smaller amounts in brain, spleen, lung, and kidney (data not shown).
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Given that CRN contains 16 TPRs, we examined whether CRN interacts with Hsp90. HA-CRN immunoprecipitates prepared from transfected HEK293T cells indeed contained endogenous Hsp90 (Fig. 5A). Furthermore, an in vitro‘pull-down’ assay revealed that a GST-CRN fusion protein specifically interacted with recombinant Hsp90 (data not shown). These results thus suggested that the TPRs of CRN serve as a binding site for Hsp90. To examine further the interactions among CYC4, CRN, and Hsp90, we performed an in vivo binding assay in HEK293T cells transfected with expression vectors for FLAG-CYC4 and HA-Hsp90. FLAG-CYC4 was found to interact specifically with HA-Hsp90 (data not shown). We also prepared polyclonal antibodies to human CYC4, and found that immunoprecipitates prepared from non-transfected HEK293T cells with these antibodies also contained Hsp90 (Fig. 5B). These results demonstrate that endogenous CYC4 constitutively interacts with endogenous Hsp90. To examine whether CRN and/or Hsp90 affects the ubiquitin ligase activity of CYC4, in vitro ubiquitylation assay using recombinant CYC4, CRN and Hsp90 was performed. The ubiquitin ligase activity of CYC4 with VCP/Hsp90 was little changed compared with that in the absence of VCP/Hsp90 (Fig. 5C). Therefore CRN/Hsp90 may not serve as a regulator for ubiquitin ligase activity of CYC4. To determine the regions of CRN responsible for binding to CYC4 and to Hsp90, we constructed various deletion mutants of CRN (Fig. 5D). In vivo binding assays in HEK293T cells revealed that CRN(1–467) and CRN(468–690), but not CRN(1–249), interacted with CYC4 (Fig. 5E), suggesting that either of two regions of CRN included within residues 250–467 and 468–690 mediate the interaction with CYC4. In contrast, CRN(1–467), but not CRN(1–249) or CRN(468–690), associated with Hsp90 (Fig. 5F), suggesting that the region spanning residues 250–467 contains the binding site for Hsp90. These results are consistent with the notion that CYC4, CRN, and Hsp90 form a trimeric complex.
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Discussion |
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The mammalian U-box protein CHIP associates with the molecular chaperones Hsp90 or Hsp70 in order to recognize abnormal forms of CFTR, the glucocorticoid receptor, luciferase, and the Pael receptor (Connell et al. 2001; Meacham et al. 2001; Murata et al. 2001; Imai et al. 2002). The combination of CHIP with Hsp90 mediates ubiquitylation of the glucocorticoid receptor, and CHIP together with Hsc70 targets immature CFTR for proteasomal degradation. Denatured firefly luciferase was also polyubiquitylated by CHIP in the presence of E1 and E2 (Ubc4 or UbcH5c) in vitro only when the unfolded substrate was captured by the COOH-terminal region of Hsp90, which also contains a CHIP binding site, or by Hsc70 and Hsp40. We have now shown that the interaction between CHIP and Hsp90 or Hsp70 requires both the TPRs and the charged region in the middle portion of CHIP but not its U-box domain.
In the present study, we also identified EKN1 as a new binding partner for CHIP and found that the TPR domain of CHIP is not required for the interaction with EKN1. Although the function of EKN1 is unknown, a structurally similar TPR-containing protein, XAP2 (hepatitis B virus X-associated protein), has previously been shown to interact functionally with CHIP (Lees et al. 2003). XAP2, which is also known as Ara9 (AhR-associated protein 9) or AIP2 (AhR-interacting protein 2), is a component of the dioxin receptor (DR) chaperone complex (Kuzhandaivelu et al. 1996). In addition to XAP2, this complex comprises the molecular chaperone Hsp90 and the cochaperone p23 (Kazlauskas et al. 1999). Over-expression of CHIP induces the degradation of the DR, whereas over-expression of XAP2 inhibits this action of CHIP, suggesting that XAP2 may directly suppress the ubiquitin ligase activity of CHIP (Lees et al. 2003). Given the structural similarity between XAP2 and EKN1, the latter protein also might inhibit CHIP activity either by direct binding or by sequestering molecular chaperones such as Hsp70.
Our present results indicate that U-box proteins other than UFD2a and CHIP also interact with molecular chaperones or cochaperones. We thus showed that UFD2b binds to DnaJc7, which also appears to interact with Hsp90 (Ohtsuka & Hata 2000). DnaJc7 possesses two functional domains related to chaperone function: a TPR domain, which interacts with the COOH-terminal region of Hsp90 or Hsp70 (EEVD motif) (Scheufler et al. 2000), and a J domain, which was first identified in DnaJ (Hsp40) of E. coli and mediates the interaction with Hsc70 (Pellecchia et al. 1996). This domain structure of DnaJc7 is similar to that of p58IPK, which serves as a negative regulator of the interferon-inducible double-stranded RNA–dependent protein serine-threonine kinase (PKR) (Tang et al. 1999; Melville et al. 2000). The p58IPK protein is a cochaperone and possibly directs Hsp70 or Hsc70 through interaction with its TPR domain to refold and thereby to inhibit the kinase activity of PKR. Several kinases other than PKR are regulated by the refolding activity of molecular chaperones, especially that of Hsp90. Given the structural similarity between DnaJc7 and p58IPK, the former also might regulate a protein kinase (or kinases) through its interaction with molecular chaperones such as Hsp90 or Hsp70. It is also possible that DnaJc7 functions as a substrate recognition module for UFD2b in the ubiquitin-dependent degradation of target proteins.
We also identified CRN as a binding partner for CYC4, suggesting that CRN may function as an adapter molecule that mediates the association between CYC4 and Hsp90. A group of four TPR-containing proteins (Clf1, Prp39, Prp42, Rna14) that contribute to premRNA splicing has recently been identified in yeast (Wang et al. 2003). One of these proteins, Clf1, is related (40% sequence identity) to Drosophila CRN (Chung et al. 1999). Furthermore, Drosophila CRN has been shown to be a component of a high molecular mass complex in embryonic nuclear extracts that contains snRNPs (Raisin-Tani & Leopold 2002). Drosophila crn mutants manifest reduced levels of cellular DNA synthesis, suggesting a role for CRN in the eukaryotic cell cycle. To date, however, few proteins with a clearly defined biochemical role in the cell cycle have been shown to be required for pre-mRNA splicing. Further studies of CRN function might provide insight into a link between cell cycle regulation and RNA splicing.
The fact that almost all U-box proteins tested interact with molecular chaperones or cochaperones suggests that U-box proteins both rely on molecular chaperones to recognize unfolded or misfolded proteins and function in the degradation of these damaged proteins. We failed to find proteins with apparent structural characteristics of chaperone or cochaperone that interact with PRP19, a U-box protein in the spliceosome complex (Hatakeyama et al. 2001). Yeast two-hybrid screen for PRP19 yielded four proteins that interact with PRP19: CRK7 (Cdc2 related protein kinase7 for splicing), Rbm6, Cdc5L (components of spliceosome), and S1-1 (RNA binding protein). Although structural characteristics of the deduced amino acid sequence of these proteins do not indicate that these molecules serve as chaperone or cochaperone, further functional analysis will be necessary to draw a final conclusion. Given that U-box proteins exhibit different subcellular distributions, the specific localization of each such protein in the cell might be important for its specific function. The preferential target molecules for each U-box protein also may be determined by the molecular chaperones or cochaperones with which it associates: VCP for UFD2a and UIP5, or Hsp90 or Hsp70 in conjunction with DnaJc7, EKN1 (or no cochaperone), or CRN for UFD2b, CHIP, and CYC4, respectively (Fig. 6). U-box proteins likely play important roles in the ubiquitylation of specific substrates, especially under conditions of cellular stress, when they may serve to link the processes of protein folding by molecular chaperones and protein degradation by the ubiquitin-proteasome pathway. The function of U box–type E3s might therefore be impaired in pathological conditions characterized by protein accumulation or deposition, including viral infections, protein metabolic diseases such as systemic amyloidosis, and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Elucidation of the target specificity of the various U-box proteins complexed with molecular chaperones should provide insight into the physiological importance of these ubiquitin ligases.
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Experimental procedures |
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HEK293T cells were cultured under an atmosphere of 5% CO2 at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% foetal bovine serum (Invitrogen).
Yeast two-hybrid screening
Complementary DNAs encoding amino acids 1–539 of mouse UFD2b, residues 129–304 of mouse CHIP, full-length mouse UIP5, or full-length human CYC4 were fused in-frame to the nucleotide sequence for the LexA domain (BD) in the yeast two-hybrid vector pBTM116. To screen for proteins that interact with each of these U-box proteins, we transfected yeast strain L40 stably expressing the corresponding pBTM116 vector with a mouse T cell lymphoma or human HeLa Matchmaker cDNA library (Clontech). Transformants were plated on 10 culture dishes (150 by 15 mm) containing synthetic medium deficient in Trp, Leu, Lys, His and uracil but supplemented with 25 mM 3-aminotriazole (3-AT) to reduce the number of false-positive colonies. After incubation of the plates for 7 days at 30 °C, growing colonies were transferred to fresh plates, incubated for an additional day, and assayed for ß-galactosidase activity. To isolate plasmids from ß-galactosidase-positive colonies, we transformed yeast strain L40 with total yeast DNA containing the respective plasmid and then plated the cells on synthetic medium deficient in Leu; total DNA was then isolated from the resulting colonies and introduced into Eschericia coli XL-1Blue (Clontech) for further manipulation.
Yeast transformation and ß-galactosidase assay
Yeast strain L40 [MATa, his3D200, trp1–901, leu2–3, 112, ade2, LYS2::(lexAop) 4-HIS3, URA3::(lexAop) 8-lacZ, gal4, gal80] (Invitrogen) was used for all transformations and assays. Yeast transformation was performed by the lithium acetate method, and cells were grown at 30 °C with appropriate synthetic complete ‘drop-out’ medium. For assay of ß-galactosidase activity, yeast colonies were transferred to filter papers and the cells were lysed by submerging the filters in liquid nitrogen for 30 s. The filters were then maintained at room temperature for 5 min, transferred to other filters that had been soaked in Z buffer (100 mM sodium phosphate (pH 7.0), 10 mM KCl, 1 mM Mg2SO4) supplemented with 50 mMß-mercaptoethanol and 5-bromo-4-chloro-3-indole ß-D-galactoside (0.07 mg/mL), and incubated for 3 h at 30 °C.
Cloning of cDNAs and plasmid construction
Expression plasmids for FLAG epitope–tagged wild-type or mutant forms of mouse UFD2a, UFD2b, CHIP, and UIP5 as well as human CYC4 were generated as previously described (Hatakeyama et al. 2001). Mouse VCP cDNA was amplified from thymus cDNA by the polymerase chain reaction (PCR) with Taq polymerase (Takara, Tokyo, Japan) and the primers 5'-TGCTCCTCCGCCTCAGCGAGTCCAG-3' and 5'-CCAAACTACGACAGAAACCGTGTG G-3'. The amplified fragment was rendered blunt-ended by T4 DNA polymerase (New England Biolabs), phosphorylated with T4 polynucleotide kinase (New England Biolabs), subcloned into pBluescriptII SK+ (Stratagene), and sequenced. It was then used as a probe to screen 1 x 106 clones of a mouse T cell cDNA library in 
ZAP (Stratagene). Clones containing the entire coding region were obtained. To generate VCP tagged with the haemagglutinin epitope (HA) at its NH2-terminus, we performed PCR with pBluescriptII SK+ containing full-length VCP cDNA as the template and subcloned the resulting product in pcDNA3 (Invitrogen). Mouse DnaJc7 cDNA was amplified by PCR with Taq polymerase from a mouse T cell lymphoma cDNA library (Clontech), subcloned into pcDNA3 for expression as a Myc epitope– or HA-tagged protein, and sequenced. Human and mouse EKN1 cDNAs were amplified by PCR with Taq polymerase from HeLa cell and mouse testis cDNA, respectively, subcloned into pcDNA3 for expression with an HA tag, and sequenced. A mouse partial CRN cDNA was excised from pACT-CRN, which was isolated as a result of yeast two-hybrid screening with a full-length CYC4 cDNA as the bait; it was then used as a probe to screen 1 x 106 clones of a mouse T cell cDNA library in ZAP. Clones containing the entire coding region were obtained. To generate full-length or truncated forms of CRN tagged with HA at their NH2-termini, we performed PCR with pBluescriptII SK+ containing CRN cDNA as the template and subcloned the amplified fragments in pcDNA3.
Production of recombinant proteins in bacteria
Glutathione S-transferase (GST) fusion proteins were expressed in E. coli strain DH5
cultured in the presence of 0.1 mM isopropyl-ß-D-thiogalactopyranoside. Bacterial cells were suspended in phosphate-buffered saline (PBS) and lysed by sonication, and cellular debris was removed by centrifugation for 20 min at 13 000 x g. Glutathione–Sepharose 4B beads (Amersham Pharmacia Biotech) were added to the resulting supernatant, and the mixture was rotated overnight at 4 °C. The beads were washed with PBS, and GST fusion proteins were eluted with 50 mM Tris-HCl (pH 8.0) containing 10 mM reduced glutathione. To generate recombinant proteins without the GST moiety, we subjected the purified fusion proteins attached to the glutathione–Sepharose 4B beads to digestion for 4 h at 4 °C with PreScission protease (Amersham Pharmacia Biotech) in a solution containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol. Hexahistidine (His6)–tagged proteins were also expressed in E. coli strain BL21(DE3)pLysS (Novagen) incubated in the presence of 0.1 mM isopropyl-ß-D-thiogalactopyranoside and were purified with the use of ProBond resin (Invitrogen).
Baculovirus expression system
The plasmid pFASTBAC HTa (Invitrogen) containing the relevant cDNA was subjected to recombination with the baculoviral genome in HB10BAC, and the resulting recombinant viral genome was introduced into Sf9 cells by transfection in order to generate recombinant baculoviruses. Baculovirus-infected Sf9 cells were lysed, and the recombinant proteins were purified as described above for bacterially expressed His6-tagged proteins. The purified recombinant proteins appeared homogeneous by electrophoresis and Coomassie blue staining.
Production of antibodies
Polyclonal antibodies to VCP or to CYC4 were generated in rabbits by standard procedures with purified recombinant His6-tagged full-length mouse VCP and human CYC4 expressed in Sf9 as antigens. Antisera were subjected to precipitation with 50% (w/v) ammonium sulphate, and the resulting pellet was lysed with PBS, dialysed against PBS and then subjected to affinity-purification on a column of protein G–Sepharose (Amersham Pharmacia Biotech). The purified antibodies were then dialysed against PBS.
Transfection, immunoprecipitation, and immunoblot analysis
Cells (HEK293T) were transfected by the calcium phosphate method and lysed in a solution containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, aprotinin (10 µg/mL), leupeptin (10 µg/mL), 1 mM phenylmethylsulphonyl fluoride, 400 µM Na3VO4, 400 µM EDTA, 1 mM EGTA, 10 mM NaF and 10 mM sodium pyrophosphate. The lysate was centrifuged at 16 000 x g for 10 min at 4 °C, and the resulting supernatant was incubated with antibodies for 2 h at 4 °C. Protein G-Sepharose equilibrated with lysis buffer was added to the mixture, which was then rotated for 1 h at 4 °C. The resin was separated by centrifugation, washed four times with lysis buffer, and then boiled in SDS sample buffer. Immunoblot analysis was performed with the following primary antibodies: anti-Myc (1 µg/mL; 9E10, Covance), anti-FLAG (1 µg/mL; M5, Sigma), anti-HA (1 µg/mL; HA.11/16B12, Babco), anti-Hsp90 (1 µg/mL; 68, Transduction Laboratory), and anti-Hsp70 (1 µg/mL; 7, Transduction Laboratory). Immune complexes were detected with horseradish peroxidase–conjugated antibodies to mouse or rabbit immunoglobulin G (1 : 10 000 dilution; Promega) and an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).
In vitro ubiquitylation assay
The in vitro ubiquitylation assay was performed as described (Hatakeyama et al. 2001). In brief, reaction mixtures (20 µL) containing 1 µg of GST-UIP5 or His6-CYC4, 1 or 5 µg of GST-CRN, His-VCP or Hsp90 (Sigma), 0.1 µg of recombinant rabbit E1 (BostonBiochem, Cambridge, MA, USA), 1 µL of crude lysate of E. coli expressing Ubc3 or Ubc4, 0.5 U of phosphocreatine kinase, 1 µg of ubiquitin (Sigma), 25 mM Tris-HCl (pH 7.5), 120 mM NaCl, 2 mM ATP, 1 mM MgCl2, 0.3 mM dithiothreitol, and 1 mM creatine phosphate were incubated for 2 h at 30 °C. The reaction was terminated by the addition of SDS sample buffer containing 4%ß-mercaptoethanol and heating at 95 °C for 5 min. Samples were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on 11% gels and then subjected to immunoblot analysis with a mouse monoclonal antibody to ubiquitin (clone 1B3; MBL) and horseradish peroxidase-conjugated rabbit polyclonal antibodies to mouse immunoglobulin (Promega). Immune complexes were detected by enhanced chemiluminescence (ECL kit; Amersham Pharmacia).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: nakayak1{at}bioreg.kyushu-u.ac.jp
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References |
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Chung, S., McLean, M.R. & Rymond, B.C. (1999) Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition. RNA 5, 1042–1054.[Abstract]
Connell, P., Ballinger, C.A., Jiang, J., et al. (2001) The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nature Cell Biol. 3, 93–96.[CrossRef][Medline]
Fang, S., Jensen, J.P., Ludwig, R.L., Vousden, K.H. & Weissman, A.M. (2000) Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J. Biol. Chem. 275, 8945–8951.
Freemont, P.S. (2000) RING for destruction? Curr. Biol. 10, R84–R87.[CrossRef][Medline]
Gmachl, M., Gieffers, C., Podtelejnikov, A.V., Mann, M. & Peters, J.M. (2000) The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex. Proc. Natl Acad. Sci. USA 97, 8973–8978.
Hatakeyama, S., Kitagawa, M., Nakayama, K., et al. (1999) Ubiquitin-dependent degradation of IB is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1. Proc. Natl Acad. Sci. USA 96, 3859–3863.
Hatakeyama, S., Yada, M., Matsumoto, M., Ishida, N. & Nakayama, K.I. (2001) U box proteins as a new family of ubiquitin-protein ligases. J. Biol. Chem. 276, 33111–33120.
Hershko, A. & Ciechanover, A. (1992) The ubiquitin system for protein degradation. Annu. Rev. Biochem. 61, 761–807.[CrossRef][Medline]
Hershko, A. & Ciechanover, A. (1998) The ubiquitin system. Annu. Rev. Biochem. 67, 425–479.[CrossRef][Medline]
Hershko, A., Heller, H., Elias, S. & Ciechanover, A. (1983) Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258, 8206–8214.
Honda, R. & Yasuda, H. (2000) Activity of MDM2, a ubiquitin ligase, toward p53 or itself is dependent on the RING finger domain of the ligase. Oncogene 19, 1473–1476.[CrossRef][Medline]
Huibregtse, J.M., Scheffner, M., Beaudenon, S. & Howley, P.M. (1995) A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc. Natl Acad. Sci. USA 92, 5249.
Imai, Y., Soda, M., Hatakeyama, S., et al. (2002) CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity. Mol. Cell 10, 55–67.[CrossRef][Medline]
Jiang, J., Ballinger, C.A., Wu, Y., et al. (2001) CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation. J. Biol. Chem. 276, 42938–42944.
Joazeiro, C.A., Wing, S.S., Huang, H., et al. (1999) The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science 286, 309–312.
Kamura, T., Conrad, M.N., Yan, Q., Conaway, R.C. & Conaway, J.W. (1999) The Rbx1 subunit of SCF and VHL E3 ubiquitin ligase activates Rub1 modification of cullins Cdc53 and Cul2. Genes Dev. 13, 2928–2933.
Kaneko, C., Hatakeyama, S., Matsumoto, M., et al. (2003) Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a. Biochem. Biophys. Res. Commun. 300, 297–304.[CrossRef][Medline]
Kazlauskas, A., Poellinger, L. & Pongratz, I. (1999) Evidence that the co-chaperone p23 regulates ligand responsiveness of the dioxin (Aryl hydrocarbon) receptor. J. Biol. Chem. 274, 13519–13524.
Kitagawa, M., Hatakeyama, S., Shirane, M., et al. (1999) An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of ß-catenin. EMBO J. 18, 2401–2410.[CrossRef][Medline]
Koegl, M., Hoppe, T., Schlenker, S., et al. (1999) A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96, 635–644.[CrossRef][Medline]
Kuzhandaivelu, N., Cong, Y.S., Inouye, C., Yang, W.M. & Seto, E. (1996) XAP2, a novel hepatitis B virus X-associated protein that inhibits X transactivation. Nucleic Acids Res. 24, 4741–4750.
Lees, M.J., Peet, D.J. & Whitelaw, M.L. (2003) Defining the role for XAP2 in stabilization of the dioxin receptor. J. Biol. Chem. 278, 35878–35888.
Leverson, J.D., Joazeiro, C.A., Page, A.M., et al. (2000) The APC11 RING-H2 finger mediates E2-dependent ubiquitination. Mol. Biol. Cell 11, 2315–2325.
Lorick, K.L., Jensen, J.P., Fang, S., et al. (1999) RING fingers mediate ubiquitin-conjugating enzyme (E2) -dependent ubiquitination. Proc. Natl Acad. Sci. USA 96, 11364–11369.
Matsumoto, M., Yada, M., Hatakeyama, S., et al. (2004) Molecular clearance of ataxin-3 is regulated by a mammalian E4. EMBO J. 23, 659–669.[CrossRef][Medline]
Meacham, G.C., Patterson, C., Zhang, W., Younger, J.M. & Cyr, D.M. (2001) The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nature Cell Biol. 3, 100–105.[CrossRef][Medline]
Melville, M.W., Katze, M.G. & Tan, S.L. (2000) p58 (IPK), novel cochaperone containing tetratricopeptide repeats and a J-domain with oncogenic potential. Cell. Mol. Life Sci. 57, 311–322.[CrossRef][Medline]
Meyer, H.H., Shorter, J.G., Seemann, J., Pappin, D. & Warren, G. (2000) A complex of mammalian Ufd1 and Npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. EMBO J. 19, 2181–2192.[CrossRef][Medline]
Murata, S., Minami, Y., Minami, M., Chiba, T. & Tanaka, K. (2001) CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein. EMBO Report 2, 1133–1138.[CrossRef][Medline]
Nakayama, K., Nagahama, H., Minamishima, Y.A., et al. (2000) Targeted disruption of Skp2 results in accumulation of cyclin E and p27Kip1, polyploidy and centrosome overduplication. EMBO J. 19, 2069–2081.[CrossRef][Medline]
Ohtsuka, K. & Hata, M. (2000) Mammalian HSP40/DNAJ homologs: cloning of novel cDNAs and a proposal for their classification and nomenclature. Cell Stress Chaperones 5, 98–112.[CrossRef][Medline]
Pellecchia, M., Szyperski, T., Wall, D., Georgopoulos, C. & Wuthrich, K. (1996) NMR structure of the J-domain and the gly/phe-rich region ofthe Eschericia coli DnaJ chaperone. J. Mol. Biol. 260, 236–250.[CrossRef][Medline]
Peters, J.M. (1998) SCF and APC: the Yin and Yang of cell cycle regulated proteolysis. Curr. Opin. Cell Biol. 10, 759–768.[CrossRef][Medline]
Raisin-Tani, S. & Leopold, P. (2002) Drosophila crooked-neck protein co-fractionates in a multiprotein complex with splicing factors. Biochem. Biophys. Res. Commun. 296, 288–292.[CrossRef][Medline]
Scheffner, M., Huibregtse, J.M., Vierstra, R.D. & Howley, P.M. (1993) The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75, 495–505.[CrossRef][Medline]
Scheffner, M., Nuber, U. & Huibregtse, J.M. (1995) Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade. Nature 373, 81–83.[CrossRef][Medline]
Scheufler, C., Brinker, A., Bourenkov, G., et al. (2000) Structure of TPR domain-peptide complexes: Critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101, 199–210.[CrossRef][Medline]
Staub, O., Gautschi, I., Ishikawa, T., et al. (1997) Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. EMBO J. 16, 6325–6336.[CrossRef][Medline]
Tang, N.M., Korth, M.J., Gale, M., et al. (1999) Inhibition of double-stranded RNA- and tumor necrosis factor alpha-mediated apoptosis by tetratricopeptide repeat protein and cochaperone p58 (IPK). Mol. Cell. Biol. 19, 4757–4765.
Wang, Q., Hobbs, K., Lynn, B. & Rymond, B.C. (2003) The Clf1p splicing factor promotes spliceosome assembly through N-terminal tetratricopeptide repeat contacts. J. Biol. Chem. 278, 7875–7883.
Waterman, H., Levkowitz, G., Alroy, I. & Yarden, Y. (1999) The RING finger of c-Cbl mediates desensitization of the epidermal growth factor receptor. J. Biol. Chem. 274, 22151–22154.
Weissman, A.M. (2001) Themes and variations on ubiquitylation. Nature Rev. Mol. Cell Biol. 2, 169–178.[CrossRef][Medline]
Xie, Y. & Varshavsky, A. (1999) The E2–E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain. EMBO J. 18, 6832–6844.[CrossRef][Medline]
Yang, Y., Fang, S., Jensen, J.P., Weissman, A.M. & Ashwell, J.D. (2000) Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli. Science 288, 874–877.
Yaron, A., Hatzubai, A., Davis, M., et al. (1998) Identification of the receptor component of the IkappaBalpha-ubiquitin ligase. Nature 396, 590–594.[CrossRef][Medline]
, Ye, Y., Meyer, H.H. & Rapoport, T.A. (2001) The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414, 652–656.[CrossRef][Medline]
Zhang, K., Smouse, D. & Perrimon, N. (1991) The crooked neck gene of Drosophila contains a motif found in a family of yeast cell cycle genes. Genes Dev. 5, 1080–1091.
Received: 16 January 2004
Accepted: 4 March 2004
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