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Originally published In Press as doi:10.1074/jbc.M701042200 on June 5, 2007

J. Biol. Chem., Vol. 282, Issue 32, 23184-23193, August 10, 2007
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Werner Helicase-interacting Protein 1 Binds Polyubiquitin via Its Zinc Finger Domain*Formula

Rebecca A. Bish1 and Michael P. Myers2

From the Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724

Received for publication, February 2, 2007 , and in revised form, May 23, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA repair is regulated on many levels by ubiquitination. In order to identify novel connections between DNA repair pathways and ubiquitin signaling, we used mass spectrometry to identify proteins that interact with lysine 6-linked polyubiquitin chains. From this proteomic screen, we identified the DNA repair protein WRNIP1 (Werner helicase-interacting protein 1), along with nucleosome assembly protein 1, as novel ubiquitin-interacting proteins. We found that a small zinc finger domain at the N terminus of WRNIP1 is sufficient and necessary for noncovalent ubiquitin binding. This ubiquitin-binding zinc finger (UBZ) domain binds polyubiquitin but not monoubiquitin and appears to show no specificity for polyubiquitin chain linkage. A homologous zinc finger domain in RAD18 also binds polyubiquitin, suggesting a wider role for the UBZ domain in DNA repair. The WRNIP1 ubiquitin-binding function, along with its previously established ATPase activity, suggests that WRNIP1 plays a role in the metabolism of ubiquitinated proteins. Supporting this model, deletion of MGS1, the yeast homolog of WRNIP1, slows the rate of ubiquitin turnover, rendering yeast resistant to cycloheximide. We also find that WRNIP1 is heavily modified with ubiquitin and SUMO, revealing complex layers in the involvement of ubiquitin pathway proteins in the regulation of DNA repair. The novel ubiquitin-binding ability of WRNIP1 sheds light on the role of UBZ domain-containing proteins in postreplication DNA repair.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ubiquitination is an abundant post-translational modification that serves to regulate a wide variety of cellular pathways (1–3). Ubiquitin, a 76-amino acid polypeptide, is conjugated to one or more lysine residues within a substrate protein via a multistep, multienzyme process (4). The consequences of ubiquitination for the substrate protein vary depending on the extent and type of ubiquitination. Monoubiquitination can lead to altered protein-protein interactions, relocalization, or the endocytosis of membrane-bound receptors (5, 6). Ubiquitin can also undergo ubiquitination, leading to the formation of a polyubiquitin chain. Conjugation to a polyubiquitin chain in which the interubiquitin linkage occurs through Lys-48 generally results in the proteasome-mediated degradation of the substrate protein (7). Other types of polyubiquitin chain linkages do not appear to result in proteasomal degradation but instead play a signaling role in pathways, such as the inflammatory response (8) and DNA repair (9–11).

Several different DNA repair pathways have been shown to be regulated by ubiquitination. For example, the proper execution of nucleotide excision repair depends upon the polyubiquitination and degradation of a subset of its component enzymes (12, 13), whereas translesion DNA synthesis is regulated by the mono- and polyubiquitination of PCNA3 (11). BRCA1, which plays an essential role in several DNA repair pathways, catalyzes the formation of an unusual type of polyubiquitin chain linked through Lys-6 (14). These Lys-6-linked polyubiquitin chains have been visualized at sites of DNA damage, perhaps serving as a scaffold for the assembly of DNA repair complexes (15).

To better understand the function of nondegradative polyubiquitin chains, we conducted a proteomic screen for proteins that interact with Lys-6-linked polyubiquitin chains. WRNIP1 (Werner helicase-interacting protein 1) and NAP1 (nucleosome assembly protein 1), were identified as candidate ubiquitin-interacting proteins through this screen.

WRNIP1 was originally identified in a yeast two-hybrid screen through its interaction with WRN (Werner syndrome protein), a helicase important for genomic stability and telomere maintenance (16–18). Annotated domains in WRNIP1 include a small Rad18 (radiation-sensitive 18)-like zinc finger at the N terminus and a AAA+ ATPase domain in the middle of the protein. The homology of WRNIP1 with the replication factor C family of clamp loader proteins, along with its ability to stimulate DNA polymerase {delta} synthesis of new DNA, suggests a possible role for WRNIP1 in replication and/or replication-dependent DNA repair (18, 19).

WRNIP1/MGS1 (maintenance of genome stability 1) has been shown to be involved in postreplication DNA repair (20). Postreplication repair is a division of the DNA damage response that ensures that DNA replication proceeds smoothly despite the presence of lesions that have evaded the timely attention of base or nucleotide excision repair (21). The RAD6-RAD18 ubiquitin ligase complex is responsible for channeling lesions into the error-free homologous recombination-dependent pathway or the error-prone pathway, in which low fidelity DNA polymerases replicate past the damage (11).

Evidence from yeast suggests that WRNIP1/Mgs1p negatively regulates postreplication repair (20). Yeasts that overexpress Mgs1p are generally healthy but exhibit marked sensitivity to the DNA-damaging agents hydroxyurea, methyl methanesulfonate, and UV radiation (22, 23). This increase in DNA damage sensitivity upon Mgs1p overexpression is dependent on the presence of RAD18, indicating that an overabundance of Mgs1p cripples the postreplication repair pathway (23).

In this study, we show that both WRNIP1 and RAD18 bind polyubiquitin through their zinc finger domains. This novel ubiquitin-binding activity suggests that WRNIP1 may exert its function in postreplication repair by modulating the turnover of ubiquitinated proteins. One phenotype exhibited by several yeast mutants deficient in pathways that regulate the turnover of ubiquitinated proteins is altered sensitivity to the protein synthesis inhibitor cycloheximide (24, 25). Under conditions of moderate translational inhibition, ubiquitin is the first protein to drop below the threshold required for continued growth (26). Any increase in ubiquitin levels, whether through ubiquitin overexpression or impaired ubiquitin degradation, results in resistance to cycloheximide (26). We find that mgs1{Delta} yeasts have an impaired turnover of ubiquitin and exhibit resistance to cycloheximide treatment. The finding that both WRNIP1 and RAD18 bind polyubiquitin and accelerate the turnover of ubiquitinated proteins highlights the intimate connection between the ubiquitin-proteasome pathway and postreplication DNA repair.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—pHA-UbLys-X series of plasmids has been previously described (27). WRNIP1 was cloned by PCR into pCMV-FLAG-MAT2 (Sigma) and pMAL-c2e (New England Biolabs) using pET-WRNIP (19) as a template. The WRNIP1 D37A mutant was generated by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene). A NAP1 cDNA (Open Biosystems, clone ID 2964631) was cloned by PCR into pEGFP-C1 (Clontech). The RAD18 and WRNIP1 UBZ domains were constructed by annealing complementary synthetic phosphorylated oligonucleotides (supplemental Table 1) for 5 min at 95 °C and then allowing the DNA to cool to room temperature over the course of 1 h. The annealed product was then ligated into pMAL-c2e digested with KpnI and XbaI. All constructs were verified by DNA sequencing or by mass spectrometry of the resulting proteins.

Yeast Strains—Wild type parental yeast (strain BY4741, genotype MATa his3{Delta}1 leu2{Delta}0 met15{Delta}0 ura3{Delta}0 and a derivative mgs1{Delta} strain (clone ID 1994) were obtained from Open Biosystems from the Saccharomyces Genome Deletion Project collection (28).

Antibodies—The following antibodies were used in this study: anti-ubiquitin and anti-beta-actin (clone P4D1 and catalog number 4967, respectively; Cell Signaling Technologies), anti-FLAG and anti-UBE1 (clones M2 and 2G2-3.5, respectively; Sigma), anti-SUMO1 (small ubiquitin-like modifier 1) (catalog number FL-101; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GFP (mix of clones 7.1 and 13.1; Roche Applied Science), anti-calmodulin (catalog number 05-173; Upstate%20Biotechnology">Upstate Biotechnology, Inc.), and an anti-beta-actin yeast loading control (catalog number ab8224; Abcam). Horseradish peroxidase-conjugated secondary antibodies against rabbit and mouse were purchased from Amersham Biosciences. Anti-mouse horseradish peroxidase-conjugated TrueBlot secondary antibody was obtained from Ebioscience. Polyclonal antisera against WRNIP1 were raised by immunizing rabbits with a mixture of two multiantigenic peptides with the sequences GETESRESYDAPPT (amino acids 103–116) and EELRGVDFFKQRRC (amino acids 652–665). The immunization protocol was carried out by Covance Research Products, Inc. (Denver, PA). Agarose beads were covalently conjugated to antibodies against the FLAG or HA epitope tags (Sigma).

Cell Culture and Transfection—HEK293 and HEK293T cells were cultured as recommended by American Type Culture Collection. Plasmid DNA was introduced into the cells by calcium phosphate transfection.

Cell Lysis—HEK293 or HEK293T cells were washed twice with phosphate-buffered saline, lysed in cold Nonidet P-40 buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, Roche Complete protease inhibitor tablet), sonicated in 0.5-s bursts for 20 s, and spun at 13,000 rpm in a microcentrifuge for 10 min to remove cell debris.

Immunoprecipitation—Cell lysate was incubated for 1 h on a rotator at 4 °C with antibody-conjugated agarose beads. The beads were washed at least five times with Nonidet P-40 buffer and subsequently analyzed by mass spectrometry or SDS-PAGE followed by immunoblotting.

Denaturing Protein Purification—Proteins were immunoprecipitated according to the protocol listed above and then eluted with the 3x FLAG peptide (Sigma) at 100 µg/ml. Solid urea was added to the eluate to a final concentration of 8 M. This denatured eluate was then incubated with Ni2+-nitrilotriacetic acid beads (Qiagen), washed, and eluted with imidazole according to the manufacturer's instructions.

Sample Preparation for Mass Spectrometry—Following immunoprecipitation, bead-bound proteins were washed an additional three times in 20 mM diammonium phosphate, pH 8.0, and then incubated with 50 ng of sequencing grade modified trypsin (Promega) for 8 h at 37 °C. The supernatant was removed from the beads, reduced by boiling for 5 min with 10 mM tris(2-carboxyethyl)phosphine (Pierce), and alkylated with 15 mM iodoacetamide for 1 h in the dark. An equal volume of 5% formic acid was added prior to sample cleanup with C18 Zip-Tips (Millipore).

Mass Spectrometry—Samples were analyzed by LC-MS/MS using an LTQ mass spectrometer (Thermo Electron) attached to a MicroTech high pressure liquid chromatograph.

Mass Spectrometry Data Analysis—LC-MS/MS data in the form of .RAW files were converted to .mzXML files by ReadW (version 1.6) and then searched against human protein data bases by the Global Proteome Machine interface to the X!Tandem algorithm (version 2006.06.01.2 [EC] ) (29). Searches were conducted using the following parameters: –2/+4 Da precursor ion mass error, 0.4 Da product ion mass error, complete carbamidomethylation of cysteines (+57 Da), partial oxidation of methionine (+16 Da), partial ubiquitination of lysine (+114.1 Da), and partial deamidation of asparagine and glutamine (–1 Da). Potential sumoylation sites were identified with SUMO-plot by Abgent (30).

Calculation of Protein Abundance Factor—The relative abundance of proteins identified by mass spectrometry was calculated by the protein abundance factor method (31). In short, the number of unique peptides identified from a particular protein is divided by that protein's molecular mass in kilodaltons and then multiplied by 104.

Recombinant Protein Expression and Purification—Recombinant proteins expressed as maltose-binding protein (MBP) fusions were purified on amylose beads in column buffer (1x phosphate-buffered saline, pH 7.4, 150 mM NaCl) according to the manufacturer's protocol (New England Biolabs). Recombinant FLAG-tagged proteins were purified on anti-FLAG EZView agarose beads (Sigma) in Nonidet P-40 buffer.

Ubiquitin Pull-down Assay—Ubiquitin binding was determined by incubating bead-bound purified recombinant proteins with 500 ng of ubiquitin (monoubiquitin or Lys-48- or Lys-63-linked polyubiquitin chains 1–7; Boston Biochem) in column buffer for 30 min at 4 °C. Beads were washed extensively in column buffer, and bead-bound protein complexes were eluted by boiling for 5 min in Laemmli buffer.

Determination of Cycloheximide Resistance—Cycloheximide resistance was determined by spotting 10-fold serial dilutions of yeast, starting with 106 cells, on YPD plates containing the indicated cycloheximide concentrations.

Ubiquitin Turnover Assay—Log phase yeast cultures were grown in YPD containing 200 µg/ml cycloheximide (Sigma), and aliquots were taken for Western blot as previously described (26). Western blot densitometry was performed on scans of the films with ImageJ (32).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Proteomic Screen for Ubiquitin-interacting Proteins—Polyubiquitin chains linked through Lys-6 have been implicated in DNA damage-related signaling pathways (14). In order to identify proteins that interact with Lys-6-linked polyubiquitin chains, we used a construct expressing HA-tagged ubiquitin in which all of the lysines except Lys-6 are mutated to arginine (pHA-UbLys-6) (27) and performed an immunoprecipitation against the HA tag from transfected HEK293 cells. This strategy results in the purification of Lys-6-linked polyubiquitin chains and any accompanying covalently or noncovalently bound proteins. Peptides were eluted by incubation of the beads with trypsin. These tryptic peptides were analyzed in a shotgun fashion by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS). Resulting spectra were then searched against a human protein data base with the Global Proteome Machine software (29).

The combined results of two independent Lys-6-linked ubiquitin immunoprecipitations lead to the identification of 53 proteins that were present only in the Lys-6-ubiquitin purification and not the untransfected control sample. Forty-eight of the 53 proteins were present in both immunoprecipitations. A protein identification is considered valid when at least two nonredundant peptides from the same protein have been assigned a statistically meaningful log(e) score less than or equal to –3.0. Identified proteins were ranked by their protein abundance factor (31).

Thirty-one of these proteins can be classified as known participants in ubiquitin metabolism (Table 1). This category of proteins includes known or predicted E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases, deubiquitinating enzymes, and proteins with previously identified ubiquitin-binding domains. Interestingly, no subunits of the proteasome were identified, despite the fact that the proteasome is an abundant multiprotein complex with several polyubiquitin receptors (33). The lack of proteasome subunits in this purification is probably related to the apparent nondegradative function of Lys-6-linked polyubiquitin chains.


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  		TABLE 1
Ubiquitin pathway proteins from Lys-6 ubiquitin immunoprecipitations

This list contains ubiquitin pathway proteins identified by mass spectrometry in immunoprecipitations of Lys-6-linked polyubiquitin chains. Total, the total number of peptides identified from this protein. Unique, the number of nonredundant peptides. PAF, protein abundance factor (see "Experimental Procedures"). DUB, deubiquitinating enzyme; bind, protein that contains a known ubiquitin-binding domain.

 
As expected, ubiquitin was the most abundant protein identified in these samples (Table 1). The N-terminal HA fusion and the lysine-to-arginine mutations allow for the discrimination of peptides derived from the pHA-UbLys-6 construct from peptides that come from endogenous ubiquitin. 301 of the 370 ubiquitin peptides identified in this screen can only originate from the pHA-UbLys-6 construct. None of the remaining peptides can be unambiguously attributed to endogenous ubiquitin, suggesting that the purified ubiquitin consists almost exclusively of the mutant ubiquitin construct in the form of monoubiquitin or Lys-6-linked polyubiquitin chains.

A further 22 proteins identified in the Lys-6-ubiquitin immunoprecipitations were specific to the transfected sample and could not be classified as ubiquitin-proteasome pathway proteins. These proteins are considered candidate ubiquitin-interacting proteins (Table 2).


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  		TABLE 2
Candidate Lys-6 ubiquitin-interacting proteins

This list contains proteins identified by mass spectrometry in immunoprecipitations of Lys-6-linked polyubiquitin chains. These proteins are not known to play a role in ubiquitin metabolism. Total, the total number of peptides identified from this protein. Unique, the number of nonredundant peptides. PAF, protein abundance factor (see "Experimental Procedures").

 
WRNIP1 and NAP1 Co-immunoprecipitate with Ubiquitin—When ranked according to the estimated abundance in the purification, the top three candidate ubiquitin-interacting proteins are calmodulin, WRNIP1, and NAP1 (Table 2 and supplemental Tables 2 and 3). To confirm that these proteins specifically co-immunoprecipitate with ubiquitin and to test whether this interaction is specific to Lys-6-linked polyubiquitin chains, we used the pHA-UbLys-X set of ubiquitin constructs (27). Each of the five HA-ubiquitin constructs contains mutations at six of the seven lysines, leaving only one lysine available for polyubiquitin chain formation. Ubiquitin purifications were prepared from HEK293T cells transfected with the pHA-UbLys-X constructs by immunoprecipitation against the HA tag. The immunoprecipitations were blotted with antibodies against calmodulin and WRNIP1 (Fig. 1A). To test the interaction of NAP1 with ubiquitin, a cDNA encoding NAP1 was cloned into pEGFP-C1 vector so that NAP1 could be detected with anti-GFP antibodies. NAP1-GFP was cotransfected with the pHA-UbLys-X constructs (Fig. 1B). As a positive control, these immunoprecipitations were also blotted with an antibody against the E1 ubiquitin-activating enzyme (UBE1), one of the most abundant ubiquitin-proteasome pathway proteins identified from the initial Lys-6-ubiquitin purifications (Table 1).

UBE1, WRNIP1, and NAP1 specifically co-immunoprecipitated with ubiquitin (Fig. 1, A and B). However, calmodulin was detected at equal levels in immunoprecipitations from transfected and untransfected cells and is therefore a nonspecific contaminant (Fig. 1A). Both WRNIP1 and NAP1 were detected in purifications of each of the five HA-ubiquitin constructs, suggesting that the ubiquitin interaction is not specific for Lys-6-linked chains.

WRNIP1 was of particular interest, because it has been implicated in the cellular response to DNA damage, but little is known about its function. WRNIP1 was the ninth most abundant specific protein in the Lys-6-ubiquitin purifications, identified by nine unique tryptic peptides (supplemental Table 2). Since WRNIP1 appears to constitute a potential link between DNA repair and ubiquitination, we decided to further investigate the WRNIP1 interaction with ubiquitin.


Figure 1
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  		FIGURE 1.
WRNIP1 interacts with polyubiquitin chains. A, WRNIP1 co-immunoprecipitates with multiple types of polyubiquitin chains. The HA-tagged ubiquitin constructs are each mutated such that only the indicated lysine (K) is available for interubiquitin conjugation. HEK293 cells were transfected, immunoprecipitated (IP), and blotted as indicated. B, NAP1 and UBE1 specifically co-immunoprecipitate with ubiquitin. HEK293 cells were co-transfected with the indicated constructs, and the lysates or immunoprecipitations (IP) were subjected to Western blot.

 
WRNIP1 Binds Polyubiquitin through its UBZ Domain—In order to test whether WRNIP1 is capable of noncovalent interactions with ubiquitin, we performed an in vitro binding assay, using recombinant FLAG-tagged WRNIP1 and wild type human ubiquitin. Bacterially expressed WRNIP1 was purified on anti-FLAG beads and then incubated with monoubiquitin or polyubiquitin chains linked through either Lys-48 or Lys-63.

WRNIP1 bound both Lys-48- and Lys-63-linked polyubiquitin chains. The noncovalent interaction between WRNIP1 and diubiquitin was weak, and no interaction with free monoubiquitin was observed (Fig. 2A). WRNIP1 robustly bound ubiquitin chains of three or more ubiquitin molecules, indicating that WRNIP1 must contain a ubiquitin-binding domain.

We had several reasons to suspect that the N-terminal zinc finger domain is responsible for the ability of WRNIP1 to bind ubiquitin. A homologous zinc finger domain is found in Rad18, a protein that functions as a ubiquitin ligase and therefore could reasonably be expected to bind ubiquitin (34, 35). Furthermore, a homolog of WRNIP1 exists in Arabidopsis thaliana (accession number NP_173839 [GenBank] .1) in which the zinc finger domain is replaced by a UBA domain, which is known to bind ubiquitin. Finally, it was recently demonstrated that similar zinc finger domains in DNA polymerases {kappa} and {eta} bind ubiquitin. The RAD18-like zinc finger domain in these polymerases was renamed the ubiquitin-binding zinc finger (UBZ) domain (36).

Based on this evidence, we mutated a conserved aspartate residue in the WRNIP1 zinc finger domain to alanine (WRNIP1-D37A). This single point mutation in the putative UBZ domain completely abolished ubiquitin binding in vitro (Fig. 2B). Similarly, ubiquitin did not co-immunoprecipitate with WRNIP1-D37A when purified from HEK293T cells (Fig. 2C). Furthermore, an MPB fusion of the WRNIP1 putative UBZ domain (residues 15–44) binds ubiquitin with the same specificity as the full-length protein (Fig. 2B). These data demonstrate that the zinc finger domain of WRNIP1 is a bona fide UBZ domain, which is both necessary and sufficient for WRNIP1 polyubiquitin binding.


Figure 2
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  		FIGURE 2.
WRNIP1 binds ubiquitin through its UBZ domain. A, WRNIP1 binds polyubiquitin chains. Ubiquitin pull-down assay using monoubiquitin (mono), Lys-48- or Lys-63-linked polyubiquitin chains. input, 200 ng of ubiquitin loaded as a marker. FLAG-BAP, FLAG-tagged bacterial alkaline phosphatase. B, the UBZ domain of WRNIP1 is sufficient and necessary for ubiquitin binding. Lys-48-linked ubiquitin pull-downs were performed with the following recombinant proteins: MBP, MBP fused to residues 17–40 of WRNIP1 (UBZ), MBP fused to full-length wild type WRNIP1 (WT), and MBP fused to full-length WRNIP1 with a mutation in the UBZ domain (D37A). C, WRNIP1 binds ubiquitin in vivo. Empty vector (–), wild type FLAG-MAT-tagged WRNIP1 (wt), or WRNIP1-FLAG-MAT with a mutation in the UBZ domain (D37A) was transfected into HEK293T cells, and lysates or immunoprecipitates (IP) were immunoblotted as indicated.

 
The Zinc Finger Domain of Rad18 Binds Polyubiquitin—Six human proteins are annotated as containing UBZ domains: WRNIP1, the translesion synthesis DNA polymerases {kappa} and {eta}, the E3 ubiquitin ligase RAD18, MTMR15, and a novel protein that we call UBZ1 (Fig. 3A).

The RAD18 UBZ domain is currently annotated as a DNA-binding domain. A recent study found that this zinc finger domain is both sufficient and necessary for the localization of RAD18 to DNA damage-induced foci (37). In light of the ubiquitin-binding function of the WRNIP1 UBZ domain as well as the clear connections of RAD18 to the ubiquitin-proteasome pathway, we tested whether the zinc finger domain in RAD18 also binds polyubiquitin. In vitro ubiquitin binding assays demonstrate that the RAD18 UBZ domain binds polyubiquitin in a manner similar to that of WRNIP1 (Fig. 3B).


Figure 3
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  		FIGURE 3.
Rad18 binds polyubiquitin through its UBZ domain. A, alignment of annotated UBZ domains from the human genome. The core cysteine and histidine residues that comprise the zinc-binding cage are marked in boldface type. The conserved aspartate residue that was mutated to alanine to make the D37A UBZ domain mutant is noted with an asterisk. Pol {kappa}, DNA polymerase {kappa} (SwissProt accession number Q9UBT6), which has two tandem UBZ domains. UBZ1, a novel protein of unknown function (Q5TE78), WRNIP1 (Q96S55), RAD18 (Q9NS91), and MTMR15 (Q86WU8) are shown. Pol {eta}, DNA polymerase {eta} (Q9Y253). B, Rad18 UBZ domain is sufficient for polyubiquitin binding in vitro. An in vitro ubiquitin pull-down was performed under the same conditions as described in the legend to Fig. 2A, using the UBZ domain of human Rad18 (residues 199–228) fused to MBP.

 
WRNIP1 Is Subject to UBZ Domain-dependent Ubiquitination—We observed that WRNIP1 always runs as a doublet or smear of higher molecular weight species on Western blots, suggesting that WRNIP1 is subject to post-translational modification (Fig. 1A). Many ubiquitin-binding proteins are also covalently conjugated to ubiquitin (5), in a process known as coupled ubiquitination. Therefore, we tested whether WRNIP1 is subject to a similar covalent linkage to ubiquitin.

We purified WRNIP1-FLAG-MAT under strongly denaturing conditions and performed an immunoblot to test for the presence of covalently conjugated ubiquitin. A smear of ubiquitin-protein conjugates was observed, starting at ~85 kDa, the expected molecular mass of monoubiquitinated WRNIP1 (Fig. 4A). The higher molecular weight ubiquitin-reactive species could represent WRNIP1, which is multiply monoubiquitinated, polyubiquitinated, or modified with both ubiquitin and a range of other post-translational modifications. The ubiquitination of WRNIP1 is abolished in the D37A UBZ domain mutant (Fig. 4A). Thus, as is the case with many other ubiquitin receptors, WRNIP1 ubiquitination is dependent on its ability to noncovalently bind ubiquitin (5).

The lysines on which WRNIP1 is ubiquitinated were identified by subjecting WRNIP1-FLAG-MAT to tryptic digestion and analysis by LC-MS/MS. Ubiquitination sites can be identified through mass spectrometry by the characteristic 114.1-Da mass shift and missed tryptic cleavage at the modified lysine (38). Data from eight WRNIP1 immunoprecipitation-mass spectrometry experiments were analyzed, yielding a total of 4249 WRNIP1-derived peptides with statistically significant scores. Of these 4249 peptides from WRNIP1, 102 peptides were observed to contain a lysine with a mass shift characteristic of covalent conjugation to ubiquitin, resulting in the identification of 12 ubiquitinated lysines (Table 3). A representative spectrum from one of the ubiquitinated peptides of WRNIP1 is shown in Fig. 4B. The ubiquitination sites are spread throughout the WRNIP1 sequence (Fig. 4C). In separate experiments, we have also observed a single ubiquitination site at lysine 231 on Mgs1p, the yeast homolog of WRNIP1 (data not shown).


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  		TABLE 3
Ubiquitination sites identified on WRNIP1 by mass spectrometry

WRNIP1-FLAG-MAT was immunoprecipitated from HEK293T cells, digested with trypsin, and analyzed by mass spectrometry. This table lists ubiquitination sites identified from eight independent experiments. Ubiquitination sites were only counted when there was a missed tryptic cleavage after the modified lysine, the mass shift of the peptide was +114.1 Da, and the peptide had a statistically significant log(e) score less than -3.0. Ubiquitinated (Ub) lysines are marked in boldface type.

 
In the course of our analysis of WRNIP1 by mass spectrometry, we also identified peptides from both SUMO1 and SUMO2/3, indicating that WRNIP1 is post-translationally modified with SUMO as well as ubiquitin. Sumoylation of WRNIP1 was confirmed by a Western blot of WRNIP1 purified under denaturing conditions (Fig. 4A). Sumoylated WRNIP1 runs as a single band with a molecular mass of ~100 kDa, a mass shift consistent with the conjugation of a single SUMO molecule. The contrast between the single discrete band representing sumoylated WRNIP1 and the smear of ubiquitinated WRNIP1 suggests that modification with different members of the ubiquitin-like protein family divides WRNIP1 into two distinct and nonoverlapping pools.

Significantly, we find that the D37A UBZ domain WRNIP1 mutant that is no longer ubiquitinated undergoes increased sumoylation (Fig. 4A). This inverse correlation of ubiquitin and SUMO levels suggests that these two modifications may antagonistically regulate WRNIP1 function. Supporting this hypothesis, the WRNIP1 sequence contains three SUMO consensus sites according to the SUMOplot algorithm (Fig. 4C) (30). The highest scoring of these sites is Lys-81, which was also identified as a ubiquitination site in this study (Table 3). Thus, the mechanism behind the antagonism between ubiquitination and sumoylation may be competition for the same lysine residue.

MGS1/WRNIP1 Accelerates the Turnover of Ubiquitinated Proteins—The combination of a AAA+ ATPase domain and a ubiquitin-binding domain occurs in other proteins, the most well studied example being valosin-containing protein. Valosin-containing protein serves to facilitate the degradation of ubiquitinated proteins by the proteasome by binding the polyubiquitin chains and performing work with its ATPase domain (39).

To test whether WRNIP1 may similarly regulate the turnover of ubiquitinated proteins, we turned to yeast for functional assays. MGS1, the yeast homolog of WRNIP1, is strongly conserved (22). High levels of homology are observed between MGS1 and WRNIP1 throughout the entire sequence, including the UBZ domain (22). Mgs1p also interacts with both the yeast WRN homolog Sgs1p (slow growth suppressor 1 protein) (22) and ubiquitin (data not shown).

One of the hallmarks of yeast strains that have deficiencies in the turnover of ubiquitinated proteins is resistance to translational inhibitors, such as cycloheximide. In this case, cycloheximide resistance results from slowed degradation of ubiquitin (25). We find that mgs1{Delta} yeasts are resistant to growth on plates containing cycloheximide (Fig. 5A). When yeasts are treated with 200 µg/ml cycloheximide, a dose that completely inhibits protein synthesis, ubiquitinated proteins appear to turn over more slowly in mgs1{Delta} yeast than in wild type yeast (Fig. 5, B and C). These data indicate that the cycloheximide resistance of mgs1{Delta} yeast results from a reduction in the rate of turnover of ubiquitin, suggesting a previously unrecognized role for WRNIP1/Mgs1p in ubiquitin metabolism.


Figure 4
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  		FIGURE 4.
Post-translational modification of WRNIP1. A, WRNIP1 is ubiquitinated and sumoylated. Empty vector (–), wild type WRNIP1-FLAG-MAT (WT), or WRNIP1-FLAG-MAT with a mutation in the UBZ domain (D37A) was affinity-purified from HEK293T cells under denaturing conditions and immunoblotted as indicated. B, a representative MS/MS spectrum identifying a ubiquitination site on WRNIP1 at lysine 301. The singly charged y ion series is marked in red, with the corresponding peptide sequence marked above the spectrum. The singly charged b ion series is blue. All other matched product ions (y or b ions that are doubly charged or have lost water or ammonia) are green. Unmatched ions are marked in black. C, WRNIP1 domain architecture, including the ubiquitination sites identified in this study ({diamondsuit}) and high confidence predicted sumoylation sites (•) at lysines 62, 81, and 482 identified by SumoPlot (30). WT, wild type.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A Proteomic Screen for Ubiquitin-interacting Proteins—In this study, we identified proteins that co-purify with Lys-6-linked polyubiquitin chains. Almost two-thirds of the proteins identified by mass spectrometry from this screen are known or predicted to function in the ubiquitin-proteasome pathway. Twenty-one of the ubiquitin pathway proteins identified are involved in the conjugation of ubiquitin to substrate proteins, whereas only two proteins were involved in the reverse deconjugation reaction. A further seven proteins contain ubiquitin-binding domains. Many of the ubiquitin pathway proteins identified here function in the assembly and disassembly of Lys-48-linked polyubiquitin chains or the binding of mono- or polyubiquitin with no known chain linkage specificity. We believe that the lack of any factors specific to Lys-6-linked polyubiquitin chains in this screen can probably be attributed to the relatively low abundance of these Lys-6-specific factors as compared with proteins that are capable of more general interactions with ubiquitin. These data may also indicate that most ubiquitin-interacting proteins exhibit little inherent chain specificity. Chain specificity may instead be generated by secondary interactions with the ubiquitinated protein or other factors, such as subcellular localization.


Figure 5
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  		FIGURE 5.
Deletion of MGS1 affects cycloheximide sensitivity and ubiquitin turnover. A, mgs1{Delta} yeast are resistant to cycloheximide. Wild type yeast (wt) and an mgs1{Delta} strain were spotted in serial dilution on YPD plates containing no drug (YPD) or 0.5 or 1.0 µg/ml cycloheximide (CHX). B, mgs1{Delta} yeasts have impaired ubiquitin turnover. Log phase wild type and mgs1{Delta} yeast were incubated with 200 µg/ml cycloheximide. Whole cell lysates were made from aliquots of yeast taken over the course of 6 h and blotted with the indicated antibodies. C, quantification by densitometry of the Western blots from B. Values plotted are the fraction of protein remaining as compared with time 0.

 
Twenty-two of the proteins identified in this screen are not currently annotated as participating in the ubiquitin-proteasome pathway. We present evidence that the most abundant candidate protein from this screen that specifically interacts with ubiquitin, WRNIP1, binds polyubiquitin through a zinc finger domain at its N terminus. Additionally, we show that NAP1 copurifies with ubiquitin, although the nature of this interaction has yet to be determined.

Six proteins on the list of potential ubiquitin-interacting proteins (TCPbeta, TCP{delta}, TCP{gamma}, TCP{eta}, TCP{theta}, and TCP{zeta}) encode subunits of the cytosolic chaperonin complex called chaperonin-containing TCP-1 (CCT). This complex is responsible for the ATP-dependent folding of a subset of cellular proteins, including actin and tubulin (40, 41). CCT is also essential for the proper assembly and activation of at least two ubiquitin ligase complexes (40, 42). Interestingly, CCT has been shown to moderate the toxicity of polyglutamine aggregates, which are heavily ubiquitinated (43, 44). The presence of the CCT complex in this ubiquitin purification, along with its previously demonstrated association with ubiquitinated protein aggregates and ubiquitination machinery, suggests the possibility that the association of CCT with its substrates may be regulated by ubiquitination. Alternatively, the apparent association of the CCT complex with ubiquitin could be explained by the ubiquitin-mediated regulation and degradation of CCT (45).

Regulation of WRNIP1 Function by Post-translational Modification—The development of mass spectrometry allows for the direct observation of protein post-translational modifications. This approach has highlighted the importance of combinatorial post-translational modifications in the regulation of protein function. Here we find that WRNIP1 is modified with ubiquitin, SUMO1, and SUMO2/3.

WRNIP1 is one of many proteins whose ubiquitin-binding domain directs its own ubiquitination. WRNIP1 is heavily ubiquitinated, with 12 sites identified. Several of these ubiquitination sites lie near critical conserved motifs within the ATPase domain, suggesting that ubiquitination may regulate WRNIP1 ATPase activity by directly interfering with nucleotide binding or hydrolysis.

Additional evidence that WRNIP1 function is regulated by post-translational modification comes from our finding that mutating the UBZ domain not only eliminates ubiquitination but concomitantly increases sumoylation. The signaling pathways that lead to the attachment of either ubiquitin or SUMO to WRNIP1 are currently unknown. Our data indicate that conjugation to SUMO excludes the addition of ubiquitin on a given molecule of WRNIP1. This inverse correlation between the two modifications suggests that ubiquitination and sumoylation may have antagonistic effects on WRNIP1 function.

One well documented mechanism for mutually exclusive ubiquitination and sumoylation is competition for the same lysine (11, 46). WRNIP1 may also experience a similar competition, since the highest scoring SUMO consensus site at Lys-81 was found in this study to undergo ubiquitination. Interestingly, the amino acid sequence surrounding Lys-81 also matches the recently described phospho-dependent sumoylation motif, with a SUMO consensus site separated by two residues from a proline-directed phosphorylation site (47). The threonine following Lys-81 in this motif was shown to be phosphorylated in a study of phosphoproteins from mouse liver (48). Since this motif is perfectly conserved from mice to humans, any potential sumoylation at Lys-81 may be regulated by phosphorylation at Thr-85, adding another layer of complexity to the post-translational modifications of WRNIP1.

Overlapping Functions of RAD18 and WRNIP1 in DNA Repair—RAD18 and WRNIP1 share similar CCHC-type zinc finger domains, originally called the RAD18-like zinc finger (ZnF_Rad18; accession number SM00734 in SMART) and now known as the UBZ domain (36). These two proteins also appear to have overlapping functions, illustrated by the fact that mgs1{Delta}rad18{Delta} yeast exhibit a severe growth defect (20). It has been suggested that the zinc finger-dependent functions of RAD18 are redundant with the function of WRNIP1 (37). In this study, we present both biochemical and mechanistic evidence supporting the hypothesis that certain functions of RAD18 and WRNIP1 may indeed overlap.

We first demonstrate that the UBZ domains of WRNIP1 and RAD18 share a common biochemical function. Although this zinc finger domain is currently annotated as binding DNA, our data indicate that the UBZ domains from both RAD18 and WRNIP1 bind polyubiquitin. In the case of RAD18, it is clear that the UBZ domain is not required for RAD18 ubiquitin ligase activity, instead directing a separate repair activity (37, 49). Thus, WRNIP1 and RAD18 may interact with similar or identical ubiquitinated structures to carry out their role in repair (see below).

Furthermore, both WRNIP1 and RAD18 accelerate protein turnover through the ubiquitin-proteasome pathway. RAD18, in conjunction with RAD6, regulates DNA repair through its ubiquitin ligase activity (49). Although the most well understood role of RAD18 in DNA repair is the monoubiquitination of PCNA (11), it is clear that RAD18 has additional PCNA-independent functions in repair (50) and that it targets some of its substrates for proteasomal degradation (51, 52).

In this study, we show that MGS1 stimulates the turnover of ubiquitinated proteins. Yeasts deleted for MGS1 degrade ubiquitinated proteins more slowly than the parental strain, resulting in cycloheximide resistance. The defect in protein turnover is moderate in mgs1{Delta} yeast, and ubiquitinated proteins are eventually degraded, albeit at a slower rate than in wild type strains. MGS1 therefore probably acts on a specific subset of proteins rather than as a general polyubiquitin receptor. The previously noted homology to replication factor C family proteins suggests that the DNA-stimulated ATPase activity of WRNIP1 may work to pry ubiquitinated proteins from the DNA at sites of DNA damage, facilitating their transfer to the proteasome. This model explains the observed negative regulation by MGS1 of DNA repair.

Thus, RAD18 and WRNIP1/MGS1 appear to share both the biochemical function of polyubiquitin binding through their UBZ domains and a common mechanism of action in accelerating the turnover of proteins through the ubiquitin-proteasome system. Given that the increased sensitivity to DNA damage upon overexpression of Mgs1p is dependent on the presence of RAD18, it is likely that the set of proteins targeted for degradation by RAD18-dependent ubiquitination at least partially overlaps with the proteins whose degradation is accelerated by WRNIP1.

The UBZ Domain and DNA Repair—In addition to WRNIP1 and RAD18, two other proteins, the translesion synthesis DNA polymerases {kappa} and {eta}, have been shown to bind ubiquitin through their UBZ domains. Interestingly, all four of these UBZ domain-containing proteins participate in the repair of a similar class of DNA lesions. Thus, every UBZ family protein with a known function participates in the same biological process. There is even tantalizing evidence that MTMR15, another UBZ domain-containing protein whose function is entirely unknown, may also be involved in DNA repair. A recent study identified MTMR15 by mass spectrometry in immunoprecipitations of the mismatch repair proteins MLH1, PMS1, and PMS2 (53).

The common DNA repair function shared by UBZ family proteins suggests that unlike most ubiquitin-binding domains, the UBZ domain is utilized specifically for post-replication DNA repair. One possibility is that the UBZ domain interacts with a DNA damage-specific ubiquitinated structure. Given the ability of the UBZ domain to bind multiple types of polyubiquitin chains, as shown in this study, the specificity is more likely conferred by the substrate protein rather than a unique polyubiquitin chain assembly. PCNA is an interesting candidate for the ubiquitinated scaffold on which UBZ domain-containing proteins assemble, since Pol{kappa}, Pol{eta}, RAD18, and WRNIP1/MGS1 all bind PCNA (11, 23, 54, 55).

One mechanism by which the UBZ domain may allow these proteins to participate in DNA repair is suggested by the observation that for RAD18, Pol{kappa}, and Pol{eta}, the UBZ domain is both sufficient and necessary for correct localization to DNA damage-induced nuclear foci (37, 56, 57). The UBZ domain may therefore function in recruitment to repair sites upon a DNA damage-specific ubiquitin-mediated signal. Further investigation into the ubiquitin-binding property of WRNIP1 and other UBZ domain-containing proteins has the potential to shed light on novel mechanisms for the regulation of post-replication DNA repair.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1–3. Back

1 Recipient of a David H. Koch Fellowship of the Watson School of Biological Sciences and a fellowship from the American Foundation for Aging Research and also supported by Grant GM 065094 from the NIGMS, National Institutes of Health. Back

2 Supported by Grants 25603521 and 25102067 from the NCI, National Institutes of Health. To whom correspondence should be addressed: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Tel.: 516-367-6806; Fax: 516-367-8873; E-mail: myers{at}cshl.edu.

3 The abbreviations used are: PCNA, proliferating cell nuclear antigen; UBZ, ubiquitin-binding zinc finger; GFP, green fluorescent protein; AAA, ATPase associated with various activities; CCT, chaperonin containing TCP1; HA, hemagglutinin; HEK293, human embryonic kidney 293; HEK293T, human embryonic kidney 293 cells with SV40 large T antigen; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; MAT, metal affinity tag; NAP1, nucleosome assembly protein 1; YPD, yeast extract/peptone/dextrose medium; MBP, maltose-binding protein; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Tomohiko Ohta, Dr. Takemi Enomoto, and Dr. Toshiki Tsurimoto for gifts of plasmids and Oliver Fregoso for critical review of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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