Identification of HHR23A as a Substrate for E6-associated Protein-mediated Ubiquitination

The human papilloma virus E6-associated protein (E6AP) functions as a ubiquitin protein ligase (E3) in the E6-mediated ubiquitination of p53. E6AP is also an E3 in the absence of E6, but its normal cellular substrates have not yet been identified. Here we report the identification of HHR23A, one of the human homologues of the yeast DNA repair protein Rad23, as an E6-independent target of E6AP. HHR23A binds E6AP and is ubiquitinated in vitro in an E6AP-dependent manner. Ubiquitinated forms of endogenous HHR23A are detectable in mammalian cells. Overexpression of wild-type E6AP in vivo enhances the ubiquitination of HHR23A, whereas a dominant negative E6AP mutant inhibits HHR23A ubiquitination. Although HHR23A is a stable protein in non-synchronized cells, its levels are regulated in a cell cycle-dependent manner, with specific degradation occurring during S phase. The S phase degradation of HHR23A could be blocked in vivo by dominant negative E6AP, providing direct evidence for the involvement of E6AP in the regulation of HHR23A. Consistent with a role of the HHR23 proteins in DNA repair, UV-induced DNA damage inhibited HHR23A degradation. Although the precise role of HHR23 proteins in DNA repair and cell cycle progression remains to be elucidated, our data suggest that E6AP-mediated ubiquitination of HHR23A may have important implications in DNA repair and cell cycle progression. The human papilloma virus E6-associated protein (E6AP) functions as a ubiquitin protein ligase (E3) in the E6-mediated ubiquitination of p53. E6AP is also an E3 in the absence of E6, but its normal cellular substrates have not yet been identified. Here we report the identification of HHR23A, one of the human homologues of the yeast DNA repair protein Rad23, as an E6-independent target of E6AP. HHR23A binds E6AP and is ubiquitinated in vitro in an E6AP-dependent manner. Ubiquitinated forms of endogenous HHR23A are detectable in mammalian cells. Overexpression of wild-type E6AP in vivo enhances the ubiquitination of HHR23A, whereas a dominant negative E6AP mutant inhibits HHR23A ubiquitination. Although HHR23A is a stable protein in non-synchronized cells, its levels are regulated in a cell cycle-dependent manner, with specific degradation occurring during S phase. The S phase degradation of HHR23A could be blocked in vivo by dominant negative E6AP, providing direct evidence for the involvement of E6AP in the regulation of HHR23A. Consistent with a role of the HHR23 proteins in DNA repair, UV-induced DNA damage inhibited HHR23A degradation. Although the precise role of HHR23 proteins in DNA repair and cell cycle progression remains to be elucidated, our data suggest that E6AP-mediated ubiquitination of HHR23A may have important implications in DNA repair and cell cycle progression. Protein ubiquitination is implicated in a variety of cellular processes, including DNA repair, cell cycle control, chromosomal organization, intracellular translocation of proteins, and apoptosis (1Finley D. Chau V. Annu. Rev. Cell Biol. 1991; 7: 25-69Crossref PubMed Scopus (421) Google Scholar, 2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1205) Google Scholar, 3Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1459) Google Scholar). Ubiquitin-dependent proteolysis is the best known aspect of the ubiquitin pathway. The covalent conjugation of multiple ubiquitin molecules to lysine residues of a target protein serves to signal its recognition and rapid degradation by the 26 S proteasome (3Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1459) Google Scholar, 4Hochstrasser M. Curr. Opin. Biol. 1995; 7: 215-223Crossref PubMed Scopus (784) Google Scholar, 5Pickart C.M. FASEB J. 1997; 11: 1055-1066Crossref PubMed Scopus (308) Google Scholar). Ubiquitination of protein substrates is a multi-step process that involves the concerted action of at least three classes of enzymes as follows: ubiquitin-activating enzyme (E1), 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzymes; E3, ubiquitin protein ligase; E6AP, E6-associated protein; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; NER, nucleotide excision repair; WGE, wheat germ extracts; CMV, cytomegalovirus; WT, wild type; HPV, human papilloma virus; HA, hemagglutinin; mAb, monoclonal antibody; XP, Xeroderma pigmentosum; XPC, xeroderma pigmentosum group C. ubiquitin-conjugating enzymes (E2s), and ubiquitin protein ligases (E3s) (3Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1459) Google Scholar). Although the biochemical mechanisms of ubiquitin transfer within the enzymatic components of the pathway and its subsequent conjugation to target proteins is now understood in considerable detail, it is still unclear how specific proteins are recognized by the ubiquitin system as substrates. E1 first activates ubiquitin in an ATP-dependent reaction through the formation of ubiquitin adenylate, followed by a thiol ester bond between the carboxyl terminus of ubiquitin and thiol group of a specific cysteine residue in E1. Ubiquitin is then transferred to a specific cysteine residue in one of several E2s (6Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar). E2 enzymes, in turn, may transfer the ubiquitin either directly to a substrate or to E3 enzymes that finally catalyze the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and the ε-amino group of lysine residues on a target protein (3Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1459) Google Scholar, 7Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar, 8Scheffner M. Nuber U. Huibregtse J. Nature. 1995; 373: 81-83Crossref PubMed Scopus (747) Google Scholar). A substrate may be multiply ubiquitinated by sequential linkage of additional ubiquitin molecules to each other through specific lysine residues (Lys-48 or Lys-63). Multi-ubiquitination of a protein leads to its recognition and consequent degradation by the 26 S proteasome (3Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1459) Google Scholar, 5Pickart C.M. FASEB J. 1997; 11: 1055-1066Crossref PubMed Scopus (308) Google Scholar, 6Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar). The mechanisms involved in the recognition of specific proteins as substrates of the ubiquitin system are not fully understood. However, it is likely that E3 ubiquitin protein ligases are the key components that provide specificity to the ubiquitin system by direct interaction with specific substrates. Although two E3 activities had previously been identified from rabbit reticulocytes (E3a and E3b) (9Heller A. Hershko A. J. Biol. Chem. 1990; 265: 6532-6535Abstract Full Text PDF PubMed Google Scholar, 10Reiss Y. Heller H. Hershko A. J. Biol. Chem. 1989; 264: 10378-10383Abstract Full Text PDF PubMed Google Scholar, 11Reiss Y. Hershko A. J. Biol. Chem. 1990; 265: 3685-3690Abstract Full Text PDF PubMed Google Scholar), it was the cloning and characterization of E6AP that revealed structural and functional features of a new class of E3 enzymes. E6AP was initially identified as a 100-kDa cellular protein that, in conjunction with the E6 oncoprotein of human papilloma virus type 16 (HPV), constituted the E3 activity in the ubiquitination of p53 (7Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar, 12Huibregtse J.M. Scheffner M. Howley P.M. EMBO J. 1991; 10: 4129-4135Crossref PubMed Scopus (695) Google Scholar, 13Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell. Biol. 1993; 13: 775-784Crossref PubMed Scopus (468) Google Scholar, 14Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text PDF PubMed Scopus (3464) Google Scholar). E6AP was also found to promote the ubiquitination of cellular proteins in the absence of E6, indicating that E6AP could function as an E3 enzyme independent of E6 (7Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar). Sequence analysis of E6AP revealed a region of approximately 350 amino acids in the carboxyl terminus that was highly conserved among a number of proteins from various organisms (15Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar). This region, subsequently termed the HECT domain, contains a conserved cysteine residue that serves as the active site for thiol ester formation with ubiquitin (15Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar). In addition to E6AP, several other Hect domain proteins have now been shown to be capable of forming thiol ester complexes, suggesting that Hect-containing proteins belong to a family of structurally related ubiquitin protein ligases (8Scheffner M. Nuber U. Huibregtse J. Nature. 1995; 373: 81-83Crossref PubMed Scopus (747) Google Scholar, 15Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar). As mentioned above, the specificity of substrate recognition by the ubiquitin system may be achieved by E3 enzymes capable of direct interaction with specific substrates. In the case of Hect proteins for instance, their divergent amino-terminal sequences may provide the necessary diversity required for substrate recognition, whereas their conserved carboxyl terminus (Hect domain) can interact with specific E2 enzymes and catalyze the ubiquitination of bound substrates (16Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell. Biol. 1993; 13: 4918-4927Crossref PubMed Scopus (349) Google Scholar, 17Huibregtse J.M. Yang J.C. Beaudenon S.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 8: 3656-3661Crossref Scopus (182) Google Scholar, 18Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). To date, only a small number of proteins have been identified as substrates of Hect E3 enzymes. The general amino acid permease Gap1 and uracil permease Fur4 have been reported to be ubiquitinated by RSP5 (NPI1), a Hect protein of Saccharomyces cerevisiae (19Galan J.M. Moreau V. Andre B. Volland C. Haguenauer-Tsapis R. J. Biol. Chem. 1996; 271: 10946-10952Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 20Hein C. Springael J. Volland C. Haguenauer-Tsapis R. Andre B. Mol. Microbiol. 1995; 18: 77-87Crossref PubMed Scopus (298) Google Scholar). The large subunit of RNA polymerase II (Rpb1) was recently identified also as an RSP5 substrate (17Huibregtse J.M. Yang J.C. Beaudenon S.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 8: 3656-3661Crossref Scopus (182) Google Scholar). In addition, theSchizosaccharomyces pombe homologue of RSP5, Pub1, has been shown to target the CDC25 phosphatase for ubiquitin-dependent degradation (21Nefsky B. Beach D. EMBO J. 1996; 15: 1301-1312Crossref PubMed Scopus (97) Google Scholar). In the case of E6AP, p53 is the only known substrate; however, the ubiquitination of p53 by E6AP is dependent upon the presence of oncogenic E6 proteins of HPV. To date no other substrate for E6AP-mediated ubiquitination has been isolated. Interestingly, E6AP was recently identified as the gene affected in Angelman syndrome, a genetic neurological disorder. A majority of the mutations in E6AP is predicted to abolish the catalytic activity of E6AP, raising the possibility that deregulation of E6AP substrates may contribute to the pathogenesis of Angelman syndrome (22Kishino T. Lalande M. Wagstaff J. Nat. Genet. 1997; 15: 70-73Crossref PubMed Scopus (1023) Google Scholar,23Matsuura T. Sutcliffe J.S. Fang P. Galjaard R.J. Jiang Y.H. Benton C.S. Rommens J.M. Beaudet A.L. Nat. Genet. 1997; 15: 1-5Crossref PubMed Scopus (684) Google Scholar). In an attempt to identify E6-independent substrates of E6AP, we isolated one of the human homologues of yeast Rad23 (HHR23A) as an E6AP-interacting protein using the yeast two-hybrid system (18Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 24Masutani C. Sugasawa K. Yanagisawa J. Sonoyama T. Ui M. Enomoto T. Takio K. Tanaka K. van der Spek P.J. Bootsma D. Hoiejmakers J.H.J. Hanaoka F. EMBO J. 1994; 13: 1831-1843Crossref PubMed Scopus (334) Google Scholar). Rad23 is involved in excision repair of UV-damaged DNA and has also been implicated in spindle pole body duplication and cell cycle progression in S. cerevisiae (25Biggins S. Ivanovska I. Rose M.D. J. Cell Biol. 1996; 133: 1331-1346Crossref PubMed Scopus (147) Google Scholar, 26Miller R.D. Prakash L. Prakash S. Mol. Gen. Genet. 1982; 188: 235-239Crossref PubMed Scopus (49) Google Scholar, 27Wang Z. Wei S. Reed S.H. Wu X. Svejstrup J.Q. Feaver W.J. Kornberg R.D. Friedberg E.C. Mol. Cell. Biol. 1997; 17: 635-643Crossref PubMed Scopus (67) Google Scholar). Two homologues of Rad23 exist in humans, HHR23A and HHR23B (24Masutani C. Sugasawa K. Yanagisawa J. Sonoyama T. Ui M. Enomoto T. Takio K. Tanaka K. van der Spek P.J. Bootsma D. Hoiejmakers J.H.J. Hanaoka F. EMBO J. 1994; 13: 1831-1843Crossref PubMed Scopus (334) Google Scholar). Both of these proteins have been reported to bind and function with the xeroderma pigmentosum group C (XPC) protein in nucleotide excision repair (NER) (28Masutani C. Araki M. Sugasawa K. van der Spek P.J. Yamada A. Uchida A. Maekawa T. Bootsma D. Hoeijmakers J.H.J. Hanaoka F. Mol. Cell. Biol. 1997; 17: 6915-6923Crossref PubMed Scopus (96) Google Scholar, 29Sugasawa K. Ng J. Masutani C. Maekawa T. Uchida A. van der Spek P.J. Eker A.P.M. Rademakers S. Visser C. Aboussekhra A. Wood R.D. Hanaoka F. Bootsma D. Hoeijmakers J.H.J. Mol. Cell. Biol. 1997; 17: 6924-6931Crossref PubMed Scopus (110) Google Scholar). Our results identify HHR23A as a novel, E6-independent substrate of E6AP. We demonstrate that HHR23A interacts with E6AP and is efficiently ubiquitinated in an E6AP-dependent manner in vitro. By using anti-HHR23A and anti-ubiquitin antibodies, a small fraction of endogenous HHR23A was found conjugated to ubiquitin. Transient expression of wild-type E6AP enhanced the ubiquitinated fraction of HHR23A, whereas a dominant negative E6AP mutant inhibited HHR23A ubiquitination in vivo. Although HHR23A appears to be a stable protein in asynchronously growing cells, we have found that HHR23A protein levels are regulated during cell cycle progression. The level of HHR23A was found to be the highest in M phase and early G1, with a consistent 3–5-fold decrease occurring during late G1 and early S phase, indicating targeted degradation of the protein at specific stages of the cell cycle. The decrease in HHR23A protein levels was completely blocked by transient expression of dominant negative E6AP, providing direct evidence for the involvement of E6AP in the cell cycle-dependent degradation of HHR23A. Significantly, treatment of cells with UV radiation also abolished HHR23A degradation, suggesting that DNA damage may regulate HHR23A stability. At present, the precise roles of HHR23 proteins in repair of UV-damaged DNA is not clear. Nevertheless, our data suggest that E6AP-mediated ubiquitination of HHR23A may be important in regulating its function in DNA repair and cell cycle progression. A modified version of the yeast two-hybrid screen used to identify E6AP-interacting proteins has been described previously (18Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Interacting clones were isolated, and their DNA sequence was determined by dideoxynucleotide sequencing. The BLAST algorithm was used to search GenBankTM data bases, and eight independent inserts of varying lengths were identified as HHR23A. Clone 15-2, containing the entire open reading frame of HHR23A, was used for further analysis. Thehhr23a and hhr23b genes were amplified by polymerase chain reaction using appropriate oligonucleotide primers and were subcloned as 5′ BamHI/3′ SalI fragments into pGem-1 (Promega) under T7 RNA polymerase orientation for in vitro transcription and translation. For expression in bacteria as GST fusion proteins, polymerase chain reaction-derived HHR23 cDNAs (5′ BamHI/3′ SalI) were inserted in frame with GST into pGex-4T-2 (Amersham Pharmacia Biotech). Bacterial expression constructs for E1 and various E2s were described previously (18Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). E6AP constructs for in vitro synthesis, baculovirus-based vectors for expression of E6AP in insect cells, and CMV-E6AP constructs for mammalian cell expression have been described (7Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar, 13Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell. Biol. 1993; 13: 775-784Crossref PubMed Scopus (468) Google Scholar, 30Talis A. Huibregtse J.M. Howley P.M. J. Biol. Chem. 1998; 273: 6439-6445Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). GST fusion proteins were expressed in E. coli HB101 and affinity purified with glutathione-Sepharose beads (Amersham Pharmacia Biotech). The relative amounts of GST-HHR23 proteins were determined against known amounts of bovine serum albumin by Coomassie Blue staining.In vitro synthesis of proteins was carried out in TNT-coupled wheat germ extracts (WGE, Promega) as per manufacturer's instructions. Preparation of E6AP from Hi5 cells infected with recombinant baculovirus expressing wild-type or mutant E6AP proteins has been described (7Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar, 15Huibregtse J. Scheffner M. Beaudenon S. Howley P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (703) Google Scholar, 31Scheffner M. Huibregtse J.M. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8797-8801Crossref PubMed Scopus (235) Google Scholar). Bacterial expression of E1 and E2 enzymes was reported previously (7Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar, 18Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). E6AP synthesized in vitro using WGE (Promega) in the presence of [35S]methionine was incubated with approximately 2 μg of GST or 1 μg each of GST-HHR23 proteins coupled to glutathione-Sepharose beads (Amersham Pharmacia Biotech) in binding buffer (100 mm Tris-HCl, pH 7.4, 120 mm NaCl, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin/leupeptin mix) for 3 h at 4°C. Reactions were washed four times in binding buffer and analyzed by SDS-PAGE and autoradiography. In vitro ubiquitination assay of HHR23A has been described previously (18Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Briefly, HHR23 proteins were synthesized in vitro in the presence of [35S]methionine for 90 min at 30°C using TNT-coupled WGE (Promega). 5-μl aliquots of in vitrotranslated HHR23 proteins were incubated with 5–10 ng of E1, approximately 100 ng of E2 (UbcH7), 200 ng each of WT or mutant (C833A) E6AP, and 4 μg of ubiquitin (Sigma) in 20 mm Tris-HCl, pH 7.6, 50 mm NaCl, 4 mm ATP, 10 mmMgCl2, and 0.2 mm dithiothreitol. Reactions were terminated after 1.5 h at 30°C by the addition of SDS sample buffer and were analyzed by SDS-PAGE and autoradiography. Early passage human diploid fibroblasts GM6419 and XPC (GM2995) cells were obtained from Coriell Cell Repository. 2C. Maki, unpublished observations. Cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (for COS.7 and U2OS cells) or 15% (for GM6419 or XPC cells) fetal bovine serum at 37°C with 5% CO2. Transfection of COS.7 cells was performed using standard procedures essentially as described (32Talis A.L. Huibregtse J.M. Howley P.M. J. Biol. Chem. 1998; 273: 6439-6445Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). 10 μg each of CMV-HA-E6AP DNA was introduced into cells by the CaCl2method, and whole cell lysates were prepared 60 h post-transfection for immunoprecipitation and immunoblot analysis. U2OS cells were transfected with 10 μg each of CMV-HA-E6AP (WT or C833A) using the FuGene6 transfection reagent as per manufacturer's instructions (Roche Molecular Biochemicals). GM6419, U2OS, and XPC cells were plated at an approximate density of 106 cells per 10-cm dish and allowed to grow for 14 h. At the end of this period, cells were refed with fresh media containing 500 ng/ml nocodazole (Sigma) (33Ludlow J.W. Glendening C.L. Livingston D.M. DeCaprio J.E. Mol. Cell. Biol. 1993; 13: 367-372Crossref PubMed Scopus (221) Google Scholar). After 20 h a mitotic shake was performed by gentle pipeting to remove loosely adherent mitotic cells. For each time course experiment, seven 10-cm dishes for U2OS cells and 14 10-cm dishes for GM6419 and XPC cells were used. Cells were collected by centrifugation and washed three times with media to remove any nocodazole. The cells were then resuspended in 7 ml of media, and 1 ml was removed as the M phase sample. Equal aliquots (1 ml each) of remaining cells were plated in six 35-mm plates for each of the time points to be analyzed (i.e. 4, 8, 12, 16, 20, and 24 h). At the end of each time point, cells were rinsed once with phosphate-buffered saline, and whole cell lysates were prepared by incubating cells in lysis buffer (100 mm Tris-HCl, pH 7.4, 120 mm NaCl, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin/leupeptin mix) for 15 min on ice followed by gentle sonication. Absorbance measurements at a wavelength of 595 nm were done to quantify the total protein amounts present in each sample. For the experiment shown in Fig. 5, U2OS cells were transfected with CMV-E6AP constructs using FuGene transfection reagent. Twenty-four hours post-transfection, cells were synchronized by nocodazole treatment for 24 h. Mitotic cells were collected and replated for indicated amounts of time, and cell extracts were prepared at the end of each time point. In experiments involving UV radiation (Fig. 6), 2 h after plating (post-mitotic shake), cells were rinsed with phosphate-buffered saline and irradiated with an 8-J UV dose using UV Stratalinker 1800 (Stratagene). The cells were immediately refed with fresh media and transferred back to 37°C, 5% CO2 incubator.Figure 6Effect of UV radiation on HHR23A protein levels. U2OS cells were treated with nocodazole (as in Fig.4 A), and mitotic cells were collected by shake-off and centrifugation. Equal numbers of cells were replated and allowed to grow for 2 h. At the end of this time period, one set of plates was UV-irradiated at a dose of 8 J (lanes 4–6) and immediately transferred back to the incubator. Cell extracts were prepared at the time points indicated, and HHR23A was detected by immunoblotting with anti-HHR23A antibodies. Protein levels were quantitated using NIH Image software (see bar graph). In the unirradiated set (lanes 1–3), a 3-fold decrease is seen in HHR23A levels at 16 h post-mitotic shake (early S; comparelanes 1 and 2). In contrast, UV radiation (lanes 4–6) completely blocked the decrease in HHR23A levels (compare lanes 4 and 5).View Large Image Figure ViewerDownload (PPT) Rabbit polyclonal sera against GST-HHR23A were generated at Babco. To detect ubiquitinated HHR23A species in mammalian cells (untransfected or transfected with E6AP constructs), whole cell extracts of COS.7 and U2OS cells were prepared in RIPA buffer (20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 150 mm NaCl, 0.25% SDS, 1% Nonidet P-40, 1% deoxycholate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/μl aprotinin/leupeptin mix). Cells from three confluent 10-cm plates were lysed, and HHR23A was immunoprecipitated from 2 mg of total cell extract in RIPA buffer at 4°C with anti-HHR23A antibodies. 40 μl of protein A-Sepharose (v/v) was added after 4 h, and samples were washed extensively in RIPA buffer to avoid co-precipitation of proteins other than HHR23A. As control, equal amounts of cell extracts (2 mg) were subjected to precipitation with preimmune sera. Immunoprecipitates were boiled for 5 min in SDS sample buffer and resolved by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Nen) using standard procedures, and immunoblot analysis was carried out with anti-HHR23A and anti-ubiquitin antibodies. Detection of proteins was performed with ECL reagents (Nen). HHR23A ubiquitination after transfection of COS.7 cells with CMV-HA-E6AP constructs was detected similarly by immunoprecipitation (in RIPA buffer) and immunoblotting of 2 mg of whole cell lysates with anti-HHR23A antibodies. Expression of E6AP (WT and C833A) in U2OS cells was detected by immunoblotting with anti-HA MAb (12CA5, Babco). For the time course analysis, GM6419, U2OS, or XPC cell extracts were prepared in lysis buffer by gentle sonication, and 100 μg of whole cell lysates were resolved by SDS-PAGE. HHR23A and cyclin A were detected by immunoblotting with anti-HHR23A or anti-cyclin A antibodies (BF683, Santa Cruz Biotechnology). Quantitation of relative protein amounts was performed by using the Gel Plotting Macros feature of NIH-Image software. In an attempt to identify potential substrates and regulators of E6AP, we used a modified version of the yeast two-hybrid system to isolate cDNA clones that encode E6AP-interacting proteins. A catalytically inactive form of E6AP in which the active site cysteine residue is substituted with alanine was used as bait to avoid potential degradation of interacting proteins (18Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). E6AP (C833A) was fused in frame with the Gal4 DNA-binding domain and introduced into the yeast reporter strain MaV103. Expression of the fusion protein was confirmed by immunoblot analysis with anti-E6AP and anti-Gal4 DNA-binding domain antibodies. The prey cDNA library, fused to the Gal4 activation domain, was derived from activated human T cells. Interacting clones were isolated by plating transformants on histidine drop-out plates containing 25 mm 3-aminotriazole (18Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Interaction positive cDNAs were rescued, and the DNA sequence of isolated clones was determined. Using the BLAST algorithm, eight independent clones consisting of cDNA inserts of varying lengths were identified as HHR23A, one of the human homologues of the yeast DNA repair protein Rad23 (24Masutani C. Sugasawa K. Yanagisawa J. Sonoyama T. Ui M. Enomoto T. Takio K. Tanaka K. van der Spek P.J. Bootsma D. Hoiejmakers J.H.J. Hanaoka F. EMBO J. 1994; 13: 1831-1843Crossref PubMed Scopus (334) Google Scholar, 26Miller R.D. Prakash L. Prakash S. Mol. Gen. Genet. 1982; 188: 235-239Crossref PubMed Scopus (49) Google Scholar, 27Wang Z. Wei S. Reed S.H. Wu X. Svejstrup J.Q. Feaver W.J. Kornberg R.D. Friedberg E.C. Mol. Cell. Biol. 1997; 17: 635-643Crossref PubMed Scopus (67) Google Scholar). Clone 15-2, containing the entire open reading frame of HHR23A, was subcloned into appropriate vectors and used in further analysis. To confirm the interaction between HHR23A and E6AP, the HHR23A cDNA was cloned into pGEX-4T-2 vector and expressed as a GST fusion protein in bacteria. We also wanted to ascertain whether the second human homologue of yeast Rad23, HHR23B, which shares over 70% sequence homology with HHR23A, would interact with E6AP (24Masutani C. Sugasawa K. Yanagisawa J. Sonoyama T. Ui M. Enomoto T. Takio K. Tanaka K. van der Spek P.J. Bootsma D. Hoiejmakers J.H.J. Hanaoka F. EMBO J. 1994; 13: 1831-1843Crossref PubMed Scopus (334) Google Scholar). Consequently, we obtained the HHR23B cDNA and expressed it as a fusion protein with GST in bacteria. Both HHR23 proteins were purified using glutathione-Sepharose beads and assayed for their ability to bind radiolabeled E6AP synthesized in vitro using wheat germ extract (which does not contain endogenous E6AP). Fig.1 A (lanes 2 and3) shows that GST-HHR23A and GST-HHR23B are both capable of efficient interaction with E6AP. The GST portion alone failed to bind E6AP, serving as negative control (lane 1). One interesting feature of the HHR23 proteins is the presence of a ubiquitin-like region in their amino terminus (24Masutani C. Sugasawa K. Yanagisawa J. Sonoyama T. Ui M. Enomoto T. Takio K. Tanaka K. van der Spek P.J. Bootsma D. Hoiejmakers J.H.J. Hanaoka F. EMBO J. 1994; 13: 1831-1843Crossref PubMed Scopus (334) Google Scholar, 34van der Spek P.J. Visser C. Hanaoka F. Smit B. Hagemeijer A. Bootsma D. Hoeijmakers J.H.J. Genomics. 1996; 31: 20-27Crossref PubMed Scopus (61) Google Scholar). This region is most likely an integral part of these proteins as it lacks the Gly-Gly sequence present in the carboxyl terminus of ubiquitin required for cleavage of ubiquitin peptides from linear molecules (3Hochstrasser M. Annu. Rev. Genet