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• W2136406488 abstract "Ubiquitin-protein ligases (E3s) of the HECT family share a conserved catalytic region that is homologous to the E6-AP Cterminus. The HECT domain defines a large E3 family, but only a handful of these enzymes have been defined with respect to substrate specificity or biological function. We showed previously that the C-terminal domain of one family member, KIAA10, catalyzes the assembly of polyubiquitin chains, whereas the N-terminal domain binds to proteasomes in vitro (You, J., and Pickart, C. M. (2001) J. Biol. Chem. 276, 19871–19878). We show here that KIAA10 also associates with proteasomes within cells but that this association probably involves additional contacts with proteasome subunits other than the one (S2/Rpn1) identified in our previous work. We report that the N-domain of KIAA10 also mediates an association with TIP120B (TATA-binding protein-interacting protein 120B), a putative transcriptional regulator. Biochemical and co-transfection studies revealed that TIP120B, but not the closely related protein TIP120A, is a specific substrate of KIAA10 in vitro and within C2C12 myoblasts but not in Cos-1 cells. KIAA10 and TIP120B are both highly expressed in human skeletal muscle, suggesting that KIAA10 may regulate TIP120B homeostasis specifically in this tissue. Ubiquitin-protein ligases (E3s) of the HECT family share a conserved catalytic region that is homologous to the E6-AP Cterminus. The HECT domain defines a large E3 family, but only a handful of these enzymes have been defined with respect to substrate specificity or biological function. We showed previously that the C-terminal domain of one family member, KIAA10, catalyzes the assembly of polyubiquitin chains, whereas the N-terminal domain binds to proteasomes in vitro (You, J., and Pickart, C. M. (2001) J. Biol. Chem. 276, 19871–19878). We show here that KIAA10 also associates with proteasomes within cells but that this association probably involves additional contacts with proteasome subunits other than the one (S2/Rpn1) identified in our previous work. We report that the N-domain of KIAA10 also mediates an association with TIP120B (TATA-binding protein-interacting protein 120B), a putative transcriptional regulator. Biochemical and co-transfection studies revealed that TIP120B, but not the closely related protein TIP120A, is a specific substrate of KIAA10 in vitro and within C2C12 myoblasts but not in Cos-1 cells. KIAA10 and TIP120B are both highly expressed in human skeletal muscle, suggesting that KIAA10 may regulate TIP120B homeostasis specifically in this tissue. Many important intracellular proteins, including cell-cycle regulators, tumor suppressors, oncoproteins, and transcription factors, are targeted for degradation by 26 S proteasomes through conjugation to Ub, 1The abbreviations used are: Ub, ubiquitin; polyUb, polyubiquitin (refers to a branched, isopeptide-linked ubiquitin chain); CD, C-terminal domain of KIAA10; E1, ubiquitin-activating enzyme; E2, ubiquitinconjugating enzyme; E3, ubiquitin-protein ligase; HECT, homologous to the E6-AP Cterminus; ND, N-terminal domain of KIAA10; RING, really interesting new gene; TBP, TATA-binding protein; TIP, TBP-interacting protein; FL, full-length; GST, glutathione S-transferase. a highly conserved protein of 76 amino acids (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Google Scholar). This proteolytic targeting function underlies the role of ubiquitin in such processes as cell cycle progression, tumorigenesis, antigen presentation, and cell death (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Google Scholar). A single Ub is an inefficient signal for degradation by proteasomes (2Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. EMBO J. 2000; 19: 94-102Google Scholar). Proteasome-bound substrates are rather marked with a polyUb chain in which successive Ubs are joined by Lys48-Gly76 isopeptide bonds (3Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Google Scholar, 4Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crooke S.T. Chau V. Mol. Cell. Biol. 1994; 14: 5501-5509Google Scholar). This modification is accomplished through the sequential actions of three classes of enzymes (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Google Scholar, 5Pickart C. Annu. Rev. Biochem. 2001; 70: 503-533Google Scholar). Ub-activating enzyme (E1) uses ATP to drive the formation of a thiol ester bond between an E1 active-site Cys and the C-terminal carboxyl of Ub (Gly76). The activated Ub is then transferred to the active site Cys of a Ub-conjugating enzyme (E2). Ub is finally transferred from the E2 to an ϵ-amino group of the substrate in a reaction that requires a specific Ub-protein ligase (E3). In many cases, the polyUb chain is probably assembled by the same E3 that recognizes the substrate, but in some cases chain assembly may require specialized, Ub-dedicated E3s (6Mastrandrea L.D. You J. Niles E.G. Pickart C.M. J. Biol. Chem. 1999; 274: 27299-27306Google Scholar, 7Hoege C. Pfander B. Moldovan G.-L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Google Scholar). The substrate-linked chain is ultimately recognized by the 19 S regulatory complex of the 26 S proteasome (2Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. EMBO J. 2000; 19: 94-102Google Scholar, 3Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Google Scholar, 4Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crooke S.T. Chau V. Mol. Cell. Biol. 1994; 14: 5501-5509Google Scholar, 8Lam Y.A. Lawson T.G. Velayutham M. Zweier J.L. Pickart C.M. Nature. 2002; 416: 763-767Google Scholar), resulting in the unfolding, translocation, and hydrolysis of the substrate polypeptide chain (9Baumeister W. Walz J. Zuhl F. Seemuller E. Cell. 1998; 92: 367-380Google Scholar). PolyUb chains linked through other lysine residues of Ub also occur within cells and in some cases may represent functionally distinct signals (for review, see Ref. 10Pickart C.M. Trends Biochem. Sci. 2000; 25: 544-548Google Scholar). The Ub conjugation system features multiple E2 and E3 enzymes, with a large array of combinatorial E2-E3 pairings allowing for the selective targeting of diverse substrates (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Google Scholar). Substrate recognition usually reflects a direct interaction between the E3 enzyme and a specific ubiquitination signal or degron of the substrate (for review, see Ref. 11Laney J.D. Hochstrasser M. Cell. 1999; 97: 427-430Google Scholar). E3 enzymes display a modular construction, with unique domains/subunits mediating substrate interaction and conserved domains/subunits responsible for catalysis. A small number of distinctive catalytic domains defines the known E3 families (for review, see Ref. 5Pickart C. Annu. Rev. Biochem. 2001; 70: 503-533Google Scholar). One family, known as the HECT E3s, employs a covalent catalytic mechanism (12Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Google Scholar). A second family, known as the RING E3s, recruits a specific E2 enzyme by means of a globular zinc binding domain (13Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Google Scholar). A subset of the RING E3s, known as SCF E3s, consists of multisubunit enzymes in which the RING and substrate-binding domains are localized to distinct subunits (13Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Google Scholar). HECT E3s share a conserved ∼350-amino acid region that is defined by its homology to the C terminus of E6-AP. This HECT domain harbors the Cys residue that forms a catalytic thiol ester with Ub (14Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Google Scholar). The prototypic family member, E6-AP, is a 100-kDa host cell protein that forms a complex with the E6 protein of oncogenic human papilloma viruses, thereby acquiring the ability to bind and ubiquitinate the p53 tumor suppressor (15Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Google Scholar). In contrast to the conserved catalytic module, HECT E3s display highly variable N-terminal domains. The recognition of p53 is mediated principally by the N terminus of E6-AP in conjunction with the viral E6 protein (16Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell. Biol. 1993; 13: 775-784Google Scholar). The current model for HECT E3 structure-function postulates that the divergent N termini of these E3s mediate specific substrate binding, whereas the conserved HECT domain supplies catalytic activity in ubiquitination (14Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Google Scholar). In principle, independent substrate binding and catalytic motifs can ensure the selective and efficient ubiquitination of different substrates. However, although databases reveal a large family of mammalian HECT E3s (14Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Google Scholar), only a handful of these enzymes has been defined with respect to substrate specificity or biological function. In previous work we used a cognate E2 affinity approach to purify and identify a rabbit E3 enzyme that uses free Ub as the substrate for assembly of unanchored polyUb chains linked through Lys29 or Lys48. This enzyme corresponded to the HECT domain E3 encoded by the human KIAA10 cDNA (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar). The C-terminal 420 amino acids of KIAA10, called the C-domain (CD), are necessary and sufficient for assembly of both types of polyUb chains, indicating that the N-terminal domain (ND) is responsible for another function(s). We found that the ND bound proteasomes in vitro (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar), suggesting that KIAA10 belongs to the small group of conjugating factors known to associate with proteasomes (18Xie Y. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2497-2502Google Scholar, 19Tongaonkar P. Chen L. Lambertson D. Ko B. Madura K. Mol. Cell. Biol. 2000; 20: 4691-4698Google Scholar, 20Kleijnen M.F. Shih A.H. Zhou P. Kumar S. Soccio R.E. Kedersha N.L. Gill G. Howley P. Mol. Cell. 2000; 6: 409-419Google Scholar). Among these factors is the HUL5-encoded HECT E3 (21Leggett D.S. Hanna J. Borodovsky A. Crosas B. Schmidt M. Baker R.T. Walz T. Plough H. Finley D. Mol. Cell. 2002; 10: 495-507Google Scholar), an apparent ortholog of KIAA10 in budding yeast (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar). The purpose of E3-proteasome interactions is uncertain, but one possibility is that they provide an independent mechanism (besides the substrate-linked chain) for targeting certain substrates to proteasomes. In the case of KIAA10, we demonstrated a robust interaction with the purified S2/Rpn1 subunit of the 19 S complex of the proteasome (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar). However, it was uncertain whether KIAA10 associates with proteasomes in vivo. Nor could we exclude that the ND mediated other functionally significant interactions. Here we report experiments probing the function of the KIAA10-ND. We show that KIAA10 indeed associates with 26 S proteasomes in mammalian cells but that this interaction is likely to depend on contacts with a subunit(s) besides S2/Rpn1. Most importantly, we provide strong evidence that TIP120B (TBP-interacting protein 120B (22Aoki T. Okada N. Ishida M. Yogosawa S. Makino Y. Tamura T. Biochem. Biophys. Res. Commun. 1999; 261: 911-916Google Scholar)) is a specific substrate that is targeted for degradation in skeletal muscle through KIAA10-catalyzed polyubiquitination. Our results confirm the utility of affinity capture methods for the identification of E3 substrates and set the stage for achieving a fuller understanding of the functions of TIP120B and KIAA10. Proteins—E1 (23Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Google Scholar), UbcH5A (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar), several versions of KIAA10 (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar), and GST-S2 (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar) were expressed and purified as in previous work. Bovine Ub and GSH beads were from Sigma. Sources of other reagents are given below. Plasmids and Cloning—pGEX* plasmids encoding full-length (FL), 655-residue N-terminal domain (ND), and 428-residue C-terminal domain (CD) of KIAA10 have been described (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar). We made pET3a-KIAA10 a template for in vitro transcription/translation and full-length enzyme expression in Escherichia coli. The KIAA10 open reading frame was amplified by PCR to introduce flanking NdeI sites and ligated into pET3a. The C1051A mutation was introduced into this plasmid by standard PCR methods. Plasmids pBluescript-TIP120A, pCDNATIP120B, and pCDNA-FLAG-TIP120B have been described (22Aoki T. Okada N. Ishida M. Yogosawa S. Makino Y. Tamura T. Biochem. Biophys. Res. Commun. 1999; 261: 911-916Google Scholar). For mammalian cell expression, the KIAA10 and KIAA10-C1051A coding sequences were amplified by PCR to introduce 5′-SalI and 3′-NotI sites and subcloned in-frame with the Myc tag in pCMV-MYC (Clontech). The sequences of all PCR-derived cDNA constructs were verified. For Northern blotting we probed a human MTN blot (Clontech) with a 32P-labeled probe complementary to the ND coding region. KIAA10-Proteasome Interaction—A series of N-terminal truncation mutants of KIAA10-ND (see Fig. 1 and “Results”) were constructed in pET3a-KIAA10 by standard PCR methods. [35S]Met-labeled in vitro translation products were produced in wheat germ extract (Promega). A 10-μl aliquot of translation mixture was mixed with 10 μl of immobilized GST-S2 or control immobilized GST (∼0.2 mg of protein/ml beads). Binding buffer (0.18 ml of 20 mm Tris-HCl (pH 7.6), 50 mm NaCl, 0.1% Nonidet P-40, 0.5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride) was added to the beads followed by 4 h of incubation at 4 °C with gentle agitation. The beads were washed with 4 × 0.3 ml of binding buffer and eluted with 20 μl of sample buffer. Aliquots of 8 μl were analyzed by SDS-PAGE and autoradiography. To assess if KIAA10 interacts with proteasomes in Cos-1 cells, pCMV-MYC vector specifying wild type or Δ132-KIAA10 was transfected into Cos-1 cells using FuGENE 6 reagent (Roche Applied Science) according to the manufacturer's instructions (plasmids used in mammalian cell transfection were purified using the EndoFree Plasmid Maxi kit from Qiagen). In all cases, 3 μg of plasmid was used to transfect 106 cells on a 10-cm dish. After 48 h, cells were harvested by scraping into 0.9 ml of radioimmune precipitation assay buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in phosphate-buffered saline) per dish. Cells were lysed by passing the suspension through a 21-gauge needle 2 or 3 times. The lysate was incubated on ice for 60 min and then clarified by centrifuging at 10,000 × g for 10 min (4 °C). Soluble extract protein (1 ml, ∼0.5 mg of protein) was mixed with 20 μl of protein A-agarose (Sigma) and 2 μl of antibody against the human S8/Rpt6 (p45) 19 S subunit or 5 μl of antibody against the S10a/Rpn7 subunit (Affiniti) and then rotated at 4 °C for 2 h. The beads were washed with 4 × 0.5 ml of radioimmune precipitation assay buffer, and bound proteins were eluted with 40 μl of sample buffer. Aliquots (10 μl) were resolved on a 10% gel. Proteins were transferred to Immobilon-P (Millipore) and blotted with c-Myc antibody (Santa Cruz A-14) to detect Myc-KIAA10 and Myc-Δ132-KIAA10 (ECL detection). Partial Purification of Full-length KIAA10—To maximize the specific activity of the recombinant E3, we partially purified the small fraction of soluble enzyme produced from pET3a-KIAA10 in E. coli. Expression in BL21(DE3)pJY2 cells (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar) at 30 °C was as described (6Mastrandrea L.D. You J. Niles E.G. Pickart C.M. J. Biol. Chem. 1999; 274: 27299-27306Google Scholar). Cell lysis and DNA digestion followed established procedures (23Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Google Scholar). The extract was centrifuged at 10,000 × g for 20 min, and the supernatant (32 ml, from 2 liters of cell suspension) was passed through a 30-ml Q-Sepharose column (Amersham Biosciences) that had been pre-equilibrated with base buffer (50 mm Tris-HCl (pH 7.5), 0.1 mm EDTA, and 0.5 mm dithiothreitol). The column was washed with three column volumes of base buffer and then eluted with a 200-ml gradient of 0–0.5 m NaCl. Fractions of 5 ml were collected and assayed for activity in K29-Ub2 synthesis (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar). The active fractions were pooled and loaded onto a MonoQ fast protein liquid chromatography column (Amersham Biosciences) equilibrated with base buffer. The column was eluted with a 0–0.6 m NaCl gradient. The pooled active fractions from this column were used as an E3 source after desalting by repeated dilution with base buffer and reconcentration (final volume, ∼1.7 ml). Characterization of C1051A-KIAA10—[35S]Met-labeled wild-type and C1051A-KIAA10 proteins were expressed by in vitro transcription/translation in reticulocyte lysate (Promega). An aliquot of each translation mixture was assayed for thiol ester formation by incubating for several min with Ub2 in the presence of E1 and UbcH5A under established conditions (23Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Google Scholar). Wild type, but not mutant, E3 formed a labeled band that was upshifted by ∼18 kDa and was detected only when the reaction was quenched in non-reducing sample buffer (use of Ub2 rather than Ub1 facilitated detection of the molecular mass shift). We attribute the upshifted band to formation of a thiol ester with Ub2 that is abolished after mutation of the HECT Cys residue (Cys1051) to Ala. In Vitro Binding of TIP120 to KIAA10—[35S]Met-labeled TIP120A and TIP120B proteins were produced by in vitro transcription/translation in wheat germ extract (Promega) using plasmids pBluescript-TIP120A and pCDNA-TIP120B, respectively. GST-KIAA10-FL, ND, and CD fusion proteins were produced in E. coli using the respective pGEX* plasmids (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar). A 20-μl aliquot of each translation mixture was mixed with 10 μl of immobilized GST-KIAA10-FL, -ND, -CD, or GST alone (∼0.5 mg of protein/ml beads). Binding buffer (0.17 ml, see “KIAA10-proteasome interaction,” above) was added to the beads followed by overnight incubation at 4 °C with gentle agitation. The beads were washed with 0.3 ml of binding buffer and eluted with 20 μl of SDS-PAGE sample buffer. An aliquot of each eluate (8 μl) was analyzed by SDS-PAGE and autoradiography. In Vitro Ubiquitination of TIP120B—[35S]Met-labeled TIP120B was produced by in vitro transcription/translation in reticulocyte lysate (Promega) using pCDNA-TIP120B. The translation product, 1 μl, was incubated in a total volume of 20 μl with 0.1 μm E1, 0.3 μm UbcH5A, and 146 μm Ub under established conditions (pH 7.6, 37 °C) (6Mastrandrea L.D. You J. Niles E.G. Pickart C.M. J. Biol. Chem. 1999; 274: 27299-27306Google Scholar) with or without 1 μl of partially purified recombinant KIAA10 (above). At 0 and 4 h, 8-μl aliquots were quenched with sample buffer and resolved on an 8% SDS-PAGE gel. TIP120B and its ubiquitination products were detected by autoradiography. Intracellular Interaction of TIP120B and KIAA10—Cos-1 cells were co-transfected with pCDNA-FLAG-TIP120B and pCMV-MYC vector specifying wild type or C1051A-KIAA10, harvested, and lysed as described (see “KIAA10-proteasome interaction,” above). Soluble extract protein (∼0.5 mg) was mixed with 10 μl of anti-FLAG M2 agarose (Sigma) and rotated at 4 °C for 2 h. The beads were washed with 4 × 0.5 ml radioimmune precipitation assay buffer, and bound proteins were eluted with 40 μl of sample buffer. Aliquots (10 μl) were resolved on an 8% gel. Proteins were transferred to Immobilon-P (Millipore) and blotted with anti-c-Myc antibody (Santa Cruz A-14) to detect the Myctagged E3 (ECL detection). Intracellular Ubiquitination of TIP120B—Myoblast C2C12 cells were from ATCC. C2C12 cells (106 cells) were co-transfected using FuGENE 6 (above) with pCDNA-FLAG-TIP120B together with pCMVMYC-KIAA10 or pCMV-MYC-KIAA10-C1051A (or an empty vector control). Each transfection employed 3 μg of each vector and was done in duplicate. After 40 h, 50 μm MG-132 (Peptides International) was added to one set of transfections. Cells were harvested 6 h later and lysed (above). TIP120B was immunoprecipitated (above), and the immunoprecipitates were screened by immunoblotting with affinity-purified Ub antibodies (produced by us according to procedures described in Haas and Bright (24Haas A.L. Bright P.M. J. Biol. Chem. 1985; 260: 12464-12473Google Scholar)). Intracellular Destabilization of TIP120B—C2C12 cells (105 cells) were co-transfected with pCDNA-FLAG-TIP120B and pCMV-MYCKIAA10 or pCMV-MYC-KIAA10 mutant (or vector control) as described above using a total of 2 μg of plasmid DNA (1 μg of each) per transfection. After 40 h, 50 μm MG-132 was added to the indicated transfections. After 6 h more, cells were harvested and lysed (above). Lysates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-FLAG antibody (Sigma) or affinity-purified anti-TIP120B antibody (22Aoki T. Okada N. Ishida M. Yogosawa S. Makino Y. Tamura T. Biochem. Biophys. Res. Commun. 1999; 261: 911-916Google Scholar). Studies of the ND-Proteasome Interaction—We have shown that KIAA10 (Fig. 1A) associates via its ND with purified 26 S proteasomes (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar). This interaction might be explained by binding of the ND to S2/Rpn1, one of the two largest subunits of the 19 S regulatory complex of the proteasome (Fig. 1B and Ref. 17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar). As a first step in evaluating the biological significance of this interaction, we carried out deletion mutagenesis to map the region of the ND responsible for its interaction with S2/Rpn1. N-terminally truncated versions of the ND were labeled by in vitro transcription/translation and tested for binding in pull-down assays with a GST-S2 fusion protein (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar). Removal of the first 88 residues of the ND had a minimal effect on the S2-ND interaction (Fig. 1, B and C), but mutants truncated by ≥132 residues displayed strongly diminished binding (Fig. 1B, compare lanes 10, 12, and 14, with 6 and 8). Versions of the ND lacking ≥132 N-terminal residues did, however, retain weak binding activity (∼18% of the intact ND, Fig. 1C). Thus, a second, minor S2 binding determinant is located elsewhere in the ND. In all cases, minimal binding to GST alone (Fig. 1B) confirmed the specificity of the ND-S2 interaction. Several considerations suggest that reduced binding of Δ132-ND to S2/Rpn1 reflects the presence of a binding epitope in the first 132 residues of the ND versus improper folding of truncated ND molecules. First, in vitro transcription/translation frequently produces folded proteins. For example, full-length KIAA10 produced by this method quantitatively forms a thiol ester with Ub, indicating full activity (see “Experimental Procedures”) even though full-length KIAA10 produced in bacteria is largely insoluble (17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar). Second, a GST-Δ132-ND fusion protein binds purified 26 S proteasomes (see the next paragraph) as well as a KIAA10-specific substrate (TIP120B, see Fig. 2 below). These findings show that N-terminal truncation is compatible with proper folding. This conclusion is confirmed by the behavior of Δ132-KIAA10 expressed in Cos-1 cells (see the next paragraph). Finally, the results of a complementary C-terminal deletion study are fully consistent with the result shown in Fig. 1B. In these experiments the ND was successively truncated at its C terminus, and a fragment consisting of residues 1–222 was found to bind efficiently to GST-S2. 2J. Blum and C. Pickart, unpublished experiments. If the S2-ND interaction is responsible for the binding of KIAA10 to 26 S proteasomes, then Δ132-KIAA10 should exhibit reduced binding to proteasomes. We used two approaches to determine if this prediction was met. First, GST-fused versions of intact or truncated (Δ132) ND were used to pull down purified 26 S proteasomes in vitro. Surprisingly, there was no detectable inhibition of the ND-proteasome interaction as a result of the truncation (data not shown). In a more physiological experiment, we transfected Myc-tagged versions of WTKIAA10 and Δ132-KIAA10 into Cos-1 cells, immunoprecipitated proteasomes with an antibody against the S8/Rpt6 ATPase subunit of the 19 S complex, and screened the immunoprecipitates for the presence of KIAA10 by Western blotting. Full-length KIAA10 was co-precipitated by the S8 antibody (Fig. 1D, middle panel). Although this interaction seemed to be more pronounced with the truncated E3, this effect could be explained by higher expression of the truncated protein (upper panel). A similar result was obtained when we used antibody against a non-ATPase 19 S subunit, S10a/Rpn7, to precipitate proteasomes (Fig. 1D, bottom panel). The agreement between the results of intracellular and in vitro interaction assays strongly suggests that KIAA10 interacts directly with proteasomes. Direct interaction also applies in the case of yeast Ufd4 (18Xie Y. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2497-2502Google Scholar, 25Xie Y. Varshavsky A. Nat. Cell Biol. 2002; 4: 1003-1007Google Scholar), whereas E6-AP interacts with proteasomes through an accessory factor (20Kleijnen M.F. Shih A.H. Zhou P. Kumar S. Soccio R.E. Kedersha N.L. Gill G. Howley P. Mol. Cell. 2000; 6: 409-419Google Scholar). These findings show for the first time that KIAA10 associates with proteasomes within cells. The in vitro and intracellular results concur in suggesting that the extreme N terminus of the E3 is not the only determinant of its interaction with proteasomes. We cannot exclude that the residual interaction between Δ132-KIAA10 and S2/Rpn1 (Fig. 1C) is sufficient for interaction with cellular proteasomes, but we consider it more likely that a distal region of the ND (beyond residue 132) also binds to a different, unidentified subunit of the 19 S complex. There is precedent for a given E3 interacting with more than one subunit of the 19 S complex (18Xie Y. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2497-2502Google Scholar, 25Xie Y. Varshavsky A. Nat. Cell Biol. 2002; 4: 1003-1007Google Scholar). Interaction of TIP120B with KIAA10—We next addressed whether the KIAA10-ND might be responsible for interacting with a specific substrate. To gain insight into the likely properties of cognate substrates, we conducted Northern blot analysis of KIAA10 mRNA levels in human tissues (data not shown). The results confirmed a previous report that KIAA10 is highly expressed in skeletal muscle (26Schwarz S.E. Rosa J.L. Scheffner M. J. Biol. Chem. 1998; 273: 12148-12154Google Scholar). Levels of KIAA10 mRNA in kidney and pancreas were <1% that of the level in muscle, whereas expression in heart, brain, placenta, lung, and liver was nearly undetectable (data not shown). Based on this expression pattern we speculated that KIAA10 might target a muscle protein(s) for degradation in the Ub-proteasome pathway. In earlier work we found that an affinity-purified preparation of KIAA10 also contained low levels of peptides conserved among members of the TIP120 family (data not shown and Ref. 17You J. Pickart C.M. J. Biol. Chem. 2001; 276: 19871-19878Google Scholar), raising the possibility that these proteins interact with UbcH5A or KIAA10. TIP120A, the original family member, was identified during a search for proteins that bound to TBP in vitro (27Yogosawa S. Makino Y. Yoshida T. Kishimoto T. Muramatsu M. Tamura T. Biochem. Biophys. Res. Commun. 1996; 229: 612-617Google Scholar). TIP120B was later cloned by virtue of its similarity to TIP120A (22Aoki T. Okada N. Ishida M. Yogosawa S. Makino Y. Tamura T. Biochem. Biophys. Res. Commun. 1999; 261: 911-916Google Scholar). In humans, TIP120B is expressed most highly in skeletal muscle, whereas TIP120A is ubiquitously expressed (22Aoki T. Okada N. Ishida M. Yogosawa S. Makino Y. Tamura T. Biochem. Biophys. Res. Commun. 1999; 261: 911-916Google Scholar, 28Aoki T. Okada N. Wakamatsu T. Tamura T. Biochem. Biophys. Res. Commun. 2002; 296: 1097-1103Google Scholar). The two TIP120 proteins are 60% identical to one another (22Aoki T. Okada N. Ishida M. Yogosawa S. Makino Y. Tamura T. Biochem. Biophys. Res. Commun. 1999; 261: 911-916Google Scholar), and both contain multiple copies of a proposed protein interaction module, the HEAT repeat (29Neuwald A.F. Hirano T. Genome Res. 2000; 10: 1445-1452Google Scholar), whose significance for TIP120 function is unknown. Both isoforms interact with TBP under physiological conditions in vitro and associate with TBP in nuclear extracts (22Aoki T. Okada N. Ishida M. Yogosawa S. Makino Y. Tamura T. Biochem. Biophys. Res. Commun. 1999; 261: 911-916Google Scholar, 27Yogosawa S. Makino Y. Yoshida T. Kishimoto T. Muramatsu M. Tamu" @default.
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• W2136406488 title "Proteolytic Targeting of Transcriptional Regulator TIP120B by a HECT Domain E3 Ligase" @default.
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• W2136406488 cites W1965520255 @default.
• W2136406488 cites W1965718518 @default.
• W2136406488 cites W1974837828 @default.
• W2136406488 cites W1984573021 @default.
• W2136406488 cites W1991930087 @default.
• W2136406488 cites W1992798036 @default.
• W2136406488 cites W1994196743 @default.
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• W2136406488 cites W2008044453 @default.
• W2136406488 cites W2008744347 @default.
• W2136406488 cites W2008798771 @default.
• W2136406488 cites W2013339424 @default.
• W2136406488 cites W2014675809 @default.
• W2136406488 cites W2020294551 @default.
• W2136406488 cites W2025450862 @default.
• W2136406488 cites W2032590709 @default.
• W2136406488 cites W2033778029 @default.
• W2136406488 cites W2035599991 @default.
• W2136406488 cites W2045350410 @default.
• W2136406488 cites W2046511938 @default.
• W2136406488 cites W2047050750 @default.
• W2136406488 cites W2061050157 @default.
• W2136406488 cites W2064850779 @default.
• W2136406488 cites W2083629798 @default.
• W2136406488 cites W2086966984 @default.
• W2136406488 cites W2094172710 @default.
• W2136406488 cites W2095424116 @default.
• W2136406488 cites W2105006635 @default.
• W2136406488 cites W2119301405 @default.
• W2136406488 cites W2120527610 @default.
• W2136406488 cites W2125134465 @default.
• W2136406488 cites W2126467039 @default.
• W2136406488 cites W2132087311 @default.
• W2136406488 cites W2132535068 @default.
• W2136406488 cites W2133500834 @default.
• W2136406488 cites W2138688363 @default.
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• W2136406488 cites W2170634115 @default.
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