TIP120A associates with cullins and modulates ubiquitin ligase activity.

The cullin-containing ubiquitin-protein isopeptide ligases (E3s) play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling. They have multisubunit modular structures in which substrate recognition and the catalysis of ubiquitination are carried out by distinct polypeptides. In a search for proteins involved in regulation of cullin-containing E3 ubiquitin ligases we immunopurified CUL4B-containing complex from HeLa cells and identified TIP120A as an associated protein by mass spectrometry. Immunoprecipitation of cullins revealed that all cullins tested specifically interacted with TIP120A. Reciprocal immunoaffinity purification of TIP120A confirmed the stable interaction of TIP120A with cullin family proteins. TIP120A formed a complex with CUL1 and Rbx1, but interfered with the binding of Skp1 and F-box proteins to CUL1. TIP120A greatly reduced the ubiquitination of phosphorylated IkappaBalpha by SCF(beta-TrCP) ubiquitin ligase. These results suggest that TIP120A functions as a negative regulator of SCF E3 ubiquitin ligases and may modulate other cullin ligases in a similar fashion.

The cullin-containing ubiquitin-protein isopeptide ligases (E3s) play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling. They have multisubunit modular structures in which substrate recognition and the catalysis of ubiquitination are carried out by distinct polypeptides. In a search for proteins involved in regulation of cullin-containing E3 ubiquitin ligases we immunopurified CUL4B-containing complex from HeLa cells and identified TIP120A as an associated protein by mass spectrometry. Immunoprecipitation of cullins revealed that all cullins tested specifically interacted with TIP120A. Reciprocal immunoaffinity purification of TIP120A confirmed the stable interaction of TIP120A with cullin family proteins. TIP120A formed a complex with CUL1 and Rbx1, but interfered with the binding of Skp1 and F-box proteins to CUL1. TIP120A greatly reduced the ubiquitination of phosphorylated IB␣ by SCF ␤-TrCP ubiquitin ligase. These results suggest that TIP120A functions as a negative regulator of SCF E3 ubiquitin ligases and may modulate other cullin ligases in a similar fashion.
The ubiquitin-dependent proteolysis provides a fundamental mechanism for regulating protein activity in various processes ranging from cell cycle and developmental switches to signal transduction (1). This process begins with the attachment of a multiubiquitin chain to a target protein and involves several enzymatic activities. A ubiquitin-activating enzyme (E1) 1 activates ubiquitin in an ATP-dependent reaction by forming a thioester bond with the C-terminal glycine of ubiquitin. The ubiquitin is then transferred to a specific sulfhydryl group on a ubiquitin-conjugating enzyme (E2). A ubiquitin-protein ligase (E3) transfers the activated ubiquitin from E2 to a lysine residue of a bound substrate, forming an isopeptide bond. Sub-strate specificity is determined mainly by E3s which bind both the protein substrate and the cognate E2. Once the multiubiquitin chain is assembled on a protein substrate by the cooperation of E1, E2, and E3 enzymes, the target protein is recognized and degraded by the 26 S proteasome (1)(2)(3).
In mammalian cells, a wide variety of E3s are found. The cullin family proteins play an important role in a group of multisubunit E3 ubiquitin ligases by associating with an Rbx1 (also known as ROC1 and Hrt1) family member of RING finger proteins to form the integral core (4). The SCF complexes are the best characterized ones of this class (5). They consist of CUL1, Rbx1, Skp1, and an F-box protein. Rbx1 contains the RING-H2 finger domain, forms a catalytic core with CUL1, and recruits the cognate E2 (6 -8). Skp1 functions as an adaptor that links an F-box protein to CUL1 (9). Substrates of the SCF complexes are bound by F-box proteins, which contain the Skp1-binding F-box motif and a variable protein-protein interaction domain that directly interacts with substrates (9, 10). Since a large number of F-box proteins are encoded by eukaryotic genomes (11)(12)(13), a variety of proteins are expected to be substrates of the SCF complexes, assuming that most of the F-box proteins form functional SCF E3 ubiquitin ligases. So far, a few SCF complexes, including SCF Skp2 , SCF ␤-TrCP , SCF-Cdc4 , and SCF Grr1 , have been demonstrated to have E3 activities for specific substrates.
CUL2 and CUL5 can also assemble multisubunit E3 ubiquitin ligases that bear a striking resemblance to SCF-type complexes. CUL2/Rbx1 and CUL5/Rbx1 form a complex with the Elongin BC heterodimer that functions as an adaptor analogously to Skp1 in the SCF complexes. The Elongin BC complex binds to a large number of proteins including the von Hippel-Lindau (VHL) tumor suppressor protein (14 -16) and members of the SOCS-box protein family (17,18), each of which contains an Elongin BC-binding site and a diverse protein-protein interaction motif (19). As a component of the VHL ubiquitin ligase complex, the VHL protein targets the ␣ subunits of the hypoxia-inducible transcription factors HIF1 and HIF2 for ubiquitination (20,21). In the case of SOCS-1, Vav and JAK2 are known to be specific substrates of the E3 complex (22,23). Most SOCS-box proteins may function as a substrate-binding subunit of an E3 ubiquitin ligase complex.
Studies on cullin/Rbx1-containing E3 ubiquitin ligases raise the possibility that other cullin family members could function as a component of ubiquitin ligases with distinct substrate specificities by forming multiprotein complexes with yet unidentified adaptors and/or substrate recognition subunits. In an effort to address this possibility, we have been purifying cullin-containing complexes from HeLa cells and identifying specifically associated proteins. In this report, we present purification of CUL4B-containing complexes and demonstrate that TIP120A specifically interacts with cullin family proteins and that it negatively regulates the activity of an SCF ubiquitin ligase by interfering with the binding of Skp1 to CUL1.
Protein Identification by Mass Spectrometry-Immunopurified pro-tein complexes were resolved on sodium dodecyl sulfate (SDS)-4 -20% gradient polyacrylamide gels (Novex). After staining gels with Sypro Ruby (Bio-Rad) and subsequently with colloidal Coomassie Blue, protein bands were excised and digested with trypsin as described previously (27). In-gel tryptic digests of proteins were analyzed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry using Voyager-DE STR (Applied Biosystems) and by nanoelectrospray ionization tandem mass spectrometry on API QSTAR Pulsar Q-TOF (Applied Biosystems). The mass spectral data were used to search the National Center for Biotechnology Information nonredundant and expressed sequence tag data bases.
Immunoprecipitations and Western Blotting-Transfection was carried out by the CaPO 4 -DNA precipitation method using Hepes or BES buffer. After 36 h, cells were lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mM phenylmethanesulfonyl fluoride, and 1.0% Nonidet P-40. Cell lysates were adjusted to 0.1% Nonidet P-40 and incubated with anti-FLAG or anti-HA antibody resin (Sigma) for 4 h at 4°C. The immune complexes were recovered by low speed centrifugation, and the resin was washed extensively with the binding buffer with 0.1% Nonidet P-40 and then eluted with buffer containing 20 mM Tris-HCl (pH 8.0) and 2% SDS. Immunoprecipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad) and visualized by Western blotting with the enhanced chemiluminescence reagents (Amersham Biosciences). For Western blotting, we used antibodies against FLAG (Sigma), HA (Babco), TIP120A (BD Biosciences), CUL1 (Lab Vision), Rbx1 (Lab Vision), Skp1 (Zymed Laboratories Inc.), and Skp2 (Zymed Laboratories Inc.).
Expression and Purification of Recombinant Proteins-His 6 -tagged yeast Uba1, human Ubc3, and UbcH5A (Ref. 28, a kind gift from J. W. Conaway) were expressed in Escherichia coli strain BL21(DE3) and purified by Ni 2ϩ -agarose beads (Qiagen). Ub containing a His 6 tag and a protein kinase C recognition site (a kind gift from Z.-Q. Pan) was purified as described previously (29). Rat TIP120A containing N-terminal FLAG and His 6 tags was expressed in Sf21 cells and purified as described previously (30). Generation of recombinant baculoviruses encoding human CUL1 with a His 6 tag, human ␤-TrCP with a FLAG tag, and human Skp1 with a His 6 tag (a generous gift from M. Pagano) were described previously (31). Human Rbx1 linked with an N-terminal FLAG tag was subcloned into a baculovirus expression vector, pFastBac (Invitrogen), and expressed in Sf21 cells. Sf21 cells were cultured at 27°C in TNM-FH (Sigma) with 5% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 g/ml). Sf21 cells were co-infected with the recombinant baculoviruses indicated in Fig. 5. Sixty hours after infection, cells were collected and lysed in ice-cold buffer containing 40 mM Hepes-NaOH (pH 7.9), 150 mM NaCl, 1 mM DTT, 0.5% (v/v) Triton X-100, 10% (v/v) glycerol, 5 g/ml leupeptin, 5 g/ml antipain, 5 g/ml pepstatin A, and 5 g/ml aprotinin. Lysates were clarified by centrifugation at 10,000 ϫ g for 20 min at 4°C. FLAG-␤-TrCP/His-Skp1 complex was purified by applying the supernatant onto an M2-agarose column (Sigma) equilibrated with the lysis buffer. After extensive washing of the column with the lysis buffer, the bound proteins were eluted with the lysis buffer containing 0.3 mg/ml FLAG peptide (Sigma). His-CUL1/FLAG-Rbx1 was purified using Co 2ϩ -agarose beads (Clontech) according to the manufacturer's instructions.

RESULTS AND DISCUSSION
Purification of CUL4B-containing Complex-To facilitate the purification of CUL4B-containing complex, we first established a HeLa-derived cell line that conditionally expressed FLAG-tagged CUL4B using an episomal expression vector. The tetracycline-regulated expression of FLAG-CUL4B in stable transfectants was tested by immunoblotting of cell extracts with anti-FLAG antibody. FLAG-CUL4B was induced upon removal of tetracycline, while it was not detected in cells grown in the presence of tetracycline (Fig. 1A). FLAG-CUL4B and its associated proteins were purified from extracts of induced and uninduced cells by a single-step immunoaffinity purification procedure, which had proved to be a gentle and efficient method (33). Several cellular proteins specifically co-purified with FLAG-CUL4B under the experimental conditions employed, as judged by SDS-polyacryamide gel electrophoresis of purified proteins from induced and uninduced cells (Fig. 1B). Bands corresponding to specifically interacting proteins were excised and analyzed by matrix-assisted laser desorption/ionization mass spectrometry and nanoelectrospray tandem mass spectrometry. The p14 band was identified as Rbx1 (data not shown), which forms a stable complex with all cullin family members (4). Another protein p50 turned out to be a novel protein of unknown function. However, since reciprocal immunoprecipitation experiments did not confirm the specificity of the interaction between p50 and CUL4B, p50 was not further studied. As shown in Fig. 2, mass spectrometric analyses of tryptic peptides derived from p120 revealed it as human TIP120A, which was shown to be a transcription factor that enhanced transcription by RNA polymerases I, II, and III (34,35). The fact that TIP120A was found in CUL4B-containing complex raised the possibility that in addition to the reported function of TIP120A in transcription, it might be involved in regulation of E3 ubiquitin ligase and/or ubiquitin-dependent proteolysis.
TIP120A Interacts with Cullin Family Proteins-To address the specificity of CUL4B-TIP120A interaction, we tested other cullin family members for their binding to TIP120A. HeLa cells were transfected with expression constructs of FLAG-tagged CUL1, CUL2, CUL3, CUL4A, or CUL4B, and anti-FLAG immunoprecipitation was carried out on the cell lysates. Western blotting of immunoprecipitates with anti-TIP120A antibodies indicated that TIP120A associated with all cullins tested (Fig.  3A). Immunoprecipitates of an unrelated protein FLAG-Chk2kd did not contain TIP120A (lane 1), indicating that the interaction between TIP120A and cullins was specific. To confirm the association of TIP120A with cullins, FLAG-TIP120A was immunopurified using anti-FLAG antibodies from cells that conditionally expressed the protein. SDS-polyacryamide gel electrophoresis of purified proteins revealed several protein bands with their molecular masses of 80 -90 kDa that were present only in the preparation derived from induced cells (Fig.  3B). Mass spectrometric analyses identified these proteins as CUL1, CUL2, CUL3, CUL4A, and CUL4B (data not shown), confirming that TIP120A specifically interacted with cullins. A protein band with the molecular mass of 14 kDa was identified as Rbx1, suggesting that TIP120A formed a trimeric complex with a cullin and Rbx1. Since TIP120A interacted with most, if not all, cullins, it might function as a global regulator of cullincontaining ubiquitin ligases. TIP120A Interferes with Binding of Skp1 and F-box Proteins to CUL1-Since CUL1 was the best studied member of the  2-6). As a negative control, an unrelated protein FLAG-tagged Chk2kd was expressed (lane 1). Cell lysates were immunoprecipitated with anti-FLAG antibody-coupled beads. The immunoprecipitates were subjected to SDS-polyacryamide gel electrophoresis and analyzed by Western blotting using anti-FLAG antibody to detect FLAG-tagged proteins (lower panel) and anti-TIP120 antibody to detect co-immunoprecipitated TIP120 (upper panel). B, immunoaffinity purification of FLAG-tagged TIP120A. Ten l each of purified FLAG-TIP120A samples from uninduced and induced cells was separated by a 4 -20% SDS-polyacrylamide gel and visualized by Sypro Ruby-Coomassie double staining. cullin family, we chose CUL1 to further characterize its interaction with TIP120A. To examine whether the interaction of TIP120A with CUL1 affects the SCF complex formation of CUL1, HeLa cells were transfected with expression constructs of epitope-tagged TIP120A, CUL1, Rbx1, Skp1, and an F-box protein Skp2 or ␤-TrCP. HA-CUL1 and FLAG-TIP120A were then immunoprecipitated with anti-HA and anti-FLAG antibodies, respectively, and co-purified proteins were probed by immunoblotting with suitable antibodies (Fig. 4A). FLAGtagged TIP120A, Rbx1, Skp1, and ␤-TrCP were specifically detected in HA-CUL1 immunoprecipitates (lanes 1 and 2). In contrast, FLAG-TIP120A immunoprecipitates contained HA-CUL1 and HA-Rbx1, but not Skp1 and Skp2 (lanes 3 and 4), indicating that FLAG-TIP120A did not form a complex with Skp1 and Skp2. To confirm that F-box proteins Skp2 and ␤-TrCP do not interact with TIP120A, immunoprecipitation of HA-Skp2 or HA-␤-TrCP was performed and associated proteins analyzed by immunoblotting (Fig. 4B). CUL1 and Skp1 were present in HA-Skp2 and HA-␤-TrCP immunoprecipitates, but TIP120A was not pulled down in the precipitates (lanes 5 and 6). Since CUL1 directly associates with Skp1 (9, 36) and TIP120A (data not shown), interactions among CUL1, Skp1, and TIP120A were examined by coupled immunoprecipitation and immunoblotting (Fig. 4C). Whereas CUL1 was immunoprecipitated with both TIP120A and Skp1, the immunoprecipitates of FLAG-TIP120A contained only CUL1, but not Skp1, and those of FLAG-Skp1 contained only CUL1, but not TIP120A (lanes 4 -6). Collectively, these data demonstrated that in cells existed at least two distinct CUL1-containing complexes either with TIP120A or with Skp1 and that the binding of TIP120A and Skp1 to CUL1 was mutually exclusive. To further test whether TIP120A and Skp1 compete for the binding to CUL1, HeLa cells were transfected with expression constructs of FLAG-CUL1, HA-TIP120A, HA-Skp1, and HA-Skp2. FLAG-CUL1 was immunoprecipitated with anti-FLAG antibodies, and HA-tagged TIP120A, Skp1, and Skp2 were probed by immunoblotting with anti-HA and anti-TIP120A antibodies (Fig. 4D). Overexpression of TIP120A reduced coprecipitation of Skp1 to 4 -18% in triplicate experiments and Skp2 to 30 -40% (lanes 5 and 6). In a similar competition experiment with TIP120A and ␤-TrCP, TIP120A inhibited the association of ␤-TrCP with CUL1 in a dose-dependent manner (lanes 12-14). These data showed that TIP120A interfered with binding of Skp1 and F-box proteins Skp2 and ␤-TrCP. It was noted that inhibition of Skp1 binding to CUL1 by TIP120A was greater than that of Skp2 or ␤-TrCP binding. Recent structural studies on SCF Skp2 showed that the F-box motif of Skp2 directly interacts with CUL1 as well as Skp1 (36). This Skp2-CUL1 interaction might not be affected by TIP120A binding  6) and immunoblotting experiments were performed as described in the legend to A. C, association of CUL1 either with Skp1 or with TIP120A. HeLa cells were transiently transfected with plasmid vectors expressing epitope-tagged or untagged TIP120A, CUL1, and Skp1 proteins as indicated. Immunoprecipitations and immunoblotting experiments were performed as described in the legend to A. D, competition of TIP120A and Skp1 for the binding to CUL1. HeLa cells were transiently transfected with plasmid vectors expressing HA-TIP120A, FLAG-CUL1, HA-Skp1, HA-Skp2, and HA-␤-TrCP proteins in combinations as indicated. Immunoprecipitations with ␣-FLAG antibodies and immunoblotting experiments were performed as described in the legend to A. and might contribute to less inhibition of Skp2 binding by TIP120A. Further investigation is required to clarify this point.
TIP120A Inhibits Ubiquitination of IB␣ by SCF ␤-TrCP -To determine the functional consequence of association of TIP120A with CUL1, we examined the effect of TIP120A on the substrate-specific ubiquitin ligase activity of SCF ␤-TrCP using IB␣ as a substrate. To devise an in vitro reconstituted system for the ubiquitination of IB␣, we produced all components used in the ubiquitination reaction as recombinant proteins by Escherichia coli or the baculovirus system. Fig. 5A shows dye staining patterns of the purified subunits of SCF ␤-TrCP after separation by SDS-PAGE. GST-IB␣ 1-54 was phosphorylated with IKK␤ S177E/S181E in the presence of [␥-32 P]ATP and incubated in the Ub ligation reaction with recombinant CUL1/Rbx1 and ␤-TrCP/Skp1 (Fig. 5B). A high molecular weight 32 P-labeled protein ladder was formed, indicating that phosphorylated GST-IB␣ 1-54 was polyubiquitinated. The ubiquitination of GST-IB␣ 1-54 was dependent not only on E1 and E2 (data not shown) but on CUL1/Rbx1 and ␤-TrCP/Skp1 (lanes 1-4), showing that intact SCF ␤-TrCP complex was required for IB␣ ubiquitination. Addition of increasing amounts of recombinant TIP120A greatly reduced the ubiquitination of IB␣ by CUL1 complex in a dose-dependent manner (lanes 4 -7). To test whether the reduction of IB␣ ubiquitination by TIP120A is correlated with the decrease of Skp1 binding to CUL1, the CUL1/Rbx1 complex in the ubiquitination reactions was immunoprecipitated with anti-Rbx1 antibodies and bound TIP120A and Skp1 monitored by Western blotting. As the association of TIP120A with CUL1 increased, the binding of Skp1 to CUL1 decreased (Fig. 5B, lower panel). These results indicated that TIP120A inhibited ubiquitin ligase activity of CUL1 by interfering with the binding of Skp1 and ␤-TrCP to CUL1.
TIP120A was initially identified as a TBP-interacting protein using in vitro affinity purification procedures (34) and subsequently shown to function as a transcriptional activator (35). The results presented here demonstrate a novel function of TIP120A, namely, negative regulation of SCF E3 ubiquitin ligases by inhibiting Skp1 binding to CUL1. Since Skp1 is an adapter subunit of SCF complexes that links the F-box protein to CUL1 (9,25,37), inhibition of Skp1 binding by TIP120A results in inhibition of association of F-box proteins and reduction of functional SCF complexes. It has been reported that TIP120A expression was up-regulated during the differentiation process of certain cells. For instance, retinoic acid treatment of P19 mouse embryonal carcinoma cells, which induces differentiation and withdrawal from the cell cycle, elevated expression of TIP120A (38). In addition, it has been shown that overexpression of TIP120A in P19 cells arrested cell growth (38). Since SCF E3 ligases play a key role in progression of cell division cycle, it is tempting to speculate that induction of TIP120A down-regulates SCF E3 ligases which may, in turn, help cells exit from cell cycle.
The function of TIP120A is not likely to be restricted to negative regulation of SCF complexes. Recent evidence suggests that SCF ubiquitin ligases may directly deliver substrate proteins to the proteasome for degradation (39,40). For instance, it has been shown that Skp1 interacts with an Snf1related protein kinase (SnRK) and ␣4 subunit of the 20 S proteasome in Arabidopsis (39), suggesting that SnRK and/or ␣4 subunit may function as a docking site for the SCF complexes on the proteasome. Thus, competitive binding of TIP120A to CUL1 against Skp1 may help release of SCF ligases from a proteasomal docking site and unloading of the substrate. Our finding that TIP120A interacts not only with CUL1 but with other cullins suggests that TIP120A has a general role common to most, if not all, cullin-containing E3 ligases. Further structural and functional dissection of TIP120A-cullin complexes is expected to further clarify the role of TIP120A in regulation of ubiquitination and proteolysis. Purified GST-IB␣ 1-54 was phosphorylated with IKK␤ S177E/S181E in the presence of [␥-32 P]ATP and incubated with recombinant His-CUL1/FLAG-Rbx1(500 ng), FLAG-␤-TrCP/His-Skp1 (300 ng), and FLAG-TIP120A (0.5, 1, and 2 g in lanes 5, 6, and 7, respectively) as indicated. Reactions were performed at 37°C for 20 min, terminated by adding Laemmli loading buffer, and resolved by SDS-PAGE followed by autoradiography to visualize the ubiquitinated IB␣ ladders. For the immunoblotting experiments shown in the lower panel, reactions were performed without the substrate and ATP, and the CUL1/Rbx1 complex was immunoprecipitated with anti-Rbx1 antibodies.