WO2005114188A2 - Cul4 e3 ligase mediators as regulators of p53 - Google Patents

Abstract

The invention relates to assays for identifying modulators of p53­ubiquitination by CUL4 E3 ligase complexes. This assays allows detection of agents and compounds that affect p53 ubiquitination and thus, cell cycle regulation and cell survival in cells. In some assays, an increase in ubiquitination, in comparison to a test sample lacking a test compound, indicates a stimulation of p53 ubiquitination activity, whereas a reduction in p53 ubiquitination indicates an inhibitor of activity. The assays provided may be suited, for example, for high-throughput screening of agents. The invention further relates to methods of modulating p53 activity in a cell, such as a cell of a mammal, by administering agents which decrease expression or activity of a member of a CUL4 E3 ligase complex, or which block the binding of p53 to a CUL4 E3 ligase complex.

Description

NOVEL MEDIATORS OF p53 LEVELS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Application No. 60/565,707, filed April 27, 2004, entitled "NOVEL MEDIATORS OF p53 LEVELS." The entire teachings of the referenced application are incorporated by reference herein.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT The invention described herein was supported, in whole or in part, by Grant No. RO1-CA72878 from the National Institutes of Health. The United States government has certain rights in the invention.

BACKGROUND OF THE INVENTION The p53 transcription factor (or "p53") is of key importance for the protection of an organism against carcinogenesis. P53 performs this function by regulating several cellular processes, the most important of which are apoptosis and cell-cycle progression. The p53 transcription factor is a nuclear phosphoprotein involved in the control of cell proliferation, and mutations in the p53 gene are commonly found to be associated with diverse type of human cancer (Levine et al., Nature 351: 453, 1991). Elevated p53 protein levels were observed in some human tumor lines. P53 plays a crucial role in the regulation of DNA replication at the Gl/S checkpoint. Wild-type p53 allows cells to arrest in GI so as to provide an opportunity for DNA repair prior to commencement of replicative DNA synthesis. P53 acts to reduce the incidence of cancers by mediating apoptosis in cells with activated oncogenes. P53 exhibits DNA-binding activity (Kern et al., Science 252: 1708, 1991) and transcriptional activation properties (Fields et al., Science 249: 1046, 1990; Raycroft et al., Science 249: 1049, 1990; Bargonetti et al., Cell 65: 1083, 1991; and Agoffet al., Science 259: 84, 1993). Tetramer formation is critical to p53 ability to activate transcription (Sakaguchi et al., Biochemistry 36: 10117-24, 1997). Point mutated forms of p53 found associated with transformed cells have been observed to have lost the sequence-specific DNA binding function (Kern et al., supra; Bargonetti et al., supra; and El-Deiry et al., Nature Genetics 1: 45, 1992). Moreover, many of the mutant p53 proteins can act as dominant negatives to inhibit this activity of wild-type p53. Some viral-encoded oncoproteins (e.g., SV40 large T antigen) inhibit the DNA- binding activity of p53 apparently as a consequence of forming complexes with the p53 protein (Bargonetti et al., supra). Modulation of p53 bioactivities (e.g., transcription regulating function) or its cellular level would affect various cellular processes and provide therapeutic means for treating a number of diseases and conditions. There is a need in the art for novel methods and compositions for modulating p53 activities and thereby inhibiting cell proliferation in tumor formation and tumor growth. The instant invention fulfills this and other needs.

SUMMARY OF THE INVENTION The invention relates to assays for identifying modulators of p53- ubiquitination by CUL4 E3 ligase complexes. This assays allows detection of agents and compounds that affect p53 ubiquitination and thus, cell cycle regulation and cell survival in cells. In some assays, an increase in ubiquitination, in comparison to a test sample lacking a test compound, indicates a stimulation of p53 ubiquitination activity, whereas a reduction in p53 ubiquitination indicates an inhibitor of activity. The assays provided may be suited, for example, for high-throughput screening of agents. The invention further relates to methods of modulating p53 activity in a cell, such as a cell of a mammal, by administering agents which decrease expression or activity of a member of a CUL4 E3 ligase complex, or which block the binding of p53 to a CUL4 E3 ligase complex. One aspect of the invention provides a method of identifying a test compound which modulates the ubiquitination of a p53 polypeptide, the method comprising (a) providing a CUL4 E3 ligase complex; (b) incubating the CUL4 E3 ligase complex with a p53 polypeptide for an amount of time sufficient for the ubiquitination of p53; (c) determining the ubiquitination of the p53 polypeptide; and (d) comparing the ubiquitination of the p53 polypeptide measured in (b) to ubiquitination of a p53 polypeptide in a mixture not contacted with the test compound to determine a difference in the ubiquitination of the p53 substrate, wherein the difference is indicative of the ability of the test compound to modulate the ubiquitination of a p53 polypeptide. In another embodiment, the CUL4 E3 ligase complex is a CUL4A E3 ligase complex or a CUL4B E3 ligase complex. Polypeptides in the CUL4 E3 ligase complex include, but are not limited to, CUL4A, CUL4B, DDB 1 , L2DTL, ROC 1 , ROC2, MDM2 and Pirh2. In a preferred embodiment, the CUL4 E3 ligase complex comprises CUL4A or CUL4B. In some embodiments, the test compound increases p53 ubiquitination, while in other embodiments it decreases p53 ubiquitination. In some embodiments, the ligase complex is incubated with a p53 polypeptide in the presence of additional components such as an ubiquitin-activating El enzyme, an ubiquitin-conjugating E2 enzyme, adenosine tri-phosphate (ATP) and ubiquitin. Ubiquitin-conjugating E2 enzymes include those selected from Cdc34, UbcHl, UbcH2, UbcH3, UbcH4, UbcH5, UbcH6, UbcH7, UbcHlO and L-UBC. In one embodiment, the UbcH5 is UbcH5A, UbcH5B or UbcH5C. In certain embodiments, the ubiquitin is a derivatized ubiquitin, such as but not limited to, ¹²⁵I-ubiquitin, a fluorescent ubiquitin, glutathione-S-transferase ubiquitin, and a biotmylated ubiquitin. In certain embodiments, providing the CUL4 E3 ligase complex comprises immunoprecipitating the complex using an antibody that binds to a CUL4A or CUL4B protein, such as an antibody that binds to a tag on the CUL4A or the CUL4B protein. In some embodiments, the antibody binds to a member of the CUL4 E3 ligase complex, such as but not limited to, CUL4A, CUL4B, DDB1, L2DTL, ROC1, ROC2, MDM2 and Pirh2. In other embodiments, the CUL4 E3 ligase complex is reconstituted from purified, or substantially purified, protein components, which may be recombinant polypeptides or naturally-occurring polypeptides. Another aspect of the invention provides A method of decreasing the level of a p53 polypeptide in a cell, the method comprising contacting the cell with an agent that: (a) increases the expression level of an mRNA encoding a CUL4-associated polypeptide in the cell; (b) increases the level of a CUL4-associated polypeptide in the cell; or (c) increases binding between a p53 polypeptide and a CUL4 E3 ligase complex; (d) increases the ubiquitination activity of a CUL4 ligase complex; (e) increases the binding between a CUL4 ligase complex and MDM2; (f) increases the binding between a CUL4 ligase complex and Pirh2; or (g) increases the activity of a CUL1 E3 ligase complex. In one embodiment, the CUL4-associated polypeptide is a CUL4A-associated polypeptide or a CUL4B-associated polypeptide. CUL4- associated polypeptides include, but are not limited to, CUL4A, CUL4B, DDB1, L2DTL, ROC 1 , ROC2, MDM2 and Pirh2. In another embodiment, the CUL4 E3 ligase complex is a CUL4A E3 ligase complex or a CUL4B E3 ligase complex. Polypeptides in the CUL4 E3 ligase complex include, but are not limited to, CUL4A, CUL4B, DDB1, L2DTL, ROC1, ROC2, MDM2 and Pirh2. In another embodiment, the method for reducing the expression level of a p53 polypeptide in a cell further comprises increasing the expression level or activity of at least one E2 conjugation enzyme. In certain embodiments, the E2 conjugation enzyme is selected from the group consisting of CDC34 E2, UbcH5B E2 and UbcH5C E2. In some embodiments, the agent increases the polyubiquitination of p53. In one preferred embodiment, the p53 is human p53. Another aspect of the invention provides A method of increasing the level of a p53 polypeptide in a cell, the method comprising contacting the cell with an agent that: (a) decreases the expression level of an mRNA encoding a CUL4-associated polypeptide in the cell; (b) decreases the level of a CUL4-associated polypeptide in the cell; (c) decreases binding between a p53 polypeptide and a CUL4 E3 ligase complex; (d) decreases the ubiquitination activity of a CUL4 ligase complex; (e) decreases the binding between a CUL4 ligase complex and MDM2; (f) decreases the binding between a CUL4 ligase complex and Pirh2; or (g) decreases the activity of a CUL1 E3 ligase complex. In one embodiment, the CUL4-associated polypeptide is a CUL4A-associated polypeptide or a CUL4B-associated polypeptide. CUL4- associated polypeptides include, but are not limited to, CUL4A, CUL4B, DDB1, L2DTL, ROC1, ROC2, MDM2 and Pirh2. In another embodiment, the CUL4 E3 ligase complex is a CUL4A E3 ligase complex or a CUL4B E3 ligase complex. Polypeptides in the CUL4 E3 ligase complex include, but are not limited to, CUL4A, CUL4B, DDB1, L2DTL, ROC1, ROC2, MDM2 and Pirlι2. In another embodiment, the method for reducing the expression level of a p53 polypeptide in a cell further comprises increasing the expression level or activity of at least one E2 conjugation enzyme. In certain embodiments, the E2 conjugation enzyme is selected from the group consisting of CDC34 E2, UbcH5B E2 and UbcH5C E2. In some embodiments, the agent increases the polyubiquitination of p53. In one preferred embodiment, the p53 is human p53. In certain embodiments of the methods for increasing p53 levels in a cell, the cell is in a mammal, such as a human. In some embodiments, the mammal has a tumor or cancer. In other embodiments, the mammal has a mutation in the p53 gene which reduces its expression level or its activity, such that increasing p53 levels restores, at least in part, normal expression levels and or p53 bioactivity. In some embodiments, the agent is administered to the subject in conjuction with other therapies. In the case of a mammal having a tumor or cancer, such therapies may include chemotherapy, hormone therapy or surgical interventions.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 A-1E show that both p53 and MDM2 interact with the CUL4A E3 ligase complex and that the interaction between p53 and CUL4 is regulated by gamma-irradiation. Figure 1A: Human CUL4A interacts withp53. Human HEK 293 cell lysates were immunoprecipitated with pre-immune (pre-immune) serum, monoclonal anti-p53 (DO1) and MDM2 (SMP14) antibodies, and anti-CULl, CUL4A, and CUL4B antibodies followed by western blotting analysis as indicated. Figure IB: The p53, MDM2, CUL4A, CUL4B, CULl , and CUL2 complexes were immunoprecipitated from human U2OS pre-treated with either dimethyl sulfoxide (DMSO) or 50 μg/ml MG132 for 2 hours as indicated. Their interactions with p53 and MDM2 were analyzed as in A by western blotting. Figure IC: The interaction between p53 and CUL4 complexes is sensitive to gamma-irradiation. A stable 293 cell line expressing p53 Flag-tagged at the amino terminus was treated with or without gamma-irradiation (10 Gy). Cells were harvested at the indicated time points and the CUL4 and MDM2 complexes were immunoprecipitated by anti-CUL4CT or anti-MDM2 antibodies, respectively. The presence of Flag-tagged p53 in the CUL4 or MDM2 immunocomplexes was detected with anti-Flag immunoblot. The total protein levels of Flag-p53 and CULl were included as controls. Figure ID: The U2OS cells were irradiated as in (C). The CUL4A complex was immunoprecipitated and the presence of p53 was detected by western blot. Figure IE: Same as (D) except some cells were treated with 10 μM Wortmannin or DMSO for 2 hours before irradiation as indicated. The dissociation of p53 from CUL4A in response to DNA damage is abolished by Wortmannin. In D and E, the cells were also treated with MG132 for 1.5 hours to stabilize p53 on CUL4A complexes.

Figures 2A-2E show that CUL4 complexes interact with Pirh2. Figure 2 A: The MDM2 and p53 double null mouse embryonic fibroblasts (MEFs) were infected with retrovirus encoding human p53. Sixty hours post-infection, the cells were harvested and analyzed for the interaction between p53 and CUL4A or CUL4B as in Figure 1 A. Figure 2B: CUL4 complexes specifically interact with Pirh2. Pirh2, CULl, CUL4A, and CUL4B complexes were immunoprecipitated from 293 cells as indicated. The interaction between Pirh2 and cullins were analyzed by western blotting with anti- Pirh2 and cullin antibodies as in figure 1A. Figures 3A-3D show that MDM2 and Pirh2 binding to CUL4A requires the CUL4A amino terminal "adaptor domain"; and MDM2, Pirh2 and p53 interact with DDB1. Figure 3 A: Alignment of adaptor domains at the amino termini of various cullins (CUL1-CUL5) with the following sequence identifiers: CULl, SEQ ID NO:l 1; CUL2, SEQ ID NO: 12; CUL5, SEQ ID NO: 13; CUL3, SEQ ID NO: 14; CUL4A, SEQ ID NO:15; CUL4B, SEQ ID NO:16. The bold letters represent the conserved residues among cullins. The CUL4A.N1 mutant (a.a. 83-761; the full length CUL4A is 761 a.a.) partially removes the "adaptor domain" while the CUL4A.N2 mutant (a.a. 145-761) deletes the entire "adaptor domain" of CUL4A. Figure 3B: The wild-type and NI and N2 mutants of CUL4A were Flag-tagged and transfected into 293 cells together with either Pirh2 or MDM2 as indicated. The binding was assayed by immunoprecipitating with anti-Flag antibodies (top two panels) followed by anti-Flag, Pirh2, and MDM2 west blotting, respectively. Total protein levels of Pirh2 and MDM2 were analyzed by western blotting of cell lysates. Figure 3C: MDM2, Pirh2 and p53 bind to DDB1. U2OS cells were treated with MG132 as described in figure 1. The lysates were immunoprecipitated with anti-p53 (DO 1 ), anti-MDM2 (OP 115), pre-immune serum (pre-im), and anti-CUL4A antibodies. The immunoprecipitates were blotted with anti-DDBl antibodies as indicated. Figure 3D: Human L2DTL binds to CUL4, DDB1, and p53. Top panel: U2OS cell lysates were immunoprecipitated by anti-human L2DTL, pre-immune (pre- im), anti-CUL4A, CUL4B, and CUL4CT antibodies. The immunoprecipitates were blotted with anti-human L2DTL or DDB1 antibodies. Lower panel: A Flag-tagged human L2DTL was transfected into 293 cells and immunoprecipitated by anti-Flag, p53, pre-immune, CUL4A, CUL4B, and CUL4CT antibodies. The immunoprecipitates were blotted with anti-Flag antibody. Figures 4A-4F shows that silencing of CUL4A expression stabilizes p53, inducing the accumulation of CDK inhibitor p21, GI cell cycle arrest, and apoptosis. Figure 4A: U2OS cells were treated with 100 nM siRNA for CUL4A and a control siRNA (luciferase) for 60 hours. Protein synthesis inhibitor cycloheximide (100 μg/ml) was then added and the protein levels of p53, CUL4A, and CULl (as a control) were determined at indicated time by western blotting. Figure 4B: Silencing of CUL4A induces p21 accumulation and GI cell cycle arrest. U87MG cells were treated with 50 nM siRNAs for luciferase (control), CUL4A, and CUL4A and CUL4B in the presence or absence of 50 nM p53 siRNA as indicated. 72 hours-post transfection, one set of cells were examined for the levels of p21, p53, CUL4A and CUL4B. Figure 4C: A parallel set of panel B was analyzed for cell cycle effect by flow-cytometry. Loss of CUL4A or CUL4A and CUL4B induces p21 accumulation and GI arrest (48% GI in control versus 57% GI cells in CUL4 deficient cells) and a corresponding decrease of S phase cells (47% to 38%). Silencing of p53 significantly suppresses the effect of CUL4 deficiency for p21 and GI cell arrest (from 57% of CUL4A to 48% of CUL4A plus p53 siRNA). Duplicate experiments were conducted in parallel and essentially the same conclusion was obtained with three experimental repeats. Figure 4D, 4E: CUL4A deficiency enhances the UV-induced apoptosis in p53 positive cells (U2OS) but not in cognate p53 negative cells expressing a dominant p53. U2OS cells were infected with an empty retrovirus (RVY) or a retrovirus containing a dominant negative p53 (CTF), as described in Methods, and stable cells were selected by hygromycin. The U2OS-RVY cells responded to UV irradiation (20 J/M²) by stabilizing p53 and inducing p21 while no changes in p53 and p21 were observed in U2OS-CTF cell (Top panels). The cells were treated with 100 nM siRNAs for luciferase, CUL4A, and CUL4A and CUL4B for 48 hours. The cells were irradiated with 20 J/M² and processed for apoptosis assay by the TUNEL assay after 24 hours. The percentage of apoptotic cells was plotted. Figure 4F: Elimination of DDB1, L2DTL, or ROC1 by siRNA also causes p53 accumulation and induces p21. U2OS cells were treated with 100 nM siRNAs for luciferase and DDB1, L2DTL, or ROC1 for 60 hours. The levels of p53 and p21 were analyzed by western blotting. The specific effects of siRNA towards their cognate proteins were also determined as indicated. Figures 5A-5H show that the CUL4A E3 ligase complexes exhibit polyubiquitination activity towards p53 which is dependent on the presence of MDM2 or Pirh2. Figure 5A: Flag-tagged p53 was transfected into 293 cells as described in Figure IC. The MDM2- and CUL4-associated Flag-p53 was immunoprecipitated by anti-MDM2 (SMP-14), CUL4A, and CUL4B antibodies. The immunoprecipitates were further incubated with 500 ng of CDC34, 50 ng of El, 10 μM ubiquitin, and 2 mM ATP for 30 minutes. The polyubiquitinated p53 was visualized by anti-Flag immunoblot. The blot was stripped and re-blotted with anti-p53, MDM2, CUL4A and CUL4B antibodies as indicated. Figure 5B: Characterization of CUL4A-mediated p53 polyubiquitination. Immunocomplexes by pre-immune serum or anti-CUL4A antibodies were immunoprecipitated from U2OS cells and ubiquitination reactions were conducted by adding Flag-p53, El, E2, ubiquitin, and ATP as indicated. Polyubiquitinated p53 was detected by the anti-Flag antibody. The absence of El or ubiquitin (last two lanes) in the ubiquitination reaction greatly abolished the polyubiquitination of p53 by CUL4A complex. Lower panel: The CUL4A immunocomplexes contain DDB1 and ROC1. The CUL4A immunoprecipitates from U2OS cells were blotted with anti-DDBl, CUL4A, and ROC1 antibodies. Figure 5C: CUL4A E3 ligase complex specifically polyubiquitinates p53. CUL2 and CUL4A complexes were immunoprecipitated from U2OS cells. The Flag-p53 was incubated with CUL2 and CUL4A complexes for polyubiquitination as assayed in B. Figure 5D: p53 is specifically polyubiquitinated by immunoprecipitated CUL4A ligase but not MDM2 in vitro. MDM2 and CUL4A complexes were immunoprecipitated by anti-MDM2 (SMP-14), pre-immune, anti-amino and anti-carboxy termini of CUL4A antibodies (CUL4A and CUL4CT). The immunoprecipitated complexes were incubated with isolated Flag-p53 and polyubiquitination reactions were conducted as in (A). The polyubiquitinated p53 was visualized by anti-Flag immunoblot. The blot was stripped and re-blotted with anti-MDM2 and CUL4A antibodies. Figure 5E: CUL4A complexes isolated from MDM2 and p53 double null mouse fibroblasts (MDM2 and p53 -/- MEFs) display significant reduction in polyubiquitination activity towards p53 as compared to CUL4A complex isolated from the wild-type cells (MDM2 and p53 +/+). The CUL4A complexes were isolated by immunoprecipitation from wild-type MEFs (MDM2+/+) and MDM2/p53 null MEFs (MDM2V- and ρ53-/-) and assayed for their polyubiquitination activity against Flag-tagged p53 as in (B). Figure 5F: Supplementation of recombinant MDM2 restores the p53 polyubiquitination activity of CUL4A complex isolated from MDM2-/- MEF cells. The MDM2 protein was fused with the glutathione-S-transferase (GST) at amino tenninus and expressed as a fusion protein in the baculovirus-expression system. The GST-MDM2 was purified by GST-affinity chromatography. The CUL4 complexes from MDM2 and p53 double null cells were supplemented with or without GST- MDM2 and assayed for Flag-tagged p53 polyubiquitination activity. Left panel: 10 ng of GST-MDM2 was added as indicated. Right panel: 0, 1 , and 10 ng of GST-MDM2 was added to CUL4A complexes immunoprecipitated from MDM2 deficient MEFs or a control immunoprecipitation using the pre-immune serum as indicated. The polyubiquitinated Flag p53 was detected by anti-Flag antibody. Figure 5G: Analysis of p53 polyubiquitination by Pirh2. Left panel: CUL4A and Pirh2 complexes were immunoprecipitated from U20S cells and p53 polyubiquitination was assayed as in Figure 5B. Right panel: Supplementation of recombinant GST-Pirh2 protein to CUL4A complexes from MDM2 and p53 double null MEFs restores the polyubiquitination activity of CUL4 towards p53. GST-Pirh2 was purified from bacteria and supplemented at two different amounts (50 and 500 ng) to immunoprecipitated CUL4A complex or pre-immune serum (pre) as indicated.

Polyubiquitination reaction was performed as in A except a mixture of both CDC34 and UBCH5C were used as E2. Figure 5H: Requirement of human L2DTL, DDB1, ROC1 in p53 polyubiquitination by CUL4 E3 ligase complexes. U2OS cells were treated with control siRNA (Luc) or siRNAs against L2DTL, DDB1, or ROC1 as described in Figure 4. After 60 hours, the CUL4A E3 ligase complexes were isolated by anti-CUL4A antibodies and their polyubiquitination activities against Flag-p53 were analyzed and compared. Figure 6 shows a schematic diagram of a method of screening for compounds which modulate ubiquitination of p53 by a CUL4 E3 ligase complex, including the detection of ubiquitinated p53. DETAILED DESCRIPTION OF THE INVENTION I. Overview The invention relates to regulators of p53 ubiquitination, methods for screening modulators of the CUL4-dependent ubiquitination of p53, and methods for modulating p53 activities in a cell. In one aspect, the invention provides methods for identifying an agent, or a compound, that modulates a p53 bioactivity. In addition, the present invention provides methods and compositions for assaying the ubiquitination of p53 by CUL4 E3 ligase complexes. By "E3" it is meant a ubiquitin ligase, as described below, comprising one or more components associated with ligation of ubiquitin to a ubiquitination substrate protein for ubiquitin-dependent proteolysis. In one embodiment, E3 is CUL4A or CUL4B. The invention also provides methods of increasing or decreasing the levels of p53 polypeptide in a cell. The present invention is directed, in part, to methods for assaying CUL4 ubiquitin ligase activity towards a p53 substrate, and in particular to in vitro methods suitable for high-throughput screening. Some of the methods provided allow the measurement of p53 ubiquitination directly where the reaction has occurred, thus obviating the need for separating ubiquitinated forms of p53 from nonubiquitinated forms by SDS PAGE, gel filtration chromatography or other similar procedures. This facilitates, for example, multi-well array analysis and high-throughput screening techniques for modulators of ubiquitination activity. In certain embodiments, the screening methods comprise combining ubiquitin and ubiquitin ligation enzymes, including a CUL4 E3 ligase complex, and measuring the amount of ubiquitin ligated to a p53 substrate protein. In certain embodiments, the ubiquitination of the p53 substrate protein itself is not measured; what is measured is poly-ubiquitin chains produced in the ligase reaction in the presence of a p53 substrate. Therefore, as used herein, "p53 substrate protein" means a p53 protein, a fragment thereof, or a p53 fusion protein, to which ubiquitin is bound through the activity of ubiquitination enzymes; "ubiquitination" and grammatical equivalents thereof means the binding of ubiquitin to a substrate protein. In certain embodiments, the CUL4 E3 ligase complex is attached to the surface of a reaction vessel, such as the well of a multi-well plate. This embodiment facilitates the separation of ligated ubiquitin from unligated ubiquitin. This embodiment allows the ubiquitin ligase reaction, detection and measurement of ligated ubiquitin to occur in the same vessel, making the assay useful for high- throughput screening applications. In another preferred embodiment, the CUL4 E3 ligase complex is free in solution. In this embodiment, ubiquitination activity is monitored using a system that produces a signal which varies with the extent of ubiquitination, such as the fluorescence resonance energy transfer (FRET) system. In a preferred embodiment, the ubiquitin is labeled, either directly or indirectly, and the amount of label is measured. This allows for easy and rapid detection and measurement of ligated ubiquitin, making the assay useful for high- throughput screening applications. In one preferred embodiment, the signal of the label varies with the extent of ubiquitination, such as a FRET system.

II. Screening Methods One aspect of the invention provides methods of identifying test agents which modulate the CUL4 E3 ligase-dependent ubiquitination of p53. One method of identifying an test compound which modulates the ubiquitination of a p53 polypeptide comprises (a) providing a CUL4 E3 ligase complex; (b) incubating the CUL4 E3 ligase complex with a p53 polypeptide for an amount of time sufficient for the ubiquitination of p53; (c) determining the ubiquitination of the p53 polypeptide; and (d) comparing the ubiquitination of the p53 polypeptide as measured in (b) to ubiquitination of a p53 polypeptide in a mixture not contacted with the test compound to determine a difference in the ubiquitination of the p53 substrate, wherein the difference is indicative of the ability of the test compound to modulate the ubiquitination of a p53 polypeptide. The present invention provides methods and compositions comprising combining or incubating, ubiquitin, p53, CUL4 E3 ligases and other components. The terms "combining" or "incubating" refer to the addition of the various components into a receptacle under conditions in which ubiquitin ligase activity or ubiquitination may take place. In a preferred embodiment, the receptacle is a well of a 96-well plate or other commercially available multiwell plate. In another embodiment, the receptacle is the reaction vessel of a FACS machine. Other receptacles useful in the present invention include, but are not limited to 384-well plates and 1536-well plates. Still other receptacles useful in the present invention will be apparent to the skilled artisan. A compound can modulate the p53-ubiquination activity of a CUL4 E3 complex by either stimulating or inhibiting the ubiquitination of p53. A compound inhibits ubiquitination if the level of thep53 substrate that is ubiquitinated is decreased as compared with the level of p53 substrate ubiquitinated in the absence of the test compound. In one embodiment, the compound inhibits ubiquitination by 25%, 50%, 60%, 70%, 80%), 90%, or 95% or more as compared to a control sample not contacted with the compound. A compound stimulates ubiquitination if the fraction of the p53 substrate that is ubiquitinated or the amount of ubiquitin incorporated into the p53 substrate is increased as compared to reactions performed in the absence of the test compound. In one embodiment, the compound stimulates ubiquitination by 25%, 50%, 60%, 70%), 80%, 90%>, or 95% or more as compared to a control sample not contacted with the compound. In one embodiment, the CUL4 E3 ligase complex comprises CUL4A (SEQ ID NO:3) or CUL4B (SEQ ID NO:4). A human CUL4A isoform of 759 amino acids is described as Genbank Accession No. NP_001008895, while a 659 amino acid human isoform is described as Genbank Accession No. NP_003580, both of which are encompassed by the term "CUL4A." The amino acid sequence of human CUL4B is available as Genbank Deposit No. NP_003579. The human p53 amino acid sequence is available as Genbank Deposit No. NP_000537 (SEQ ID NO:2). In some embodiments, the screening methods use polypeptides having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% amino acid sequence identity to the wild-type p53 sequence as set forth in SEQ ID NO:2, or naturally occurring variants of p53. Naturally occurring variants of p53, such as mutated p53 genes found in many human cancers, are well known in the art. In one embodiment, the CUL4 E3 ligase complex comprises one or more of the polypeptides selected from the group consisting of DDB1 (SEQ ID NO:7), L2DTL (SEQ ID NO:8), ROC1 (SEQ ID NO:5), ROC2 (SEQ ID NO:6), MDM2 (SEQ ID NO:9) and Pirh2 (SEQ ID NO: 10). The amino acid sequence of human damage-specific DNA binding protein 1 (DDB1) is available as Genbank Deposit No. NP_ 001914. The amino acid sequence of L2DTL, a human WD-40 repeat gene homolog of the Drosophila lethal (2) denticleless heat shock gene, is available as

Genbank Deposit No. AAF35182. The amino acid sequence of ROC1, also known as RING-box protein 1 (Rbxl), Regulator of cullins 1, RING finger protein 75, or ZYP protein, is available as Genbank Deposit No. NP_055063. The amino acid sequence of ROC2, also known as RING-box protein 2 (Rbx2), RING finger protein 7, Regulator of cullins 2, CKII beta-binding protein 1 or CKBBPl, is available as Genbank Deposit No. Q9UBF6. The amino acid sequence of MDM2, known as transformed 3T3 cell double minute 2, is available as Genbank Deposit No. NP_071328. p53 -induced protein, ring-h2 domain-containing (Pirh2) is also known as androgen receptor N-terminal domain-interacting protein (ARNIP), and Zinc-Finger Protein 363 (ZFP363) (see Beitel et al. (2002) J. Molec. Endocr. 29: 41-60; and Leng et al. (2003) Cell 112: 779-791, 2003). The amino acid sequences of three isoforms of PirH2, are described as Genbank Deposit Nos. NP_056251, NP_001008925 and NP_001009922. In some embodiments, the CUL4 E3 ligase complex is incubated with a p53 polypeptide in the presence of one or more of ubiquitin, ubiquitin-activating El enzyme, an ubiquitin-conjugating E2 enzyme, adenosine tri-phosphate (ATP) and ubiquitin. In on preferred embodiments of the methods provided herein, incubating the CUL4 E3 ligase complex with a p53 polypeptide is performed in the presence of ubiquitin. As used herein, "ubiquitin" refers to a polypeptide which is ligated to another polypeptide by ubiquitin ligase enzymes. The ubiquitin can be from any species of organism, preferably a eukaryotic species. Preferably, the ubiquitin is mammalian. More preferably, the ubiquitin is human ubiquitin. In certain embodiments, the methods of the invention utilize the 76 amino acid human ubiquitin (SEQ ID NO:l). Other embodiments utilize variants of ubiquitin. Also encompassed by "ubiquitin" are naturally occurring alleles and man-made variants of the 76 amino acid polypeptide. In certain embodiments, variants of ubiquitin have an overall amino acid sequence identity of preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90% of the amino acid sequence of human ubiquitin. In some embodiments the sequence identity will be as high as about 93 to 95 or 98%. As is known in the art, a number of different programs can be used to identify whether a protein (or nucleic acid as discussed below) has sequence identity or similarity to a known sequence such as to a p53 (SEQ ID NO. "2) or ubiquitin sequence (SEQ ID NO:l). Sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics

Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably using the default settings, or by inspection. Preferably, percent identity may be calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc. An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266: 460- 480 (1996); http:^last.wustl/edu/blast/README.html]. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=l, overlap fraction=0.125, word threshold (T)=l 1. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. A percent amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored). Ubiquitin proteins of the present invention may be shorter or longer than the

76 amino acid ubiquitin protein of SEQ ID NO: 1. Thus, in a preferred embodiment, included within the definition of ubiquitin are portions or fragments of the amino acid sequence depicted in SEQ ID NO: 1. In one embodiment herein, fragments of ubiquitin are considered ubiquitin proteins if they are ligated to another polypeptide by ubiquitin ligase enzymes. In addition, as is more fully outlined below, ubiquitin can be made longer than the amino acid sequence depicted in SEQ ID NO: 1 ; for example, by the addition of tags, the addition of other fusion sequences, or the elucidation of additional coding and non-coding sequences. In certain embodiments, an ubiquitin peptide is fused to a fluorescent peptide, such as Green Fluorescent Peptide (GFP). The ubiquitin protein, as well as other proteins of the present invention, may be recombinant. A "recombinant protein" is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid in a cell. In some embodiments, ubiquitin, or any other protein components of the reaction mixture, contains a label to facilitate its detection. By "label" is meant a molecule that can be directly (i.e., a primary label) or indirectly (i.e., a secondary label) detected; for example a label can be visualized and/or measured or otherwise identified so that its presence or absence can be known. As will be appreciated by those in the art, the manner in which this is done will depend on the label. Preferred labels include, but are not limited to, fluorescent labels, label enzymes and radioisotopes. By "fluorescent label" is meant any molecule that may be detected via its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, ftuorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland, hereby expressly incoφorated by reference. Suitable fluorescent labels also include, but are not limited to, green fluorescent protein (GFP; Chalfie, et al, Science 263(5148):802-805 (Feb. 11, 1994); and EGFP; Clontech-Genbank Accession Number U55762), blue fluorescent protein (BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1 J9; 2. Stauber, R. H. Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein (EYFP; 1. Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303), luciferase (Ichiki, et al., J. Immunol. 150(12): 5408-5417 (1993)), /?-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8) :2603-2607 (April 1988)) and Renilla WO 92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S. Pat. Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558) All of the above-cited references are expressly incorporated herein by reference. In some instances, multiple fluorescent labels are employed. In a preferred embodiment, at least two fluorescent labels are used which are members of a fluorescence resonance energy transfer (FRET) pair. FRET is phenomenon known in the art wherein excitation of one fluorescent dye is transferred to another without emission of a photon. A FRET pair consists of a donor fluorophore and an acceptor fluorophore. The fluorescence emission spectrum of the donor and the fluorescence absorption spectrum of the acceptor must overlap, and the two molecules must be in close proximity. The distance between donor and acceptor at which 50% of donors are deactivated (transfer energy to the acceptor) is defined by the Forster radius (R_o), which is typically 10-100 angstroms. Changes in the fluorescence emission spectrum comprising FRET pairs can be detected, indicating changes in the number of that are in close proximity (i.e., within 100 angstrom of each other). This will typically result from the binding or dissociation of two molecules, one of which is labeled with a FRET donor and the other of which is labeled with a FRET acceptor, wherein such binding brings the FRET pair in close proximity. Binding of such molecules will result in an increased fluorescence emission of the acceptor and/or quenching of the fluorescence emission of the donor. FRET pairs (donor/acceptor) useful in the invention include, but are not limited to, EDANS/fluorescien, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy 5, fluorescein/Cy 5.5 and fluorescein/LC Red 705. In another aspect of FRET, a fluorescent donor molecule and a nonfluorescent acceptor molecule ("quencher") may be employed. In this application, fluorescent emission of the donor will increase when quencher is displaced from close proximity to the donor and fluorescent emission will decrease when the quencher is brought into close proximity to the donor. Useful quenchers include, but are not limited to, DABCYL, QSY 7 and QSY 33. Useful fluorescent donor/quencher pairs include, but are not limited to EDANS/DABCYL, Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL and fluorescein/QSY 7 dye. The skilled artisan will appreciate that FRET and fluorescence quenching allow for monitoring of binding of labeled molecules over time, providing continuous information regarding the time course of binding reactions. By "label enzyme" it is meant an enzyme which may be reacted in the presence of a label enzyme substrate which produces a detectable product. Suitable label enzymes for use in the present invention include but are not limited to, horseradish peroxidase, alkaline phosphatase and glucose oxidase. Methods for the use of such substrates are well known in the art. The presence of the label enzyme is generally revealed through the enzyme's catalysis of a reaction with a label enzyme substrate, producing an identifiable product. Such products may be opaque, such as the reaction of horseradish peroxidase with tetramethyl benzedine, and may have a variety of colors. Other label enzyme substrates, such as Luminol (available from Pierce Chemical Co.), have been developed that produce fluorescent reaction products. Methods for identifying label enzymes with label enzyme substrates are well known in the art and many commercial kits are available. Examples and methods for the use of various label enzymes are described in Savage et al., Previews 247:6-9 (1998), Young, J. Virol. Methods 24:227-236 (1989), which are each hereby incorporated by reference in their entirety. By "radioisotope" is meant any radioactive molecule. Suitable radioisotopes for use in the invention include, but are not limited to ¹⁴C, ³H, ³²P, ³³P, ³⁵S, ¹²⁵I, and ¹³¹I. The use of radioisotopes as labels is well known in the art. Some embodiments of the invention include combining or incubating El in the reaction mixture. "El" refers to an ubiquitin activating enzyme. In a certain embodiment, El is capable of transferring ubiquitin to an E2 (as defined below) in the reaction mixture. In a certain embodiment, El binds ubiquitin. In one embodiment, El forms a high energy thiolester bond with ubiquitin, thereby "activating" the ubiquitin. El enzyme is well known to one of skill in the art (e.g., Hershko et al., Ann. Rev. Biochem. 61:761-807, 1992, and Monia et al., Biotechnol. 8: 209-215, 1990, herein incorporated by reference). El enzyme initiates the ubiquitination process by activating ubiquitin. Any of the El enzymes known in the art are suitable for use in the invention method. Exemplary El enzymes include, but are not limited to, those ©having the amino acid sequences disclosed in ATCC accession numbers A38564, S23770, AAA61246, P22314, CAA40296 and BAA33144, incorporated herein by reference. Preferably El is human El. El is commercially available from Affiniti Research Products (Exeter, U.K.). Some embodiments of the invention include combining or incubating E2 in the reaction mixture. "E2" refers to an ubiquitin carrier enzyme (also known as a ubiquitin conjugating enzyme). In certain embodiments, ubiquitin is transferred from El to E2. In a preferred embodiment, the transfer results in a thiolester bond formed between E2 and ubiquitin. In a preferred embodiment, E2 is capable of transferring ubiquitin to an E3, defined below. In certain embodiments, the ubiquitination substrate protein is ubiquitin. Exemplary E2 proteins that may be used in the methods described herein include, but are not limited to, those having the amino acid sequences disclosed in ATCC accession numbers AAC37534, P49427, CAA82525, AAA58466, AAC41750, P51669, AAA91460, AAA91461, CAA63538, AAC50633, P27924, AAB36017, Q16763, AAB86433, AAC26141, CAA04156, BAA11675, Q16781, NP_003333, BAB18652, AAH00468, CAC16955, CAB76865, CAB76864, NP_05536, 000762, XP_009804, XP_009488, XP_006823, XP_006343, XP_005934, XP_002869, XP_003400, XP_009365, XP_010361, XP 004699, XP_004019, 014933, P27924, P50550, P52485, P51668, P51669, P49459, P37286, P23567, P56554, and CAB45853, each of which is incorporated herein by reference. In certain embodiments the sequences disclosed in ATCC accession numbers NP003331, NP003330, NP003329, P49427, AAB53362, NP008950, XP009488 or AAC41750 are used, also incorporated by reference. The skilled artisan will appreciate that many different E2 proteins and isozymes are known and may be used in the present invention, provided that the E2 has ubiquitin conjugating activity. Also specifically included within the term "E2" are variants of E2. In certain embodiments, E2 is one of Ubc5 (Ubch5), Ubc3 (Ubch3), Ubc4 (Ubch4) and UbcX (UbclO, UbchlO). In one embodiment, E2 is Ubc5c. Suitable ubiquitin conjugating enzymes that can be employed in the invention method include Cdc34, UbcHl, UbcH2, UbcH3, UbcH4, UbcH5, UbcH6, UbcH7, UbcHIO, L-UBC, and the like (see Kaiser, et al, FEBS Letts 350:1-4, 1994; Kaiser, et al, FEBS Letts 377:193-196, 1995; Nuber, et al, J Biol Chem 271:2795-2800, 1996; Jensen, et al, J Biol Chem 270:30408-30414, 1995; Robinson, et al, Mamm Genome 6:725-731 , 1995; and Plon et al., Proc. Natl. Acad. Sci. USA 90:10484-10488, all of which are herein incoφorated by reference). "Cdc34" refers to a ubiquitin-conjugating enzyme isolated from yeast. In one specific embodiment, the ubiquitin-conjugating E2 enzyme is selected from the group consisting of Cdc34, UbcHl, UbcH2, UbcH3, UbcH4, UbcH5, UbcH6, UbcH7, UbcHIO and L-UBC. In one embodiment, the ubiquitin-conjugating E2 enzyme UbcH5 is UbcH5A, UbcH5B or UbcH5C. In one embodiment, the methods of the present invention use variant proteins, such as, for example ubiquitin, CUL4A, CUL4B, p53, DDB1, L2DTL, ROC1, ROC2, MDM2, Pirh2, El, E2 and/or E3 variants. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding a protein of the present compositions, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below. The polypeptides used in the methods described herein, such as CUL4A,

CUL4B, p53, DDB1, L2DTL, ROC1, ROC2, MDM2, Pirh2, El, E2 and/or E3 variants, may be naturally-occurring polypeptides or recombinant polypeptides. Recombinant polypeptides of the present invention may be produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding the protein, under the appropriate conditions to induce or cause expression of the protein. The conditions appropriate for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield. Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melanogaster cells, Pichia pastoris and P. methanolica, Saccharomyces cerevisiae and other yeasts, Bacillus subtilis, E. coli, SF9 cells, SF21 cells, C129 cells, Saos-2 cells, Hi-5 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells. The polypeptides may also be made as a fusion proteins, using techniques well known in the art. Thus, for example, the p53 protein may be made as a fusion protein to increase expression, to facilitate its detection, or for other reasons. For example, when the protein is a peptide, the nucleic acid encoding the peptide may be linked to other nucleic acid for expression puφoses. Similarly, proteins of the invention can be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc. In certain embodiments, the protein is purified or isolated after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the ubiquitin protein may be purified using a standard anti-ubiquitin antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer- Verlag, NY (1982). The degree of purification necessary will vary depending on the use of the protein. In some instances no purification will be necessary. In some embodiments, the CUL4 E3 ligase complex is immunoprecipitated, such as from cell lysate of mammalian or insect cells, using an antibody that binds to a CUL4A or CUL4B protein, or to a tag on a CUL4A or a CUL4B protein. In some embodiments, CUL4 complexes are affinity purified by immunoprecipitation with antibodies against any component of the CUL4 E3 ligase complexes, including but not limited to CUL4A and CUL4B, DDB1, L2DTL, ROC1, ROC2, MDM2 or Pirh2. The proteins in the CUL4 complexes can also be tagged with epitope tags such as Flag, histidine, HA, Myc, T7, maltose binding protein, glutathione-S-transferase tags, etc. These tagged proteins can be either expressed alone or co-expressed to facilitate the purification of CUL4 complexes after they are expressed in human, mouse, rat cells, or other expression systems by their corresponding affinity matrix. It is also possible to use epitope-tagged recombinant proteins expressed in insect cells such as SF9-baculovirus expression system or bacteria to promote the isolation of the CUL4 complexes for p53 polyubiquitination. In some embodiments, the protein components of the p53 ubiquitination system are protein that are substantially purified and reconstituted into the reaction system. The term "substantially purified" as used herein refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In one embodiment the substantially purified polypeptide comprises at least 80% dry weight, preferably 95-99% dry weight of a polypeptide ofinterest. One skilled in the art can purify polypeptides, such as CUL4A or MDM2, using standard techniques for protein purification. The substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be determined by amino-terminal amino acid sequence analysis. In addition, a variety of other reagents may be included in the assay systems. These include reagents like salts, solvents, buffers, neutral proteins, e.g. albumin, detergents, etc. which may be used to facilitate optimal ubiquitination enzyme activity and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The compositions will also preferably include adenosine tri-phosphate (ATP). The mixture of components may be added in any order that promotes ubiquitin ligase activity or optimizes identification of candidate modulator effects. In certain embodiments, ubiquitin is provided in a reaction buffer solution, followed by addition of the ubiquitination enzymes with or without p53. In certain embodiment, ubiquitin is provided in a reaction buffer solution, a candidate modulator is then added, followed by addition of the ubiquitination enzymes and p53. Once combined, preferred methods of the invention comprise measuring the amount of ubiquitin bound to p53. As will be understood by one of ordinary skill in the art, the mode of measuring will depend on the specific tag, if any, that is attached to the ubiquitin. As will also be apparent to the skilled artisan, the amount of ubiquitin bound will encompass not only the particular ubiquitin protein bound directly to p53, but also the ubiquitin proteins bound to that particular ubiquitin in a polyubiquitin chain. The effect of the test compound on the activity of a CUL4 ubiquitin ligase complex is quantified by measuring the ubiquitination of p53. In one embodiment, the ubiquitin used in the method of the invention is a derivatized ubiquitin. A "derivatized ubiquitin" is a ubiquitin molecule including a label that is readily identified. For example, the derivatized ubiquitin can be an ¹²⁵I-ubiquitin, a fluorescent ubiquitin, a glutathione S-transferase conjugated ubiquitin and a biotinylated ubiquitin. Using assays well known in the art, the presence of the label, and thus the amount of derivatized ubiquitination, can be identified. Ubiquitination results in an increase in the molecular weight of the p53 substrate. Thus any assay which measures molecular weight of p53, such as SDS-poly acrylamide gel electrophoresis, can be used to measure ubiquitination. This assay can be readily adapted to the large scale screening of compound libraries by converting it to a solid phase format. In one specific nonlimiting example, ubiquitination assays can be performed with an appropriately engineered substrate in microtiter plate in the presence of a derivatized ubiquitin. For example, the ubiquitination of a chimeric substrate, such as a chimeric p53 can be measured. A "chimeric p53" is comprised of (i) a p53 polypeptide, or fragment thereof capable of been ubiquitinated, and (ii) one or more heterologous polypeptides. Thus, in one embodiment, the chimeric p53 is a maltose binding protein(MBP) p53 chimera containing a myc epitope-hexahistidine tag at the C-terminus (MBP- p53mycHis6). Following the contacting of the components of the reaction, aliquots of the ubiquitination assays are transferred from a first microtiter plate to a second microtiter plate with an appropriate surface. In one embodiment, reactions are transferred to a microtiter plate whose wells have been coated with a reagent that can capture p53 (e.g., for MBP-p53, wells coated with amylose, anti-MBP antibody, anti- myc antibody, anti-p53 antibody, or NiNTA). After washing away unbound proteins, p53-coated wells can be directly imaged (e.g., for reactions performed with fluorescent or radio-labeled ubiquitin). Alternatively, wells can be contacted with an appropriate reagent to capture derivatized ubiquitin (e.g., biotin-Ub, GST-Ub etc.) covalently linked to substrate MBP-p53mycHis6p. The wells would then be probed with reagents directed against p53 (anti-MBP, anti-p53) to detect the extent of p53-ubiquitin conjugates formed, or alternatively, a labeled p53 substrate (fluorescent, radioactive) would be used and imaged directly. These assays are similar in design but yield distinct information, either of which can be used with the method of the invention. The first assay measures the total amount of ubiquitin incoφorated into substrate, and the second measures the total fraction of substrate that becomes covalently linked to at least one ubiquitin molecule. Both of these assays can be used to differentiate between compounds that block the formation of p53-ubiquitin linkage versus those compounds that interfere with the elaboration of p53-linked polyubiquitin chains. All of the assays can be used to identify compounds that modulate the activities of an ubiquitin ligase. Another assay for ubiquitination is a scintillation proximity assay. This assay uses beads containing a fluorescent substrate that emits light when activated by radioactive substances, and a means of conjugating the bead to ubiquitin. In one embodiment, the bead containing a fluorescent substrate is avidinated, and is contacted with biotinylated ubiquitin. A radiolabeled p53 substrate is incubated with the beads in the presence of the reaction components. Ubiquitinated p53 is quantified by measuring bead fluorescence, which occurs only upon ubiquitination of the labeled p53. (See Bosworth, N. et al., Nature 341 : 167-168, 1989), incoφorated herein by reference. U.S. Patent Nos. 6,413,725 and 6,737,244 describe methods of detecting ubiquitination of a substrate in vitro that may be adapted to the use of p53 and a CUL4 E3 ligase complex in accordance with the methods described herein. In another aspect of the invention, a method of assaying ubiquitination enzyme activity is provided. This method comprises combining tagl -ubiquitin and tag2- ubiquitin, El, E2 and E3 under conditions in which ubiquitination can take place and measuring the amount or rate of ubiquitination. In this embodiment, tagl and tag2 constitute a FRET pair or tagl is a fluorescent label and tag2 is a quencher of tagl. In one embodiment, the method includes combining a candidate ubiquitination modulator with the other components. In a preferred embodiment of this method, measuring is by measuring the fluorescent emission spectrum from the combination, preferably continuously or at specific time points following combining the components. These measurements may be compared to the fluorescent emission spectrum of unbound tagl and tag2 ubiquitin. One prefeπed screening method using the detection strategy illustrated in

Figure 6 and exemplified in Example 13. In one embodiment of this methods, two FRET partners are each immobilized on separate beads. One type of bead binds to ubiquitin and the other to p53, such that a signal is detected when p53 is ubiquitinated. Test compounds that can be screened for modulation of p53 ubiquitination with methods of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, polypeptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Some test compounds are synthetic molecules, and others natural molecules. Test compounds may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-by-step fashion. Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642. Peptide libraries can also be generated by phage display methods (see, e.g., Devlin, WO 91/18980). Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field. Known pharmacological agents can be subject to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs. Combinatorial libraries of peptides or other compounds can be fully randomized, with no sequence preferences or constants at any position. Alternatively, the library can be biased, i.e., some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in some cases, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, or to purines. The test compounds can be naturally occurring proteins or their fragments.

Such test compounds can be obtained from a natural source, e.g., a cell or tissue lysate. Libraries of poljφeptide agents can also be prepared, e.g., from a cDNA library commercially available or generated with routine methods. The test compounds can also be peptides, e.g., peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly prefeπed. The peptides can be digests of naturally occurring proteins, random peptides, or "biased" random peptides. In some methods, the test compounds are polypeptides or proteins. The test compounds can also be nucleic acids. Nucleic acid test compounds can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be similarly used as described above for proteins. In some prefeπed methods, the test compounds are small molecules (e.g., molecules with a molecular weight of not more than about 1,000). Preferably, high throughput assays are adapted and used to screen for such small molecules. In some methods, combinatorial libraries of small molecule test compounds as described above can be readily employed to screen for small molecule modulators of p53. A number of assays are available for such screening, e.g., as described in Schultz (1998) Bioorg Med Chem Lett 8:2409-2414; Weller (1997) Mol Divers. 3:61-70; Fernandes (1998) Cuπ Opin Chem Biol 2:597-603; and Sittampalam (1997) Cuπ Opin Chem Biol 1:384-91. Libraries of test compounds to be screened with the claimed methods can also be generated based on structural studies of the CUL4 E3 ligase complex members, including MDM2 and pirH2, their fragments or analogs. Such structural studies allow the identification of test compounds that are more likely to bind to the CUL4 E3 ligase complex members. The three-dimensional structure of a CUL4 E3 ligase complex member can be studied in a number of ways, e.g., crystal structure and molecular modeling. Methods of studying protein structures using X-ray crystallography are well known in the literature. See Physical Bio-chemistry, Van Holde, K. E. (Prentice-Hall, New Jersey 1971), pp. 221-239, and Physical Chemistry with Applications to the Life Sciences, D. Eisenberg & D. C. Crothers (Benjamin Cummings, Menlo Park 1979). Computer modeling of the structure of CUL4 E3 ligase complex members provides another means for designing test compounds for screening p53 modulators. Methods of molecular modeling have been described in the literature, e.g., U.S. Pat. No. 5,612,894 entitled "System and method for molecular modeling utilizing a sensitivity factor", and U.S. Pat. No. 5,583,973 entitled "Molecular modeling method and system". In addition, protein structures can also be determined by neutron diffraction and nuclear magnetic resonance (NMR). See, e.g., Physical Chemistry, 4th Ed. Moore, W. J. (Prentice-Hall, New Jersey 1972), and NMR of Proteins and Nucleic Acids, K. Wuthrich (Wiley-Interscience, New York 1986). In addition, the crystal Structure of human Mdm2 bound to an imidazoline inhibitor is known (see MMDB Accession No. 26209 and Chen et al. (2003) "MMDB: Entrez's 3D-structure database", Nucleic Acids Res; 31(1): 474-7. Modulators of the present invention also include antibodies that specifically bind to a CUL4 E3 ligase complex member, such as those which specifically bind to CUL4A, CUL4B, DDB1, L2DTL, ROC1, ROC2, MDM2 or Pirh2. Such antibodies can be monoclonal or polyclonal. Such antibodies can be generated using methods well known in the art. For example, the production of non-human monoclonal antibodies, e.g., murine or rat, can be accomplished by, for example, immunizing the animal with a CUL4 E3 ligase complex member or fragment thereof (See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.). Such an immunogen can be obtained from a natural source, by peptides synthesis or by recombinant expression. Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861. Human antibodies can be obtained using phage-display methods. See, e.g., Dower et al., WO 91/17271 ; McCafferty et al., WO 92/01047. In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to a CUL4 E3 ligase complex member of the present invention. Human antibodies against a CUL4 E3 ligase complex member can also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus. See, e.g., Lonberg et al., WO93/12227 (1993); Kucherlapati, WO 91/10741 (1991). Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using a CUL4 E3 ligase complex member or its fragment.

III. Regulation of p53 Levels in a Cell The invention further provides methods of modulating the expression level of p53 in a cell, preferably a human cell and human p53. One aspect of the invention provides a method of reducing the expression level of a p53 polypeptide in a cell, the method comprising contacting the cell with an agent that: (a) increases the expression level of an mRNA encoding a CUL4-associated polypeptide in the cell; (b) increases the level of a CUL4-associated polypeptide in the cell; (c) increases binding between a p53 polypeptide and a CUL4 E3 ligase complex; (d) increases the ubiquitination activity of a CUL4 ligase complex; (e) increases the binding between a CUL4 ligase complex and MDM2; (f) increases the binding between a CUL4 ligase complex and Pirh2; (g) increases the activity of a CULl E3 ligase complex. In one embodiment, CUL4 is CUL4A or CUL4B. Some embodiments further comprise the step of increasing the expression level or activity of at least one E2 conjugation enzyme, such as one selected from CDC34 E2, UbcH5B E2 and UbcH5C E2. In some embodiments, the agent increases the polyubiquitination of p53. In one embodiment, the CUL4-associated polypeptide is selected from CUL4A, CUL4B, DDB 1 , L2DTL, ROC1, ROC2, MDM2 and Pirh2. In another embodiment, the CUL4 ligase complex comprises one or more polypeptides selected from CUL4A, CUL4B, DDB1, L2DTL, ROC1, ROC2, MDM2 and Pirh2. Another aspect of the invention provides methods of increasing the expression of p53 in a cell, preferably of human p53 in a human cell. One methods comprises contacting the cell with an agent that: (a) decreases the expression level of an mRNA encoding a CUL4-associated polypeptide in the cell; (b) decreases the level of a CUL4-associated polypeptide in the cell; (c) decreases binding between a p53 polypeptide and a CUL4 E3 ligase complex; (d) decreases the ubiquitination activity of a CUL4 ligase complex; (e) decreases the binding between a CUL4 ligase complex and MDM2; (f) decreases the binding between a CUL4 ligase complex and Pirh2; (g) decreases the activity of a CULl E3 ligase complex. In one embodiment, CUL4 is CUL4A or CUL4B. In another embodiment, the CUL4-associated polypeptide is selected from the group consisting of CUL4A, CUL4B, DDBl, L2DTL, ROC1, ROC2, MDM2 and Pirh2. In another embodiment, the CUL4 ligase complex comprises one or more polypeptides selected from the group consisting of CUL4A, CUL4B, DDB 1 , L2DTL, ROC 1 , ROC2, MDM2 and Pirh2. In another embodiment, the agent decreases the polyubiquitination of p53. In a prefeπed embodiment, the cell is in a mammal, such as a rodent or a human. In one embodiment, the mammal, such as a human, has a hypeφlastic conditions, such as a tumor or cancer. The methods described herein are not limited to a human with any particular hypeφlastic condition. In specific embodiments, the individual is afflicted with at least one form of renal cell cancer, Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer, sarcoma, pancreatic cancer, leukemia, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, lymphoma, mastocytoma, lung cancer, mammary adenocarcinoma, pharyngeal squamous cell carcinoma, testicular cancer, gastrointestinal cancer, or stomach cancer. In some embodiments, agent for increasing the expression of p53 in a cell is an antisense polynucleotide directed to a CUL4-associated polypeptide or a CUL4 E3 ligase complex. The antisense polynucleotide may be directed for example, to a gene encoding one or more of the following polypeptides: CULl , CUL4A, CUL4B, DDB 1 , L2DTL, ROC1, ROC2, MDM2 and Pirh2. The term "polynucleotide" as refeπed to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms. Polynucleotides may comprise or consist of sequences that regulate gene expression, such as antisense polynucleotides, ribozymes, siRNA, shRNA and the like, and/or encode a gene product such as an mRNA or polypeptide product. As used herein, the term "small interfering RNA" ("siRNA") (also refeπed to in the art as "short interfering RNAs") refers to an RNA (or RNA analog) comprising or consisting of between about 10-50 nucleotides (or nucleotide analogs) that are capable of directing or mediating RNA interference. Preferably, a siRNA comprises or consists of between about 15-30 nucleotides or nucleotide analogs, more preferably between about 16-25 nucleotides (or nucleotide analogs), even more preferably between about 18-23 nucleotides (or nucleotide analogs), and even more preferably between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to a siRNA comprising or consisting of about 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term "long" siRNA refers to a siRNA comprising or consisting of about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, e.g., enzymatic processing, to a short siRNA. Examples 6 and 7 exemplify the use of siRNAs specific for CUL4A, DDBl, L2DTL and ROC1 to increase p53 levels. The term "short hairpin RNA" or shRNA refers to an RNA molecule comprising at least two complementary portions hybridized or capable of hybridizing to form a double-stranded (duplex) structure sufficiently long to mediate RNAi (typically at least 19 base pairs in length), and at least one single-stranded portion, typically between approximately 1 and 10 nucleotides in length that forms a loop. The duplex portion may, but typically does not, contain one or more bulges consisting of one or more unpaired nucleotides. As described further below, shRNAs are thought to be processed into siRNAs by the conserved cellular RNAi machinery. Thus shRNAs are precursors of siRNAs and are, in general, similarly capable of inhibiting expression of a target transcript. In some embodiments, agent for increasing the expression of p53 in a cell is an antibody directed to a CUL4-associated polypeptide or a CUL4 E3 ligase complex. The antibody may be specific for example, to one of the following polypeptides: CULl, CUL4A, CUL4B, DDBl, L2DTL, ROC1, ROC2, MDM2 and Pirh2. The term "antibody" as used herein is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragments thereof which are also specifically reactive with a vertebrate, e.g., mammalian, protein. Antibodies can be fragmented using conventional techniques and the fragments screened for utility and/or interaction with a specific epitope ofinterest. Thus, the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein. Non-limiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab' , Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker. The scFv's may be covalently or non-covalently linked to form antibodies having two or more binding sites. The term antibody also includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant (e.g. chimeric and other derivatized) antibodies. The generation of antibodies based on the known protein sequences of CULl , CUL4A, CUL4B, DDB 1 , L2DTL, ROC 1 , ROC2, MDM2 and Pirh2 is routine for one skilled in the art. Furthermore, the Examples describe the use of antibodies against MDM2, CUL4A, CUL4B and PirH2, among others. Additional inhibitors of MDM2 that may be used in the methods for increasing levels of p53 are described in U.S. Patent Publication No. 2003-0060432. In some embodiments, the agents decrease binding between various components of the CUL4 E3 ligase complex with each other or with p53. Exemplary agents which may be used to block such binding interactions include peptides and poljφeptide which competitively compete for binding. Such agents include nonfunctional mutants or fragments of CULl , CUL4A, CUL4B, DDB 1 , L2DTL, ROC1, ROC2, MDM2 and Pirh2. In some embodiments, the agents comprise a fragment of MDM2 which competes for binding of MDM2 to p53 or MDM2 to CUL4A B. In another embodiment, the agents comprise a fragment of PirH2 which competes for binding of PirH2 to p53 or PirH2 to CUL4A/B. Agents which block the binding of MDM2 and p53 include the family of Nutlins, as described in Vassilev et al. (2004) Cell Cycle;3(4):419-21. In addition, Zhong et al. (2005) Proteins.; 58(l):222-34 describes the design of inhibitors of MDM2 binding to p53 based on the structures of the two proteins. As similar approach may also be used to design inhibitors of pirH2/p53 interactions. When the agent for increasing p53 levels is a polypeptide, it may be adminstered to a cell in a mammal though an expression constructs encoding the agent in any biologically effective carrier, e.g. any formulation or composition capable of effectively transfecting cells in vivo with a recombinant fusion gene. Approaches include insertion of the subject fusion gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and heφes simplex virus- 1 , or recombinant bacterial or eukaryotic plasmids. Viral vectors can be used to transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation carried out in vivo. It will be appreciated that because transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e.g. locally or systemically. When the agent for increasing or decreasing the p53 levels in a cell in a mammal, the toxicity and therapeutic efficacy of the agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices are prefeπed. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine more accurately useful doses in mammal including humans. Levels of the desired therapeutic or diagnostic agent delivered to the plasma may be measured, for example, by high performance liquid chromatography. In one embodiment of the methods described herein, the effective amount of the agent is between about lmg and about 50mg per kg body weight of the individual. In one embodiment, the effective amount of the agent is between about 2mg and about 40mg per kg body weight of the individual. In one embodiment, the effective amount of the agent is between about 3mg and about 30mg per kg body weight of the individual. In one embodiment, the effective amount of the agent is between about 4mg and about 20mg per kg body weight of the individual. In another embodiment, the effective amount of the agent is between about 5mg and about lOmg per kg body weight of the individual. In one embodiment of the methods described herein, the agent is administered at least once per day. In another embodiment, the agent is administered daily. In yet another embodiment, the agent is administered every other day, every 6-8 days, or weekly. As for the amount of the compound and/or agent for administration to the individual, one skilled in the art knows how to determine empirically the appropriate amount. As used herein, a dose or amount would be one in sufficient quantities to either inhibit the disorder, treat the disorder, treat the individual or prevent the individual from becoming afflicted with the disorder or condition to be ameliorated. This amount may be considered an effective amount. One of ordinary skill in the art can perform simple titration experiments to determine what amount is required to treat the individual. The dose of the composition of the invention will vary depending on the individual and upon the particular route of administration used. In one embodiment, the dosage may range from about 0.1 to about 100,000 ug/kg body weight of the individual. Based upon the composition, the dose can be delivered continuously, such as by continuous pump, or at periodic intervals. For example, on one or more separate occasions. Desired time intervals of multiple doses of a particular composition can be determined by one skilled in the art. The effective amount may be based upon, among other things, the size of the compound, the biodegradability of the compound, the bioactivity of the compound and the bioavailability of the compound. If the compound does not degrade quickly, is bioavailable and highly active, a smaller amount will be required to be effective. The effective amount will be known to one of skill in the art; it will also be dependent upon the form of the compound, the size of the compound and the bioactivity of the compound. One of skill in the art may routinely perform empirical activity tests for a compound to determine the bioactivity in bioassays and thus determine the effective amount. In one embodiment of the above methods, the effective amount of the compound comprises from about 1.0 ng/kg to about 100 mgkg body weight of the individual. In another embodiment of the above methods, the effective amount of the compound comprises from about 100 ng/kg to about 50 mg/kg body weight of the individual. In another embodiment of the above methods, the effective amount of the compound comprises from about 1 μg/kg to about 10 mg/kg body weight of the individual. In another embodiment of the above methods, the effective amount of the compound comprises from about 100 μg/kg to about 1 mg/kg body weight of the individual. As for when the compound, compositions and/or agent is to be administered, one skilled in the art can determine when to administer such compound and/or agent. The administration may be constant for a certain period of time or periodic and at specific intervals. The compound may be delivered hourly, daily, weekly, monthly, yearly (e.g. in a time release form) or as a one time delivery. The delivery may be continuous delivery for a period of time, e.g. intravenous delivery. The delivery may be local, such as at the site of a tumor, or may be systemic. In some embodiments, the agents which increase p53 levels are administered to mammals as formulation with a pharmaceutical acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art. Such pharmaceutically acceptable carriers may include but are not limited to aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electroryte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. The agent may be administered to the mammal (e.g. human) using any of the methods known to one skilled in the art. The compound may be administered by various routes including but not limited to aerosol, intravenous, oral or topical route. The administration may comprise intralesional, intraperitoneal, subcutaneous, intramuscular or intravenous injection; infusion; liposome-mediated delivery; topical, infrathecal, gingival pocket, per rectum, intrabronchial, nasal, transmucosal, intestinal, oral, ocular or otic delivery. In a further embodiment, the administration includes intrabronchial administration, anal, intrathecal administration or transdermal delivery. The compounds and or agents of the subject invention may be delivered locally via a capsule which allows sustained release of the agent or the peptide over a period of time. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the methods of the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines) and the agent coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors. Other embodiments incoφorate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral. Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited" to. The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise. The term "such as" is used herein to mean, and is used interchangeably with the phrase, "such as but not limited to".

EXEMPLIFICATION The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for puφoses of illustration of certain aspects and embodiments of the present invention and are not intended to be limiting in any way. The contents of any patents, patent applications, patent publications, or scientific articles referenced anywhere in this application are herein incoφorated by reference in their entirety.

Description of Experimental Procedures Human HEK293, HeLa, U2OS, H1299, SAOS2, U87MG cells and mouse embryonic fibroblasts were cultured as described previously (18). The CUL4A, CUL4B, and p53 siRNAs were sj thesized and the transfection of the siRNAs was conducted as previously described (5, 18). For stable Flag-tagged p53 expression cell line, Flag-p53 was cloned under the CMV promoter control (pCMV-2B) and after transfection, the stable expressing lines are selected by G418. The p53 retrovirus was expressed in Phenix cells, harvested, and used as described previously (32). The RVY- and CTF-U2OS cells were constructed by infecting an empty retrovirus vector (RVY) or the retrovirus containing a dominant negative p53 (CTF, kindly provided by Dr. Daniel DiMaio, Yale University, Connecticut) and selected by hygromycin resistance (19). The gamma-iπadiation and cell cycle analysis after bromodeoxyuridine incoφoration by flow-cytometry were conducted as described (18, 32). Apoptosis was analyzed using APO-BRDU kit from BD Biosciences PharMingen, California. Antibodies, plasmids, and p53 polyubiquitination reaction: Anti-p53 (DO1) and MDM2 (SMP14 and D12) antibodies were purchased from Santa Cruz, California. Mouse monoclonal antibody OPl 15 against MDM2 was obtained from Calbiochem. The anti-p53 BD antibody was obtained from BD Biosciences PharMingen. The CUL2, CUL4A, CUL4B, and CUL4CT antibodies were used as described (18, 55). Anti-Pirh2 antibody was made by raising a rabbit polyclonal antibody using the GST-Pirh2 protein. We also purchased an anti-Pirh2 antibody from Bethyl Laboratories, Texas. The anti-human DDBl antibodies were kindly provided by Drs. Betty Slagle (Baylor College of Medicine, Texas) and Yue Xiong (University of North Carolina, North Carolina). The DDBl cDNA and the full length human Pirh2 cDNA were purchased from ATCC. Immunoprecipitation and western blotting were conducted as described before (18). For polyubiquitination of p53 by MDM2 and CUL4 ligase complexes, Flag-tagged p53 was expressed in 293 cells and immunoprecipitated by anti-MDM2, CUL4A, or CUL4CT antibodies. The polyubiquitination of p53 associated with MDM2 or CUL4 complexes was assayed by addition of purified El, E2 (CDC34, UBCH5C, or a combination of CDC34 and UBCH5C), in the presence of ubiquitin and ATP. Alternatively, Flag-p53 protein was expressed in 293 cells, isolated by anti-Flag antibody and protein A Sepharose beads, eluted from the beads by Flag peptide, and used as a substrate for polyubiquitination by MDM2 and CUL4 complexes. In some experiments, recombinant proteins of MDM2 and Pirh2 were used. The recombinant GST-Pirh2 protem was constructed in pGEX and expressed in bacteria while a GST-MDM2 baculovirus was constructed to produce the GST-MDM2 recombinant fusion protein in insect SF9 cells (18, 48).

Example 1 : CUL4A complex interacts with p53 and'MDM2 in both human 293 and U2OS cells: Our previous studies indicated that abnormal activation of the replication licensing factor CDTl leads to chromosome polyploidy (37). CDTl is also rapidly proteolyzed by the CUL4/ROC1 E3 ligase in response to DNA damage (18). Since p53 is activated in response to DNA damage and replication perturbation and loss of p53 is associated with genome instability (26, 34, 53), we examined the activation of p53 by CDTl -mediated replication process and the CUL4 E3 ligase. Suφrisingly, we found that p53 is associated with the CUL4A ligase complex (Figure 1).

Immunoprecipitation of CUL4A complexes from human embryonic kidney 293 cells revealed that p53 is present in CUL4A immunocomplexes by two independent CUL4A antibodies (anti-amino terminal or anti-carboxy terminal peptide antibodies, CUL4A or CUL4CT, respectively, Figures 1 A and C). Since 293 cells are transformed by adenovirus and since CUL5/Elongin B/Elongin C E3 ligase has been shown to be involved in the degradation of p53 mediated by adenovirus E4orf6 and E1B55K proteins (42), we also examined the binding of p53 and MDM2 to CUL4 complex in U2OS cells which contain no known viral proteins (13). Examination of CUL4A complex isolated from U2OS cells indicated that p53 also binds to CUL4A complex and this binding is enhanced in the presence of MG132, an inhibitor of the 26S proteosome, suggesting this binding is not mediated by adenovirus proteins. Conversely, CUL4A can also be detected in p53 immunoprecipitates by two independent anti-p53 antibodies (data not shown). These studies show that the CUL4A ubiquitin E3 ligase complexes interact with p53. Ubiquitin-dependent ρ53 proteolysis is regulated in part by the oncoprotein MDM2 (35, 44, 53). However, although MDM2 is implicated in targeting p53 for polyubiquitination in vivo, MDM2 can only monoubiquitinate p53 in vitro at physiologic levels, suggesting MDM2 requires additional proteins to polyubiquitinate p53 (35, 44, 53). To test whether CUL4A E3 ligase complexes interact with MDM2, endogenous MDM2 was examined for its binding to CUL4A. In both 293 and U2OS cells, MDM2 can be detected by both anti-CUL4A and anti-CUL4CT immunoprecipitates (Figure 1A and B, Figure 5D). The interactions between the CUL4A E3 ligase complex, p53 and MDM2 were further confirmed by the association of p53 and MDM2 with other components of the CUL4A E3 ligase complex (see below, Fig. 3C).

Example 2: The interaction between p53 and CUL4A complex is regulated by gamma-iπadiation: The p53 protein is stabilized by DNA damage (53). This regulation is in part mediated through the phosphorylation of the multiple serines at the amino terminus of p53 by the ATM/ATR checkpoint kinases. Phosphorylation of these serine residues in p53 causes its dissociation from MDM2 and promotes p53 protein stability (4, 45, 47). We found that the interaction between the CUL4A complex and p53 is rapidly reduced in response to gamma-iπadiation in both 293 and U2OS cells (Figure IC and D). The association between p53 and MDM2 was also abolished by gamma- iπadiation (Figure IC). To further determine the regulation of p53 dissociation, we treated cells with Wortmannin, an inhibitor of the ATM/ATR kinase family (12). Prior treatment of cells with Wortmannin suppressed the dissociation of p53 from the CUL4A complex in response to iπadiation (Figure IE), suggesting the involvement of ATM/ATR kinase family members in regulating the p53-CUL4A complex interaction. The dissociation of p53 from both CUL4A and MDM2 in response to gamma-iπadiation also suggests CUL4A may cooperate with MDM2 in regulating p53 in response to checkpoint activation.

Example 3: Pirh2 interacts with CUL4 E3 ligase complex In these experiments, we have noticed that sometimes there is significant interaction between p53 and the CUL4A complex even in the presence of gamma- iπadiation (Figure 1C-E). These observations suggest that MDM2 may not be the only cellular protein that mediates complex formation between p53 and CUL4A ligase. To examine whether CUL4A ligase complex can interact with p53 in the absence of MDM2, we expressed p53 in the MDM2 and p53 double null MEFs (MDM-/- and p53-/-) by retrovirus-mediated infection. We found that under these conditions, the ectopically expressed p53 can bind to CUL4A, and to lesser extent CUL4B, a human paralogue of CUL4A, in the absence of MDM2 (Figure 2). In a search of additional proteins that associate with CUL4 complexes and regulate p53, we found that Pirh2, a RING protein that binds p53 and is implicated in regulating p53 stability (27), is also present in CUL4 immunocomplexes (Figure 2B and 5G). This observation suggests that CUL4 complexes may also cooperate with Pirh2 to regulate p53 protein levels.

Example 4: The binding of MDM2 and Pirh2 requires the adaptor domain at CUL4A amino terminus. CUL4 belongs to the cullin E3 ligase family (18, 23). Cullin-containing ubiquitin E3 ligases use various substrate-targeting subunits (F-box proteins for SCF, SOCS proteins for Elongin B/C-CUL2, and BTB/POZ proteins for CUL3) to recognize different substrates for polyubiquitination (24). The substrate-targeting subunits bind to a conserved amino terminal "adaptor domain" of cullins through an adaptor protein (SKP1 for CULl in SCF and Elongin C for CUL2 and CUL5). CUL4A, as well as CUL4B, contains the conserved adaptor domain at its amino terminus for the binding of the putative adaptor protein(s) and thus the substrate- specificity subunits (Figure 3A)(41). To test whether the interaction between CUL4A and MDM2 or Pirh2 requires the CUL4A adaptor domain, we analyzed the binding of MDM2 and Pirh2 to various CUL4A "adaptor domain" mutants (Figure 3A and B). Deletion analysis of the amino terminus of CUL4A shows that the binding of both MDM2 and Pirh2 requires the presence of the adaptor domain of CUL4A (Figure 3 A and B), suggesting that they may act as the substrate-specificity components of CUL4 E3 ligase complexes.

Example 5 : MDM2 and Pirh2 bind to DDB 1. a putative adaptor protein of CUL4A E3 ligase. It has been shown that the DNA damage binding protein 1 (DDB 1) binds to the adaptor region in CUL4A and may serve as an adaptor protein for CUL4 (20, 39). Our deletion analysis prompted us to test whether MDM2 and Pirh2 also interact with DDBl. Westem-blot analysis of MDM2 and Pirh2 immunocomplexes revealed that DDBl is associated with both MDM2 and Pirh2 as well as p53 (Figure 3C). We have recently isolated several CUL4 associated proteins, including DDBl and ROC1. The human L2DTL, a WD40 repeat-containing protein which shares homology with the Drosophila embryonic lethal mutant, lethal(2)denticleless (25), was also identified. Our analysis indicates that the endogenous human L2DTL binds to DDBl and CUL4 in vivo (Figure 3D). The Human L2DTL protein also interacts with p53 in vivo when it is expressed in 293 cells (Figure 3D). These studies suggest that DDBl and human L2DTL may be involved in p53 regulation.

Example 6: Silencing of CUL4A induces p53 accumulation. p21 induction, GI cell cycle aπest and apoptosis in response to UV damage. The binding of p53 to the CUL4 E3 ligase complex suggests that the CUL4 E3 ligase may regulate p53 stability in vivo. To test this possibility, we used the siRNA method (11) to silence the expression of CUL4A and measured the effect on p53 protein levels. To determine whether CUL4A alters the degradation rate of p53 protein, we also treated cells with the protein sjmthesis inhibitor, cycloheximide, and assayed p53 levels at various times during the treatment. Silencing of CUL4A caused a significant accumulation of p53 and impeded p53 protein decay (Figure 4A), indicating that p53 stability is regulated by the CUL4A ligase in vivo. Increased levels of p53 have been shown to induce the transcriptional activation of CDK inhibitor p21, GI cell cycle aπest, and apoptosis (34, 53). Silencing of CUL4A expression is sufficient to induce accumulation of p21 (Figure 4B). We also reproducibly observed that silencing of CUL4A expression causes GI cell cycle aπest in a number of cell lines including U87MG glioblastoma cells (Figure 4C). Since CUL4A is known to target various other proteins (such as replication licensing factor CDTl, c-Jun, and DDB2) for polyubiquitination (18, 39, 50), silencing of CUL4A may not only affect the GI cell cycle progression. We found that the p21 induction and GI cell cycle aπest are associated with elevated levels of p53 (Figure 4B). Co-silencing of p53 and CUL4A suppressed the increase of p21 level and GI cell cycle aπest, suggesting these CUIAA-deficiency induced processes result from the elevated levels of p53 (Figure 4B and C). Ablation of CUL4A expression by siRNA also sensitizes p53 positive U2OS cells to undergo apoptosis in response to UV-iπadiation (Figures 4D, 4E) whereas U2OS cells which stably express dominant negative p53 mutant (CTF) (19) are much more resistant to CUL4A silencing under these conditions (Figures 4D, 4E).

Example 7: Silencing of DDBl, L2DTL, and ROC1 induces p53 accumulation. Since DDBl binds to CUL4A, ρ53, MDM2, and Pirh2 (Figure 3C), we tested whether DDBl is also involved in regulating p53. Silencing of DDBl is sufficient to cause p53 accumulation and p21 induction (Figure 4F), similar to the effect of CUL4A silencing (Figure 4A-D). We also examined the effects of silencing L2DTL and the Ring finger protein ROC1, on p53 levels. We found that siRNA-mediated silencing of both L2DTL and ROC1 led to the accumulation of p53 (Figure 4F). These studies suggest that the L2DTL-DDB 1 -CUL4-ROC 1 E3 ligase complex regulates p53 protein levels, which in turn control cell cycle progression, DNA damage checkpoints, and cell survival. Example 8: The isolated DDB1-CUL4A-ROC1 E3 ligase complex contains an intrinsic, specific, and robust polyubiquitination activity towards p53. Our studies suggest that CUL4A regulates p53 protein stability in vivo. To further determine whether p53 serves as a substrate of CUL4A E3 ligase, we examined whether the CUL4-associated p53 can be directly ubiquitinated by the CUL4 complexes in vitro. A Flag-epitope tagged p53 was transfected into 293 cells and immunoprecipitated by anti-MDM2, CUL4A, or CUL4B antibodies. We found that the transfected and thus ectopically expressed p53 can be immunoprecipitated by antibodies against MDM2 and CUL4A, and weakly by CUL4B (Figure 5A). The p53 protein associated with the MDM2 and CUL4 immunocomplexes was further incubated in a polyubiquitination reaction containing CDC34, an E2 ubiquitin conjugating enzyme, and El, the ubiquitin activating enzyme, in the presence of ubiquitin and ATP (18). The MDM2- and CUL4A-associated ρ53 could be poljTibiquitinated by both MDM2 and CUL4A complexes (Figure 5 A), suggesting that CUL4 complexes and MDM2 can polyubiquitinate p53 once it is bound by these E3 ligases. Intriguingly, while the anti-MDM2 antibody (SMP-14) immunoprecipitated much more MDM2 and its bound p53, only weak p53 polyubiquitination activity was observed in MDM2 immunoprecipitation. Notably, this MDM2 antibody co-immunoprecipitates very little CUL4. In contrast, we found that even though CUL4A E3 ligase complexes contain much less MDM2, the polj xbiquitination activity of CUL4 complexes for p53 is much stronger than that of MDM2 under these conditions (Figure 5A). Thus, under our assay conditions, the ability to polj biquitinate p53 appears to coπelate with the association of MDM2 with CUL4A complex. To further analyze p53 polyubiquitination by the CUL4 complex, we developed a p53 polyubiquitination assay using isolated p53 protein (Figure 5B-G). To isolate p53, a stable 293 cell line expressing a Flag-tagged p53 was established. The Flag-p53 protein was isolated from this 293 line by anti-Flag antibodies and then eluted from protein A Sepharose beads using the Flag-peptide (18). The purified p53 was used as a substrate for the isolated CUL4 ubiquitin E3 ligase or MDM2 by specific anti-CUL4 or MDM2 antibodies. Using this assay, we found that p53 can be robustly poljαibiquitinated by the CUL4A E3 ligase complex, which contains CUL4A, ROC1, L2DTL and DDBl (Figure 5B). This reaction is dependent on the exogenously added recombinant El ubiquitin activating enzyme and ubiquitin and is inhibited by methylated ubiquitin (mUb), a chain terminator for polyubiquitination (Figure 5B) (18). To determine the specificity of p53 polyubiquitination reaction by CUL4A E3 ligase, the ability of various cullin E3 ligases to polyubiquitinate p53 was compared. We found that while CUL4A E3 ligase can specifically and robustly polyubiquitinate p53, other cullin ligases such as CUL2 E3 ligase cannot (Figure 5C). Thus the ability to polyubiquitinate p53 is specific to the CUL4A E3 ligase under these assay conditions.

Example 9: MDM2 displays poor polyubiquitination activity towards p53 Using this assay, we found that while CUL4A E3 ligase complexes isolated by two independent CUL4A antibodies can poljαibiquitinate p53, MDM2 displays very poor polyubiquitination activity for p53 (Figure 5D). This activity is not due to the lack of p53 binding to MDM2, since MDM2 complex contains substantial amount of p53 (Figure 5 A). The low of p53 polyubiquitination activity of MDM2 in vitro is consistent with many previously published reports (34, 35, 53).

Example 10: CUL4A E3 ligase requires MDM2 for polyubiquitination of p53 in vitro Our studies indicate that p53 is a substrate of the CUL4A E3 ligase complexes. Since MDM2 interacts with the CUL4A ligase complexes (Figure 1), we asked whether MDM2 status affects CUL4A ligase activity. To address this question, we compared the p53 polyubiquitination activity of CUL4 complexes isolated from mouse embryonic fibroblasts (MEFs) containing either the wild-type MDM2 (+/+) or the MDM2 null mutation (MDM2 -/-) (38). Since MDM2 null MEFs cannot grow due to the accumulation of p53, the MDM2 deficient MEFs are also p53 null (p53 -/-) (38). While the CUL4A E3 ligase complexes isolated from the wild-type MEFs (MDM2 +/+) displayed a robust poljαibiquitination activity against p53, the CUL4A ligase complex from the MDM2 deficient MEFs (MDM2 -/-) contained a substantially lower ubiquitination activity for p53 (Figure 5E), suggesting that CUL4A E3 ligase activity is dependent on the presence of MDM2 in these cells. To further test whether MDM2 protein is required for CUL4A E3 ligase activity towards p53, we supplemented recombinant GST-MDM2 protein to the CUL4A complexes isolated from the MDM2 and p53 double deficient cells (MDM2-/- and p53-/-) to determine whether it can restore the polyubiquitination activity of CUL4A complexes. We found addition of the recombinant MDM2 protein greatly stimulated and significantly restored the p53 polyubiquitination activity of CUL4A complexes (Figure 5F). Under these conditions, the recombinant MDM2 protein did not exhibit polyubiquitination activity towards p53 in the absence of the CUL4A complexes (Figure 5F), consistent with previous reports and our analysis using the MDM2 protein isolated from cells (Figure 5A, B and 5D) (35).

Example 11 : Recombinant Pirh2 protein complements CUL4A E3 ligase for p53 polyubiquitination in vitro In MDM2 null MEFs, p53 has also been inactivated to prevent lethality caused by the elevated p53 levels (21). Since Pirh2 is transcriptionally regulated by p53 (27), the reduced level of Pirh2 in the MDM2 and p53 double null MEFs may further contribute to the low p53 polj ibiquitination activity of CUL4A complexes isolated from these cells (Figure 5E and F). Similar to MDM2, immunoprecipitated Pirh2 protein from U2OS cells did not have substantial polyubiquitination activity towards p53 under our assay conditions (Figure 5G, left panel). To test whether Pirh2 protein is also required for CUL4A E3 ligase activity towards p53, we supplemented recombinant GST-Pirh2 protein to the CUL4A complexes isolated from the MDM2 and p53 double deficient MEFs. Addition of the recombinant Pirh2 protein to the CUL4A complex induced a robust p53 polyubiquitination activity of CUL4A E3 ligase (Figure 5G, right panel). Under these conditions, the recombinant Pirh2 protein did not exhibit polj ibiquitination activity towards p53 in the absence of the CUL4A complexes (Figure 5G, right panel). Like MDM2, it has been shown that the recombinant GST-Pirh2 has very low ubiquitination activity toward p53 in a purified system (27, 35, 53). These observations indicate that CUL4A E3 ligase cooperates with both MDM2 and Pirh2 to regulate p53 polyubiquitination and protein stability. Example 12: Polyubiquitination of p53 by CUL4 E3 ligase complex requires DDBL L2DTL. and ROCl. Since DDBl, L2DTL, and ROC1 bind to CUL4, we further examined the involvement of these proteins in polyubiquitination of p53 by CUL4 E3 ligase complex. The CUL4A E3 ligase complexes were isolated by immunoprecipitation from cells treated with either control siRNA (luciferase, Luc) or siRNAs against DDBl, L2DTL, and ROC1 (Figure 5H). The CUL4 E3 ligase complexes from these cells were assayed for their ability to polj ibiquitinate p53. We found that the poljTxbiquitination activity of CUL4 E3 ligase complexes is greatly diminished in DDB 1 , L2DTL, and ROC 1 siRNA-treated cells (Figure 5H), as compared with the complex isolated from control Luc siRNA treated cells. These studies indicate that DDBl, L2DTL, and ROC1 are required for p53 polyubiquitination by the CUL4 E3 ligase complexes.

Example 13: Protocol for p53 Polvubiquitination Figure 6 diagrams a high-throughput method for identifying agents which modulate the CUL4-dependent ubiquitination of p53. A Flag-tagged p53 is expressed in 293 or other cells. When expressed in 253 cells, p53 retains its ability to bind to CUL4 complex and MDM2 or Pirh2. The p53-CUL4 complexes are isolated by immunoprecipitation with anti-MDM2, CUL4A, CUL4B, or CUL4CT antibodies. The poljTibiquitmation of p53 associated with MDM2 or CUL4 complexes was assayed by addition of purified ubiquitin activating enzyme El, ubiquitin conjugating E2 enzyme (CDC34, TJBCH5B or 5C, or a combination of CDC34 and UBCH5B or 5C), in the presence of ubiquitin and ATP. Alternatively, Flag-p53 protein was expressed in 293 or other cells, isolated by anti-Flag antibody and protein A

Sepharose beads, eluted from the beads by Flag peptide, and used as a substrate for polj ibiquitination by MDM2 and CUL4 complexes as described above. We also used a recombinant GST-tagged p53 expressed in baculovirus expression system as the substrate for CUL4 complex. It is also possible to use other forms of tagged or untagged recombinant p53, expressed in organisms such as bacteria, insect cells including SF9 cells, or other species that can serve as the substrate for polyubiquitination by CUL4 complexes. To adapt p53 polyubiquitination for high throughput screen (HTS), tagged-p53 (Flag, histidine, HA, Myc, T7, maltose binding protein, glutathione-S-transferase tags, etc.) expressed in 293 cells or other cells can be immunoprecipitated with anti-CUL4 peptide antibodies and protein A beads. The p53-CUL4 complexes can be eluted from the beads by CUL4 peptide. The complex can be aliquoted into 96 or 384 wells for p53 polyubiquitination. The poljαibiquitination is initiated by incubation of ubiquitin activating enzyme El, ubiquitin conjugating enzyme E2 (CDC34, UBCH5B or 5C, or a combination of CDC34 and UBCH5B or 5C), ubiquitin and ATP. To detect the ubiquitinated or poljTibiquitinated p53 species in the reaction with high sensitivity, we will use the Alphascreen or FRET methods. For Alphascreen , ubiquitin is conjugated with biotin. The biotinylated ubiquitin will be added to reaction. After 30-90 min, the reaction will be terminated by EDTA. Anti-p53 antibody, Streptavidin-Donor Beads and protein A Acceptor beads (both from Perkin- Elmer) will be added to the mix. While the biotinylated and ubiquitinated p53 will be recognized by Streptavidin-Donor, p53 will be recognized by anti-p53 antibody and the p53/anti-p53 immunocomplex can bind to protein A Acceptor beads. Thus the biotinylated and ubiquitinated p53 serves as a bridge between Streptavidin-Donor and protein A Acceptor Beads. As the Streptavidin-Donor and protein A Acceptor Beads are each coated with light-sensitive chemicals, the close physical proximity of Donor and Acceptor beads will allow a photo-chemical reaction to take place. The light signal thus generated can be read and quantified by a luminometer. Similar assays can be developed using the FRET method.

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Sequence Listing

SEQ ID NO: 1; (Human Ubiquitin Protein)

MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGR TLSDYNIQKESTLHLVLRLRGG

SEQ ID NO:2; (Human p53 Protein)

MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQW

FTEDPGPDEAPRMPEAAPRVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGS YGFRLGFLHSGTAKS VTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVR AMAIΥKQSQHMTEWRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDR NTFRHSVWPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSG NLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSS PQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSS HLKSKKGQSTSRHKKLMFKTEGPDSD

SEQ ID NO:3 (Human CUL4A Protein)

MADEAPRKGSFSALVGRTNGLTKPAALAAAPAKPGGAGGSKKLVIKNFRDR PRLPDNYTQDTWRKLHEAVRAVQSSTSIRYNLEELYQAVENLCSHKVSPMLY KQLRQACEDHVQAQILPFREDSLDSVLFLKKLNTCWQDHCRQMIMIRSIFLFL DRTYVLQNSTLPSΓWDMGLELFRTHIISDKMVQSKTΓDGILLLIERERSGEAVD RSLLRSLLGMLSDLQVYKDSFELKFLEETNCLYAAEGQRLMQEREVPEYLNH VSKRLEEEGDRVITYLDHSTQKPLIACVEKQLLGEHLTAILQKGLDHLLDENR VPDLAQMYQLFSRVRGGQQALLQHWSEYIKTFGTAIVINPEKDKDMVQDLL DFKDKVDHVIEVCFQKNEPJVNLMKESFETFI KRPNKPAELIAKHVDSKLRA GNKEATDEELERTLDKIMILFRFIHGKDVFEAFYKKDLAKRLLVGKSASVDAE KSMLSKLKHECGAAFTSKLEGMFKDMELSKDIMVHFKQHMQNQSDSGPIDL TVNILTMGYWPTYTPMEVHLTPEMIKLQEVFKAFYLGKHSGRKLQWQTTLG HAVLKAEFKEGKKEFQVSLFQTLVLLMFNEGDGFSFEEIKMATGIEDSELRRT LQSLACGKARVLIKSPKGKEVEDGDKFJFNGEFKHKLFRIKΓNQIQMKETVEE QVSTTER QDRQYQIDAAΓVRIMKMRKTLGHNLLVSELYNQLKFPVKPGDL KKRIESLIDRDYMERDKDNPNQYHYVA

SEQ ID NO:4 (Human CUL4B Protein):

MFPTGFSSPSPSAAAAAQEVRSATDGNTSTTPPTSAKKRKLNSSSSSSSNSSNE REDFDSTSSSSSTPPLQPRDSASPSTSSFCLGVSVAASSHVPIQKKLRFEDTLEF VGFDAKMAEESSSSSSSSSPTAATSQQQQLKNKSILISS VAS VHHANGLAKSST

TVSSFANSKPGSAKKLVIKNFKDKPKLPENYTDETWQKLKEAVEAIQNSTSIK YNLEELYQAVENLCSYKISANLYKQLRQICEDHIKAQIHQFREDSLDSVLFLK KIDRCWQNHCRQMIMIRSIFLFLDRTYVLQNSMLPSIWDMGLELFRAHIISDQ KVQNKTIDGILLLIEP^RNGEAIDRSLLRSLLSMLSDLQΓYQDSFEQRFLEE'TNR LYAAEGQKLMQERE VPEYLHHVNKRLEEEADRLITYLDQTTQKSLIATVEKQ LLGEHLTAILQKGLNNLLDENRIQDLSLLYQLFSRVRGGVQVLLQQWLEYIKA FGSTIVINPEKDKTMVQELLDFKDKVDHJXDICFLKNEKFINAMKEAFETFΓNK PJ'NKPAELIAKYVDSKLRAGNKEATDEELEKMLDKIMIIFRFIYGKDVFEAFY KKDLAKRLLVGKSASVDAEKSMLSKLKHECGAAFTSKLEGMFKDMELSKDI MIQFKQYMQNQNVPGNIELTVNILTMGYWPTYVPMEVHLPPEMVKLQEIFKT FYLGKHSGRKLQWQSTIGHCVLKAEFKEGKKELQVSLFQTLVLLMFNEGEEF SLEEIKQATGIEDGELRRTLQSLACGKARVLAKNPKGKDIEDGDKFICNDDFK HKLFMK QIQMKETVEEQASTTER QDRQYQIDAAIVPJMKMRKTLSHNL LVSEVYNQLKFPVKPADLKKRIESLIDRDYMERDKENPNQYNYIA

SEQ ID NO:5 (Human ROC1 Protein)

MAAAMDVDTPSGTNSGAGKKRFEVKKWNAVALWAWDIWDNCAICRNHI MDLCIECQANQASATSEECTVAWGVCNHAFHFHCISRWLKTRQVCPLDNRE WEFQKYGH

SEQ ID NO:6 (Human ROC2 Protein) MADVEDGEETCALASHSGSSGSKSGGDKMFSLKKWNAVAMWSWDVECDT CAICRVQVMDACLRCQAENKQEDCVVVWGECNHSFHNCCMSLWVKQNNR CPLCQQDWWQRIGK

5 SEQ ID NO:7 (Human DDBl Protein): MSYNYVVTAQKPTAVNGCVTGHFTSAEDLNLLIAKNTRLEIYVVTAEGLRPV KEVGMYGKIAVMELFRPKGESKDLLFILTAKYNACILEYKQSGESIDIITRAHG NVQDRIGRPSETGIIGIIDPECRMIGLRLYDGLFKVIPLDRDNKELKAFNIRLEE LHVIDVKFLYGCQAPTICFVYQDPQGRHVKTYEVSLREKEFNKGPWKQENVE l o AEASMVIAVPEPFGGAIIIGQESITYHNGDKYLAIAPPIIKQSTIVCHNRVDPNG SRYLLGDMEGRLFMLLLEKEEQMDGTVTLKDLRVELLGETSIAECLTYLDNG VWVGSRLGDSQLVKLNVDSNEQGSYVVAMETFTNLGPΓVDMCVVDLERQG QGQLVTCSGAFKEGSLRIIRNGIGIHEHASIDLPGIKGLWPLRSDPNRETDDTL VLSFVGQTRVLMLNGEEVEETELMGFVDDQQTFFCGNVAHQQLIQITSASVR

15 LVSQEPKALVSEWKEPQAKNISVASCNSSQVWAVGRALYYLQIHPQELRQIS HTEMEHEVACLDITPLGDSNGLSPLCAIGLWTDISARILKLPSFELLHKEMLGG EIIPRSILMTTFESSHYLLCALGDGALFYFGLNIETGLLSDRKKVTLGTQPTVLR TFRSLSTTN ACSDRPTVTYSSNHKLVFSNVNLKEVNYMCPLNSDGYPDSLA LANNSTLTIGTIDEIQKLHIRTVPLYESPRKICYQEVSQCFGVLSSRIEVQDTSG 0 GTTALRPSASTQALSSSVSSSKLFSSSTAPHETSFGEEVEVHNLLIIDQHTFEVL HAHQFLQNEYALSLVSCKLGKDPNTYFIVGTAMVYPEEAEPKQGPJVVFQYS DGKLQTVAEKEVKGAVYSMVEFNGKLLASLNSTVRLYEWTTEKELRTECNH YNNIMALYLKTKGDFILVGDLMRSVLLLAYKPMEGNFEEIARDFNPNWMSA VEILDDDNFLGAENAFNLFVCQKDSAATTDEERQHLQEVGLFHLGEFVNVFC 5 HGSLVMQNLGETSTPTQGSVLFGTVNGMIGLVTSLSESWYNLLLDMQNRLN KVIKSVGKIEHSFWRSFHTERKTEPATGFIDGDLIESFLDISRPKMQEWANLQ YDDGSGMKREATADDLIKWEELTRIH

SEQ ID NO: 8 (Human L2DTL Protein) 0 MLFNSVLRQPQLGVLRNGWSSQYPLQSLLTGYQCSGNDEHTSYGETGVPVPP FGCTFSSAPNMEHVLAVANEEGFVRLYNTESQSFRKKCFKEWMAHvVNAVFD LAWVPGELKLVTAAGDQTAKFWDVKAGELIGTCKGHQCSLKSVAFSKFEKA WCTGGRI)GNIMVWDTRCNKKDGFYRQVNQISGAHNTSDKQTPSKPKKKQN SKGLAPSVDFQQSVTVVLFQDENTLVSAGAVDGIIKVWDLRKNYTAYRQEPI ASKSFLYPGSSTRKLGYSSLILDSTGSTLFANCTDDNIYMFNMTGLKTSPVAIF NGHQNSTFYVKSSLSPDDQFLVSGSSDEAAYTWKVSTPWQPPTVLLGHSQEV TSVCWCPSDFTKIATCSDDNTLKIWRLNTGLEEKPGGDKLSTVGWASQKKKE SRPGLVTVTSSQSTPAKAPRVKCNPSNSSPSSAACAPSCAGDLPLPSNTPTFSIK TSPAKARSPINRRGSVSSVSPKPPSSFKMSIRNWVTRTPSSSPPITPPASETKIMS PRKALIPVSQKSSQAEACSESRNRVKRRLDSSCLESVKQKCVKSCNCVTELDG QVENLHLDLCCLAGNQEDLSKDSLGPTKSSKIEGAGTSISEPPSPISPYASESCG TLPLPLRPCGEGSEMVGKENSSPENKNWLLAMAAKRKAENPSPRSPSSQTPNS RRQSGKTLPSPVTITPSSMRKICTYFHRKSQEDFCGPEHSTEL

SEQ ID NO:9 (Human MDM2 Protein)

MDRYLLLVIWGEGKFPSAASREAEHGPEVSSGEGTENQPDFTAANVYHLLKR SISASINPEDSTFPACSVGGIPGSKKWFFAVQAIYGFYQFCSSDWQEIHFDTEK DKIEDVLQTNIEECLGAVECFEEEDSNSRESLSLADLYEEAAENLHQLSDKLP APGRAMVDIILLLSDKDPPKLKDYLPTVGALKHLREWYSAKITIAGNHCEΓNC QKIAEYLSANVVSLEDLRNVIDSKELWRGKIQΓWERKFGFEISFPEFCLKGVTL KNFSTSNLNTDFLAKKIIPSKDKNILPKVFHYYGPALEFVQMIKLSDLPSCYMS DIEFELGLTNSTKQNSVLLLEQISSLCSKVGALFVLPCTISNILIPPPNQLSSRKW KEYIAKKPKTISVPDVEVKGECSSYYLLLQGNGNRRCKATLIHSANQINGSFA LNLIHGKMKTKTEEAKLSFPFDLLSLPHFSGEQIVQREKQLANVQVLALEECL

KRPVKLAKQPETVSVAELKSLLVLTRKHFLDYFDAVIPKMILRKMDKIKTFNIL NDFSPVEPNSSSLMETNPLEWPERHVLQNLETFEKTKQKMRTGSLPHSSEQLL GHKEGPRDSITLLDAKELLKYFTSDGLPIGDLQPLPIQKGEKTFVLTPELSPGK LQVLPFEKASVCHYHGIEYCLDDRKALERDGGFSELQSRLIRYETQTTCTRES FPVPTVLSPLPSPWSSDPGSVPDGEVLQNELRTEVSRLKRRSKDLNCLYPRKR LVKSESSESLLSQTTGNSNHYHHHVTSRKPQTERSLPVTCPLVPIPSCETPKLA TKTSSGQKSMHESKTSRQIKESRSQKHTRILKEVVTETLKKHSITETHECFTAC SQRLFEISKFYLKDLKTSRGLFEEMKKTA NNAVQVIDWVLEKTSKK

SEQ ID NO: 10 (Human PirH2 Protein) MAATAREDGASGQERGQRGCEHYDRGCLLKAPCCDKLYTCRLCHDNNEDH QLDRFKVKEVQCLNCEKIQHAQQTCEECSTLFGEYYCDICHLFDKDKKQYHC ENCGICRIGPKEDFFHCLKCNLCLAMNLQGRHKCIENVSRQNCPICLEDIHTSR WAHVLPCGHLLHRTCYEEMLKEGYRCPLCMHSALDMTRYWRQLDDEVAQ TPMPSEYQNMTVDILCNDCNGRSTVQFHILGMKCKICESYNTAQAGGRRISL DQQ

Claims

We claim:

1. A method of identifying an test compound which modulates the ubiquitination of a p53 polypeptide, the method comprising (a) providing a CUL4 E3 ligase complex; (b) incubating the CUL4 E3 ligase complex with a p53 polypeptide for an amount of time sufficient for the ubiquitination of p53; (c) determining the ubiquitination of the p53 polypeptide; and (d) comparing the ubiquitination of the p53 poljφeptide measured in (b) to ubiquitination of a p53 polypeptide in a mixture not contacted with the test compound to determine a difference in the ubiquitination of the p53 substrate, wherein the difference is indicative of the ability of the test compound to modulate the ubiquitination of a p53 polypeptide.

2. The method of claim 1 , wherein the CUL4 E3 ligase complex comprises CUL4A or CUL4B.

3. The method of claim 2, wherein the CUL4 E3 ligase complex comprises one or more of the polypeptides selected from the group consisting of CUL4A, CUL4B, DDBl, L2DTL, ROC1, ROC2, MDM2 and Pirh2.

4. The method of claim 3, wherein the CUL4 E3 ligase complex comprises Pirh2.

5. The method of claim 1, wherein the test compound increases p53 ubiquitination.

6. The method of claim 1 , wherein the test compound decreases p53 ubiquitination.

7. The method of claim 1 , wherein the ligase complex is incubated with a p53 polypeptide in the presence of one or more of an ubiquitin-activating El enzyme, an ubiquitin-conjugating E2 enzyme, adenosine tri-phosphate (ATP) and ubiquitin.

8. The method of claim 7, wherein ubiquitin-conjugating E2 enzyme is selected from the group consisting of Cdc34, UbcHl, UbcH2, UbcH3, UbcH4, UbcH5, UbcH6, UbcH7, UbcHl 0 and L-UBC.

9. The method of claim 8, wherein UbcH5 is UbcH5A, UbcH5B or UbcH5C.

10. The method of claim 1 , wherein the ubiquitin is a derivatized ubiquitin.

11. The method of claim 10, wherein said derivatized ubiquitin is selected from the group consisting of a ¹²⁵I-ubiquitin, a fluorescent ubiquitin, glutathione-S- transferase ubiquitin, and a biotinylated ubiquitin.

12. The method of claim 1 , wherein providing the CUL4 E3 ligase complex comprises immunoprecipitating the complex using an antibody that binds to a CUL4A or CUL4B protein.

12. The method of claim 12, wherein the antibody binds to a tag on the CUL4A or the CUL4B protein.

13. The method of claim 1, wherein the CUL4 E3 ligase complex comprises a recombinant CUL4A or a CUL4B protein.

14. The method of claim 1 , wherein the p53 is human p53.

15. A method of decreasing the level of a p53 polypeptide in a cell, the method comprising contacting the cell with an agent that: (a) increases the expression level of an mRNA encoding a CUL4- associated polypeptide in the cell; (b) increases the level of a CUL4-associated polypeptide in the cell; or (c) increases binding between a p53 polypeptide and a CUL4 E3 ligase complex; (d) increases the ubiquitination activity of a CUL4 ligase complex; (e) increases the binding between a CUL4 ligase complex and MDM2; (f) increases the binding between a CUL4 ligase complex and Pirh2; or (g) increases the activity of a CULl E3 ligase complex.

16. The method of claim 15, wherein CUL4 is CUL4A or CUL4B.

17. The method of claim 15, further comprising increasing the expression level or activity of at least one E2 conjugation enzyme.

18. The method of claim 17, wherein the E2 conjugation enzyme is selected from the group consisting of CDC34 E2, UbcH5B E2 and UbcH5C E2.

19. The method of claim 15, wherein the agent increases the polj ibiquitination of p53.

20. The method of claim 15, wherein the p53 is mammalian p53.

21. The method of claim 7b, wherein the mammalian p53 is human, mouse, rat, guinea pig monkey, monkey, hamster, dog, cat, cow, pig or sheep p53.

23. The method of claim 15, wherein the CUL4-associated polypeptide is selected from the group consisting of CUL4A, CUL4B, DDBl, L2DTL, ROCl, ROC2, MDM2 and Pirh2.

24. The method of claim 15, wherein the CUL4 ligase complex comprises one or more porypeptides selected from the group consisting of CUL4A, CUL4B, DDBl, L2DTL, ROCl, ROC2, MDM2 and Pirh2.

25. A method of increasing the level of a p53 polypeptide in a cell, the method comprising contacting the cell with an agent that: (a) decreases the expression level of an mRNA encoding a CUL4- associated polypeptide in the cell; (b) decreases the level of a CUL4-associated polypeptide in the cell; (c) decreases binding between a p53 polypeptide and a CUL4 E3 ligase complex; (d) decreases the ubiquitination activity of a CUL4 ligase complex; (e) decreases the binding between a CUL4 ligase complex and MDM2; (f) decreases the binding between a CUL4 ligase complex and Pirh2; or (g) decreases the activity of a CULl E3 ligase complex.

26. The method of claim 25, wherein CUL4 is CUL4A or CUL4B.

27. The method of claim 25, wherein the CUL4-associated polypeptide is selected from the group consisting of CUL4A, CUL4B, DDBl, L2DTL, ROCl, ROC2, MDM2 and Pirh2.

28. The method of claim 25, wherein the agent decreases the polj ibiquitination of p53.

29. The method of claim 25, wherein the p53 is human p53.

30. The method of claim 25, wherein the cell is in a vertebrate.

31. The method of claim 30, wherein the vertebrate is in a mammal.

32. The method of claim 31 , wherein the mammal is a human.

33. The method of claim 32, wherein the human has a tumor or cancer.

34. The method of claim 25, wherein the CUL4 ligase complex comprises one or more polypeptides selected from the group consisting of CUL4A, CUL4B, DDBl, L2DTL, ROCl, ROC2, MDM2 and Pirh2.