A high-throughput screen measuring ubiquitination of p53 by human mdm2

Tumor suppressor p53 is typically maintained at low levels in normal cells. In response to cellular stresses, such as DNA damage, p53 is stabilized and can stimulate responses leading to cell cycle arrest or apoptosis. Corresponding to its central role in preventing propagation of damaged cells, mutation or deletion of p53 is found in nearly 50% of all human tumors. Mdm2 (mouse-d-minute 2) and its human ortholog (hmdm2 or hdm2) catalyze the ubiquitination of p53, targeting it for degradation via the proteosome. Thus, the activity of mdm2 is inversely correlated with p53 levels. Based on this, inhibition of human mdm2 activity by a small-molecule therapeutic will lead to net stabilization of p53 and be the basis for development of a novel cancer therapeutic. Previous high-throughput screening assays of mdm2 measured the autoubiquitination activity of mdm2, which occurs in the absence of an acceptor substrate such as p53. The major drawback to this approach is that inhibitors of mdm2 autoubiquitination may lead to a net stabilization of mdm2 and thus have the opposite effect of inhibitors that interfere with p53 ubiquitination. The authors describe the development, validation, and execution of a high-throughput screening measuring the ubiquitination of p53 by mdm2, with p53 labeled with europium and the other substrate (Ub-UbcH5b) labeled with a Cy5 on the ubiquitin. After confirming that known inhibitors are detected with this assay, it was successfully automated and used to query >600,000 compounds from the GlaxoSmithKline collection for mdm2 inhibitors.     

Human mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization

The mdm2 gene product is an important regulator of p53 function and stability. mdm2 is an E3 ubiquitin ligase for p53 and the RING finger domain of mdm2 is critical for ligase activity. Ubiquitin (Ub) conjugation is a general targeting modification and poly-ubiquitin chains specifically target proteins to the proteasome for degradation. In this report, we show that the multistep cascade of mdm2-mediated p53 ubiquitination can be reduced to three purified recombinant proteins: ubiquitin-conjugated E2, mdm2, and p53. This simplification allows enzymatic analysis of the isolated ligase reaction. The simplified reaction recapitulates the ubiquitination of p53 observed with individual components and the p53-Ub((n)) is qualitatively similar to p53-Ub((n)) detected in lactacystin-treated cells. Surprisingly, we find that p53 is modified with multiple mono-ubiquitin moieties as opposed to a poly-ubiquitin chain. Finally, kinetic analysis indicates the transfer reaction proceeds either through a modified Ping Pong mechanism involving requisite enzyme isomerization steps, or through a Rapid Equilibrium Random Bi Bi mechanism involving very large anti-cooperative interactions between the two substrate binding pockets on the enzyme, mediated through allosteric changes in enzyme structure.     

Arsenic disrupts cellular levels of p53 and mdm2: a potential mechanism of carcinogenesis.

The antitumor protein p53 plays a critical role in DNA repair. Inorganic arsenic exposure is associated with a wide variety of human tumors, particularly of the skin. To investigate how inorganic arsenic might interfere with DNA repair and lead to greater incidence of hyperkeratosis and skin tumors, we exposed human keratinocytes (HaCaT) to environmentally relevant concentrations of arsenite for 14 days. Arsenite reduced p53 levels while concomitantly increasing the p53 regulatory protein mdm2 levels in a dose- and time-dependent manner. We propose the disruption of the p53-mdm2 loop regulating cell cycle arrest as a model for arsenic-related skin carcinogenesis and it may be important in tumors with elevated mdm2 levels.     

Defective p53 post-translational modification required for wild type p53 inactivation in malignant epithelial cells with mdm2 gene amplification

Mdm2 gene amplification occurs in benign and chemotherapy-responsive malignant tumors with wtp53 genes as well as in breast and epithelial cancers. Mdm2 amplification in benign tumors suggests that it is not sufficient for p53 inactivation in cancer, implying that other defects in the p53 pathway are required for malignancy. We investigated mechanisms of wtp53 protein inactivation in malignant conversion of epithelial cells by comparing clonally related initiated cells with their derivative cancerous cells that have mdm2 amplification. Deficiencies in p53 accumulation and activities in response to DNA damage were not due simply to Mdm2 destabilization of p53 protein, but to continued association of DNA-bound p53 with Mdm2 protein and lack of binding and acetylation by p300 protein. The aberrant interactions were not because of mdm2 amplification alone, because DNA-bound p53 protein from initiated cells failed to bind ectopically expressed Mdm2 or endogenous overexpressed Mdm2 from cancerous cells. Phosphorylations of endogenous p53 at Ser18, -23, or -37 were insufficient to dissociate Mdm2, because each was induced by UV in cancerous cells. Interestingly, phospho-mimic p53-T21E did dissociate the Mdm2 protein from DNA-bound p53 and recovered p300 binding and p21 induction in the cancerous cells. Thus wtp53 in malignant cells with mdm2 amplification can be inactivated by continued association of DNA-bound p53 protein with Mdm2 and failure of p300 binding and acetylation, coupled with a defect in p53 phosphorylation at Thr21. These findings suggest therapeutic strategies that address both p53/Mdm2 interaction and associated p53 protein defects in human tumors that have amplified mdm2 genes.     

Structural details on mdm2-p53 interaction

Mdm2 is a cellular antagonist of p53 that keeps a balanced cellular level of p53. The two proteins are linked by a negative regulatory feedback loop and physically bind to each other via a putative helix formed by residues 18-26 of p53 transactivation domain (TAD) and its binding pocket located within the N-terminal 100-residue domain of mdm2 (Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A. J., and Pavletich, N. P. (1996) Science 274, 948-953). In a previous report we demonstrated that p53 TAD in the mdm2-freee state is mostly unstructured but contains two nascent turns in addition to a "preformed" helix that is the same as the putative helix mediating p53-mdm2 binding. Here, using heteronuclear multidimensional NMR methods, we show that the two nascent turn motifs in p53 TAD, turn I (residues 40-45) and turn II (residues 49-54), are also capable of binding to mdm2. In particular, the turn II motif has a higher mdm2 binding affinity ( approximately 20 mum) than the turn I and targets the same site in mdm2 as the helix. Upon mdm2 binding this motif becomes a well defined full helix turn whose hydrophobic face formed by the side chains of Ile-50, Trp-53, and Phe-54 inserts deeply into the helix binding pocket. Our results suggest that p53-mdm2 binding is subtler than previously thought and involves global contacts such as multiple "non-contiguous" minimally structured motifs instead of being localized to one small helix mini-domain in p53 TAD.     

Opposing effects of Ras on p53: transcriptional activation of mdm2 and induction of p19ARF

Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. Here, we show that the mdm2 gene is also regulated by the Ras-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdm2 induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19ARF. This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19ARF. Levels of p53 are therefore determined by opposing effects of Raf-induced p19ARF and Mdm2.     

Hoiamide D, a marine cyanobacteria-derived inhibitor of p53/MDM2 interaction

Bioassay-guided fractionation of two cyanobacterial extracts from Papua New Guinea has yielded hoiamide D in both its carboxylic acid and conjugate base forms. Hoiamide D is a polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS)-derived natural product that features two consecutive thiazolines and a thiazole, as well as a modified isoleucine residue. Hoiamide D displayed inhibitory activity against p53/MDM2 interaction (EC(50)=4.5 μM), an attractive target for anticancer drug development.     

SAC1 encodes a regulated lipid phosphoinositide phosphatase, defects in which can be suppressed by the homologous Inp52p and Inp53p phosphatases

The yeast protein Sac1p is involved in a range of cellular functions, including inositol metabolism, actin cytoskeletal organization, endoplasmic reticulum ATP transport, phosphatidylinositol-phosphatidylcholine transfer protein function, and multiple-drug sensitivity. The activity of Sac1p and its relationship to these phenotypes are unresolved. We show here that the regulation of lipid phosphoinositides in sac1 mutants is defective, resulting in altered levels of all lipid phos- phoinositides, particularly phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. We have identified two proteins with homology to Sac1p that can suppress drug sensitivity and also restore the levels of the phosphoinositides in sac1 mutants. Overexpression of truncated forms of these suppressor genes confirmed that suppression was due to phosphoinositide phosphatase activity within these proteins. We have now demonstrated this activity for Sac1p and have characterized its specificity. The in vitro phosphatase activity and specificity of Sac1p were not altered by some mutations. Indeed, in vivo mutant Sac1p phosphatase activity also appeared unchanged under conditions in which cells were drug-resistant. However, under different growth conditions, both drug sensitivity and the phosphatase defect were manifest. It is concluded that SAC1 encodes a novel lipid phosphoinositide phosphatase in which specific mutations can cause the sac1 phenotypes by altering the in vivo regulation of the protein rather than by destroying phosphatase activity.     

MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p, and Mad2p

We show that MAD3 encodes a novel 58-kD nuclear protein which is not essential for viability, but is an integral component of the spindle checkpoint in budding yeast. Sequence analysis reveals two regions of Mad3p that are 46 and 47% identical to sequences in the NH(2)-terminal region of the budding yeast Bub1 protein kinase. Bub1p is known to bind Bub3p (Roberts et al. 1994) and we use two-hybrid assays and coimmunoprecipitation experiments to show that Mad3p can also bind to Bub3p. In addition, we find that Mad3p interacts with Mad2p and the cell cycle regulator Cdc20p. We show that the two regions of homology between Mad3p and Bub1p are crucial for these interactions and identify loss of function mutations within each domain of Mad3p. We discuss roles for Mad3p and its interactions with other spindle checkpoint proteins and with Cdc20p, the target of the checkpoint.