The p53 tumor suppressor is activated after DNA damage to maintain genomic stability and prevent transformation. Rapid activation of p53 by ionizing radiation is dependent on signaling by the ATM kinase. MDM2 and MDMX are important p53 regulators and logical targets for stress signals. We found that DNA damage induces ATMdependent phosphorylation and degradation of MDMX. Phosphorylated MDMX is selectively bound and degraded by MDM2 preceding p53 accumulation and activation. Reduction of MDMX level by RNAi enhances p53 response to DNA damage. Loss of ATM prevents MDMX degradation and p53 stabilization after DNA damage. Phosphorylation of MDMX on S342, S367, and S403 were detected by mass spectrometric analysis, with the first two sites confirmed by phosphopeptide-specific antibodies. Mutation of MDMX on S342, S367, and S403 each confers partial resistance to MDM2-mediated ubiquitination and degradation. Phosphorylation of S342 and S367 in vivo require the Chk2 kinase. Chk2 also stimulates MDMX ubiquitination and degradation by MDM2. Therefore, the E3 ligase activity of MDM2 is redirected to MDMX after DNA damage and contributes to p53 activation.

The p53 tumor suppressor is regulated by MDM2-mediated ubiquitination and degradation. Mitogenic signals activate p53 by induction of ARF expression, which inhibits p53 ubiquitination by MDM2. Recent studies showed that the MDM2 homolog MDMX is also an important regulator of p53. We present evidence that MDM2 promotes MDMX ubiquitination and degradation by the proteasomes. This effect is stimulated by ARF and correlates with the ability of ARF to bind MDM2. Promotion of MDM2-mediated MDMX ubiquitination requires the N-terminal domain of ARF, which normally inhibits MDM2 ubiquitination of p53. An intact RING domain of MDM2 is also required, both to interact with MDMX and to provide E3 ligase function. Increase of MDM2 and ARF levels by DNA damage, recombinant ARF adenovirus infection, or inducible MDM2 expression leads to proteasome-mediated down-regulation of MDMX levels. Therefore, MDMX and MDM2 are coordinately regulated by stress signals. The ARF tumor suppressor differentially regulates the ability of MDM2 to promote p53 and MDMX ubiquitination and activates p53 by targeting both members of the MDM2 family.The p53 protein is regulated by multiple mechanisms, which is critical for its ability to respond to stress and function as a tumor suppressor. p53 turnover is regulated by MDM2, which functions as a ubiquitin E3 ligase to promote p53 ubiquitination and degradation by proteasomes (33). Stress signals such as DNA damage induce p53 accumulation by phosphorylation (22). Mitogenic signals activate p53 by induction of the ARF tumor suppressor encoded by an alternative open reading frame in the p16INK4a locus, which inhibits the ability of MDM2 to ubiquitinate p53 (24,30,33).MDMX is a recently identified homolog of MDM2 (28). MDMX shares strong homology to MDM2 at the amino acid sequence level and can bind to p53 and inhibit its transcription function in transient-transfection assays. However, unlike MDM2, MDMX does not promote p53 ubiquitination or degradation in vivo (9, 29). Furthermore, expression of MDMX is not induced by DNA damage (23,28). MDM2 is well established as an important regulator of p53 activity during embryonic development. Knock out of MDM2 in mice results in embryonic lethality due to hyperactivation of p53 (20). However, recent studies showed that MDMX-null mice also die in utero in a p53-dependent fashion, which can be rescued by crossing into the p53-null background (5,19,21). Therefore, MDMX is also an important regulator of p53 during embryonic development, having a function that cannot be substituted by endogenous MDM2.MDM2 is a ubiquitin E3 ligase that promotes ubiquitination of itself, p53, and several other cellular proteins, including androgen receptor, Tip60, glucocorticoid receptor, and beta 2-adrenergic receptor (12,14,25,27). Ubiquitination of p53 and MDM2 itself requires the integrity of the C-terminal RING domain, which in other RING-containing E3 ligases is involved in direct interaction with the E2 ubiquitin-conjugating enzyme (11). Efficient ubiquitination of p53 also requi...

Breast cancer resistance protein (BCRP/ABCG2) is a molecular determinant of pharmacokinetic properties of many drugs in humans. To understand post-transcriptional regulation of ABCG2 and the role of microRNAs (miRNAs) in drug disposition, we found that microRNA-328 (miR-328) might readily target the 3Ј-untranslated region (3Ј-UTR) of ABCG2 when considering target-site accessibility. We then noted 1) an inverse relation between the levels of miR-328 and ABCG2 in MCF-7 and MCF-7/MX100 breast cancer cells and 2) that miR-328 levels could be rescued in MCF-7/MX100 cells by transfection with miR-328 plasmid. Luciferase reporter assays showed that ABCG2 3Ј-UTR-luciferase activity was decreased more than 50% in MCF-7/MX100 cells after transfection with miR-328 plasmid, the activity was increased over 100% in MCF-7 cells transfected with a miR-328 antagomir, and disruption of miR-328 response element within ABCG2 3Ј-UTR led to a 3-fold increase in luciferase activity. Furthermore, the level of ABCG2 protein was down-regulated when miR-328 was over-expressed, and the level was up-regulated when miR-328 was inhibited by selective antagomir. Altered ABCG2 protein expression was associated with significantly declined or elevated levels of ABCG2 3Ј-UTR and coding sequence mRNAs, suggesting possible involvement of the mechanism of mRNA cleavage. Finally, miR-328-directed down-regulation of ABCG2 expression in MCF-7/MX100 cells resulted in an increased mitoxantrone sensitivity, as manifested by a significantly lower IC 50 value (2.46 Ϯ 1.64 M) compared with the control (151 Ϯ 32 M). Together, these findings suggest that miR-328 targets ABCG2 3Ј-UTR and, consequently, controls ABCG2 protein expression and influences drug disposition in human breast cancer cells.Breast cancer resistance protein BCRP/ABCG2 is an ATPbinding cassette membrane transporter expressed ubiquitously in humans, controlling the absorption, distribution and clearance of numerous xenobiotics, including pharmaceutical agents, dietary carcinogens and conjugated metabolites (Mao and Unadkat, 2005;van Herwaarden and Schinkel, 2006;Vore and Leggas, 2008). In addition, overexpression of ABCG2 and other drug transporters in tumorigenic stem cells represents an important mechanism for multidrug resistance (Dean et al., 2005). Because ABCG2 was discovered in drugresistant human cancer cells (e.g., MCF-7/AdrVp and S1MI80), these cell lines have been widely used for studying the function and regulation of ABCG2 and defining its role in drug disposition and multidrug resistance. In particular, gene amplification (Ross et al., 1999;Knutsen et al., 2000;Volk et al., 2002) has been shown to be an important mechanism for elevated ABCG2 expression in drug-resistant cancer cells. Recent studies have demonstrated that transcriptional factors [i.e., nuclear receptors (Ee et al.,