PSD-95 is a major scaffolding protein of the postsynaptic density, tethering NMDA- and AMPA-type glutamate receptors to signaling proteins and the neuronal cytoskeleton. Here we show that PSD-95 is regulated by the ubiquitin-proteasome pathway. PSD-95 interacts with and is ubiquitinated by the E3 ligase Mdm2. In response to NMDA receptor activation, PSD-95 is ubiquitinated and rapidly removed from synaptic sites by proteasome-dependent degradation. Mutations that block PSD-95 ubiquitination prevent NMDA-induced AMPA receptor endocytosis. Likewise, proteasome inhibitors prevent NMDA-induced AMPA receptor internalization and synaptically induced long-term depression. This is consistent with the notion that PSD-95 levels are an important determinant of AMPA receptor number at the synapse. These data suggest that ubiquitination of PSD-95 through an Mdm2-mediated pathway is critical in regulating AMPA receptor surface expression during synaptic plasticity.

The p53-MDM2 feedback loop is vital for cell growth control and is subjected to multiple regulations in response to various stress signals. Here we report another regulator of this loop. Using an immunoaffinity method, we purified an MDM2-associated protein complex that contains the ribosomal protein L23. L23 interacted with MDM2, forming a complex independent of the 80S ribosome and polysome. The interaction of L23 with MDM2 was enhanced by treatment with actinomycin D but not by gamma-irradiation, leading to p53 activation. This activation was inhibited by small interfering RNA against L23. Ectopic expression of L23 reduced MDM2-mediated p53 ubiquitination and also induced p53 activity and G 1 arrest in p53-proficient U2OS cells but not in p53-deficient Saos-2 cells. These results reveal that L23 is another regulator of the p53-MDM2 feedback regulation.

The CDK inhibitor p21waf1/cip1 is degraded by a ubiquitin-independent proteolytic pathway. Here, we show that MDM2 mediates this degradation process. Overexpression of wild-type or ring finger-deleted, but not nuclear localization signal (NLS)-deleted, MDM2 decreased p21waf1/cip1 levels without ubiquitylating this protein and affecting its mRNA level in p53(-/-) cells. This decrease was reversed by the proteasome inhibitors MG132 and lactacystin, by p19(arf), and by small interfering RNA (siRNA) against MDM2. p21waf1/cip1 bound to MDM2 in vitro and in cells. The p21waf1/cip1-binding-defective mutant of MDM2 was unable to degrade p21waf1/cip1. MDM2 shortened the half-life of both exogenous and endogenous p21waf1/cip1 by 50% and led to the degradation of its lysine-free mutant. Consequently, MDM2 suppressed p21waf1/cip1-induced cell growth arrest of human p53(-/-) and p53(-/-)/Rb(-/-)cells. These results demonstrate that MDM2 directly inhibits p21waf1/cip1 function by reducing p21waf1/cip1 stability in a ubiquitin-independent fashion.

Inhibition of the MDM2-p53 feedback loop is critical for p53 activation in response to cellular stresses. The ribosomal proteins L5, L11, and L23 can block this loop by inhibiting MDM2-mediated p53 ubiquitination and degradation in response to ribosomal stress. Here, we show that L11, but not L5 and L23, leads to a drastic accumulation of ubiquitinated and native MDM2. This effect is dependent on the ubiquitin ligase activity of MDM2, but not p53, and requires the central MDM2 binding domain (residues 51-108) of L11. We further show that L11 inhibited 26 S proteasomemediated degradation of ubiquitinated MDM2 in vitro and consistently prolonged the half-life of MDM2 in cells. These results suggest that L11, unlike L5 and L23, differentially regulates the levels of ubiquitinated p53 and MDM2 and inhibits the turnover and activity of MDM2 through a postubiquitination mechanism.The tumor suppressor protein p53 is a transcription factor activated in response to stress to induce expression of its target genes. The proteins encoded by these genes then mediate multiple cellular responses, such as cell cycle arrest, apoptosis, differentiation, cell senescence, or DNA repair (1). Also, p53 can directly trigger mitochondria-mediated apoptosis in response to DNA damage (2-4). The tumor suppression function of p53 is well reflected in the fact that more than half of human tumors harbor mutations in the p53 gene, and many others retain impaired function of the p53 pathway (5,6). Because of its inhibitory effect on cell growth, p53 is maintained at a low steady-state level and in an inert form in physiological conditions. This duty is mainly fulfilled by the E3 2 ubiquitin ligase MDM2 that mediates p53 constant degradation through a ubiquitin-dependent proteasome pathway (7,8). The mdm2 gene itself is a downstream target of p53, thus forming a tight autoregulatory feedback loop (9-11). Consistent with this notion, gene amplification and overexpression of MDM2 have also been shown in a variety of tumors, particularly in soft tissue sarcomas, lymphomas, and breast and lung cancers (12)(13)(14)(15)(16). Interfering with the MDM2-p53 feedback loop leads to p53 activation, ultimately preventing neoplasia. One example of this regulation is alternative reading frame (ARF) (p14 ARF in human, p19 ARF in mouse)-mediated inhibition of this loop in response to * This work was supported in part by NCI, National Institutes of Health Grants CA93614, CA095441, and CA079721 (to H. L.) and CA107532 (to S. R. G.). 1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, OR Health and Science University, 3181 SW Sam Jackson Park Rd., Portland,; E-mail: luh@ohsu.edu.. 2 The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; ARF, alternative reading frame; E1, ubiquitin activating enzyme (UBA); E2, ubiquitin carrier protein; HA, hemagglutinin; Ni-NTA, nickel nitrilotriacetic acid; GFP, green fluorescent protein; MEF, murine embryonic fibroblast cell. 18). Also, in response to DNAdamaging agen...

It has been shown that MDMX inhibits the activity of the tumor suppressor p53 by primarily cooperating with the p53 feedback regulator MDM2. Here, our study shows that this inhibition can be overcome by 14-3-3c and Chk1. 14-3-3c was identified as an MDMX-associated protein via an immuno-affinity purification-coupled mass spectrometry. Consistently, 14-3-3c directly interacted with MDMX in vitro, and this interaction was stimulated by MDMX phosphorylation in vitro and in cells. Interestingly, in response to UV irradiation, the wild-type, but not the kinase-dead mutant, Chk1 phosphorylated MDMX at serine 367, enhanced the 14-3-3c-MDMX binding and the cytoplasmic retaining of MDMX. The Chk1 specific inhibitor UCN-01 repressed all of these effects. Moreover, overexpression of 14-3-3c, but not its mutant K50E, which did not bind to MDMX, suppressed MDMX-enhanced p53 ubiquitination, leading to p53 stabilization and activation. Finally, ablation of 14-3-3c by siRNA reduced UV-induced p53 level and G1 arrest. Thus, these results demonstrate 14-3-3c and Chk1 as two novel regulators of MDMX in response to UV irradiation.

c-myc and p53 networks control proliferation, differentiation, and apoptosis and are responsive to, and cross-regulate a variety of stresses and metabolic and biosynthetic processes. At c-myc, the far upstream element binding protein (FBP) and FBP-interacting repressor (FIR) program transcription by looping to RNA polymerase II complexes engaged at the promoter. Another FBP partner, JTV1/AIMP2, a structural subunit of a multi-aminoacyl-tRNA synthetase (ARS) complex, has also been reported to stabilize p53 via an apparently independent mechanism. Here, we show that in response to oxidative stress, JTV1 dissociates from the ARS complex, translocates to the nucleus, associates with FBP and co-activates the transcription of a new FBP target, ubiquitin-specific peptidase 29 (USP29). A previously uncharacterized deubiquitinating enzyme, USP29 binds to, cleaves poly-ubiquitin chains from, and stabilizes p53. The accumulated p53 quickly induces apoptosis. Thus, FBP and JTV1 help to coordinate the molecular and cellular response to oxidative stress.

Background: How glucocorticoids affect ER-positive breast cancer cell proliferation is unclear.Results: GR occupies ERα-binding regions (EBRs) via tethering to AP1 and ERα.Conclusion: Interaction of GR with EBRs via ERα and AP1 inhibits E2-ERα activity.Significance: Breast tumors with ERα and AP1 expression will be responsive to glucocorticoid therapy; the study establishes a foundation for personalized medicine for BC.

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