Inherently disparate cell growth and division, which are intimately coupled through a delicate network of intracellular and extracellular signaling, require ribosomal biogenesis. A number of events imparting instability to ribosomal biogenesis can cause nucleolar stress. In response to this stress, several ribosomal proteins bind to MDM2 and block MDM2-mediated p53 ubiquitination and degradation, resulting in p53-dependent cell cycle arrest. By doing so, the ribosomal proteins play a crucial role in connecting deregulated cell growth with inhibition of cell division. The ribosomal protein-MDM2-p53 signaling pathway provides a molecular switch that may constitute a surveillance network monitoring the integrity of ribosomal biogenesis.

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.

Although ribosomal proteins are known for playing an essential role in ribosome assembly and protein translation, their ribosome-independent functions have also been greatly appreciated. Over the past decade, more than a dozen of ribosomal proteins have been found to activate the tumor suppressor p53 pathway in response to ribosomal stress. In addition, these ribosomal proteins are involved in various physiological and pathological processes. This review is composed to overview the current understanding of how ribosomal stress provokes the accumulation of ribosome-free ribosomal proteins, as well as the ribosome-independent functions of ribosomal proteins in tumorigenesis, immune signaling, and development. We also propose the potential of applying these pieces of knowledge to the development of ribosomal stress-based cancer therapeutics.

The oncoprotein MDM2 associates with ribosomal proteins L5, L11, and L23. Both L11 and L23 have been shown to activate p53 by inhibiting MDM2-mediated p53 suppression. Here we have shown that L5 also activates p53. Overexpression of L5 stabilized ectopic p53 in H1299 cells and endogenous p53 in U2OS cells. Consequently, L5 enhanced p53 transcriptional activity and induced p53-dependent G 1 cell cycle arrest. Furthermore, like L11 and L23, L5 also remarkably inhibited MDM2-mediated p53 ubiquitination. The interaction of L5 with MDM2 was also enhanced by treatment with a low dose of actinomycin D. Actinomycin D-induced p53 was inhibited by small interference RNA against L5. By reciprocal co-immunoprecipitation, we further showed that there were at least two MDM2-ribosomal protein complexes in cells: MDM2-L5-L11-L23 and p53-MDM2-L5-L11-L23. We propose that the MDM2-L5-L11-L23 complex functions to inhibit MDM2-mediated p53 ubiquitination and thus activates p53.

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 protein p73 is a structural and functional homologue of the p53 tumour-suppressor protein but, unlike p53, it is not induced in response to DNA damage. The tyrosine kinase c-Abl is activated by certain DNA-damaging agents and contributes to the induction of programmed cell death (apoptosis) by p53-dependent and p53-independent mechanisms. Here we show that c-Abl binds to p73 in cells, interacting through its SH3 domain with the carboxy-terminal homo-oligomerization domain of p73. c-Abl phosphorylates p73 on a tyrosine residue at position 99 both in vitro and in cells that have been exposed to ionizing radiation. Our results show that c-Abl stimulates p73-mediated transactivation and apoptosis. This regulation of p73 by c-Abl in response to DNA damage is also demonstrated by a failure of ionizing-radiation-induced apoptosis after disruption of the c-Abl-p73 interaction. These findings show that p73 is regulated by a c-Abl-dependent mechanism and that p73 participates in the apoptotic response to DNA damage.

The newly identified p53 homolog p73 can mimic the transcriptional activation function of p53. We investigated whether p73, like p53, participates in an autoregulatory feedback loop with MDM2. p73 bound to MDM2 both in vivo and in vitro. Wild-type but not mutant MDM2, expressed in human p53 null osteosarcoma Saos-2 cells, inhibited p73- and p53-dependent transcription driven by the MDM2 promoter-derived p53RE motif as measured in transient-transfection and chloramphenicol acetyltransferase assays and also inhibited p73-induced apoptosis in p53-null human lung adenocarcinoma H1299 cells. MDM2 did not promote the degradation of p73 but instead disrupted the interaction of p73, but not of p53, with p300/CBP by competing with p73 for binding to the p300/CBP N terminus. Both p73alpha and p73beta stimulated the expression of the endogenous MDM2 protein. Hence, MDM2 is transcriptionally activated by p73 and, in turn, negatively regulates the function of this activator through a mechanism distinct from that used for p53 inactivation.

The two forms of RNA polymerase H that exist in vivo, phosphorylated (HO) and nonphosphorylated (HA), were purified to apparent homogeneity from HeLa cells. The nonphosphorylated form preferentially binds to the preinitiation complex. RNA polymerase H in the complex was converted by a cellular protein kinase to the phosphorylated form.Purified RNA polymerase II cannot accurately initiate transcription from class II promoters in vitro unless it is supplemented with general transcription initiation factors (1-3). Seven human general transcription factors (TFIIA, -IIB, -IID, -lIE, -IIF, -IIG, and -IIH) that, together with RNA polymerase II are sufficient for specific transcription, have been identified (O.F. and D.R., unpublished results).Despite progress in the purification of the general transcription factors, determination of their individual activities and contributions to the formation of a transcriptioncompetent complex remains obscure. Complicating these analyses is the existence in eukaryotic cells of two different forms of RNA polymerase II, IIA and IIO, which differ in the level of phosphorylation of a highly conserved heptapeptide repeat present at the carboxyl terminus of the largest subunit (carboxyl-terminal domain; CTD) (4-6). The heptapeptide repeat is essential for viability (7-10); nevertheless, a third species ofRNA polymerase II lacking the CTD (IIB) has been observed in vitro (11)(12)(13)(14). Photoaffinity labeling experiments demonstrated that nascent RNA transcripts crosslink almost exclusively to the phosphorylated IIO form in vivo and in vitro (5,15,16), suggesting that it is the IIO polymerase that elongates RNA chains. Monoclonal antibodies against the nonphosphorylated IIA form inhibited specific transcription initiation (17, 18), suggesting that RNA polymerase IIA was more active than IIO during specific transcription initiation in vitro. Therefore, the CTD phosphorylation state may regulate the transition from initiation to elongation (19).We have purified human RNA polymerases IIO and IIA to apparent homogeneity and analyzed their roles in transcription. We demonstrate that the IIA form associates preferentially with the preinitiation complex where it is then converted by a cellular protein kinase to the phosphorylated 11O form. MATERIALS AND METHODSTranscription Factors, Transcription Reaction Mixtures, and DNA Binding Assays. Transcription reactions and DNA binding assays were performed as described (20). Transcription factor IIA (TFIIA) (P. Cortes and D.R., unpublished data), TFIIB (22), TFIIE (23), and TFIIF (24) were purified from HeLa cell nuclear extracts as described. TFIID was purified to homogeneity as described (20) Purification of the Phosphorylated (HO) and Nonphosphorylated (HA) Forms of RNA Polymerase H. RNA polymerase II was purified from HeLa cell nuclear pellets (7.5 x 1010 cells). Enzyme solubilization and chromatography on DE-52 were as described (26). Active fractions ofthe DE-52 column, between 0.2 and 0.3 M salt, were pooled (109 mg of protein, 170 ml...