MDMX has emerged as a negative regulator of p53 transcriptional activity following DNA damage, loss of ribosomal integrity, and aberrant mitogenic signaling. Disruption of rRNA biogenesis by ribosomal stress activates p53 by releasing ribosomal proteins from nucleoli which bind MDM2 and inhibit p53 degradation. We found that p53 activation by ribosomal stress requires degradation of MDMX by MDM2. This occurs by L11 binding to the acidic domain of MDM2 which promotes its E3 ligase function preferentially towards MDMX. Further, unlike DNA damage which regulates MDMX stability through ATM-dependent phosphorylation events, ribosomal stress does not require MDMX phosphorylation suggesting p53 may be more sensitive to suppression by MDMX under these conditions. Indeed, we find that tumor cells overexpressing MDMX are less sensitive to ribosomal stress-induced growth arrest by the addition of actinomycin D due to formation of inactive p53-MDMX complexes that fail to transcriptionally activate downstream targets such as p21. Knockdown of MDMX increases sensitivity to actinomycin D, whereas MDMX overexpression abrogates p53 activation. Furthermore, MDMX expression promotes resistance to the chemotherapeutic agent 5-fluorouracil (5-FU), which at low concentrations activates p53 by inducing ribosomal stress without significant DNA damage signaling. Knockdown of MDMX abrogates HCT116 tumor xenograft formation in nude mice. MDMX overexpression does not accelerate tumor growth but increases resistance to 5-FU treatment in vivo. In addition to MDMX regulation at the protein level, we found that regulation of cellular MDMX levels, like MDM2, can occur at the transcriptional level by inducing the Ras/Raf/MEK/ERK pathway. We found MDMX levels in tumor cell lines closely correlate with promoter activity and mRNA level. Activated K-Ras and growth factor IGF-1 induce MDMX expression at the transcriptional level through mechanisms that involve the MAPK kinase and c-Ets-1 transcription factors. Pharmacological inhibition of MEK results in down-regulation of MDMX in tumor cell lines. MDMX overexpression is detected in ~50% of human colon tumors and showed strong correlation with increased Erk phosphorylation. Taken together, the data show that MDMX has multiple modes of regulation, which ultimately determine the overall extent of p53 activation.

SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...

SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...

PML is involved in many cellular processes. It organizes nuclear structures PML nuclear bodies (PML NBs) and it associates with nucleolus in response to ribosomal stress to form PML nucleolar associations (PNAs). The function of PNAs is unclear. To elucidate this question, one can attempt to identify proteins interacting with PML at nucleolus. The common method is co- immunoprecipitation, however, this approach cannot be used for PML due to its low solubility. To defeat this, an alternative way of proximity-dependent biotin labelling could be used. The goal of this work was to explore a suitability of biotin labelling for identification of PML nucleolar partners. For this purpose I prepared constructs of wild type or mutated PML with GFP and biotin ligase for transient and stable expression and analysed their propensity to form PML NBs and doxorubicin-induced PNAs, and biotinylate their vicinity. In transient expression, both fusion proteins formed PML NBs and only wild type but not mutated PML IV formed PNAs after doxorubicin treatment with preserved biotinylation capability. In stable expression of fusion proteins in cells with PML knockout the number and composition of PML NBs was aberrant and no PNAs were observed. However, this system was utilized for optimization of solubilisation of biotinylated...