Expression of the p53 Gene and Activation of p53-Dependent Transcription in Melanoma Cell Lines

Malignant melanoma has poor prognosis because of its high metastatic potential and resistance to chemotherapy. A possible approach to more effective therapy is induction of p53-dependent apoptosis. This approach is promising, since the wild-type _p53 is expressed in most melanomas. An attempt was made to estimate the functional activity of p53 in several malignant melanoma cell lines. Most lines were characterized by a high protein level and nuclear localization of p53. All cell lines expressing the wild-type p53 showed stabilization of p53, its translocation into the nucleus, and activation of several target genes in response to DNA-damaging agents, suggesting that p53 was functionally active. A high-molecular-weight protein localized in the cytoplasm and mimicking a p53 epitope was found in several cell lines. It was shown that the DO-1 epitope is not derived from p53, ruling out cytoplasmic retention of p53 in melanoma cell lines. A mechanism of camptothecin-induced stabilization of p53 by decreasing the level of the HDM2 mRNA was described for melanoma cells but not for normal melanocytes, suggesting a differential effect of camptothecin on tumor-derived and primary cells.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 445–456.Original Russian Text Copyright © 2005 by Razorenova, Agapova, Chumakov.     

Phosphorylation of p53 by TAF1 Inactivates p53-Dependent Transcription in the DNA Damage Response

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Identification of p53 Sequence Elements That Are Required for MDM2-Mediated Nuclear Export

It has been demonstrated that MDM2 can differentially regulate subcellular distribution of p53 and its close structural homologue p73. In contrast to MDM2-mediated p53 nuclear export, p73 accumulates in the nucleus as aggregates that colocalize with MDM2. Distinct distribution patterns of p53 and p73 suggest the existence of unique structural elements in the two homologues that determine their MDM2-mediated relocalization in the cell. Using a series of p53/p73 chimeric proteins, we demonstrate that three regions of p53 are involved in the regulation of MDM2-mediated nuclear export. The DNA binding domain (DBD) is involved in the maintenance of a proper conformation that is required for functional activity of the nuclear export sequence (NES) of p53. The extreme C terminus of p53 harbors several lysine residues whose ubiquitination by MDM2 appears to be the initial event in p53 nuclear export, as evidenced by the impaired nucleocytoplasmic shuttling of p53 mutants bearing simultaneous substitutions of lysines 370, 372, 373, 381, 382, and 386 to arginines (6KR) or alanines (6KA). Finally, the region between the DBD and the oligomerization domain of p53, specifically lysine 305, also plays a critical role in fully revealing p53NES. We conclude that MDM2-mediated nuclear export of p53 depends on a series of ubiquitination-induced conformational changes in the p53 molecule that lead to the activation of p53NES. In addition, we demonstrate that the p53NES may be activated without necessarily disrupting the p53 tetramer.     

p53-Dependent subcellular proteome localization following DNA damage

The nucleolus is involved in regulating several aspects of stress responses and cell cycle arrest through the tumor suppressor p53. Under normal conditions, p53 is a short-lived protein that is present in cells at a barely detectable level. Upon exposure of cells to various forms of exogenous stress, such as DNA damage, there is a stabilization of p53 which is then responsible for an ensuing cascade of events. To further investigate the effect of p53 activation, we used a MS-based proteomics method to provide an unbiased, quantitative and high-throughput approach for measuring the subcellular distribution of the proteome that is dependent on p53. The spatial proteomics method analyses a whole cell extract created by recombining differentially labeled subcellular fractions derived from cells in which proteins have been mass labeled with heavy isotopes [Boisvert, F.-M., Lam, Y. W., Lamont, D., Lamond, A. I., Mol. Cell. Proteomics 2010, 9, 457-470]. This was used here to measure the relative distribution between cytoplasm, nucleus and nucleolus of around 2000 proteins in HCT116 cells that are either expressing wild-type p53 or null for p53. Spatial proteomics also facilitates a proteome-wide comparison of changes in protein localization in response to a wide range of physiological and experimental perturbations. We used this method to study differences in protein localization in HCT116 cells either with or without p53, and studied the differences in cellular response to DNA damage following treatment of HCT116 cells with etoposide in both p53 wild-type and null genetic backgrounds.     

FlhF and Its GTPase Activity Are Required for Distinct Processes in Flagellar Gene Regulation and Biosynthesis in Campylobacter jejuni

FlhF proteins are putative GTPases that are often necessary for one or more steps in flagellar organelle development in polarly flagellated bacteria. In Campylobacter jejuni, FlhF is required for σ⁵⁴-dependent flagellar gene expression and flagellar biosynthesis, but how FlhF influences these processes is unknown. Furthermore, the GTPase activity of any FlhF protein and the requirement of this speculated activity for steps in flagellar biosynthesis remain uncharacterized. We show here that C. jejuni FlhF hydrolyzes GTP, indicating that these proteins are GTPases. C. jejuni mutants producing FlhF proteins with reduced GTPase activity were not severely defective for σ⁵⁴-dependent flagellar gene expression, unlike a mutant lacking FlhF. Instead, these mutants had a propensity to lack flagella or produce flagella in improper numbers or at nonpolar locations, indicating that GTP hydrolysis by FlhF is required for proper flagellar biosynthesis. Additional studies focused on elucidating a possible role for FlhF in σ⁵⁴-dependent flagellar gene expression were conducted. These studies revealed that FlhF does not influence production of or signaling between the flagellar export apparatus and the FlgSR two-component regulatory system to activate σ⁵⁴. Instead, our data suggest that FlhF functions in an independent pathway that converges with or works downstream of the flagellar export apparatus-FlgSR pathway to influence σ⁵⁴-dependent gene expression. This study provides corroborative biochemical and genetic analyses suggesting that different activities of the C. jejuni FlhF GTPase are required for distinct steps in flagellar gene expression and biosynthesis. Our findings are likely applicable to many polarly flagellated bacteria that utilize FlhF in flagellar biosynthesis processes.Flagellar biosynthesis in bacteria is a complex process that requires expression of more than 50 genes in a sequential manner to ensure that the encoded proteins are secreted and interact in a proper order to construct a flagellar organelle (8). Formation of a flagellum to impart swimming motility is often an essential determinant for many bacteria to infect hosts or reside in an environmental niche. As such, flagella and flagellar motility are required for Campylobacter jejuni to initiate and maintain a harmless intestinal colonization in many wild and agriculturally important animals (16, 17, 19, 35, 47, 49), which leads to large reservoirs of the bacterium in the environment and the human food supply (13). In addition, flagellar motility is essential for the bacterium to infect human hosts to cause a diarrheal disease, which can range from a mild, watery enteritis to a severe, bloody diarrheal syndrome (4). Due to its prevalence in nature and in the food supply, C. jejuni is a leading cause of enteritis in humans throughout the world (7).C. jejuni belongs to a subset of motile bacteria that produce polarly localized flagella, which includes important pathogens of humans, such as Helicobacter, Vibrio, and Pseudomonas species. These bacteria have some commonalities in mechanisms for flagellar gene expression and biosynthesis, such as using both alternative σ factors, σ²⁸ and σ⁵⁴, for expression of distinct sets of flagellar genes (1, 6, 9, 11, 18, 20-22, 26, 36, 40, 44, 45, 49). In addition, these bacteria produce the putative FlhF GTPase, which is required in each bacterium for at least one of the following: expression of a subset of flagellar genes, biosynthesis of flagella, or the polar placement of the flagella. For instance, FlhF is required for expression of some σ⁵⁴- and σ²⁸-dependent flagellar genes and for production of flagella in the classical biotype of Vibrio cholerae (10). However, V. cholerae flhF mutants of another biotype can produce a flagellum in a minority of cells, but the flagellum is at a lateral site (14). Similar lateral flagella were found in flhF mutants of Pseudomonas aeruginosa and Pseudomonas putida (34, 37). FlhF of Vibrio alginolyticus may also be involved in the polar formation of flagella and may possibly influence the number of flagella produced (28, 29). Demonstration that FlhF is polarly localized in some of these species and the fact that FlhF has been observed to assist the early flagellar MS ring protein, FliF, in localizing to the old pole in one biotype of V. cholerae give credence that FlhF may be involved in the polar placement of flagella in the respective organisms (14, 29, 34).Bioinformatic analysis indicates that the FlhF proteins belong to the SIMIBI class of NTP-binding proteins (30). More specifically, the GTPase domains of FlhF proteins are most similar to those of the signal recognition particle (SRP) pathway GTPases, such as Ffh and FtsY. Because of the homology of the GTPase domains, these three proteins may form a unique subset within the SIMIBI proteins. Whereas the GTPase activities of the interacting Ffh and FtsY proteins have been extensively characterized (32, 38, 39, 42), little is known about the GTP hydrolysis activity of FlhF. Structural determination of FlhF of Bacillus subtilis indicates that the potential GTPase activity of FlhF is likely varied relative to those of Ffh and FtsY (2). However, no biochemical analysis has been performed to verify or characterize the ability of an FlhF protein to hydrolyze GTP. As such, no studies have correlated the biochemical activity of FlhF in relation to GTP hydrolysis with the role that FlhF performs in flagellar gene expression or biosynthesis.Through previous work, we have delineated the regulatory cascades governing flagellar gene expression in C. jejuni. We have found that formation of the flagellar export apparatus (FEA), a multiprotein inner membrane complex (consisting of the proteins FlhA, FlhB, FliF, FliO, FliP, FliQ, and FliR) that secretes most of the flagellar proteins out of the cytoplasm to form the flagellum, is required to activate the FlgS sensor kinase to begin a phosphorelay to the cognate FlgR response regulator (23, 24). Once activated by phosphorylation, FlgR likely interacts with σ⁵⁴ in RNA polymerase to initiate expression of many flagellar genes encoding components of the flagellar basal body, rod, and hook (20, 24). After formation of the hook, flaA, encoding the major flagellin, is expressed via σ²⁸ and RNA polymerase to generate the flagellar filament and complete flagellar biosynthesis (6, 18, 20, 21, 49). In two separate genetic analyses, we found that flhF mutants of C. jejuni are nonmotile and show a more than 10-fold reduction in expression of σ⁵⁴-dependent flagellar genes, indicating that FlhF is required for both flagellar gene expression and biosynthesis (20). However, it is unclear how FlhF influences expression of σ⁵⁴-dependent flagellar genes. Furthermore, it is unknown if the GTPase activity of FlhF is required for flagellar gene expression or biosynthesis in C. jejuni.We have performed experiments to determine that C. jejuni FlhF specifically hydrolyzes GTP, confirming that FlhF is a GTPase. Whereas the FlhF protein is required for motility, flagellar biosynthesis, and expression of σ⁵⁴-dependent flagellar genes, the GTPase activity of the protein significantly influences only proper biosynthesis of flagella. These results suggest that multiple biochemical activities of FlhF (including GTPase activity and likely other, as yet uncharacterized activities mediated by other domains) are required at distinct steps in flagellar gene expression and biosynthesis. In addition, we provide biochemical and genetic evidence that FlhF likely functions in a pathway separate from the FEA-FlgSR pathway in C. jejuni to influence expression of σ⁵⁴-dependent flagellar genes. This study provides corroborative genetic and biochemical analysis of FlhF to indicate that FlhF has multiple inherent activities that function at different steps in development of the flagellar organelle, which may be applicable to many polarly flagellated bacteria.     

Insertional Mutagenesis and Deep Profiling Reveals Gene Hierarchies and a Myc/p53-Dependent Bottleneck in Lymphomagenesis

Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC, -Runx2) develop rapid onset tumours that can be accelerated and rendered polyclonal by neonatal Moloney murine leukaemia virus (MoMLV) infection. RIM/DS analysis of 28 polyclonal lymphomas identified 771 common insertion sites (CISs) defining a ‘progression network’ that encompassed a remarkably large fraction of known MoMLV target genes, with further strong indications of oncogenic selection above the background of MoMLV integration preference. Progression driven by RIM was characterised as a Darwinian process of clonal competition engaging proliferation control networks downstream of cytokine and T-cell receptor signalling. Enhancer mode activation accounted for the most efficiently selected CIS target genes, including Ccr7 as the most prominent of a set of chemokine receptors driving paracrine growth stimulation and lymphoma dissemination. Another large target gene subset including candidate tumour suppressors was disrupted by intragenic insertions. A second RIM/DS screen comparing lymphomas of wild-type and parental transgenics showed that CD2-MYC tumours are virtually dependent on activation of Runx family genes in strong preference to other potent Myc collaborating genes (Gfi1, Notch1). Ikzf1 was identified as a novel collaborating gene for Runx2 and illustrated the interface between integration preference and oncogenic selection. Lymphoma target genes for MoMLV can be classified into (a) a small set of master regulators that confer self-renewal; overcoming p53 and other failsafe pathways and (b) a large group of progression genes that control autonomous proliferation in transformed cells. These findings provide insights into retroviral biology, human cancer genetics and the safety of vector-mediated gene therapy.