Quantitative model for inferring dynamic regulation of the tumour suppressor gene p53

Background: The availability of various "omics" datasets creates a prospect of performing the study of genome-wide genetic regulatory networks. However, one of the major challenges of using mathematical models to infer genetic regulation from microarray datasets is the lack of information for protein concentrations and activities. Most of the previous researches were based on an assumption that the mRNA levels of a gene are consistent with its protein activities, though it is not always the case. Therefore, a more sophisticated modelling framework together with the corresponding inference methods is needed to accurately estimate genetic regulation from "omics" datasets. Results: This work developed a novel approach, which is based on a nonlinear mathematical model, to infer genetic regulation from microarray gene expression data. By using the p53 network as a test system, we used the nonlinear model to estimate the activities of transcription factor (TF) p53 from the expression levels of its target genes, and to identify the activation/inhibition status of p53 to its target genes. The predicted top 317 putative p53 target genes were supported by DNA sequence analysis. A comparison between our prediction and the other published predictions of p53 targets suggests that most of putative p53 targets may share a common depleted or enriched sequence signal on their upstream non-coding region. Conclusions: The proposed quantitative model can not only be used to infer the regulatory relationship between TF and its down-stream genes, but also be applied to estimate the protein activities of TF from the expression levels of its target genes

P19 H-Ras Induces G1/S Phase Delay Maintaining Cells in a Reversible Quiescence State

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Three functional c-ras genes, known as c-H-ras, c-K-ras, and c-N-ras, have been largely studied in mammalian cells with important insights into normal and tumorigenic cellular signal transduction events. Two K-Ras mRNAs are obtained from the same pre-mRNA by alternative splicing. H-Ras pre-mRNA can also be alternatively spliced in the IDX and 4A terminal exons, yielding the p19 and p21 proteins, respectively. However, despite the Ras gene family’s established role in tumorigenic cellular signal transduction events, little is known about p19 function. Previous results showed that p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multi-protein complexes in different signaling pathways (Cancer Res 2003, 63 p5178). This observation suggests that p19 and p21 play differential and complementary roles in the cell.[Principal Findings]: We found that p19 regulates telomerase activity through its interaction with p73a/b proteins. We also found that p19 overexpression induces G1/S phase delay; an observation that correlates with hypophosphorylation of both Akt and p70SK6. Similarly, we also observed that FOXO1 is upregulated when p19 is overexpressed. The three observations of (1) hypophosphorylation of Akt, (2) G1/S phase delay and (3) upregulation of FOXO1 lead us to conclude that p19 induces G1/S phase delay, thereby maintaining cells in a reversible quiescence state and preventing entry into apoptosis. We then assessed the effect of p19 RNAi on HeLa cell growth and found that p19 RNAi increases cell growth, thereby having the opposite effect of arrest of the G1/S phase or producing a cellular quiescence state.[Significance]: Interestingly, p19 induces FOXO1 that in combination with the G1/S phase delay and hypophosphorylation of both Akt and p70SK6 leads to maintenance of a reversible cellular quiescence state, thereby preventing entry into apoptosis.This work was supported by Fundacion de Investigacion Medica Mutua Madrileña Automovilista (Fundacion MMA), the Plan Nacional (MEC) BFU2005-00701 and the Fundacion Eugenio Rodriguez Pascual. M.C. was a recipient of a Fmed MMA fellowship.Peer reviewe

Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling

Mitochondrial Alterations in PINK1 Deficient Cells Are Influenced by Calcineurin-Dependent Dephosphorylation of Dynamin-Related Protein 1

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential

The P53 pathway : role of telomerase and identification of novel targets : acts of a master regulator of tumor suppression

A key role of the p53 protein in tumor suppression is reflected by its frequent mutations in human tumors. p53 is activated by various types stress conditions such as DNA damage, hypoxia and oncogene activation that results in p53-mediated cellular responses including DNA repair, cell cycle arrest, and apoptosis. p53 is a transcription factor that upon stabilization regulates expression of multiple genes involved in the above cellular responses. Thus, depiction of novel p53 targets is important for a better understanding of its tumor suppressive function. In addition, p53 also induces transcription-independent apoptotic activity. The catalytic subunit of human telomerase, hTERT involved in telomere length maintenance is repressed in normal cells but is highly expressed in most human tumors. We have previously shown that hTERT is downregulated upon activation of wtp53 suggesting that p53-mediated repression of hTERT is important for p53-induced apoptosis. We investigated the effects of constitutive expression of hTERT on p53-dependent apoptosis using two different cell systems, the BL41-tsp53 and HCT116 wtp53+/+ and wtp53-/-. We showed that hTERT expression antagonizes p53-induced apoptosis in both cells. This inhibitory effect was independent of hTERT telomerase activity as expression of catalytically inactive hTERT efficiently blocked apoptosis. To further explore the mechanism of hTERT anti-apoptotic function we studied the mitochondrial pathway and found that hTERT acts upstreams of the mitochondria. hTERT expression specifically inhibits activation of Bax. Thus, hTERT exerts its anti-apoptotic function by inhibition of Bax. Our data suggests that p53-dependent downregulation of hTERT is important for p53-induced Bax activation and apoptosis. Identification of novel p53 targets is important for elucidation of p53 function. Several studies have addressed p53-dependent gene expression by microarray analysis. However, analysis of p53-dependent expression at the protein level can help to identify targets that are regulated by transcription-independent mechanisms. We examined the effects of p53 activation on the proteome using 2D gel electrophoresis analysis of mitomycin C-treated HCT116 colon carcinoma cells and identified 55 novel proteins, differentially expressed in a p53-dependent manner by mass spectrometry. The proteins identified are involved in different cellular processes. Several of them lack putative p53-binding sites and thus are likely to be regulated independently of p53-mediated transcription. This could be due to posttranslational modifications such as phosphorylation of these targets. To further investigate this possibility, we analyzed p53-dependent phosphorylation of proteins using a fluorescent phosphoprotein dye and 2DE and mass spectrometry. Forty-four proteins showed changes in phosphorylation in a p53-dependent manner. This suggests that active p53 not only regulates gene expression but also triggers posttranslational modification of proteins. This thesis provides two new aspects of p53 function, namely the significance of p53-mediated downregulation of hTERT for induction of apoptosis and regulation of protein expression by different mechanisms that further demonstrate the multiple dimensions of p53’s role as tumor suppressor

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