Interaction of JLP with Plk1 recruits FoxK1 to interact and form a ternary complex

JLP (JNK associated Leucine zipper protein) is a scaffolding protein, which has been shown to interact with and activate JNK/p38MAPK pathway. Its interaction with various signaling proteins is associated with coordinated regulation of cellular process such as endocytosis, motility, neurite outgrowth, cell proliferation and apoptosis. Here we identified a mitotic Serine/Threonine kinase, Polo like kinase 1 (Plk1), as a novel interaction partner of JLP through a mass spectrometry based approach. We show that the N-terminal domain of JLP interacts with the polo-box domain (PBD) of Plk1 in a phosphorylation-dependent manner. Our results indicate that, JLP is phospho-primed on Thr 351 residue on its Nterminus, which is recognized by the PBD of Plk1 leading to phosphorylation of JLP at additional sites. Moreover, treatment of cells with the Plk1 inhibitor, BI2536 affects the interaction demonstrating the importance of Plk1 kinase activity in this process. Since JLP is a scaffolding protein that recruits proteins to mediate specific cell signaling events, we hypothesized that the interaction of JLP with Plk1 might result in the recruitment of other proteins to this complex. To test this hypothesis, we carried out SILAC labeling of proteins in mitotic cells in the presence or absence of BI2536. Through mass-spectrometry we identified the transcription factor, FoxK1 as a Plk1-dependent JLP-interacting protein. Furthermore, we show that JLP, Plk1 and FoxK1 form a ternary complex, which occurs only during mitosis. Knockdown of Plk1 and not JLP, affected the interaction between JLP and FoxK1 indicating that the formation of the ternary complex is dependent on Plk1. FoxK1 has been previously characterized as a transcriptional repressor of cyclin dependent kinase inhibitor, p21/WAF1. We observed that knockdown of JLP in U2OS cells results in increased protein levels of FoxK1 and a reduction of p21 expression. Moreover, immunofluorescence studies in asynchronous cells showed that FoxK1 is excluded from the nucleus during mitosis. Based on our observations, we propose that formation of the ternary complex between JLP, Plk1 and FoxK1 regulates the stability and/or localization of FoxK1

A Role for Non-Covalent SUMO Interaction Motifs in Pc2/CBX4 E3 Activity

Background: Modification of proteins by the small ubiquitin like modifier (SUMO) is an essential process in mammalian cells. SUMO is covalently attached to lysines in target proteins via an enzymatic cascade which consists of E1 and E2, SUMO activating and conjugating enzymes. There is also a variable requirement for non-enzymatic E3 adapter like proteins, which can increase the efficiency and specificity of the sumoylation process. In addition to covalent attachment of SUMO to target proteins, specific non-covalent SUMO interaction motifs (SIMs) that are generally short hydrophobic peptide motifs have been identified. Methodology/Principal Findings: Intriguingly, consensus SIMs are present in most SUMO E3s, including the polycomb protein, Pc2/Cbx4. However, a role for SIMs in SUMO E3 activity remains to be shown. We show that Pc2 contains two functional SIMs, both of which contribute to full E3 activity in mammalian cells, and are also required for sumoylation of Pc2 itself. Pc2 forms distinct sub-nuclear foci, termed polycomb bodies, and can recruit partner proteins, such as the corepressor CtBP. We demonstrate that mutation of the SIMs in Pc2 prevents Pc2-dependent CtBP sumoylation, and decreases enrichment of SUMO1 and SUMO2 at polycomb foci. Furthermore, mutational analysis of both SUMO1 and SUMO2 reveals that the SIM-interacting residues of both SUMO isoforms are required for Pc2-mediated sumoylation and localization to polycomb foci

A randomised controlled trial of oxygen therapy on growth and development of preterm infants

Background: Physiological studies have shown that many preterm infants and infants with chronic lung disease may suffer chronic hypoxaemia, which possibly leads to poor growth and development. Anecdotal reports indicate that there is a drive to increase the oxygen saturation target range to a higher level in these infants due primarily to perceived benefits derived from clinical experience and from uncontrolled observational studies of babies discharged on home oxygen. Objective The BOOST (Benefits Of Oxygen Saturation Targeting) trial is the first randomised trial to assess the long-term benefits and harms of two different oxygen saturation target ranges. Methods: BOOST was a multicentre, double blinded, randomised controlled trial that enrolled 358 infants born at less than 30 weeks� gestation who remained oxygen-dependent at 32 weeks postmenstrual age. They were randomly assigned to target either a functional oxygen saturation range of 91-94% (standard or control group) or 95-98% (higher or treatment group). The primary outcomes were growth and neurodevelopmental measures at 12 months corrected age. Secondary outcomes included length of hospital stay, retinopathy of prematurity, health service utilisation, parental stress, and infant temperament. Results: Prognostic baseline characteristics did not differ between the two groups. Mean birth weight and gestational age of enrolled infants was 917g and 26.5 weeks respectively. The rate of antenatal corticosteroid use was 83%

Narrative-based computational modelling of the Gp130/JAK/STAT signalling pathway.

BACKGROUND: Appropriately formulated quantitative computational models can support researchers in understanding the dynamic behaviour of biological pathways and support hypothesis formulation and selection by "in silico" experimentation. An obstacle to widespread adoption of this approach is the requirement to formulate a biological pathway as machine executable computer code. We have recently proposed a novel, biologically intuitive, narrative-style modelling language for biologists to formulate the pathway which is then automatically translated into an executable format and is, thus, usable for analysis via existing simulation techniques. RESULTS: Here we use a high-level narrative language in designing a computational model of the gp130/JAK/STAT signalling pathway and show that the model reproduces the dynamic behaviour of the pathway derived by biological observation. We then "experiment" on the model by simulation and sensitivity analysis to define those parameters which dominate the dynamic behaviour of the pathway. The model predicts that nuclear compartmentalisation and phosphorylation status of STAT are key determinants of the pathway and that alternative mechanisms of signal attenuation exert their influence on different timescales. CONCLUSION: The described narrative model of the gp130/JAK/STAT pathway represents an interesting case study showing how, by using this approach, researchers can model biological systems without explicitly dealing with formal notations and mathematical expressions (typically used for biochemical modelling), nevertheless being able to obtain simulation and analysis results. We present the model and the sensitivity analysis results we have obtained, that allow us to identify the parameters which are most sensitive to perturbations. The results, which are shown to be in agreement with existing mathematical models of the gp130/JAK/STAT pathway, serve us as a form of validation of the model and of the approach itself

Regulation of expression of the rat orthologue of mouse double minute 2 (MDM2) by H2O2-induced oxidative stress in neonatal rat cardiac myocytes.

The Mdm2 ubiquitin ligase is an important regulator of p53 abundance and p53-dependent apoptosis. Mdm2 expression is frequently regulated by a p53 Mdm2 autoregulatory loop whereby p53 stimulates Mdm2 expression and hence its own degradation. Although extensively studied in cell lines, relatively little is known about Mdm2 expression in heart where oxidative stress (exacerbated during ischemia-reperfusion) is an important pro-apoptotic stimulus. We demonstrate that Mdm2 transcript and protein expression are induced by oxidative stress (0.2 mm H(2)O(2)) in neonatal rat cardiac myocytes. In other cells, constitutive Mdm2 expression is regulated by the P1 promoter (5' to exon 1), with inducible expression regulated by the P2 promoter (in intron 1). In myocytes, H(2)O(2) increased Mdm2 expression from the P2 promoter, which contains two p53-response elements (REs), one AP-1 RE, and two Ets REs. H(2)O(2) did not detectably increase expression of p53 mRNA or protein but did increase expression of several AP-1 transcription factors. H(2)O(2) increased binding of AP-1 proteins (c-Jun, JunB, JunD, c-Fos, FosB, and Fra-1) to an Mdm2 AP-1 oligodeoxynucleotide probe, and chromatin immunoprecipitation assays showed it increased binding of c-Jun or JunB to the P2 AP-1 RE. Finally, antisense oligonucleotide-mediated reduction of H(2)O(2)-induced Mdm2 expression increased caspase 3 activation. Thus, increased Mdm2 expression is associated with transactivation at the P2 AP-1 RE (rather than the p53 or Ets REs), and Mdm2 induction potentially represents a cardioprotective response to oxidative stress

Recent advances in the structural molecular biology of Ets transcription factors: interactions, interfaces and inhibition

The Ets family of eukaryotic transcription factors is based around the conserved Ets DNA-binding domain. Although their DNA-binding selectivity is biochemically and structurally well characterized, structures of homodimeric and ternary complexes point to Ets domains functioning as versatile protein-interaction modules. In the present paper, we review the progress made over the last decade to elucidate the structural mechanisms involved in modulation of DNA binding and protein partner selection during dimerization. We see that Ets domains, although conserved around a core architecture, have evolved to utilize a variety of interaction surfaces and binding mechanisms, reflecting Ets domains as dynamic interfaces for both DNA and protein interaction. Furthermore, we discuss recent advances in drug development for inhibition of Ets factors, and the roles structural biology can play in their future

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O-2

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2

Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: Determinants of DNA Binding and Redox Regulation by Disulfide Bond Formation.

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors

ELK1 Uses Different DNA Binding Modes to Regulate Functionally Distinct Classes of Target Genes

Eukaryotic transcription factors are grouped into families and, due to their similar DNA binding domains, often have the potential to bind to the same genomic regions. This can lead to redundancy at the level of DNA binding, and mechanisms are required to generate specific functional outcomes that enable distinct gene expression programmes to be controlled by a particular transcription factor. Here we used ChIP–seq to uncover two distinct binding modes for the ETS transcription factor ELK1. In one mode, other ETS transcription factors can bind regulatory regions in a redundant fashion; in the second, ELK1 binds in a unique fashion to another set of genomic targets. Each binding mode is associated with different binding site features and also distinct regulatory outcomes. Furthermore, the type of binding mode also determines the control of functionally distinct subclasses of genes and hence the phenotypic response elicited. This is demonstrated for the unique binding mode where a novel role for ELK1 in controlling cell migration is revealed. We have therefore uncovered an unexpected link between the type of binding mode employed by a transcription factor, the subsequent gene regulatory mechanisms used, and the functional categories of target genes controlled