Serum response factor cleavage by caspases 3 and 7 linked to apoptosis in human BJAB cells

Apoptosis involves the cessation of cellular processes, the breakdown of intracellular organelles, and, finally, the nonphlogistic clearance of apoptotic cells from the body. Important for these events is a family of proteases, caspases, which are activated by a proteolytic cleavage cascade and drive apoptosis by targeting key proteins within the cell. Here, we demonstrate that serum response factor (SRF), a transcription factor essential for proliferative gene expression, is cleaved by caspases and that this cleavage occurs in proliferating murine fibroblasts and can be induced in the human B-cell line BJAB. We identify the two major sites at which SRF cleavage occurs as Asp245 and Asp254, the caspases responsible for the cleavage and generate a mutant of SRF resistant to cleavage in BJAB cells. Investigation of the physiological and functional significance of SRF cleavage reveals that it correlates with the loss of e-fos expression, whereby neither SRF cleavage fragment retains transcriptional activity. Moreover, the expression of a noncleavable SRF in BJAB cells suppresses apoptosis induced by Fas cross-linking. These results suggest that for apoptosis to proceed, the transcriptional events promoting cell survival and proliferation, in which SRF is involved, must first be inactivated

ELK-1 ubiquitination status and transcriptional activity are modulated independently of F-Box protein FBXO25

The mitogen-responsive, ETS-domain transcription factor ELK-1 stimulates the expression of immediate early genes at the onset of the cell cycle and participates in early developmental programming. ELK-1 is subject to multiple levels of posttranslational control, including phosphorylation, SUMOylation, and ubiquitination. Recently, removal of monoubiquitin from the ELK-1 ETS domain by the Ubiquitin Specific Protease USP17 was shown to augment ELK-1 transcriptional activity and promote cell proliferation. Here we have used coimmunoprecipitation experiments, protein turnover and ubiquitination assays, RNA-interference and gene expression analyses to examine the possibility that USP17 acts antagonistically with the F-box protein FBXO25, an E3 ubiquitin ligase previously shown to promote ELK-1 ubiquitination and degradation. Our data confirm that FBXO25 and ELK-1 interact in HEK293T cells and that FBXO25 is active toward Hand1 and HAX1, two of its other candidate substrates. However, our data indicate that FBXO25 neither promotes ubiquitination of ELK-1 nor impacts on its transcriptional activity and suggest that an E3 ubiquitin ligase other than FBXO25 regulates ELK-1 ubiquitination and function

De-ubiquitination of ELK-1 by USP17 potentiates mitogenic gene expression and cell proliferation

ELK-1 is a transcription factor involved in ERK-induced cellular proliferation. Here we show that its transcriptional activity is modulated by ubiquitination at lysine 35 (K35). The level of ubiquitinated ELK-1 rises in mitogen-deprived cells and falls upon mitogen stimulation or oncogene expression. Ectopic expression of USP17, a cell cycle-dependent deubiquitinase, decreases ELK-1 ubiquitination and up-regulates ELK-1 target-genes with a concomitant increase in cyclin D1 expression. In contrast, USP17 depletion attenuates ELK-1-dependent gene expression and slows cell proliferation. The reduced rate of proliferation upon USP17 depletion appears to be a direct effect of ELK-1 ubiquitination because it is rescued by an ELK-1(K35R) mutant refractory to ubiquitination. Overall, our results show that ubiquitination of ELK-1 at K35, and its reversal by USP17, are important mechanisms in the regulation of nuclear ERK signalling and cellular proliferation. Our findings will be relevant for tumours that exhibit elevated USP17 expression and suggest a new target for intervention

Strategy for tumor selective disruption of androgen receptor function in the spectrum of prostate cancer

Purpose: Testosterone suppression in prostate cancer (PC) is limited by serious side effects and resistance via restoration of androgen receptor (AR) functionality. ELK1 is required for ARdependent growth in various hormone-dependent and castration resistant PC models. The amino terminal domain of AR docks at two sites on ELK1 to co-activate essential growth genes. This study explores the ability of small molecules to disrupt the ELK1-AR interaction in the spectrum of PC, inhibiting AR activity in a manner that would predict functional tumor selectivity. Experimental design: Small molecule drug discovery and extensive biological characterization of a lead compound. Results: We have discovered a lead molecule (KCI807) that selectively disrupts ELK1-dependent promoter activation by wild-type and variant ARs without interfering with ELK1 activation by ERK. KCI807 has an obligatory flavone scaffold and functional hydroxyl groups on C5 and C3'. KCI807 binds to AR, blocking ELK1 binding, and selectively blocks recruitment of AR to chromatin by ELK1. KCI807 primarily affects a subset of AR target growth genes selectively suppressing AR-dependent growth of PC cell lines with a better inhibitory profile than enzalutamide. KCI807 also inhibits in vivo growth of castration/enzalutamide-resistant cell line-derived and patient-derived tumor xenografts. In the rodent model, KCI807 has a plasma half-life of 6h and maintenance of its antitumor effect is limited by self-induced metabolism at its 3'-hydroxyl. Conclusions: The results offer a mechanism-based therapeutic paradigm for disrupting the AR growth-promoting axis in the spectrum of prostate tumors while reducing global suppression of testosterone actions. KCI807 offers a good lead molecule for drug development

Dimer formation and conformational flexibility ensure cytoplasmic stability and nuclear accumulation of Elk-1

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin–proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs

Radial structure, inflow and central mass of stationary radiative galaxy clusters

We analyse the radial structure of self-gravitating spheres consisting of multiple interpenetrating fluids, such as the X-ray emitting gas and the dark halo of a galaxy cluster. In these dipolytropic models, the adiabatic dark matter sits in equilibrium, while the gas develops a gradual, smooth, quasi-stationary cooling flow. Both affect and respond to the collective gravitational field. We find that all subsonic, radially continuous, steady solutions require a non-zero minimum central point mass. For Mpc-sized haloes with 7–10 effective degrees of freedom (F2), the minimum central mass is compatible with observations of supermassive black holes. Smaller gas mass influxes enable smaller central masses for wider ranges of F2. The halo comprises a sharp spike around the central mass, embedded within a core of nearly constant density (at 101–102.5 kpc scales), with outskirts that attenuate and naturally truncate at finite radius (several Mpc). The gas density resembles a broken power law in radius, but the temperature dips and peaks within the dark core. A finite minimum temperature occurs due to gravitational self-warming, without cold mass dropout nor needing regulatory heating. X-ray emission from the intracluster medium mimics a β-model plus bright compact nucleus. Near-sonic points in the gas flow are bottlenecks to the allowed steady solutions; the outermost are at kpc scales. These sites may preferentially develop cold mass dropout during strong perturbations off equilibrium. Within the sonic point, the profile of gas specific entropy is flatter than s∝r1/2, but this is a shallow ramp and not an isentropic core. When F2 is large, the inner halo spike is only marginally Jeans stable in the central parsec, suggesting that a large non-linear disturbance could trigger local dark collapse on to the central object

Derivation of irrigation requirements for radiological impact assessments

When assessing the radiological impacts of radioactive waste disposal, irrigation using groundwater contaminated with releases from the disposal system is a principal means of crop and soil contamination. In spite of their importance for radiological impact assessments, irrigation data are scarce and often associated with considerable uncertainty for several reasons including limited obligation to measure groundwater abstraction and differences in measuring methodologies. Further uncertainty arises from environmental (e.g. climate and landscape) change likely to occur during the assessment long time frame. In this paper, we derive irrigation data using the crop growth AquaCrop model relevant to a range of climates, soils and crops for use in radiological impact assessments. The AquaCrop estimates were compared with actual irrigation data reported in the literature and with estimates obtained from simple empirical methods proposed for use in radiological impact assessments. Further, the AquaCrop irrigation data were analysed using mixed effects modelling to investigate the effects of climate, soil and crop type on the irrigation requirement. Irrigation estimates from all models were within a reasonable range of the measured values. The AquaCrop estimates, however, were at the higher end of the range and higher than those from the empirical methods. Nevertheless, they may be more appropriate for conservative radiological assessments. The use of mixed effects modelling allowed for the characterisation of crop-specific variability in the irrigation data, and in contrast to the empirical methods, the AquaCrop and the mixed effects models accounted for the soil effect on the irrigation requirement. The approach presented in this paper is relevant for obtaining irrigation data for a specific site under different climatic conditions as well as for generic dose assessments. To the best of our knowledge, this is one of the most comprehensive analyses of irrigation data in the context of radiological impact assessment currently available

Insight into consumer experience on UK train transportation services

Customers’ experiences are significant in a rapidly changing service context, and this is shaped by the quality of service provided. With social media changing the way consumers engage with service providers, experiences are shared online. This study carried out three analyses of brand-related conversations on Twitter with the aim of exploring consumers’ attitudes to and experiences of train operating companies. Firstly, Python was used for the tweet mining and sentiment analysis (n = 1,914,494 tweets) to investigate the polarity between the opinions of commuters. Secondly, tweets were thematically analysed and grouped to understand how consumers experience the service quality. Lastly, content analysis of the tweets was carried out to identify the variations in service quality. Results indicated that there is overall positive customer experience, however, there are variations in service quality dimension across the different train groups, highlight the need to improve service quality at different touchpoints, especially the tangible features of the trains and presence of responsive and emphatic staff. This study further broadens the context of customer experience through eWOM on social media for service brands, contribute towards related literature on sentiment analysis and service brands, providing significant theoretical and practical implications for researchers and managers

Actomyosin-based Retrograde Flow of Microtubules in the Lamella of Migrating Epithelial Cells Influences Microtubule Dynamic Instability and Turnover and Is Associated with Microtubule Breakage and Treadmilling

We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally “pioneering” MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 μm/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione–2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that ∼80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends

Mitogen-induced recruitment of ERK and MSK to SRE promoter complexes by ternary complex factor Elk-1

Many eukaryotic genes are acutely regulated by extra-cellular signals. The c-fos serum response element (SRE) mediates transcriptional activation in response to mitogens through serum response factor (SRF)-dependent recruitment of Elk-1, a mitogen-activated protein kinase (MAPK)-responsive transcription factor. How subsequent events at SRE promoters stimulate initiation of transcription has yet to be fully resolved. Here we show that extra-cellular signal-regulated kinase (ERK) and mitogen and stress-activated kinase (MSK) are recruited to SRE promoter complexes in vitro and in vivo. Their recruitment in vitro correlates with Elk-1 binding and for ERK the D domain/KIM of Elk-1 is specifically involved. In vivo, recruitment of ERK and MSK is stimulated by mitogens, correlates with histone H3 phosphorylation and is impaired by Elk-1 knockdown. Immunocytochemistry and confocal microscopy reveal that ERK appears to associate to some extent with initiating rather than elongating RNA polymerase II. Taken together, our data add to the body of evidence implying that ERK and related MAPKs may fulfil a generic role at the promoters of acutely regulated genes