The PKARI�� Subunit of Protein Kinase A Modulates the Activation of p90RSK1 and Its Function

Previously, we showed that interactions between p90^RSK1 (RSK1) and the subunits of type I protein kinase A (PKA) regulate the activity of PKA and cellular distribution of active RSK1 (Chaturvedi, D., Poppleton, H. M., Stringfield, T., Barbier, A., and Patel, T. B. (2006) Mol. Cell Biol. 26, 4586–4600). Here we examined the role of the PKARIα subunit of PKA in regulating RSK1 activation and cell survival. In mouse lung fibroblasts, silencing of the PKARIα increased the phosphorylation and activation of RSK1, but not of RSK2 and RSK3, in the absence of any stimulation. Silencing of PKARIα also decreased the nuclear accumulation of active RSK1 and increased its cytoplasmic content. The increased activation of RSK1 in the absence of any agonist and changes in its subcellular redistribution resulted in increased phosphorylation of its cytoplasmic substrate BAD and increased cell survival. The activity of PKA and phosphorylation of BAD (Ser-155) were also enhanced when PKARIα was silenced, and this, in part, contributed to increased cell survival in unstimulated cells. Furthermore, we show that RSK1, PKA subunits, D-AKAP1, and protein phosphatase 2A catalytic subunit (PP2Ac) exist in a complex, and dissociation of RSK1 from D-AKAP1 by either silencing of PKARIα, depletion of D-AKAP1, or by using a peptide that competes with PKARIα for binding to AKAPs, decreased the amount of PP2Ac in the RSK1 complex. We also demonstrate that PP2Ac is one of the phosphatases that dephosphorylates RSK, but not ERK1/2. Thus, in unstimulated cells, the increased phosphorylation and activation of RSK1 after silencing of PKARIα or depletion of D-AKAP1 are due to decreased association of PP2Ac in the RSK1 complex.Cyclic AMP-dependent protein kinase (PKA)³ plays a pivotal role in manifesting an array of biological actions ranging from cell proliferation and tumorigenesis to increased inotropic and chronotropic effects in the heart as well as regulation of long term potentiation and memory. The PKA holoenzyme is a heterotetramer and consists of two catalytic (PKAc) subunits bound to a dimer of regulatory subunits. To date, four isoforms of the PKAc (PKAcα, PKAcβ, PKAcγ, and PKAcδ) and four isoforms of the regulatory subunits (RIα, RIβ, RIIα, and RIIβ) have been described (1). The various isoforms of PKA subunits are expressed differently in a tissue- and cell-specific manner (2). In addition to binding and inhibiting the activity of PKAc via their pseudo substrate region (3–6), the R subunits also interact with PKA-anchoring proteins (AKAPs) and facilitate the localization of PKA in specific subcellular compartments (7, 8). More than 50 AKAP family members have been described, and although most of these have a higher affinity for the RII subunits (9), certain AKAPs such as D-AKAP1 and D-AKAP2 preferentially bind the PKARIα subunit (10–12). Because the AKAPs also bind other signaling molecules such as phosphatases (PP2B) and kinases (protein kinase C), they act as scaffolds to organize and integrate specific signaling events within specific compartments in the cells (7, 8, 13, 14).We have shown that the PKARIα and PKAcα subunits of PKA interact with the inactive and active forms of p90^RSK1 (RSK1), respectively (15). Binding of inactive RSK1 to PKARIα decreases the interactions between PKARIα and PKAc, whereas the association of active RSK1 with PKAc increases interactions between PKARIα and PKAc such that larger amounts of cAMP are required to activate PKAc in the presence of active RSK1 (15). Moreover, the indirect (via subunits of PKA) interaction of RSK1 with AKAPs is required for the nuclear localization of active RSK1 (15), and disruption of the interactions of RSK1·PKA complex from AKAPs results in increased cytoplasmic distribution of active RSK1 with a concomitant increase in phosphorylation of its cytosolic substrates such as BAD and reduced cellular apoptosis (15). These findings show the functional and biological significance of RSK1·PKA·AKAP interactions.Besides inhibiting PKAc activity, the physiological role of PKARIα is underscored by the findings that mutations in the PKAR1A gene that result in haploinsufficiency of PKARIα are the underlying cause of Carney complex (CNC) (16, 17). CNC is an autosomal dominant multiple neoplasia syndrome in which myxomas of the skin, heart, and/or vicera are recurrent and also associated with high incidence of endocrine and ovarian tumors as well as Schwannomas (18–20). The majority of patients with the multiple neoplasia CNC syndrome harbor mutations in the PKAR1A gene (21) that result in PKARIα haploinsufficiency. Importantly, however, loss of heterozygosity or alterations in PKA activity may not contribute toward the tumorigenicity in either CNC patients or mouse model of CNC (21). This suggests that loss of function(s) of PKARIα other than inhibition of PKA activity is(are) involved in the enhanced tumorigenicity in CNC patients and in the murine CNC model.Because RSK1 regulates cell growth, survival, and tumorigenesis (22–27), and because its subcellular localization and ability to inhibit apoptosis is regulated by its interactions via PKARIα with AKAPs (15), we reasoned that in conditions such as CNC where PKARIα levels are decreased, the increase in tumorigenicity may emanate from aberrant regulation of the activity and/or subcellular localization of RSK1. Therefore, herein we have investigated whether PKARIα regulates the activation of RSK1 and its biological functions. Decreasing expression of PKARIα by small interfering RNA (siRNA) enhanced the activation of RSK1, but not RSK2 or RSK3, in the absence of an agonist such as EGF. This was accompanied by an increase in the cytoplasmic localization of the active RSK1 and enhanced cell survival in the absence of any growth factor. Silencing of PKARIα also increased PKAc activity and while part of the anti-apoptotic response could be attributed to an increase in PKAc activity, activation of RSK1 under basal conditions contributed significantly to cell survival. The elevation in RSK1 activity upon PKARIα silencing was not due to increased PKAc activity. Rather the activation of RSK1 in the absence of PKARIα was due to a decrease in PP2A in the RSK1 complex. These findings demonstrate a novel role for PKARIα in the regulation of RSK1 activation, a key enzyme that mediates the downstream actions of the ERK1/2 cascade.     

The endogenous production of hydrogen sulphide in intrauterine tissues

Background  

Hydrogen sulphide is a gas signalling molecule which is produced endogenously from L-cysteine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The possible role of hydrogen sulphide in reproduction has not yet been fully investigated. It has been previously demonstrated that hydrogen sulphide relaxes uterine smooth muscle in vitro. The aim of the present study was to investigate the endogenous production of hydrogen sulphide in rat and human intrauterine tissues in vitro.     

Amino acid recognition and gene regulation by riboswitches

Riboswitches specifically control expression of genes predominantly involved in biosynthesis, catabolism and transport of various cellular metabolites in organisms from all three kingdoms of life. Among many classes of identified riboswitches, two riboswitches respond to amino acids lysine and glycine to date. Though these riboswitches recognize small compounds, they both belong to the largest riboswitches and have unique structural and functional characteristics. In this review, we attempt to characterize molecular recognition principles employed by amino acid-responsive riboswitches to selectively bind their cognate ligands and to effectively perform a gene regulation function. We summarize up-to-date biochemical and genetic data available for the lysine and glycine riboswitches and correlate these results with recent high-resolution structural information obtained for the lysine riboswitch. We also discuss the contribution of lysine riboswitches to antibiotic resistance and outline potential applications of riboswitches in biotechnology and medicine.     

Genome Analysis of the Anaerobic Thermohalophilic Bacterium Halothermothrix orenii

Halothermothirx orenii is a strictly anaerobic thermohalophilic bacterium isolated from sediment of a Tunisian salt lake. It belongs to the order Halanaerobiales in the phylum Firmicutes. The complete sequence revealed that the genome consists of one circular chromosome of 2578146 bps encoding 2451 predicted genes. This is the first genome sequence of an organism belonging to the Haloanaerobiales. Features of both Gram positive and Gram negative bacteria were identified with the presence of both a sporulating mechanism typical of Firmicutes and a characteristic Gram negative lipopolysaccharide being the most prominent. Protein sequence analyses and metabolic reconstruction reveal a unique combination of strategies for thermophilic and halophilic adaptation. H. orenii can serve as a model organism for the study of the evolution of the Gram negative phenotype as well as the adaptation under thermohalophilic conditions and the development of biotechnological applications under conditions that require high temperatures and high salt concentrations.     

Role of CBP and SATB-1 in Aging,Dietary Restriction,and Insulin-Like Signaling

How dietary restriction (DR) increases lifespan and decreases disease burden are questions of major interest in biomedical research. Here we report that hypothalamic expression of CREB-binding protein (CBP) and CBP-binding partner Special AT-rich sequence binding protein 1 (SATB-1) is highly correlated with lifespan across five strains of mice, and expression of these genes decreases with age and diabetes in mice. Furthermore, in Caenorhabditis elegans, cbp-1 is induced by bacterial dilution DR (bDR) and the daf-2 mutation, and cbp-1 RNAi specifically in adults completely blocks lifespan extension by three distinct protocols of DR, partially blocks lifespan extension by the daf-2 mutation but not of cold, and blocks delay of other age-related pathologies by bDR. Inhibiting the C. elegans ortholog of SATB-1 and CBP-binding partners daf-16 and hsf-1 also attenuates lifespan extension by bDR, but not other protocols of DR. In a transgenic Aβ42 model of Alzheimer''s disease, cbp-1 RNAi prevents protective effects of bDR and accelerates Aβ42-related pathology. Furthermore, consistent with the function of CBP as a histone acetyltransferase, drugs that enhance histone acetylation increase lifespan and reduce Aβ42-related pathology, protective effects completely blocked by cbp-1 RNAi. Other factors implicated in lifespan extension are also CBP-binding partners, suggesting that CBP constitutes a common factor in the modulation of lifespan and disease burden by DR and the insulin/IGF1 signaling pathway.     

Target Cell Cyclophilins Facilitate Human Papillomavirus Type 16 Infection

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV–induced diseases.     

Massive Fluid Requirements and an Unusual BUN/Creatinine Ratio for Pre-Renal Failure in Patients with Cholera

Background

Cholera is an important infectious cause of secretory diarrhea. The primary symptom of infection is the sudden onset of watery diarrhea with subsequent volume depletion causing renal insufficiency. The objective of this research is to study the level of dehydration at presentation and subsequent fluid management in patients with cholera.

Methods

This study was conducted on 191 patients of Cholera admitted at a tertiary care hospital in Karachi, Pakistan during the period of 5 years. Medical charts were evaluated retrospectively for initial hydration status, baseline lab investigations on admission and discharge and fluid therapy given to all the patients while their stay in the hospital and the data was analyzed on SPSS 15.0.

Results

Out of the 191 patients, 83(43%) were males and 108 (57%) were females with mean age of 42.3 years (SD±18.34). The average duration of symptoms was 3.75 days (SD±2.04). Of 191 patients, 175 (92.1%) presented with dehydration, 80 (42.3%) were given Ringer''s Lactate (R/L) + Normal Saline (N/S), 45 (24%) patients were given R/L + N/S + Oral Rehydration Therapy (ORS), 27 (14.3%) of the patients were kept on R/L only and remaining were given various combinations of R/L, N/S, ORS and Dextrose Saline (D/S). On admission mean Blood Urea Nitrogen (BUN) was 24.54 (SD±16.6), mean creatinine was 2.47 (SD±2.35) and mean BUN/Creatinine ratio was 11.63 (SD±5.7).

Conclusion

Aggressive fluid rehydration remains the cornerstone of management of cholera. Instead of presenting with a classical BUN/Creatinine ratio of >20∶1, patients with pre-renal failure in cholera may present with a BUN/Creatinine ratio of <15∶1.     

The Scottish Structural Proteomics Facility: targets, methods and outputs

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.