Interrogating cAMP-dependent Kinase Signaling in Jurkat T Cells via a Protein Kinase A Targeted Immune-precipitation Phosphoproteomics Approach

In the past decade, mass-spectrometry-based methods have emerged for the quantitative profiling of dynamic changes in protein phosphorylation, allowing the behavior of thousands of phosphorylation sites to be monitored in a single experiment. However, when one is interested in specific signaling pathways, such shotgun methodologies are not ideal because they lack selectivity and are not cost and time efficient with respect to instrument and data analysis time.Here we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the cAMP-dependent protein kinase (PKA) consensus motif. This targeted phosphoproteomic strategy is used to profile temporal quantitative changes of potential PKA substrates in Jurkat T lymphocytes upon prostaglandin E₂ (PGE₂) stimulation, which increases intracellular cAMP, activating PKA. Our method combines ultra-high-specificity motif-based immunoaffinity purification with cost-efficient stable isotope dimethyl labeling. We identified 655 phosphopeptides, of which 642 (i.e. 98%) contained the consensus motif [R/K][R/K/X]X[pS/pT]. When our data were compared with a large-scale Jurkat T-lymphocyte phosphoproteomics dataset containing more than 10,500 phosphosites, a minimal overlap of 0.2% was observed. This stresses the need for such targeted analyses when the interest is in a particular kinase.Our data provide a resource of likely substrates of PKA, and potentially some substrates of closely related kinases. Network analysis revealed that about half of the observed substrates have been implicated in cAMP-induced signaling. Still, the other half of the here-identified substrates have been less well characterized, representing a valuable resource for future research.The identification and quantification of protein phosphorylation under system perturbations is an integral part of systems biology (1, 2). The combination of phosphopeptide enrichment (3–6), stable isotope labeling, and high-resolution mass spectrometry (MS) methods (7–9) has become the method of choice for the identification of novel phosphorylation sites and for the quantitation of temporal dynamics within signaling networks (10, 11), allowing the behavior of thousands of phosphorylation sites to be studied in a single experiment (10, 12, 13). Nowadays, one of the most commonly adopted high-throughput phosphoproteomics strategies utilizes two consecutive separation steps: (i) an initial fractionation to reduce the sample complexity, and (ii) a phosphopeptide-specific affinity purification. Such techniques include strong cation exchange fractionation under acidic conditions (3), followed by a chelation-based method with the use of metal ions (i.e. immobilized metal ion affinity chromatography (4), metal oxide affinity chromatography (10, 14), or Ti⁴⁺ immobilized metal ion affinity chromatography (6)). Alternatives to strong cation exchange for the first sample fractionation step have also been reported, including the use of electrostatic repulsion liquid chromatography (15, 16), which is well suited for the identification of multiply phosphorylated peptides, or hydrophilic interaction chromatography (17).Although the number of detected phosphorylated peptides is nowadays impressive, these kinds of methodologies are still inclined to identify/quantify the more abundant phosphoproteins present in a sample. For example, phosphotyrosine peptides are underrepresented because of their relatively lower abundance.In order to analyze key signaling events that may occur on less abundant phosphoproteins, more targeted approaches, focused on a specific pathway or a specific post-translational modification, are thus still essential. Studies examining post-translational modifications are often based on immunoaffinity purification at the protein or peptide level using dedicated antibodies. Recent examples include the selective enrichment of acetylated lysines (18) and phosphorylated tyrosines (19, 20). More recently, the first specific methods targeting serine/threonine phosphorylation motifs using immune-affinity assays have emerged (21, 22). The advantages of targeted approaches are their potentially higher sensitivity and more specific throughput with, as a consequence, relatively faster and easier data interpretation, which make them attractive for many systems biology applications.Immunoaffinity enrichment can be applied at both the protein and the peptide level, and both have been explored to study protein tyrosine phosphorylation (23). The first one results mainly in information on total protein phosphorylation levels. The detection of the actual phosphoresidue might be hampered by the high content of unmodified peptides derived from the immune-purified phosphoprotein and its binding partners. Immunoprecipitation at the peptide level (20, 24, 25), in contrast, leads to improved phosphosite characterization, with the identification of hundreds of sites, albeit with the loss (generally) of information regarding total protein expression.To profile the dynamic regulation of phosphorylation events via mass spectrometry, stable isotope labeling is often implemented, either with the use of amino acids in cell culture (10) or via chemical peptide labeling of the proteolytic digests (26, 27). To identify low-abundant signaling events, phosphoprotein/phosphopeptide immunoprecipitation is typically performed on several milligrams of material because of the substoichiometric abundance of post-translational modifications. This may hamper the use of expensive isotope-labeling reagents such as iTRAQ or tandem mass tag reagents, given the large amount of chemicals needed. Boersema et al. (28) introduced an alternative sensitive and accurate triplex labeling approach using inexpensive reagents (i.e. formaldehyde) that is much less limited in terms of the sample type or amount. We combined this latter stable-isotope dimethyl labeling approach (27–29) with highly specific antibodies raised against a set of cAMP-dependent protein kinase (PKA) phosphorylated substrates as based on the current literature (11, 30–34). It is generally accepted that PKA phosphorylates sites with the reasonably stringent consensus motif [R/K][R/K/X]X[pS/pT]. It should be noted that this consensus motif resembles somewhat the motifs of other AGC kinases (e.g. Akt, PKG, PKC).The basicity of the PKA motifs may hamper their analysis via MS-based proteomics, especially when trypsin is used as a protease, as the peptides may become too small to be sequenced. The use of trypsin is also unfavorable in the approach presented here when attempting to immunoprecipitate peptides bearing the PKA motif. Therefore, we decided to use Lys-C in order to keep the (dominant (RRX[pS/pT])) phosphorylated motif intact. To enhance identification, we applied decision-tree MS/MS technology (9), which makes use of HCD and ETD for more efficient fragmentation, higher mass accuracy in tandem MS mode, and less background noise (35).We applied this method to screen the response of Jurkat T cells to prostaglandin E₂ (PGE₂) treatment. PGE₂ is a potent inflammatory mediator that plays an important role in several immune-regulatory actions (36). It is produced by many different cell types, including tumor cells, where carcinogenesis is associated with chronic inflammatory responses (37). PGE₂ signaling in T cells is initiated by its binding to the G protein–coupled receptors EP1, -2, -3, and -4. Signaling pathways that are initiated by PGE₂ are for the most part under control of the second messenger cyclic adenosine monophosphate (cAMP),¹ which is generated from ATP by adenylyl cyclase when PGE₂ binds to EP2 or EP4 receptors. One of the primary targets of cAMP is PKA—cAMP binding releases the catalytic subunit activating the kinase. In the current study, we efficiently enriched close to 650 phosphopeptides containing the [R/K][R/K/X]X[pS/pT] consensus motif. Almost all these sites were absent in a recently reported comprehensive phosphoproteomics dataset of Jurkat T cells (12), compiled using shotgun strong cation exchange–immobilized metal ion affinity chromatography analysis and containing ∼10,500 phosphorylation sites, illustrative of the complementarity and selectivity of our approach. The qualitative and quantitative data presented here provide a wide-ranging and credible resource of likely PKA substrates. Network analysis confirmed several established cAMP-dependent signaling nodes in our dataset, although most identified potential PKA substrates are “novel” (i.e. not previously reported and/or linked to PKA). Therefore, the dataset presented here can be considered as a comprehensive and reliable resource for future research into cAMP-related signaling.     

The Use of Climatic Niches in Screening Procedures for Introduced Species to Evaluate Risk of Spread: A Case with the American Eastern Grey Squirrel

Species introduction represents one of the most serious threats for biodiversity. The realized climatic niche of an invasive species can be used to predict its potential distribution in new areas, providing a basis for screening procedures in the compilation of black and white lists to prevent new introductions. We tested this assertion by modeling the realized climatic niche of the Eastern grey squirrel Sciurus carolinensis. Maxent was used to develop three models: one considering only records from the native range (NRM), a second including records from native and invasive range (NIRM), a third calibrated with invasive occurrences and projected in the native range (RCM). Niche conservatism was tested considering both a niche equivalency and a niche similarity test. NRM failed to predict suitable parts of the currently invaded range in Europe, while RCM underestimated the suitability in the native range. NIRM accurately predicted both the native and invasive range. The niche equivalency hypothesis was rejected due to a significant difference between the grey squirrel’s niche in native and invasive ranges. The niche similarity test yielded no significant results. Our analyses support the hypothesis of a shift in the species’ climatic niche in the area of introductions. Species Distribution Models (SDMs) appear to be a useful tool in the compilation of black lists, allowing identifying areas vulnerable to invasions. We advise caution in the use of SDMs based only on the native range of a species for the compilation of white lists for other geographic areas, due to the significant risk of underestimating its potential invasive range.     

CARD14/CARMA2sh and TANK differentially regulate poly(I:C)–induced inflammatory reaction in keratinocytes

CARD14/CARMA2sh (CARMA2sh) is a scaffold protein whose mutations are associated with the onset of human genetic psoriasis and other inflammatory skin disorders. Here we show that the immunomodulatory adapter protein TRAF family member-associated NF-κB activator (TANK) forms a complex with CARMA2sh and MALT1 in a human keratinocytic cell line. We also show that CARMA2 and TANK are individually required to activate the nuclear factor κB (NF-κB) response following exposure to polyinosinic-polycytidylic (poly [I:C]), an agonist of toll-like receptor 3. Finally, we present data indicating that TANK is essential for activation of the TBK1/IRF3 pathway following poly (I:C) stimulation, whereas CARMA2sh functions as a repressor of it. More important, we report that two CARMA2sh mutants associated with psoriasis bind less efficiently to TANK and are therefore less effective in suppressing the TBK1/IRF3 pathway. Overall, our data indicate that TANK and CARMA2sh regulate TLR3 signaling in human keratinocytes, which could play a role in the pathophysiology of psoriasis.     

Application of Abscisic Acid (S-ABA) to ‘Crimson Seedless’ Grape Berries in a Mediterranean Climate: Effects on Color, Chemical Characteristics, Metabolic Profile, and S-ABA Concentration

‘Crimson Seedless’ is a table grape cultivar that often fails to develop adequate red color in Mediterranean climates. Application of abscisic acid (S-ABA) may be an aid for improving color, but its potential effects on overall quality and S-ABA concentration of the berry should be also considered. We tested two concentrations (200 and 400 mg/L) and different times of application (from 1 week after veraison up to 9 days before harvest) of a commercial formulation of S-ABA (ProTone^®) to verify the effect on harvestable bunches, color, chemical characteristics, metabolic profile, and S-ABA concentration in the berry. It was found that either the application of S-ABA at 400 mg/L one week after veraison or the application of S-ABA at 400 mg/L one week and four weeks after veraison positively affected the berry skin color, shifting the hue (h°) from 20 to a more red-violet hue (h° = 11–12). In general, the application of S-ABA, with the exception of the late treatments, enhanced coloration of the berries and increased the amount of harvestable bunches at the first pick because it promoted the skin-coloring process. S-ABA did not affect berry firmness but reduced the berry detachment force. Nevertheless, the values remained sufficiently high and the general quality of the bunch was not compromised. Ripening parameters (°Brix, pH, titratable acidity) were not affected by S-ABA applications, and even the primary metabolite profile was not influenced by the treatments as ascertained by multivariate statistical analyses [principal component analyses (PCA) and partial least squares discriminant analysis (PLS-DA)] applied to nuclear magnetic resonance (NMR) data. The S-ABA concentration in the berry, when treatments were performed around veraison, was within the natural range for grape (10–400 ng/g f.w.), whereas when late treatments were applied (few days before harvest), the concentration was higher (more than 1,000 ng/g f.w.). The best results for yield, quality, and S-ABA concentration in the berry were observed for the treatments performed a few days after veraison at the dose of 400 mg/L. This study gives new information about the positive effects of S-ABA on color without any particular change in the metabolic profile of the berry.     

Discovery,synthesis, selectivity modulation and DMPK characterization of 5-azaspiro[2.4]heptanes as potent orexin receptor antagonists

Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor antagonist class has been discovered. SAR and Pharmacokinetic optimization of this series is herein disclosed. Lead compound 15 exhibits potent activity against orexin 1 and orexin 2 receptors along with low cytochrome P450 inhibition potential, good brain penetration and oral bioavailability in rats.     

The glutathione S-transferase gene superfamily: an in silico approach to study the post translational regulation

The use of plants to reclaim contaminated soils and groundwater, known as phytoremediation, is a promising biotechnological strategy which has gained a lot of attention in the last few years. Plants have evolved sophisticated detoxification systems against the toxin chemicals: following the uptake, the compounds are activated so that certain functional groups can conjugate hydrophilic molecules, such as thiols. The resulting conjugates are recognized by the tonoplast transporters and sequestered into the vacuoles. The xenobiotic conjugation with glutathione is mediated by enzymes which belong to the superfamily of glutathione S-transferases (GSTs) catalyzing the nucleophylic attack of the sulphur of glutathione on the electrophilic groups of the cytotoxic substrates therefore playing a crucial role in their degradation. This study was designed to identify the putative correlation between structural and functional characteristics of plant GST classes belonging to different plant species. Consequently, the protein sequences of the expressed GSTs have been retrieved from UniGene, classified and then analyzed in order to assess the evolutionary trend and to predict secondary structure. Moreover, the fingerprint analysis was performed with SCAN Prosite in the attempt to correlate meaningful signature profile and biological information. The results evidenced that all the soluble GSTs have a tendency to assume the α-helix secondary structure followed by random coil and β-sheet. The fingerprint analysis revealed that specific signature profiles related mainly to protein phosphorylation are in the GST classes of all considered species thus suggesting that they might be subjected to reversible activation by phosphorylation-mediated regulation. This approach provides the knowledge of the relationship between presence of conserved signature profile and biological function in the view of future selection of GSTs which might be employed in either mutagenesis or genetic engineering studies.     

Putative spermine synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine

Polyamines are involved in many fundamental cellular processes. Common polyamines are putrescine, spermidine and spermine. Spermine is synthesized by transfer of an aminopropyl residue derived from decarboxylated S-adenosylmethionine to spermidine. Thermospermine is an isomer of spermine and assumed to be synthesized by an analogous mechanism. However, none of the recently described spermine synthases was investigated for their possible activity as thermospermine synthases. In this work, putative spermine synthases from the diatom Thalassiosira pseudonana and from Arabidopsis thaliana could be identified as thermospermine synthases. These findings may explain the previous result that two putative spermine synthase genes in Arabidopsis produce completely different phenotypes in knock-out experiments. Likely, part of putative spermine synthases identifiable by sequence comparisons represents in fact thermospermine synthases.     

Alterations in linker flexibility suppress DNA topoisomerase I mutant-induced cell lethality

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent camptothecin (CPT). Crystallographic studies of the 70-kDa C terminus of human Top1p bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix. The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended linker domain, and the conserved C-terminal active site Tyr domain. CPT bound to the covalent Top1p-DNA complex limits linker flexibility, allowing structural determination of this domain. We previously reported that mutation of Ala(653) to Pro in the linker increases the rate of enzyme-catalyzed DNA religation, thereby rendering Top1A653Pp resistant to CPT (Fiorani, P., Bruselles, A., Falconi, M., Chillemi, G., Desideri, A., and Benedetti P. (2003) J. Biol. Chem. 278, 43268-43275). Molecular dynamics studies suggested mutation-induced increases in linker flexibility alter Top1p catalyzed DNA religation. To address the functional consequences of linker flexibility on enzyme catalysis and drug sensitivity, we investigated the interactions of the A653P linker mutation with a self-poisoning T718A mutation within the active site of Top1p. The A653P mutation suppressed the lethal phenotype of Top1T718Ap in yeast, yet did not restore enzyme sensitivity to CPT. However, the specific activity of the double mutant was decreased in vivo and in vitro, consistent with a decrease in DNA binding. These findings support a model where changes in the flexibility or orientation of the linker alter the geometry of the active site and thereby the kinetics of DNA cleavage/religation catalyzed by Top1p.