1,721,015 research outputs found
AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity
No full textMurine thymoma viral oncogene homolog (AKT) kinases target both cytosolic and nuclear substrates for phosphorylation. Whereas the cytosolic substrates are known to be closely associated with the regulation of apoptosis and autophagy or metabolism and protein synthesis, the nuclear substrates are, for the most part, poorly understood. To better define the role of nuclear AKT, potential AKT substrates were isolated from the nuclear lysates of leukemic cell lines using a phosphorylated AKT substrate antibody and identified in tandem mass spectrometry. Among the proteins identified was adenosine deaminase acting on RNA (ADAR)1p110, the predominant nuclear isoform of the adenosine deaminase acting on double-stranded RNA. Coimmunoprecipitation studies and in vitro kinase assays revealed that AKT-1, -2, and -3 interact with both ADAR1p110 and ADAR2 and phosphorylate these RNA editases. Using site-directed mutagenesis of suspected AKT phosphorylation sites, AKT was found to primarily phosphorylate ADAR1p110 and ADAR2 on T738 and T553, respectively, and overexpression of the phosphomimic mutants ADAR1p110 (T738D) and ADAR2 (T553D) resulted in a 50-100% reduction in editase activity. Thus, activation of AKT has a direct and major impact on RNA editing.-Bavelloni, A., Focaccia, E., Piazzi, M., Raffini, M., Cesarini, V., Tomaselli, S., Orsini, A., Ratti, S., Faenza, I., Cocco, L., Gallo, A., Blalock, W. L. AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity
PLC-beta2 activity on actin-associated polyphosphoinositides promotes migration of differentiating tumoral myeloid precursors.
No full textDuring both maturation and function, neutrophils are subjected to reorganization of the actin cytoskeleton. Among the molecules that influence cytoskeletal architecture, the amount and subcellular localization of phosphoinositides, regulated by specific kinases and phosphatases, may play a crucial role. In neutrophils, PLC-beta2 is a major phosphoinositide-dependent phospholipase C isoform activated in response to chemoattractants, even though its role in the modifications of cell morphology and motility that occur during the inflammatory process has not been fully elucidated. In APL-derived promyelocytes induced to complete their maturation program, we have found that the expression levels of PLC-beta2 positively correlate with the degree of the reached neutrophil differentiation. Here, we demonstrate that PLC-beta2 modulates the migration capability of promyelocytes induced to differentiate with ATRA. In differentiating cells, the association of PLC-beta2 with actin, mediated by the PH domain, seems crucial for catalytic activity. We conclude that phosphodiesterase activity of PLC-beta2 on the actin-associated PIP2 may be responsible, by modifying the phosphoinositide pools, for the modifications of cytoskeleton architecture that take place during motility of differentiating promyelocytes
A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins.
No full textPURPOSE: To explore the potential of a chip-based miniaturized capillary gel electrophoresis device in a quantitative evaluation of the human tear protein profile and to validate the method.
METHODS: A total of 5 μl of tears were collected from 25 patients diagnosed as having mild to moderate dry eye according to Dry Eye Workshop guidelines and from 20 matched normal volunteers. Protein analysis was performed with the 2100 Bioanalyzer; different protein kit assays were evaluated (Protein 80 kit, Protein 230 kit, High Sensitivity Protein 250 kit) for sizing and quantifying protein samples from 5 to 80 kDa, 14 to 230 kDa, and 5 to 250 kDa, respectively. A standard protein ladder was loaded on each chip to allow an estimation of the appropriate molecular weight of the separated proteins; a sample buffer containing a lower and an upper marker was used to check the correct alignment of each lane. Virtual bands generated by the Bioanalyzer were identified and validated as follows: tear samples were run in parallel and proteins separated by one-dimensional and two-dimensional sodium dodecyl sulfate-PAGE and characterized by immunoblotting, enzymatic digestion, and analysis with liquid chromatography-mass spectrometry followed by a search of the SProt human protein database.
RESULTS: Analyses were successfully performed by using as small as a 2 μl tear sample. The Protein 230 kit was selected as the best chip kit, able to differentiate all the proteins of interest. The measurement noise parameters were low, and reproducibility and repeatability exhibited high accuracy (0.998 and 0.995, respectively) and precision (0.974 and 0.977, respectively). The coefficient of variability was slightly higher than that declared by the manufacturer (6.2% versus 5.0%). Total protein content and the following proteins were recognized in all samples: lipophilin A lysozyme C, tear lipocalin-1, zinc-alpha-2-glycoprotein, serotransferrin, lactotransferrin, and exudated serum albumin.
CONCLUSIONS: Our data demonstrate that this chip-based tear protein analysis is a reliable method of instrumental diagnosis in daily clinical activity and may provide supporting evaluation parameters for diagnosing and managing tear-based disorders
Signal transduction in ribosome biogenesis: A recipe to avoid disaster
Full text linkEnergetically speaking, ribosome biogenesis is by far the most costly process of the cell and, therefore, must be highly regulated in order to avoid unnecessary energy expenditure. Not only must ribosomal RNA (rRNA) synthesis, ribosomal protein (RP) transcription, translation, and nuclear import, as well as ribosome assembly, be tightly controlled, these events must be coordinated with other cellular events, such as cell division and differentiation. In addition, ribosome biogenesis must respond rapidly to environmental cues mediated by internal and cell surface receptors, or stress (oxidative stress, DNA damage, amino acid depletion, etc.). This review examines some of the well-studied pathways known to control ribosome biogenesis (PI3K-AKT-mTOR, RB-p53, MYC) and how they may interact with some of the less well studied pathways (eIF2α kinase and RNA editing/splicing) in higher eukaryotes to regulate ribosome biogenesis, assembly, and protein translation in a dynamic manner
Revisiting the role of GSK3, a modulator of innate immunity, in idiopathic inclusion body myositis
Full text linkIdiopathic or sporadic inclusion body myositis (IBM) is the leading age-related (onset > 50 years of age) autoimmune muscular pathology, resulting in significant debilitation in affected individuals. Once viewed as primarily a degenerative disorder, it is now evident that much like several other neuro-muscular degenerative disorders, IBM has a major autoinflammatory component resulting in chronic inflammation-induced muscle destruction. Thus, IBM is now considered primarily an inflammatory pathology. To date, there is no effective treatment for sporadic inclusion body myositis, and little is understood about the pathology at the molecular level, which would offer the best hopes of at least slowing down the degenerative process. Among the previously examined potential molecular players in IBM is glycogen synthase kinase (GSK)-3, whose role in promoting TAU phosphorylation and inclusion bodies in Alzheimer’s disease is well known. This review looks to re-examine the role of GSK3 in IBM, not strictly as a promoter of TAU and Abeta inclusions, but as a novel player in the innate immune system, discussing some of the recent roles discovered for this well-studied kinase in inflammatory-mediated pathology
Alternative Splicing, RNA Editing, and the Current Limits of Next Generation Sequencing
Full text linkThe advent of next generation sequencing (NGS) has fostered a shift in basic analytic strategies of a gene expression analysis in diverse pathologies for the purposes of research, pharmacology, and personalized medicine. What was once highly focused research on individual signaling pathways or pathway members has, from the time of gene expression arrays, become a global analysis of gene expression that has aided in identifying novel pathway interactions, the discovery of new therapeutic targets, and the establishment of disease-associated profiles for assessing progression, stratification, or a therapeutic response. But there are significant caveats to this analysis that do not allow for the construction of the full picture. The lack of timely updates to publicly available databases and the “hit and miss” deposition of scientific data to these databases relegate a large amount of potentially important data to “garbage”, begging the question, “how much are we really missing?” This brief perspective aims to highlight some of the limitations that RNA binding/modifying proteins and RNA processing impose on our current usage of NGS technologies as relating to cancer and how not fully appreciating the limitations of current NGS technology may negatively affect therapeutic strategies in the long run
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