34 research outputs found
Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cβ2
BACKGROUND: Two main genes encoding the catalytic subunits Cα and Cβ of cyclic AMP dependent protein kinase (PKA) have been identified in all vertebrates examined. The murine, bovine and human Cβ genes encode several splice variants, including the splice variant Cβ2. In mouse Cβ2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cβ2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues. RESULTS: We identified a novel 47 kDa splice variant encoded by the mouse Cβ gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cβ2. Based on the present results and the existence of a human brain-specifically expressed Cβ splice variant designated Cβ4 that is identical to the former mouse Cβ2 splice variant, the mouse splice variant has now been renamed mouse Cβ4. CONCLUSION: Murine lymphoid tissues express a protein that is a homologue of human and bovine Cβ2. The murine Cβ gene encodes the splice variants Cβ1, Cβ2, Cβ3 and Cβ4, as is the case with the human Cβ gene
Increased Basal cAMP-dependent Protein Kinase Activity Inhibits the Formation of Mesoderm-derived Structures in the Developing Mouse Embryo
A targeted disruption of the RIalpha isoform of protein kinase A (PKA) was created by using homologous recombination in embryonic stem cells. Unlike the other regulatory and catalytic subunits of PKA, RIalpha is the only isoform that is essential for early embryonic development. RIalpha homozygous mutant embryos fail to develop a functional heart tube at E8.5 and are resorbed at approximately E10.5. Mutant embryos show significant growth retardation and developmental delay compared with wild type littermates from E7.5 to E10.5. The anterior-posterior axis of RIalpha mutants is well developed, with a prominent head structure but a reduced trunk. PKA activity measurements reveal an increased basal PKA activity in these embryos. Brachyury mRNA expression in the primitive streak of RIalpha mutants is significantly reduced, consistent with later deficits in axial, paraxial, and lateral plate mesodermal derivatives. This defect in the production and migration of mesoderm can be completely rescued by crossing RIalpha mutants to mice carrying a targeted disruption in the Calpha catalytic subunit, demonstrating that unregulated PKA activity rather than a specific loss of RIalpha is responsible for the phenotype. Primary embryonic fibroblasts from RIalpha mutant embryos display an abnormal cytoskeleton and an altered ability to migrate in cell culture. Our results demonstrate that unregulated PKA activity negatively affects growth factor-mediated mesoderm formation during early mouse development
Dynamic Axonal Translation in Developing and Mature Visual Circuits.
Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.This work was supported by Wellcome Trust Programme Grant (085314/Z/08/Z), European Research Council Advanced Grant (322817) to CEH , Cambridge Wellcome Trust PhD programme in Developmental Biology (PMAG/406; BT-B), Gates Cambridge Scholarship (JQL), Basic Science Research Program (2013R1A1A1009625 & 2014K2A7A1036305), Biomedical Technology Development Program (2013M3A9D5072551), & Brain Research Program (2015M3C7A1028396) funded through the NRF by the Korean government (MSIP), Yonsei University Future-leading Research Initiative of 2015 (2015-22-0095), and a faculty research grant from Yonsei University College of Medicine for 2013 (6-2013-0064-2-1) to HJ.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Cell Press
Cell-Type Specific Expression of a Dominant Negative PKA Mutation in Mice
We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology
Targeting adipose tissue
Two different types of adipose tissues can be found in humans enabling them to respond to starvation and cold: white adipose tissue (WAT) is generally known and stores excess energy in the form of triacylglycerol (TG), insulates against cold, and serves as a mechanical cushion. Brown adipose tissue (BAT) helps newborns to cope with cold. BAT has the capacity to uncouple the mitochondrial respiratory chain, thereby generating heat rather than adenosine triphosphate (ATP). The previously widely held view was that BAT disappears rapidly after birth and is no longer present in adult humans. Using positron emission tomography (PET), however, it was recently shown that metabolically active BAT occurs in defined regions and scattered in WAT of the adult and possibly has an influence on whole-body energy homeostasis. In obese individuals adipose tissue is at the center of metabolic syndrome. Targeting of WAT by thiazolidinediones (TZDs), activators of peroxisome proliferator-activated receptor γ (PPARγ) a ‘master’ regulator of fat cell biology, is a current therapy for the treatment of type 2 diabetes. Since its unique capacity to increase energy consumption of the body and to dissipate surplus energy as heat, BAT offers new perspectives as a therapeutic target for the treatment of obesity and associated diseases such as type 2 diabetes and metabolic syndrome. Recent discoveries of new signaling pathways of BAT development give rise to new therapeutic possibilities in order to influence BAT content and activity
Cre recombinase<i>-</i>mediated activation of the RIαB allele in ES cells decreases PKA activity and forskolin-stimulated CRE-luciferase reporter expression.
<p><i>A</i>, Transfection of ES cells with a <i>Cre</i> expression vector (pOG231) results in the expression of mutant RNA transcripts. Levels of mutant transcripts were determined by RT-PCR, restriction digestion, and Southern analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018772#s4" target="_blank">Materials and Methods</a>. The 53 bp fragment indicates WT and the 140 bp fragment indicates mutant RIα transcripts. The small upper band present in lane 1 (WT) is due to incomplete digestion with BstN1, and was subtracted from lane 2 (RIαB) to determine the level of mutant RNA in these cells (<2%). The level of mutant transcript in RIαB/Cre ES cells is ∼49%. <i>B</i>, Detection of Cre-mediated recombination events in RIαB/Cre ES cells was determined by PCR using primers 1 and 2 (as shown in the figure) to amplify across the specified regions. Lane 1 is a WT control, Lane 2 is positive control for recombination. Lane 3 represents RIαB ES cells, which contain a single band due to presence of single WT RIα allele (the PCR conditions did not allow amplification through the floxed-neo<sup>r</sup> cassette). Lane 4 represents RIαB/Cre ES cells, which contain a recombined allele (356 bp) and WT allele (309 bp). <i>C</i>, Kinase assay of basal and total activity in WT, RIαB, and RIαB/Cre ES cells. All samples were done in triplicate in the absence (basal PKA activity) or presence of 5 µΜ cAMP (total PKA activity) using Kemptide as substrate. Kinase activity that was not PKA-specific was measured in the presence of PKI and subtracted from basal and total values. Data values are represented as mean ± SEM. <i>D,</i> Representative CRE-luciferase assay. WT, RIαB, and RIαB/Cre ES cell lines were transfected with the CRE-dependent α168-luciferase reporter and then stimulated with forskolin (10 uM) for 14 h before an assay for luciferase activity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018772#s4" target="_blank">Materials and Methods</a>. Transfections were done in triplicate.</p
RIαB/Alb-<i>cre</i> mice exhibit increases in glucose disposal.
<p><i>A</i>, Blood glucose levels were determined in 24 hr fasted WT, RIαB, and RIαB/Alb-<i>cre</i> mice. n = 10. <i>B</i>, Glucose tolerance test was performed in 24 hr fasted WT, RIαB, and RIαB/Alb-<i>cre</i> mice injected i.p. with 2 mg of glucose per gram of body weight. Blood glucose levels were determined at the indicated time points. Over the first 60 min, the glucose disposal of RIαB/Alb-cre was significantly enhanced compared to RIαB alone (p<.001). n = 7. <i>C</i>, WT, RIαB, and RIαB/Alb-<i>cre</i> mice were injected with 0.75 mU of insulin per kilogram of body weight at time 0 and blood glucose levels were determined at the indicated time points. Values are presented as means ± SEM. n = 10.</p
Liver-specific PKA inhibition does not alter fasting-regulated gene expression.
<p>mRNA levels of enzymes required for gluconeogenesis in the 24 hr fasted state (Fast) or after being fasted for 24 hr then allowed access to food for 6 hr (Fed) in WT, RIαB<b>,</b> and RIαB/Alb-<i>cre</i> mice. Levels of each mRNA were measured by quantitative real time PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018772#s4" target="_blank">Materials and Methods</a>. Data are expressed relative to the results of a fasted WT control animal. Each value represents the mean ± SEM; n = 3 animals per group. No significant differences were found in gene induction. PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1α), PEPCK (phospho<i>enol</i>pyruvate carboxykinase), G6Pase (glucose-6-phosphatase), GCK (glucokinase), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), and GLUT2 (glucose transporter type 2).</p
Phenotypes Observed by Activating the RIαB Allele with Various Cre Driver Mouse Lines.
<p>Phenotypes Observed by Activating the RIαB Allele with Various Cre Driver Mouse Lines.</p
RiboTag analysis of actively translated mRNAs in Sertoli and Leydig cells in vivo
Male spermatogenesis is a complex biological process that is regulated by hormonal signals from the hypothalamus (GnRH), the pituitary gonadotropins (LH and FSH) and the testis (androgens, inhibin). The two key somatic cell types of the testis, Leydig and Sertoli cells, respond to gonadotropins and androgens and regulate the development and maturation of fertilization competent spermatozoa. Although progress has been made in the identification of specific transcripts that are translated in Sertoli and Leydig cells and their response to hormones, efforts to expand these studies have been restricted by technical hurdles. In order to address this problem we have applied an in vivo ribosome tagging strategy (RiboTag) that allows a detailed and physiologically relevant characterization of the "translatome" (polysome-associated mRNAs) of Leydig or Sertoli cells in vivo. Our analysis identified all previously characterized Leydig and Sertoli cell-specific markers and identified in a comprehensive manner novel markers of Leydig and Sertoli cells; the translational response of these two cell types to gonadotropins or testosterone was also investigated. Modulation of a small subset of Sertoli cell genes occurred after FSH and testosterone stimulation. However, Leydig cells responded robustly to gonadotropin deprivation and LH restoration with acute changes in polysome-associated mRNAs. These studies identified the transcription factors that are induced by LH stimulation, uncovered novel potential regulators of LH signaling and steroidogenesis, and demonstrate the effects of LH on the translational machinery in vivo in the Leydig cell