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

Protein Kinase A Regulatory Subunits in Human Adipose Tissue: Decreased R2B Expression and Activity in Adipocytes From Obese Subjects

OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects

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

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

Protein Kinase A Regulatory Subunits in Human Adipose Tissue: Decreased R2B Expression and Activity in Adipocytes From Obese Subjects

OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects

Gravitational tidal effects on galactic open clusters

We have investigated the 2-D stellar distribution in the outer parts of three nearby open clusters: NGC 2287 (=M41), NGC 2516, and NGC 2548 (=M48). Wide-field star counts have been performed in two colours on pairs of digitized ESO and SRC Schmidt plates, allowing us to select likely cluster members in the colour-magnitude diagrams. Cluster tidal extensions were emphasized using a wavelet transform. Taking into account observational biases, namely the galaxy clustering and differential extinction in the Galaxy, we have associated these stellar overdensities with real open cluster structures stretched by the galactic gravitational field. As predicted by theory and simulations, and despite observational limitations, we detected a general elongated (prolate) shape in a direction parallel to the galactic Plane, combined with tidal tails extended perpendicularly to it. This geometry is due both to the static galactic tidal field and the heating up of the stellar system when crossing the Disk. The time varying tidal field will deeply affect the cluster dynamical evolution, and we emphasize the importance of adiabatic heating during the Disk-shocking. In the case of NGC 2548, our dating of the last shocking with the Plane (based on a tidal clump) is consistent with its velocity. During the 10-20 Z-oscillations experienced by a cluster before its dissolution in the Galaxy, crossings through the galactic Disk contribute to at least 15% of the total mass loss. Using recent age estimations published for open clusters, we find a destruction time-scale of about 600 Myr for clusters in the solar neighbourhood.Comment: To be published in A&A (accepted July 2001). 10 pages, 10 figures. Version with full resolution figures available at http://www.cai-mama.obspm.fr/pagesPersonnelles/bergond/img/referee.ps.g

A photometric long-term study of CP stars in open clusters

Photometric variability of chemically peculiar (CP) stars of the upper main sequence is closely connected to their local stellar magnetic field and their rotational period. Long term investigations, as presented here, help us to identify possible stellar cycles (as in the Sun). Furthermore, these data provide a basis for detailed surface mapping techniques. Photoelectric Stroemgren uvby time series for 27 CP stars within the boundaries of open clusters are presented. In addition, Hipparcos photometric data (from 1989 to 1993) are used for our analysis. Our observations cover a time period of about six years (1986 to 1992) with typically fifteen measurements for each objects. These observations help us to determine the rotational periods of these objects. A standard reduction procedure was applied to the data. When possible, we merged our data sets with already published ones to obtain a more significant result. A detailed time series analysis was performed, involving five different methods to minimize spurious detections. We established, for the first time, variability for fourteen CP stars. For additional two stars, a merging of already published data sets, resulted in more precise periods, whereas for six objects, the published periods could be confirmed. Last, but not least, no significant variations were found for five stars. Apart from six stars, all targets seem to be members of their host open clusters.Comment: 10 pages, 3 figures, accepted by Astronomy and Astrophysic

Erythropoietin signaling regulates heme biosynthesis

Heme is required for survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for many cellular processes, how it is coupled to cellular differentiation is unknown. Here, using zebrafish, murine, and human models, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, AKAP10, regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane. This integrated pathway culminates with the direct phosphorylation of the crucial heme biosynthetic enzyme, ferrochelatase (FECH) by protein kinase A (PKA). Biochemical, pharmacological, and genetic inhibition of this signaling pathway result in a block in hemoglobin production and concomitant intracellular accumulation of protoporphyrin intermediates. Broadly, our results implicate aberrant PKA signaling in the pathogenesis of hematologic diseases. We propose a unifying model in which the erythroid transcriptional program works in concert with post-translational mechanisms to regulate heme metabolism during normal development

ApoB100-LDL Acts as a Metabolic Signal from Liver to Peripheral Fat Causing Inhibition of Lipolysis in Adipocytes

International audienceBACKGROUND: Free fatty acids released from adipose tissue affect the synthesis of apolipoprotein B-containing lipoproteins and glucose metabolism in the liver. Whether there also exists a reciprocal metabolic arm affecting energy metabolism in white adipose tissue is unknown. METHODS AND FINDINGS: We investigated the effects of apoB-containing lipoproteins on catecholamine-induced lipolysis in adipocytes from subcutaneous fat cells of obese but otherwise healthy men, fat pads from mice with plasma lipoproteins containing high or intermediate levels of apoB100 or no apoB100, primary cultured adipocytes, and 3T3-L1 cells. In subcutaneous fat cells, the rate of lipolysis was inversely related to plasma apoB levels. In human primary adipocytes, LDL inhibited lipolysis in a concentration-dependent fashion. In contrast, VLDL had no effect. Lipolysis was increased in fat pads from mice lacking plasma apoB100, reduced in apoB100-only mice, and intermediate in wild-type mice. Mice lacking apoB100 also had higher oxygen consumption and lipid oxidation. In 3T3-L1 cells, apoB100-containing lipoproteins inhibited lipolysis in a dose-dependent fashion, but lipoproteins containing apoB48 had no effect. ApoB100-LDL mediated inhibition of lipolysis was abolished in fat pads of mice deficient in the LDL receptor (Ldlr(-/-)Apob(100/100)). CONCLUSIONS: Our results show that the binding of apoB100-LDL to adipocytes via the LDL receptor inhibits intracellular noradrenaline-induced lipolysis in adipocytes. Thus, apoB100-LDL is a novel signaling molecule from the liver to peripheral fat deposits that may be an important link between atherogenic dyslipidemias and facets of the metabolic syndrome