Isoform-specific subcellular localization and function of protein kinase A identified by mosaic imaging of mouse brain.

Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different regulatory (R) subunits. Deficiency in either the RIβ or the RIIβ subunit results in distinct neuronal phenotypes. Although RIβ contributes to synaptic plasticity, it is the least studied isoform. Using isoform-specific antibodies, we generated high-resolution large-scale immunohistochemical mosaic images of mouse brain that provided global views of several brain regions, including the hippocampus and cerebellum. The isoforms concentrate in discrete brain regions, and we were able to zoom-in to show distinct patterns of subcellular localization. RIβ is enriched in dendrites and co-localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron microscopy, we confirmed the mitochondrial and nuclear localization of RIβ in cultured neurons. To show the functional significance of nuclear localization, we demonstrated that downregulation of RIβ, but not of RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform specificity is defined by precise localization

GPR61 anchoring of PKA consolidates GPCR and cAMP signaling

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein–protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein–protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions

Postfeminist stylistics, work femininities and coaching: a multimodal study of a website

The aim of this paper is to examine representations of work femininities on a British website offering coaching specifically aimed at women. It builds on and contributes to studies of postfeminist representations but with a specific focus on work femininities and coaching webpages. Although studies on postfeminist representation have analysed the way young women’s, embodied and sexualised femininities are depicted across a wide variety of mainstream media, there has not been a study that focuses on the representation of work femininities on coaching websites. My approach matter because feminist authors critique popular psychology and link it to postfeminism and neoliberalism but as yet studies have focused on self-help books and magazines and not new media. Furthermore, coaching websites are an important medium for circulating postfeminist work femininities and psychological advice, produced through the digital labour of women entrepreneurs. Through my analysis of one website, influenced by feminist social semiotic multimodality literature, the paper contributes to postfeminist theory and organisation studies by explaining how ‘postfeminist stylistics’ reproduce postfeminist tropes and depictions of relational and individualised entrepreneurial femininities visually and textually (Lewis, 2014)

Angiotensin II Requires Zinc and Downregulation of the Zinc Transporters ZnT3 and ZnT10 to Induce Senescence of Vascular Smooth Muscle Cells

Senescence, a hallmark of mammalian aging, is associated with the onset and progression of cardiovascular disease. Angiotensin II (Ang II) signaling and zinc homeostasis dysfunction are increased with age and are linked to cardiovascular disease, but the relationship among these processes has not been investigated. We used a model of cellular senescence induced by Ang II in vascular smooth muscle cells (VSMCs) to explore the role of zinc in vascular dysfunction. We found that Ang II-induced senescence is a zinc-dependent pathway mediated by the downregulation of the zinc transporters ZnT3 and ZnT10, which work to reduce cytosolic zinc. Zinc mimics Ang II by increasing reactive oxygen species (ROS), activating NADPH oxidase activity and Akt, and by downregulating ZnT3 and ZnT10 and inducing senescence. Zinc increases Ang II-induced senescence, while the zinc chelator TPEN, as well as overexpression of ZnT3 or ZnT10, decreases ROS and prevents senescence. Using HEK293 cells, we found that ZnT10 localizes in recycling endosomes and transports zinc into vesicles to prevent zinc toxicity. Zinc and ZnT3/ZnT10 downregulation induces senescence by decreasing the expression of catalase. Consistently, ZnT3 and ZnT10 downregulation by siRNA increases ROS while downregulation of catalase by siRNA induces senescence. Zinc, siZnT3 and siZnT10 downregulate catalase by a post-transcriptional mechanism mediated by decreased phosphorylation of ERK1/2. These data demonstrate that zinc homeostasis dysfunction by decreased expression of ZnT3 or ZnT10 promotes senescence and that Ang II-induced senescence is a zinc and ROS-dependent process. Our studies suggest that zinc might also affect other ROS-dependent processes induced by Ang II, such as hypertrophy and migration of smooth muscle cells

A novel fast mechanism for GPCR-mediated signal transduction—control of neurotransmitter release

In addition to calcium influx, charge movement in the G protein–coupled M2-muscarinic receptor is required for the control of acetylcholine release

Derivation of Xeno-Free and GMP-Grade Human Embryonic Stem Cells – Platforms for Future Clinical Applications

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs

A Mouse Model of Acrodermatitis Enteropathica: Loss of Intestine Zinc Transporter ZIP4 (Slc39a4) Disrupts the Stem Cell Niche and Intestine Integrity

Loss-of-function of the zinc transporter ZIP4 in the mouse intestine mimics the lethal human disease acrodermatitis enteropathica. This is a rare disease in humans that is not well understood. Our studies demonstrate the paramount importance of ZIP4 in the intestine in this disease and reveal that a root cause of lethality is disruption of the intestine stem cell niche and impaired function of the small intestine. This, in turn, leads to dramatic weight loss and death unless treated with exogenous zinc

Regulation of GSK-3 Activity as A Shared Mechanism in Psychiatric Disorders

Serin/Treonin kinaz ailesinin üyelerinden bir kinaz olarak ilk kez glikojen sentaz’ı inhibe ettiği keşfedilen glikojen sentaz kinaz-3 (GSK-3), günümüzde bilinen 50’den fazla substratı ile birçok hücre içi düzenleyici mekanizmada görev alan geniş etki spektrumlu bir enzim olarak kabul edilmektedir. GSK-3’ün memelilerde GSK-3α ve GSK-3β olmak üzere yapısal olarak yüksek homoloji gösteren iki izoformu bulunmaktadır. Her iki izoform birçok dokuda yaygın dağılım göstermekle beraber, en yüksek oranda beyinde bulunmakta ve genellikle benzer fonksiyonlar göstermektedirler. Diğer protein kinazların aksine GSK-3 uyarılmamış hücrede yapısal olarak aktif yani defosforile halde bulur. Protein kinaz A (PKA), protein kinaz B (PKB;AKT) ve protein kinaz C (PKC) gibi diğer protein kinazlarla fosforilasyona uğrayarak olarak inaktive edilir. Günümüzde artmış GSK-3 aktivitesinin major depresyon, bipolar bozukluk, hiperaktivite bozuklukları gibi hastalıklar ve şizofreni oluşumunda rol oynayabileceğine ilişkin önemli bulgular mevcuttur. Bu nedenle söz konusu psikiyatrik hastalıklarda arttığı gösterilen GSK-3 aktivitesinin azaltılmasının tedavide umut verici bir yaklaşım olabileceği kabul edilebilir. Bu gözden geçirme çalışmasında yukarıda sözü edilen psikiyatrik hastalıkların oluşmasında görev alan GSK-3 aracılı mekanizmalara kısaca değinilerek GSK-3’ün aktivitesinin düzenlenmesinde rol oynadığı gösterilen klinikte kullanılan ilaçlara yer verilmiştir. Anahtar sözcükler: GSK-3, depresyon, bipolar bozukluk, şizofren