Dichotomy of Tyrosine Hydroxylase and Dopamine Regulation between Somatodendritic and Terminal Field Areas of Nigrostriatal and Mesoaccumbens Pathways

Measures of dopamine-regulating proteins in somatodendritic regions are often used only as static indicators of neuron viability, overlooking the possible impact of somatodendritic dopamine (DA) signaling on behavior and the potential autonomy of DA regulation between somatodendritic and terminal field compartments. DA reuptake capacity is less in somatodendritic regions, possibly placing a greater burden on de novo DA biosynthesis within this compartment to maintain DA signaling. Therefore, regulation of tyrosine hydroxylase (TH) activity may be particularly critical for somatodendritic DA signaling. Phosphorylation of TH at ser31 or ser40 can increase activity, but their impact on L-DOPA biosynthesis in vivo is unknown. Thus, determining their relationship with L-DOPA tissue content could reveal a mechanism by which DA signaling is normally maintained. In Brown-Norway Fischer 344 F1 hybrid rats, we quantified TH phosphorylation versus L-DOPA accumulation. After inhibition of aromatic acid decarboxylase, L-DOPA tissue content per recovered TH protein was greatest in NAc, matched by differences in ser31, but not ser40, phosphorylation. The L-DOPA per catecholamine and DA turnover ratios were significantly greater in SN and VTA, suggesting greater reliance on de novo DA biosynthesis therein. These compartmental differences reflected an overall autonomy of DA regulation, as seen by decreased DA content in SN and VTA, but not in striatum or NAc, following short-term DA biosynthesis inhibition from local infusion of the TH inhibitor α-methyl-p-tyrosine, as well as in the long-term process of aging. Such data suggest ser31 phosphorylation plays a significant role in regulating TH activity in vivo, particularly in somatodendritic regions, which may have a greater reliance on de novo DA biosynthesis. Thus, to the extent that somatodendritic DA release affects behavior, TH regulation in the midbrain may be critical for DA bioavailability to influence behavior

Immunohistochemical Characterisation of Cell-Type Specific Expression of CK1δ in Various Tissues of Young Adult BALB/c Mice

BACKGROUND: Casein kinase 1 delta (CK1delta) phosphorylates many key proteins playing important roles in such biological processes as cell growth, differentiation, apoptosis, circadian rhythm and vesicle transport. Furthermore, deregulation of CK1delta has been linked to neurodegenerative diseases and cancer. In this study, the cell specific distribution of CK1delta in various tissues and organs of young adult BALB/c mice was analysed by immunohistochemistry. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical staining of CK1delta was performed using three different antibodies against CK1delta. A high expression of CK1delta was found in a variety of tissues and organ systems and in several cell types of endodermal, mesodermal and ectodermal origin. CONCLUSIONS: These results give an overview of the cell-type specific expression of CK1delta in different organs under normal conditions. Thus, they provide evidence for possible cell-type specific functions of CK1delta, where CK1delta can interact with and modulate the activity of key regulator proteins by site directed phosphorylation. Furthermore, they provide the basis for future analyses of CK1delta in these tissues

Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

Background: Armadillo, the Drosophila orthologue of vertebrate beta-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex'' is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets. Methodology/Principal Findings: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional'' and "adhesive'' pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10) displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, Delta NArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-Delta NArm(1-155) produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10). These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin. Conclusions/Significance: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling. Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-CadherinPublisher PDFPeer reviewe

Isoform specific phosphorylation of p53 by protein kinase CK1

The ability of three isoforms of protein kinase CK1 (alpha, gamma(1), and delta) to phosphorylate the N-terminal region of p53 has been assessed using either recombinant p53 or a synthetic peptide reproducing its 1-28 sequence. Both substrates are readily phosphoylated by CK1delta and CK1alpha, but not by the gamma isoform. Affinity of full size p53 for CK1 is 3 orders of magnitude higher than that of its N-terminal peptide (K (m) 0.82 muM vs 1.51 mM). The preferred target is S20, whose phosphorylation critically relies on E17, while S6 is unaffected despite displaying the same consensus (E-x-x-S). Our data support the concept that non-primed phosphorylation of p53 by CK1 is an isoform-specific reaction preferentially affecting S20 by a mechanism which is grounded both on a local consensus and on a remote docking site mapped to the K(221)RQK(224) loop according to modeling and mutational analysis

Chemical dissection of the APC repeat 3 multistep phosphorylation by the concerted action of protein kinases CK1 and GSK3

A crucial event in machinery controlled by Wnt signaling is the association of beta-catenin with the adenomatous polyposis coli (APC) protein, which is essential for the degradation of beta-catenin and requires the multiple phosphorylation of APC at six serines (1501, 1503, 1504, 1505, 1507, and 1510) within its repeat three (R3) region. Such a phosphorylation is believed to occur by the concerted action of two protein kinases, CK1 and GSK3, but its mechanistic aspects are a matter of conjecture. Here, by combining the usage of variably phosphorylated peptides reproducing the APC R3 region and Edman degradation assisted localization of residues phosphorylated by individual kinases, we show that the process is initiated by CK1, able to phosphorylate S1510 and S1505, both specified by non-canonical determinants. Phosphorylation of S1505 primes subsequent phosphorylation of S1501 by GSK3. In turn, phospho-S1501 triggers the hierarchical phosphorylation of S1504 and S1507 by CK1. Once phosphorylated, S1507 primes the phosphorylation of both S1510 and S1503 by CK1 and GSK3, respectively, thus completing all six phosphorylation steps. Our data also rule out the intervention of CK2 despite the presence of a potential CK2 phosphoacceptor site, S1510LDE, in the R3 repeat. S1510 is entirely unaffected by CK2, while it is readily phosphorylated even in the unprimed peptide by CK1delta but not by CK1gamma. This discloses a novel motif significantly different from non-canonical sequences phosphorylated by CK1 in other proteins, which appears to be specifically recognized by the delta isoform of CK1

Environmental Governance for the Coastal Marine Ecosystem Services of Chiloé Island (Southern Chile)

The Chiloé Island, located in Southern Chile, is worldwide known for its culture, originated in the syncretism between ancestral groups and colonizers, and molded through the interactions with the isolated ecosystems and extreme weather conditions. In this chapter, we analyze the state of the island’s social-ecological systems, the drivers (direct and indirect), and the impacts. We also analyze the current modes of governance related to coastal marine ecosystems and the social effects of an event that occurred during the year 2016, known as Chilote’s May.Fil: Delgado, Luisa E.. Universidad de Chile.; ChileFil: Marín, Victor H.. Universidad de Chile.; ChileFil: Asún, Rodrigo. Universidad de Chile.; ChileFil: Zúñiga, Claudia. Universidad de Chile.; ChileFil: Natenzon, Claudia Eleonor. Universidad de Buenos Aires. Facultad de Filosofía y Letras. Instituto de Geografía "Romualdo Ardissone"; ArgentinaFil: Castro Diaz, Ivan Ricardo. Universidad Nacional de Entre Ríos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Paredes, Lorena D.. Universidad de Chile.; ChileFil: Caprioli, Franco. Universidad de Chile.; Chil

Phosphorylation At Ser244 By Ck1 Determines Nuclear Localization And Substrate Targeting Of Pkd2

Protein kinase D2 (PKD2), a member of the PKD family of serine/threonine kinases, is localized in various subcellular compartments including the nucleus where the kinase accumulates upon activation of G-protein-coupled receptors. We define three critical post-translational modifications required for nuclear accumulation of PKD2 in response to activation of the CCK2 receptor (CCK2R): phosphorylation at Ser706 and Ser710 within the activation loop by PKC eta leading to catalytic activity and phosphorylation at Ser244 within the zinc-finger domain, which is crucial for blocking nuclear export of active PKD2 by preventing its interaction with the Crm-1 export machinery. We identify CK1delta and epsilon as upstream activated kinases by CCK2R that phosphorylate PKD2 at Ser244. Moreover, nuclear accumulation of active PKD2 is a prerequisite for efficient phosphorylation of its nuclear substrate, HDAC7. Only nuclear, active PKD2 mediates CCK2R-induced HDAC7 phosphorylation and Nur77 expression. Thus, we define a novel, compartment-specific signal transduction pathway downstream of CCK2R that phosphorylates PKD2 at three specific sites, results in nuclear accumulation of the active kinase and culminates in efficient phosphorylation of nuclear PKD2 substrates in human gastric cancer cells

Integral Prospection of Andean Microbial Ecosystem Project

When microbial ecosystems first started to be reported 10 years ago, nobody reallyhad a notion of the relevance they would have in the Central Andean region.Consequently, the heritage of the microbialites reported in El Peinado, LagunaNegra, Laguna Pozo Bravo, Laguna La Brava, etc. promises to position the Andes asreservoirs of the most relevant modern microbialites on the planet (Table 17.1,Fig. 17.1). Furthermore, the number of different ecosystems is worth paying closeattention to, as it gives rise to questions such as: What favors the development ofthese ecosystems? What are the conditions that influence the precipitation of a carbonaceousor a gypsum system at such a short distance and under similar environmentalconditions, such as at the Atacama salt flat? Why are oncolites distributed so? Untilnow, it has been possible only to survey the systems and to carry out more in-depthstudies in some of them to try to achieve their preservation. Throughout the prospection of the Andean microbial ecosystems (AMEs), some shared characteristics have beenfound from the geological, physical, and chemical points of view [(1) active volcanicincidence: all of the microbial ecosystems that have been found are in some way connectedto areas where active volcanoes are present; (2) underground water input;(3) mixed zones with different salinities: underground low-conductivitywater andsalar thalassic water; (4) two kinds of microbialites found: oncolites (at LagunaNegra, Tres Quebradas, Las Quinoas, etc.) or domes with thrombolites at the bottomand stromatolites at the top surface (at La Brava, Pozo Bravo, Ojos Bravos, and ElPeinado)] and from the biological point of view [(5) predominance of diatoms, themain component in all studied systems; (6) predominance of anaerobic over aerobicphotosynthetic microorganisms; (7) microbial rhodopsin as the main system for producing adenosine triphosphate (ATP); (8) arsenicresistance and bioenergetic mechanisms;and (9) predominance of Carbon fixation pathways other than the Calvincycle]. The biological aspects of these are being studied thoroughly in our lab and arebriefly discussed below.Fil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Villafañe, Patricio Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Lencina, Agustina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Catamarca. Universidad Nacional de Catamarca. Centro de Investigaciones y Transferencia de Catamarca; Argentin