Shoc2/Sur8 protein regulates neurite outgrowth

This is an openaccess article distributed under the terms of the Creative Commons Attribution License.-- et al.The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.GL, TG and LMD were recipients of fellowships from the Ministerio de Educación y Ciencia (MEC) (to GL, TG), and Fondo de Investigaciones Sanitarias (FIS) (to LMD). LSR held a postdoctoral research contract from CIBERNED. This work was supported by FIS grant (PI10/00815) to JLO; CIBERNED to MC; SAF2008-01951, Comunidad Autónoma de Madrid (CAM) SSAL-0202-2006-01 and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) to TI; FIS grant PI12/00775 and ISCIII-RETIC (Red Temática de Investigación Cooperativa en Cáncer) RD12/0036/0027 from the Instituto de Salud Carlos III to PSG; and FIS grants (PI09/0562 and PI13/00703), ISCIIIRETIC (RD06/0020/0003 and RD12/0036/0021), and the Spanish Association Against Cancer (AECC) to JMR.Peer Reviewe

Localización y función de la proteína quinasa D y kidins220 en células neuronales: participación de proteínas PDZ

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 5 de Septiembre de 200

Light Microscopy and Image Analysis Unit

Poster.-- Close Encounters IIM (3rd Kind), Vigo, 23 June 2022Light microscopy and image analysis unit (SEMAIM) provides assistance for all groups and visitors throughout the process of microscopic imaging. The staff assists researchers in the planning of light microscopy experiments and provides training for the operation of the microscopes and for speciffic imaging techniques. Additionally, support is provided in the processing, rendering and analysis of the acquired datasetsN

Light Microscopy and Image Analysis Unit

Poster.-- Encontros IIM Primeira fase, 12 de decembro de 2019Peer reviewe

Lipid raft disruption triggers protein kinase C and Src-dependent protein kinase D activation and Kidins220 phosphorylation in neuronal cells

Kidins220 (kinase D-intracting substrate of 220 kDa) is a novel neurospecific protein recently cloned as the first substrate for the Ser/Thr kinase protein kinase D (PKD). Herein we report that Kidins220 is constitutively associated to lipid rafts in PC12 cells, rat primary cortical neurons, and brain synaptosomes. Immunocytochemistry and confocal microscopy together with sucrose gradient fractionation show co-localization of Kidins220 and lipid raft-associated proteins. In addition, cholesterol depletion of cell membranes with methyl-β-cyclodextrin dramatically alters Kidins220 localization and detergent solubility. By studying the putative involvement of lipid rafts in PKD activation and signaling we have found that active PKD partitions in lipid raft fractions after sucrose gradient centrifugation and that green fluorescent protein-PKD translocates to lipid raft microdomains at the plasma membrane after phorbol ester treatment. Strikingly, lipid rafts disruption by methyl-β-cyclodextrin delays green fluorescent protein-PKD translocation, as determined by live cell confocal microscopy, and activates PIM, increasing Kidins220 phosphorylation on Ser919 by a mechanism involving PKCε and the small soluble tyrosine kinase Src. Collectively, these results reveal the importance of lipid rafts on PKD activation, translocation, and downstream signaling to its substrate Kidins220.This work was supported by Ministerio de Ciencia y Tecnología Grant SAF2001-1703 and “Comunidad de Madrid” Grant CAM 08.5/0038/2001.1 (to T. I.).Peer Reviewe

Kidins220/ARMS modulates the activity of microtubule-regulating proteins and controls neuronal polarity and development

In order for neurons to perform their function, they must establish a highly polarized morphology characterized, in most of the cases, by a single axon and multiple dendrites. Herein we find that the evolutionarily conserved protein Kidins220 (kinase D-interacting substrate of 220-kDa), also known as ARMS (ankyrin repeat-rich membrane spanning), a downstream effector of protein kinase D and neurotrophin and ephrin receptors, regulates the establishment of neuronal polarity and development of dendrites. Kidins220/ARMS gain and loss of function experiments render severe phenotypic changes in the processes extended by hippocampal neurons in culture. Although Kidins220/ARMS early overexpression hinders neuronal development, its down-regulation by RNA interference results in the appearance of multiple longer axon-like extensions as well as aberrant dendritic arbors. We also find that Kidins220/ARMS interacts with tubulin and microtubule-regulating molecules whose role in neuronal morphogenesis is well established (microtubule-associated proteins 1b, 1a, and 2 and two members of the stathmin family). Importantly, neurons where Kidins220/ARMS has been knocked down register changes in the phosphorylation activity of MAP1b and stathmins. Altogether, our results indicate that Kidins220/ARMS is a key modulator of the activity of microtubule-regulating proteins known to actively regulate neuronal morphogenesis and suggest a mechanism by which it contributes to control neuronal development.This work was supported by “Ministerio de Ciencia e Innovación” Grant SAF2008-01951, “Neurodegmodels” Grant CAM S-SAL-0202-2006-01 from “Comunidad de Madrid,” and CIBERNED from “Instituto de Salud Carlos III” (Spain) (to T. I.).Peer Reviewe

Protein kinase D activity controls endothelial nitric oxide synthesis

Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone.This work was supported by the Mineco [grant numbers SAF2011-26233 to T.I., BFU2009-10442 and BFU2012-37934 to I.R.-C.]; Comunidad de Madrid [grant number P2010/BMD-2331-Neurodegmodels-CM to T.I.]; and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, to T.I. L.S.-R. was funded by research contracts from CIBERNED; C.A.-R. was a recipient of a FPU predoctoral fellowship from Mineco.Peer Reviewe

Risk factor-based clustering of Listeria monocytogenes in food processing environments using principal component analysis

13 pages, 8 figures, 1 table.-- Under a Creative Commons licenseListeria monocytogenes has a range of strategies that allow it to persist as biofilms in food processing environments (FPE), making it a pathogen of concern to the food industry. The properties of these biofilms are highly variable among strains, and this significantly affects the risk of food contamination. The present study therefore aims to conduct a proof-of-concept study to cluster strains of L. monocytogenes by risk potential using principal component analysis, a multivariate approach. A set of 22 strains, isolated from food processing environments, were typed by serogrouping and pulsed-field gel electrophoresis, showing a relatively high diversity. They were characterized in terms of several biofilm properties that might pose a potential risk of food contamination. The properties studied were tolerance to benzalkonium chloride (BAC), the structural parameters of biofilms (biomass, surface area, maximum and average thickness, surface to biovolume ratio and roughness coefficient) measured by confocal laser scanning microscopy and (3) transfer of biofilm cells to smoked salmon. The PCA correlation circle revealed that the tolerance of biofilms to BAC was positively correlated with roughness, but negatively with biomass parameters. On the contrary, cell transfers were not related to three-dimensional structural parameters, which suggests the role of other variables yet unexplored. Additionally, hierarchical clustering grouped strains into three different clusters. One of them included the strains with high tolerance to BAC and roughness. Another one consisted of strains with enhanced transfer ability, whereas the third cluster contained those that stood out for the thickness of biofilms. The present study represents a novel and effective way to classify L. monocytogenes strains according to biofilm properties that condition the potential risk of reaching the consumer through food contamination. It would thus allow the selection of strains representative of different worst-case scenarios for future studies in support of QMRA and decision-making analysisThis research was financially supported by the Spanish Ministry of Science and Innovation (ASEQURA, PID2019-10 8420RB-C31)Peer reviewe

Spectral fingerprinting of biobanked fish-borne parasites

Poster.-- 2nd Biospecimen Research Symposium: Focus on Quality and Standards, Berlin, February 5-6, 2019Marine parasites are an important health and quality threat in fishery products. There are a growing number of fish-borne parasitic infections and hypersensitivity reactions following intake of viable parasites. Anisakidae (Anisakis, Pseudoterranova and Contracaecum) are by far the most prevalent macroparasites in fish products worldwide and causes human parasitic infections commonly associated to this consumption.Peer reviewe

Calcium-dependent expression of TNF-α in neural cells is mediated by the calcineurin/NFAT pathway

We report induction of TNF-α via the calcium/calcineurin/NFAT pathway in PC12 neural cells. In PC12, expression of TNF-α mRNA, protein and TNF-α gene promoter activity was induced by co-stimulation with phorbol ester and either calcium ionophore A23187 or the L-type Voltage Gated Calcium Channel agonist Bay K 8644. Pre-treatment with calcineurin inhibitors CsA or FK506 inhibited the dominant calcium-dependent component of this induction, limiting it to the level achieved with phorbol ester alone. Promoter activation by Bay was abolished by nifedipine, a specific inhibitor of L-type Voltage Gated Calcium Channels. Exogenous NFAT protein transactivated the TNF-α promoter, and the peptide VIVIT-a specific inhibitor of calcineurin/NFAT binding-blocked calcium-inducible transactivation of the TNF-α promoter. Given proposed functions of TNF-α in spatial learning, memory and the pathogenesis of neurodegenerative diseases, the data presented suggest an important role for calcineurin/NFAT signaling in these key neurological processes. © 2006 Elsevier Inc. All rights reserved.This work was supported by grant 08.5/0039.1/2003 from the Comunidad Autónoma de Madrid (CAM) to E. Cano and by grants to J.M. Redondo from the Ministerio de Ciencia y Tecnología (SAF 2003-02920), the RECAVA programme of the Ministerio de Sanidad y Consumo, the European Union LSHM-CT-2004-005033 (EICOSANOX) and the Fundación La Marato (TV3). A. Canellada was supported by a fellowship from the Ministerio de Educación y Ciencia and the Fundación Carolina of Spain. E. Cano is a recipient of a Ramon y Cajal contract from the Ministerio de Educación y Ciencia.Peer Reviewe