BBA - Molecular Cell Research (v.1853, #11PA)

Differential tyrosine phosphorylation controls the function of CNK1 as a molecular switch in signal transduction by Adrian Fischer; Tilman Brummer; Bettina Warscheid; Gerald Radziwill (2847-2855).
Scaffold proteins are multidomain proteins without enzymatic function that play a central role in coordinating signaling processes. The scaffold protein CNK1 interacts with pathway-specific signaling proteins and thereby regulates these respective pathways. Here, we revealed tyrosine phosphorylation as a critical regulation mechanism to control the function of CNK1. We identified Tyr 26 as a PDGF-induced and, additionally, Tyr 519 and Tyr 665 as SRC-induced tyrosine phosphorylation sites. Phosphomimetic mutants indicate that phosphorylation of Tyr 519 recruits CNK1 to the nucleus and additional phosphorylation of Tyr 26 enables CNK1 to promote SRE-dependent gene expression. Contrary, mutants preventing tyrosine phosphorylation promote matrix metalloproteinase MMP14 promoter activity. CNK1-driven cell proliferation partially depends on its tyrosine phosphorylation. Upon PDGF stimulation, CNK1 is recruited to the plasma membrane mediated by SRC. Knock down of CNK1 prevents PDGF-induced SRE-dependent gene expression, MMP14 promoter activity and cell proliferation. Thus, tyrosine phosphorylation is an important mechanism to control the subcellular localization of CNK1 and its distinct biological functions.
Keywords: CNK1; Platelet-derived growth factor; Scaffold protein; Signal transduction; SRC; Tyrosine phosphorylation;

Eps15 homology domain containing protein of Plasmodium falciparum (PfEHD) associates with endocytosis and vesicular trafficking towards neutral lipid storage site by Vandana Thakur; Mohd Asad; Shaifali Jain; Mohammad E. Hossain; Akanksha Gupta; Inderjeet Kaur; Sumit Rathore; Shakir Ali; Nida J. Khan; Asif Mohmmed (2856-2869).
The human malaria parasite, Plasmodium falciparum, takes up numerous host cytosolic components and exogenous nutrients through endocytosis during the intra-erythrocytic stages. Eps15 homology domain-containing proteins (EHDs) are conserved NTPases, which are implicated in membrane remodeling and regulation of specific endocytic transport steps in eukaryotic cells. In the present study, we have characterized the dynamin-like C-terminal Eps15 homology domain containing protein of P. falciparum (PfEHD). Using a GFP-targeting approach, we studied localization and trafficking of PfEHD in the parasite. The PfEHD-GFP fusion protein was found to be a membrane bound protein that associates with vesicular network in the parasite. Time-lapse microscopy studies showed that these vesicles originate at parasite plasma membrane, migrate through the parasite cytosol and culminate into a large multi-vesicular like structure near the food-vacuole. Co-staining of food vacuole membrane showed that the multi-vesicular structure is juxtaposed but outside the food vacuole. Labeling of parasites with neutral lipid specific dye, Nile Red, showed that this large structure is neutral lipid storage site in the parasites. Proteomic analysis identified endocytosis modulators as PfEHD associated proteins in the parasites. Treatment of parasites with endocytosis inhibitors obstructed the development of PfEHD-labeled vesicles and blocked their targeting to the lipid storage site. Overall, our data suggests that the PfEHD is involved in endocytosis and plays a role in the generation of endocytic vesicles at the parasite plasma membrane, that are subsequently targeted to the neutral lipid generation/storage site localized near the food vacuole.
Keywords: Malaria; Plasmodium falciparum; Eps15 homology domain (EHD); Endocytosis; Food-vacuole; Lipid storage;

Dominant negative Ras attenuates pathological ventricular remodeling in pressure overload cardiac hypertrophy by Manuel Ramos-Kuri; Kleopatra Rapti; Hind Mehel; Shihong Zhang; Perundurai S. Dhandapany; Lifan Liang; Alejandro García-Carrancá; Regis Bobe; Rodolphe Fischmeister; Serge Adnot; Djamel Lebeche; Roger J. Hajjar; Larissa Lipskaia; Elie R. Chemaly (2870-2884).
The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2 weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy.
Keywords: Heart failure; Cardiac hypertrophy; Ras oncogene; Pathological hypertrophy; Physiological hypertrophy; Ras inhibition;

Modulatory role of the anti-apoptotic protein kinase CK2 in the sub-cellular localization of Fas associated death domain protein (FADD) by Valérie Vilmont; Odile Filhol; Anne-Marie Hesse; Yohann Couté; Christophe Hue; Léa Rémy-Tourneur; Sylvie Mistou; Claude Cochet; Gilles Chiocchia (2885-2896).
The Fas associated death domain protein (FADD) is the key adaptor molecule of the apoptotic signal triggered by death receptors of the TNF-R1 superfamily. Besides its crucial role in the apoptotic machinery, FADD has proved to be important in many biological processes like tumorigenesis, embryonic development or cell cycle progression. In a process to decipher the regulatory mechanisms underlying FADD regulation, we identified the anti-apoptotic kinase, CK2, as a new partner and regulator of FADD sub-cellular localization. The blockade of CK2 activity induced FADD re-localization within the cell. Moreover, cytoplasmic FADD was increased when CK2β was knocked down. In vitro kinase and pull down assays confirmed that FADD could be phosphorylated by the CK2 holoenzyme. We found that phosphorylation is weak with CK2α alone and optimal in the presence of stoichiometric amounts of CK2α catalytic and CK2β regulatory subunit, showing that FADD phosphorylation is undertaken by the CK2 holoenzyme in a CK2β-driven fashion. We found that CK2 can phosphorylate FADD on the serine 200 and that this phosphorylation is important for nuclear localization of FADD. Altogether, our results show for the first time that multifaceted kinase, CK2, phosphorylates FADD and is involved in its sub-cellular localization. This work uncovered an important role of CK2 in stable FADD nuclear localization.
Keywords: FADD; Regulation; Phosphorylation; CK2; Nuclear localization;

Mitochondrial DNA has a pro-inflammatory role in AMD by Bernard Dib; Haijiang Lin; Daniel E. Maidana; Bo Tian; John B. Miller; Peggy Bouzika; Joan W. Miller; Demetrios G. Vavvas (2897-2906).
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly of industrialized nations, and there is increasing evidence to support a role for chronic inflammation in its pathogenesis. Mitochondrial DNA (mtDNA) has been recently reported to be pro-inflammatory in various diseases such as Alzheimer's and heart failure. Here, we report that intracellular mtDNA induces ARPE-19 cells to secrete inflammatory cytokines IL-6 and IL-8, which have been consistently associated with AMD onset and progression. The induction was dependent on the size of mtDNA, but not on specific sequence. Oxidative stress plays a major role in the development of AMD, and our findings indicate that mtDNA induces IL-6 and IL-8 more potently when oxidized. Cytokine induction was mediated by STING (Stimulator of Interferon Genes) and NF-κB as evidenced by abrogation of the cytokine response with the use of specific inhibitors (siRNA and BAY 11-7082, respectively). Finally, mtDNA primed the NLRP3 inflammasome. This study contributes to our understanding of the potential pro-inflammatory role of mtDNA in the pathogenesis of AMD.Display Omitted
Keywords: Age-related macular degeneration; Retinal pigment epithelium; Mitochondrial DNA; Inflammation; NLRP3 inflammasome;

Cx43 increases serum induced filopodia formation via activation of p21-activated protein kinase 1 by Petra Kameritsch; Felizitas Kiemer; Heike Beck; Ulrich Pohl; Kristin Pogoda (2907-2917).
In a previous study we could show that connexin 43 (Cx43) expression increased the migration of cells in a channel-independent manner involving the MAPK p38. We analyzed here the mechanism by which Cx43 enhanced p38 activation and migration related changes of the actin cytoskeleton. HeLa cells were used as a model system for the controlled expression of Cx43 and truncated Cx43 proteins. The expression of Cx43 altered the actin cytoskeleton organization in response to serum stimulation. Cx43 expressing HeLa cells had significantly more filopodial protrusions per cell than empty-vector transfected control cells. The expression of the channel incompetent carboxyl tail of Cx43 was sufficient to enhance the filopodia formation whereas the N-terminal, channel-building part, had no such effect. The enhanced filopodia formation was p38 dependent since the p38 blocker SB203580 significantly diminished it. Immunoprecipitation revealed an interaction of the upstream regulator of p38, p21-activated protein kinase 1 (PAK1), with Cx43 resulting in an enhanced phosphorylation of PAK1. Moreover, p38 activation, filopodia formation and cell migration were significantly reduced by blocking the PAK1 activity with its pharmacological inhibitor, IPA-3. The p38 target Hsp27, which favors the actin polymerization in its phosphorylated form, was significantly more phosphorylated characterizing it as a potential candidate molecule to enhance the serum-induced actin polymerization in Cx43 expressing cells. Our results provide a novel mechanism by which Cx43 can modify actin cytoskeletal dynamics and may thereby enhance cell migration.
Keywords: Connexin; Gap junction independent; Actin cytoskeleton; Migration; p38;

Fe65 negatively regulates Jagged1 signaling by decreasing Jagged1 protein stability through the E3 ligase Neuralized-like 1 by Hye-Jin Lee; Ji-Hye Yoon; Ji-Seon Ahn; Eun-Hye Jo; Mi-Yeon Kim; Young Chul Lee; Jin Woo Kim; Eun-Jung Ann; Hee-Sae Park (2918-2928).
Fe65 is a highly conserved adaptor protein that interacts with several binding partners. Fe65 binds proteins to mediate various cellular processes. But the interacting partner and the regulatory mechanisms controlled by Fe65 are largely unknown. In this study, we found that Fe65 interacts with the C-terminus of Jagged1. Furthermore, Fe65 negatively regulates AP1-mediated Jagged1 intercellular domain transactivation in a Tip60-independent manner. We found that Fe65 triggers the degradation of Jagged1, but not the Jagged1 intracellular domain (JICD), through both proteasome and lysosome pathways. We also showed that Fe65 promotes recruitment of the E3 ligase Neuralized-like 1 (Neurl1) to membrane-tethered Jagged1 and monoubiquitination of Jagged1. These three proteins form a stable trimeric complex, thereby decreasing Jagged1 targeting by ubiquitin-mediated degradation. Consequently, Jagged1 is a novel binding partner of Fe65, and Fe65 may act as a novel effector of Jagged1 signaling.
Keywords: Fe65; Jagged1; Neuralized-like 1; Proteasome; Lysosome; Protein degradation;

Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation by Jiaxin Li; Xiaobing Dou; Songtao Li; Ximei Zhang; Yong Zeng; Zhenyuan Song (2929-2936).
Nicotinamide (NAM) is the amide of nicotinic acid and a predominant precursor for NAD+ biosynthesis via the salvage pathway. Sirt1 is a NAD+-dependent deacetylase, playing an important role in regulating cellular functions. Although hepatoprotective effect of NAM has been reported, the underlying mechanism remains elusive. ER stress, induced by saturated fatty acids, in specific palmitate, plays a pathological role in the development of nonalcoholic fatty liver disease. This study aims to determine the effect of NAM on palmitate-induced ER stress in hepatocytes and to elucidate molecular mechanisms behind. Both HepG2 cells and primary mouse hepatocytes were exposed to palmitate (conjugated to BSA at a 2:1 M ratio), NAM, or their combination for different durations. Cellular NAD+ level, Sirt1 expression/activity, ER stress, as well as cAMP/PKA/CREB pathway activation were determined. NAM increased Sirt1 expression and enzymatic activity, which contributes to the ameliorative effect of NAM on palmitate-triggered ER stress. NAM increased intracellular NAD+ level in hepatocytes, however, blocking the salvage pathway, a pathway for NAD+ synthesis from NAM, only partially prevented NAM-induced Sirt1 upregulation while completely prevented NAD+ increase in response to NAM. Further mechanistic investigations revealed that NAM elevated intracellular cAMP level via suppressing PDE activity, leading to downstream PKA and CREB activation. Importantly, cAMP/PKA/CREB pathway blockade abolished not only NAM-induced Sirt1 upregulation, but also its protective effect against ER stress. Our results demonstrate that NAM protects hepatocytes against palmitate-induced ER stress in hepatocytes via upregulating Sirt1. Activation of the cAMP/PKA/CREB pathway plays a key role in NAM-induced Sirt1 upregulation.
Keywords: Nicotinamide; Sirt1; NAD+;  Deacetylation; CAMP; PKA;

The roles of IP3 receptor in energy metabolic pathways and reactive oxygen species homeostasis revealed by metabolomic and biochemical studies by He Wen; Wen Jun Xu; Xing Jin; Sehyun Oh; Chau Hong Duc Phan; Jayoung Song; Sang Kook Lee; Sunghyouk Park (2937-2944).
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are calcium channels modulating important calcium-mediated processes. Recent studies implicate IP3R in cell metabolism, but specific evidence is missing regarding IP3R's effects on actual metabolic pathways and key energy metabolites. Here, we applied metabolomics and molecular biology to compare DT40 cell lines devoid of IP3R (KO) and its wild-type (WT) counterpart. NMR and LC–MS metabolomic data showed that the KO cell line has a very different basic energy metabolism from the WT cell line, showing enhanced Warburg effect. In particular, the KO cells exhibited significant perturbation in energy charge, reduced glutathione and NADPH ratios with slower cellular growth rate. Subsequent flow cytometry results showed that the KO cell line has a higher level of general reactive oxygen species (ROS) but has a lower level of peroxynitrites. This ROS disturbance could be explained by observing lower expression of superoxide dismutase 2 (SOD2) and unchanged expression of catalase. The higher ROS seems to be involved in the slower growth rate of the KO cells, with an ROS scavenger increasing their growth rate. However, the KO and WT cell lines did not show any noticeable differences in AMPK and phosphorylated AMPK levels, suggesting possible saturation of AMPK-mediated metabolic regulatory circuit in both cells. Overall, our study reveals IP3R's roles in ROS homeostasis and metabolic pathways as well as the effects of its KO on cellular phenotypes.
Keywords: DT40; IP3R; Metabolomics; ROS; Energy metabolism;

8-Dehydrosterols induce membrane traffic and autophagy defects through V-ATPase dysfunction in Saccharomyces cerevisae by Agustín Hernández; Gloria Serrano-Bueno; José Román Perez-Castiñeira; Aurelio Serrano (2945-2956).
8-Dehydrosterols are present in a wide range of biologically relevant situations, from human rare diseases to amine fungicide-treated fungi and crops. However, the molecular bases of their toxicity are still obscure. We show here that 8-dehydrosterols, but not other sterols, affect yeast vacuole acidification through V-ATPases. Moreover, erg2Δ cells display reductions in proton pumping rates consistent with ion-transport uncoupling in vitro. Concomitantly, subunit Vph1p shows conformational changes in the presence of 8-dehydrosterols. Expression of a plant vacuolar H+-pumping pyrophosphatase as an alternative H+-pump relieves Vma -like phenotypes in erg2Δ-derived mutant cells. As a consequence of these acidification defects, endo- and exo-cytic traffic deficiencies that can be alleviated with a H+-pumping pyrophosphatase are also observed. Despite their effect on membrane traffic, 8-dehydrosterols do not induce endoplasmic reticulum stress or assembly defects on the V-ATPase. Autophagy is a V-ATPase dependent process and erg2Δ mutants accumulate autophagic bodies under nitrogen starvation similar to Vma mutants. In contrast to classical Atg mutants, this defect is not accompanied by impairment of traffic through the CVT pathway, processing of Pho8Δ60p, GFP-Atg8p localisation or difficulties to survive under nitrogen starvation conditions, but it is concomitant to reduced vacuolar protease activity. All in all, erg2Δ cells are autophagy mutants albeit some of their phenotypic features differ from classical Atg defective cells. These results may pave the way to understand the aetiology of sterol-related diseases, the cytotoxic effect of amine fungicides, and may explain the tolerance to these compounds observed in plants.
Keywords: V-ATPase; H+-pumping pyrophosphatase; Abnormal sterols; Vacuole; Autophagy; Endocytosis;

The Na+/K+-ATPase and the amyloid-beta peptide aβ1–40 control the cellular distribution, abundance and activity of TRPC6 channels by Sylvain Chauvet; Marielle Boonen; Mireille Chevallet; Louis Jarvis; Addis Abebe; Mohamed Benharouga; Peter Faller; Michel Jadot; Alexandre Bouron (2957-2965).
The Na+/K+-ATPase interacts with the non-selective cation channels TRPC6 but the functional consequences of this association are unknown. Experiments performed with HEK cells over-expressing TRPC6 channels showed that inhibiting the activity of the Na+/K+-ATPase with ouabain reduced the amount of TRPC6 proteins and depressed Ca2 + entry through TRPC6. This effect, not mimicked by membrane depolarization with KCl, was abolished by sucrose and bafilomycin-A, and was partially sensitive to the intracellular Ca2 + chelator BAPTA/AM. Biotinylation and subcellular fractionation experiments showed that ouabain caused a multifaceted redistribution of TRPC6 to the plasma membrane and to an endo/lysosomal compartment where they were degraded. The amyloid beta peptide Aβ1–40, another inhibitor of the Na+/K+-ATPase, but not the shorter peptide Aβ1–16, reduced TRPC6 protein levels and depressed TRPC6-mediated responses. In cortical neurons from embryonic mice, ouabain, veratridine (an opener of voltage-gated Na+ channel), and Aβ1–40 reduced TRPC6-mediated Ca2 + responses whereas Aβ1–16 was ineffective. Furthermore, when Aβ1–40 was co-added together with zinc acetate it could no longer control TRPC6 activity. Altogether, this work shows the existence of a functional coupling between the Na+/K+-ATPase and TRPC6. It also suggests that the abundance, distribution and activity of TRPC6 can be regulated by cardiotonic steroids like ouabain and the naturally occurring peptide Aβ1–40 which underlines the pathophysiological significance of these processes.
Keywords: TRPC6; Trafficking; Na/K pump; Neurons; Aβ peptides; Lysosomes;

New insights into circulating FABP4: Interaction with cytokeratin 1 on endothelial cell membranes by Paula Saavedra; Josefa Girona; Alba Bosquet; Sandra Guaita; Núria Canela; Gemma Aragonès; Mercedes Heras; Lluís Masana (2966-2974).
Fatty acid-binding protein 4 (FABP4) is an adipose tissue-secreted adipokine that is involved in the regulation of energetic metabolism and inflammation. Increased levels of circulating FABP4 have been detected in individuals with cardiovascular risk factors. Recent studies have demonstrated that FABP4 has a direct effect on peripheral tissues, specifically promoting vascular dysfunction; however, its mechanism of action is unknown. The objective of this work was to assess the specific interactions between exogenous FABP4 and the plasma membranes of endothelial cells. Immunofluorescence assays showed that exogenous FABP4 localized along the plasma membranes of human umbilical vein endothelial cells (HUVECs), interacting specifically with plasma membrane proteins. Anti-FABP4 immunoblotting revealed two covalent protein complexes containing FABP4 and its putative receptor; these complexes were approximately 108 kDa and 77 kDa in size. Proteomics and mass spectrometry experiments revealed that cytokeratin 1 (CK1) was the FABP4-binding protein. An anti-CK1 immunoblot confirmed the presence of CK1. FABP4-CK1 complexes were also detected in HAECs, HCASMCs, HepG2 cells and THP-1 cells. Pharmacological FABP4 inhibition by BMS309403 results in a slight decrease in the formation of these complexes, indicating that fatty acids may play a role in FABP4 functionality. In addition, we demonstrated that exogenous FABP4 crosses the plasma membrane to enter the cytoplasm and nucleus in HUVECs. These findings indicate that exogenous FABP4 interacts with plasma membrane proteins, specifically CK1. These data contribute to our current knowledge regarding the mechanism of action of circulating FABP4.
Keywords: FABP4; Endothelial cell; Cell surface receptor; Cytokeratin 1; FABP4 inhibition;