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BBA - Molecular Cell Research (v.1773, #12)

Cumulative Contents (pp. i-viii).

Editorial Board (pp. ii).

Regulation of mitochondrial oxidative phosphorylation through cell signaling by Maik Hüttemann; Icksoo Lee; Lobelia Samavati; Hong Yu; Jeffrey W. Doan (pp. 1701-1720).

The mitochondrial oxidative phosphorylation (OxPhos) system plays a key role in energy production, the generation of free radicals, and apoptosis. A lack of cellular energy, excessive radical production, and dysregulated apoptosis are found alone or in combination in most human diseases, including neurodegenerative diseases, stroke, cardiovascular disorders, ischemia/reperfusion, and cancer. In the context of its relevance to human disease, this article reviews current knowledge about the regulation of OxPhos with a focus on cell signaling and discusses identified phosphorylation sites with the aid of crystal structures of OxPhos complexes. Several recent studies have shown that all OxPhos components can be phosphorylated; even the small electron carrier cytochrome c is tyrosine phosphorylated in vivo. We propose that in higher organisms, in contrast to bacteria, cell signaling pathways are the main regulator of energy production, triggered for example by hormones. Pathways that have been identified to act on OxPhos include protein kinases A and C and growth factor activated receptor tyrosine kinase signaling. Present knowledge about kinases and phosphatases that execute signals at the level of the mitochondrial OxPhos system, and newly emerging concepts, such as the translocation of kinases to the mitochondria upon stimulation of a signaling pathway, are discussed.

Keywords: NADH-dehydrogenase; bc; ₁; complex; Cytochrome; c; oxidase; ATP synthase; Cancer; Neurodegenerative disease

Up-regulation of cyclooxygenase-2 by cobalt chloride-induced hypoxia is mediated by phospholipase D isozymes in human astroglioma cells by Bong-Hyun Ahn; Mi Hee Park; Young Han Lee; Taeg Kyu Kwon; Do Sik Min (pp. 1721-1731).

Cyclooxygenase-2 (COX-2) is an isoform of prostaglandin H synthase induced by hypoxia and has been implicated in the growth and progression of a variety of human cancers. In the present study, we investigated the role of phospholipase D (PLD) isozymes in cobalt chloride (CoCl₂)-induced hypoxia-driven COX-2 expression in U87 MG human astroglioma cells. CoCl₂ stimulated PLD activity and synthesis of COX-2 protein in a dose and time-dependent manner. Moreover, elevated expression of PLD1 and PLD2 increased hypoxia-induced COX-2 expression and prostaglandin E2 (PGE₂) production. Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed CoCl₂-induced COX-2 expression and PGE₂ formation. In addition, evidence that PLD activity was involved in the stimulation of COX-2 expression was provided by the observations that overexpression of wild type PLD isozymes, but not catalytically inactive PLD isozymes, stimulated CoCl₂-induced COX-2 expression and PGE₂ production. PLD1 enhanced COX-2 expression by CoCl₂ via reactive oxygen species (ROS), p38 MAPK kinase, PKC-δ, and PKA, but not ERK, whereas PLD2 enhanced CoCl₂-induced COX-2 expression via ROS and p38 MAPK, but not ERK, PKC-δ, and PKA. Differential regulation of COX-2 expression mediated through PLD isozymes was comparable with that of CoCl₂-induced PLD activity in these two PLD isozymes. Taken together, our results demonstrate for the first time that PLD1 and PLD2 isozymes enhance CoCl₂-induced COX-2 expression through differential signaling pathways in astroglioma cells.

Keywords: Cyclooxygenase-2; Prostaglandin E2; Cobalt chloride; Hypoxia; Phospholipase D

ERβ shifts from mitochondria to nucleus during estrogen-induced neoplastic transformation of human breast epithelial cells and is involved in estrogen-induced synthesis of mitochondrial respiratory chain proteins by Jin-Qiang Chen; Patricia A. Russo; Carol Cooke; Irma H. Russo; Jose Russo (pp. 1732-1746).

Both estrogen receptors (ER) alpha (ERα) and beta (ERβ) are localized in the nucleus, plasma membrane, and mitochondria, where they mediate the different physiological effects of estrogens. It has been observed that the relative subcellular localization of ERs is altered in several cancer cells. We have demonstrated that MCF-10F cells, the immortal and non-tumorigenic human breast epithelial cells (HBEC) that are ERα-negative and ERβ-positive, are transformed in vitro by 17β-estradiol (E₂), generating highly invasive cells that are tumorigenic in severe combined immunodeficient mice. E₂-transformed MCF-10F (trMCF) cells exhibit progressive loss of ductulogenesis, invasive (bsMCF) and tumorigenic (caMCF) phenotypes. Immunolocalization of ERβ by confocal fluorescent microscopy and electron microscopy revealed that ERβ is predominantly localized in mitochondria of MCF-10F and trMCF cells. Silencing ERβ expression with ERβ-specific small interference RNA (siRNA-ERβ) markedly diminishes both nuclear and mitochondrial ERβ in MCF-10F cells. The ERβ shifts from its predominant localization in the mitochondria of MCF-10F and trMCF cells to the nucleus of bsMCF cells, becoming predominantly nuclear in caMCF cells. Furthermore, we demonstrated that the mitochondrial ERβ in MCF-10F cells is involved in E₂-induced expression of mitochondrial DNA (mtDNA)-encoded respiratory chain (MRC) proteins. This is the first report of an association of changes in the subcellular localization of ERβ with various stages of E₂-induced transformation of HBEC and a functional role of mitochondrial ERβ in mediating E₂-induced MRC protein synthesis. Our findings provide a new insight into one of the potential roles of ERβ in human breast cancer.

Keywords: Estrogen receptor β; Human breast epithelial cell; Mitochondrial estrogen receptor; Mitochondrial respiratory chain protein; Specific siRNA for ERβ

The tolerance property of human D3 dopamine receptor is determined by specific amino acid residues in the second cytoplasmic loop by Ligia Westrich; Eldo V. Kuzhikandathil (pp. 1747-1758).

The D2 and D3 dopamine receptor subtypes are structurally homologous and couple to the same signal transduction pathways. Nevertheless, their evolutionary conservation suggests that the two subtypes might exhibit unique signaling characteristics. We previously determined that D3 but not D2S dopamine receptor exhibits a tolerance property in which the D3 receptor-activated G-protein coupled inward rectifier potassium currents progressively decreases upon repeated agonist stimulation. In this paper, using AtT-20 neuroendocrine cells stably expressing either human D3 or D2S receptor, we show that the tolerance property is also observed in the D3 receptor-adenylyl cyclase and D3 receptor-mitogen-activated protein kinase signaling pathways. We have previously shown that the second cytoplasmic loop of D3 receptor is required for tolerance. Here, using site-directed mutagenesis, we identified the specific amino acids in the D3 second cytoplasmic loop involved in the tolerance property. The results show that substitution of a non-conserved cysteine residue at position 147 with positively-charged lysine or arginine residues abolishes tolerance. Interestingly, the cysteine 147 residue is embedded in a putative phosphorylation site adjacent to two serine residues. Mutation of these serine residues to alanine also attenuates tolerance. Taken together, these structural studies suggest a role for phosphorylation in D3 receptor tolerance property.

Keywords: Desensitization; Potassium channels; Adenylyl cyclase; MAP kinase; Signaling; Dyskinesia

Visualizing Smad1/4 signaling response to bone morphogenetic Protein-4 activation by FRET biosensors by Kira V. Gromova; Mike Friedrich; Andrey Noskov; Gregory S. Harms (pp. 1759-1773).

Smad proteins are the major signal transducers for the Transforming Growth Factor superfamily of cytokines and their serine/threonine kinase receptors. Smads mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 stimulates phosphorylation of Smad1, which interacts with Smad4. This complex translocates into the nucleus and regulates transcription of target genes. Here, we report our development of cellular fluorescence biosensors for direct visualization of Smad signaling in live mammalian cells. Fluorescence resonance energy transfer between cyan and yellow fluorescent proteins fused to the Smad1 and Smad4 proteins was used to unravel the temporal aspects of BMP/Smad signaling. A rate-limiting delay of 2–5 min occurred between BMP activation and Smad1 activity. A similar delay was observed in the Smad1/Smad4 complexation. Further experimentation indicated that the delay is dependent on the MH1 domain and linker of Smad1. These results give new insights into the dynamics of the BMP receptor –Smad1/4 signaling process and provide a new tool for studying Smads.

Keywords: BMP; Smad; GFP; FRET; Biosensor

The Kelch protein NS1-BP interacts with alpha-enolase/MBP-1 and is involved in c-Myc gene transcriptional control by Giovanni Perconti; Arianna Ferro; Felice Amato; Patrizia Rubino; Davide Randazzo; Thorsten Wolff; Salvatore Feo; Agata Giallongo (pp. 1774-1785).

Alpha-enolase is a key glycolytic enzyme that plays a functional role in several physiological processes depending on the cellular localization. The enzyme is mainly localized in the cytoplasm whereas an alternative translated form, named MBP-1, is predominantly nuclear. The MBP-1 protein has been characterized as a c-Myc promoter binding protein that negatively controls transcription. In the present study, we identified the kelch protein NS1-BP as one of the alpha-enolase/MBP-1 partners by using a yeast two-hybrid screening. Although NS1-BP has been originally described as a protein mainly localized in the nucleus, we provide evidence that NS1-BP also interacts with actin in human cells, as reported for most kelch-containing proteins. Here we showed that alpha-enolase and MBP-1 associate with NS1-BP in vitro and in vivo by GST pull-down assays and coimmunoprecipitation experiments; subsequent immunofluorescent staining confirmed colocalization of the proteins within the cells. Furthermore, functional analyses performed by cotransfection assays revealed that NS1-BP enhances the inhibitory effect exerted by MBP-1 on c-Myc promoter. In mammalian cells, the overexpression of both proteins resulted in an increased repression of basal c-Myc transcription and consistently affected the steady state levels of endogenous c-Myc mRNA. These findings further support the distinct roles of alpha-enolase and its MBP-1 variant in maintaining cell homeostasis. Moreover, our data suggest a novel function for NS1-BP in the control of cell proliferation.

Keywords: Kelch proteins; Enolase; Glycolysis; c-Myc transcription; Yeast two-hybrid assay

Identification of PatL1, a human homolog to yeast P body component Pat1 by Nicoletta Scheller; Patricia Resa-Infante; Susana de la Luna; Rui Pedro Galao; Mario Albrecht; Lars Kaestner; Peter Lipp; Thomas Lengauer; Andreas Meyerhans; Juana Díez (pp. 1786-1792).

In yeast, the activators of mRNA decapping, Pat1, Lsm1 and Dhh1, accumulate in processing bodies (P bodies) together with other proteins of the 5′-3′-deadenylation-dependent mRNA decay pathway. The Pat1 protein is of particular interest because it functions in the opposing processes of mRNA translation and mRNA degradation, thus suggesting an important regulatory role. In contrast to other components of this mRNA decay pathway, the human homolog of the yeast Pat1 protein was unknown. Here we describe the identification of two human PAT1 genes and show that one of them, PATL1, codes for an ORF with similar features as the yeast PAT1. As expected for a protein with a fundamental role in translation control, PATL1 mRNA was ubiquitously expressed in all human tissues as were the mRNAs of LSM1 and RCK, the human homologs of yeast LSM1 and DHH1, respectively. Furthermore, fluorescence-tagged PatL1 protein accumulated in distinct foci that correspond to P bodies, as they co-localized with the P body components Lsm1, Rck/p54 and the decapping enzyme Dcp1. In addition, as for its yeast counterpart, PatL1 expression was required for P body formation. Taken together, these data emphasize the conservation of important P body components from yeast to human cells.

Keywords: Pat1; Decapping; mRNA turnover; Processing bodies; P bodies

Rit mutants confirm role of MEK/ERK signaling in neuronal differentiation and reveal novel Par6 interaction by Jennifer L. Rudolph; Geng-Xian Shi; Eda Erdogan; Alan P. Fields; Douglas A. Andres (pp. 1793-1800).

Rit is a novel member of the Ras superfamily of small GTP-binding proteins that regulates signaling pathways controlling cellular fate determination. Constitutively activated mutants of Rit induce terminal differentiation of pheochromocytoma (PC6) cells resulting in a sympathetic neuron-like phenotype characterized by the development of highly-branched neurites. Rit signaling has been found to activate several downstream pathways including MEK/ERK, p38 MAPK, Ral-specific guanine nucleotide exchange factors (GEFs), and Rit associates with the Par6 cell polarity machinery. In this study, a series of Rit effector loop mutants was generated to test the importance of these cellular targets to Rit-mediated neuronal differentiation. We find that Rit-mediated neuritogenesis is dependent upon MEK/ERK MAP kinase signaling but independent of RalGEF activation. In addition, in vivo binding studies identified a novel mechanism of Par6 interaction, suggesting that the cell polarity machinery may serve to spatially restrict Rit signaling.

Keywords: Neuronal differentiation; PC12 cell; Rit; GTPase; Ras; ERK MAP kinase; Par6

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BBA - Molecular Cell Research (v.1773, #12)

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BBA - Molecular Cell Research (v.1773, #12)