BBA - Molecular Cell Research (v.1783, #6)

Extracellular annexin A5: Functions of phosphatidylserine-binding and two-dimensional crystallization by Hugo O. van Genderen; Heidi Kenis; Leo Hofstra; Jagat Narula; Chris P.M. Reutelingsperger (953-963).
In normal healthy cells phosphatidylserine is located in the inner leaflet of the plasma membrane. However, on activated platelets, dying cells and under specific circumstances also on various types of viable leukocytes phosphatidylserine is actively externalized to the outer leaflet of the plasma membrane. Annexin A5 has the ability to bind in a calcium-dependent manner to phosphatidylserine and to form a membrane-bound two-dimensional crystal lattice. Based on these abilities various functions for extracellular annexin A5 on the phosphatidylserine-expressing plasma membrane have been proposed. In this review we describe possible mechanisms for externalization of annexin A5 and various processes in which extracellular annexin A5 may play a role such as blood coagulation, apoptosis, phagocytosis and formation of plasma membrane-derived microparticles. We further highlight the recent discovery of internalization of extracellular annexin A5 by phosphatidylserine-expressing cells.
Keywords: Annexin A5; Phosphatidylserine; Plasma membrane; Microparticle; Exocytosis; Endocytosis;

Structural model of a complex between the heterotrimeric G protein, Gsα, and tubulin by Brian T. Layden; Witchuda Saengsawang; Robert J. Donati; Shuo Yang; Debbie C. Mulhearn; Michael E. Johnson; Mark M. Rasenick (964-973).
A number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block of microtubules, and G proteins. The α subunits of some heterotrimeric G proteins, including Gsα, have been shown to interact strongly with tubulin. Binding of Gα to tubulin results in increased dynamicity of microtubules due to activation of GTPase of tubulin. Tubulin also activates Gsα via a direct transfer of GTP between these molecules. Structural insight into the interaction between tubulin and Gsα was required, and was determined, in this report, through biochemical and molecular docking techniques. Solid phase peptide arrays suggested that a portion of the amino terminus, α2–β4 (the region between switch II and switch III) and α3–β5 (just distal to the switch III region) domains of Gsα are important for interaction with tubulin. Molecular docking studies revealed the best-fit models based on the biochemical data, showing an interface between the two molecules that includes the adenylyl cyclase/Gβγ interaction regions of Gsα and the exchangeable nucleotide-binding site of tubulin. These structural models explain the ability of tubulin to facilitate GTP exchange on Gα and the ability of Gα to activate tubulin GTPase.
Keywords: G protein; Microtubule; Crystal structure; GTPase; Cytoskeleton;

The sodium pump Ena1p provides mechanistic insight into the salt sensitivity of vacuolar protein sorting mutants by Katarina Logg; Jonas Warringer; Sayed Hossein Hashemi; Mikael Käll; Anders Blomberg (974-984).
The vacuolar/endosomal network has an important but as yet undefined role in the cellular tolerance to salt stress. We hypothesized that the mechanistic basis for the importance of vacuolar protein sorting (vps) components in salt tolerance is the targeting of the crucial sodium exporter Ena1p to the plasma membrane. The link between Ena1p and the vps components was established by the observation that overexpression of Ena1p could suppress the salt sensitivity of the ESCRT knockouts vps20Δ, snf7/vps32Δ and snf8/vps22Δ. To further investigate this functional interaction, fluorescence microscopy was utilized to monitor localization of GFP-tagged Ena1p. For all analyzed vps mutants, Ena1p seemed properly localized to the plasma membrane, even during saline growth. However, quantitative differences in plasma membrane localized Ena1p were recorded; e.g. the highly salt sensitive pep12Δ mutant exhibited substantially enhanced Ena1p levels. In addition, the kinetics of Ena1p localization to the plasma membrane was severely delayed in several vps mutants, and this delay correlated to the salt specific growth defect. This paper discusses potential mechanistic hypotheses, like Ena1p transporter activity or localization kinetics, or ESCRT component's influence on signaling, for linking endosomal sorting functions to cellular salt sensitivity.
Keywords: Vacuolar protein sorting; Sodium export; Yeast; GFP; vps deletion mutant; Phenotype;

Galectin-3 regulates RasGRP4-mediated activation of N-Ras and H-Ras by Ruby Shalom-Feuerstein; Ran Levy; Victoria Makovski; Avraham Raz; Yoel Kloog (985-993).
Galectin-3 (Gal-3) is a pleiotropic β-galactoside-binding protein expressed at relatively high levels in human neoplasms. Its carbohydrate recognition domain (CRD) contains a hydrophobic pocket that can accommodate the farnesyl moiety of K-Ras. Binding of K-Ras to Gal-3 stabilizes K-Ras in its active (GTP-bound) state. Gal-3, which does not interact with N-Ras, was nevertheless shown to reduce N-Ras-GTP in BT-549 cells by an unknown mechanism that we explored here. First, comparative analysis of various cancer cell lines (glioblastomas, breast cancer cells and ovarian carcinomas) showed a positive correlation between low N-Ras-GTP/high K-Ras-GTP phenotype and Gal-3 expression levels. Next we found that epidermal growth factor-stimulated GTP loading of N-Ras, but not of K-Ras, is blocked in cells expressing high levels of Gal-3. Activation of Ras guanine nucleotide releasing proteins (RasGRPs) by phorbol 12-myristate 13-acetate (PMA) or downregulation of Gal-3 by Gal-3 shRNA increased the levels of N-Ras-GTP in Gal-3 expressing cells. We further show that the N-terminal domain of Gal-3 interacts with and inhibits RasGRP4-mediated GTP loading on N-Ras and H-Ras proteins. Growth of BT-549 cells stably expressing the Gal-3 N-terminal domain was strongly attenuated. Overall, these experiments demonstrate a new control mechanism of Ras activation in cancer cells whereby the Gal-3 N-terminal domain inhibits activation of N-Ras and H-Ras proteins.
Keywords: Ras; Galectin-3; RasGRP4; Cancer;

Insulin neuroprotection against oxidative stress is mediated by Akt and GSK-3β signaling pathways and changes in protein expression by Ana I. Duarte; Paulo Santos; Catarina R. Oliveira; Maria S. Santos; A. Cristina Rego (994-1002).
Previously we demonstrated that insulin protects against neuronal oxidative stress by restoring antioxidants and energy metabolism. In this study, we analysed how insulin influences insulin- (IR) and insulin growth factor-1 receptor (IGF-1R) intracellular signaling pathways after oxidative stress caused by ascorbate/Fe2+ in rat cortical neurons. Insulin prevented oxidative stress-induced decrease in tyrosine phosphorylation of IR and IGF-1R and Akt inactivation. Insulin also decreased the active form of glycogen synthase kinase-3β (GSK-3β) upon oxidation. Since phosphatidylinositol 3-kinase (PI-3K)/Akt-mediated inhibition of GSK-3β may stimulate protein synthesis and decrease apoptosis, we analysed mRNA and protein expression of “candidate” proteins involved in antioxidant defense, glucose metabolism and apoptosis. Insulin prevented oxidative stress-induced increase in glutathione peroxidase-1 and decrease in hexokinase-II expression, supporting previous findings of changes in glutathione redox cycle and glycolysis. Moreover, insulin precluded Bcl-2 decrease and caspase-3 increased expression. Concordantly, insulin abolished caspase-3 activity and DNA fragmentation caused by oxidative stress. Thus, insulin-mediated activation of IR/IGF-1R stimulates PI-3K/Akt and inhibits GSK-3β signaling pathways, modifying neuronal antioxidant defense-, glucose metabolism- and anti-apoptotic-associated protein synthesis. These and previous data implicate insulin as a promising neuroprotective agent against oxidative stress associated with neurodegenerative diseases.
Keywords: Cortical neurons; ERK1/2; GSK-3β; Insulin; Oxidative stress; PI-3K/Akt;

The Hsp70–Hsp90 complex is implicated in the folding and regulation of numerous signaling proteins, and Hop, the Hsp70–Hsp90 Organizing Protein, facilitates the association of this multichaperone machinery. Phosphatase treatment of mouse cell extracts reduced the number of Hop isoforms compared to untreated extracts, providing the first direct evidence that Hop was phosphorylated in vivo. Furthermore, surface plasmon resonance (SPR) spectroscopy showed that a cdc2 kinase phosphorylation mimic of Hop had reduced affinity for Hsp90 binding. Hop was predominantly cytoplasmic, but translocated to the nucleus in response to heat shock. A putative bipartite nuclear localization signal (NLS) has been identified within the Hsp90-binding domain of Hop. Although substitution of residues within the major arm of this proposed NLS abolished Hop–Hsp90 interaction as determined by SPR, this was not sufficient to prevent the nuclear accumulation of Hop under leptomycin-B treatment and heat shock conditions. These results showed for the first time that the subcellular localization of Hop was stress regulated and that the major arm of the putative NLS was not directly important for nuclear translocation but was critical for Hop–Hsp90 association in vitro. We propose a model in which the association of Hop with Hsp90 and the phosphorylated status of Hop both play a role in the mechanism of nucleo-cytoplasmic shuttling of Hop.
Keywords: Hop; Heat shock protein; Phosphorylation; Hsp90; Co-chaperone; Nuclear localization signal;

Polo-box domains confer target specificity to the Polo-like kinase family by Barbara C.M. van de Weerdt; Dene R. Littler; Rob Klompmaker; Angelina Huseinovic; Alex Fish; Anastassis Perrakis; René H. Medema (1015-1022).
Polo-like kinases (Plks) contain a conserved Polo-box domain, shown to bind to phosphorylated Ser-pSer/pThr-Pro motifs. The Polo-box domain of Plk-1 mediates substrate interaction and plays an important role in subcellular localization. Intriguingly, the major interactions between the PBD and the optimal recognition peptide are mediated by highly conserved residues in the PBD, suggesting there is little target specificity conveyed by the various PBDs. However, here we show that the affinity of the purified Plk1-3 PBDs to both a physiological Cdc25C derived phospho-peptide and an optimal recognition phospho-peptide differs significantly among family members. To decipher the role of the PBDs and kinase domains in inferring Plk specificity, we exchanged the PBD of Plk1 (PBD1) with the PBD of Plk2, 3, or 4 (PBD2–4). The resulting hybrid proteins can restore bipolar spindle formation and centrosome maturation in Plk1-depleted U2OS cells to various degrees. In these experiments PBD2 was most efficient in complementing PBD-function. Using the MPM2 antibody that recognizes a large set of mitotic phospho-proteins, we could show that PBD1 and PBD2 display some limited overlap in target recognition. Thus, PBDs convey a significant deal of target specificity, indicating that there is only a limited amount of functional redundancy possible within the Plk family.
Keywords: Mitosis; Spindle assembly; Kinase; Phospho-peptide binding domain;

The effects of Ankrd2 alteration indicate its involvement in cell cycle regulation during muscle differentiation by Camilla Bean; Nicola Facchinello; Georgine Faulkner; Gerolamo Lanfranchi (1023-1035).
Ankrd2 is a member of the Muscle Ankyrin Repeat Protein family (MARPs), consisting of sarcomere-associated proteins that can also localize in the nucleus. There are indications that MARPs might function as shuttle proteins between the cytoplasm and nucleus, likely sending information to the nucleus concerning the changes in the structure or function of the contractile machinery. Even though recent findings suggest that the MARP gene family is not essential for the basal functioning of skeletal muscle, its influence on the gene expression program of skeletal muscle cells was highlighted. To investigate this regulatory role we produced and examined both morphological and functional features of myocytes stable overexpressing or silencing the Ankrd2 protein. The transcriptional profiles of the myocytes revealed that the molecular pathways perturbed by changes in Ankrd2 protein level are congruent with the morpho-physiological and biochemical data obtained in Ankrd2-modified myoblasts induced to differentiate. Our results suggest that Ankrd2 gives an important contribution to the coordination of proliferation and apoptosis during myogenic differentiation in vitro, mainly through the p53 network.
Keywords: Myogenic differentiation; C2C12; Cell cycle regulation; Transcriptional profiling;

Novel tyrosine phosphorylated and cardiolipin-binding protein CLPABP functions as mitochondrial RNA granule by Etsuko Sano; Shigeichi Shono; Kyoko Tashiro; Hiroaki Konishi; Emiko Yamauchi; Hisaaki Taniguchi (1036-1047).
We identified a new protein containing the pleckstrin homology (PH) domain through tyrosine phosphoproteomics using epidermal growth factor-stimulated cells. The tandem PH domains of this protein can bind to mitochondria-specific phospholipid, cardiolipin or its dehydro product, phosphatidic acid; therefore, we have designated this protein as cardiolipin and phosphatidic acid-binding protein (CLPABP). In this study, we show that CLPABP is localized on the tubulin network and the mitochondrial surface in the granular form along with other proteins and RNA. The affinity of CLPABP to mitochondria is elevated depending on the extent of tyrosine phosphorylation. The CLPABP complex contains various proteins related to cytoplasmic mRNA metabolism. The unique subcellular localization of CLPABP requires its PH domains and a multifunctional protein, SF2p32, as its binding protein. The CLPABP granule also contains the cytochrome c transcript, which may be mediated by the RNA-binding protein HuR. Immunofluorescence staining reveals that the CLPABP granule is colocalized with cytochrome c and various ribosomal proteins that are present in the CLPABP complex. Therefore, the CLPABP RNA–protein complex may play a role in transporting cytochrome c mRNA and its translated product to the mitochondria.
Keywords: RNA granule; Mitochondria; PH domain; Tyrosine phosphorylation; Cytochrome c;

Previously, we demonstrated that nuclear localization of the Angiotensin II AT1A receptor was associated with the activation of transcription for the COX-2 gene, PTGS-2. The hypothesis of the present study is that AT1AR internalization from the plasma membrane is a first step in the nuclear localization of the endogenous AT1AR of rat aortic vascular smooth muscle cells and the resultant increase of COX-2 protein expression. Angiotensin II produced both a time- and concentration-dependent increase in COX-2 protein expression in these cells. Treatment with sucrose or phenylarsine oxide, inhibitors of receptor internalization, significantly inhibited AT1AR internalization and abolished the increase in COX-2 protein produced by Angiotensin II without affecting COX-2 expression on its own. Sucrose pre-treatment of rat aortic vascular smooth muscle cells resulted in an increase in p42/44 and p38 activation, while phenylarsine oxide pre-treatment activated only p38 kinase without inhibiting activation of p42/44 produced by Angiotensin II. These results demonstrate that inhibiting the internalization of the AT1AR results in a loss of ability of Angiotensin II to increase the protein expression of COX-2, thus supporting previous work showing a relationship between AT1AR nuclear localization and activation of COX-2 gene expression. Surprisingly, in contrast to other studies, the data also indicates that activation of p42/44 and/or p38 does not correlate with the increased expression of COX-2.
Keywords: Angiotensin; G protein-coupled receptor; Cyclooxygenase 2; Signal transduction; Endocytosis;

Caspase-8 is frequently deleted or silenced in neuroblastoma and other solid tumor such as medulloblastoma and small cell lung carcinoma. Caspase-8 expression can be re-established in neuroblastoma cell lines by treatment with demethylating agents or with IFN-γ. Here we show that four different retinoic acid (RA) derivatives also increase caspase-8 protein expression in neuroblastoma, medulloblastoma and small cell lung carcinoma cell lines. This increase in protein expression is mirrored by an increase in RNA expression in NB cells. However, the promoter region of the caspase-8 gene was not responsible for the induction of caspase-8 expression. Rather, we identified another intronic region containing a CREB binding site that was required for maximal induction of caspase-8 via RA. DNA–protein interaction assays revealed increased phospho-CREB binding to this response element in RA-treated NB cells. Furthermore, mutations of the CREB binding site completely blocked caspase-8 induction in the luciferase reporter system assay and transfection of dominant-negative form of CREB repressed the up-regulation of caspase-8 by RA. Importantly, RA-released cells maintained caspase-8 expression for at least 2–5 days and were more sensitive to doxorubicin and TNFα. Thus, RA treatment in conjunction with TNFα and/or subsets of cytotoxic agents may have therapeutic benefits.
Keywords: Caspase-8; Neuroblastoma; Medulloblastoma; Small cell lung carcinoma; Retinoid; Retinoic acid; CREB; Apoptosis; Transcription;

Nuclear export of LEI/L-DNase II by Crm1 is essential for cell survival by Chloé Leprêtre; Yves Fleurier; Elisabeth Martin; Alicia Torriglia (1068-1075).
LEI/L-DNase II is the key protein of a caspase-independent pathway activated by serine proteases. LEI (Leukocyte elastase inhibitor), L-DNase II precursor, is a member of the clade B serpins (also called serpin b1). In its native conformation it inhibits several intracellular proteases and has an anti-apoptotic activity. Following a metabolic stress and the increase of protease activity in the cell, LEI is cleaved and transformed into L-DNase II (LEI-derived DNase II). This transformation is due to a conformational modification that exposes a nuclear localization signal and an endonuclease active site. In this paper we show that LEI can bind the exportin Crm1, and we identify on LEI a nuclear export signal involved in the control of LEI/L-DNase II nuclearization in healthy cells. Point mutation of this site increases the accumulation of the molecule in the nucleus and triggers cell death.
Keywords: LEI; L-DNase II; NES; Apoptosis; Caspase-independent; Serpin;

The deletion of amino acids 114–121 in the TM1 domain of mouse prion protein stabilizes its conformation but does not affect the overall structure by Bastian Thaa; Ralph Zahn; Ulrich Matthey; Peter M.H. Kroneck; Alexander Bürkle; Günter Fritz (1076-1084).
A mutant of mouse prion protein (PrPC) carrying a deletion of residues 114–121 (PrPΔ114–121) has previously been described to lack convertibility into the scrapie-associated isoform of PrP (PrPSc) and to exhibit a dominant-negative effect on the conversion of wild-type PrPC into PrPSc in living cells. Here we report the characterization of recombinantly expressed PrPΔ114–121 by Fourier-transformation infrared spectroscopy (FTIR) and circular dichroism (CD) spectroscopy. The analysis of spectra revealed an increased antiparallel β-sheet content in the deletion mutant compared to wild-type PrPC. This additional short β-sheet stabilized the fold of the mutant protein by ΔΔG 0′ = 3.4 ± 0.3 kJ mol− 1 as shown by chemical unfolding experiments using guanidine hydrochloride. Secondary structure predictions suggest that the additional β-sheet in PrPΔ114–121 is close to the antiparallel β-sheet in PrPC. The high-affinity Cu2+-binding site outside the octarepeats, which is located close to the deletion and involves His110 as a ligand, was not affected, as detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting that Cu2+ binding does not contribute to the protection of PrPΔ114–121 from conversion into PrPSc. We propose that the deletion of residues 114–121 stabilizes the mutant protein. This stabilization most likely does not obstruct the interaction of PrPΔ114–121 with PrPSc but represents an energy barrier that blocks the conversion of PrPΔ114–121 into PrPSc.
Keywords: Prion; Deletion mutant; Fourier transformed infrared spectoscopy FTIR; Circular dichrosim CD; Copper; EPR;

p53 phosphorylation is involved in vascular cell death induced by the catalytic activity of membrane-bound SSAO/VAP-1 by Montse Solé; Mar Hernandez-Guillamon; Mercè Boada; Mercedes Unzeta (1085-1094).
Semicarbazide sensitive amine oxidase (SSAO) is a multifunctional enzyme present mainly in adipocytes, endothelial and smooth muscle cells. It metabolizes primary aliphatic and aromatic amines generating products able to contribute to cellular oxidative stress. SSAO is expressed in a membrane-bound form and is also present as a soluble enzyme in plasma. Both isoforms are increased in several pathologies, and the catalytic products generated by the soluble enzymatic activity can induce cytotoxicity of vascular cells in culture. We have analyzed whether the transmembrane form of the enzyme is able to produce a cytotoxic effect through methylamine oxidation. Since cells in culture lose the expression of this enzyme, we used an SSAO stably transfected smooth muscle cell line. Herein we report that cell treatment with the substrate methylamine induced a dose and time dependent cytotoxic effect. The tumor suppressor protein p53 played an important role in the molecular pathway involved in this cell death. Moreover, we also observed the induction of PUMA-α expression with mitochondrial Bcl-2 family proteins being affected, and final effector caspases being activated.
Keywords: Semicarbazide sensitive amine oxidase; Transmembrane protein; Methylamine; Apoptosis; p53; Smooth muscle cells;

Macrophage mediated protein hydroperoxide formation and lipid oxidation in low density lipoprotein are inhibited by the inflammation marker 7,8-dihydroneopterin by Carole A. Firth; Elizabeth M. Crone; Elizabeth A. Flavall; Justin A. Roake; Steven P. Gieseg (1095-1101).
The formation of oxidised low density lipoprotein (LDL) within the atherosclerotic plaque appears to be a factor in the development of advanced atherosclerotic plaques. LDL oxidation is dependent on the balance of oxidants and antioxidants within the intima. In addition to producing various oxidants, human macrophages release 7,8-dihydroneopterin which in vivo is oxidised to the inflammation marker neopterin. Using macrophage-like THP-1 cells and human monocyte-derived macrophages, we demonstrate that 7,8-dihydroneopterin is a potent inhibitor of cell-mediated LDL oxidation. 7,8-Dihydroneopterin scavenges the chain propagating lipid peroxyl radical, inhibiting both lipid and protein hydroperoxide formation. A significant amount of the hydroperoxide formed during cell-mediated LDL oxidation was protein hydroperoxide. 7,8-Dihydroneopterin oxidation to 7,8-dihydroxanthopterin was only observed in the presence of both cells and LDL, showing that 7,8-dihydroneopterin had no effect on initiating oxidant generation by the cells. 7,8-Dihydroneopterin did not regenerate α-tocopherol but competed with it for the lipid peroxyl radical. Although stimulation of both cell types with γ-interferon failed to produce sufficient 7,8-dihydroneopterin to inhibit LDL oxidation in tissue culture, analysis of advanced atherosclerotic plaque removed from patients showed that total neopterin levels could reach low micromolar concentrations. This suggests that 7,8-dihydroneopterin synthesis by macrophages could play a significant role in the development of atherosclerotic plaques.
Keywords: Macrophage; Low density lipoprotein; Protein oxidation; Lipid oxidation; Neopterin; Antioxidant; Inflammation; Atherosclerosis;

Localized cytosolic alkalization and its functional impact in ciliary cells by Liubov Lemberskiy-Kuzin; Michal Fainshtein; Polina Fridman; Elena Passwell; Alex Braiman; Zvi Priel (1102-1110).
Using confocal microscopy we demonstrate that ciliary cells from airway epithelium maintain two qualitatively distinct cytosolic regions in terms of pH regulation. While the bulk of the cytosol is stringently buffered and is virtually insensitive to changes in extracellular pH (pHo), the values of cytosolic pH in the vicinity of the ciliary membrane is largely determined by pHo. Variation of pHo from 6.2 up to 8.5 failed to affect ciliary beat frequency (CBF). Application of NH4Cl induced profound localized alkalization near cilia, which did not depress ciliary activity, but resulted in strong and prolonged enhancement of CBF. Calmodulin and protein kinase A (PKA) functionality was essential for the alkalization-induced CBF enhancement. We suggest that the ability of airway epithelium to sustain unusually strong but localized cytosolic alkalization near cilia facilitates CBF enhancement through altering the binding constants of Ca2+ to calmodulin and promotion of Ca2+–calmodulin complex formation. The NH4Cl-induced elevations in cytosolic pH and Ca2+ concentration act synergistically to activate calmodulin-dependent processes, cAMP pathway, and, thereby, stimulate CBF.
Keywords: Mucociliary; Cytosolic gradient; Airway epithelium; Acid-base balance; Ammonia; Ciliary beating;

Direct Thy-1/αVβ3 integrin interaction mediates neuron to astrocyte communication by Tamara Hermosilla; Daniel Muñoz; Rodrigo Herrera-Molina; Alejandra Valdivia; Nicolás Muñoz; Sang-Uk Nham; Pascal Schneider; Keith Burridge; Andrew F.G. Quest; Lisette Leyton (1111-1120).
Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a β3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the α subunit partner of β3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that αv forms an αvβ3 heterodimer in DI TNC1 astrocytes was obtained. In neuron–astrocyte association assays, the presence of either anti-αv or anti-β3 integrin antibodies reduced cell–cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron–astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with αvβ3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the αvβ3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.
Keywords: Thy-1; Integrins; Brain cells; Cell–cell interaction; Adhesion molecules;

Pin1 interacts with c-Myb in a phosphorylation-dependent manner and regulates its transactivation activity by E. Pani; M. Menigatti; S. Schubert; D. Hess; B. Gerrits; K-H. Klempnauer; S. Ferrari (1121-1128).
Activity and stability of the proto-oncogene c-Myb are regulated by post-translational modifications, though the molecular mechanisms underlying such control are only partially understood. Here we describe the functional interaction of c-Myb with Pin1, an isomerase that binds to phosphorylated Ser/Thr-Pro motifs. We found that co-expression of c-Myb and Pin1 led to a net increase of c-Myb transactivation activity, both on reporter constructs as well as on an endogenous target gene. DNA-binding studies revealed that Pin1 did not increase the association of c-Myb with its response element in DNA. The increase of c-Myb transactivation activity was strictly dependent on the presence of an active catalytic center in Pin1. We provide evidence that c-Myb and Pin1 physically interacted, both upon ectopic expression of the proteins in HEK-293 cells as well as in the more physiological setting of HL60 cells, where c-Myb and Pin1 are resident proteins. By point mutating each individual Ser/Thr-Pro motif in c-Myb as well as by using deletion mutants we show that S528 in the EVES-motif was the docking site for Pin1. Mass spectrometry confirmed that S528 is phosphorylated in vivo. Finally, functional studies showed that mutation of S528 to alanine almost abolished the increase of transactivation activity by Pin1. This study reveals a new paradigm by which phosphorylation controls c-Myb function.
Keywords: c-Myb; Phosphorylation site; Pin1; Transcription;

A presynaptically toxic secreted phospholipase A2 is internalized into motoneuron-like cells where it is rapidly translocated into the cytosol by Zala Jenko Pražnikar; Lidija Kovačič; Edward G. Rowan; Rok Romih; Paola Rusmini; Angelo Poletti; Igor Križaj; Jože Pungerčar (1129-1139).
The molecular mechanism of the presynaptic toxicity of secreted phospholipase A2 (sPLA2) neurotoxins, including that of ammodytoxin A (AtxA), has not been resolved. Here we report the action of AtxA on mouse motoneuron-like cells, on which it induced characteristic neurotoxic effects on synaptic vesicles and on the reorganization of F-actin. AtxA also released fatty acids from the plasmalemma. Its significantly less neurotoxic V31W mutant showed similar effects on cells but with a much higher rate of hydrolysis than the wild-type, indicating that high enzymatic activity alone is not sufficient for the observed effects. The neurotoxic action was observed by confocal microscopy of a fluorescently labelled AtxA and by electron microscopy of a nanogold-labelled toxin. The Atx-binding proteins were tagged by a photo-cross-linking reagent conjugated to the toxin. AtxA was taken up rapidly by the cells, where it interacted within minutes with calmodulin and 14-3-3 proteins in the cytosol. These data demonstrate, for the first time, the translocation of an sPLA2 from the extracellular space into the cytosol of a cell. Such an event may thus be important in explaining the action of a range of homologous endogenous sPLA2 enzymes in mammals whose roles in various cellular processes are not yet completely understood.
Keywords: Ammodytoxin; Cytosolic protein; Motoneuron; Neurotoxicity; Secreted phospholipase A2; Synaptic vesicle;

Localization of A20 to a lysosome-associated compartment and its role in NFκB signaling by Lianyun Li; Dale W. Hailey; Nia Soetandyo; Wei Li; Jennifer Lippincott-Schwartz; Hong-bing Shu; Yihong Ye (1140-1149).
A20 is a tumor necrosis factor (TNF)-inducible zinc finger protein that contains both ubiquitinating and deubiquitinating activities. A20 negatively regulates NFκB (nuclear factor κB) signaling induced by TNF receptor family and Toll-like receptors, but the mechanism of A20 action is poorly defined. Here we show that a fraction of endogenous and ectopically expressed A20 is localized to an endocytic membrane compartment that is in association with the lysosome. The lysosomal association of A20 requires its carboxy terminal zinc finger domains, but is independent of its ubiquitin-modifying activities. Interestingly, A20 mutants defective in membrane association also contain reduced NFκB inhibitory activity. These findings suggest the involvement of a lysosome-associated mechanism in A20-dependent termination of NFκB signaling.
Keywords: A20; Lysosomal degradation; Ubiquitin; NFκB; Deubiquitinating enzyme;

Tenascin-C induction by cyclic strain requires integrin-linked kinase by Silke Maier; Roman Lutz; Laurent Gelman; Ana Sarasa-Renedo; Susanne Schenk; Carsten Grashoff; Matthias Chiquet (1150-1162).
Induction of tenascin-C mRNA by cyclic strain in fibroblasts depends on RhoA and Rho dependent kinase (ROCK). Here we show that integrin-linked kinase (ILK) is required upstream of this pathway. In ILK-deficient fibroblasts, RhoA was not activated and tenascin-C mRNA remained low after cyclic strain; tenascin-C expression was unaffected by ROCK inhibition. In ILK wild-type but not ILK −/− fibroblasts, cyclic strain-induced reorganization of actin stress fibers and focal adhesions, as well as nuclear translocation of MAL, a transcriptional co-activator that links actin assembly to gene expression. These findings support a role for RhoA in ILK-mediated mechanotransduction. Rescue of ILK −/− fibroblasts by expression of wild-type ILK restored these responses to cyclic strain. Mechanosensation is not entirely abolished in ILK −/− fibroblasts, since cyclic strain activated Erk-1/2 and PKB/Akt, and induced c-fos mRNA in these cells. Conversely, lysophosphatidic acid stimulated RhoA and induced both c-fos and tenascin-C mRNA in ILK −/− cells. Thus, the signaling pathways controlling tenascin-C expression are functional in the absence of ILK, but are not triggered by cyclic strain. Our results indicate that ILK is selectively required for the induction of specific genes by mechanical stimulation via RhoA-mediated pathways.
Keywords: Mechanotransduction; Tenascin-C; RhoA/ROCK; Integrin-linked kinase; c-fos;

Endogenously expressed human canonical transient receptor potential 1 (hTRPC1) and human canonical transient receptor potential 6 (hTRPC6) have been shown to play a role in store-operated Ca2+ entry (SOCE) in human platelets, where two mechanisms for SOCE, regulated by the dense tubular system (DTS) or the acidic granules, have been identified. In cells preincubated for 1 min with 100 µM flufenamic acid we show that hTRPC6 is involved in SOCE activated by both mechanisms, as demonstrated by selective depletion of the DTS or the acidic stores, using thapsigargin (TG) (10 nM) or 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) (20 µM), respectively, although it is more relevant after acidic store depletion. Co-immunoprecipitation experiments indicated that depletion of both stores separately results in time-dependent interaction between hTRPC1 and hTRPC6, and also between both hTRPCs and the type II IP3 receptor (IP3RII). The latter was greater after treatment with TG. TBHQ-induced coupling between hTRPC1 and 6 was transient and decreased after 30s of treatment, while that induced by TG increased for at least 3 min. TBHQ induced association between SERCA3, located in the acidic stores, hTRPC1, hTRPC6 and Orai1. TBHQ also evoked coupling between SERCA3 and IP3RII, presumably located in the DTS, thus suggesting interplay between both Ca2+ stores. Similarly, TG induces the interaction of SERCA2b with hTRPC1 and 6 and the IP3RII. The interactions between hTRPC1, hTRPC6, IP3RII and SERCA3 were impaired by disruption of the microtubules, supporting a role for microtubules in Ca2+ homeostasis. In conclusion, the present data demonstrate for the first time that hTRPC1, hTRPC6, IP3RII and SERCA3 are parts of a macromolecular protein complex activated by depletion of the intracellular Ca2+ stores in human platelets.
Keywords: Ca2+ influx; hTRPC1; hTRPC6; Platelet; TG; TBHQ; SERCA; IP3 receptor; Microtubule; Orai1;

FAK-independent αvβ3 integrin-EGFR complexes rescue from anoikis matrix-defective fibroblasts by Nicoletta Zoppi; Sergio Barlati; Marina Colombi (1177-1188).
Extracellular matrix (ECM) binding to integrin receptors regulates cell cycle progression and survival. In adherent cells, ECM disassembly induces anoikis, the apoptotic pathway switched on by loss of adhesion. ECM-deficient Ehlers–Danlos syndrome (EDS) fibroblasts, to adhere to rare fibronectin (FN) fibrils, and to proliferate, only organize, as FN receptor, the αvβ3 integrin. We report that in EDS cells the αvβ3 integrin is bound to talin and vinculin, but not to tensin, and that actin cytoskeleton is disorganized. Furthermore, in EDS cells Bcl-2 is down-regulated and caspases are active. We provide evidence that the antibody-mediated αvβ3 integrin or the FN inhibition induces anoikis in EDS cells. The αvβ3 integrin transduces survival signals to pp60src-mediated tyrosine phosphorylated paxillin, instead than to FAK, and interacts with EGF receptor (EGFR). This complex, when activated by EGF and FN, signals for the rescue of EDS cells from anoikis. Therefore, EDS cells, through the αvβ3 integrin–EGFR complexes, engage a paxillin- but not FAK-mediated pathway of cell survival.
Keywords: αvβ3 integrin; Fibronectin; anoikis; Paxillin; Epidermal growth factor receptor; FAK; Ehlers–Danlos syndrome;

CSN5 binds to misfolded CFTR and promotes its degradation by Gaëlle Tanguy; Loïc Drévillon; Nicole Arous; Afia Hasnain; Alexandre Hinzpeter; Janine Fritsch; Michel Goossens; Pascale Fanen (1189-1199).
Cystic fibrosis is mainly caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The aim of this study was to find cellular proteins interacting with CFTR and regulating its processing. We have used a genetic screen in yeast to identify such proteins and identified CSN5 that interacted with the third cytoplasmic loop of CFTR. CSN5 is the 5th component of the COP9 signalosome, a complex of eight subunits that shares significant homologies to the lid subcomplex of the 26S proteasome and controls the stability of many proteins. The present study shows that CSN5 associates with the core-glycosylated form of CFTR and suggests that this association targets misfolded CFTR to the degradative pathway. Identifying CSN5 as a new component of the degradative pathway is an important step towards the goal of unraveling the sorting between misfolded and correctly folded CFTR proteins.
Keywords: CFTR; CSN5; COP9 signalosome; Interaction and degradation;

Drosophila Hsp27 is a small heat shock protein displaying exclusive nuclear localization both before and after heat shock. However, the mechanism implicated in this nuclear localization as well as the required sequences, are undefined. This study identifies the Hsp27 sequences mediating its nuclear localization. The generation of chimeric fusions between Hsp27 and Hsp23, a small heat shock protein displaying exclusive cytoplasmic localization, delineated a stretch of 15 amino acids containing a nuclear-targeting activity. Site-directed mutagenesis within this region unveiled the implication of three arginine residues (R54–R55–R56), which differentially combine to form a novel kind of nuclear localization signal (NLS). Abrogation of the nuclear localization signal activity indicated that Drosophila Hsp27 could still enter the nucleus to associate with nuclear speckles in a NLS-independent fashion. Mutagenesis of a putative nuclear export signal unveiled two leucine residues (L50 and L52) specifically involved in the association of Hsp27 to nuclear speckles and revealed novel nuclear structures formed by this Hsp27 mutant. The present study identifies two distinct sets of sequences respectively mediating the nuclear import of Hsp27 and its association to nuclear speckles. These two phenomena are uncoupled and can be separately abrogated.
Keywords: Heat shock protein; Arginine-rich nuclear localization signal; Nuclear speckle;

Regulation of EP4 expression via the Sp-1 transcription factor: Inhibition of expression by anti-cancer agents by Atsushi Kambe; Genzo Iguchi; Yuseok Moon; Hideki Kamitani; Takashi Watanabe; Thomas E. Eling (1211-1219).
For glioblastomas, COX-2 expression is linked to poor survival. COX-2 effects are mediated by the receptors EP2 and EP4, whose regulation is poorly understood. The expression of EP4, and activation or inhibition of EP4 activity in human glioblastoma T98G cells, was found to correlate with growth on soft agar. Chemoprevention drugs, troglitazone (TGZ) and some COX inhibitors, significantly suppressed EP4 expression in T98G cells in a dose dependant manner. Specificity protein 1 (Sp-1) binding sites, located within region − 197 to − 160 of the human EP4 promoter, are important for the transcription initiation of the human EP4 gene and are responsible for the EP4 suppression by TGZ. Mutation in the Sp-1 sites altered the promoter activity of luciferase constructs and TGZ effects on the promoter. The inhibitory effect of TGZ on EP4 expression was reversed by PD98059, a MEK-1/Erk inhibitor. Immunoprecipitation–Western blot analysis detected Sp-1 phosphorylation that was dependent on TGZ-induced Erks activation. ChIP assay confirmed that Sp-1 phosphorylation decreases its binding to DNA and as a result, leads to the suppression of EP4 expression. Thus, we propose that the expression of EP4 is regulated by Sp-1, but phosphorylation of Sp-1 induced by TGZ suppresses this expression. This represents a new and unique mechanism for the regulation of the EP4 receptor expression.
Keywords: Sp-1 phosphorylation; PPAR; Prostaglandin EP4 receptor; Glioblastoma; Erks activation;

The estrogen receptor-interacting protein HPIP increases estrogen-responsive gene expression through activation of MAPK and AKT by Xiaohui Wang; Zhihong Yang; Hao Zhang; Lihua Ding; Xiru Li; Cui Zhu; Yiqiong Zheng; Qinong Ye (1220-1228).
Estrogen receptors (ERα and ERβ) are estrogen-regulated transcription factors that play important roles in the development and progression of breast cancer. The biological function of ERs has been shown to be modulated by ER-interacting proteins. However, the ER-interacting proteins that not only activate MAPK and AKT, two important growth regulatory protein kinases, but also increase growth related estrogen-responsive gene expression remain unknown. Here, we report that hematopoietic PBX-interacting protein (HPIP) interacts both with ERα and with ERβ, and increases ERα target gene expression through activation of MAPK and AKT and enhanced ERα phosphorylation. ERβ inhibits ERα target gene expression, possibly by competition of ERβ with ERα for binding to HPIP, and by a decrease in available ERα for HPIP binding through the interaction of ERβ with ERα. Furthermore, HPIP increases breast cancer cell growth. These data suggest that HPIP may be an important regulator in ER signaling and that the relative ratio of ERβ to ERα may be important for HPIP function.
Keywords: Estrogen receptor; Hematopoietic PBX-interacting protein (HPIP); Interaction; Mitogen-activated protein kinase (MAPK); Protein kinase B/AKT;

Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: Effects of endothelin-1, oxidative stress and cytokines by Timothy E. Cullingford; Matthew J. Butler; Andrew K. Marshall; El Li Tham; Peter H. Sugden; Angela Clerk (1229-1236).
Krüppel-like transcription factors (Klfs) modulate fundamental cell processes. Cardiac myocytes are terminally-differentiated, but hypertrophy in response to stimuli such as endothelin-1. H2O2 or cytokines promote myocyte apoptosis. Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes. We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes. In response to endothelin-1, Klf2 mRNA expression was rapidly increased (∼ 9-fold; 15–30 min) with later increases in expression of Klf4 and Klf6 (∼ 5-fold; 30–60 min). All were regulated as immediate early genes (cycloheximide did not inhibit the increases in expression). Klf5 expression was increased at 1–2 h (∼ 13-fold) as a second phase response (cycloheximide inhibited the increase). These increases were transient and attenuated by U0126. H2O2 increased expression of Klf2, Klf4 and Klf6, but interleukin-1β or tumor necrosis factor α downregulated Klf2 expression with no effect on Klf4 or Klf6. Of the Klfs which repress transcription, endothelin-1 rapidly downregulated expression of Klf3, Klf11 and Klf15. The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that, as a family, they are actively involved in regulating phenotypic responses (hypertrophy and apoptosis) to extracellular stimuli.
Keywords: Cardiac myocytes; Endothelin-1; Immediate early genes; Krüppel-like factors; Gene expression; Cytokines;

Calreticulin is a lectin chaperone essential for intracellular calcium homeostasis. Deletion of calreticulin gene compromises the overall quality control within the endoplasmic reticulum (ER) leading to activation of the unfolded protein response. However, the ER structure of calreticulin deficient cells (crt−/) is not altered due to accumulation of misfolded proteins. Therefore, the aim of this study was to determine whether the ubiquitin–proteasome pathway is activated in crt−/ cells as a compensatory mechanism for cell survival. Here we show a significant increase in the expression of genes involved in ER associated degradation and activation of the ubiquitin–proteasome system in crt−/ cells. We also demonstrated that the ubiquitination of two proteins processed in ER, connexin 43 and A1AT NHK (α1-antitrypsin mutant) are increased in crt−/ cells. Furthermore, we showed that the increased proteasome activity in the crt−/ cells could be rescued upon re-introduction of calreticulin or calsequestrin (a muscle calcium binding protein). We also illustrated that increased cytosolic Ca2+ enhances the proteasome activity. Interestingly, suppression of calnexin function using siRNA further elevated the proteasome activity in crt−/ cells. This is the first report to show that loss of calreticulin function enhances the ubiquitin–proteasome activity which could function as a compensatory mechanism for cell survival.
Keywords: Calreticulin; Proteasome; Endoplasmic reticulum stress; Calcium; Protein degradation;

Light-dependent CK2-mediated phosphorylation of centrins regulates complex formation with visual G-protein by Philipp Trojan; Sebastian Rausch; Andreas Gieβl; Clementine Klemm; Eberhard Krause; Alexander Pulvermüller; Uwe Wolfrum (1248-1260).
Centrins are Ca2+-binding EF-hand proteins. All four known centrin isoforms are expressed in the ciliary apparatus of photoreceptor cells. Cen1p and Cen2p bind to the visual G-protein transducin in a strictly Ca2+-dependent way, which is thought to regulate light driven movements of transducin between photoreceptor cell compartments. These relatively slow motile processes represent a novel paradigm in light adaptation of photoreceptor cells.Here we validated specific phosphorylation as a novel regulator of centrins in photoreceptors. Centrins were differentially phosphorylated during photoreceptor dark adaptation. Inhibitor treatments revealed protein kinase CK2 as the major protein kinase mediating phosphorylation of Cen1p, Cen2p and Cen4p, but not Cen3p, at a specific target sequence. CK2 and ciliary centrins co-localize in the photoreceptor cilium. Direct binding of CK2 and centrins to ciliary microtubules may spatially integrate the enzyme–substrate specificity in the cilium. Kinetic light-scattering assays revealed decreased binding affinities of phosphorylated centrins to transducin. Furthermore, we show that this decrease is based on the reduction of Ca2+-binding affinities of centrins. Present data describe a novel regulatory mechanism of reciprocal regulation of stimulus dependent distribution of signaling molecules.
Keywords: Cytoskeleton; Ca2+-binding proteins; Molecular translocation; Heterotrimeric G-protein; Signal transduction; Vision;

Role of the carboxyl terminal di-leucine in phosphorylation and internalization of C5a receptor by Elena S. Suvorova; Jeannie M. Gripentrog; Martin Oppermann; Heini M. Miettinen (1261-1270).
The carboxyl tail of G protein-coupled receptors contains motifs that regulate receptor interactions with intracellular partners. Activation of the human neutrophil complement fragment C5a receptor (C5aR) is terminated by phosphorylation of the carboxyl tail followed by receptor internalization. In this study, we demonstrated that bulky hydrophobic residues in the membrane-proximal region of the C5aR carboxyl tail play an important role in proper structure and function of the receptor: Substitution of leucine 319 with alanine (L319A) resulted in receptor retention in the endoplasmic reticulum, whereas a L318A substitution allowed receptor transport to the cell surface, but showed slow internalization upon activation, presumably due to a defect in phosphorylation by both PKC and GRK. Normal agonist-induced activation of ERK1/2 and intracellular calcium release suggested that the L318A mutation did not affect receptor signaling. Binding of GRK2 and PKCβII to intracellular loop 3 of C5aR in vitro indicated that mutagenesis of L318 did not affect kinase binding. Limited proteolysis with trypsin revealed a conformational difference between wild type and mutant receptor. Our studies support a model in which the L318/L319 stabilizes an amphipathic helix (Q305–R320) in the membrane-proximal region of C5aR.
Keywords: Chemoattractant receptor; Endocytosis; Protein folding; Cytoplasmic helix 8;