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BBA - Molecular Cell Research (v.1813, #1)

Reviewer Acknowledgment (pp. iii-v).

Editorial Board (pp. i).

The GTPase RAB20 is a HIF target with mitochondrial localization mediating apoptosis in hypoxia by Thomas Hackenbeck; Regina Huber; Ruth Schietke; Karl X. Knaup; Juliana Monti; Xiaoqing Wu; Bernd Klanke; Benjamin Frey; Udo Gaipl; Bernd Wullich; Didier Ferbus; Gérard Goubin; Christina Warnecke; Kai-Uwe Eckardt; Michael S. Wiesener (pp. 1-13).

Hypoxia is a common pathogenic stress, which requires adaptive activation of the Hypoxia-inducible transcription factor (HIF). In concert transcriptional HIF targets enhance oxygen availability and simultaneously reduce oxygen demand, enabling survival in a hypoxic microenvironment. Here, we describe the characterization of a new HIF-1 target gene, Rab20, which is a member of the Rab family of small GTP-binding proteins, regulating intracellular trafficking and vesicle formation. Rab20 is directly regulated by HIF-1, resulting in rapid upregulation of Rab20 mRNA as well as protein under hypoxia. Furthermore, exogenous as well as endogenous Rab20 protein colocalizes with mitochondria. Knockdown studies reveal that Rab20 is involved in hypoxia induced apoptosis. Since mitochondria play a key role in the control of cell death, we suggest that regulating mitochondrial homeostasis in hypoxia is a key function of Rab20. Furthermore, our study implicates that cellular transport pathways play a role in oxygen homeostasis. Hypoxia-induced Rab20 may influence tissue homeostasis and repair during and after hypoxic stress.► The small GTP-binding protein Rab20 is induced by hypoxia on mRNA and protein level. ► Hypoxic induction of Rab20 is mediated by HIF-1α via direct promoter binding. ► Rab20 colocalizes with mitochondria in different cell lines. ► Rab20 is involved in hypoxia induced apoptosis.

Keywords: Hypoxia; HIF; Apoptosis; Transcription factor; tumor; Cellular transport

Membrane depolarization regulates AMPA receptor subunit expression in cerebellar granule cells in culture by Salvatore Incontro; Ramirez-Franco Jorge Ramírez-Franco; Sanchez-Prieto José Sánchez-Prieto; Magdalena Torres (pp. 14-26).

The physiological responses of AMPA receptors can be modulated through the differential expression of their subunits and by modifying their number at the cell surface. Here we have studied the expression of AMPA receptor subunits (GluR1–4) mRNAs in cerebellar granule cells grown in depolarizing (25mMK⁺) medium, and we have evaluated the effect of decreasing the [K⁺] in the culture medium for 24 h on both GluR1–4 expression (both mRNA and protein) and their presence at the plasma membrane. The expression of the four AMPAR subunits increases as the [K⁺] decreases, although the increase in GluR2 and GluR3 was only observed in the cell soma but not in the dendrites. Calcium entry through L-type calcium channel and CaMKIV activation are responsible for the reduction in the expression of AMPA receptor subunits in cells cultured in depolarizing conditions. Indeed, prolonged reduction of extracellular [K⁺] or blockage of L-type calcium channels enhanced both the surface insertion of the four AMPAR subunits and the AMPA response measured through intracellular calcium increase. These findings reveal a balanced increase in functional AMPA receptors at the surface of cells that can trigger strong increases in calcium in response to the persistent reduction of calcium entry.►Developing rat cerebellar granule cells express the four subunits of AMPARs. ►Culture depolarizing conditions affect the expression of AMPA receptors subunits. ►Intracellular Ca²⁺ increase downregulates AMPARs expression via CaMKIV activation. ►Reduction of calcium influx increases insertion of AMPAR in the plasma membrane.

Keywords: Abbreviations; AMPA; α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate; AMPAR; AMPA receptor; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CaMKIV; Ca; ²⁺; /calmodulin-dependent protein kinase type IV; CGC; cerebellar granule cell; DAPI; 4′,6-diamino-phenylindole dihydrochloride; DIV; days in vitro; FIC; freshly isolated cells; GFAP; green fibrillary acidic protein; IRD; near-infrared fluorescent dye; KA; kainate; NBQX; 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline; MK-801; (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate; NMDAR; NMDA receptor; ROI; region of interest; TBE buffer; Tris borate EDTA buffer; TES; N; -Tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid; VSCCs; voltage-sensitive calcium channelsCalcium-dependent homeostatic changes; AMPA receptors; CaMKIV; Cerebellar granule cells

Nucleolar localization/retention signal is responsible for transient accumulation of histone H2B in the nucleolus through electrostatic interactions by Yana R. Musinova; Olga M. Lisitsyna; Sergey A. Golyshev; Alexander I. Tuzhikov; Vladimir Y. Polyakov; Eugene V. Sheval (pp. 27-38).

The majority of known nuclear proteins are highly mobile. The molecular mechanisms by which they accumulate inside stable compartments that are not separated from the nucleoplasm by membranes are obscure. The compartmental retention of some proteins is associated with their biological function; however, some protein interactions within distinct nuclear structures may be non-specific. The non-specific retention may lead to the accumulation of proteins in distinct structural domains, even if the protein does not function inside this domain. In this study, we have shown that histone H2B-EGFP initially accumulated in the nucleolus after ectopic expression, and then gradually incorporated into the chromatin to leave only a small amount of nucleolus-bound histone that was revealed by removing chromatin-bound proteins with DNase I treatment. Nucleolar histone H2B had several characteristics: (i) it preferentially bound to granular component of the nucleolus and interacted with RNA or RNA-containing nucleolar components; (ii) it freely exchanged between the nucleolus and nucleoplasm; (iii) it associated with the nuclear matrix; and (iv) it bound to interphase prenuclear bodies that formed after hypotonic treatment. The region in histone H2B that acts as a nucleolar localization/retention signal (NoRS) was identified. This signal overlapped with a nuclear localization signal (NLS), which appears to be the primary function of this region. The NoRS activity of this region was non-specific, but the molecular mechanism was probably similar to the NoRSs of other nucleolar proteins. All known NoRSs are enriched with basic amino acids, and we demonstrated that positively charged motifs (nona-arginine (R9) and nona-lysine (K9)) were sufficient for the nucleolar accumulation of EGFP. Also, the correlation between measured NoRS activity and the predicted charge was observed. Thus, NoRSs appear to achieve their function through electrostatic interactions with the negatively charged components of the nucleolus. Though these interactions are non-specific, the functionally unrelated retention of a protein can increase the probability of its interaction with specific and functionally related binding sites.►Histone H2B-GFP initially accumulated in the nucleolus after ectopic expression. ►The region that acts as a nucleolar retention signal (NoRS) was identified. ►This signal overlapped with a nuclear localization signal (NLS). ►The interaction of histone H2B with the nucleolus was non-specific. ►NoRSs appear to achieve their function through simple electrostatic interactions.

Keywords: Nucleolus; Histone H2B; Nucleolar localization/retention signal; Electrostatic interaction

The cell surface structure of tumor endothelial marker 8 (TEM8) is regulated by the actin cytoskeleton by Mi Young Yang; Amit Chaudhary; Steven Seaman; Jill Dunty; Janine Stevens; Mohammed K. Elzarrad; Arthur E. Frankel; Brad St. Croix (pp. 39-49).

Tumor endothelial marker 8 (TEM8) is an integrin-like cell surface protein upregulated on tumor blood vessels and a potential vascular target for cancer therapy. Here, we found that the ability of an anti-TEM8 antibody, clone SB5, to recognize the extracellular domain of TEM8 on the cell surface depends on other host-cell factors. By taking advantage of SB5's ability to distinguish different forms of cell surface TEM8, we identified alpha-smooth muscle actin and transgelin, an actin binding protein, as intracellular factors able to alter TEM8 cell surface structure. Overexpression of either of these proteins in cells converted TEM8 from an SB5-exposed to an SB5-masked form and protected cells from SB5-saporin immunotoxins. Because the predominant form of TEM8 on the cell surface is not recognized by SB5, we also developed a new monoclonal antibody, called AF334, which is able to recognize both the SB5-exposed and the SB5-masked forms of TEM8. AF334-saporin selectively killed TEM8-positive cells independent of TEM8 cell surface structure. These studies reveal that TEM8 exists in different forms at the cell surface, a structure dependent on interactions with components of the actin cytoskeleton, and should aid in the rational design of the most effective diagnostic and therapeutic anti-TEM8 monoclonal antibodies.►TEM8, like integrins, can exist in two different forms on the cell surface. ►Binding of TEM8 to the actin cytoskeleton alters the structure of cell surface TEM8. ►TEM8 expression is regulated epigenetically by histone acetylation. ►The first mAb that recognizes native TEM8 independent of its structure is described.

Keywords: Abbreviations; TEM8; tumor endothelial marker 8; CMG2; capillary morphogenesis protein 2; IgG; immunoglobulin G; mAbs; monoclonal antibodies; α-SMA; alpha-smooth muscle actin; PA; protective antigenAngiogenesis; Endothelial; Actin cytoskeleton; TEM8; ANTXR1

Calpain digestion and HSP90-based chaperone protection modulate the level of plasma membrane F508del-CFTR by Monica Averna; Roberto Stifanese; Raffaella Grosso; Marco Pedrazzi; Roberta De Tullio; Franca Salamino; Bianca Sparatore; Sandro Pontremoli; Edon Melloni (pp. 50-59).

We are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca²⁺-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients.►A calpain-cleaved CFTR form is present in control PBMC and in higher amounts in PBMC of CF patients. ►The calpain-cleaved ΔF508 CFTR is localized exclusively in vesicle membranes ►Intracellular calpain inhibition promotes ΔF508 CFTR recovery at the plasma membrane. ►ΔF508 CFTR digestion by calpain is due to a decrease affinity of channel for HSP90. ►Ca²⁺-dependent proteolysis can be a target for new therapeutic approaches for cystic fibrosis.

Keywords: Abbreviations; ER; endoplasmic reticulum; ERAD; ER-associated degradation; CFTR; cystic fibrosis transmembrane conductance regulator; CF; cystic fibrosis; C.I.2; calpain inhibitor 2F508del-CFTR; Ca; ²⁺; -dependent proteolysis; Calpain; HSP90 protection

Hypoxia-mediated control of HIF/ARNT machinery in epidermal keratinocytes by Lynda Weir; Douglas Robertson; Irene M. Leigh; J. Keith Vass; Andrey A. Panteleyev (pp. 60-72).

Transcriptional activity of hypoxia-induced factor 1 (HIF1) – a heterodimer of HIF1α and ARNT (HIF1β) – is essential for cellular adaptation to environmental stress and plays an important role in skin development, wound healing, tumorigenesis and barrier function. Using primary mouse and human epidermal keratinocytes at ambient or hypoxic (1% O₂) conditions we studied effects of hypoxia upon HIF protein expression. Significant nuclear levels of ARNT and HIF1α along with high HIF1 activity in normoxic keratinocytes suggest an as yet uncharacterised oxygen-independent role for HIF pathway in the epidermis. Acute hypoxia results in an instant but transient increase of HIF1α protein accompanied by a gradual decrease in its mRNA, while ARNT expression remains unchanged. In prolonged (chronic) hypoxia both HIF1α and Arnt are downregulated along with decline of HIF1 activity. However, expression of classical HIF1 targets such as Selenbp1 and Vegfa remains high. Thus, keratinocytes respond to acute hypoxia with immediate block of HIF1α protein degradation and concomitant increase of HIF activity, while under chronic hypoxia pro-angiogenic signalling is maintained through HIF1-independent pathway(s). Decline of HIF1α during chronic exposure is controlled at both mRNA and protein levels, while Arnt is downregulated post-translationally. Distinct transcription levels of Hif1α and Hif3α splice variants under normoxia and their differential response to hypoxia suggest functional diversity of Hif-α isoforms and highlight the complexity of HIF machinery control in epidermal keratinocytes.► Hif1α and Arnt proteins have hypoxia-independent functions in the epidermis. ► The keratinocytes respond to acute and chronic hypoxia through different mechanisms. ► Hif1 activity in keratinocytes is regulated by hypoxia on transcriptional level as well. ► Protein-coding splice variants of Hif1α and Hif3α are differentially regulated by hypoxia. ► Hif1 activity in keratinocytes is regulated by hypoxia through multiple transcriptional and translational mechanisms.

Keywords: Abbreviations; ARNT; aryl hydrocarbon receptor nuclear translocator; PMK; primary mouse keratinocytes; PAS; Period/Arnt/Single-minded; VEGA; Vertebrate Genome AnnotationARNT; HIF1a; HIF3a; Hypoxia; Keratinocyte; Splicing

Akt-mediated signaling is induced by cytokines and cyclic adenosine monophosphate and suppresses hepatocyte inducible nitric oxide synthase expression independent of MAPK P44/42 by Baochun Zhang; Suping Li; Brian G. Harbrecht (pp. 73-79).

Cyclic AMP inhibits the expression of nitric oxide synthase (Harbrecht et al., 1995 ) in hepatocytes but the mechanism for this effect is incompletely understood. Cyclic AMP can activate several intracellular signaling pathways in hepatocytes including Protein Kinase A (PKA), cAMP regulated guanine nucleotide exchange factors (cAMP-GEFs), and calcium-mediated Protein Kinases. There is considerable overlap and cross-talk between many of these signaling pathways, however, and how these cascades regulate hepatocyte iNOS is not known. We hypothesized that Akt mediates the effect of cAMP on hepatocyte iNOS expression. Hepatocytes cultured with cytokines and dbcAMP increased Akt phosphorylation up to 2h of culture. Akt phosphorylation was inhibited by the PI3K inhibitor LY294002 (10μM), farnyltranferase inhibitor FTI-276, or transfection with a dominant negative Akt. The cyclic AMP-induced suppression of cytokine-stimulated iNOS was partially reversed by LY294002 and FTI-276. LY294002 also increased NFκB nucleus translocation by Western blot analysis in nuclear extracts. Cyclic AMP increased phosphorylation of Raf1 at serine 259 which was blocked by LY294002 and associated with decreased MAPK P44/42 phosphorylation. However, inhibition of MAPK P44/42 signaling with PD98059 failed to suppress cytokine-induced hepatocyte iNOS expression and did not enhance the inhibitory effect of dbcAMP on iNOS production. A constitutively active MAPK P44/42 plasmid had no effect on cytokine-stimulated NO production. These data demonstrate that dbcAMP regulates hepatocyte iNOS expression through an Akt-mediated signaling mechanism that is independent of MAPK P44/42.►Cytokine- and cAMP-induced Akt phosphorylation involve Ras and PI3K signaling. ►cAMP regulates hepatocyte iNOS expression through an Akt-mediated signaling mechanism ►Suppression of MAPK p44/42 signaling by cAMP do not affect on iNOS expression. ►Akt decreased NF-kB translocation and may partially mediate cAMP's effects on iNOS

Keywords: Akt; Nitric oxide synthase; Cyclic adenosine monophosphate; Hepatocyte

Involvement of caspase-9 in autophagy-mediated cell survival pathway by Hyo-Soon Jeong; Hye Yeon Choi; Eung-Ryoung Lee; Jung-Hyun Kim; Kilsoo Jeon; Hyun-Joo Lee; Ssang-Goo Cho (pp. 80-90).

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been considered for use in the prevention and treatment of cancer malignancy. FR122047 (FR) is known to have an anti-inflammatory effect, but the anticancer activity of the chemical has not yet been identified. In the present study, we could find that treatment of breast cancer MCF-7 cells with FR led to apoptosis accompanying with apparent activation of caspases. Treatment of caspase-specific inhibitors revealed that FR-induced apoptosis was caspase-8-dependent and inhibition of caspase-9 activity resulted in unexpected, marked enhancement of cell death. Knockdown of caspase-9 expression by specific siRNA caused increased susceptibility to FR-induced cell death, consistent with the results obtained with treatment of caspase-9 inhibitor. Inhibition of caspase-9 blocked the autophagic process by modulating lysosomal pH and acid-dependent cathepsin activities and augmented cell death due to blockage of cytoprotective autophagy. MCF-7 cells treated with sulforaphane, an autophagy-inducing drug, also showed marked accumulation of LC3-II, and co-treatment with caspase-9 inhibitor brought about increased susceptibility to sulforaphane-induced cell death. Different from the cases with FR or sulforaphane, etoposide- or doxorubicin-induced cell death was suppressed with co-treatment of caspase-9 inhibitor, and the drugs failed to induce significant autophagy in MCF-7 cells. Taken together, our data originally suggest that inhibition of caspase-9 may block the autophagic flux and enhance cell death due to blockage of cytoprotective autophagy.Display Omitted►FR122047 (FR), a nonsteroidal anti-inflammatory drug, led to caspase-8-dependent apoptosis. ►Inhibition of caspase-9 blocked the autophagic process and augmented cell death due to blockage of cytoprotective autophagy. ►Different from the cases with FR, etoposide- or doxorubicin-induced cell death was suppressed with co-treatment of caspase-9 inhibitor, and the drugs failed to induce significant autophagy. ►Caspase-9 may be involved in the autophagic process by modulating lysosomal pH and acid-dependent cathepsin activities.

Keywords: NSAIDs; Caspase; Apoptosis; Autophagy; MCF-7

Bcs1p can rescue a large and productive cytochrome bc₁ complex assembly intermediate in the inner membrane of yeast mitochondria by Laura Conte; Bernard L. Trumpower; Vincenzo Zara (pp. 91-101).

The yeast cytochrome bc₁ complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc₁ complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc₁ assembly and the formation of a functionally inactive bc₁ core structure of about 500-kDa. This immature bc₁ core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc₁ core structure leading to the formation of the functional homodimeric bc₁ complex. Following Bcs1p expression, the mature bc₁ complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc₁ complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc₁ complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc₁ core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc₁ complex and gives new insights into the molecular mechanisms involved in the last steps of bc₁ assembly.► Bcs1p is a chaperone involved in the cytochrome bc1 complex assembly. ► The absence of Bcs1p causes the formation of a 500-kDa bc1 subcomplex. ► This subcomplex is as a true intermediate of bc1 assembly. ► Bcs1p can rescue the 500-kDa subcomplex to the mature cytochrome bc1 complex. ► Point mutations in the Bcs1p affect its capability to restore the mature bc1 complex.

Keywords: Abbreviations; Qcr6p, Qcr7p, Qcr8p, Qcr9p, and Qcr10p; subunits 6, 7, 8, 9, and 10 of the yeast; bc; ₁; complex, respectively; Cox6bp; subunit 6b of the yeast cytochrome; c; oxidase complexCytochrome; bc; ₁; complex; Cytochrome; bc; ₁; assembly; Bcs1p; BCS1L; Yeast deletion mutants; Yeast mitochondria

Role of hepatocyte nuclear factor 4α in controlling copper-responsive transcription by Min Ok Song; Jonathan H. Freedman (pp. 102-108).

Previous global transcriptome and interactome analyses of copper-treated HepG2 cells identified hepatocyte nuclear factor 4α (HNF4α) as a potential master regulator of copper-responsive transcription. Copper exposure caused a decrease in the expression of HNF4α at both mRNA and protein levels, which was accompanied by a decrease in the level of HNF4α binding to its consensus DNA binding sequence. qRT-PCR and RNAi studies demonstrated that changes in HNF4α expression ultimately affected the expressions of its down-stream target genes. Analysis of upstream regulators of HNF4α expression, including p53 and ATF3, showed that copper caused an increase in the steady-state levels of these proteins. These results support a model for copper-responsive transcription in which the metal affects ATF3 expression and stabilizes p53 resulting in the down-regulation of HNF4α expression. In addition, copper may directly affect p53 protein levels. The suppression of HNF4α activity may contribute to the molecular mechanisms underlying the physiological and toxicological consequences of copper toxicity in hepatic-derived cells.► Copper affects the transcription of several thousand genes. ► HNF4α is a central regulator of hepatocyte transcription. ► Copper affects the steady-state level of HNF4α protein and mRNA. ► ATF3 and p53 negatively regulates HNF4α expression. ► Copper controls HNF4α levels via activation of ATF3 and p53.

Keywords: Abbreviations; HNF4α; hepatocyte nuclear factor 4α; ATF3; activating transcription factor 3; IPA; ingenuity pathway analysis; RNAi; RNA interference; siRNA; small interfering RNA; qRT-PCR; quantitative real-time PCR; SREBP-2; sterol regulatory-element binding protein 2Copper; HNF4α; HepG2 cell; Transcription; p53; Gene expression; ATF3

PNRC accumulates in the nucleolus by interaction with B23/nucleophosmin via its nucleolar localization sequence by Yuanzhong Wang; Bin Chen; Yuping Li; Dujin Zhou; Shiuan Chen (pp. 109-119).

PNRC (proline-rich nuclear receptor coregulatory protein) was primarily identified as a coactivator of nuclear receptors (NRs) by our laboratory, which enhances NR-mediated transcription by RNA polymerase II. Recent study has shown that PNRC also stimulates RNA polymerase III-dependent transcription through interaction with the subunit RPC39 of RNA polymerase III. Here, we report that PNRC accumulates in the nucleolus and its depletion by small interfering RNA (siRNA) impairs pre-rRNA transcription by RNA polymerase I. We identified the sequence at position 94–101 (⁹⁴PKKRRKKK¹⁰¹) of PNRC as its nucleolar localization sequence (NoLS). Fusion of this sequence to GFP directed GFP to the nucleolus. Characterization of the NoLS revealed that the stretches of six successive basic residues are sufficient to function as a NoLS. Through co-immunoprecipitation assay, we demonstrated that the NoLS is necessary and sufficient to mediate the association of PNRC with B23/nucleophosmin. Moreover, B23 depletion by siRNA disrupted the accumulation of PNRC in the nucleolus. Together, our study indicates that PNRC is a novel nucleolar protein that might be involved in regulation of pre-rRNA synthesis, and it localizes to the nucleolus by interaction with B23 via its NoLS. Our study also suggests that the stretches of six successive basic residues (lysine and/or arginine) could function as NoLS.►PNRC is a novel nucleolar protein. ►PNRC accumulates in the nucleolus by interaction with B23 via its NoLS. ►PNRC is involved in regulation of pre-rRNA transcription. ►The stretches of six successive basic residues could function as NoLS

Keywords: Abbreviations; aa; amino acid; DAPI; 4′,6-diamidino-2-phenylindole; GFP; green fluorescence protein; NLS; nuclear localization sequence; NoLS; nucleolar localization sequence; NR; nuclear receptor; PNRC; proline-rich nuclear receptor coregulatory protein; siRNA; small interfering RNA; SV40 T; SV40 large T antigenPNRC; NoLS; B23/nucleophosmin; Nucleolus; Coactivator

Etoposide induces apoptosis and upregulation of TACE/ADAM17 and ADAM10 in an in vitro male germ cell line model by Carlos Lizama; Andreas Ludwig; Ricardo D. Moreno (pp. 120-128).

Etoposide is a widely used anticancer drug in the treatment of different tumors. Etoposide is known to activate a wide range of intracellular signals, which may in turn induce cellular responses other than apoptosis. ADAM10 and TACE/ADAM17 belong to a family of transmembrane extracellular metalloproteinases involved in paracrine/juxtacrine regulation of many signaling pathways. The aim of this work was to evaluate if etoposide induces upregulation of ADAM10 or TACE/ADAM17 in two cell lines (GC-1 and GC-2) derived from male germ cells. Results showed that etoposide induced apoptosis in a dose–response manner in both GC-1 and GC-2 cells. Apoptosis started to increase 6h after etoposide addition in GC-2 cells, whereas the same was observed 18h after addition to the GC-1 cells. Protein and mRNA levels of ADAM10 and TACE/ADAM17 increased 18h after etoposide was removed from the GC-1 cells. In GC-2 cells, the protein levels of both proteins increased 12h after etoposide was removed. ADAM10 mRNA increased after 3h and then steadily decreased up to 12h after removal, whereas TACE/ADAM17 mRNA decreased after etoposide removal. Finally, apoptosis was prevented in GC-1 and GC-2 cells by the addition of pharmacological inhibitors of ADAM10 and TACE/ADAM17 to the culture medium of etoposide-treated cells. Our results show for the first time that etoposide upregulates ADAM10 and TACE/ADAM17 mRNA and protein levels. In addition, we also show that ADAM10 and TACE/ADAM17 have a role in etoposide-induced apoptosis.► Etoposide induces apoptosis in two cell lines (GC-1 and GC-2) derived from male germ cells. ► Etoposide induces upregulation of TACE/ADAM17 and ADAM10. ► Pharmacological inhibition of TACE/ADAM17 or ADAM10 prevents apoptosis.

Keywords: Apoptosis; Testis; Spermatogenesis; Etoposide; TACE

The maximal cytoprotective function of the heat shock protein 27 is dependent on heat shock protein 70 by R. Sreedharan; M. Riordan; G. Thullin; S. Van Why; N.J. Siegel; M. Kashgarian (pp. 129-135).

Endogenous heat shock proteins (HSPs) 70 and 25/27 are induced in renal cells by injury from energy depletion. Transfected over-expression of HSPs 70 or 27 (human analogue of HSP25), provide protection against renal cell injury from ATP deprivation. This study examines whether over-expressed HSP27 depends on induction of endogenous HSPs, in particular HSP70, to afford protection against cell injury. LLC-PK1 cells transfected with HSP27 (27OE cells) were injured by ATP depletion for 2h and recovered for 4h in the presence of HSF decoy, HSP70 specific siRNA (siRNA-70) and their respective controls. Injury in the presence of HSF decoy, a synthetic oligonucleotide identical to the heat shock element, the nuclear binding site of HSF, decreased HSP70 induction by 80% without affecting the over-expression of transfected HSP27. The HSP70 stress response was completely ablated in the presence of siRNA-70. Protection against injury, provided by over-expression of HSP27, was reduced by treatment with HSF decoy and abolished by treatment with siRNA-70. Immunoprecipitation studies demonstrated association of HSP27 with actin that was not affected by either treatment with HSF decoy or siRNA. Therefore, HSP27 is dependent on HSP70 to provide its maximal cytoprotective effect, but not for its interaction with actin. This study suggests that, while it has specific action on the cytoskeleton, HSP 25/27 must have coordinated activity with other HSP classes, especially HSP70, to provide the full extent of resistance to injury from energy depletion.►Endogenous HSP 70 and 25 are induced to a lesser degree in porcine renal tubule epithelial cells over-expressing human HSP27. ►HSP27 does not depend on induction of HSP70 for its interaction with the cytoskeletal protein, actin. ►Maximal cytoprotective effect of HSP27 is dependent on HSP70 induction.

Keywords: Heat shock protein; Acute kidney injury; Cytoskeletal stability; Gene silencing

The iNOS/Src/FAK axis is critical in Toll-like receptor-mediated cell motility in macrophages by Ming-Chei Maa; Miao Ying Chang; Jiarung Li; Yu-Yun Li; Ming-Yu Hsieh; Ching-Jau Yang; Yen-Jen Chen; Yahan Li; Hui-Chen Chen; Wei Erh Cheng; Ching-Yun Hsieh; Chun-Wen Cheng; Tzeng-Horng Leu (pp. 136-147).

The Toll-like receptors (TLRs) play a pivotal role in innate immunity for the detection of highly conserved, pathogen-expressed molecules. Previously, we demonstrated that lipopolysaccharide (LPS, TLR4 ligand)-increased macrophage motility required the participation of Src and FAK, which was inducible nitric oxide synthase (iNOS)-dependent. To investigate whether this iNOS/Src/FAK pathway is a general mechanism for macrophages to mobilize in response to engagement of TLRs other than TLR4, peptidoglycan (PGN, TLR2 ligand), polyinosinic–polycytidylic acid (polyI:C, TLR3 ligand) and CpG-oligodeoxynucleotides (CpG, TLR9 ligand) were used to treat macrophages in this study. Like LPS stimulation, simultaneous increase of cell motility and Src (but not Fgr, Hck, and Lyn) was detected in RAW264.7, peritoneal macrophages, and bone marrow-derived macrophages exposed to PGN, polyI:C and CpG. Attenuation of Src suppressed PGN-, polyI:C-, and CpG-elicited movement and the level of FAK Pi-Tyr861, which could be reversed by the reintroduction of siRNA-resistant Src. Besides, knockdown of FAK reduced the mobility of macrophages stimulated with anyone of these TLR ligands. Remarkably, PGN-, polyI:C-, and CpG-induced Src expression, FAK Pi-Tyr861, and cell mobility were inhibited in macrophages devoid of iNOS, indicating the importance of iNOS. These findings corroborate that iNOS/Src/FAK axis occupies a central role in macrophage locomotion in response to engagement of TLRs.►Ligand-activated TLR induces the expression of iNOS and Src. ►iNOS further upregulates Src expression and activity via NO production. ►Activated Src phosphorylates FAK and promotes macrophage migration.

Keywords: Abbreviations; AG; aminoguanidine hemisulfate; BMDMs; bone marrow-derived macrophages; FAK; focal adhesion kinase; iNOS; inducible nitric oxide synthase; LPS; lipopolysaccharide; ODN; oligodeoxynucleotide; PAMPs; pathogen-associated molecular patterns; PEMs; peritoneal macrophages; PGN; peptidoglycan; polyI:C; polyinosinic:polycytidylic acid; PP2; 4-amino-5-(4-chlorophenyl)-7-(; t; -butyl)pyrazolo[3,4-; d; ]pyrimidine; SFKs; Src family kinases; TLRs; Toll-like receptors; WT; wild typePeptidoglycan; Polyinosinic–polycytidylic acid; CpG; Src; Macrophage migration

SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover by Michael Skalski; Namit Sharma; Karla Williams; Andrew Kruspe; Marc G. Coppolino (pp. 148-158).

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK–Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.►Inhibition of SNARE function impaired phosphorylation of FAK in CHO cells. ►Blocking SNARE function perturbed trafficking of Src and FAK–Src interaction. ►Rac activation, focal adhesion turnover and cell spreading were all impaired. ►Plasma membrane-targeted Src rescued cell spreading and focal adhesion turnover.

Keywords: Abbreviations; CHO; Chinese hamster ovary; ECM; extracellular matrix; FN; fibronectin; FA; focal adhesions; FAK; focal adhesion kinase; NSF; N; -ethylmaleimide-sensitive fusion protein; PI3K; phosphatidylinositol 3-kinase; PIP; ₃; phosphatidylinositol 3,4,5-trisphosphate; SNARE; soluble NSF attachment protein receptorFocal adhesion; FAK; Src; SNARE; SNAP23; Membrane traffic

Role of hypoxia and EGF on expression, activity, localization and phosphorylation of carbonic anhydrase IX in MDA-MB-231 breast cancer cells by Ying Li; Hai Wang; Chingkuang Tu; Kathleen T. Shiverick; David N. Silverman; Susan C. Frost (pp. 159-167).

Carbonic anhydrase IX (CAIX) is a zinc metalloenzyme that catalyzes the reversible hydration of CO₂. CAIX is overexpressed in many types of cancer, including breast cancer, but is most frequently absent in corresponding normal tissues. CAIX expression is strongly induced by hypoxia and is significantly associated with tumor grade and poor survival. Herein, we show that hypoxia induces a significant increase in CAIX protein in MDA-MB-231 breast cancer cells. Using a unique mass spectrophotometric assay, we demonstrate that CAIX activity in plasma membranes isolated from MDA-MB-231 is correlated with CAIX content. We also show that CAIX exists predominantly as a dimeric, high-mannose N-linked glycoprotein. While there is some evidence that the dimeric form resides specifically in lipid rafts, our data do not support this hypothesis. EGF, alone, did not affect the distribution of CAIX into lipid rafts. However, acute EGF treatment in the context of hypoxia increased the amount of CAIX in lipid rafts by about 5-fold. EGF did not stimulate tyrosine phosphorylation of CAIX, although EGFR and down-stream signaling pathways were activated by EGF. Interestingly, hypoxia activated Akt independent of EGF action. Together, these data demonstrate that the active form of CAIX in the MDA-MB-231 breast cancer cell line is dimeric but that neither lipid raft localization nor phosphorylation are likely required for its dimerization or activity.►CAIX expression in hypoxic MDA-MB-231 cells is correlated with carbonic anhydrase activity. ►CAIX exists in a high mannose, dimeric glycoprotein. ►EGF, alone, does not stimulate CAIX translocation to lipid rafts but in the hypoxic environment increases the association of CAIX with lipid rafts by 5-fold. ►EGF does not stimulate the phosphorylation of CAIX, but otherwise activates the traditional downstream kinases. ►Hypoxia increases Akt phosphorylation.

Keywords: Carbonic anydrase IX; MDA-MB-231 breast cancer cell; Hypoxia; EGF; Lipid raft; Dimerization

Cellular FLIP_L plays a survival role and regulates morphogenesis in breast epithelial cells by Rosario Yerbes; Carmen Palacios; Mauricio J. Reginato; Lopez-Rivas Abelardo López-Rivas (pp. 168-178).

Strong evidences support the inhibitory activity of cellular FLICE-inhibitory protein (FLIP) in the apoptotic signalling by death receptors in tumor cells. However, little is known about the role of FLIP in the regulation of apoptosis in non-transformed cells. In this report, we demonstrate that FLIP_L plays an important role as a survival protein in non-transformed breast epithelial cells. Silencing of FLIP_L by siRNA methodology enhances TRAIL-R2 expression and activates a caspase-dependent cell death process in breast epithelial cells. This cell death requires the expression of TRAIL, TRAIL-R2, FADD and procaspase-8 proteins. A mitochondria-operated apoptotic pathway is partially required for FLIP_L siRNA-induced apoptosis. Interestingly, FLIP_L silencing markedly abrogates formation of acinus-like structures in a three-dimensional basement membrane culture model (3D) of the human mammary MCF-10A cell line through a caspase-8 dependent process. Furthermore, over-expression of FLIP_L in MCF-10A cells delayed lumen formation in 3D cultures. Our results highlight the central role of FLIP in maintaining breast epithelial cell viability and suggest that the mechanisms regulating FLIP levels should be finely controlled to prevent unwanted cell demise.►FLIP_L knockdown activates apoptosis in non-transformed breast epithelial cells. ►Apoptosis by FLIP_L knockdown depends on the expression of TRAIL, TRAIL-R2, FADD and caspase-8. ►FLIP_L regulates formation of acinus-like structures in 3D cultures of breast epithelial cells.

Keywords: Abbreviations; TRAIL; tumor necrosis factor-related apoptosis-inducing ligand; TNF; tumor necrosis factor; DISC; death-inducing signalling complex; FADD; Fas-associated death domain; z-VAD.FMK; benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketoneApoptosis; TRAIL; FLIP; Morphogenesis

Susceptibility of Hep3B cells in different phases of cell cycle to tBid by Shi-Hong Ma; George G. Chen; Caiguo Ye; Billy C.S. Leung; Rocky L.K. Ho; Paul B.S. Lai (pp. 179-185).

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC.► Pro-apoptotic tBid primarily targets/kills the HCC cells at G0/G1 phase of cell cycle whereas 5-fluorouracil mainly arrests tumor cells at the G0/G1 phase. ► The complementary effects of tBid and 5-fluorouracil on different phases of the cell cycle significantly induce much more HCC cell death. ► The phase-specific effect of tBid points to a possible new therapeutic option for HCC.

Keywords: Hepatocellular carcinoma; tBid; Apoptosis; Cell cycle; Synchronization

DNA repair in organelles: Pathways, organization, regulation, relevance in disease and aging by Pierre Boesch; Frédérique Weber-Lotfi; Noha Ibrahim; Vladislav Tarasenko; Anne Cosset; François Paulus; Robert N. Lightowlers; André Dietrich (pp. 186-200).

Both endogenous processes and exogenous physical and chemical sources generate deoxyribonucleic acid (DNA) damage in the nucleus and organelles of living cells. To prevent deleterious effects, damage is balanced by repair pathways. DNA repair was first documented for the nuclear compartment but evidence was subsequently extended to the organelles. Mitochondria and chloroplasts possess their own repair processes. These share a number of factors with the nucleus but also rely on original mechanisms. Base excision repair remains the best characterized. Repair is organized with the other DNA metabolism pathways in the organelle membrane-associated nucleoids. DNA repair in mitochondria is a regulated, stress-responsive process. Organelle genomes do not encode DNA repair enzymes and translocation of nuclear-encoded repair proteins from the cytosol seems to be a major control mechanism. Finally, changes in the fidelity and efficiency of mitochondrial DNA repair are likely to be involved in DNA damage accumulation, disease and aging. The present review successively addresses these different issues.► Endogenous and exogenous sources generate organelle DNA damage. ► Mitochondria and chloroplasts possess specific DNA repair pathways. ► Functional DNA repair in organelles is associated with the membrane compartment. ► Mitochondrial DNA repair is impaired in disease and aging. ► Regulation of organelle DNA repair may rely on relocalization of repair proteins.

Keywords: Aging; Chloroplast; Disease; DNA damage; DNA repair; Mitochondrion

Regulation of the lymphatic endothelial cell cycle by the PROX1 homeodomain protein by Shannon A. Baxter; David Y. Cheung; Patricia Bocangel; Hae K. Kim; Krista Herbert; Josette M. Douville; Jaganmohan R. Jangamreddy; Shunzhen Zhang; David D. Eisenstat; Jeffrey T. Wigle (pp. 201-212).

The homeobox transcription factor PROX1 is essential for the development and maintenance of lymphatic vasculature. How PROX1 regulates lymphatic endothelial cell fate remains undefined. PROX1 has been shown to upregulate the expression of Cyclin E, which mediates the G₁ to S transition of the cell cycle. Here we demonstrate that PROX1 activates the mouse Cyclin E1 ( Ccne1) promoter via two proximal E2F-binding sites. We have determined that the N-terminal region of PROX1 is sufficient to activate a 1-kb Ccne1 promoter, whereas the homeodomain is dispensable for activation. We have identified that the Prospero domain 1 (PD1) is required for the nuclear localization of PROX1. Our comparison of two DNA-binding-deficient constructs of PROX1 showed a cell-type-specific difference between these two proteins in both their localization and function. We demonstrated that siRNA-mediated knockdown of PROX1 in lymphatic endothelial cells decreases progression from G₁ to S phase of the cell cycle. We conclude that PROX1 activates the Ccne1 promoter independent of DNA binding, and our results illustrate a novel role for PROX1 in the regulation of lymphatic endothelial cell proliferation.► PROX1 activation of Ccne1 transcription is DNA-binding-independent. ► PROX1-mediated transcription of Ccne1 requires both E2F-binding sites. ► The PD1 domain is required for nuclear localization of PROX1. ► Knockdown of PROX1 in primary LECs decreases CCNE1 protein levels.

Keywords: Lymphatic endothelial cell; Cyclin E1; Transcription; Cell cycle

HSP90 modulates actin dynamics: Inhibition of HSP90 leads to decreased cell motility and impairs invasion by Aftab Taiyab; Ch. Mohan Rao (pp. 213-221).

HSP90, a major molecular chaperone, plays an essential role in the maintenance of several signaling molecules. Inhibition of HSP90 by inhibitors such as 17-allylamino-demethoxy-geldanamycin (17AAG) is known to induce apoptosis in various cancer cells by decreasing the activation or expression of pro-survival molecules such as protein kinase B (Akt). While we did not observe either decrease in expression or activation of pro-survival signaling molecules in human breast cancer cells upon inhibiting HSP90 with 17AAG, we did observe a decrease in cell motility of transformed cells, and cell motility and invasion of cancer cells. We found a significant decrease in the number of filopodia and lamellipodia, and in the F-actin bundles upon HSP90 inhibition. Our results show no change in the active forms or total levels of FAK and Pax, or in the activation of Rac-1 and Cdc-42; however increased levels of HSP90, HSP90α and HSP70 were observed upon HSP90 inhibition. Co-immuno-precipitation of HSP90 reveals interaction of HSP90 with G-actin, which increases upon HSP90 inhibition. FRET results show a significant decrease in interaction between actin monomers, leading to decreased actin polymerization upon HSP90 inhibition. We observed a decrease in the invasion of human breast cancer cells in the matrigel assay upon HSP90 inhibition. Over-expression of αB-crystallin, known to be involved in actin dynamics, did not abrogate the effect of HSP90 inhibition. Our work provides the molecular mechanism by which HSP90 inhibition delays cell migration and should be useful in developing cancer treatment strategies with known anti-cancer drugs such as cisplatin in combination with HSP90 inhibitors.►Inhibition of HSP90 leads to decrease in the number of filopodia and lamellipodia. ►Decrease in the number of filopodia and lamellipodia is due to the decrease in the cortical actin bundles. ►Decreased actin tread-milling is responsible for the decrease in cortical actin bundles. > Inhibition of HSP90 leads to decrease in the expression of surface HSP90 alpha. ►Decreased actin tread-milling and expression of surface HSP90 alpha leads to decrease in cell motility and invasion.

Keywords: Actin dynamics; Cell migration; HSP90; 17AAG; Invasion

ZBP-89 enhances Bak expression and causes apoptosis in hepatocellular carcinoma cells by Ann K.Y. To; George G. Chen; Ursula P.F. Chan; Caiguo Ye; Jing P. Yun; Rocky L.K. Ho; Art Tessier; Juanita L. Merchant; Paul B.S. Lai (pp. 222-230).

ZBP-89 can enhance tumor cells to death stimuli. However, the molecular mechanism leading to the inhibitory effect of ZBP-89 is unknown. In this study, 4 liver cell lines were used to screen for the target of ZBP-89 on cell death pathway. The identified Bak was further analyzed for its role in ZBP-89-mediated apoptosis. The result showed that ZBP-89 significantly and time-dependently induced apoptosis. It significantly upregulated the level of pro-apoptotic Bak. ZBP-89 targeted a region between -457 and -407 of human Bak promoter to stimulate Bak expression based on the findings of Bak promoter luciferase report gene assay and electrophoretic mobility shift assay. ZBP-89-induced Bak increase and ZBP-89-mediated apoptosis were markedly suppressed by Bak siRNA, confirming that Bak was specifically targeted by ZBP-89 to facilitate apoptosis. In conclusion, this study demonstrated that ZBP-89 significantly induced apoptosis of HCC cells via promoting Bak level.►ZBP-89 induces apoptosis of liver cancer cells. ►ZBP-89 stimulates Bak expression. ►ZBP-89 targets a region between −457 and −407 of Bak promoter.

Keywords: ZBP-89; Hepatocellular carcinoma; Bak; Apoptosis; Proliferation

Phosphorylation/dephosphorylation of human SULT4A1: Role of Erk1 and PP2A by Deanne J. Mitchell; Neville J. Butcher; Rodney F. Minchin (pp. 231-237).

SULT4A1 is a cytosolic sulfotransferase that shares little homology with other human sulfotransferases but is highly conserved between species. It is found in neurons located in several regions of the brain. Recently, the stability of SULT4A1 was shown to be regulated by Pin1, a peptidyl-prolyl cis-trans isomerase implicated in several neurodegenerative diseases. Since Pin1 binds preferentially to phosphoproteins, these findings suggested that SULT4A1 is post-translationally modified. In this study, we show that the Thr¹¹ residue of SULT4A1, which is involved in Pin1 binding is phosphorylated. MEK inhibition was shown to abolish Pin1 mediated degradation of SULT4A1 while in vitro phosphorylation assays using alanine substitution mutants of SULT4A1 demonstrated phosphorylation of Thr¹¹ by ERK1. We also show that dephosphorylation was catalyzed by the protein phosphatase 2A. The PP2A regulatory subunit, Bβ was identified from a yeast-2-hybrid screen of human brain cDNA as a SULT4A1 interacting protein. This was further confirmed by GST pull-downs and immunoprecipitation. Other members of the B subunit (αδγ) did not interact with SULT4A1. Taken together, these studies indicate that SULT4A1 stability is regulated by post-translational modification that involves the ERK pathway and PP2A. The phosphorylation of SULT4A1 allows interaction with Pin1, which then promotes degradation of the sulfotransferase.►ERK1 phosphorylates SULT4A1. ►Dephosphorylation of ser/thr residues on SULT4A1 is mediated by PP2A. ►SULT4A1 stability is regulated by post-translational modifications involving ERK1 and PP2A.

Keywords: Abbreviations; SULT; cytosolic sulfotransferase; PP2A; protein phosphatase 2A; HA; hemagglutinin A; GST; glutathione s-transferase; MAPK; mitogen activated protein kinase; ERK; extracellular signal-regulated kinaseSulfotransferase; PP2A; ERK1; Phosphorylation

Anti-apoptosis and cell survival: A review by Liam Portt; Grant Norman; Caitlin Clapp; Matthew Greenwood; Michael T. Greenwood (pp. 238-259).

Type I programmed cell death (PCD) or apoptosis is critical for cellular self-destruction for a variety of processes such as development or the prevention of oncogenic transformation. Alternative forms, including type II (autophagy) and type III (necrotic) represent the other major types of PCD that also serve to trigger cell death. PCD must be tightly controlled since disregulated cell death is involved in the development of a large number of different pathologies. To counter the multitude of processes that are capable of triggering death, cells have devised a large number of cellular processes that serve to prevent inappropriate or premature PCD. These cell survival strategies involve a myriad of coordinated and systematic physiological and genetic changes that serve to ward off death. Here we will discuss the different strategies that are used to prevent cell death and focus on illustrating that although anti-apoptosis and cellular survival serve to counteract PCD, they are nevertheless mechanistically distinct from the processes that regulate cell death.► Anti-apoptosis is mechanistically distinct from apoptosis. ► Anti-apoptosis processes are commonly observed in apoptotic resistant cells. ► Multiple physiological and genetic processes mediate anti-apoptosis and cell survival. ►Autophagy can sometimes prevent and sometimes initiate cell death. ► Pro-survival processes may serve to inhibit some forms of necrosis.

Keywords: Cell death; Anti-death; Pre-condition; Stress response; Regulation of apoptosis; Survival

Mitochondrial longevity pathways by Anna Raffaello; Rosario Rizzuto (pp. 260-268).

Production of reactive oxygen species (ROS) is a tightly regulated process, and increased levels of ROS within mitochondria are the principal trigger not only for mitochondrial dysfunctions but, more in general, for the diseases associated with aging, thus representing a powerful signaling molecules. One of the key regulators of ROS production, mitochondrial dysfunction, and aging is the 66-kDa isoform of the growth factor adapter shc (p66^shc) that is activated by stress and generates ROS within the mitochondria, driving cells to apoptosis. Accordingly, p66^shc knockout animals are one of the best characterized genetic model of longevity.On the other hand, caloric restriction is the only non-genetic mechanism that is shown to increase life span. Several studies have revealed a complex network of signaling pathways modulated by nutrients, such as IGF-1, TOR, sirtuins, AMP kinase, and PGC-1α that are connected and converge to inhibit oxidative stresses within the mitochondria. Animal models in which components of these signaling pathways are induced or silenced present a general phenotype characterized by the deceleration of the aging process. This review will summarize the main findings in the process that link mitochondria to longevity and the connections between the different signaling molecules involved in this intriguing relationship.►Mitochondrial ROS are potent signal molecules able to regulate the aging process. ►ROS production within mitochondria is regulated by intracellular signaling cascades. ►The life span determinant p66shc triggers ROS production within mitochondria. ►Other signaling cascades (e.g., IGF-1) converge on ROS production to control aging.

Keywords: Mitochondria; Reactive oxygen species; Life span; Oxidative stress; Signaling pathways; Aging

N-acetylglucosamine transferase is an integral component of a kinesin-directed mitochondrial trafficking complex by Kieran Brickley Karine Pozo; F. Anne Stephenson (pp. 269-281).

Trafficking kinesin proteins (TRAKs) 1 and 2 are kinesin-associated proteins proposed to function in excitable tissues as adaptors in anterograde trafficking of cargoes including mitochondria. They are known to associate with N-acetylglucosamine transferase and the mitochondrial rho GTPase, Miro. We used confocal imaging, Förster resonance energy transfer and immunoprecipitations to investigate association between TRAKs1/2, N-acetylglucosamine transferase, the prototypic kinesin-1, KIF5C, and Miro. We demonstrate that in COS-7 cells, N-acetylglucosamine transferase, KIF5C and TRAKs1/2 co-distribute. Förster resonance energy transfer was observed between N-acetylglucosamine transferase and TRAKs1/2. Despite co-distributing with KIF5C and immunoprecipitations demonstrating a TRAK1/2, N-acetylglucosamine transferase and KIF5C ternary complex, no Förster resonance energy transfer was detected between N-acetylglucosamine transferase and KIF5C. KIF5C, N-acetylglucosamine transferase, TRAKs1/2 and Miro formed a quaternary complex. The presence of N-acteylglucosamine transferase partially prevented redistribution of mitochondria induced by trafficking proteins 1/2 and KIF5C. TRAK2 was a substrate for N-acetylglucosamine transferase with TRAK2 (S562) identified as a site of O-N-acetylglucosamine modification. These findings substantiate trafficking kinesin proteins as scaffolds for the formation of a multi-component complex involved in anterograde trafficking of mitochondria. They further suggest that O-glycosylation may regulate complex formation.► nOGT co-immunoprecipitates with kinesin and TRAKs from brain extracts. ► Association of nOGT with kinesin is indirect and mediated via TRAK proteins. ► The TRAK/kinesin/nOGT complex associates with endogenous mitochondrial Miro. ► TRAK/kinesin mitochondrial redistribution is impaired by nOGT. ► O-Glycosylation regulates formation of the kinesin/TRAK/Miro trafficking complex.

Keywords: Abbreviations; CaMKII; calcium-calmodulin-dependent protein kinase II; EAA1; early endosome antigen; ECFP; enhanced cyan fluorescent protein; EYFP; enhanced yellow fluorescent protein; GABA; γ-aminobutyric acid; GlcNAc; N-acetylglucosamine; GRIF-1; GABA; _A; receptor interacting factor-1; HAP-1; huntingtin-associated protein; HEK; human embryonic kidney; hrs; hepatocyte growth factor-regulated tyrosine kinase substrate; KHC; kinesin heavy chain; KIF; kinesin superfamily protein; PC; pheochromocytoma; PVDF; polyvinylidene fluoride; OGT; β O-linked GlcNAc transferase; OIP; OGT interacting protein; TRAK; trafficking kinesin proteinTRAK; Milton; Mitochondrial trafficking; Mitochondrial transport; N-acetylglucosamine transferase (OGT); Kinesin

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BBA - Molecular Cell Research (v.1813, #1)