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BBA - Molecular Cell Research (v.1773, #3)

Editorial Board (pp. ii).

Principles of hepatic organic anion transporter regulation during cholestasis, inflammation and liver regeneration by Andreas Geier; Martin Wagner; Christoph G. Dietrich; Michael Trauner (pp. 283-308).

Hepatic uptake and biliary excretion of organic anions (e.g., bile acids and bilirubin) is mediated by hepatobiliary transport systems. Defects in transporter expression and function can cause or maintain cholestasis and jaundice. Recruitment of alternative export transporters in coordination with phase I and II detoxifying pathways provides alternative pathways to counteract accumulation of potentially toxic biliary constituents in cholestasis. The genes encoding for organic anion uptake ( NTCP, OATPs), canalicular export ( BSEP, MRP2) and alternative basolateral export (MRP3, MRP4) in liver are regulated by a complex interacting network of hepatocyte nuclear factors (HNF1, 3, 4) and nuclear (orphan) receptors (e.g., FXR, PXR, CAR, RAR, LRH-1, SHP, GR). Bile acids, proinflammatory cytokines, hormones and drugs mediate causative and adaptive transporter changes at a transcriptional level by interacting with these nuclear factors and receptors. Unraveling the underlying regulatory mechanisms may therefore not only allow a better understanding of the molecular pathophysiology of cholestatic liver diseases but should also identify potential pharmacological strategies targeting these regulatory networks. This review is focused on general principles of transcriptional basolateral and canalicular transporter regulation in inflammation-induced cholestasis, ethinylestradiol- and pregnancy-associated cholestasis, obstructive cholestasis and liver regeneration. Moreover, the potential therapeutic role of nuclear receptor agonists for the management of liver diseases is highlighted.

Keywords: Abbreviations; ABC; ATP-binding cassette; CAR (NR1I3); constitutive androstane receptor; CBDL; common bile duct ligation; Cyp; cytochrome p450; FXR (NR1H4); farnesoid X receptor/bile acid receptor; HNF; Hepatocyte nuclear factor; IL-1β; interleukin 1 beta; LPS; lipopolysaccharide; Mdr; multidrug resistance gene; Mrp; multidrug resistance-associated protein; Ntcp (Slc10a1); Na; ⁺; /taurocholate cotransporter; Oatp (Slc21a); organic anion transporter; Ost; organic solute transporter; PPARα (NR1C1); peroxisome proliferator activated receptor alpha; PPARγ (NR1C3); peroxisome proliferator activated receptor gamma; PXR (NR1I2); pregnane X receptor; RARα (NR1B1); retinoic acid receptor; RXRα (NR2B1); retinoid X receptor; SHP (NR0B2); short heterodimer partner; TNFα; tumor necrosis factor alphaCholestasis; Liver regeneration; Endotoxin; Cytokines; Nuclear (orphan) receptor; Hepatocyte-enriched transcription factor

Signaling pathways implicated in oncostatin M-induced aggrecanase-1 and matrix metalloproteinase-13 expression in human articular chondrocytes by Mohammed El Mabrouk; Judith Sylvester; Muhammad Zafarullah (pp. 309-320).

Molecular mechanisms of oncostatin M (OSM)-stimulated cartilage extracellular matrix catabolism and signaling pathways were investigated in human arthritic chondrocytes. OSM, alone or with Interleukin-1 (IL-1β), increased glycosaminoglycan release and induced ADAMTS-4 and MMP-13 protein expression in human cartilage explants. OSM dose- and time-dependently increased ADAMTS-4 mRNA and MMP-13 protein expression in human femoral head chondrocytes. Extracellular signal-regulated kinases (ERK1/2)-MAPK pathway inhibitor, U0126, down-regulated ADAMTS-4 and MMP-13 induction by OSM. Janus kinase 2 (JAK2) inhibitor, AG490, suppressed OSM-induced ADAMTS-4 mRNA expression but did not affect MMP-13 levels while JAK3 pharmacological inhibitor and siRNA transfection suppressed both. Parthenolide, a signal transducer and activator of transcription (STAT1 and STAT3) phosphorylation inhibitor, reduced OSM-induced ADAMTS-4 and MMP-13 gene expression and prevented STAT1/3 DNA binding activity. Additionally, OSM-enhanced ADAMTS-4 mRNA and MMP-13 expression was down-regulated by phosphatidylinositol 3-kinase (PI3K) and Akt/PKB inhibitors, LY294002 and NL-71-101. Furthermore, JAK3 inhibition time-dependently down-regulated Akt but not ERK1/2 phosphorylation suggesting that Akt is a downstream target of JAK3. These results suggest that OSM-stimulated ADAMTS-4 and MMP-13 expression is mediated by ERK1/2, JAK3/STAT1/3 and PI3K/Akt and by cross talk between these pathways. The inhibitors of these cascades could block OSM-evoked degeneration of cartilage by ADAMTS-4 and MMP-13.

Keywords: Arthritis; Human chondrocytes; Matrix metalloproteinase; ADAMTS; Oncostatin M; Cell signaling

Functional studies of aldo-keto reductases in Saccharomyces cerevisiae by Qing Chang; Terry A. Griest; Theresa M. Harter; J. Mark Petrash (pp. 321-329).

We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast.

Keywords: Abbreviations; AKR; aldo-keto reductase; AR; aldose reductase; GPD; glyceraldehyde 3-phosphate dehydrogenase; IEC; ion exchange chromatography; ORF; open reading frame; YPD; yeast peptone dextrose; synthetic completeAldo-keto reductase; Aldose reductase; Saccharomyces cerevisiae; Mutagenesis; Heat shock

Functional studies of aldo-keto reductases in Saccharomyces cerevisiae by Qing Chang; Terry A. Griest; Theresa M. Harter; J. Mark Petrash (pp. 321-329).

RCAN3, a novel calcineurin inhibitor that down-regulates NFAT-dependent cytokine gene expression by M . Carme Mulero; Anna Aubareda; Agatha Schlüter; Mercè Pérez-Riba (pp. 330-341).

The regulators of calcineurin (RCAN) proteins, previously known as calcipressins, have been considered to be a well conserved family from yeast to human based on the conservation of their FLISPP motif. Here, after performing a RCAN comparative genomic analysis we propose the existence of a novel functionally closely related RCAN subfamily restricted to vertebrates, the other RCAN proteins being considered only as distantly related members of the family. In addition, while three paralogous RCAN genes are found in vertebrates, there is only one in the other members of Eukarya. Moreover, besides the FLISPP motif, these paralogous genes have two others conserved motifs, the Cn-inhibitor RCAN (CIC) and the PxIxxT, which are restricted to vertebrates. In humans, RCAN1 and RCAN2 bind and inhibit Cn through their C-terminal region. Given the high amino acid identity in this region among human RCANs, authors in the field have hypothesized a role for RCAN3 in inhibiting Cn activity. Here, we demonstrate for the first time that human RCAN3, encoded by the RCAN3 (also known as DSCR1L2) gene, interacts physically and functionally with Cn. This interaction takes place only through the RCAN3 CIC motif. Overexpression of this sequence inhibits Cn activity towards the nuclear factor of activated T cells (NFAT) transcription factors and down-regulates NFAT-dependent cytokine gene expression in activated human Jurkat T cells.

Keywords: RCAN3; Calcineurin; Cytokine; DSCR1L2; NFAT; Immunosuppression

RCAN3, a novel calcineurin inhibitor that down-regulates NFAT-dependent cytokine gene expression by M . Carme Mulero; Anna Aubareda; Agatha Schlüter; Mercè Pérez-Riba (pp. 330-341).

Keywords: RCAN3; Calcineurin; Cytokine; DSCR1L2; NFAT; Immunosuppression

Necrotic death without mitochondrial dysfunction-delayed death of cardiac myocytes following oxidative stress by Tammy M. Casey; Peter G. Arthur; Marie A. Bogoyevitch (pp. 342-351).

Oxidative stress has been implicated in cell death in range of disease states including ischemia/reperfusion injury of the heart and heart failure. Here we have investigated the mechanisms of cell death following chronic exposure of cardiac myocytes to oxidative stress initiated by hydrogen peroxide. This exposure induced a delayed form of cell death with ultrastructural changes typical of necrosis, and that was accompanied by the release of lactate dehydrogenase and increased lipid peroxidation. However, this delayed death was not accompanied by the loss of mitochondrial membrane potential or caspase-3 activation. Furthermore, we could demonstrate that this delayed necrosis was at least partially prevented by pre-treatment with the hypertrophic stimuli endothelin-1 or leukemic inhibitory factor. Our results suggest that this delayed form necrosis may also comprise an ordered series of events involving pathways amenable to therapeutic modulation.

Keywords: Necrosis; Energy stress; Caspase activity; Lipid peroxidation; Antioxidants

Necrotic death without mitochondrial dysfunction-delayed death of cardiac myocytes following oxidative stress by Tammy M. Casey; Peter G. Arthur; Marie A. Bogoyevitch (pp. 342-351).

Keywords: Necrosis; Energy stress; Caspase activity; Lipid peroxidation; Antioxidants

Protein kinase C inhibits binding of diacylglycerol kinase-ζ to the retinoblastoma protein by Alrik P. Los; John de Widt; Matthew K. Topham; Wim J. van Blitterswijk; Nullin Divecha (pp. 352-357).

We previously showed that the retinoblastoma protein (pRB), a key regulator of G1 to S-phase transition of the cell cycle, binds to and stimulates diacylglycerol kinase-ζ (DGKζ) to phosphorylate the lipid second messenger diacylglycerol into phosphatidic acid. pRB binds to the MARCKS phosphorylation-site domain of DGKζ that can be phosphorylated by protein kinase C (PKC). Here, we report that activation of PKC by phorbol ester inhibits DGKζ binding to pRB. Ro 31-8220, a specific inhibitor of PKC, alleviated this inhibition of binding. Mimicking of PKC phosphorylation of serine residues (by S/D but not S/N mutations) within the DGKζ-MARCKS phosphorylation-site domain also prevented DGKζ binding to pRB, suggesting that PKC phosphorylation of these residues negatively regulates the interaction between DGKζ and pRB. In PKC overexpression studies, it appeared that activation of particularly the (wild-type) PKCα isoform inhibits DGKζ binding to pRB, whereas dominant-negative PKCα neutralized this inhibition. PKCα activation thus prevents DGKζ regulation by pRB, which may have implications for nuclear diacylglycerol and phosphatidic acid levels during the cell cycle.

Keywords: Abbreviations; DAG; diacylglycerol; DGK; diacylglycerol kinase; GST; glutathion-; S; -transferase; MARCKS; myristoylated alanine-rich C-kinase substrate; PSD; phosphorylation-site domain; PKC; protein kinase C; pRB; retinoblastoma proteinDiacylglycerol kinase; Retinoblastoma protein; Protein kinase C; Cell cycle; Diacylglycerol; Phosphatidic acid

Protein kinase C inhibits binding of diacylglycerol kinase-ζ to the retinoblastoma protein by Alrik P. Los; John de Widt; Matthew K. Topham; Wim J. van Blitterswijk; Nullin Divecha (pp. 352-357).

Expression of a mutant p193/CUL7 molecule confers resistance to MG132- and etoposide-induced apoptosis independent of p53 or Parc binding by Joshua D. Dowell; Shih-Chong Tsai; Dora C. Dias-Santagata; Hidehiro Nakajima; Zhuo Wang; Wuqiang Zhu; Loren J. Field (pp. 358-366).

p193/CUL7 is an E3 ubiquitin ligase initially identified as an SV40 Large T Antigen binding protein. Expression of a dominant interfering variant of mouse p193/CUL7 (designated 1152stop) conferred resistance to MG132- and etoposide-induced apoptosis in U2OS cells. Immune precipitation/Western analyses revealed that endogenous p193/CUL7 formed a complex with Parc (a recently identified parkin-like ubiquitin ligase) and p53. Apoptosis resistance did not result from 1152stop-mediated disruption of the endogenous p193/CUL7 binding partners. Moreover, 1152stop molecule did not directly bind to endogenous p193/CUL7, Parc or p53. These data suggested a role for p193/CUL7 in the regulation of apoptosis independently of p53 and Parc activity.

Keywords: E3 ligase; BH-3 only; SV40 T-Antigen

Activation of p38MAPK by repetitive low-grade oxidative stress leads to pro-survival effects by Prosenjit Sen; Prabir Kumar Chakraborty; Sanghamitra Raha (pp. 367-374).

V79 lung fibroblasts were subjected to repetitive oxidative stress in culture through exposures to 30 μM H₂O₂ for 4 weeks. Within the first week of treatment p38MAPK became dually phosphorylated and became increasingly phosphorylated during the 4-week stress period. Akt also became phosphorylated on Ser⁴⁷³ and Thr³⁰⁸ after the second week of treatment and remained phosphorylated throughout the study. NFκB p65 and IκB kinase (IKK) became phosphorylated and NFκB transcriptional activity became augmented during repetitive stress. Treatment of the cells concurrently with SB203580, a specific p38MAPK inhibitor, robustly blocked activation of NFκB transcriptional activity, phosphorylation of p65, and IKK but only partially blocked Akt phosphorylation. Similar simultaneous treatment with PI-3 kinase inhibitor LY294002 prominently blocked Akt phosphorylation. Pre-exposure to short interfering RNA (si RNA ) to p38MAPK resulted in a complete blockage of the NFκB p65 and IKK phosphorylations as well as the anti-apoptotic influence induced by a single low dose of H₂O₂ but produced a partial obstruction of Akt phosphorylation. Repetitively stressed cells were found to be significantly resistant to apoptosis-inducing agents such as ultraviolet radiation (UVR) and mM H₂O₂. Concurrent treatment with SB203580 almost completely restored the normal apoptotic response such as DNA fragmentation after UVR and mM H₂O₂. LY294002, a PI-3 kinase inhibitor and SN-50, an inhibitor of NFκB, produced partial restorations of the apoptotic response. We conclude that activation of p38MAPK by repetitive oxidative stress is the key event which through its command over down-stream survival elements such as Akt and NFκB controls the anti-apoptotic environment of the repetitively H₂O₂-stressed cells.

Keywords: Abbreviations; NFκB; nuclear factor kappa B; CREB; cAMP response element binding protein; AP1; activator protein 1; iNOS; inducible nitric oxide synthase; MAPK; mitogen activated protein kinasep38MAPK; Akt; NFκB; IKK; p65; Repetitive stress; Apoptosis

RETRACTED: Activation of p38MAPK by repetitive low-grade oxidative stress leads to pro-survival effects by Prosenjit Sen; Prabir Kumar Chakraborty; Sanghamitra Raha (pp. 367-374).

This article has been removed consistent with Elsevier Policy on Article Withdrawal. Please seehttp://www.elsevier.com/locate/withdrawalpolicy.The Publisher apologizes for any inconvenience this may cause.Reason: It was brought to our attention that loading control scans in two sets of figures among a total of 14 sets of figures presented in the article (BBA-MCR 1773:367-374, 2007) bear similarities. The first author, who was solely responsible for these experiments, regrets that due to inadequate archiving of the primary scans, dating back a number of years, he is unable to provide original unedited scans of the two sets of loading control blots at this point of time to conclusively establish the authenticity of the loading control scans. The authors therefore retract the article at this time. However, the actual experimental data, the conclusions and the resulting model are absolutely sound and the authors have reproduced most of the data. The authors fully intend to republish the data.Prosenjit SenPrabir Kumar ChakrabortySanghamitra Raha

Glycogen synthase kinase-3β binds to E2F1 and regulates its transcriptional activity by Gisela García-Alvarez; Verònica Ventura; Oriol Ros; Rosa Aligué; Joan Gil; Albert Tauler (pp. 375-382).

GSK3β and E2F1 play an important role in the control of proliferation and apoptosis. Previous work has demonstrated that GSK3β indirectly regulates E2F activity through modulation of cyclin D1 levels. In this work we show that GSK3β phosphorylates human E2F1 in vitro at serine 403 and threonine 433, both residues localized at its transactivation domain. This phosphorylation was not detected in vivo. However, co-immunoprecipitation experiments do reveal in vivo binding of these proteins. Moreover, uninhibitable and catalitycally inactive GSK3β forms inhibit the transcriptional activity of a fusion protein containing E2F1 transactivation domain. Both forms of GSK3β inhibit E2F1 with similar efficiency. Interestingly the effect was independent of the mutation of serine 403 and threonine 433 to alanine. This suggests that this transcriptional modulation is independent of GSK3β kinase activity and phosphorylation state of serine 403 and threonine 433. The re-targeting of these GSK3β forms to the nucleus results in a higher capacity to regulate E2F1 transcriptional activity. Depletion of the levels of GSK3β protein using siRNA activates E2F1 transcriptional activity. The data presented in this study offer a new mechanism of regulation of E2F1 by direct binding of GSK3β to its transactivation domain.

Keywords: Abbreviations; E2F; E2 factor; pRb; retinoblastoma protein; PI 3-kinase/PKB; phosphatidylinositol 3-kinase/protein kinase B; GSK3β; glycogen synthase kinase-3β; hE2F; human E2F1; GST; glutathione; S; -transferase; DRB; 5,6-dichloro-1-β-; d; -ribofuranosylbenzimidazole; siRNA; small interfering RNA; cdk7; cyclin-dependent kinase 7; MS; mass spectrometryGSK3β; E2F1; Protein–protein interaction; Transcriptional activity; Transcription factor

Glycogen synthase kinase-3β binds to E2F1 and regulates its transcriptional activity by Gisela García-Alvarez; Verònica Ventura; Oriol Ros; Rosa Aligué; Joan Gil; Albert Tauler (pp. 375-382).

Curcumin-induced degradation of ErbB2: A role for the E3 ubiquitin ligase CHIP and the Michael reaction acceptor activity of curcumin by Yunjin Jung; Wanping Xu; Heejung Kim; Namchul Ha; Len Neckers (pp. 383-390).

We investigated the molecular mechanism underlying curcumin depletion of ErbB2 protein. Curcumin induced ErbB2 ubiquitination but pretreatment with proteasome inhibitors neither prevented curcumin depletion of ErbB2 protein nor further accumulated ubiquitinated ErbB2. Curcumin increased association of endogenous and ectopically expressed CHIP, a chaperone-dependent ubiquitin ligase, with ErbB2. In COS7 cells cotransfected with ErbB2 and various CHIP plasmids followed by curcumin treatment, CHIP-H260Q (a mutant lacking ubiquitin ligase activity) promoted less curcumin-induced ErbB2 ubiquitination than did wild type CHIP, and CHIP-K30A (a mutant incapable of binding Hsp90 and Hsp70) neither associated with ErbB2 nor promoted its ubiquitination. ErbB2 mutants lacking the kinase domain failed to associate with CHIP and were completely resistant to ubiquitination and depletion induced by curcumin. Finally, curcumin's Michael reaction acceptor functionality was required for both covalent association of curcumin with ErbB2 and curcumin-mediated ErbB2 depletion. These data suggest (1) that CHIP-dependent ErbB2 ubiquitination is implicated in curcumin-stimulated ErbB2 depletion, and (2) that covalent modification of ErbB2 by curcumin is the proximal signal which initiates this process.

Keywords: Curcumin; ErbB2; Michael reaction; E3 ubiquitin ligase; Molecular chaperones

Serotonin 5-HT_1A receptor stimulates c-Jun N-terminal kinase and induces apoptosis in Chinese hamster ovary fibroblasts by Justin H. Turner; Maria N. Garnovskaya; John R. Raymond (pp. 391-399).

The 5-HT_1A receptor is a prototypical member of the large and diverse serotonin receptor family. One key role of this receptor is to stimulate cell proliferation and differentiation via the extracellular signal regulated protein kinase (ERK) mitogen activated protein (MAP) kinase. There are few reports on the ability of the 5-HT_1A receptor to modulate other MAP kinases such as c-Jun N-terminal kinase (JNK), which is activated by various extracellular stimuli, resulting in cell growth, differentiation, and programmed cell death. We report here for the first time that the 5-HT_1A receptor stimulates JNK. JNK stimulation was Pertussis toxin-sensitive and was mediated by Rho family low molecular weight GTPases. The 5-HT_1A receptor also increased apoptosis, which was mimicked by the MEK inhibitor PD98059, and blocked by the JNK inhibitor SP600125. These results suggest that the 5-HT_1A receptor stimulates both ERK-dependent anti-apoptotic pathways and JNK-dependent pro-apoptotic pathways in CHO cells.

Keywords: Abbreviations; 5-HT; 5-hydroxytryptamine; CHO; Chinese hamster ovary; ERK; extracellular signal-regulated protein kinase; GPCR; G protein-coupled receptor; JNK; c-Jun N-terminal kinase; MAPK; mitogen-activated protein kinase; MEK; mitogen-activated protein kinases kinase; MKK; mitogen-activated protein kinase kinase; PBS; phosphate buffered saline; PKC; protein kinase C; PLC; phospholipase C; PTX; Pertussis; toxin; PVDF; polyvinylidene fluoride5-hydroxytryptamine; Serotonin; Apoptosis; Programmed cell death; G protein; Phosphorylation

Serotonin 5-HT_1A receptor stimulates c-Jun N-terminal kinase and induces apoptosis in Chinese hamster ovary fibroblasts by Justin H. Turner; Maria N. Garnovskaya; John R. Raymond (pp. 391-399).

Detection of MMP activity in living cells by a genetically encoded surface-displayed FRET sensor by Jie Yang; Zhihong Zhang; Juqiang Lin; Jinling Lu; Bi-feng Liu; Shaoqun Zeng; Qingming Luo (pp. 400-407).

Matrix metalloproteinases (MMPs) are secretory endopeptidases. They have been associated with invasion by cancer-cell and metastasis. Previous studies have demonstrated that proteolytic activity could be detected using fluorescence resonance energy transfer (FRET) with mutants of GFP. To monitor MMP activity, we constructed vectors that encoded a MMP Substrate Site (MSS) between YFP and CFP. In vitro, YFP–MSS–CFP can be used to detect MMP activity and 1,10-phenathroline inhibition of MMP activity. In living cells, MMPs are secreted proteins and act outside of the cell, and therefore YFP–MSS–CFP^display was anchored on the cellular surface to detect extracellular MMP. A pDisplay-YC vector expressing the YFP–MSS–CFP^display on the cellular surface was transfected into MCF-7 cells that expressed low levels of MMP. Efficient transfer of energy from excited CFP to YFP within the YFP–MSS–CFP^display molecule was observed, and real-time FRET was declined when MCF-7 was incubated with MMP2. However, no such transfer of energy was detected in the YFP–MSS–CFP^display expressing MDA-MB 435s cells, in which high secretory MMP2 were expressed. The FRET sensor YFP–MSS–CFP^display can sensitively and reliably monitor MMP activation in living cells and can be used for high-throughput screening of MMP inhibitors for anti-cancer treatments.

Keywords: Abbreviations; MMP; Matrix metalloproteinase; FRET; fluorescence resonance energy transfer; ECFP; enhanced cyan fluorescence protein; EYFP; enhanced yellow fluorescence protein; MSS; MMP Substrate Site; PDGFR; platelet derived growth factor receptorMMP; Fluorescence Resonance Energy Transfer (FRET); Fluorescence assay; Secretory; MMP inhibitor

Functional characterization of APOBEC-1 complementation factor phosphorylation sites by David M. Lehmann; Chad A. Galloway; Celeste MacElrevey; Mark P. Sowden; Joseph E. Wedekind; Harold C. Smith (pp. 408-418).

ApoB mRNA editing involves site-specific deamination of cytidine 6666 producing an in-frame translation stop codon. Editing minimally requires APOBEC-1 and APOBEC-1 complementation factor (ACF). Metabolic stimulation of apoB mRNA editing in hepatocytes is associated with serine phosphorylation of ACF localized to editing competent, nuclear 27S editosomes. We demonstrate that activation of protein kinase C (PKC) stimulated editing and enhanced ACF phosphorylation in rat primary hepatocytes. Conversely, activation of protein kinase A (PKA) had no effect on editing. Recombinant PKC efficiently phosphorylated purified ACF64 protein in vitro, whereas PKA did not. Mutagenesis of predicted PKC phosphorylation sites S154 and S368 to alanine inhibited ethanol-stimulated induction of editing suggesting that these sites function in the metabolic regulation of editing. Consistent with this interpretation, substitution of S154 and S368 with aspartic acid stimulated editing to levels comparable to ethanol treatment in control McArdle RH7777 cells. These data suggest that phosphorylation of ACF by PKC may be a key regulatory mechanism of apoB mRNA editing in rat hepatocytes.

Keywords: Abbreviations; APOBEC-1; apolipoprotein B editing catalytic subunit 1; ACF; APOBEC-1 complementation factor; apoB; apolipoprotein B; RRM; RNA recognition motif; PP1; protein phosphatase 1; PKC; protein kinase C; PKA; protein kinase A; NT; N-terminal; CT; C-terminalapoB; RNA editing; APOBEC-1 Complementation factor; Phosphorylation; Regulation

Mitochondrial targeting of farnesyl diphosphate synthase is a widespread phenomenon in eukaryotes by David Martín; María-Dolors Piulachs; Núria Cunillera; Albert Ferrer; Xavier Bellés (pp. 419-426).

The isoprenoid pathway is responsible for the generation of a wide range of products that are crucial for cellular processes; namely, cholesterol synthesis, protein glycosylation, growth control and synthesis of several hormones. Farnesyl diphosphate synthase (FPS), a key enzyme in this pathway, is usually considered to be cytosolic/peroxisomal. However, significant enzymatic activity has also been detected in rat liver mitochondria, although none of the mammalian FPS genes characterized to date contain sequences coding for mitochondrial transit peptides. Here, we describe the genomic organization of the human FPS gene and demonstrate that one of the two mRNAs expressed from this gene encodes an isoform with a 66 amino acid N-terminal extension containing a peptide that targets it to mitochondria. Previous studies suggested that the N-terminal extension of FPS in the plant Arabidopsis thaliana contains a mitochondrial targeting sequence. In this study, database analysis reveals that this is also the case in a number of mammals and insects. Finally, we provide functional proofs that the N-terminal sequence of Drosophila melanogaster FPS targets the protein to mitochondria. Taken together, these data suggest that mitochondrial targeting of FPS may be widespread among eukaryotes.

Keywords: Abbreviations; DmFPS; Drosophila melanogaster; farnesyl diphosphate synthase; EGFP; enhance green fluorescent protein; FPS; farnesyl diphosphate synthase; GP; gold particles; HsFPS; human farnesyl diphosphate synthase; WSR; mutant strain of; Saccharomyces cerevisiae; carrying a disrupted; COXIV; geneIsoprenoid; Cholesterol; Juvenile hormone; Mitochondria; Drosophila

TGFβ1 regulation of vimentin gene expression during differentiation of the C2C12 skeletal myogenic cell line requires Smads, AP-1 and Sp1 family members by Yongzhong Wu; Xueping Zhang; Morgan Salmon; Xia Lin; Zendra E. Zehner (pp. 427-439).

Vimentin exhibits a complex pattern of developmental and tissue-specific expression regulated by such growth factors as TGFβ1, PDGF, FGF, EGF and cytokines. Vimentin is expressed in the more migratory, mesenchymal cell and its expression is often down-regulated to make way for tissue-specific intermediate filaments proteins such as desmin in muscle. Here, we suggest a mechanism to explain how TGFβ1 contributes to the up-regulation of vimentin expression while blocking myogenesis. TGFβ1 binds to serine/threonine kinase receptors resulting in the phosphorylation of Smad2 and Smad3, followed by formation of a heteromeric complex with Smad4. The translocation of this complex to the nucleus modulates transcription of selected genes such as vimentin. However, the vimentin gene lacks a consensus TGFβ1 response element. By transient transfection analysis of vimentin's various promoter elements fused to the CAT reporter gene, we have determined that tandem AP-1 sites surrounded by GC-boxes are required for TGFβ1 induction. Mutations within this region eliminated the ability of Smad3 to induce reporter gene expression. DNA precipitation and ChIP assays suggest that c-Jun, c-Fos, Smad3 and Sp1/Sp3 interact over this region, but this interaction changes during myogenesis with TGFβ1 induction.

Keywords: Smad3; c-Jun; c-Fos; AP-1 family; Sp1; TGFβ1; Vimentin; Myogenesis

Selective roles for α-PKC in positive signaling for O₂⁻ generation and calcium mobilization but not elastase release in differentiated HL60 cells by Helen M. Korchak; Lindsay B. Dorsey; Haiying Li; DeMauri Mackie; Laurie E. Kilpatrick (pp. 440-449).

Protein kinase C (PKC) isotypes and Ca²⁺ mobilization have been implicated in phagocytic cell functions such as O₂⁻ generation. Ca/DG-dependent α-PKC and β-PKC have similar substrate specificities and cofactor requirements in vitro. However it is not known if these isotypes play redundant or unique roles in the intact cell. In the present study, a role for α-PKC in positive signaling for fMet-Leu-Phe- and PMA-activated O₂⁻ generation was probed using an siRNA strategy in HL60 cells differentiated to a neutrophilic phenotype (dHL60 cells). A selective decrease in α-PKC in dHL60 cells attenuated O₂⁻ generation but not degranulation, and reduced ligand-induced phosphorylation of p47^phox as previously shown for β-PKC. However α-PKC, unlike β-PKC, was a positive regulator of fMet-Leu-Phe-triggered Ca²⁺ uptake via SOCC (Store Operated Calcium Channels). The ability of a selective SOCC inhibitor, MRS1845, to decrease fMet-Leu-Phe induced Ca²⁺ uptake and O₂⁻ generation confirmed that Ca²⁺ uptake via SOCC was required for O₂⁻ generation. These results indicate that α-PKC and β-PKC are required for optimal O₂⁻ generation, but play different roles in Ca²⁺ signaling for phagocytic responses such as O₂⁻ generation.

Keywords: Abbreviations; dHL60 cells; HL60 cells differentiated to a neutrophilic phenotype; f-Met-Leu-Phe; N formyl methionyl leucyl phenylalanine; fMLP; f-Met-Leu-Phe; O; ₂; ⁻; superoxide anion; PKC; protein kinase C; SOCC; store operated Ca; ²⁺; channel; PMA; phorbol myristate acetate; EGTA; ethylene glycol bis(-aminoethyl ether)-; N,N,N′,N′; -tetra acetic acid; AEBSF; 4-(2-aminoethyl)-benzenesulfonyl fluorideα-PKC; O; ₂; ⁻; generation; Degranulation; p47phox; Ca; ²⁺; signaling

Distinct activities of the related protein kinases Cdk1 and Ime2 by Kara E. Sawarynski; Alexander Kaplun; Guri Tzivion; George S. Brush (pp. 450-456).

In budding yeast, commitment to DNA replication during the normal cell cycle requires degradation of the cyclin-dependent kinase (CDK) inhibitor Sic1. The G1 cyclin–CDK complexes Cln1–Cdk1 and Cln2–Cdk1 initiate the process of Sic1 removal by directly catalyzing Sic1 phosphorylation at multiple sites. Commitment to DNA replication during meiosis also appears to require Sic1 degradation, but the G1 cyclin–CDK complexes are not involved. It has been proposed that the meiosis-specific protein kinase Ime2 functionally replaces the G1 cyclin–CDK complexes to promote Sic1 destruction. To investigate this possibility, we compared Cln2–Cdk1 and Ime2 protein kinase activities in vitro. Both enzyme preparations were capable of catalyzing phosphorylation of a GST–Sic1 fusion protein, but the phosphoisomers generated by the two activities had significantly different electrophoretic mobilities. Furthermore, mutation of consensus CDK phosphorylation sites in Sic1 affected Cln2–Cdk1- but not Ime2-dependent phosphorylation. Phosphoamino acid analysis and phosphopeptide mapping provided additional evidence that Cln2–Cdk1 and Ime2 targeted different residues within Sic1. Examination of other substrates both in vitro and in vivo also revealed differing specificities. These results indicate that Ime2 does not simply replace G1 cyclin–CDK complexes in promoting Sic1 degradation during meiosis.

Keywords: Cyclin-dependent kinase; G1 cyclin; Meiosis; Phosphorylation; Replication protein A; Sic1

Distinct activities of the related protein kinases Cdk1 and Ime2 by Kara E. Sawarynski; Alexander Kaplun; Guri Tzivion; George S. Brush (pp. 450-456).

Keywords: Cyclin-dependent kinase; G1 cyclin; Meiosis; Phosphorylation; Replication protein A; Sic1

Implications on zinc binding to S100A2 by Michael Koch; Shibani Bhattacharya; Torsten Kehl; Mario Gimona; Milan Vašák; Walter Chazin; Claus W. Heizmann; Peter M.H. Kroneck; Günter Fritz (pp. 457-470).

Human S100A2 is an EF-hand calcium-binding S100 protein that is localized mainly in the nucleus and functions as tumor suppressor. In addition to Ca²⁺ S100A2 binds Zn²⁺ with a high affinity. Studies have been carried out to investigate whether Zn²⁺ acts as a regulatory ion for S100A2, as in the case of Ca²⁺. Using the method of competition with the Zn²⁺ chelator 4-(2-pyridylazo)-resorcinol, an apparent K_d of 25 nM has been determined for Zn²⁺ binding to S100A2. The affinity lies close to the range of intracellular free Zn²⁺ concentrations, suggesting that S100A2 is able to bind Zn²⁺ in the nucleus. Two Zn²⁺-binding sites have been identified using site directed mutagenesis and several spectroscopic techniques with Cd²⁺ and Co²⁺ as probes. In site 1 Zn²⁺ is bound by Cys21 and most likely by His 17. The binding of Zn²⁺ in site 2 induces the formation of a tetramer, whereby the Zn²⁺ is coordinated by Cys2 from each subunit. Remarkably, only binding of Zn²⁺ to site 2 substantially weakens the affinity of S100A2 for Ca²⁺. Analysis of the individual Ca²⁺-binding constants revealed that the Ca²⁺ affinity of one EF-hand is decreased about 3-fold, whereas the other EF-hand exhibits a 300-fold decrease in affinity. These findings imply that S100A2 is regulated by both Zn²⁺ and Ca²⁺, and suggest that Zn²⁺ might deactivate S100A2 by inhibiting response to intracellular Ca²⁺ signals.

Keywords: Abbreviations; ANS; 4,4′-dianilino-1,1′-binaphtyl-5,5′-sulfonic acid; PAR; 4-(2-pyridylazo)-resorcinolS100; S100A2; Zinc; Calcium; Cobalt

Erratum to “Biphasic electric current stimulates proliferation and induces VEGF production in osteoblasts” [Biochim. Biophys. Acta 1763 (2006) 907–916] by In Sook Kim; Jong Keun Song; Yu Lian Zhang; Tae Hyung Lee; Tae Hyung Cho; Yun Mi Song; Do Kyun Kim; Sung June Kim; Soon Jung Hwang (pp. 471-471).

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BBA - Molecular Cell Research (v.1773, #3)

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BBA - Molecular Cell Research (v.1773, #3)