BBA - Molecular Cell Research (v.1833, #5)

Enough is enough: TatA demand during Tat-dependent protein transport by René Steffen Hauer; René Schlesier; Kathleen Heilmann; Julia Dittmar; Mario Jakob; Ralf Bernd Klösgen (957-965).
The twin-arginine translocation (Tat1) pathway is unique with respect to its property to translocate proteins in a fully folded conformation across ion-tight membranes. In chloroplasts and Gram-negative bacteria, Tat translocase consists of the integral subunits TatB and TatC, which are assumed to constitute the membrane receptor, and TatA, a bitopic membrane protein being responsible in a yet unknown manner for the membrane translocation step. Antibody inhibition of intrinsic thylakoidal TatA activity and recovery of transport by heterologously expressed, purified TatA allowed to exactly quantify the amount of TatA required to catalyse membrane transport of the model Tat substrate 16/23. We can show that TatA concentrations in the 100 nM range are sufficient to efficiently catalyse membrane transport of the protein, which corresponds well to the amount of TatA identified in thylakoids. Furthermore, TatA shows cooperativity in its catalytic activity suggesting that Tat translocase operates as an allosteric enzyme complex.Display Omitted► TatA is purified in functional form after heterologous expression. ► Soluble TatA can fully replace thylakoidal TatA in catalysing protein transport. ► TatA is required only at concentrations in the 100 nM range. ► TatA shows cooperativity in its catalytic activity.
Keywords: Twin-arginine translocation; Thylakoid membrane; Transport mechanism; Cooperative effect; Heterologous overexpression; Protein purification;

CCN4 induces vascular cell adhesion molecule-1 expression in human synovial fibroblasts and promotes monocyte adhesion by Ju-Fang Liu; Sheng-Mou Hou; Chun-Hao Tsai; Chun-Yin Huang; Chin-Jung Hsu; Chih-Hsin Tang (966-975).
CCN4 is a cysteine-rich protein that belongs to the Cyr61, CTGF, Nov family of matricellular proteins. Here, we investigated the intracellular signaling pathways involved in CCN4-induced vascular cell adhesion molecule-1 expression in human osteoarthritis synovial fibroblasts. Stimulation of OASFs with CCN4 induced VCAM-1 expression. CCN4-induced VCAM-1 expression was attenuated by αvβ5 or α6β1 integrin antibody, Syk inhibitor, PKCδ inhibitor (rottlerin), JNK inhibitor (SP600125), and AP-1 inhibitors (curcumin and tanshinone). Stimulation of cells with CCN4 increased Syk, PKCδ, and JNK activation. Treatment of OASFs with CCN4 also increased c-Jun phosphorylation, AP-1-luciferase activity, and c-Jun binding to the AP-1 element in the VCAM-1 promoter. Moreover, up-regulation of VCAM-1 increased the adhesion of monocytes to OASF monolayers, and this adhesion was attenuated by transfection with a VCAM-1 siRNA. Our results suggest that CCN4 increases VCAM-1 expression in human OASFs via the Syk, PKCδ, JNK, c-Jun, and AP-1 signaling pathways. The CCN4-induced VCAM-1 expression promoted monocyte adhesion to human OASFs.► CCN4 was highly expressed in OA synovial fibroblasts as compared to normal SFs. ► CCN4 induces the monocyte adhesion in OASFs by up-regulation of VCAM-1 expression. ► CCN4 acts through the αvβ5/α6β1 integrin, Syk, PKCδ, and JNK pathways to induce VCAM-1. ► AP-1 is key transcription factor of CCN4-induced VCAM-1 expression.
Keywords: CCN4; OA; VCAM-1; Syk; Integrin;

MEK-1 activates C-Raf through a Ras-independent mechanism by Deborah T. Leicht; Vitaly Balan; Jun Zhu; Alexander Kaplun; Agnieszka Bronisz; Ajay Rana; Guri Tzivion (976-986).
C-Raf is a member of the Ras–Raf–MEK–ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using 32P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating the C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf–MEK interaction is enhanced due to the down-regulation of RKIP and MST2.► MEK overexpression induces C-Raf hyper-phosphorylation and activation. ► Co-expression of wildtype C-Raf and MEK constitutively activates ERK MAPK. ► MEK-induced C-Raf activation is Ras independent. ► MEK-induced C-Raf activation requires C-Raf–MEK interaction. ► MEK is a novel, Ras-independent C-Raf activator.
Keywords: Raf; Ras; MEK; ERK; MAPK; Phosphorylation;

Breast cancer is the second most common cancer with a high incidence rate worldwide. One of the promising therapeutic approaches on breast cancer is to use the drugs that target the estrogen receptor (ER). In the present investigation, marmorin, a type I ribosome inactivating protein from the mushroom Hypsizigus marmoreus, inhibited the survival of breast cancer in vitro and in vivo. It evinced more potent cytotoxicity toward estrogen receptor (ER)-positive MCF7 breast cancer cells than ER-negative MDA-MB-231 cells. Further study disclosed that marmorin undermined the expression level of estrogen receptor α (ERα) and significantly inhibited the proliferation of MCF7 cells induced by 17β-estradiol. Knockdown of ERα in MCF7 cells significantly attenuated the inhibitory effect of marmorin on proliferation, suggesting that the ERα-mediated pathway was implicated in the suppressive action of marmorin on ER-positive breast cancer cells. Moreover, marmorin induced time-dependent apoptosis in both MCF7 and MDA-MB-231 cells. It brought about G2/M-phase arrest, mitochondrial membrane potential depolarization and caspase-9 activation in MCF7 cells, and to a lesser extent in MDA-MB-231 cells. Marmorin triggered the death receptor apoptotic pathway (e.g. caspase-8 activation) and endoplasmic reticulum stress (ERS, as evidenced by phosphorylation of PERK and IRE1α, cleavage of caspase-12, and up-regulation of CHOP expression) in both MCF7 and MDA-MB-231 cells. In summary, marmorin exhibited inhibitory effect on breast cancer partially via diminution of ERα and apoptotic pathways mediated by mitochondrial, death receptor and ERS. The results advocate that marmorin is a potential candidate for breast cancer therapy.► Marmorin inhibited the proliferation of breast cancer cells in vitro and in vivo. ► MCF7 and MDA-MB-231 cells showed differentiated sensitivity to marmorin. ► Marmorin down-regulates estrogen receptor α expression in MCF7 cells. ► MCF7 and MDA-MB-231 cells shared discrepancy of apoptotic pathways caused by marmorin.
Keywords: Marmorin; Ribosome inactivating protein; Breast cancer; Apoptosis; Estrogen receptor α; Endoplasmic reticulum stress;

Yeast protective response to arsenate involves the repression of the high affinity iron uptake system by Liliana Batista-Nascimento; Michel B. Toledano; Dennis J. Thiele; Claudina Rodrigues-Pousada (997-1005).
Arsenic is a double-edge sword. On the one hand it is powerful carcinogen and on the other it is used therapeutically to treat acute promyelocytic leukemia. Here we report that arsenic activates the iron responsive transcription factor, Aft1, as a consequence of a defective high-affinity iron uptake mediated by Fet3 and Ftr1, whose mRNAs are drastically decreased upon arsenic exposure. Moreover, arsenic causes the internalization and degradation of Fet3. Most importantly, fet3ftr1 mutant exhibits increased arsenic resistance and decreased arsenic accumulation over the wild-type suggesting that Fet3 plays a role in arsenic toxicity. Finally we provide data suggesting that arsenic also disrupts iron uptake in mammals and the link between Fet3, arsenic and iron, can be relevant to clinical applications.► Iron homeostasis ► Arsenic toxicity ► Adapted yeast response ► Iron uptake, disruption in mammals
Keywords: Arsenic uptake; Fet3–Ftr1; Iron deficiency;

Sphingosine kinase 1 expression is downregulated during differentiation of Friend cells due to decreased c-MYB by N. Mizutani; M. Kobayashi; S. Sobue; M. Ichihara; H. Ito; K. Tanaka; S. Iwaki; S. Fujii; Y. Ito; K. Tamiya-Koizumi; A. Takagi; T. Kojima; T. Naoe; M. Suzuki; M. Nakamura; Y. Banno; Y. Nozawa; T. Murate (1006-1016).
Sphingosine kinase 1 (SPHK1) overexpression in malignant cells has been reported. Mouse Friend cells showed higher SPHK1 but not SPHK2 expression compared with other mouse cell lines. A Sphk1 promoter analysis demonstrated the region between − 53 bp and the first exon as the minimal promoter. Further promoter truncation revealed the importance of a MYB-binding site. EMSA using this region as the probe demonstrated one band containing c-MYB protein, and its intensity decreased during erythroid differentiation with hexamethylane bisacetamide (HMBA), a potent inducer of erythroid differentiation of Friend cells. ChIP assay also revealed in vivo binding of c-MYB. c-MYB overexpression and siRNA for c-Myb affected SPHK1 expression, confirming the important regulatory role of c-MYB in SPHK1 expression. HMBA reduced c-MYB expression rapidly. Induced differentiation by HMBA caused a marked and rapid reduction of SPHK1 mRNA, protein and enzyme activity leading to the rapid decrease of cellular sphingosine 1-phosphate level. Moreover, terminally differentiated cells did not resume SPHK1 expression. Compared with original Friend cells, stable overexpression of wild-type SPHK1 showed higher cell proliferation, resistance to cell death by serum depletion. Interestingly, HMBA-induced differentiation of these cells was delayed but not completely suppressed. In contrast, SPHK inhibitor and its siRNA inhibited cell growth and enhanced HMBA-induced differentiation significantly, suggesting that SPHK1 delayed HMBA-induced differentiation by its cell proliferation-promoting activity. Effects of pertussis toxin, a G-protein-coupled receptor inhibitor, and S1P receptor antagonist on Friend cell growth and differentiation were negligible, suggesting the importance of the intracellular SPHK1/S1P signaling in Friend cells.► SPHK1 but not SPHK2 was overexpressed in a mouse Friend erythroeukemia cell line. ► SPHK1 expression of Friend cells affects their proliferation and differentiation. ► SPHK1 expression decreased during HMBA-induced differentiation. ► 5′-promoter between − 53 bp and the first exon is important for SPHK1 transcription. ► C-MYB regulates SPHK1 transcription by its binding with the 5′-promoter region.
Keywords: Friend cell; SPHK1; c-MYB; Promoter analysis; ChIP; HMBA-induced differentiation;

A structural perspective of the MAVS-regulatory mechanism on the mitochondrial outer membrane using bioluminescence resonance energy transfer by Osamu Sasaki; Takuma Yoshizumi; Misa Kuboyama; Takeshi Ishihara; Emiko Suzuki; Shun-ichiro Kawabata; Takumi Koshiba (1017-1027).
In most eukaryotic cells, mitochondria have various essential roles for proper cell function, such as energy production, and in mammals mitochondria also act as a platform for antiviral innate immunity. Mitochondrial-mediated antiviral immunity depends on the activation of the cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling pathway, and on the participation of mitochondrial antiviral signaling (MAVS), which is localized on the mitochondrial outer membrane. After RNA virus infection, RLRs translocate to the mitochondrial surface to interact with MAVS, and the adaptor protein undergoes a conformational change that is essential for downstream signaling, although its structural features are poorly understood. Here we examined the MAVS-regulatory mechanism on the mitochondrial outer membrane using bioluminescence resonance energy transfer (BRET) in live cells. Using a combination of BRET and functional analysis, we found that the activated MAVS conformation is a highly ordered oligomer, at least more than three molecules per complex unit on the membrane. Hepatitis C virus NS3/4A protease and mitofusin 2, which are known MAVS inhibitors, interfere with MAVS homotypic oligomerization in a distinct manner, each differentially altering the active conformation of MAVS. Our results reveal structural features underlying the precise regulation of MAVS signaling on the mitochondrial outer membrane, and may provide insight into other signaling systems involving organelles.► In mammals, MAVS is involved in the antiviral innate immunity. ► We examined the MAVS-regulatory mechanism in live cells using BRET system. ► Activated MAVS conformation is a highly ordered oligomer. ► MAVS inhibitors differentially affect the MAVS homotypic oligomerization. ► We provide structural insight into mitochondrial-mediated antiviral signaling.
Keywords: Antiviral innate immunity; BRET; MAVS; Mitochondria; Signal transduction;

Gefitinib is an Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitor, approved for patients with non-small cell lung cancer (NSCLC). In this report we demonstrate that gefitinib loaded PLGA nanoparticles (GNPs), in comparison to gefitinib, exhibited higher anti-cancer activity on A549 lung carcinoma cells and A431 skin carcinoma cells. Increased inhibition of pEGFR in both the cell types explains its higher anti-cancer activity. Interestingly, gefitinib resistant, H1975 (T790M EGFR mutant) lung carcinoma cells was also found to be sensitive to GNPs. Our data shows that GNPs hyperacetylate histone H3 in these cells, either directly or indirectly, which may account for the augmented cell death. GNPs were proficient in activating histone acetyltransferases (p300/CBP), which in turn induces the expression of p21 and cell cycle arrest. Furthermore, inhibition of histone acetyltransferases by garcinol results in alleviation of cell death caused by GNPs. In addition to this, nuclear intrusion of GNPs results in the inhibition of NO production in nucleus, possibly through nuclear EGFR, which might be responsible for preventing cell proliferation in resistant cells. To best of our knowledge, we provide first evidence that GNPs potentiate cell death by activating p300/CBP histone acetyltransferases.► The study highlights the higher efficacy of nano-gefitinib over gefitinib alone. ► GNPs caused higher cell death in A431 and A549 cells as compared to gefitinib. ► GNPs also prove to be effective in gefitinib resistant cells (H1975). ► Histone H3 acetylation played a pivotal role in higher potency of nano-gefitinib. ► Role of histone acetylation was confirmed by inhibiting HATs(p300/CBP) by garcinol.
Keywords: Gefitinib; EGFR; T790M; Histone; p300/CBP;

Biphasic Erk1/2 activation sequentially involving Gs and Gi signaling is required in beta3-adrenergic receptor-induced primary smooth muscle cell proliferation by Tarik Hadi; Marina Barrichon; Pascal Mourtialon; Maeva Wendremaire; Carmen Garrido; Paul Sagot; Marc Bardou; Frédéric Lirussi (1041-1051).
The beta3 adrenergic receptor (B3-AR) reportedly induces cell proliferation, but the signaling pathways that were proposed, involving either Gs or Gi coupling, remain controversial. To further investigate the role of G protein coupling in B3-AR induced proliferation, we stimulated primary human myometrial smooth muscle cells with SAR150640 (B3-AR agonist) in the absence or presence of variable G-protein inhibitors. Specific B3-AR stimulation led to an Erk1/2 induced proliferation. We observed that the proliferative effects of B3-AR require two Erk1/2 activation peaks (the first after 3 min, the second at 8 h). Erk1/2 activation at 3 min was mimicked by forskolin (adenylyl-cyclase activator), and was resistant to pertussis toxin (Gi inhibitor), suggesting a Gs protein signaling. This first signaling also required the downstream Gs signaling effectors PKA and Src. However, Erk1/2 activation at 8 h turned out to be pertussis toxin-dependent, and PKA-independent, indicating a Gi signaling pathway in which Src and PI3K were required. The pharmacological inhibition of both the Gs and Gi pathway abolished B3-AR-induced proliferation. Altogether, these data indicate that B3-AR-induced proliferation depends on the biphasic activation of Erk1/2 sequentially induced by the Gs/PKA/Src and Gi/Src/PI3K signaling pathways.► The beta-3 adrenergic receptor induces primary smooth muscle cell proliferation. ► Biphasic Erk1/2 activation, at 3 and 8 h, is required for proliferation. ► This biphasic activation depends upon distinct Gs/cAMP/PKA and Gi/Src/PI3K pathways. ► We identified a novel G-protein coupled receptor sequential signaling pathway.
Keywords: Proliferation; Erk1/2; G protein coupled receptor; Beta-3 adrenergic receptor; Cyclins; Protein kinases;

Oestradiol signalling through the Akt–mTORC1–S6K1 by O. Varea; M. Escoll; H. Diez; J.J. Garrido; F. Wandosell (1052-1064).
The oestradiol plays an important role in normal brain development and exerts neuroprotective actions. Oestradiol is mainly produced in the ovary and in addition is locally synthesised in the brain. Most of the oestradiol functions have been associated with its capacity to directly bind and dimerize “classical oestrogen receptors” (ERs), alpha and beta. The ERs' actions have been classified as “genomic” and “non-genomic” depending on whether protein synthesis occurs through ER driven transcription or not. Indeed, recent evidence suggests that oestrogen may also act as a more general “trophic factor”. Hence, we have studied the capacity of oestradiol to activate the PI3K/Akt pathway and its implication in axonal growth and neuronal morphogenesis. Our data show that when oestrogen receptors are blocked the axonal and dendritic lengths are reduced in mouse primary neurons. We found that Akt/Rheb/mTORC1 responds to ER activation in neurons and that some elements of this pathway are able to restore a normal neuronal morphology even in the presence of oestrogen receptor antagonist. All these data demonstrate a new mechanism regulated by oestradiol, at least in neuronal morphogenesis.► Oestradiol potentiated axonal and dendritic growth. ► Oestradiol antagonist (ICI) prevented axonal growth. ► Oestradiol antagonist (ICI) inhibited S6K activity as reported by pS6. ► Overexpression of Rheb or S6K on ICI-treated neurons restores pS6 and axonal growth.
Keywords: Estrogen; Rheb activation; Estradiol cytoplasmic signalling; S6 kinase activation;

Clathrin-mediated hemoglobin endocytosis is essential for survival of Leishmania by Shruti Agarwal; Ruchir Rastogi; Deepika Gupta; Nitin Patel; Manoj Raje; Amitabha Mukhopadhyay (1065-1077).
Leishmania is auxotroph for heme. Previously, we have shown that Leishmania acquire heme from the degradation of endocytosed hemoglobin via a specific receptor located in the flagellar pocket. Here, we report the cloning and expression of clathrin heavy chain from Leishmania (Ld-CHC) and provide evidences that Ld-CHC is localized in flagellar pocket and regulates Hb-endocytosis in Leishmania. Kinetic analysis of Hb trafficking in GFP-Ld-CHC overexpressed Leishmania reveals that Hb is internalized through Ld-CHC coated region and remains associated with Ld-CHC containing vesicles at early time points of internalization and subsequently starts dissociating from Hb-containing vesicles at later time points indicating that clathrin-coating and uncoating regulate Hb trafficking in Leishmania. Interestingly, overexpression of dominant negative mutant of clathrin heavy chain of Leishmania (GFP-Ld-CHC-Hub) blocks the Hb internalization and causes severe growth defect in parasite. Moreover, we have shown that chlorpromazine, a pharmacological agent, blocks Hb internalization in Leishmania by depolymerizing Ld-CHC and thereby inhibits the growth of the parasites. Taken together, our results have shown that Hb endocytosis in Leishmania is a clathrin dependent process and is essential for the survival of the parasites.► Cloning and expression of clathrin heavy chain from Leishmania ► Dominant negative mutant of clathrin inhibits hemoglobin endocytosis in Leishmania. ► Chlorpromazine, a drug that kills Leishmania, depolymerizes clathrin and blocks Hb endocytosis. ► Clathrin-mediated Hb endocytosis is essential for survival of Leishmania.
Keywords: Leishmania; Hemoglobin; Trafficking; Clathrin; Survival; Chlorpromazine;

CAND1-dependent control of cullin 1-RING Ub ligases is essential for adipogenesis by Dawadschargal Dubiel; Maria Elka Gierisch; Xiaohua Huang; Wolfgang Dubiel; Michael Naumann (1078-1084).
Cullin-RING ubiquitin (Ub) ligases (CRLs) are responsible for ubiquitinylation of approximately 20% of all proteins degraded by the Ub proteasome system (UPS). CRLs are regulated by the COP9 signalosome (CSN) and by Cullin-associated Nedd8-dissociated protein 1 (CAND1). The CSN is responsible for removal of Nedd8 from cullins inactivating CRLs. CAND1 modulates the assembly of F-box proteins into cullin 1–RING Ub ligases (CRL1s). We show that CAND1 preferentially blocks the integration of Skp2 into CRL1s. Suppression of CAND1 expression in HeLa cells leads to an increase of the Skp2 assembly into CRL1s and to significant reduction of the cyclin-dependent kinase (CDK) inhibitor p27. In contrary, CAND1 overexpression causes elevation of p27. The observed CAND1-dependent effects and CAND1 expression are independent of the CSN as demonstrated in CSN1 knockdown cells. Increase of p27 is a hallmark of preadipocyte differentiation to adipocytes (adipogenesis). We demonstrate that the accumulation of p27 is associated with an increase of CAND1 and a decrease of Skp2 during adipogenesis of human LiSa-2 preadipocytes. CAND1 knockdown reduces p27 and blocks adipogenesis. Due to the impact of CAND1 on Skp2 control, CAND1 could represent an important effector molecule in adipogenesis, but also in cancer development.► CAND1 preferentially blocks Skp2 integration into CRL1s in human cells. ► CAND1 knockdown causes Skp2 assembly into CRL1s and a significant decrease of p27. ► CAND1 increases in preadipocyte differentiation associated with elevation of p27. ► Knockdown of CAND1 in preadipocytes blocks adipogenesis.
Keywords: COP9 signalosome; F-box proteins; Preadipocytes; p27; Skp2;

NF-κB activation fails to protect cells to TNFα-induced apoptosis in the absence of Bcl-xL, but not Mcl-1, Bcl-2 or Bcl-w by Elisenda Casanelles; Raffaella Gozzelino; Fernando Marqués-Fernández; Victoria Iglesias-Guimarais; Mercè Garcia-Belinchón; María Sánchez-Osuna; Carme Solé; Rana S. Moubarak; Joan X. Comella; Victor J. Yuste (1085-1095).
TNFα can promote either cell survival or cell death. The activation of NF-κB plays a central role in cell survival while its inhibition makes TNFα-triggered cytotoxicity possible. Here, we report that the overexpression of a non-degradable mutant of the inhibitor of NF-κB (super-repressor (SR)-IκBα) sensitizes HeLa cells towards TNFα-induced apoptosis, involving caspases activation and cytocrome C release from the mitochondria. Interestingly, we describe that the specific knockdown of Bcl-xL, but not that of Bcl-2, Bcl-w or Mcl-1, renders cells sensitive to TNFα-induced apoptosis. This cytotoxic effect occurs without altering the activation of NF-κB. Then, the activation of the NF-κB pathway is not sufficient to protect Bcl-xL-downregulated cells from TNFα-induced cell death, meaning that TNFα is not able to promote cell survival in the absence of Bcl-xL. In addition, Bcl-xL silencing does not potentiate the cytotoxicity afforded by the cytokine in SR-IκBα-overexpressing cells. This indicates that TNFα-induced apoptosis in SR-IκBα-overexpressing cells relies on the protein levels of Bcl-xL. We have corroborated these findings using RD and DU-145 cells, which also become sensitive to TNFα-induced apoptosis after Bcl-xL knockdown despite that NF-κB remains activated. Altogether, our results point out that the impairment of the anti-apoptotic function of Bcl-xL should make cells sensitive towards external insults circumventing the TNFα-triggered NF-κB-mediated cytoprotective effect. Hence, the specific inhibition of Bcl-xL could be envisaged as a promising alternative strategy against NF-κB-dependent highly chemoresistant proliferative malignancies.► NF-κB super-repressor overexpression sensitizes HeLa cells to TNFα-induced apoptosis. ► The specific knockdown of Bcl-xL sensitizes cells to TNFα-triggered apoptosis. ► The NF-κB pathway activated by TNFα is still functional in Bcl-xL-downregulated cells. ► Bcl-xL silencing does not increase TNFα-induced apoptosis in NF-κB-repressed cells. ► TNFα-induced NF-κB-mediated cell survival relies on the anti-apoptotic role of Bcl-xL.
Keywords: Apoptosis; Bcl-xL; Bcl-2 family; NF-κB; TNFα;

A subset of FG-nucleoporins is necessary for efficient Msn5-mediated nuclear protein export by Erin M. Finn; Elise P. DeRoo; George W. Clement; Sheila Rao; Sarah E. Kruse; Kate M. Kokanovich; Kenneth D. Belanger (1096-1103).
The transport of proteins between the cytoplasm and nucleus requires interactions between soluble transport receptors (karyopherins) and phenylalanine–glycine (FG) repeat domains on nuclear pore complex proteins (nucleoporins). However, the role of specific FG repeat-containing nucleoporins in nuclear protein export has not been carefully investigated. We have developed a novel kinetic assay to investigate the relative export kinetics mediated by the karyopherin Msn5/Kap142 in yeast containing specific FG-Nup mutations. Using the Msn5 substrate Crz1 as a marker for Msn5-mediated protein export, we observe that deletions of NUP100 or NUP2 result in decreased rates of Crz1 export, while nup60Δ and nup42Δ mutants do not vary significantly from wild type. The decreased Msn5 export rate in nup100Δ was confirmed using Mig1-GFP as a transport substrate. A nup100ΔGLFG mutant shows defects in nuclear export kinetics similar to a nup100Δ deletion. Removal of FG-repeats from Nsp1 also decreases export kinetics, while a loss of Nup1 FXFGs does not. To confirm that our export data reflected functional differences in protein localization, we performed Crz1 transcription activation assays using a CDRE::LacZ reporter gene that is upregulated upon increased transcription activation by Crz1 in vivo. We observe that expression from this reporter increases in nup100ΔGLFG and nsp1ΔFGΔFXFG strains that exhibit decreased Crz1 export kinetics but resembles wild-type levels in nup1ΔFXFG strains that do not exhibit export defects. These data provide evidence that the export of Msn5 is likely mediated by a specific subset of FG-Nups and that the GLFG repeat domain of Nup100 is important for Msn5-mediated nuclear protein export.► A novel approach to study nuclear protein export kinetics is described. ► Cells lacking the nucleoporins Nup100 or Nup2 exhibit slowed export. ► GLFG repeats of Nup100 are necessary for efficient protein export. ► FXFG repeats in Nsp1 but not Nup1 affect nuclear export kinetics. ► Nup mutations that affect export alter Crz1 transcription factor activity.
Keywords: Nuclear pore complex; Nuclear export; Nucleoporin; Karyopherin; Exportin;

Nek7 kinase accelerates microtubule dynamic instability by Sivan Cohen; Adva Aizer; Yaron Shav-Tal; Amiel Yanai; Benny Motro (1104-1113).
The NIMA-related kinases (NRK or Nek) are emerging as conserved and crucial regulators of mitosis and cilia formation. The microtubule (MT) network has long been suspected as a major target of the Neks. However, the underlying mechanism remains unclear. Using the PlusTipTracker software, recently developed by the Danuser group, we followed the consequences of alterations in Nek7 levels on MT dynamic instability. siRNA-mediated downregulation of Nek7 in HeLa cells resulted in lower speeds of MT growth and catastrophe, reduction of the relative time spent in catastrophe, and considerably lowered the overall MT dynamicity. Co-expression of Nek7 with the siRNA treatment rescued the MT phenotypes, while ectopic overexpression of Nek7 yielded inverse characteristics compared to Nek7 downregulation. MT dynamics in mouse embryonic fibroblasts derived from targeted null mutants for Nek7 recapitulated the siRNA downregulation phenotypes. Precise MT dynamic instability is critical for accurate shaping of the mitotic spindle and for cilium formation, and higher MT dynamicity is associated with tumorigenicity. Thus, our results can supply a mechanistic explanation for Nek involvement in these processes.► Microtubule (MT) dynamic instability was followed using the PlusTipTracker software. ► Nek7 kinase downregulation or absence reduces MT growth and shrinkage speeds. ► Nek7 downregulation reduces the relative time spent in shrinkage and MT dynamicity. ► Ectopic overexpression of Nek7 yields inverse dynamic characteristics. ► Our results provide a mechanistic explanation for Nek7 impact on mitosis and cancer.
Keywords: NIMA kinase; Nek7; Microtubule dynamics; siRNA; Gene targeting;

CTGF induces monocyte chemoattractant protein-1 expression to enhance monocyte migration in human synovial fibroblasts by Shan-Chi Liu; Chin-Jung Hsu; Yi-Chin Fong; Show-Mei Chuang; Chih-Hsin Tang (1114-1124).
Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. CTGF is abundantly expressed in osteoarthritis (OA). Migration and infiltration of mononuclear cells to inflammatory sites are playing important roles during OA pathogenesis. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is the key chemokine that regulates migration and infiltration of monocytes. However, the effect of CTGF on MCP-1 expression and monocyte migration is largely unknown. Our results showed that MCP-1 was highly expressed in OA synovial fibroblasts (OASFs) as compared with normal SFs. Directly applying OASFs with CTGF increased MCP-1 expression in a concentration- and a time-dependent manner. CTGF mediated MCP-1 production was attenuated by αvβ5 integrin neutralized antibody. Pretreatment with focal adhesion kinase (FAK), MEK, AP-1, and NF-κB inhibitors also inhibited the potentiating action of CTGF. CTGF-mediated increase of NF-κB and AP-1 luciferase activity was inhibited by FAK, MEK, and ERK inhibitors or mutants. In vitro chemotaxis assay showed that OA synovial fluid and supernatants from CTGF treated OASFs increased migration of monocyte. In addition, CTGF-mediated migration was inhibited by the FAK and MEK inhibitors. Taken together, our results indicated that CTGF enhances the migration of monocyte cells by increasing MCP-1 expression through the αvβ5 integrin, FAK, MEK, ERK, and NF-κB/AP-1 signal transduction pathway.► MCP-1 was highly expressed in OA synovial fibroblasts as compared with normal SFs. ► CTGF mediates the monocyte migration in OASFs by up-regulation of MCP-1 production. ► CTGF acts through the αvβ5 integrin, FAK, MEK, and ERK to induce MCP-1 activation. ► NF-κB and AP-1 are two key transcription factors of CTGF-induced MCP-1 activation.
Keywords: CTGF; Migration; MCP-1; OA;

Emerging role of tyrosine phosphatase, TCPTP, in the organelles of the early secretory pathway by Madhavi Muppirala; Vijay Gupta; Ghanshyam Swarup (1125-1132).
T-cell protein tyrosine phosphatase, TCPTP, is a ubiquitously expressed non-receptor type tyrosine phosphatase. There are two splice variants of TCPTP, TC48 and TC45, which differ in their sub-cellular localizations and functions. TC45 is a nuclear protein, which has both nuclear and cytoplasmic substrates, and is involved in many signaling events including endocytic recycling of platelet-derived growth factor β-receptor. TC48 is a predominantly endoplasmic reticulum (ER)-localizing protein, which dephosphorylates some of the substrates of TC45 at the ER. However, recently few specific substrates for TC48 have been identified. These include C3G (RapGEF1), syntaxin 17 and BCR-Abl. TC48 moves from the ER to post-ER compartments, the ER–Golgi intermediate compartment (ERGIC) and Golgi, and it is retrieved back to the ER. The retrieval of ER proteins from post-ER compartments is generally believed as a mechanism of targeting these proteins to the ER. However, it is possible that this shuttling of TC48 serves to regulate signaling in the early secretory pathway. For example, TC48 dephosphorylates phosphorylated C3G at the Golgi and inhibits neurite outgrowth. TC48 interacts with and dephosphorylates syntaxin 17, which is an ER and ERGIC-localizing protein involved in vesicle transport. A yeast two-hybrid screen identified several unique interacting partners of TC48 belonging to two groups — proteins involved in vesicle trafficking and proteins involved in cell adhesion. These interacting proteins could be substrates or regulators of TC48 function and localization. Thus, the role of TC48 seems to be more diverse, which is still to be explored.► TC48 cycles between the ER and Golgi, and functions as a phosphatase at the Golgi. ► TC48 interacts with several proteins involved in membrane vesicle trafficking. ► TC48 dephosphorylates syntaxin 17, a SNARE protein involved in vesicle traffic. ► We suggest that TC48 regulates signaling at the ER and Golgi.
Keywords: TCPTP; Tyrosine phosphatase TC48; Early secretory pathway; Vesicle trafficking; Tyrosine phosphorylation; Tyrosine dephosphorylation;

Glycosylation regulates the function and membrane localization of KCC4 by Tzu-Yu Weng; Wen-Tai Chiu; Hsiao-Sheng Liu; Hung-Chi Cheng; Meng-Ru Shen; David B. Mount; Cheng-Yang Chou (1133-1146).
Glycosylation plays a role in regulating many biological activities, including protein folding and cell surface expression of biomolecules. However, the importance of glycosylation for KCC4 function has not previously been demonstrated. Site-directed mutagenesis was performed on the four putative extracellular N-linked glycosylation sites of KCC4 to determine the role of these sites in KCC4 half-life, cell surface expression, and transporter activity, as well as in KCC4-dependent tumor formation. We showed that triple (N312/331/344/Q) and quadruple (N312/331/344/360/Q) mutations of N-linked glycosylation sites disrupt the N-linked glycosylation of KCC4, resulting in the accumulation of KCC4, predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Further investigation indicated that mutations of the central two (N331/344/Q) N-linked glycosylation sites inhibit the membrane trafficking of KCC4. Our data suggest that the glycan moieties at the N331 and N344 sites were Endo H-resistant, complex-form structures, and that the N312 and N360 sites were Endo H-sensitive, high mannose-containing structures. Under hypotonic stress conditions, the ability to adapt to changes in intracellular chloride ion concentrations and RVD (regulatory volume decrease) activities were less efficient in cells containing the deglycosylated form of KCC4 that were not expressed at the cell surface. Deglycosylated forms of KCC4 also demonstrated decreased tumor formation and lung colonization in mouse xenografts. The difference in glycan complexity may account for the differential impact of each branch on the biological effects of KCC4. We propose that glycosylation is essential for the surface expression, stabilization, and bioactivity of KCC4.► N-linked glycosylation is important for KCC4 membrane trafficking and function. ► The central two sites of N-linked glycan are important for KCC4 membrane trafficking. ► The complexity of KCC4 N-linked glycan is crucial for KCC4-regulated bioactivities.
Keywords: KCC4; N-linked glycosylation; Glycan complexity; Membrane expression; Tumor formation;

The Warburg effect is known to be crucial for cancer cells to acquire energy. Nutrient deficiencies are an important phenomenon in solid tumors, but the effect on cancer cell metabolism is not yet clear. In this study, we demonstrate that starvation of HeLa cells by incubation with Hank's buffered salt solution (HBSS) induced cell apoptosis, which was accompanied by the induction of reactive oxygen species (ROS) production and AMP-activated protein kinase (AMPK) phosphorylation. Notably, HBSS starvation increased lactate production, cytoplasmic pyruvate content and decreased oxygen consumption, but failed to change the lactate dehydrogenase (LDH) activity or the glucose uptake. We found that HBSS starvation rapidly induced pyruvate dehydrogenase kinase (PDK) activation and pyruvate dehydrogenase (PDH) phosphorylation, both of which were inhibited by compound C (an AMPK inhibitor), NAC (a ROS scavenger), and the dominant negative mutant of AMPK. Our data further revealed the involvement of ROS production in AMPK activation. Moreover, DCA (a PDK inhibitor), NAC, and compound C all significantly decreased HBSS starvation-induced lactate production accompanied by enhancement of HBSS starvation-induced cell apoptosis. Not only in HeLa cells, HBSS-induced lactate production and PDH phosphorylation were also observed in CL1.5, A431 and human umbilical vein endothelial cells. Taken together, we for the first time demonstrated that a low-nutrient condition drives cancer cells to utilize glycolysis to produce ATP, and this increases the Warburg effect through a novel mechanism involving ROS/AMPK-dependent activation of PDK. Such an event contributes to protecting cells from apoptosis upon nutrient deprivation.► Nutrient deprivation induced ROS production which is upstream molecule of AMPK. ► Nutrient deprivation induced PDK activity. ► Nutrient deprivation induced Warburg effect through ROS/AMPK/PDK/PDH pathway. ► Warburg effect protected cells from apoptosis upon nutrient deprivation.
Keywords: Starvation; ROS; AMPK; PDK; Warburg effect; Apoptosis;

Loss of Neil3, the major DNA glycosylase activity for removal of hydantoins in single stranded DNA, reduces cellular proliferation and sensitizes cells to genotoxic stress by Veslemøy Rolseth; Silje Zandstra Krokeide; David Kunke; Christine Gran Neurauter; Rajikala Suganthan; Yngve Sejersted; Gunn Annette Hildrestrand; Magnar Bjørås; Luisa Luna (1157-1164).
7,8-Dihydro-8-oxoguanine (8-oxoG) is one of the most common oxidative base lesions in normal tissues induced by a variety of endogenous and exogenous agents. Hydantoins are products of 8-oxoG oxidation and as 8-oxoG, they have been shown to be mutagenic lesions. Oxidative DNA damage has been implicated in the etiology of various age-associated pathologies, such as cancer, cardiovascular diseases, arthritis, and several neurodegenerative diseases. The mammalian endonuclease VIII-like 3 (Neil3) is one of the four DNA glycosylases found to recognize and remove hydantoins in the first step of base excision repair (BER) pathway. We have generated mice lacking Neil3 and by using total cell extracts we demonstrate that Neil3 is the main DNA glycosylase that incises hydantoins in single stranded DNA in tissues. Using the neurosphere culture system as a model to study neural stem/progenitor (NSPC) cells we found that lack of Neil3 impaired self renewal but did not affect differentiation capacity. Proliferation was also reduced in mouse embryonic fibroblasts (MEFs) derived from Neil3−/− embryos and these cells were sensitive to both the oxidative toxicant paraquat and interstrand cross-link (ICL)-inducing agent cisplatin. Our data support the involvement of Neil3 in removal of replication blocks in proliferating cells.► Neil3 is the major enzyme in tissues removing hydantoins from single stranded DNA. ► Neurospheres derived from newborn Neil3−/− forebrain display reduced proliferation but normal differentiation capability. ► Neil3−/− derived MEFs exhibit reduced proliferation and are cisplatin sensitive.
Keywords: Oxidative DNA damage; Base excision repair; DNA glycosylase; Neil3; Hydantoins; Cisplatin;

Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2–3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2's effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.► Sex specific membrane responses of rat chondrocytes to E2 are estrogen receptor α and β dependent. ► Female chondrocytes expressed more ERα and ERβ on the membrane and caveolae fraction than male. ► Male chondrocytes showed no membrane-response to E2, but had the same active downstream pathway. ► Palmitoylation of the membrane receptors might contribute to their sex-specific responses to E2.
Keywords: Sex-specific response to 17β-estradiol; Estrogen receptor; Distribution of ERα and ERβ in male and female growth plate chondrocytes;

Luteinizing hormone receptor undergoes downregulation during preovulatory Luteinizing hormone surge through a post-transcriptional mechanism involving an RNA binding protein designated as LRBP. The present study examined the mechanism by which LRBP induces the degradation of Luteinizing hormone receptor mRNA, specifically the role of decapping of Luteinizing hormone receptor mRNA and the translocation of LRBP-bound Luteinizing hormone receptor mRNA to degradative machinery. Immunoprecipitation of the complex with the 5′cap structure antibody followed by real time PCR analysis showed progressive loss of capped Luteinizing hormone receptor mRNA during downregulation suggesting that Luteinizing hormone receptor mRNA undergoes decapping prior to degradation. RNA immunoprecipitation analysis confirmed dissociation of eukaryotic initiation factor 4E from the cap structure, a step required for decapping. Furthermore, RNA immunoprecipitation analysis using antibody against the p body marker protein, DCP1A showed that Luteinizing hormone receptor mRNA was associated with the p bodies, the cytoplasmic foci that contain RNA degradative enzymes and decapping complex. Immunohistochemical studies using antibodies against LRBP and DCP1A followed by confocal analysis showed colocalization of LRBP with DCP1A during downregulation. This was further confirmed by co-immunoprecipitation of LRBP with DCP1A. The association of LRBP and Luteinizing hormone receptor mRNA in the p bodies during downregulation was further confirmed by examining the association of a second p body component, rck/p54, using immunoprecipitation and RNA immunoprecipitation respectively. These data suggest that the association of LRBP with Luteinizing hormone receptor mRNA results in the translocation of the messenger ribonucleoprotein complex to the p bodies leading to decapping and degradation.► Accumulation of LHR mRNA in p bodies during downregulation ► Role of LRBP in LHR mRNA accumulation in p bodies ► Decapping of LHR mRNA in p bodies leads to mRNA degradation.
Keywords: Luteinizing hormone receptor; mRNA decay; mRNA binding protein; p bodies; Decapping;

RNA helicase Ddx5 and the noncoding RNA SRA act as coactivators in the Notch signaling pathway by Claudia Jung; Gerhard Mittler; Franz Oswald; Tilman Borggrefe (1180-1189).
Notch signaling plays a pivotal role in embryonic and postnatal development.Upon binding of a Notch ligand, proteolytic cleavage events liberate the Notch-intracellular domain (NICD) that migrates into the nucleus. In order to activate target genes, NICD associates with the transcription factor RBP-J (also known as CSL), Mastermind and the acetyltransferase p300.Here, we identify the DEAD-box RNA helicase Ddx5 as a novel component of the RBP-J/NICD complex utilizing a biotinylation-tagging approach followed by mass-spectrometry. Biochemical assays confirm a direct interaction of Ddx5 with RBP-J. We show that Ddx5 localizes at RBP-J binding sites within the Notch target genes preTCRα, Hes1 and CD25 in a Notch-dependent manner. Moreover, knockdown of Ddx5 also downregulates a subset of Notch target genes in a murine pre T-cell model. Interestingly, also knockdown/overexpression of the RNA coactivator SRA, a cofactor of Ddx5, downregulates Hes1 and preTCRα. Using Chromatin-IP, we show that this effect is accompanied with a loss of p300 occupancy at Notch target genes and decreased histone acetylation. Together, our data demonstrate that Ddx5 and SRA function as coactivators of Notch signaling.► RNA helicase Ddx5 physically interacts with the RBP-J/NICD complex. ► RNA helicase Ddx5 is found at Notch target genes in vivo. ► RNA helicase Ddx5 acts as a co-activator in Notch signaling. ► The long non-coding RNA SRA associates with Ddx5 and NICD in T-cells and is a positive regulator of Notch signaling. ► Occupancy of p300 and histone acetylation is lost upon SRA knockdown and overexpression at Notch target genes.
Keywords: Notch signaling; Transcriptional regulation; Coactivator; Lymphocyte development; Non-coding RNA;

Control of mitogenic and motogenic pathways by miR-198, diminishing hepatoma cell growth and migration by Natalia Elfimova; Elisabeth Sievers; Hannah Eischeid; Monika Kwiecinski; Andrea Noetel; Heike Hunt; Diana Becker; Peter Frommolt; Maria Quasdorff; Hans Michael Steffen; Peter Nürnberg; Reinhard Büttner; Andreas Teufel; Hans-Peter Dienes; Uta Drebber; Margarete Odenthal (1190-1198).
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths, worldwide. MicroRNAs, inhibiting gene expression by targeting various transcripts, are involved in genomic dysregulation during hepatocellular tumorigenesis. In previous studies, microRNA-198 (miR-198) was shown to be significantly downregulated in HCV-positive hepatocellular carcinoma (HCC). Herein, the function of miR-198 in hepatocellular carcinoma cell growth and gene expression was studied.In hepatoma cell-types with low levels of liver-specific transcription factor HNF1α indicating a low differentiation grade, miR-198 expression was most downregulated. However, miR-198 treatment did not restore the expression of the liver-specific transcription factors HNF1α or HNF4α. Importantly, overexpression of miR-198 in Pop10 hepatoma cells markedly reduced cell growth. In agreement, comprehensive gene expression profiling by microarray hybridisation and real-time quantification revealed that central signal transducers of proliferation pathways were downregulated by miR-198. In contrast, genes mediating cellular adherence were highly upregulated by miR-198. Thus, the low expression of E-cadherin and claudin-1, involved in cell adhesion and cell-cell contacts, was abolished in hepatoma cells after miR-198 overexpression. This definite induction of both proteins by miR-198 was shown to be accompanied by a significantly impaired migration activity of hepatoma Pop10 cells.In conclusion, miR-198 acts as a tumor suppressor by repression of mitogenic and motogenic pathways diminishing cell growth and migration.Display Omitted► In hepatoma cell-types with low differentiation grade, miR-198 expression was prominently reduced. ► Central signal transducers of proliferation pathways were controlled by miR-198. ► miR-198 upregulates synthesis of E-cadherin and claudin-1, involved in cell adhesion and cell-cell contacts. ► miR-198 acts as a tumor suppressor by repression of mitogenic and motogenic activities.
Keywords: miRNA; Hepatocellular carcinoma; Expression profiles; Cell proliferation and apoptosis; Cell migration; Claudin-1;

The highly conserved, N-terminal (RXXX)8 motif of mouse Shadoo mediates nuclear accumulation by E. Tóth; P.I. Kulcsár; E. Fodor; F. Ayaydin; L. Kalmár; A.É. Borsy; L. László; E. Welker (1199-1211).
The prion protein (PrP)—known for its central role in transmissible spongiform encephalopathies—has been reported to possess two nuclear localization signals and localize in the nuclei of certain cells in various forms. Although these data are superficially contradictory, it is apparent that nuclear forms of the prion protein can be found in cells in either the healthy or the diseased state. Here we report that Shadoo (Sho)—a member of the prion protein superfamily—is also found in the nucleus of several neural and non-neural cell lines as visualized by using an YFP-Sho construct. This nuclear localization is mediated by the (25-61) fragment of mouse Sho encompassing an (RXXX)8 motif. Bioinformatic analysis shows that the (RXXX)n motif (n = 7-8) is a highly conserved and characteristic part of mammalian Shadoo proteins. Experiments to assess if Sho enters the nucleus by facilitated transport gave no decisive results: the inhibition of active processes that require energy in the cell, abolishes nuclear but not nucleolar accumulation. However, the (RXXX)8 motif is not able to mediate the nuclear transport of large fusion constructs exceeding the size limit of the nuclear pore for passive entry. Tracing the journey of various forms of Sho from translation to the nucleus and discerning the potential nuclear function of PrP and Sho requires further studies.► Tagged forms of Shadoo show dual, plasma membrane and nuclear localization. ► The (RXXX)8 motif of Shadoo is capable of directing the protein to the nucleus. ► The arginines of the (RXXX)8 motif have a critical role in nuclear localization. ► Shadoo has a not yet identified binding partner in the nucleolus.
Keywords: Shadoo; Prion protein; RGG-box; (RXXX)n motif; Nucleic acid binding; Nuclear localization signal;

The pp32 (ANP32A) gene acts as a tumor suppressor while its closely related homologue pp32r1 (ANP32C) is oncogenic and is overexpressed in breast, prostate and pancreatic tumors. The transduction of p53wt cell lines (ACHN and HeLa) with pp32r1 or pp32r1Y140H lentivirus increased the proliferation of p53wt cell lines compared to the untransduced control cells while transduction of the p53R248W MiaPaCa2 cell line had no effect. Cell cycle analysis of transduced ACHN cells by PI staining and BrdU incorporation illustrated a pronounced shift toward the S-phase of the cell cycle in cells overexpressing the pp32r1 and pp32r1Y140H proteins. Confocal microscopy and western blotting demonstrated that pp32r1 and the pp32r1Y140H mutant protein reside predominantly in the cytoplasm in constrast to pp32 which is a nuclear/cytoplasmic shuttling protein. To determine the effects of pp32r1 or pp32r1Y140H overexpression at the proteomic level we performed a comprehensive proteome analysis on ACHN, ACHN-pp32r1 and ACHN-pp32r1Y140H cell lysates using the isotope-coded protein label (ICPL) method. Among those proteins with > 40% regulation were Macrophage Capping protein (CAPG) and Chromodomain Helicase DNA binding protein 4 (CHD4) proteins which were significantly upregulated by pp32r1 and pp32r1Y140H overexpression. This increase in CHD4 also appears to influence a number of cell cycle regulator genes including; p53, p21 and cyclinD1 as judged by western blotting. Silencing of CHD4 in ACHN-pp32r1Y140H cells using specific shRNA reverted the cell cycle dysregulation caused by pp32r1Y140H expression to that of the untransduced ACHN cell line, suggesting that CHD4 is the prominant effector of the pp32r1/pp32r1Y140H phenotype.► Differential sub-cellular (cytoplasmic) localization of pp32r1 compared to pp32. ► Up regulation of G1/S phase (50%) in pp32r1Y140H over expressing cells. ► Whole proteomic analysis (ICPL) identifies CHD4 as significantly dysregulated. ► CHD4 knockdown studies confirm the importance of CHD4, p21 and cyclinD1.
Keywords: pp32r1; Cell cycle; CHD4; p21; Cancer; ICPL;

Suppression of Androgen Receptor Enhances the Self-renewal of Mesenchymal Stem Cells Through Elevated Expression of EGFR by Chiung-Kuei Huang; Meng-Yin Tsai; Jie Luo; Hong-Yo Kang; Soo Ok Lee; Chawnshang Chang (1222-1234).
Bone marrow derived mesenchymal stem cells (BM-MSCs) have been widely applied in several clinical trials of diseases, such as myocardial infarction, liver cirrhosis, neurodegenerative disease, and osteogenesis imperfecta. Although most studies demonstrated that transplantation of BM-MSCs did exert a temporary relief and short-term therapeutic effects, eventually all symptoms recur, therefore it is essential to improve the therapeutic efficacy of transplantation by either elevating the self-renewal of BM-MSCs or enhancing their survival rate. Herein we demonstrated that the BM-MSCs and adipocyte derived mesenchymal stem cells (ADSCs) isolated from the androgen receptor (AR) knockout mice have higher self-renewal ability than those obtained from the wild-type mice. Knockdown of AR in MSC cell lines exhibited similar results. Mechanistic dissection studies showed that the depletion of AR resulted in activation of Erk and Akt signaling pathways through epidermal growth factor receptor (EGFR) activation or pathway to mediate higher self-renewal of BM-MSCs. Targeting AR signals using ASC-J9® (an AR degradation enhancer), hydroxyflutamide (antagonist of AR), and AR-siRNA all led to enhanced self-renewal of MSCs, suggesting the future possibility of using these anti-AR agents in therapeutic approaches.► Knockout of AR enhances self-renewal potential in MSCs through EGFR/ERK/AKT. ► AR inhibits self-renewal potentials of MSCs. ► Targeting AR in MSCs might lead to better transplantation therapeutic efficacy.
Keywords: Epidermal growth factor receptor; Mesenchymal stem cells; Self-renewal; Androgen receptor; Androgen receptor knockout;

PTPA activates protein phosphatase-2A through reducing its phosphorylation at tyrosine-307 with upregulation of protein tyrosine phosphatase 1B by Yu Luo; Yun-Juan Nie; Hai-Rong Shi; Zhong-Fei Ni; Qun Wang; Jian-Zhi Wang; Gong-Ping Liu (1235-1243).
Protein phosphatase-2A (PP2A), an important phosphatase in dephosphorylating tau and preserving synapse, is significantly suppressed in Alzheimer's disease (AD), but the mechanism is not well understood. Here, we studied whether phosphotyrosyl phosphatase activator (PTPA) could activate PP2A by reducing its inhibitory phosphorylation at tyrosine 307 (P-PP2AC). We found that overexpression of PTPA activated PP2A by decreasing the level of P-PP2AC with reduced phosphorylation of tau, while knockdown of PTPA inhibited PP2A by increasing the level of P-PP2AC with enhanced tau phosphorylation. We also observed that expression of PTPA could upregulate the protein and mRNA levels of protein tyrosine phosphatase 1B (PTP1B) and simultaneous downregulation of PTP1B eliminated PTPA-induced PP2A activation. Importantly, we also found that the protein level of PTPA is downregulated in the brains of AD patients, and the AD transgenic mouse models with expression of mutant human amyloid precursor protein (hAPP) or the longest human tau (htau), respectively. Our data indicate that PTPA may activate PP2A through activating PTP1B and thus reducing the level of P-PP2AC, therefore upregulation of PTPA may represent a potential strategy in rescuing PP2A and arresting tau pathology in AD.► PTPA activated PP2A by decreasing P-PP2AC level with reduced tau phosphorylation. ► PTPA modulates the phosphorylation of PP2AC by PTP1B. ► PTPA level decreases in the brains of AD patients and transgenic mice models.
Keywords: Protein phosphatase-2A phosphatase activator; Protein phosphatase-2A; Protein tyrosine phosphatase 1B; Phosphorylation; Tau; Alzheimer disease;

A role for annexin A7 (A7) is postulated in the obligatory fusion between lamellar bodies and the plasma membrane during surfactant secretion in alveolar type II cells. This study investigated if surfactant secretagogues increase cell surface A7, which could support A7 insertion into plasma membrane as annexin proteins reportedly lack membrane penetration ability. In vivo trafficking of A7 to cell surface was determined by immuno-staining after non-permeabilizing fixation of alveolar type II cells. Stimulation with various secretagogues increased protein kinase-dependent staining for A7 and ABCA3 in comparison to control cells. Biotin-labeling of surface proteins showed ~ 4% of total A7 in control cells, which increased ~ 3–4 folds in stimulated type II cells. Increased cell surface A7 was also observed by protein cross-linking studies showing ~ 70 kDa A7-adduct in the membranes but not in the cytosol fraction of PMA- or A23187-stimulated cells. In vitro phosphorylation increased the Ca2 +-dependent binding of recombinant A7 to lung plasma membranes; and subsequent cross-linking showed increased levels of ~ 70 kDa A7-adduct. PMA-stimulation of type II cells increased A7 trafficking to lipid rafts suggesting that the latter are involved in A7 trafficking to the cell surface. However, in vitro membrane insertion of recombinant A7 and its tryptophan mutants as determined by fluorescence quenching with doxylPC suggested only shallow membrane insertion by A7. Together, our studies support in vivo association between surfactant secretion and cell surface A7 occurring by insertion into plasma membrane and by fusion of A7 containing lamellar bodies.► Suggested planar configuration can limit membrane insertion by annexin proteins. ► Stimulation of surfactant secretion increases annexin A7 on type II cell surface. ► Protein kinase activation increases cell surface annexin A7 possibly via lipid rafts. ► Recombinant annexin A7, however, failed to show insertion into artificial membranes. ► Phosphorylation could promote in vivo membrane penetration by annexin A7.
Keywords: Lung surfactant secretion; Protein cross-linking; Fluorescence quenching; Biotin-labeling of proteins; Immuno-fluorescence;

New insights into the function and regulation of mitochondrial fission by Hidenori Otera; Naotada Ishihara; Katsuyoshi Mihara (1256-1268).
Mitochondrial morphology changes dynamically by coordinated fusion and fission and cytoskeleton-based transport. Cycles of outer and inner membrane fusion and fission are required for the exchange of damaged mitochondrial genome DNA, proteins, and lipids with those of healthy mitochondria to maintain robust mitochondrial structure and function. These dynamics are crucial for cellular life and death, because they are essential for cellular development and homeostasis, as well as apoptosis. Disruption of these functions leads to cellular dysfunction, resulting in neurologic disorders and metabolic diseases. The cytoplasmic dynamin-related GTPase Drp1 plays a key role in mitochondrial fission, while Mfn1, Mfn2 and Opa1 are involved in fusion reaction. Here, we review current knowledge regarding the regulation and physiologic relevance of Drp1-dependent mitochondrial fission: the initial recruitment and assembly of Drp1 on the mitochondrial fission foci, regulation of Drp1 activity by post-translational modifications, and the role of mitochondrial fission in cell pathophysiology.► Mitochondrial morphology is controlled by fusion and fission. ► Dynamin-related protein 1 (Drp1) mediates mitochondrial fission. ► Mitochondrial fission plays an important role in apoptosis and mitochondrial quality control. ► Drp1 is regulated by various post-translational modifications. ► Mitochondrial fission deficiencies cause various human diseases.
Keywords: Mitochondrial fission; Drp1; Apoptosis; Mitophagy; Post-translational modification; Neurodegenerative disease;

The CATS (FAM64A) protein is a substrate of the Kinase Interacting Stathmin (KIS) by Leticia Fröhlich Archangelo; Philipp A. Greif; Alexandre Maucuer; Valérie Manceau; Naresh Koneru; Carolina L. Bigarella; Fernanda Niemann; Marcos Tadeu dos Santos; Jörg Kobarg; Stefan K. Bohlander; Sara Teresinha Olalla Saad (1269-1279).
The CATS protein (also known as FAM64A and RCS1) was first identified as a novel CALM (PICALM) interactor that influences the subcellular localization of the leukemogenic fusion protein CALM/AF10. CATS is highly expressed in cancer cell lines in a cell cycle dependent manner and is induced by mitogens. CATS is considered a marker for proliferation, known to control the metaphase-to-anaphase transition during the cell division. Using CATS as a bait in a yeast two-hybrid screen we identified the Kinase Interacting Stathmin (KIS or UHMK1) protein as a CATS interacting partner. The interaction between CATS and KIS was confirmed by GST pull-down, co-immunopreciptation and co-localization experiments. Using kinase assay we showed that CATS is a substrate of KIS and mapped the phosphorylation site to CATS serine 131 (S131). Protein expression analysis revealed that KIS levels changed in a cell cycle-dependent manner and in the opposite direction to CATS levels. In a reporter gene assay KIS was able to enhance the transcriptional repressor activity of CATS, independent of CATS phophorylation at S131. Moreover, we showed that CATS and KIS antagonize the transactivation capacity of CALM/AF10.In summary, our results show that CATS interacts with and is a substrate for KIS, suggesting that KIS regulates CATS function.► CATS is a phospho-protein. ► CATS-KIS represents a novel kinase–substrate pair. ► KIS is a critical modulator of CATS function. ► KIS enhances the transcriptional repressor activity of CATS. ► CATS and KIS antagonize the transactivation capacity of CALM/AF10.
Keywords: CATS; FAM64A; KIS; UHMK1; Phosphorylation; CALM/AF10-leukemia;

Corrigendum to “Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin” [Biochim. Biophys. Acta 1823 (2012) 1686–1696] by Brigitte Gerard; Matthew A. Sanders; Daniel W. Visscher; Larry Tait; Malathy P.V. Shekhar (1280-1281).