BBA - Molecular Cell Research (v.1853, #10PA)

Modulation of focal adhesion constituents and their down-stream events by EGF: On the cross-talk of integrins and growth factor receptors by Philipp Eberwein; Dougal Laird; Simon Schulz; Thomas Reinhard; Thorsten Steinberg; Pascal Tomakidi (2183-2198).
Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the effects of GFRs on focal adhesions (FAs). Therefore, we tested the hypothesis whether EGF can modulate constituents of FAs and subsequent down-stream events. To this end, EGF-treated keratinocytes were subjected to combined fluorescence imaging and western blotting, to quantify expression and/or activation of molecules, involved in integrin GFR cross-talk, and receptor proximal and distal signaling events. Generally, EGF response revealed an amplified redistribution or activation of molecules under study, which will be explained in detail from the plasma membrane to the cell interior. In addition to significant activation of EGF receptor (EGFR) at tyrosine Tyr845, a remarkable redistribution was detectable for the focal adhesion constituents, integrin ß1 and ß3, and zyxin. Increased activation also applied to focal adhesion kinase (FAK) by phosphorylation at Tyr397, Tyr576, and Src at Tyr418, while total FAK remained unchanged. Risen activity was seen as well for the analyzed distal down-stream events, p190RhoGAP and MAP kinases p42/44. Intriguingly, Src-specific inhibitor Herbimycin A abrogated the entire EGF response except FAK Tyr397 phosphorylation, independent of EGF presence. Mechanistically, our results show that EGF modulates adhesion in a dual fashion, by firstly redistributing focal adhesion constituents to adhesion sites, but also by amplifying levels of activated RhoA antagonist p190RhoGAP, important for cell motility. Further, the findings suggest that the observed EGF response underlies an EGFR integrin cross-talk under recruitment of receptor proximal FAK and Src, and MAP kinase and p190RhoGAP as receptor distal events.
Keywords: Integrins; Zyxin; Focal adhesions; Growth factor receptors; Epidermal growth factor; Cell motility; Mechanotransduction;

The use of high linear energy transfer radiations in the form of carbon ions in heavy ion beam lines or alpha particles in new radionuclide treatments has increased substantially over the past decade and will continue to do so due to the favourable dose distributions they can offer versus conventional therapies. Previously it has been shown that exposure to heavy ions induces pan-nuclear phosphorylation of several DNA repair proteins such as H2AX and ATM in vitro. Here we describe similar effects of alpha particles on ex vivo irradiated primary human peripheral blood lymphocytes. Following alpha particle irradiation pan-nuclear phosphorylation of H2AX and ATM, but not DNA-PK and 53BP1, was observed throughout the nucleus. Inhibition of ATM, but not DNA-PK, resulted in the loss of pan-nuclear phosphorylation of H2AX in alpha particle irradiated lymphocytes. Pan-nuclear gamma-H2AX signal was rapidly lost over 24 h at a much greater rate than foci loss. Surprisingly, pan-nuclear gamma-H2AX intensity was not dependent on the number of alpha particle induced double strand breaks, rather the number of alpha particles which had traversed the cell nucleus. This distinct fluence dependent damage signature of particle radiation is important in both the fields of radioprotection and clinical oncology in determining radionuclide biological dosimetry and may be indicative of patient response to new radionuclide cancer therapies.
Keywords: Alpha particles; Complex damage; DNA double strand break; Pan-nuclear intensity;

Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells by Lisa Schwind; Nadine Wilhelm; Sabine Kartarius; Mathias Montenarh; Erwin Gorjup; Claudia Götz (2207-2216).
CK2 is a serine/threonine protein kinase, which is so important for many aspects of cellular regulation that life without CK2 is impossible. Here, we analysed CK2 during adipogenic differentiation of human mesenchymal stem cells (hMSCs). With progress of the differentiation CK2 protein level and the kinase activity decreased. Whereas CK2α remained in the nucleus during differentiation, the localization of CK2β showed a dynamic shuttling in the course of differentiation. Over the last years a large number of inhibitors of CK2 kinase activity were generated with the idea to use them in cancer therapy. Our results show that two highly specific inhibitors of CK2, CX-4945 and quinalizarin, reduced its kinase activity in proliferating hMSC with a similar efficiency. CK2 inhibition by quinalizarin resulted in nearly complete inhibition of differentiation whereas, in the presence of CX-4945, differentiation proceeded similar to the controls. In this case, differentiation was accompanied by the loss of CX-4945 inhibitory function. By analysing the subcellular localization of PPARγ2, we found a shift from a nuclear localization at the beginning of differentiation to a more cytoplasmic localization in the presence of quinalizarin. Our data further show for the first time that a certain level of CK2 kinase activity is required for adipogenic stem cell differentiation and that inhibition of CK2 resulted in an altered localization of PPARγ2, an early regulator of differentiation.Display Omitted
Keywords: Stem cells; Protein kinase CK2; Adipogenic differentiation; Kinase inhibitors;

Stress triggers mitochondrial biogenesis to preserve steroidogenesis in Leydig cells by Igor A. Gak; Sava M. Radovic; Aleksandra R. Dukic; Marija M. Janjic; Natasa J. Stojkov-Mimic; Tatjana S. Kostic; Silvana A. Andric (2217-2227).
Adaptability to stress is a fundamental prerequisite for survival. Mitochondria are a key component of the stress response in all cells. For steroid-hormones-producing cells, including also Leydig cells of testes, the mitochondria are a key control point for the steroid biosynthesis and regulation. However, the mitochondrial biogenesis in steroidogenic cells has never been explored. Here we show that increased mitochondrial biogenesis is the adaptive response of testosterone-producing Leydig cells from stressed rats. All markers of mitochondrial biogenesis together with transcription factors and related kinases are up-regulated in Leydig cells from rats exposed to repeated psychophysical stress. This is followed with increased mitochondrial mass. The expression of PGC1, master regulator of mitochondrial biogenesis and integrator of environmental signals, is stimulated by cAMP-PRKA, cGMP, and β-adrenergic receptors. Accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. Here we propose that all events induced by acute stress, the most common stress in human society, provoke adaptive response of testosterone-producing Leydig cells and activate PGC1, a protein required to make new mitochondria but also protector against the oxidative damage. Given the importance of mitochondria for steroid hormones production and stress response, as well as the role of steroid hormones in stress response and metabolic syndrome, we anticipate our result to be a starting point for more investigations since stress is a constant factor in life and has become one of the most significant health problems in modern societies.Display Omitted
Keywords: Mitochondrial biogenesis; Leydig cells; Stress; Testosterone; PGC1;

Prion protein (PrP) mislocalized in the cytosol has been presumed to be the toxic entity responsible for the neurodegenerative process in transmissible spongiform encephalopathies (TSE), also called prion diseases. The mechanism underlying the neurotoxicity of cytosolic PrP (cytoPrP) remains, however, unresolved. In this study we analyze toxic effects of the cell-penetrating PrP fragment, PrP1–30 — encompassing residues responsible for binding and aggregation of tubulin. We have found that intracellularly localized PrP1-30 disassembles microtubular cytoskeleton of primary neurons, which leads to the loss of neurites and, eventually, necrotic cell death. Accordingly, stabilization of microtubules by taxol reduced deleterious effects of cytosolic PrP1–30. Furthermore, we have demonstrated that decreased phosphorylation level of microtubule-associated proteins (MAPs), which also increases stability of microtubular cytoskeleton, protects neurons from the toxic effects of PrP1–30. CHIR98014 and LiCl — inhibitors of glycogen synthase kinase 3 (GSK-3), a major kinase responsible for phosphorylation of MAPs, inhibited PrP1-30-induced disruption of microtubular cytoskeleton and increased viability of peptide-treated neurons. We have also shown that the N-terminal fragment of cytoPrP may cause the loss of dendritic spines. PrP1–30-induced changes at the level of spines have also been prevented by stabilization of microtubules by taxol as well as LiCl. These observations indicate that the neurotoxicity of cytoPrP is tightly linked to the disruption of microtubular cytoskeleton. Importantly, this study implies that lithium, the commonly used mood stabilizer, may be a promising therapeutic agent in TSE, particularly in case of the disease forms associated with accumulation of cytoPrP.Display Omitted
Keywords: Prion protein; Tubulin; Microtubule-associated proteins; Glycogen synthase kinase 3; Prion disease; Lithium;

Lipocalin 2 promotes the migration and invasion of esophageal squamous cell carcinoma cells through a novel positive feedback loop by Ze-Peng Du; Bing-Li Wu; Yang-Min Xie; Ying-Li Zhang; Lian-Di Liao; Fei Zhou; Jian-Jun Xie; Fa-Min Zeng; Xiu-E Xu; Wang-Kai Fang; En-Min Li; Li-Yan Xu (2240-2250).
Lipocalin 2 (LCN2) is a poor prognostic factor in esophageal squamous cell carcinoma (ESCC), however its functional roles and molecular mechanisms of action remain to be clarified. Here, we described the functions and signaling pathways for LCN2 in ESCC. Overexpression of LCN2 in ESCC cells accelerated cell migration and invasion in vitro, and promoted lung metastasis in vivo. Blocking LCN2 expression inhibited its pro-oncogenic effect. Either overexpression of LCN2 or treatment with recombinant human LCN2 protein enhanced the activation of MEK/ERK pathway, which in turn increases endogenous LCN2 to increase MMP-9 activity. The decreased p-cofilin and increased p-ERM induced by pERK1/2 cause the cytoskeleton F-actin rearrangement and alter the behavior of ESCC cells mediated by LCN2. As a consequence, activation of MMP-9 and the rearrangement of F-actin throw light on the mechanisms for LCN2 in ESCC. These results imply that LCN2 promotes the migration and invasion of ESCC cells through a novel positive feedback loop.
Keywords: Esophageal squamous cell carcinoma; Lipocalin 2; Positive feedback; Migration and invasion of cancer cells;

Biphasic effect of PTK7 on KDR activity in endothelial cells and angiogenesis by Won-Sik Shin; Hye-Won Na; Seung-Taek Lee (2251-2260).
Protein tyrosine kinase 7 (PTK7) is a member of the defective receptor protein tyrosine kinase family which lacks catalytic activity. Expression of PTK7 is increased in various cancers but its role in carcinogenesis is not well understood. We previously showed that disruption of PTK7 function suppresses VEGF-induced angiogenic phenotypes in HUVECs and mice. Here, we investigated molecular mechanisms for modulating VEGF-induced physiological effects by PTK7. Treatment with a high concentration of extracellular domain of PTK7 (soluble PTK7; sPTK7) or knockdown of PTK7 inhibited VEGF-induced phosphorylation of kinase insert domain receptor (KDR) but did not inhibit phosphorylation of fms-related tyrosine kinase 1 (FLT-1) in HUVECs. PTK7, more specifically sPTK7, interacted with KDR but not with FLT-1 in HUVECs and HEK293 cells. In vitro binding assay showed that sPTK7 formed oligomers with the extracellular domain of KDR (sKDR) up to an approximately 1:3 molar ratio, and vice versa. sPTK7 at lower molar ratios than sKDR enhanced the binding of VEGF to sKDR. At the same or higher molar ratios, it reduced the binding of VEGF to sKDR. Increasing concentrations of sPTK7 or increasing levels of PTK7 expression first increased and then decreased VEGF-induced KDR phosphorylation, migration, and capillary-like tube formation of HUVECs, as well as in vivo angiogenesis. Taken together, our data demonstrates that PTK7 regulates the activity of KDR biphasically by inducing oligomerization of KDR molecules at lower concentrations and by surrounding KDR molecules at higher concentrations.
Keywords: Defective receptor protein tyrosine kinase; PTK7; KDR; VEGF; Angiogenesis;

Targeting of multiple oncogenic signaling pathways by Hsp90 inhibitor alone or in combination with berberine for treatment of colorectal cancer by Yen-Hao Su; Wan-Chun Tang; Ya-Wen Cheng; Peik Sia; Chi-Chen Huang; Yi-Chao Lee; Hsin-Yi Jiang; Ming-Heng Wu; I-Lu Lai; Jun-Wei Lee; Kuen-Haur Lee (2261-2272).
There is a wide range of drugs and combinations under investigation and/or approved over the last decade to treat colorectal cancer (CRC), but the 5-year survival rate remains poor at stages II–IV. Therefore, new, more-efficient drugs still need to be developed that will hopefully be included in first-line therapy or overcome resistance when it appears, as part of second- or third-line treatments in the near future. In this study, we revealed that heat shock protein 90 (Hsp90) inhibitors have high therapeutic potential in CRC according to combinative analysis of NCBI's Gene Expression Omnibus (GEO) repository and chemical genomic database of Connectivity Map (CMap). We found that second generation Hsp90 inhibitor, NVP-AUY922, significantly downregulated the activities of a broad spectrum of kinases involved in regulating cell growth arrest and death of NVP-AUY922-sensitive CRC cells. To overcome NVP-AUY922-induced upregulation of survivin expression which causes drug insensitivity, we found that combining berberine (BBR), a herbal medicine with potency in inhibiting survivin expression, with NVP-AUY922 resulted in synergistic antiproliferative effects for NVP-AUY922-sensitive and -insensitive CRC cells. Furthermore, we demonstrated that treatment of NVP-AUY922-insensitive CRC cells with the combination of NVP-AUY922 and BBR caused cell growth arrest through inhibiting CDK4 expression and induction of microRNA-296-5p (miR-296-5p)-mediated suppression of Pin1–β-catenin–cyclin D1 signaling pathway. Finally, we found that the expression level of Hsp90 in tumor tissues of CRC was positively correlated with CDK4 and Pin1 expression levels. Taken together, these results indicate that combination of NVP-AUY922 and BBR therapy can inhibit multiple oncogenic signaling pathways of CRC.
Keywords: Colorectal cancer; Connectivity Map; Heat shock protein 90 inhibitor; Berberine; miR-296-5p;

Cell–cell fusion induced by the Ig3 domain of receptor FGFRL1 in CHO cells by Lei Zhuang; Amit V. Pandey; Peter M. Villiger; Beat Trueb (2273-2285).
FGFRL1 is a single-pass transmembrane protein with three extracellular Ig domains. When overexpressed in CHO cells or related cell types, it induces cell–cell fusion and formation of large, multinucleated syncytia. For this fusion-promoting activity, only the membrane-proximal Ig domain (Ig3) and the transmembrane domain are required. It does not matter whether the transmembrane domain is derived from FGFRL1 or from another receptor, but the distance of the Ig3 domain to the membrane is crucial. Fusion can be inhibited with soluble recombinant proteins comprising the Ig1–Ig2–Ig3 or the Ig2–Ig3 domains as well as with monoclonal antibodies directed against Ig3. Mutational analysis reveals a hydrophobic site in Ig3 that is required for fusion. If a single amino acid from this site is mutated, fusion is abolished. The site is located on a β-sheet, which is part of a larger β-barrel, as predicted by computer modeling of the 3D structure of FGFRL1. It is possible that this site interacts with a target protein of neighboring cells to trigger cell–cell fusion.Display Omitted
Keywords: Fibroblast growth factor (FGF); Fibroblast growth factor receptor (FGFR); Fusion protein; Fusogen; Syncytia; Molecular modeling;

JMJD5 interacts with p53 and negatively regulates p53 function in control of cell cycle and proliferation by Xiaobin Huang; Shuilian Zhang; Hongyan Qi; Zhengyang Wang; Hong-Wu Chen; Jimin Shao; Jing Shen (2286-2295).
JMJD5 is a Jumonji C domain-containing demethylase/hydroxylase shown to be essential in embryological development, osteoclastic maturation, circadian rhythm regulation and cancer metabolism. However, its role and underlying mechanisms in oncogenesis remain unclear. Here, we demonstrate that JMJD5 forms complex with the tumor suppressor p53 by interacting with p53 DNA-binding domain (DBD), and negatively regulates its activity. Downregulation of JMJD5 resulted in increased expression of multiple p53 downstream genes, such as the cell cycle inhibitor CDKN1A and DNA repair effector P53R2, only in p53-proficient lung cancer cells. Upon DNA damage, the JMJD5–p53 association decreased, and thereby, promoted p53 recruitment to the target genes and stimulated its transcriptional activity. Furthermore, JMJD5 facilitated the cell cycle progression in a p53-dependent manner under both normal and DNA damage conditions. Depletion of JMJD5 inhibited cell proliferation and enhanced adriamycin-induced cell growth suppression in the presence of p53. Collectively, our results reveal that JMJD5 is a novel binding partner of p53 and it functions as a positive modulator of cell cycle and cell proliferation mainly through the repression of p53 pathway. Our study extends the mechanistic understanding of JMJD5 function in cancer development and implicates JMJD5 as a potential therapeutic target for cancer.
Keywords: JMJD5; p53 tumor suppressor; Cell cycle; Gene transcription; Cancer development;

TRIM29 regulates the p63-mediated pathway in cervical cancer cells by Yasushi Masuda; Hidehisa Takahashi; Shigetsugu Hatakeyama (2296-2305).
Cell invasion and adhesion play an important role in cancer metastasis and are orchestrated by a complicated network of transcription factors including p63. Here, we show that a member of the tripartite motif protein family, TRIM29, is required for regulation of the p63-mediated pathway in cervical cancer cells. TRIM29 knockdown alters the adhesion and invasion activities of cervical cancer cells. TRIM29 knockdown and overexpression cause a significant decrease and increase of TAp63α expression, respectively. TRIM29 knockdown alters the expression pattern of integrins and increases ZEB1 expression. TRIM29 is required for suppression of an increase in the adhesion activity of cells by TAp63α. These findings suggest that TRIM29 regulates the p63-mediated pathway and the behavior of cervical cancer cells.
Keywords: TRIM29; Cell adhesion; Cervical cancer; p63; Integrin;

Oncostatin M (OSM), a cytokine in the interleukin-6 (IL-6) family, has been proposed to play a protective role in the central nervous system, such as attenuation of excitotoxicity induced by N-methyl-D-aspartate (NMDA) and glutamate. However, the potential neuroprotective effects of OSM against mitochondrial dysfunction have never been reported. In the present study, we tested the hypothesis that OSM may confer neuronal resistance against 3-nitropropionic acid (3-NP), a plant toxin that irreversibly inhibits the complex II of the mitochondrial electron transport chain, and characterized the underlying molecular mechanisms. We found that OSM preconditioning dose- and time-dependently protected cortical neurons against 3-NP toxicity. OSM stimulated expression of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 family member expressed in differentiating myeloid cells, that required prior phosphorylation of Janus kinase-1 (JAK1), JAK2, extracellular signal-regulated kinase-1/2 (ERK1/2), signal transducer and activator of transcription-3 (STAT3), STAT1, and cAMP-response element-binding protein (CREB). Pharmacological inhibitors of JAK1, JAK2, ERK1/2, STAT3, STAT1, and CREB as well as the siRNA targeting at STAT3 and Mcl-1 all abolished OSM-dependent 3-NP resistance. Finally, OSM-dependent Mcl-1 induction contributed to the enhancements of mitochondrial bioenergetics including increases in spare respiratory capacity and ATP production. In conclusion, our findings indicated that OSM induces Mcl-1 expression via activation of ERK1/2, JAK1/2, STAT1/3, and CREB; furthermore, OSM-mediated Mcl-1 induction contributes to bioenergetic improvements and neuroprotective effects against 3-NP toxicity in cortical neurons. OSM may thus serve as a novel neuroprotective agent against mitochondrial dysfunction commonly associated with pathogenic mechanisms underlying neurodegeneration.
Keywords: cAMP-response element-binding protein (CREB); Extracellular signal-regulated kinase (ERK); Janus kinase (JAK); Myeloid cell leukemia-1 (Mcl-1); 3-nitropropionic acid (3-NP); signal transducer and activator of transcription (STAT);

Identification and functional characterization of Trypanosoma brucei peroxin 16 by Vishal C. Kalel; Wolfgang Schliebs; Ralf Erdmann (2326-2337).
Protozoan parasites of the family Trypanosomatidae infect humans as well as livestock causing devastating diseases like sleeping sickness, Chagas disease, and Leishmaniasis. These parasites compartmentalize glycolytic enzymes within unique organelles, the glycosomes. Glycosomes represent a subclass of peroxisomes and they are essential for the parasite survival. Hence, disruption of glycosome biogenesis is an attractive drug target for these Neglected Tropical Diseases (NTDs). Peroxin 16 (PEX16) plays an essential role in peroxisomal membrane protein targeting and de novo biogenesis of peroxisomes from endoplasmic reticulum (ER). We identified trypanosomal PEX16 based on specific sequence characteristics and demonstrate that it is an integral glycosomal membrane protein of procyclic and bloodstream form trypanosomes. RNAi mediated partial knockdown of Trypanosoma brucei PEX16 in bloodstream form trypanosomes led to severe ATP depletion, motility defects and cell death. Microscopic and biochemical analysis revealed drastic reduction in glycosome number and mislocalization of the glycosomal matrix enzymes to the cytosol. Asymmetry of the localization of the remaining glycosomes was observed with a severe depletion in the posterior part. The results demonstrate that trypanosomal PEX16 is essential for glycosome biogenesis and thereby, provides a potential drug target for sleeping sickness and related diseases.Display Omitted
Keywords: Peroxisome; Peroxin; PEX16; Trypanosomatids; Glycosome; Trypanosoma brucei;

Oncogenic acidic nuclear phosphoproteins ANP32C/D are novel clients of heat shock protein 90 by Yuliia Yuzefovych; Rainer Blasczyk; Trevor Huyton (2338-2348).
The acidic nuclear phosphoproteins (ANP32A-H) are an evolutionarily conserved family of proteins with diverse and sometimes opposing cellular functions. Here we show that the oncogenic family members ANP32C and ANP32D are associated in complexes containing the molecular chaperone Hsp90. The oncogenic ANP32C protein appears to be highly unstable with a rapid degradation (t1/2  > 30 min) occurring upon treatment of cells with cycloheximide. ANP32C was also found to be associated with oncogenic Hsp90 complexes by virtue of its ability to interact and be immunoprecipitated by the Hsp90 inhibitor PU-H71. Further studies treating cells with the Hsp90 inhibitors PU-H71 and 17-AAG showed atypical increased protein stability and prevention of ANP32C degradation compared to the Hsp90 client AKT. Cells overexpressing ANP32C or its mutant ANP32CY140H showed enhanced sensitivity to treatment with PU-H71 as demonstrated by CCK-8 and colony formation assays. Our results highlight that certain malignancies with ANP32C/D overexpression or mutation might be specifically targeted using Hsp90 inhibitors.Display Omitted
Keywords: ANP32C; Hsp90; Cell cycle; Cancer;

PAK1 modulates a PPARγ/NF-κB cascade in intestinal inflammation by Kyle Dammann; Vineeta Khare; Michaela Lang; Thierry Claudel; Felix Harpain; Nicolas Granofszky; Rayko Evstatiev; Jonathan M. Williams; D. Mark Pritchard; Alastair Watson; Christoph Gasche (2349-2360).
P21-activated kinases (PAKs) are multifunctional effectors of Rho GTPases with both kinase and scaffolding activity. Here, we investigated the effects of inflammation on PAK1 signaling and its role in colitis-driven carcinogenesis. PAK1 and p-PAK1 (Thr423) were assessed by immunohistochemistry, immunofluorescence, and Western blot. C57BL6/J wildtype mice were treated with a single intraperitoneal TNFα injection. Small intestinal organoids from these mice and from PAK1-KO mice were cultured with TNFα. NF-κB and PPARγ were analyzed upon PAK1 overexpression and silencing for transcriptional/translational regulation. PAK1 expression and activation was increased on the luminal intestinal epithelial surface in inflammatory bowel disease and colitis-associated cancer. PAK1 was phosphorylated upon treatment with IFNγ, IL-1β, and TNFα. In vivo, mice administered with TNFα showed increased p-PAK1 in intestinal villi, which was associated with nuclear p65 and NF-κB activation. p65 nuclear translocation downstream of TNFα was strongly inhibited in PAK1-KO small intestinal organoids. PAK1 overexpression induced a PAK1–p65 interaction as visualized by co-immunoprecipitation, nuclear translocation, and increased NF-κB transactivation, all of which were impeded by kinase-dead PAK1. Moreover, PAK1 overexpression downregulated PPARγ and mesalamine recovered PPARγ through PAK1 inhibition. On the other hand PAK1 silencing inhibited NF-κB, which was recovered using BADGE, a PPARγ antagonist. Altogether these data demonstrate that PAK1 overexpression and activation in inflammation and colitis-associated cancer promote NF-κB activity via suppression of PPARγ in intestinal epithelial cells.
Keywords: PAK1; NF-κB; PPARγ; Inflammation; Ulcerative colitis; Colitis-associated cancer;

Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway by Kazuhiro Katayama; Khyati Kapoor; Shinobu Ohnuma; Atish Patel; William Swaim; Indu S. Ambudkar; Suresh V. Ambudkar (2361-2370).
P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7 ± 1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1 ± 0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39–50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.
Keywords: P-glycoprotein; Endosome; Degradation; Half-life; Proteasome; Lysosome;

P-21-activated kinases (PAKs) are serine/threonine kinases comprising six isoforms divided in two groups, group-I (PAK1–3)/group-II (PAK4–6) which play important roles in cell cytoskeletal dynamics, survival, secretion and proliferation and are activated by diverse stimuli. However, little is known about PAKs ability to be activated by gastrointestinal (GI) hormones/neurotransmitters/growth-factors. We used rat pancreatic acini to explore the ability of GI-hormones/neurotransmitters/growth-factors to activate Group-I-PAKs and the signaling cascades involved. Only PAK2 was present in acini. PAK2 was activated by some pancreatic growth-factors [EGF, PDGF, bFGF], by secretagogues activating phospholipase-C (PLC) [CCK, carbachol, bombesin] and by post-receptor stimulants activating PKC [TPA], but not agents only mobilizing cellular calcium or increasing cyclic AMP. CCK-activation of PAK2 required both high- and low-affinity-CCK1-receptor-state activation. It was partially reduced by PKC- or Src-inhibition, but not with PI3K-inhibitors (wortmannin, LY294002) or thapsigargin. IPA-3, which prevents PAK2 binding to small-GTPases partially inhibited PAK2-activation, as well as reduced CCK-induced ERK1/2 activation and amylase release induced by CCK or bombesin. This study demonstrates pancreatic acini, possess only one Group-I-PAK, PAK2. CCK and other GI-hormones/neurotransmitters/growth-factors activate PAK2 via small GTPases (CDC42/Rac1), PKC and SFK but not cytosolic calcium or PI3K. CCK-activation of PAK2 showed several novel features being dependent on both receptor-activation states, having PLC- and PKC-dependent/independent components and small-GTPase-dependent/independent components. These results show that PAK2 is important in signaling cascades activated by numerous pancreatic stimuli which mediate their various physiological/pathophysiological responses and thus could be a promising target for the development of therapies in some pancreatic disorders such as pancreatitis.
Keywords: PAK2 activation; Pancreatic acini; CCK; Signaling; Pancreatic growth factor; PKC;

Cyclin C interacts with steroid receptor coactivator 2 and upregulates cell cycle genes in MCF-7 cells by Olivera Bozickovic; Tuyen Hoang; Ingvild S. Fenne; Thomas Helland; Linn Skartveit; Mamoru Ouchida; Gunnar Mellgren; Jørn V. Sagen (2383-2391).
Steroid receptor coactivator 2 (SRC-2) is a coactivator that regulates nuclear receptor activity. We previously reported that SRC-2 protein is degraded through the action of cAMP-dependent protein kinase A (PKA) and cAMP response element binding protein (CREB). In the study presented here, we aimed to identify proteins that interact with and thereby regulate SRC-2. We isolated cyclin C (CCNC) as an interacting partner with the SRC-2 degradation domain aa 347-758 in a yeast two-hybrid assay and confirmed direct interaction in an in vitro assay. The protein level of SRC-2 was increased with CCNC overexpression in COS-1 cells and decreased with CCNC silencing in COS-1 and MCF-7 cells. In a pulse-chase assay, we further show that silencing of CCNC resulted in a different SRC-2 degradation pattern during the first 6 h after the pulse. Finally, we provide evidence that CCNC regulates expression of cell cycle genes upregulated by SRC-2. In conclusion, our results suggest that CCNC temporarily protects SRC-2 against degradation and this event is involved in the transcriptional regulation of SRC-2 cell cycle target genes.
Keywords: GRIP1; TIF2; NCoA2; CycC; Mediator;

Micro-RNAs miR-29a and miR-330-5p function as tumor suppressors by targeting the MUC1 mucin in pancreatic cancer cells by Solange Tréhoux; Fatima Lahdaoui; Yannick Delpu; Florence Renaud; Emmanuelle Leteurtre; Jérôme Torrisani; Nicolas Jonckheere; Isabelle Van Seuningen (2392-2403).
MUC1 is an oncogenic mucin overexpressed in several epithelial cancers, including pancreatic ductal adenocarcinoma, and is considered as a potent target for cancer therapy. To control cancer progression, miRNAs became very recently, major targets and tools to inhibit oncogene expression. Inhibiting MUC1 using miRNAs appears thus as an attractive strategy to reduce cancer progression. However, potent miRNAs and associated mechanisms regulating MUC1 expression remain to be identified. To this aim, we undertook to study MUC1 regulation by miRNAs in pancreatic cancer cells and identify those with tumor suppressive activity. MiRNAs potentially targeting the 3′-UTR, the coding region, or the 5′-UTR of MUC1 were selected using an in silico approach. Our invitro and invivo experiments indicate that miR-29a and miR-330-5p are strong inhibitors of MUC1 expression in pancreatic cancer cells through direct binding to MUC1 3′-UTR. MUC1 regulation by the other selected miRNAs (miR-183, miR-200a, miR-876-3p and miR-939) was found to be indirect. MiR-29a and miR-330-5p are also deregulated in human pancreatic cancer cell lines and tissues and in pancreatic tissues of KrasG12D mice. In vitro, miR-29a and miR-330-5p inhibit cell proliferation, cell migration, cell invasion and sensitize pancreatic cancer cells to gemcitabine. In vivo intra-tumoral injection of these two miRNAs in xenografted pancreatic tumors led to reduced tumor growth. Altogether, we have identified miR-29a and miR-330-5p as two new tumor suppressive miRNAs that inhibit the expression of MUC1 oncogenic mucin in pancreatic cancer cells.
Keywords: MUC1 mucin; Pancreas; Cancer; MiRNA; Chemoresistance;

Augmented EGF receptor tyrosine kinase activity impairs vascular function by NADPH oxidase-dependent mechanism in type 2 diabetic mouse by Modar Kassan; Karima Ait-Aissa; Maha Ali; Mohamed Trebak; Khalid Matrougui (2404-2410).
We previously determined that augmented EGFR tyrosine kinase (EGFRtk) impairs vascular function in type 2 diabetic mouse (TD2). Here we determined that EGFRtk causes vascular dysfunction through NADPH oxidase activity in TD2.Mesenteric resistance arteries (MRA) from C57/BL6 and db/db mice were mounted in a wired myograph and pre-incubated for 1 h with either EGFRtk inhibitor (AG1478) or exogenous EGF. The inhibition of EGFRtk did not affect the contractile response to phenylephrine-(PE) and thromboxane-(U46619) or endothelium-dependent relaxation (EDR) to acetylcholine in MRA from control group. However, in TD2 mice, AG1478 reduced the contractile response to U46619, improved vasodilatation and reduced p22phox-NADPH expression, but had no effect on the contractile response to PE. The incubation of MRA with exogenous EGF potentiated the contractile response to PE in MRA from control and diabetic mice. However, EGF impaired the EDR and potentiated the vasoconstriction to U46619 only in the control group. Interestingly, NADPH oxidase inhibition in the presence of EGF restored the normal contraction to PE and improved the EDR but had no effect on the potentiated contraction to U46619. Vascular function improvement was associated with the rescue of eNOS and Akt and reduction in phosphorylated Rho-kinase, NOX4 mRNA levels, and NADPH oxidase activity. MRA from p47phox −/− mice incubated with EGF potentiated the contraction to U46619 but had no effect to PE or ACh responses.The present study provides evidence that augmented EGFRtk impairs vascular function by NADPH oxidase-dependent mechanism. Therefore, EGFRtk and oxidative stress should be potential targets to treat vascular dysfunction in TD2.
Keywords: EGF; Type 2 diabetes; Oxidative stress; ER stress; Vascular dysfunction;

Intracellular dynamics and fate of polystyrene nanoparticles in A549 Lung epithelial cells monitored by image (cross-) correlation spectroscopy and single particle tracking by Sarah Deville; Rozhin Penjweini; Nick Smisdom; Kristof Notelaers; Inge Nelissen; Jef Hooyberghs; Marcel Ameloot (2411-2419).
Novel insights in nanoparticle (NP) uptake routes of cells, their intracellular trafficking and subcellular targeting can be obtained through the investigation of their temporal and spatial behavior. In this work, we present the application of image (cross-) correlation spectroscopy (IC(C)S) and single particle tracking (SPT) to monitor the intracellular dynamics of polystyrene (PS) NPs in the human lung carcinoma A549 cell line. The ensemble kinetic behavior of NPs inside the cell was characterized by temporal and spatiotemporal image correlation spectroscopy (TICS and STICS). Moreover, a more direct interpretation of the diffusion and flow detected in the NP motion was obtained by SPT by monitoring individual NPs. Both techniques demonstrate that the PS NP transport in A549 cells is mainly dependent on microtubule-assisted transport. By applying spatiotemporal image cross-correlation spectroscopy (STICCS), the correlated motions of NPs with the early endosomes, late endosomes and lysosomes are identified. PS NPs were equally distributed among the endolysosomal compartment during the time interval of the experiments. The cotransport of the NPs with the lysosomes is significantly larger compared to the other cell organelles. In the present study we show that the complementarity of ICS-based techniques and SPT enables a consistent elaborate model of the complex behavior of NPs inside biological systems.
Keywords: Polystyrene nanoparticles; Image correlation spectroscopy; Single particle tracking; Colocalization; Diffusion;

Involvement of the heterodimeric interface region of the nucleotide binding domain-2 (NBD2) in the CFTR quaternary structure and membrane stability by Julien Micoud; Sylvain Chauvet; Klaus Ernst Ludwig Scheckenbach; Nadia Alfaidy; Marc Chanson; Mohamed Benharouga (2420-2431).
The cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the ATP-binding cassette (ABC) superfamily that functions as a chloride channel. The predicted structure of CFTR protein contains two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD1 and NBD2). The opening of the Cl channel is directly linked to ATP-driven tight dimerization of CFTR's NBD1 and NBD2 domains. The presence of a heterodimeric interfaces (HI) region in NBD1 and NBD2 generated a head to tail orientation necessary for channel activity. This process was also suggested to promote important conformational changes in the associated transmembrane domains of CFTR, which may impact the CFTR plasma membrane stability. To better understand the role of the individual HI region in this process, we generated recombinant CFTR protein with suppressed HI-NBD1 and HI-NBD2. Our results indicate that HI-NBD2 deletion leads to the loss of the dimerization profile of CFTR that affect its plasma membrane stability. We conclude that, in addition to its role in Cl transport, HI-NBD2 domain confers membrane stability of CFTR by consolidating its quaternary structure through interactions with HI-NBD1 region.
Keywords: CFTR; NBD2; Dimerization; Membrane stability; Quaternary structure; Cystic fibrosis;

Phosphorylation of phosphatidylinositol 4-phosphate 5-kinase γ by Akt regulates its interaction with talin and focal adhesion dynamics by Oanh Thi Tu Le; Oh Yeon Cho; Mai Hoang Tran; Jung Ah Kim; Sunghoe Chang; Ilo Jou; Sang Yoon Lee (2432-2443).
The type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family members and their lipid product, phosphatidylinositol 4,5-bisphosphate (PIP2) are important regulators of actin cytoskeleton. PIP5Kγ 90 kDa (PIP5Kγ90), an isoform of PIP5K, localizes to focal adhesions (FAs) and is activated via its interaction with the cytoskeletal protein, talin. Currently, regulatory signaling pathways of talin–PIP5Kγ90 interaction related to FA dynamics and cell motility are not well understood. Considering the presence of Akt consensus motifs in PIP5Kγ90, we examined a potential link of Akt activation to talin–PIP5Kγ90 interaction. We found that Akt phosphorylated PIP5Kγ90 specifically at serine 555 (S555) in vitro and in epidermal growth factor (EGF)-treated cells phosphoinositide 3-kinase-dependently. EGF treatment suppressed talin–PIP5Kγ90 interaction and PIP2 levels. Similarly, a phosphomimetic mutant (S555D), but not non-phosphorylatable mutant (S555A), of PIP5Kγ90 had reduced talin binding affinity, lowered PIP2 levels, and was dislocated from FAs. The S555D mutant also caused decreases in actin stress fibers and vinculin-positive FAs. Moreover, assembly and disassembly of FAs were enhanced by S555D expression and EGF-induced cell migration was relatively low in S555A-expressing cells compared to wild-type-expressing cells. PIP5Kγ87, a PIP5Kγ splice variant lacking the talin binding motif, was phosphorylated by Akt, which, however, hardly affected PIP2 levels. Taken together, our results suggested that Akt-mediated PIP5Kγ90 S555 phosphorylation is a novel regulatory point for talin binding to control PIP2 level at the FAs, thereby modulating FA dynamics and cell motility.Display Omitted
Keywords: Phosphatidylinositol 4-phosphate 5-kinase γ; Phosphatidylinositol 4,5-bisphosphate; Akt; Talin; Phosphorylation; Focal adhesion;

Differential expression, distinct localization and opposite effect on Golgi structure and cell differentiation by a novel splice variant of human PRMT5 by Muhammad Sohail; Manli Zhang; David Litchfield; Lisheng Wang; Sam Kung; Jiuyong Xie (2444-2452).
Alternative splicing contributes greatly to the proteomic diversity of metazoans. Protein arginine methyltransferase 5 (PRMT5) methylates arginines of Golgi components and other factors exerting diverse effects on cell growth/differentiation, but the underlying molecular basis for its subcellular distribution and diverse roles has not been fully understood. Here we show the detailed properties of an evolutionarily emerged splice variant of human PRMT5 (PRMT5S) that is distinct from the original isoform (PRMT5L). The isoforms are differentially expressed among mammalian cells and tissues. The PRMT5S is distributed all over the cell but PRMT5L mainly colocalizes with Giantin, a Golgi marker. PRMT5 knockdown led to an enlarged Giantin pattern, which was prevented by the expression of either isoform. Rescuing PRMT5S also increased the percentage of cells with an interphase Giantin pattern compacted at one end of the nucleus, consistent with its cell cycle-arresting effect, while rescuing PRMT5L increased that of the mitotic Giantin patterns of dynamically fragmented structures. Moreover, the isoforms are differentially expressed during neuronal or dendritic cell differentiation, and their ectopic expression showed an opposite effect on dendritic cell differentiation. Furthermore, besides their differential regulation of gene expression, both isoforms also similarly regulate over a thousand genes particularly those involved in apoptosis and differentiation. Taking these properties together, we propose that their differential expression and subcellular localization contribute to spatial and temporal regulation of arginine methylation and gene expression to exert different effects. The novel PRMT5S likely contributes to the observed diverse effects of PRMT5 in cells.
Keywords: PRMT5; Alternative splicing; Giantin; Golgi structure; Cell cycle; Dendritic cell;

HGF/c-Met signaling promotes liver progenitor cell migration and invasion by an epithelial–mesenchymal transition-independent, phosphatidyl inositol-3 kinase-dependent pathway in an in vitro model by A. Suárez-Causado; D. Caballero-Díaz; E. Bertrán; C. Roncero; A. Addante; M. García-Álvaro; M. Fernández; B. Herrera; A. Porras; I. Fabregat; A. Sánchez (2453-2463).
Oval cells constitute an interesting hepatic cell population. They contribute to sustain liver regeneration during chronic liver damage, but in doing this they can be target of malignant conversion and become tumor-initiating cells and drive hepatocarcinogenesis. The molecular mechanisms beneath either their pro-regenerative or pro-tumorigenic potential are still poorly understood. In this study, we have investigated the role of the HGF/c-Met pathway in regulation of oval cell migratory and invasive properties. Our results show that HGF induces c-Met-dependent oval cell migration both in normal culture conditions and after in vitro wounding. HGF-triggered migration involves F-actin cytoskeleton reorganization, which is also evidenced by activation of Rac1. Furthermore, HGF causes ZO-1 translocation from cell–cell contact sites to cytoplasm and its concomitant activation by phosphorylation. However, no loss of expression of cell–cell adhesion proteins, including E-cadherin, ZO-1 and Occludin-1, is observed. Additionally, migration does not lead to cell dispersal but to a characteristic organized pattern in rows, in turn associated with Golgi compaction, providing strong evidence of a morphogenic collective migration. Besides migration, HGF increases oval cell invasion through extracellular matrix, a process that requires PI3K activation and is at least partly mediated by expression and activation of metalloproteases. Altogether, our findings provide novel insights into the cellular and molecular mechanisms mediating the essential role of HGF/c-Met signaling during oval cell-mediated mouse liver regeneration.Display Omitted
Keywords: c-Met; Liver progenitor cell; Migration; Invasion; Epithelial–mesenchymal transition; PI3K;

Different states of integrin LFA-1 aggregation are controlled through its association with tetraspanin CD9 by Raquel Reyes; Alicia Monjas; María Yánez-Mó; Beatriz Cardeñes; Giulia Morlino; Alvaro Gilsanz; Yesenia Machado-Pineda; Esther Lafuente; Peter Monk; Francisco Sánchez-Madrid; Carlos Cabañas (2464-2480).
The tetraspanin CD9 has been shown to interact with different members of the β1 and β3 subfamilies of integrins, regulating through these interactions cell adhesion, migration and signaling. Based on confocal microscopy co-localization and on co-immunoprecipitation results, we report here that CD9 associates with the β2 integrin LFA-1 in different types of leukocytes including T, B and monocytic cells. This association is resistant to stringent solubilization conditions which, together with data from chemical crosslinking, in situ Proximity Ligation Assays and pull-down experiments, suggest a primary/direct type of interaction mediated by the Large Extracellular Loop of the tetraspanin. CD9 exerts inhibitory effects on the adhesive function of LFA-1 and on LFA-1-dependent leukocyte cytotoxic activity. The mechanism responsible for this negative regulation exerted by CD9 on LFA-1 adhesion does not involve changes in the affinity state of this integrin but seems to be related to alterations in its state of aggregation.
Keywords: Tetraspanin; Integrin; CD9; LFA-1; Adhesion; Cytotoxicity;

Identification of lipid droplet structure-like/resident proteins in Caenorhabditis elegans by Huimin Na; Peng Zhang; Yong Chen; Xiaotong Zhu; Yi Liu; Yangli Liu; Kang Xie; Ningyi Xu; Fuquan Yang; Yong Yu; Simon Cichello; Ho Yi Mak; Meng C. Wang; Hong Zhang; Pingsheng Liu (2481-2491).
The lipid droplet (LD) is a cellular organelle that stores neutral lipids in cells and has been linked with metabolic disorders. Caenorhabditis elegans has many characteristics which make it an excellent animal model for studying LDs. However, unlike in mammalian cells, no LD structure-like/resident proteins have been identified in C. elegans, which has limited the utility of this model for the study of lipid storage and metabolism. Herein based on three lines of evidence, we identified that MDT-28 and DHS-3 previously identified in C. elegans LD proteome were two LD structure-like/resident proteins. First, MDT-28 and DHS-3 were found to be the two most abundant LD proteins in the worm. Second, the proteins were specifically localized to LDs and we identified the domains responsible for this targeting in both proteins. Third and most importantly, the depletion of MDT-28 induced LD clustering while DHS-3 deletion reduced triacylglycerol content (TAG). We further characterized the proteins finding that MDT-28 was ubiquitously expressed in the intestine, muscle, hypodermis, and embryos, whereas DHS-3 was expressed mainly in intestinal cells. Together, these two LD structure-like/resident proteins provide a basis for future mechanistic studies into the dynamics and functions of LDs in C. elegans.
Keywords: Lipid droplet; Structure-like/Resident Proteins; Triacylglycerol (TAG); MDT-28; Perilipin family;

Glucose deprivation occurs in several human diseases, including infarctions and solid tumors, and leads to cell death. In this article, we investigate the role of the pseudokinase Tribbles homolog 3 (TRIB3) in the cellular stress response to glucose starvation using cell lines derived from HEK293, which is highly glycolytic under standard conditions. Our results show that TRIB3 mRNA and protein levels are strongly upregulated in glucose-deprived cells via the induction of activating transcription factor 4 (ATF4) by the endoplasmic reticulum (ER) stress sensor kinase PERK. Cell survival in glucose-deficient conditions is enhanced by TRIB3 overexpression and reduced by TRIB3 knockdown. Genome-wide gene expression profiling uncovered approximately 40 glucose deprivation-responsive genes that are affected by TRIB3, including several genes involved in signaling processes and metabolism. Based on transcription factor motif analysis, the majority of TRIB3-downregulated genes are target genes of ATF4, which TRIB3 is known to inhibit. The gene most substantially upregulated by TRIB3 is insulin-like growth factor binding protein 2 (IGFBP2). IGFBP2 mRNA and protein levels are downregulated in cells subjected to glucose deprivation, and reduced IGFBP2 expression aggravates cell death during glucose deficiency, while overexpression of IGFBP2 prolongs cell survival. Moreover, IGFBP2 silencing abrogates the pro-survival effect of TRIB3. Since TRIB3 augments IGFBP2 expression in glucose-starved cells, the data indicate that IGFBP2 contributes to the attenuation of cell death by TRIB3. These results implicate TRIB3 and IGFBP2, both of which are known to be overexpressed in several types of cancers, as pro-survival modulators of cell viability in nutrient-deficient microenvironments.
Keywords: Tribbles; TRB3; IGFBP2; Cell death; Glucose deprivation; Gene expression;

cAMP-mediated secretion of brain-derived neurotrophic factor in developing airway smooth muscle by Michael A. Thompson; Rodney D. Britt; Ine Kuipers; Alecia Stewart; James Thu; Hitesh C. Pandya; Peter MacFarlane; Christina M. Pabelick; Richard J. Martin; Y.S. Prakash (2506-2514).
Moderate hyperoxic exposure in preterm infants contributes to subsequent airway dysfunction and to risk of developing recurrent wheeze and asthma. The regulatory mechanisms that can contribute to hyperoxia-induced airway dysfunction are still under investigation. Recent studies in mice show that hyperoxia increases brain-derived neurotrophic factor (BDNF), a growth factor that increases airway smooth muscle (ASM) proliferation and contractility. We assessed the mechanisms underlying effects of moderate hyperoxia (50% O2) on BDNF expression and secretion in developing human ASM. Hyperoxia increased BDNF secretion, but did not alter endogenous BDNF mRNA or intracellular protein levels. Exposure to hyperoxia significantly increased [Ca2 +]i responses to histamine, an effect blunted by the BDNF chelator TrkB-Fc. Hyperoxia also increased ASM cAMP levels, associated with reduced PDE4 activity, but did not alter protein kinase A (PKA) activity or adenylyl cyclase mRNA levels. However, 50% O2 increased expression of Epac2, which is activated by cAMP and can regulate protein secretion. Silencing RNA studies indicated that Epac2, but not Epac1, is important for hyperoxia-induced BDNF secretion, while PKA inhibition did not influence BDNF secretion. In turn, BDNF had autocrine effects of enhancing ASM cAMP levels, an effect inhibited by TrkB and BDNF siRNAs. Together, these novel studies suggest that hyperoxia can modulate BDNF secretion, via cAMP-mediated Epac2 activation in ASM, resulting in a positive feedback effect of BDNF-mediated elevation in cAMP levels. The potential functional role of this pathway is to sustain BDNF secretion following hyperoxic stimulus, leading to enhanced ASM contractility and proliferation.
Keywords: Lung; Development; Hyperoxia; Neurotrophin; Cyclic nucleotide; Epac;

The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia by Julia D. Knopf; Stefan Tholen; Maria M. Koczorowska; Olivier De Wever; Martin L. Biniossek; Oliver Schilling (2515-2525).
Fibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.
Keywords: Fibroblast activation protein alpha; Cancer associated fibroblasts; Interactome; Lipid rafts;

PAK1 is involved in sensing the orientation of collagen stiffness gradients in mouse fibroblasts by V.I. Pinto; H. Mohammadi; W.S. Lee; A.H. Cheung; C.A. McCulloch (2526-2538).
Migrating cells sense variations of stiffness in connective tissue matrices but how cells detect and respond to stiffness orientation is not defined. We examined cell extension formation on collagen with underlying support (vertical stiffness gradient) or on collagen laterally supported by nylon (lateral stiffness gradient). At 6 h after plating, cells plated on laterally-supported collagen exhibited > 2-fold more abundant and ~ 2-fold longer cell extensions than cells plated on collagen with underlying support. We examined whether p21-activated kinase 1 (PAK1) influences extension formation that is dependent on the orientation of support. At 6 h after plating on collagen with underlying support, wild-type cell extensions were 40% shorter than PAK1 knockdown cells. In contrast, on laterally-supported collagen, wild-type cell extensions were 2-fold longer than PAK1 knockdown cells. In cells plated on laterally-supported collagen, there were ~ 2-fold reductions of collagen fiber alignment and compaction in PAK1 knockdown cells compared with wild-type cells. PAK1 knockdown did not affect collagen fiber alignment or compaction by cells plated on collagen with underlying support. Wild-type cells with lateral support of collagen exhibited 3-fold increases of phospho-myosin staining at 6 h, which was 2-fold lower in PAK1 knockdown cells. In contrast, cells on collagen with underlying support showed no increase of phospho-myosin staining at any times. PAK1 knockdown did not affect α2 or β1 integrin expression or function. We conclude that PAK1 is involved in the ability of cells to sense the orientation of stiffness in collagen substrates and generate contractile forces that affect cell extension formation.
Keywords: Physical boundaries; Cell extensions; p21-Activated kinase; Constrained matrices; Collagen remodeling;

Identification of Tpr and α-actinin-4 as two novel SLK-interacting proteins by Aala Jaberi; Erika Hooker; Julie Guillemette; Joan Papillon; Arnold S. Kristof; Andrey V. Cybulsky (2539-2552).
Expression and activity of the Ste20-like kinase, SLK, are increased during kidney development and recovery from ischemia–reperfusion injury. SLK mediates apoptosis in various cells, and can regulate cell cycle progression and cytoskeletal remodeling. In cells, SLK is detected in a high molecular mass complex, suggesting that SLK is a dimer/oligomer, or is in tight association with other proteins. To better understand the regulation, localization and function of SLK, we sought to identify proteins in this high molecular mass complex. Analysis by mass spectroscopy identified the nucleoporin, translocated promoter region (Tpr), and the cytoskeletal protein, α-actinin-4, as potential SLK-interacting proteins. Using a protein complementation assay, we showed that the 350 amino acid C-terminal, coiled-coil domain of SLK was responsible for homodimerization, as well as interaction with Tpr and α-actinin-4. The association of SLK with Tpr and α-actinin-4, respectively, was confirmed by co-immunoprecipitation. Subsets of total cellular SLK colocalized with Tpr at the nuclear envelope, and α-actinin-4 in the cytoplasm. Expression of Tpr attenuated activation-specific autophosphorylation of SLK, and blocked SLK-induced apoptosis and AP-1 activity. In contrast to the effect of Tpr, autophosphorylation of SLK was not affected by α-actinin-4. Thus, SLK interacts with Tpr and α-actinin-4 in cells, and these protein–protein interactions may control the subcellular localization and the biological activity of SLK.
Keywords: Fluorescence; Phosphorylation; Protein complementation assay; Protein kinases; Protein–protein interactions; Subcellular organelles;

One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136–S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136–S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress.
Keywords: Antisense RNA; σB; Stress response; Bacillus subtilis; Primary binding protein; Ribosome;

Regions outside of conserved PxxPxR motifs drive the high affinity interaction of GRB2 with SH3 domain ligands by Rebekah R. Bartelt; Jonathan Light; Aldo Vacaflores; Alayna Butcher; Madhana Pandian; Piers Nash; Jon C.D. Houtman (2560-2569).
SH3 domains are evolutionarily conserved protein interaction domains that control nearly all cellular processes in eukaryotes. The current model is that most SH3 domains bind discreet PxxPxR motifs with weak affinity and relatively low selectivity. However, the interactions of full-length SH3 domain-containing proteins with ligands are highly specific and have much stronger affinity. This suggests that regions outside of PxxPxR motifs drive these interactions. In this study, we observed that PxxPxR motifs were required for the binding of the adaptor protein GRB2 to short peptides from its ligand SOS1. Surprisingly, PxxPxR motifs from the proline rich region of SOS1 or CBL were neither necessary nor sufficient for the in vitro or in vivo interaction with full-length GRB2. Together, our findings show that regions outside of the consensus PxxPxR sites drive the high affinity association of GRB2 with SH3 domain ligands, suggesting that the binding mechanism for this and other SH3 domain interactions may be more complex than originally thought.
Keywords: SH3 domains; GRB2; SOS1; CBL; Signal transduction;

TRAP1 controls cell migration of cancer cells in metabolic stress conditions: Correlations with AKT/p70S6K pathways by Ilenia Agliarulo; Danilo Swann Matassa; Maria Rosaria Amoroso; Francesca Maddalena; Lorenza Sisinni; Leandra Sepe; Maria Carla Ferrari; Diana Arzeni; Rosario Avolio; Giovanni Paolella; Matteo Landriscina; Franca Esposito (2570-2579).
Cell motility is a highly dynamic phenomenon that is essential to physiological processes such as morphogenesis, wound healing and immune response, but also involved in pathological conditions such as metastatic dissemination of cancers. The involvement of the molecular chaperone TRAP1 in the regulation of cell motility, although still controversial, has been recently investigated along with some well-characterized roles in cancer cell survival and drug resistance in several tumour types. Among different functions, TRAP1-dependent regulation of protein synthesis seems to be involved in the migratory behaviour of cancer cells and, interestingly, the expression of p70S6K, a kinase responsible for translation initiation, playing a role in cell motility, is regulated by TRAP1. In this study, we demonstrate that TRAP1 silencing enhances cell motility in vitro but compromises the ability of cells to overcome stress conditions, and that this effect is mediated by the AKT/p70S6K pathway. In fact: i) inhibition of p70S6K activity specifically reduces migration in TRAP1 knock-down cells; ii) nutrient deprivation affects p70S6K activity thereby impairing cell migration only in TRAP1-deficient cells; iii) TRAP1 regulates the expression of both AKT and p70S6K at post-transcriptional level; and iii) TRAP1 silencing modulates the expression of genes involved in cell motility and epithelial–mesenchymal transition. Notably, a correlation between TRAP1 and AKT expression is found in vivo in human colorectal tumours. These results provide new insights into TRAP1 role in the regulation of cell migration in cancer cells, tumour progression and metastatic mechanisms.
Keywords: TRAP1; AKT/p70S6K pathway; Colorectal carcinoma; Metastasis; Wound healing;

ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells by Natalie Al-Furoukh; Alessandro Ianni; Hendrik Nolte; Soraya Hölper; Marcus Krüger; Sjoerd Wanrooij; Thomas Braun (2580-2591).
Proteostasis is crucial for life and maintained by cellular chaperones and proteases. One major mitochondrial protease is the ClpXP complex, which is comprised of a catalytic ClpX subunit and a proteolytic ClpP subunit. Based on two separate observations, we hypothesized that ClpX may play a leading role in the cellular function of ClpXP. Therefore, we analyzed the effect of ClpX overexpression on a myoblast proteome by quantitative proteomics.ClpX overexpression results in the upregulation of markers of the mitochondrial proteostasis pathway, known as the “mitochondrial unfolded protein response” (UPRmt). Although this pathway is described in detail in Caenorhabditis elegans, it is not clear whether it is conserved in mammals. Therefore, we compared features of the classical nematode UPRmt with our mammalian ClpX-triggered UPRmt dataset. We show that they share the same retrograde mitochondria-to-nucleus signaling pathway that involves the key UPRmt transcription factor CHOP (also known as Ddit3, CEBPZ or GADD153).In conclusion, our data confirm the existence of a mammalian UPRmt that has great similarity to the C. elegans pathway. Furthermore, our results illustrate that ClpX overexpression is a good and simple model to study the underlying mechanisms of the UPRmt in mammalian cells.
Keywords: UPRmt (mitochondrial unfolded protein response); ClpXP; Proteomics; SILAC; Myogenesis; CHOP/Ddit3/CEBPZ/GADD153;

Rab is a small GTP-binding protein family that regulates various pathways of vesicular transport. Although more than 60 Rab proteins are targeted to specific organelles in mammalian cells, the mechanisms underlying the specificity of Rab proteins for the respective organelles remain unknown. In this study, we reconstituted the Golgi targeting of Rab6A in streptolysin O (SLO)-permeabilized HeLa cells in a cytosol-dependent manner and investigated the biochemical requirements of targeting. Golgi-targeting assays identified Bicaudal-D (BICD)2, which is reportedly involved in the dynein-mediated transport of mRNAs during oogenesis and embryogenesis in Drosophila, as a cytosolic factor for the Golgi targeting of Rab6A in SLO-permeabilized HeLa cells. Subsequent immunofluorescence analyses indicated decreased amounts of the GTP-bound active form of Rab6 in BICD2-knockdown cells. In addition, fluorescence recovery after photobleaching (FRAP) analyses revealed that overexpression of the C-terminal region of BICD2 decreased the exchange rate of GFP–Rab6A between the Golgi membrane and the cytosol. Collectively, these results indicated that BICD2 facilitates the binding of Rab6A to the Golgi by stabilizing its GTP-bound form. Moreover, several analyses of vesicular transport demonstrated that Rab6A and BICD2 play crucial roles in Golgi tubule fusion with the endoplasmic reticulum (ER) in brefeldin A (BFA)-treated cells, indicating that BICD2 is involved in coat protein I (COPI)-independent Golgi-to-ER retrograde vesicular transport.
Keywords: Rab6; BICD2; Semi-intact cell; Golgi-targeting assay; Retrograde transport;

PodocyteTRPC6 channels have been implicated in glomerular diseases. Syndecan-4 (Sdc4) is a membrane proteoglycan that can be cleaved to release a soluble ectodomain capable of paracrine and autocrine signaling. We have confirmed that overexpression of Sdc4 core protein increases surface abundance of TRPC6 channels in cultured podocytes, whereas Sdc4 knockdown has the opposite effect. Exposure to soluble Sdc4 ectodomain also increased the surface abundance of TRPC6, and increased cationic currents evoked by a diacylglycerol analog in podocytes. Sdc4 ectodomain increased generation of reactive oxygen species (ROS), reduced activation of RhoA, increased activation of Rac1, increased nuclear abundance of NFATc1, and increased total β3-integrin. The effects of Sdc4 ectodomain on cell-surface TRPC6 were blocked by the ROS quencher TEMPOL, and by the Rac1 inhibitor NSC-23766, but were not blocked by inhibition of calcineurin–NFATc1 signaling. The Sdc4 core protein co-immunoprecipitates with β3-integrin in cultured podocytes. Moreover, effects of Sdc4 ectodomain on TRPC6, ROS generation, Rac1 and RhoA modulation, and NFATc1 activation were blocked by cilengitide, a selective inhibitor of outside-in signaling through αv-containing integrins. Exposure to TNF, or serum from three patients with recurrent FSGS in relapse, increased shedding of podocyte Sdc4 ectodomains into the surrounding medium. This was also observed after treating podocytes with the metalloproteinase ADAM17 or after overexpression of the Sdc4 core protein. Increased concentrations of Sdc4 ectodomain were detected in urine of rats during acute puromycin aminonucleoside nephrosis. Locally generated Sdc4 may play a role in regulating TRPC6 channels, and may contribute to glomerular pathology.
Keywords: Inflammation; FSGS; Oxidative stress; Proteoglycan; Calcium; Metalloproteinase;

Effect of extracellular calcium on regucalcin expression and cell viability in neoplastic and non-neoplastic human prostate cells by Cátia V. Vaz; Daniel B. Rodrigues; Sílvia Socorro; Cláudio J. Maia (2621-2628).
Extracellular calcium (Ca2 + o) and its receptor, the Ca2 +-sensing receptor (CaSR), play an important role in prostate physiology, and it has been shown that the deregulation of Ca2 + homeostasis and the overexpression of CaSR are involved in prostate cancer (PCa). Regucalcin (RGN), a Ca2 +-binding protein that plays a relevant role in intracellular Ca2 + homeostasis, was identified as an under-expressed protein in human PCa. Moreover, RGN was associated with suppression of cell proliferation, suggesting that the loss of RGN may favor development and progression of PCa. This work aims to unveil the role of Ca2 + o on RGN expression and viability of non-neoplastic (PNT1A) and neoplastic (LNCaP) prostate cell lines. It was demonstrated that Ca2 + o up-regulates RGN expression in both cell lines, but important differences were found between cells for dose- and time-responses to Ca2 + o treatment. It was also shown that high [Ca2 +]o triggers different effects on cell proliferation of neoplastic and non-neoplastic PCa cells, which seems to be related with RGN expression levels. This suggests the involvement of RGN in the regulation of cell proliferation in response to Ca2 + o treatment. Also, the effect of Ca2 + o on CaSR expression seems to be dependent of RGN expression, which is strengthened by the fact that RGN-knockdown in PNT1A cells increases the CaSR expression, whereas transgenic rats overexpressing RGN exhibit low levels of CaSR. Overall, our results highlighted the importance of RGN as a regulatory protein in Ca2 +-dependent signaling pathways and its deregulation of RGN expression by Ca2 + o may contribute for onset and progression of PCa.
Keywords: PNT1A; LNCaP; Prostate cancer; Regucalcin; Calcium; CaSR;

C3G (RapGEF1), a regulator of actin dynamics promotes survival and myogenic differentiation of mouse mesenchymal cells by K. Sasi Kumar; A. Ramadhas; S.C. Nayak; S. Kaniyappan; K. Dayma; V. Radha (2629-2639).
RapGEF1 (C3G) is a ubiquitously expressed protein that is essential for mammalian embryonic development. We have shown earlier that C3G regulates cytoskeletal dynamics and is required for neuronal differentiation. To determine if C3G plays a wider role in differentiation of multiple tissue types, we examined its role in skeletal muscle differentiation using the model system of C2C12 cells in culture. C3G protein is highly expressed in mouse skeletal muscle and its transcript and protein levels increase as C2C12 cells are induced to differentiate. Increase in C3G was predominantly seen in the nuclei of myotubes. Ectopic expression of C3G promoted myotube formation when cells were cultured in growth as well as differentiation medium and, enhanced MHC levels were associated with C3G expression. C3G induced differentiation required its catalytic and protein interaction domains and was dependent on the function of cellular R-Ras. Knockdown of cellular C3G using small hairpin RNA reduced expression of muscle specific markers and β-catenin, resulting in impaired differentiation. Disabling C3G function also resulted in enhanced cell death suggesting that cellular C3G is required for cell survival. In cells grown in growth medium, over-expressed C3G increased Akt activity, and C3G knockdown reduced it. C3G expression also suppressed cyclin D1 levels, and induced p27 expression, molecules involved in regulating cell proliferation. Endogenous C3G localizes to focal adhesions in myotubes and C3G expressing cells show distinct stress fibers, elongation and parallel alignment. Expression of a dominant negative construct of C3G, disrupts actin cytoskeleton and formation of focal adhesions resulting in detachment of cells from the substratum and inhibition of differentiation. Our results provide evidence that C3G plays an important role in myogenic differentiation by coordinating cell cycle exit, actin dynamics and survival signaling.
Keywords: C3G; RapGEF1; C2C12 cells; Myogenic differentiation; Adhesion; Survival;

In situ Fourier transform infrared analysis of live cells' response to doxorubicin by Pedro L. Fale; Ali Altharawi; K.L. Andrew Chan (2640-2648).
The study of the response of cancer cells to chemotherapy drugs is of high importance due to the specificity of some drugs to certain types of cancer and the resistance of some specific cancer types to chemotherapy drugs. Our aim was to develop and apply the label-free and non-destructive Fourier transform infrared (FTIR) method to determine the sensitivity of three different cancer cell-lines to a common anti-cancer drug doxorubicin at different concentrations and to demonstrate that information about the mechanism of resistance to the chemotherapy drug can be extracted from spectral data. HeLa, PC3, and Caco-2 cells were seeded and grown on an attenuated total reflection (ATR) crystal, doxorubicin was applied at the clinically significant concentration of 0.1–20 μM, and spectra of the cells were collected hourly over 20 h. Analysis of the amide bands was correlated with cell viability, which had been cross validated with MTT assays, allowing to determine that the three cell lines had significantly different resistance to doxorubicin. The difference spectra and principal component analysis (PCA) highlighted the subtle chemical changes in the living cells under treatment. Spectral regions assigned to nucleic acids (mainly 1085 cm− 1) and carbohydrates (mainly 1024 cm− 1) showed changes that could be related to the mode of action of the drug and the mechanism of resistance of the cell lines to doxorubicin. This is a cost-effective method that does not require bioassay reagents but allows label-free, non-destructive and in situ analysis of chemical changes in live cells, using standard FTIR equipment adapted to ATR measurements.
Keywords: Living cells; Chemotherapy; Drug resistance; Multi-bounce ATR; Drug screening; Infrared spectroscopy;

Wounding leads not only to plasma membrane disruption, but also to compromised cytoskeleton structures. This results not only in unwarranted exchanges between the cytosol and extracellular milieu, but also in loss of tensegrity, which may further endanger the cell. Tensegrity can be described as the interplay between the tensile forces generated by the apparent membrane tension, actomyosin contraction, and the cytoskeletal structures resisting those changes (e.g., microtubules). It is responsible for the structural integrity of the cell and for its ability to sense mechanical signals. Recent reviews dealing with single-cell healing mostly focused on the molecular machineries controlling the traffic and fusion of specific vesicles, or their role in different pathologies. In this review, we aim to take a broader view of the different modes of single cell repair, while focussing on the different ways the changes in plasmalemma surface area and composition, plasmalemma tension, and cytoskeletal dynamics may influence and affect single-cell repair.
Keywords: Tensegrity; Single-cell wound repair; Plasmalemma; Cytoskeleton dynamics; Membrane tension; Tether force;

Myeloid leukemia cells maintain a high intracellular ROS level and use redox signals for survival. The metabolism of ROS also affects cell fate, including cell death and differentiation. Superoxide dismutases (SODs) are major antioxidant enzymes that have high levels of expression in myeloid leukemia cells. However, the role of SODs in the regulation of myeloid leukemia cells' biological function is still unclear. To investigate the function of SODs in myeloid leukemia cell death and differentiation, we used myeloid leukemia cell lines K562, MEG-01, TF-1, and HEL cells for this study. We found that PMA-induced megakaryocytic differentiation in myeloid leukemia cells is accompanied by cell death and SOD1 down-regulation, while SOD2 expression is not affected. The role of SOD1 is verified when ATN-224, a SOD1 specific inhibitor, inhibits cell proliferation and promotes cell death in myeloid leukemia cells without PMA treatment. Moreover, inhibition or silencing of SODs further increases cell death and decreases polyploidization induced by PMA while they were partially reversed by SOD1 overexpression. Thus, SOD1 expression is required for myeloid leukemia cell fate determination. In addition, the knockdown of PKD2 reduces cell death and promotes polyploidization induced by PMA. PMA/PKD2-mediated necrosis via PARP cleavage involves both SOD1-dependent and -independent pathways. Finally, ATN-224 enhanced the inhibition of cell proliferation by Ara-C. Taken together, the results demonstrate that SOD1 regulates cell death and differentiation in myeloid leukemia cells. ATN-224 may be beneficial for myeloid leukemia therapy.
Keywords: Myeloid leukemia; Reactive oxygen species (ROS); Superoxide dismutase 1 (SOD1); Protein kinase D2 (PKD2); ATN-224;

Functional characterization of importin α8 as a classical nuclear localization signal receptor by Chihiro Kimoto; Tetsuji Moriyama; Akira Tsujii; Yoshinobu Igarashi; Chikashi Obuse; Yoichi Miyamoto; Masahiro Oka; Yoshihiro Yoneda (2676-2683).
Importin α8 has recently been identified as an importin α family member based on its primary structure and binding ability to importin β1 and to several karyophilic proteins. However, there has been no experimental evidence that importin α8 actually functions in the nuclear transport of classical nuclear localization signal (cNLS)-containing cargo. Here, using an in vitro transport assay, we demonstrate that purified recombinant importin α8 can transport SV40T antigen cNLS-containing cargo into the nucleus of HeLa cells, in conjunction with importin β1. Pull-down assays, followed by mass spectrometry analysis, identified 179 putative importin α8-binding proteins, only 62 of which overlap with those of importin α1, the closest importin α family member. Among the importin α8-binding candidates, we showed that DNA damage-binding protein 2 (DDB2) was actually transported into the nucleus via the importin α8/β1 pathway. Furthermore, we found that the other subtypes of importin α, which were also identified as importin α8-binding candidates, indeed form heterodimers with importin α8. Notably, we found that these importin α8-containing heterodimers were more stable in the presence of cNLS-substrates than heterodimers containing importin α1. From these findings, we propose that importin α8 functions as a cNLS receptor with distinct cargo specificity, and that heterodimerization by importin α8 is a novel regulatory mode of cNLS binding, in addition to the autoinhibitory regulation by the importin β binding domain.
Keywords: Importin α; KPNA; Nuclear localization signal; DDB2; Mass spectrometry; Dimer;

FKBP25 and FKBP38 regulate non-capacitative calcium entry through TRPC6 by Esther Lopez; Alejandro Berna-Erro; Gines M. Salido; Juan A. Rosado; Pedro C. Redondo (2684-2696).
Non-capacitative calcium entry (NCCE) contributes to cell activation in response to the occupation of G protein-coupled membrane receptors. Thrombin administration to platelets evokes the synthesis of diacylglycerol downstream of PAR receptor activation. Diacylglycerol evokes NCCE through activating TRPC3 and TRPC6 in human platelets. Although it is known that immunophilins interact with TRPCs, the role of immunophilins in the regulation of NCCE remains unknown. Platelet incubation with FK506, an immunophilin antagonist, reduced OAG-evoked NCCE in a concentration-dependent manner, an effect that was independent on the inactivation of calcineurin (CaN). FK506 was unable to reduce NCCE evoked by OAG in platelets from TRPC6−/− mice. In HEK-293 cells overexpressing TRPC6, currents through TRPC6 were altered in the presence of FK506. We have found interaction between FKBP38 and other FKBPs, like FKBP25, FKBP12, and FKBP52 that were not affected by FK506, as well as with calmodulin (CaM). FK506 modified the pattern of association between FKBP25 and TRPCs as well as impaired OAG-evoked TRPC3 and TRPC6 coupling in both human and mouse platelets. By performing biotinylation experiments we have elucidated that FKBP25 and FKBP38 might be found at different cellular location, the plasma membrane and the already described intracellular locations. Finally, FKBP25 and FKBP38 silencing significantly inhibits OAG-evoked NCCE in MEG-01 and HEK293 cells, while overexpression of FKBP38 does not modify NCCE in HEK293 cells. All together, these findings provide strong evidence for a role of immunophilins, including FKBP25 and FKBP38, in NCCE mediated by TRPC6.
Keywords: TRPC6; FK506; Platelets; NCCE; FKBP25; FKBP38;

CLOCK-BMAL1 is a key transcription factor complex of the molecular clock system that generates circadian gene expression and physiology in mammals. Here, we demonstrate that sumoylation of BMAL1 mediates the rapid activation of CLOCK-BMAL1 by CREB-binding protein (CBP) in nuclear foci and also the resetting of the circadian clock. Under physiological conditions, a bimolecular fluorescence complementation-based fluorescence resonance energy transfer (BiFC-FRET) assay revealed that CLOCK-BMAL1 rapidly dimerized and formed a ternary complex with CBP in discrete nuclear foci in response to serum stimuli. We found that the formation of this ternary complex requires sumoylation of BMAL1 by SUMO3. These processes were abolished by both the ectopic expression of the SUMP2/3-specific protease, SUSP1, and mutation of the major sumoylation site (Lys259) of BMAL1. Moreover, molecular inhibition of BMAL1 sumoylation abrogated acute Per1 transcription and severely dampened the circadian gene oscillation triggered by clock synchronization stimuli. Taken together, these findings suggest that sumoylation plays a critical role in the spatiotemporal co-activation of CLOCK-BMAL1 by CBP for immediate-early Per induction and the resetting of the circadian clock.
Keywords: Circadian clock; BMAL1; CBP; Sumoylation; Nuclear body; BiFC-based FRET; SUSP1; Clock resetting;

Fast endocytic recycling determines TRPC1–STIM1 clustering in ER–PM junctions and plasma membrane function of the channel by Lorena Brito de Souza; Hwei Ling Ong; Xibao Liu; Indu S. Ambudkar (2709-2721).
Stromal interaction molecule 1 (STIM1) senses depletion of ER–Ca2 + store and clusters in ER–PM junctions where it associates with and gates Ca2 + influx channels, Orai1 and TRPC1. Clustering of TRPC1 with STIM1 and Orai1 in these junctions is critical since Orai1-mediated Ca2 + entry triggers surface expression of TRPC1 while STIM1 gates the channel. Thus, plasma membrane function of TRPC1 depends on the delivery of the channel to the sites where STIM1 puncta are formed. This study examines intracellular trafficking mechanism(s) that determine plasma membrane expression and function of TRPC1 in cells where Orai1 and TRPC1 are endogenously expressed and contribute to Ca2 + entry. We report that TRPC1 is internalized by Arf6-dependent pathway, sorted to Rab5-containing early endosomes, and trafficked to ER–PM junctions by Rab4-dependent fast recycling. Overexpression of Arf6, or Rab5, but not the respective dominant negative mutants, induced retention of TRPC1 in early endosomes and suppressed TRPC1 function. Notably, cells expressing Arf6 or Rab5 displayed an inwardly rectifying ICRAC current that is mediated by Orai1 instead of TRPC1-associated ISOC, demonstrating that Orai1 function was not altered. Importantly, expression of Rab4, but not STIM1, with Rab5 rescued surface expression and function of TRPC1, restoring generation of ISOC. Together, these data demonstrate that trafficking via fast recycling endosomes determines TRPC1–STIM1 clustering within ER–PM junctions following ER–Ca2 + store depletion which is critical for the surface expression and function of the channel. Ca2 + influx mediated by TRPC1 modifies Ca2 +-dependent physiological response of cells.
Keywords: TRPC1; Orai1; STIM1; Store-operated calcium entry; Endocytic recycling; Surface expression;

DNAJB1 negatively regulates MIG6 to promote epidermal growth factor receptor signaling by Soo-Yeon Park; Hyo-Kyoung Choi; Jae Sung Seo; Jung-Yoon Yoo; Jae-Wook Jeong; Youngsok Choi; Kyung-Chul Choi; Ho-Geun Yoon (2722-2730).
Mitogen-inducible gene 6 (MIG6) is a tumor suppressor implicated in the development of human cancers; however, the regulatory mechanisms of MIG6 remain unknown. Here, using a yeast two-hybrid screen, we identified DnaJ homolog subfamily B member I (DNAJB1) as a novel MIG6-interacting protein. We found that DNAJB1 binds to and decreases MIG6 protein, but not mRNA, levels. DNAJB1 overexpression dosage-dependently decreased MIG6 protein levels. Conversely, DNAJB1 knockdown increased MIG6 protein levels. DNAJB1 destabilizes MIG6 by enhancing K48-linked ubiquitination of MIG6. However, knocking-down of DNAJB1 reduced the ubiquitination of MIG6. DNAJB1 positively regulates the epidermal growth factor receptors (EGFR) signaling pathway via destabilization of MIG6; however, DNAJB1 knockdown diminishes activation of EGFR signaling as well as elevation of MIG6. Importantly, the increased levels of MIG6 by DNAJB1 knockdown greatly enhanced the gefitinib sensitivity in A549 cells. Thus, our study provides a new molecular mechanism to regulate EGFR signaling through modulation of MIG6 by DNAJB1 as a negative regulator.
Keywords: DNAJB1; MIG6; EGFR; Gefitinib; Lung cancer;

Tfp1 is required for ion homeostasis, fluconazole resistance and N-Acetylglucosamine utilization in Candida albicans by Chang Jia; Kai Zhang; Qilin Yu; Bing Zhang; Chenpeng Xiao; Yijie Dong; Yulu Chen; Biao Zhang; Laijun Xing; Mingchun Li (2731-2744).
The vacuolar-type H+-ATPase (V-ATPase) is crucial for the maintenance of ion homeostasis. Dysregulation of ion homeostasis affects various aspects of cellular processes. However, the importance of V-ATPase in Candida albicans is not totally clear. In this study, we demonstrated the essential roles of V-ATPase through Tfp1, a putative V-ATPase subunit. Deletion of TFP1 led to generation of an iron starvation signal and reduced total iron content, which was associated with mislocalization of Fet34p that was finally due to disorders in copper homeostasis. Furthermore, the tfp1∆/∆ mutant exhibited weaker growth and lower aconitase activity on nonfermentable carbon sources, and iron or copper addition partially rescued the growth defect. In addition, the tfp1∆/∆ mutant also showed elevated cytosolic calcium levels in normal or low calcium medium that were relevant to calcium release from vacuole. Kinetics of cytosolic calcium response to an alkaline pulse and VCX1 (VCX1 encodes a putative vacuolar Ca2 +/H+ exchanger) overexpression assays indicated that the cytosolic calcium status was in relation to Vcx1 activity. Spot assay and concentration-kill curve demonstrated that the tfp1∆/∆ mutant was hypersensitive to fluconazole, which was attributed to reduced ergosterol biosynthesis and CDR1 efflux pump activity, and iron/calcium dysregulation. Interestingly, carbon source utilization tests found the tfp1∆/∆ mutant was defective for growth on N-Acetylglucosamine (GlcNAc) plate, which was associated with ATP depletion due to the decreased ability to catabolize GlcNAc. Taken together, our study gives new insights into functions of Tfp1, and provides the potential to better exploit V-ATPase as an antifungal target.
Keywords: Candida albicans; V-ATPase; Tfp1; Ion homeostasis; Fluconazole; GlcNAc;

A Tat ménage à trois — The role of Bacillus subtilis TatAc in twin-arginine protein translocation by Vivianne J. Goosens; Alba De-San-Eustaquio-Campillo; Rut Carballido-López; Jan Maarten van Dijl (2745-2753).
The twin-arginine translocation system (Tat) is a protein transport system that moves fully folded and cofactor-containing proteins across membranes of bacteria, archaea and thylakoids. The minimal Tat pathway is composed of two subunits, TatA and TatC. In some organisms TatA has been duplicated and evolved to form a third specialized subunit, TatB. The Bacillus subtilis genome encodes two TatC subunits (TatCd and TatCy) and three TatA subunits (TatAd, TatAy and TatAc). These subunits combine to form two parallel minimal pathways, TatAy-TatCy and TatAd-TatCd. The purpose and role of the third TatA component, TatAc, has remained ambiguous. In this study we examined the translocation of two natively expressed TatAy-TatCy-dependent substrates, EfeB and QcrA, in various Tat-deficient genetic backgrounds. More specifically, we examined the ability of different mutated TatAy subunits to complement for the absence of wild-type TatAy. We further detailed a graded growth phenotype associated with the functional translocation of EfeB. We found that in various instances where specific amino acid substitutions were made in TatAy, a definite TatAc-associated growth phenotype occurred in genetic backgrounds lacking TatAc. Altogether, our findings show that TatAy and TatAc interact and that this TatAy–TatAc interaction, although not essential, supports the translocation of the Tat substrate EfeB when TatAy function is compromised. This implies that the third TatA-like protein in B. subtilis could represent an intermediate evolutionary step in TatA-TatB specialization.
Keywords: Twin-arginine translocation; TatA; TatB; TatAc; EfeB; Bacillus subtilis;

Corrigendum to “Recruitment and activation of SLK at the leading edge of migrating cells requires Src family kinase activity and the LIM-only protein 4” [Biochim. Biophys. Acta 1853/7 (2015) 1683–1692] by Kyla D. Baron; Khalid Al-Zahrani; Jillian Conway; Cédrik Labrèche; Christopher J. Storbeck; Jane E. Visvader; Luc A. Sabourin (2754).