BBA - Molecular Cell Research (v.1823, #12)
Editorial Board (i).
A role of DNA-dependent protein kinase for the activation of AMP-activated protein kinase in response to glucose deprivation by Parmeshwar Narayan Amatya; Hong-Beum Kim; Seon-Joo Park; Cha-Kyung Youn; Jin-Won Hyun; In-Youb Chang; Jung-Hee Lee; Ho Jin You (2099-2108).
The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an essential role in double-strand break repair by initially recognizing and binding to DNA breaks. Here, we show that DNA-PKcs interacts with the regulatory γ1 subunit of AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that has been proposed to function as a “fuel gauge” to monitor changes in the energy status of cells and is controlled by the upstream kinases LKB1 and Ca2+/calmodulin-dependent kinase kinase (CaMKK). In co-immunoprecipitation analyses, DNA-PKcs and AMPKγ1 interacted physically in DNA-PKcs-proficient M059K cells but not in DNA-PKcs-deficient M059J cells. Glucose deprivation-stimulated phosphorylation of AMPKα on Thr172 and of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, is substantially reduced in M059J cells compared with M059K cells. The inhibition or down-regulation of DNA-PKcs by the DNA-PKcs inhibitors, wortmannin and Nu7441, or by DNA-PKcs siRNA caused a marked reduction in AMPK phosphorylation, AMPK activity, and ACC phosphorylation in response to glucose depletion in M059K, WI38, and IMR90 cells. In addition, DNA–DNA-PKcs−/− mouse embryonic fibroblasts (MEFs) exhibited decreased AMPK activation in response to glucose-free conditions. Furthermore, the knockdown of DNA-PKcs led to the suppression of AMPK (Thr172) phosphorylation in LKB1-deficient HeLa cells under glucose deprivation. Taken together, these findings support the positive regulation of AMPK activation by DNA-PKcs under glucose-deprived conditions in mammalian cells.► We identify AMPKγ1 as an endogenous interacting partner for DNA-PKcs. ► Phospho-AMPK and AMPK activity is decreased in DNA-PK-deficient cells. ► Inhibition or knockdown of DNA-PK decreased AMPK activity. ► DNA-PK plays a critical role in the regulation of AMPK activation.
Keywords: DNA-PKcs; AMPK; ACC; Glucose depletion;
Tyrosine phosphorylation of a SNARE protein, Syntaxin 17: Implications for membrane trafficking in the early secretory pathway by Madhavi Muppirala; Vijay Gupta; Ghanshyam Swarup (2109-2119).
The T-cell protein tyrosine phosphatase is expressed as two splice variants — TC45, a nuclear protein, and TC48, which is localized predominantly in the ER (endoplasmic reticulum). Yeast two-hybrid screening revealed direct interaction of TC48 with Syntaxin17, a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein localized predominantly in the ER and to some extent in the ER-Golgi intermediate compartment. Syntaxin 17 did not interact with TC45. C-terminal 40 amino acids of TC48 were sufficient for interaction with syntaxin 17. Overexpressed syntaxin 17 was phosphorylated at tyrosine upon pervanadate treatment (a tyrosine phosphatase inhibitor/tyrosine kinase activator) of COS-1 cells. Mutational analysis identified Tyr156 in the cytoplasmic domain as the major site of phosphorylation. Endogenous syntaxin 17 was phosphorylated by pervanadate treatment in CHO and MIN6 cells but was not phosphorylated in a variety of other cell lines tested. c-Abl was identified as one of the kinases, which phosphorylates syntaxin 17 in MIN6 cells. Phosphorylation of endogenous and overexpressed syntaxin 17 was reduced in the presence of IGF receptor and EGF receptor kinase inhibitors. Serum depletion reduced pervanadate-induced phosphorylation of endogenous syntaxin 17. TC48 coexpression reduced phosphorylation of syntaxin 17 by pervanadate and purified TC48 directly dephosphorylated syntaxin 17. β-COP dispersal by overexpressed syntaxin 17 was reduced after pervanadate-induced phosphorylation. A phospho-mimicking mutant (Y156E) of syntaxin 17 showed reduced interaction with COPI vesicles. These results suggest that tyrosine phosphorylation of syntaxin 17 is likely to have a role in regulating syntaxin 17 dependent membrane trafficking in the early secretory pathway.► Syntaxin 17, a SNARE protein, gets phosphorylated on tyrosine 156. ► Tyrosine phosphatase TC48 interacts with syntaxin 17 and dephosphorylates it. ► c-Abl was identified as one of the kinases, which phosphorylates syntaxin 17. ► A phospho-mimicking mutant of syntaxin 17 shows reduced binding with beta COP. ► Results indicate a role of tyrosine phosphorylation in the early secretory pathway.
Keywords: Tyrosine phosphatase TC48; Syntaxin 17; Tyrosine phosphorylation; Tyrosine dephosphorylation; Membrane trafficking;
Novel involvement of leukotriene B4 receptor 2 through ERK activation by PP2A down-regulation in leukotriene B4-induced keratin phosphorylation and reorganization of pancreatic cancer cells by Mi Kyung Park; Youngran Park; Jaegal Shim; Hye Ja Lee; Sanghee Kim; Chang Hoon Lee (2120-2129).
Perinuclear reorganization via phosphorylation of specific serine residues in keratin is involved in the deformability of metastatic cancer cells. The level of leukotriene B4 is high in pancreatic cancers. However, the roles of LTB4 and its cognate receptors in keratin reorganization of pancreatic cancers are not known. LTB4 dose-dependently induced phosphorylation and reorganization of Keratin 8 (K8) and these processes were reversed by LY255283 (BLT2 antagonist). BLT2 agonists such as Comp A and 15(S)-HETE also induced phosphorylation of serine 431 in K8. Moreover, Comp A-induced K8 phosphorylation and reorganization were blocked by LY255283. Gene silencing of BLT2 suppressed Comp A-induced K8 phosphorylation and reorganization in PANC-1 cells. Over-expression of BLT2 promoted K8 phosphorylation. Comp A promoted the migration of PANC-1 cells in a dose-dependent manner, and LY255283 blocked Comp A-induced migration, respectively. PD98059 (ERK inhibitor) suppressed Comp A-induced phosphorylation of serine 431 and reorganization of K8. Gene silencing of BLT2 suppressed the expression of pERK, and over-expression of BLT2 increased the expression of pERK even without Comp A. Comp A induced the expression of active ERK (pERK) and BLT2. These inductions were blocked by PD98059. Comp A decreased PP2A expression and hindered the binding of PP2A to the K8, leading to the activation of ERK. PD98059 suppressed the Comp A-induced migration of PANC-1 cells and BLT2 over-expression-induced migration of PANC-1 cells. Overall, these results suggest that BLT2 is involved in LTB4‐induced phosphorylation and reorganization through ERK activation by PP2A downregulation, leading to increased migration of PANC‐1 cells.Display Omitted► Leukotriene B4 (LTB4) induces phosphorylation and reorganization of keratin 8 (K8). ► LTB4 receptor BLT2 is involved in LTB4-induced phosphorylation and reorganization of K8. ► ERK kinase mediates the LTB4-induced 431 serine phosphorylation of keratin 8. ► Compound A decreases the expression of protein phosphatase 2A leading to ERK activation. ► Compound A induces the expression of BLT2 and this induction was blocked by PD98059.
Keywords: Leukotriene B4; Phosphorylation of keratin-8; Perinuclear reorganization of keratin-8; LY255283; Leukotriene B4 receptor 2; PP2A;
Telomerase antagonist imetelstat inhibits esophageal cancer cell growth and increases radiation-induced DNA breaks by Xuping Wu; Shirin Smavadati; Katarina Nordfjäll; Krister Karlsson; Fredrik Qvarnström; Martin Simonsson; Michael Bergqvist; Sergei Gryaznov; Simon Ekman; Ylva Paulsson-Karlsson (2130-2135).
Telomerase is mainly active in human tumor cells, which provides an opportunity for a therapeutic window on telomerase targeting. We sought to evaluate the potential of the thio-phosphoramidate oligonucleotide inhibitor of telomerase, imetelstat, as a drug candidate for treatment of esophageal cancer. Our results showed that imetelstat inhibited telomerase activity in a dose-dependent manner in esophageal cancer cells. After only 1 week of imetelstat treatment, a reduction of colony formation ability of esophageal cancer cells was observed. Furthermore, long-term treatment with imetelstat decreased cell growth of esophageal cancer cells with different kinetics regarding telomere lengths. Short-term imetelstat treatment also increased γ-H2AX and 53BP1 foci staining in the esophageal cancer cell lines indicating a possible induction of DNA double strand breaks (DSBs). We also found that pre-treatment with imetelstat led to increased number and size of 53BP1 foci after ionizing radiation. The increase of 53BP1 foci number was especially pronounced during the first 1 h of repair whereas the increase of foci size was prominent later on. This study supports the potential of imetelstat as a therapeutic agent for the treatment of esophageal cancer.► Imetelstat inhibits telomerase activity in esophageal cancer cells. ► Imetelstat decreases cell proliferation and colony formation ability. ► Imetelstat increases the number and size of radiation induced DNA breaks.
Keywords: Telomerase; Esophageal cancer; Imetelstat; DNA double strand break; γ-H2AX and 53BP1 foci;
Distinctive roles of PLD signaling elicited by oxidative stress in synaptic endings from adult and aged rats by Melina V. Mateos; Norma M. Giusto; Gabriela A. Salvador (2136-2148).
The role of iron in oxidative injury in the nervous system has been extensively described. However, little is known about the role of lipid signal transduction in neurodegeneration processes triggered by iron overload. The purpose of this work was to characterize the regulation and the crosstalk between phosphatidylcholine (PC)-derived diacylglycerol (DAG) and cannonical signaling pathways during iron-induced oxidative stress in cerebral cortex synaptic endings (Syn) obtained from adult (4 months old) and aged (28 months old) rats. DAG production was increased in Syn exposed to iron. This rise in DAG formation was due to phospholipase D1 (PLD1) and PLD2 activations. In adult rats, PKD1, ERK1/2 and PKCα/βII activations were PLD1 and PLD2 dependent. In contrast, in senile rats, DAG formation catalyzed by PLDs did not participate in PKD1, ERK1/2 and PKCα/βII regulations, but it was dependent on ERK and PKC activities. Iron-induced oxidative stress promoted an increased localization of PLD1 in membrane rafts, whereas PLD2 was excluded from these domains and appeared to be involved in glutamate transporter function. Our results show a differential regulation and synaptic function of DAG generated by PLDs during iron-induced oxidative stress as a consequence of aging.► PC-derived DAG generation in synaptic endings from adult and aged rats exposed to oxidative stress has been characterized. ► Synaptic PLD 1 and PLD2 activities and their crosstalk with PKD1, ERK1/2 and PKCα/βII were studied. ► PLD activities and their localization in membrane rafts were evaluated during synaptic oxidative stress. ► The activity of Glutamate and GABA transporters and their regulation by PLDs and the ERK pathway were also evaluated. ► We advanced in new knowledge on the role of PLD during neurodegenerative processes triggered by oxidative stress.
Keywords: Phospholipase D; Protein kinase D; Oxidative injury; Aging; Diacylglycerol; Membrane microdomain;
ATF4 interacts with Abro1/KIAA0157 scaffold protein and participates in a cytoprotective pathway by Camilla T. Ambivero; Lucia Cilenti; Antonis S. Zervos (2149-2156).
Abro1 (Abraxas brother 1), also known as KIAA0157, is a scaffold protein that recruits various polypeptides to assemble the BRISC (BRCC36 isopeptide) deubiquitinating enzyme (DUB) complex. The BRISC enzyme has a Lys63-linked deubiquitinating activity and is comprised of four known subunits: MERIT40 (mediator of Rap80 interactions and targeting 40 kDa), BRE (brain and reproductive organ-expressed), BRCC36 (BRCA1/BRCA2-containing complex, subunit 3) and Abro1. We have previously shown that Abro1 has a cytoprotective role that involves the BRISC DUB complex acting on specific Lys63-linked polyubiquitinated substrates. In this report we identify three members of the AP-1 (activating protein-1) family, the ATF4, ATF5 (activating transcription factor) and JunD proteins, as specific interactors of Abro1. The function of ATF4–Abro1 interaction was investigated under normal conditions as well as under cellular stress. Abro1 is predominantly cytoplasmic, but during cellular stress it enters the nucleus and co-localizes with ATF4. Furthermore, this interaction with ATF4 is necessary and essential for the cytoprotective function of Abro1 following oxidative stress. The ability of Abro1 to specifically interact with a number of transcription factors suggests a new mechanism of regulation of the BRISC DUB complex. This regulation involves the participation of at least three known members of the AP-1 family of transcription factors.► ATF4 is identified as a specific interactor of Abro1 scaffold protein. ► Abro1 is the scaffold protein and it has a cytoprotective function. ► ATF4 interacts with Abro1 during oxidative stress. ► ATF4 binding to Abro1 causes the protein to translocate to the cell nucleus. ► The cytoprotective role of Abro1 is dependent on its interaction with ATF4.
Keywords: Activator protein-1 (AP-1) transcription factor; Activating transcription factor 4 (ATF4); Abraxas brother 1 (Abro1/KIAA0157); BRCC36 isopeptidase (BRISC) complex; Protein–protein interaction;
Noradrenergic activity regulated dexamethasone-induced increase of 5-HT3 receptor-mediated glutamate release in the rat's prelimbic cortex by Xiaoqin Zhang; Qingfang Kan; Yingmei Fu; Shanshan Liu; Zeping Dai; Yi Dong (2157-2167).
Stress hormone, glutamatergic system, serotonergic system and the noradrenergic system are involved in depressive disorders. However, the relationship among these is still unclear. The present study examined the effect of dexamethasone (DEX) on the presynaptic glutamate release of synaptosomes from the rat's prelimbic cortex by using biochemical methods combined with pharmacological approaches. The results showed that dexamethasone increased the glutamate release of synaptosomes in a dose-dependent manner. The concentration–response relationship of this effect of DEX was inverse U-shaped with a maximum at 3 μm. Further study showed that glucocorticoid receptor (GR) antagonist and GR siRNA had no effect on the DEX-induced glutamate release but 5-HT3 receptor antagonist could block the DEX-induced glutamate release which suggested that DEX produced the increased effect on the glutamate release not by GR, but through the activation of the 5-HT3 receptors which led to the influx of extrasynaptosomal Ca2 +. Moreover, β3 adrenergic receptor agonist could block the DEX-induced glutamate release. This result suggested that the effect of DEX on the glutamate release could be regulated by noradrenergic system. The mechanism study showed that β3 adrenergic receptors regulated the DEX-induced glutamate release via Gs protein–adenylate cyclase (AC)–protein kinase A (PKA) signal transduction pathway.► DEX increased the glutamate release of synaptosomes in a dose-dependent manner. ► DEX exerts its effect not by GR, but through the activation of the 5-HT3 receptors. ► The activation of β3R inhibits the DEX-induced glutamate release by Gs–AC–PKA pathway.
Keywords: Dexamethasone; Glutamate release; β3 receptor; 5-HT3 receptor; Synaptosome;
The emerging multiple roles of nuclear Akt by Alberto M. Martelli; Giovanna Tabellini; Daniela Bressanin; Andrea Ognibene; Kaoru Goto; Lucio Cocco; Camilla Evangelisti (2168-2178).
Akt is a central player in the signal transduction pathways activated in response to many growth factors, hormones, cytokines, and nutrients and is thought to control a myriad of cellular functions including proliferation and survival, autophagy, metabolism, angiogenesis, motility, and exocytosis. Moreover, dysregulated Akt activity is being implicated in the pathogenesis of a growing number of disorders, including cancer. Evidence accumulated over the past 15 years has highlighted the presence of active Akt in the nucleus, where it acts as a fundamental component of key signaling pathways. For example, nuclear Akt counteracts apoptosis through a block of caspase-activated DNase: deoxyribonuclease and inhibition of chromatin condensation, and is also involved in cell cycle progression control, cell differentiation, mRNA: messenger RNA export, DNA repair, and tumorigenesis. In this review, we shall summarize the most relevant findings about nuclear Akt and its functions.► Discussed the latest research on nuclear Akt ► Special emphasis is placed on anti-apoptotic signaling controlled by nuclear Akt. ► Nuclear Akt-interacting proteins control key processes. ► Intranuclear p-Akt levels are emerging as a predictor of tumor prognosis.
Keywords: Akt; Nucleus; Cell survival; Cell proliferation; Tumorigenesis; mRNA export;
Endoplasmic reticulum stress induces the expression of COX-2 through activation of eIF2α, p38-MAPK and NF-κB in advanced glycation end products stimulated human chondrocytes by Zafar Rasheed; Tariq M. Haqqi (2179-2189).
During aging, advanced glycation end products (AGEs) accumulate in articular cartilage. In this study we determined whether AGEs induce endoplasmic reticulum (ER) stress and studied the ER stress-activated pathways that stimulate cyclooxygenase-2 (COX-2) expression in human chondrocytes.Chondrocytes were stimulated with AGE-BSA. Gene expression was determined by quantitative PCR and protein expression was studied by immunoblotting. Studies to elucidate involved pathways were executed using siRNAs and specific inhibitors of eukaryotic initiation factor-2α (eIF2α), MAPKs and NF-κB.AGE-BSA induced expression of GRP78 with concomitant increase in COX-2 expression was observed in human chondrocytes. In addition, expression of Bag-1, an ER stress marker was also increased by AGE-BSA. RAGE knockdown inhibited AGE-BSA-induced expression of GRP78 and COX-2. Treatment with eIF2α inhibitor or eIF2α knockdown inhibited AGE-BSA-induced expression of GRP78 and COX-2 with decreased PGE2 production. Treatment with SB202190 inhibited AGE-BSA-induced expression of GRP78 and COX-2, while treatment with PD98051 inhibited AGE-BSA-induced GRP78 protein expression but had no effect on COX-2 protein expression. SP600125 had no effect on either GRP78 or COX-2 protein expression. Bay 11-7082 suppressed AGE-BSA-induced GRP78 and COX-2 expression. AGE-BSA-induced activation of NF-κB was inhibited by treatment with SB202190 and by eIF2α knockdown, but was not inhibited when chondrocytes were treated with SP600125 or PD98059.This study demonstrates that AGEs induce ER stress and stimulate the expression of COX-2 through eIF2α, p38-MAPK and NF-κB pathways in human chondrocytes. Our results provide important insights into cartilage degradation in osteoarthritis associated with latent ER stress.► AGEs induce ER stress in human chondrocytes. ► RAGE mediated ER stress stimulates COX-2 expression. ► ER stress signaling involves RAGE → eIF2α → p38MAPK → NF-κB pathways.
Keywords: ER stress; Chondrocyte; OA; eIF2-α; p38-MAPK; NF-κB;
Bax inhibitor-1 enhances survival and neuronal differentiation of embryonic stem cells via differential regulation of mitogen-activated protein kinases activities by Kilsoo Jeon; Hyejin Lim; Jung-Hyun Kim; Dawoon Han; Eung-Ryoung Lee; Gwang-Mo Yang; Min-Kyoung Song; Jin-Hoi Kim; Ssang-Goo Cho (2190-2200).
Bax inhibitor-1 (BI-1), a member of the BI-1 family of integral membrane proteins, was originally identified as an inhibitor of stress-induced cell death in mammalian cells. Previous studies have shown that the withdrawal of leukemia inhibitory factor (LIF) results in differentiation of the majority of mouse embryonic stem (mES) cells into various cell lineages, while some ES cells die within 3 days. Thus, to investigate the function of BI-1 in ES cell survival and neuronal differentiation, we generated mES cell lines that overexpress BI-1 or a carboxy-terminal BI-1ΔC mutant. Overexpression of BI-1 in mES cells significantly increased cell viability and resistance to apoptosis induced by LIF withdrawal, while the control vector or BI-1ΔC-overexpressing mES cells had no effect. Moreover, overexpression of BI-1 produced significant inhibition of the p38 mitogen-activated protein kinases (MAPK) pathway in response to LIF withdrawal, while activity of the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) MAPK pathway was increased. Interestingly, we found that BI-1-overexpressing cells showed higher expression levels of neuroectodermal markers (Otx1, Lmx1b, En1, Pax2, Wnt1, Sox1, and Nestin) and greater neuronal differentiation efficiency than control or BI-1ΔC-overexpressing mES cells did. Considering these findings, our results indicated that BI-1-modulated MAPK activity plays a key role in protecting mES cells from LIF-withdrawal-induced apoptosis and in promoting their differentiation toward neuronal lineages.► BI-1 overexpression reduces LIF withdrawal-induced apoptosis in mES cells. ► BI-1 is originally identified to induce neuroectodermal differentiation in mES cells. ► Overexpression of BI-1 leads to differential modulation of MAPK activities. ► BI-1-mediated MAPK regulation may play a key role in neuronal differentiation.
Keywords: Bax inhibitor-1; Mouse embryonic stem cell; Mitogen-activated protein kinase; Neuronal differentiation;
Resistance to butyrate impairs bile acid-induced apoptosis in human colon adenocarcinoma cells via up-regulation of Bcl-2 and inactivation of Bax by Juan I. Barrasa; Angélica Santiago-Gómez; Nieves Olmo; María Antonia Lizarbe; Javier Turnay (2201-2209).
A critical risk factor in colorectal carcinogenesis and tumor therapy is the resistance to the apoptotic effects of different compounds from the intestinal lumen, among them butyrate (main regulator of colonic epithelium homeostasis). Insensitivity to butyrate-induced apoptosis yields resistance to other agents, as bile acids or chemotherapy drugs, allowing the selective growth of malignant cell subpopulations. Here we analyze bile acid-induced apoptosis in a butyrate-resistant human colon adenocarcinoma cell line (BCS-TC2.BR2) to determine the mechanisms that underlay the resistance to these agents in comparison with their parental butyrate-sensitive BCS-TC2 cells. This study demonstrates that DCA and CDCA still induce apoptosis in butyrate-resistant cells through increased ROS production by activation of membrane-associated enzymes and subsequent triggering of the intrinsic mitochondrial apoptotic pathway. Although this mechanism is similar to that described in butyrate-sensitive cells, cell viability is significantly higher in resistant cells. Moreover, butyrate-resistant cells show higher Bcl‐2 levels that confer resistance to bile acid-induced apoptosis sequestering Bax and avoiding Bax-dependent pore formation in the mitochondria. We have confirmed that this resistance is reverted using the Bcl-2 inhibitor ABT-263, thus demonstrating that the lower sensitivity of butyrate-resistant cells to the apoptotic effects of bile acids is mainly due to increased Bcl-2 levels.► Bile acids increase ROS by activation of membrane enzymes in colon cancer cells. ► ROS trigger the MPT and the intrinsic apoptotic pathway followed by Bax activation. ► Bile-acid induced apoptosis is impaired in butyrate-resistant colon cancer cells. ► Resistance is due to overexpression of Bcl-2 and blockage of Bax activation. ► Resistance can be reverted by inhibition of Bcl-2 using ABT-263 (navitoclax).
Keywords: ABT-263; Apoptosis; Bcl-2; Bile acid; Butyrate resistance; Human colon adenocarcinoma;
Diacylglycerol kinase δ1 transiently translocates to the plasma membrane in response to high glucose by Masato Takeuchi; Shizuka Sakiyama; Takako Usuki; Hiromichi Sakai; Fumio Sakane (2210-2216).
The type II diacylglycerol kinases (DGKs) contain several functional domains such as a pleckstrin homology (PH) domain, two C1 domains and a sterile α-motif (SAM) domain. It was previously revealed that DGKδ contributes to hyperglycemia-induced peripheral insulin resistance and thereby exacerbate the severity of type 2 diabetes. Moreover, a high extracellular concentration of glucose activated DGKδ in skeletal muscle cells, which was followed by a reduction in the intracellular diacylglycerol levels and the inactivation of protein kinase Cα, the enzyme that phosphorylates and inactivates the insulin receptor. However, the intracellular behavior of DGKδ upon high glucose stimulation remains unclear. In this study, we found that DGKδ1, but not a splice variant DGKδ2 or the other type II DGKη1/2, translocated from the cytoplasm to the plasma membrane in human embryonic kidney HEK293 and mouse myoblast C2C12 cells within 5 min in response to high glucose levels. The translocation was inhibited by phosphatidylinositol 3-kinase inhibitors, LY294002 and GDC-0941, suggesting that the event is regulated via the phosphatidylinositol 3-kinase pathway. Moreover, we revealed that the PH and C1 domains are responsible for the plasma membrane translocation and that the SAM domain negatively regulates the translocation. These results indicate that DGKδ1 is the sole type II DGK isoform that responds rapidly and dynamically to high glucose levels.► We revealed the high glucose-dependent translocation of DGKδ1 to the plasma membrane. ► The plasma membrane translocation of DGKδ1 is rapid and transient. ► The translocation is a δ1 isoform-specific event among type II DGKs. ► The PH and C1 domains are responsible for the translocation. ► The SAM domain negatively regulates the translocation.
Keywords: Diacylglycerol kinase; Glucose; Plechstrin homology domain; C1 domain; Plasma membrane; Translocation;
Selenoprotein W promotes cell cycle recovery from G2 arrest through the activation of CDC25B by Yong Hwan Park; Yeong Ha Jeon; Ick Young Kim (2217-2226).
Selenoprotein W (SelW) contains a highly reactive selenocysteine (Sec; U) in the CXXU motif corresponding to the CXXC motif in thioredoxin (Trx) and thus it appears to be involved in regulating the cellular redox state. Recent reports on the interaction between SelW and 14-3-3 suggest that SelW may be redox dependently involved in the cell cycle. However, the precise function of SelW has not yet been elucidated. Here, we show that SelW is involved in the G2–M transition, especially in the recovery from G2 arrest after deoxyribonucleic acid (DNA) damage. Knockdown of SelW significantly accumulated phosphorylated cyclin‐dependent kinase (Cdk1), which eventually led to a delay in recovery from G2 arrest. We also found that inactive Cdk1 is caused by the sustained inactivation of CDC25B, which removes the inhibitory phosphate from Cdk1. Our observation from this study reveals that SelW activated CDC25B by promoting the dissociation of 14-3-3 from CDC25B through the reduction of the intramolecular disulfide bond during recovery. We suggest that SelW plays an important role in the recovery from G2 arrest by determining the dissociation of 14-3-3 from CDC25B in a redox-dependent manner.► SelW is involved in recovery from G2 arrest induced by DNA damage. ► SelW promotes the dissociation of 14-3-3 from CDC25B. ► SelW reduces the intramolecular disulfide bond of CDC25B. ► Sensitivity to anticancer drugs is increased in SelW knockdown cells.
Keywords: Selenoprotein W; 14-3-3; Cell cycle; G2 arrest; Cdk1; CDC25B;
Involvement of the prostaglandin D2 signal pathway in retinoid-inducible gene 1 (RIG1)-mediated suppression of cell invasion in testis cancer cells by Chang-Chieh Wu; Rong-Yaun Shyu; Chun-Hua Wang; Tzung-Chieh Tsai; Lu-Kai Wang; Mao-Liang Chen; Shun-Yuan Jiang; Fu-Ming Tsai (2227-2236).
Retinoid-inducible gene 1 (RIG1), also called tazarotene-induced gene 3, belongs to the HREV107 gene family, which contains five members in humans. RIG1 is expressed in high levels in well-differentiated tissues, but its expression is decreased in cancer tissues and cancer cell lines. We found RIG1 to be highly expressed in testicular cells. When RIG1 was expressed in NT2/D1 testicular cancer cells, neither cell death nor cell viability was affected. However, RIG1 significantly inhibited cell migration and invasion in NT2/D1 cells. We found that prostaglandin D2 synthase (PTGDS) interacted with RIG1 using yeast two-hybrid screens. Further, we found PTGDS to be co-localized with RIG1 in NT2/D1 testis cells. In RIG1-expressing cells, elevated levels of prostaglandin D2 (PGD2), cAMP, and SRY-related high-mobility group box 9 (SOX9) were observed. This indicated that RIG1 can enhance PTGDS activity. Silencing of PTGDS expression significantly decreased RIG1-mediated cAMP and PGD2 production. Furthermore, silencing of PTGDS or SOX9 alleviated RIG1-mediated suppression of migration and invasion. These results suggest that RIG1 will suppress cell migration/invasion through the PGD2 signaling pathway. In conclusion, RIG1 can interact with PTGDS to enhance its function and to further suppress NT2/D1 cell migration and invasion. Our study suggests that RIG1-PGD2 signaling might play an important role in cancer cell suppression in the testis.► Both RIG1 and PTGDS are highly expressed in the testicular normal cells. ► RIG1 suppresses migration and invasion of NT2/D1 testicular cells. ► RIG1 immunoprecipitates and co-localizes with PTGDS. ► Increasing PTGDS activity when cell expressing of RIG1 ► RIG1-induced cell invasion suppression is done by PGD2 bound DP1 receptor.
Keywords: Retinoid-inducible gene 1; Prostaglandin D2 synthase; Testis; HREV107 type II tumor suppressor;
Impaired c-src activation and motility defects in PEA3-null fibroblasts by Ziad Y. Chaar; Laura Hastings; Roshan Sriram; Marlene McKay; Lilia Antonova; John A. Hassell; Luc A. Sabourin (2237-2242).
Null mutations in the pea3 allele compromise the capacity of mammary tumors to metastasize in MMTV-Neu/ErbB2/HER2 transgenic mice, indicating a motility defect in PEA3-null cells. Cellular and biochemical analyses of established PEA3-null fibroblasts show impaired motility and aberrant localization of adhesion proteins in spreading cells. Our results show that PEA3 −/− cells express normal levels of key adhesion components, but that spreading PEA3-null cells fail to activate c-src and to downregulate phospho-FAK(Y397), suggesting that focal adhesion signaling is impaired. Supporting this, biochemical analysis revealed that adhesion complex-associated proteins such as p130Cas failed to undergo tyrosine phosphorylation and dissociated from the adhesion complex with delayed kinetics. Overall our data show that the motility defects observed in PEA3-null cells are due to altered adhesion signaling.► We show a reduction in focal adhesions and actin stress fibers in PEA3-null cells. ► Our study show motility defects in these cells on fibronectin-coated substrates. ► We show that these cells have defects in c-src activation and FAK tyrosine phosphorylation. ► Our data show that these cells have impaired adhesion complex activation and disassembly.
Keywords: c-src; PEA3; Adhesion complex; p130Cas; Paxillin; FAK;
Fluorescent ligand-directed co-localization of the parathyroid hormone 1 receptor with the brush-border scaffold complex of the proximal tubule reveals hormone-dependent changes in ezrin immunoreactivity consistent with inactivation by Jun Guo; Lige Song; Minlin Liu; Matthew J. Mahon (2243-2253).
Through binding to parathyroid hormone (PTH), PTH1R interacts with kidney-specific scaffold proteins, including the sodium hydrogen exchanger regulatory factors 1 and 2 (NHERFs), and ezrin. To facilitate in vivo localization, tetramethylrhodamine-labeled PTH (PTH-TMR) was used as a fluorescent probe. In mice, PTH-TMR localizes to luminal surfaces of tubular S1 segments that overlap PTH1R immunostaining, but does not directly overlap with megalin-specific antibodies. PTH-TMR staining directly overlaps with Npt2a in nascent, endocytic vesicles, marking the location of transporter regulation. PKA substrate antibodies display marked staining increases in segments labeled with PTH-TMR, demonstrating a functional effect. In the presence of secondary hyperparathyroidism, PTH-TMR staining is markedly reduced and shifts to co-localizing with megalin. At 15 min post-injection, PTH-TMR-labeled vesicles do not co-localize with either NHERF or ezrin, suggesting PTH1R dissociation from the scaffold complex. At the 5 min time point, PTH-TMR stains the base of microvilli where it localizes with both NHERF2 and ezrin, and only partially with NHERF1. Strikingly, the bulk of ezrin protein becomes undetectable with the polyclonal, CS3145 antibody, revealing a PTH-induced conformational change in the scaffold. A second ezrin antibody (3C12) is capable of detecting the altered ezrin protein. The CS3145 antibody only binds to the active form of ezrin and fails to recognize the inactive form, while the 3C12 reagent can detect either active or inactive ezrin. Here we show that the PTH1R is part of the ezrin scaffold complex and that acute actions of PTH suggest a rapid inactivation of ezrin in a spatially defined manner.Display Omitted► Parathyroid hormone-TMR (a fluorescent dye) marks the location of the PTH1R in kidney. ► PTH1R is prominently expressed on the luminal side of proximal convoluted tubules. ► Apical PTH1R is functional, including activation of PKA and NPT2a regulation. ► PTH-TMR/PTH1R directly co-localizes to a sub-population of ezrin. ► PTH induces a conformational change in bulk of ezrin consistent with inactivation.
Keywords: Parathyroid hormone; Parathyroid hormone receptor; Ezrin; NHERF1; NHERF2; Megalin;
Arabidopsis heterotrimeric G protein β subunit interacts with a plasma membrane 2C-type protein phosphatase, PP2C52 by Daisuke Tsugama; Hua Liu; Shenkui Liu; Tetsuo Takano (2254-2260).
Heterotrimeric G proteins (Gα, Gβ, Gγ) play important roles in signal transduction among various eukaryotic species. G proteins transmit signals by regulating the activities of effector proteins, but only a few Gβ-interacting effectors have been identified in plants. Here we show by a yeast two-hybrid screen that a putative myristoylated 2C-type protein phosphatase, PP2C52, is an Arabidopsis Gβ (AGB1)-interacting partner. The interaction between AGB1 and PP2C52 was confirmed by an in vitro pull-down assay and a bimolecular fluorescence complementation assay. PP2C52 transcripts were detected in many tissues. PP2C52 was localized to the plasma membrane and a mutation in the putative myristoylation site of PP2C52 disrupted its plasma membrane localization. Our results suggest that PP2C52 interacts with AGB1 on the plasma membrane and transmits signals via dephosphorylation of other proteins.► Arabidopsis G protein β subunit, AGB1, interacts with PP2C52. ► PP2C52 is localized to the plasma membrane, where it interacts with AGB1. ► The activity of PP2C52 is not affected by AGB1 and the γ subunit, AGG1.
Keywords: Abscisic acid; Arabidopsis; Heterotrimeric G protein β subunit; Myristoylation; PP2C; Protein–protein interaction;
Neuroglobin involvement in respiratory chain function and retinal ganglion cell integrity by Christophe Lechauve; Sébastien Augustin; Hélène Cwerman-Thibault; Aïcha Bouaita; Valérie Forster; Chantal Célier; Pierre Rustin; Michael C. Marden; José-Alain Sahel; Marisol Corral-Debrinski (2261-2273).
Neuroglobin is a member of the globin superfamily expressed in vertebrate brain and retina. The protein is thought to be involved in neuronal protection from hypoxia or oxidative stress and could represent a key element of Alzheimer disease pathogenesis. Our aim was to determine whether neuroglobin could be directly associated with mitochondrial metabolism and integrity. We identified three different forms of neuroglobin in the retina, varying in their apparent molecular masses; all forms are abundant in mitochondrial fractions. This indicates that a significant fraction of the protein localizes within the organelle either in the matrix or in the matrix side of the inner membrane. Since neuroglobin was especially abundant in the ganglion cell layer, we transduced retinal ganglion cells with an anti-neuroglobin short hairpin RNA using in vivo electroporation. Neuroglobin knockdown leads to reduced activities of respiratory chain complexes I and III, degeneration of retinal ganglion cells, and impairment of visual function. The deleterious effect on cell survival was confirmed in primary retinal ganglion cells subjected to inhibition of neuroglobin expression. Hence, neuroglobin should be considered as a novel mitochondrial protein involved in respiratory chain function which is essential for retinal ganglion cell integrity.► Neuroglobin localizes within mitochondria either in the matrix or in the matrix side of the inner membrane. ► Neuroglobin knockdown in primary ganglion cell cultures compromises cell survival. ► Retinal ganglion cells and their axons degenerate when neuroglobin is down regulated. ► Respiratory chain complex I and III activities in optic nerves depend on the presence of neuroglobin.
Keywords: Neuroglobin; Mitochondrion; Respiratory chain; Retinal ganglion cell; Visual function;
Autophagy regulates trans fatty acid-mediated apoptosis in primary cardiac myofibroblasts by Saeid Ghavami; Ryan H. Cunnington; Behzad Yeganeh; Jared J.L. Davies; Sunil G. Rattan; Krista Bathe; Morvarid Kavosh; Marek J. Los; Darren H. Freed; Thomas Klonisch; Grant N. Pierce; Andrew J. Halayko; Ian M.C. Dixon (2274-2286).
Trans fats are not a homogeneous group of molecules and less is known about the cellular effects of individual members of the group. Vaccenic acid (VA) and elaidic acid (EA) are the predominant trans monoenes in ruminant fats and vegetable oil, respectively. Here, we investigated the mechanism of cell death induced by VA and EA on primary rat ventricular myofibroblasts (rVF). The MTT assay demonstrated that both VA and EA (200 μM, 0–72 h) reduced cell viability in rVF (P < 0.001). The FACS assay confirmed that both VA and EA induced apoptosis in rVF, and this was concomitant with elevation in cleaved caspase-9, -3 and -7, but not caspase-8. VA and EA decreased the expression ratio of Bcl2:Bax, induced Bax translocation to mitochondria and decrease in mitochondrial membrane potential (Δψ). BAX and BAX/BAK silencing in mouse embryonic fibroblasts (MEF) inhibited VA and EA-induced cell death compared to the corresponding wild type cells. Transmission electron microscopy revealed that VA and EA also induced macroautophagosome formation in rVF, and immunoblot analysis confirmed the induction of several autophagy markers: LC3-β lipidation, Atg5–12 accumulation, and increased beclin-1. Finally, deletion of autophagy genes, ATG3 and ATG5 significantly inhibited VA and EA-induced cell death (P < 0.001). Our findings show for the first time that trans fat acid (TFA) induces simultaneous apoptosis and autophagy in rVF. Furthermore, TFA-induced autophagy is required for this pro-apoptotic effect. Further studies to address the effect of TFA on the heart may reveal significant translational value for prevention of TFA-linked heart disease.► Trans fat acids (TFAs) induce apoptosis on rat ventricular myofibroblasts (rVF). ► TFA apoptosis depends on Bcl2 family protein. ► Decrease of mitochondrial membrane potential is involved in TFA apoptosis pathway. ► TFAs provoke autophagy on rVF. ► Autophagy is necessary for TFA apoptosis induction on rVF.
Keywords: Myofibroblast; Trans fat; Vaccenic acid; Elaidic acid; Autophagy; Apoptosis;
SIAH-mediated ubiquitination and degradation of acetyl-transferases regulate the p53 response and protein acetylation by Inna Grishina; Katherina Debus; Carmen García-Limones; Constanze Schneider; Amit Shresta; Carlos García; Marco A. Calzado; M. Lienhard Schmitz (2287-2296).
Posttranslational modification of proteins by lysine acetylation regulates many biological processes ranging from signal transduction to chromatin compaction. Here we identify the acetyl-transferases CBP/p300, Tip60 and PCAF as new substrates for the ubiquitin E3 ligases SIAH1 and SIAH2. While CBP/p300 can undergo ubiquitin/proteasome-dependent degradation by SIAH1 and SIAH2, the two other acetyl-transferases are exclusively degraded by SIAH2. Accordingly, SIAH-deficient cells show enhanced protein acetylation, thus revealing SIAH proteins as indirect regulators of the cellular acetylation status. Functional experiments show that Tip60/PCAF-mediated acetylation of the tumor suppressor p53 is antagonized by the p53 target gene SIAH2 which mediates ubiquitin/proteasome-mediated degradation of both acetyl-transferases and consequently diminishes p53 acetylation and transcriptional activity. The p53 kinase HIPK2 mediates hierarchical phosphorylation of SIAH2 at 5 sites, which further boosts its activity as a ubiquitin E3 ligase for several substrates and therefore dampens the late p53 response.Display Omitted► The ubiquitin E3 ligase Siah controls the stability of various acetyl-transferases. ► HIPK2-mediated phosphorylation of Siah2 regulates its ubiquitinating activity. ► Siah2 contributes to the downregulation of the p53 response.
Keywords: SIAH; Ubiquitin E3 ligase; p53; Protein acetylation; HIPK2;
Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner by Magdalena Frank; Stéphane Duvezin-Caubet; Sebastian Koob; Angelo Occhipinti; Ravi Jagasia; Anton Petcherski; Mika O. Ruonala; Muriel Priault; Bénédicte Salin; Andreas S. Reichert (2297-2310).
Mitochondrial dysfunction is linked to apoptosis, aging, cancer, and a number of neurodegenerative and muscular disorders. The interplay between mitophagy and mitochondrial dynamics has been linked to the removal of dysfunctional mitochondria ensuring mitochondrial quality control. An open question is what role mitochondrial fission plays in the removal of mitochondria after mild and transient oxidative stress; conditions reported to result in moderately elevated reactive oxygen species (ROS) levels comparable to physical activity. Here we show that applying such conditions led to fragmentation of mitochondria and induction of mitophagy in mouse and human cells. These conditions increased ROS levels only slightly and neither triggered cell death nor led to a detectable induction of non-selective autophagy. Starvation led to hyperfusion of mitochondria, to high ROS levels, and to the induction of both non-selective autophagy and to a lesser extent to mitophagy. We conclude that moderate levels of ROS specifically trigger mitophagy but are insufficient to trigger non-selective autophagy. Expression of a dominant-negative variant of the fission factor DRP1 blocked mitophagy induction by mild oxidative stress as well as by starvation. Taken together, we demonstrate that in mammalian cells under mild oxidative stress a DRP1-dependent type of mitophagy is triggered while a concomitant induction of non-selective autophagy was not observed. We propose that these mild oxidative conditions resembling well physiological situations are thus very helpful for studying the molecular pathways governing the selective removal of dysfunctional mitochondria.► Mitophagy is specifically induced by mild and transient oxidative stress. ► Moderate levels of reactive oxygen species do not trigger non-selective autophagy. ► ROS and starvation induced mitophagy occurs in a DRP1-dependent manner. ► Starvation induced hyperfusion of mitochondria counteracts mitophagy. ► Physiological conditions for the specific induction of mitophagy are established.
Keywords: Mitophagy; ROS signaling; Mitochondrial dynamics; Hyperfusion;