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Showing papers in "Molecular and Cellular Biology in 2006"


Journal ArticleDOI
TL;DR: It is found that the autophagy system is activated as a novel signaling pathway in response to ER stress and played important roles in cell survival after ER stress.
Abstract: Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 “dots”), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.

1,757 citations


Journal ArticleDOI
TL;DR: The contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate supports the contention that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of NRF2.
Abstract: The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.

789 citations


Journal ArticleDOI
TL;DR: Investigation finds that inhibiting NF-κB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner, and suggests that ER stress induces two signals, namely TNF-α induction and TRAF2 down-regulation, which work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.
Abstract: NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.

716 citations


Journal ArticleDOI
TL;DR: It is proposed that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central α-helix in a correct position to benefit ubiquitin signaling.
Abstract: The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central alpha-helix followed by a mini antiparallel beta-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the beta-propeller structure of Keap1. In contrast, the central alpha-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two beta-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central alpha-helix in a correct position to benefit ubiquitin signaling.

639 citations


Journal ArticleDOI
TL;DR: A critical role for ATX in vascular development is revealed, it is indicated that ATX is the major LPA-producing enzyme in vivo, and the vascular defects in ATX-deficient embryos may be explained by loss of LPA signaling through Gα13.
Abstract: Autotaxin (ATX), or nucleotide pyrophosphatase-phosphodiesterase 2, is a secreted lysophospholipase D that promotes cell migration, metastasis, and angiogenesis. ATX generates lysophosphatidic acid (LPA), a lipid mitogen and motility factor that acts on several G protein-coupled receptors. Here we report that ATX-deficient mice die at embryonic day 9.5 (E9.5) with profound vascular defects in yolk sac and embryo resembling the Galpha13 knockout phenotype. Furthermore, at E8.5, ATX-deficient embryos showed allantois malformation, neural tube defects, and asymmetric headfolds. The onset of these abnormalities coincided with increased expression of ATX and LPA receptors in normal embryos. ATX heterozygous mice appear healthy but show half-normal ATX activity and plasma LPA levels. Our results reveal a critical role for ATX in vascular development, indicate that ATX is the major LPA-producing enzyme in vivo, and suggest that the vascular defects in ATX-deficient embryos may be explained by loss of LPA signaling through Galpha13.

541 citations


Journal ArticleDOI
TL;DR: It is proposed that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites.
Abstract: The chromodomain (CD) of the Drosophila Polycomb protein exhibits preferential binding affinity for histone H3 when trimethylated at lysine 27. Here we have investigated the five mouse Polycomb homologs known as Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8. Despite a high degree of conservation, the Cbx chromodomains display significant differences in binding preferences. Not all CDs bind preferentially to K27me3; rather, some display affinity towards both histone H3 trimethylated at K9 and H3K27me3, and one CD prefers K9me3. Cbx7, in particular, displays strong affinity for both H3K9me3 and H3K27me3 and is developmentally regulated in its association with chromatin. Cbx7 associates with facultative heterochromatin and, more specifically, is enriched on the inactive X chromosome. Finally, we find that, in vitro, the chromodomain of Cbx7 can bind RNA and that, in vivo, the interaction of Cbx7 with chromatin, and the inactive X chromosome in particular, depends partly on its association with RNA. We propose that the capacity of this mouse Polycomb homolog to associate with the inactive X chromosome, or any other region of chromatin, depends not only on its chromodomain but also on the combination of histone modifications and RNA molecules present at its target sites.

541 citations


Journal ArticleDOI
TL;DR: It is revealed that microRNA Mir-17-5p has a role as a tumor suppressor in breast cancer cells and completely abrogated the insulin-like growth factor 1-mediated, anchorage-independent growth of breast cancers cells.
Abstract: MicroRNAs are an extensive family of approximately 22-nucleotide-long noncoding RNAs expressed in a wide range of eukaryotes, including humans, and they are important in development and disease. We found that microRNA Mir-17-5p has extensive complementarity to the mRNA of AIB1 (named for "amplified in breast cancer 1"). Cell culture experiments showed that AIB1 expression was downregulated by Mir-17-5p, primarily through translational inhibition. Expression of Mir-17-5p was low in breast cancer cell lines. We also found that downregulation of AIB1 by Mir-17-5p resulted in decreased estrogen receptor-mediated, as well as estrogen receptor-independent, gene expression and decreased proliferation of breast cancer cells. Mir-17-5p also completely abrogated the insulin-like growth factor 1-mediated, anchorage-independent growth of breast cancer cells. Our results reveal that Mir-17-5p has a role as a tumor suppressor in breast cancer cells.

536 citations


Journal ArticleDOI
TL;DR: These findings provide the first demonstration of a molecular mechanism by which the Gli2 transcriptional activity is regulated by Shh signaling and in addition to being processed, Gli 2 full-length protein is readily degraded.
Abstract: Gli2 and Gli3 are the primary transcription factors that mediate Sonic hedgehog (Shh) signals in the mouse. Gli3 mainly acts as a transcriptional repressor, because the majority of full-length Gli3 protein is proteolytically processed. Gli2 is mostly regarded as a transcriptional activator, even though it is also suggested to have a weak repressing activity. What the molecular basis for its possible dual function is and how its activity is regulated by Shh signaling are largely unknown. Here we demonstrate that unlike the results seen with Gli3 and Cubitus Interruptus, the fly homolog of Gli, only a minor fraction of Gli2 is proteolytically processed to form a transcriptional repressor in vivo and that in addition to being processed, Gli2 full-length protein is readily degraded. The degradation of Gli2 requires the phosphorylation of a cluster of numerous serine residues in its carboxyl terminus by protein kinase A and subsequently by casein kinase 1 and glycogen synthase kinase 3. The phosphorylated Gli2 interacts directly with betaTrCP in the SCF ubiquitin-ligase complex through two binding sites, which results in Gli2 ubiquitination and subsequent degradation by the proteasome. Both processing and degradation of Gli2 are suppressed by Shh signaling in vivo. Our findings provide the first demonstration of a molecular mechanism by which the Gli2 transcriptional activity is regulated by Shh signaling.

534 citations


Journal ArticleDOI
TL;DR: It is demonstrated that SUN1 is an integral membrane protein located at the inner nuclear membrane, and the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain.
Abstract: Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.

483 citations


Journal ArticleDOI
TL;DR: Results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.
Abstract: Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.

449 citations


Journal ArticleDOI
TL;DR: Evidence that AMPK is activated in authentic hypoxic tumor microenvironments is obtained and that this activity overlaps with regions of hypoxia detected by a chemical probe, which implies that HIF-1 and AMPK are components of a concerted cellular response to maintain energy homeostasis in low-oxygen or ischemic-tissue microen environments.
Abstract: We have been studying the relationship between the activity of hypoxia-inducible factor 1 (HIF-1), the primary transcriptional regulator of the response of mammalian cells to oxygen deprivation (e.g., see references 21, 43, and 50) and the regulation of c-Jun/AP-1 transcription factors (31, 32). We determined that c-Jun N-terminal phosphorylation is induced by low-oxygen conditions (hypoxia or anoxia; called hypoxia hereafter) in an HIF-1-dependent manner (31) and showed that this HIF-1-dependent c-Jun phosphorylation absolutely requires extracellular glucose utilization (32). Together, these findings suggest that enhanced glucose absorption and/or glycolytic activity mediated by HIF-1 in response to hypoxia activates c-Jun/AP-1, as well as other targets of c-Jun N-terminal kinases. To further investigate this potential mechanism, we focused on determining the contribution of bioenergetics—ATP depletion—to hypoxia-inducible c-Jun phosphorylation in wild-type (WT) and HIF-1-null mouse embryo fibroblasts (MEFs). While exploring cellular mechanisms of ATP regulation, we observed that 5′-AMP-activated protein kinase (AMPK) activity was induced in both cell types, particularly under conditions of hypoxia and glucose deprivation. This observation suggested the hypothesis that AMPK is important for the adaptive responses of energetically stressed cells in the hypoxic and glucose-deprived microenvironments present in solid tumors (e.g., reviewed in references 35 and 59). AMPK activity is defined by a class of evolutionarily conserved serine/threonine kinases that are sensitive to various environmental stresses, especially those that perturb cellular energy status (reviewed in references 9, 19, and 47). Different members of the AMPK catalytic subunit subfamily have been characterized; the α subunits (collectively, AMPKα1 and -α2) are the most widely expressed in mammalian cells (36). AMPK is a heterotrimeric complex consisting of an α subunit and β and γ regulatory subunits, each of which is encoded by distinct genes (α1 and α2; β1 and β2; γ1, γ2, and γ3) (19). In terms of a role in ATP regulation, decreased cellular ATP levels promote AMPK activation through the allosteric binding of AMP, which in effect enables AMPK to sense increases in the cellular [AMP]/[ATP] ratio. Full activation of AMPK also requires specific phosphorylation within the activation loop of the catalytic domain of the α subunit (at Thr172 in humans and mice) by LKB1, a serine/threonine protein kinase and tumor suppressor (36, 37, 52). LKB1 is thus an AMPK kinase. Recently, mammalian Ca2+/calmodulin-dependent kinase kinases have also been identified as AMPK kinases (reviewed in reference 6). Activated AMPK phosphorylates diverse targets, including many that are directly involved in controlling cellular energy metabolism (22, 34). In cells exposed to an energy-depleting stress, AMPK is believed to function as an energy sensor that inhibits ATP-consuming processes and stimulates ATP-producing processes to optimize total cellular ATP levels for maintaining critical physiological functions (or for survival in response to extreme stress) (19). For example, in cells exposed to hypoxic or ischemic conditions that significantly deplete total ATP, activated AMPK can stimulate ATP generation by increasing both glucose absorption and glycolysis (e.g., see references 2, 19, and 22). AMPK can also generate ATP by phosphorylating and inhibiting the metabolic enzymes acetyl coenzyme A (acetyl-CoA) carboxylases 1 and 2 (ACC1/2), which synthesize malonyl-CoA (19). Malonyl-CoA synthesized by ACC1 is necessary for de novo fatty acid synthesis, whereas that synthesized by ACC2 inhibits fatty acid transport into the mitochondrion, the site of ATP production by the process of fatty acid β oxidation (18). Thus, AMPK-dependent inhibition of ACC1/2 can divert cellular metabolism from consuming ATP during fatty acid biosynthesis to producing ATP by oxidizing fatty acid stores. In the present study, we found that the combination of hypoxia and glucose deprivation decreased total cellular ATP levels to the same extent in both WT and HIF-1α-null cells. This finding supports our previous conclusion that increased intracellular glucose, rather than decreased ATP levels, is responsible for the stimulation of c-Jun N-terminal kinase activity in WT cells exposed to hypoxia. AMPK activity, conventionally defined by phosphorylation of AMPK target sites on the metabolic enzymes ACC1/2 (19), was strongly activated in both WT and HIF-1α-null cells under the same conditions of hypoxia and glucose deprivation, which is consistent with its function as a sensor of ATP depletion. However, AMPK activity was also rapidly induced in both cell types following exposure to hypoxia in the presence of glucose, even though total cellular ATP was not significantly depleted. By using genetically manipulated MEFs nullizygous for AMPK expression, we directly demonstrated that AMPK activity is sensitive to a wide range of low-oxygen conditions, at least in mesenchymal cells. To determine whether these hypoxia-inducible responses of AMPK also occur in vivo, we prepared tumor xenografts from transformed derivatives of the same WT and HIF-1α-null cells, and exposed tumor-bearing mice to the hypoxia probe pimonidazole (3, 10, 44, 45). Immunohistochemical analysis of these tumors indicated that AMPK activity was prevalent in hypoxic regions of both tumor types, especially in viable areas near necrosis. By using tumor xenografts prepared from identically transformed WT and AMPKα-null cells, we determined that the absence of AMPK activity greatly inhibited the growth of this experimental tumor type. We propose that activation of AMPK in hypoxic or ischemic microenvironments may be critical for cell survival and thus would represent a novel protective mechanism for metabolically depressed or ATP-deficient cells.

Journal ArticleDOI
TL;DR: A novel inhibitory element located immediately downstream of the 5′ splice site in intron 7 is described, which underscores for the first time the profound impact of an evolutionarily nonconserved intronic element on SMN2 exon 7 splicing.
Abstract: Humans have two nearly identical copies of the Survival Motor Neuron (SMN) gene, SMN1 and SMN2. In spinal muscular atrophy (SMA), SMN2 is not able to compensate for the loss of SMN1 due to exclusion of exon 7. Here we describe a novel inhibitory element located immediately downstream of the 5' splice site in intron 7. We call this element intronic splicing silencer N1 (ISS-N1). Deletion of ISS-N1 promoted exon 7 inclusion in mRNAs derived from the SMN2 minigene. Underlining the dominant role of ISS-N1 in exon 7 skipping, abrogation of a number of positive cis elements was tolerated when ISS-N1 was deleted. Confirming the silencer function of ISS-N1, an antisense oligonucleotide against ISS-N1 restored exon 7 inclusion in mRNAs derived from the SMN2 minigene or from endogenous SMN2. Consistently, this oligonucleotide increased the levels of SMN protein in SMA patient-derived cells that carry only the SMN2 gene. Our findings underscore for the first time the profound impact of an evolutionarily nonconserved intronic element on SMN2 exon 7 splicing. Considering that oligonucleotides annealing to intronic sequences do not interfere with exon-junction complex formation or mRNA transport and translation, ISS-N1 provides a very specific and efficient therapeutic target for antisense oligonucleotide-mediated correction of SMN2 splicing in SMA.

Journal ArticleDOI
TL;DR: A comparative assessment of all six known positions of histone lysine methylation indicates that H4K20 and H3K27 monomethylation are versatile and dynamic with respect to gene activity, suggesting the existence of novel site-specific methyltransferases and demethylases coupled to the transcription cycle.
Abstract: Complex patterns of histone lysine methylation encode distinct functions within chromatin. We previously reported that trimethylation of lysine 9 of histone H3 (H3K9) occurs at both silent heterochromatin and at the transcribed regions of active mammalian genes, suggesting that the extent of histone lysine methylation involved in mammalian gene activation is not completely defined. To identify additional sites of histone methylation that respond to mammalian gene activity, we describe here a comparative assessment of all six known positions of histone lysine methylation and relate them to gene transcription. Using several model loci, we observed high trimethylation of H3K4, H3K9, H3K36, and H3K79 in the transcribed region, consistent with previous findings. We identify H4K20 monomethylation, a modification previously linked with repression, as a mark of transcription elongation in mammalian cells. In contrast, H3K27 monomethylation, a modification enriched at pericentromeric heterochromatin, was observed broadly distributed throughout all euchromatic sites analyzed, with selective depletion in the vicinity of the transcription start sites at active genes. Together, these results underscore that similar to other described methyl-lysine modifications, H4K20 and H3K27 monomethylation are versatile and dynamic with respect to gene activity, suggesting the existence of novel site-specific methyltransferases and demethylases coupled to the transcription cycle.

Journal ArticleDOI
TL;DR: Findings provide the first evidence that GRP78 is essential for embryonic cell growth and pluripotent cell survival.
Abstract: GRP78, also known as BiP, is a central regulator of endoplasmic reticulum (ER) homeostasis due to its multiple functional roles in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. ER stress induction of GRP78/BiP represents a major prosurvival arm of the unfolded protein response (UPR). However, the physiological role of GRP78 in development is not known. Using a transgenic approach, we discovered that the Grp78 promoter is activated in both the trophectoderm and inner cell mass (ICM) of embryos at embryonic day 3.5 via a mechanism requiring the ER stress elements. To reveal the function of the GRP78 in vivo, we created a tri-loxP Grp78 mutant allele, which was further crossed with EIIA-cre to create a knockout allele. The Grp78+/- mice, which express 50% of the wild-type level of the GRP78 protein, are viable. Interestingly, the heterozygous Grp78 cells up-regulate the ER proteins GRP94 and protein disulfide isomerase at both the transcript and protein levels, while other UPR targets such as CHOP and XBP-1 are not affected. Further studies revealed that mouse embryonic fibroblasts from Grp78+/- mice are capable of responding to ER stress. However, Grp78-/- embryos that are completely devoid of GRP78 lead to peri-implantation lethality. These embryos do not hatch from the zona pellucida in vitro, fail to grow in culture, and exhibit proliferation defects and a massive increase in apoptosis in the ICM, which is the precursor of embryonic stem cells. These findings provide the first evidence that GRP78 is essential for embryonic cell growth and pluripotent cell survival.

Journal ArticleDOI
TL;DR: In vivo requirements for YAP are revealed in the developmental processes of yolk sac vasculogenesis, chorioallantoic attachment, and embryonic axis elongation.
Abstract: YAP is a multifunctional adapter protein and transcriptional coactivator with several binding partners well described in vitro and in cell culture. To explore in vivo requirements for YAP, we generated mice carrying a targeted disruption of the Yap gene. Homozygosity for the Yap(tm1Smil) allele (Yap-/-) caused developmental arrest around E8.5. Phenotypic characterization revealed a requirement for YAP in yolk sac vasculogenesis. Yolk sac endothelial and erythrocyte precursors were specified as shown by histology, PECAM1 immunostaining, and alpha globin expression. Nonetheless, development of an organized yolk sac vascular plexus failed in Yap-/- embryos. In striking contrast, vasculogenesis proceeded in both the allantois and the embryo proper. Mutant embryos showed patterned gene expression domains along the anteroposterior neuraxis, midline, and streak/tailbud. Despite this evidence of proper patterning and tissue specification, Yap-/- embryos showed developmental perturbations that included a notably shortened body axis, convoluted anterior neuroepithelium, caudal dysgenesis, and failure of chorioallantoic fusion. These results reveal a vital requirement for YAP in the developmental processes of yolk sac vasculogenesis, chorioallantoic attachment, and embryonic axis elongation.

Journal ArticleDOI
TL;DR: It is demonstrated that nutrients suppress phosphatidylinositol 3 (PI3)-kinase/Akt signaling via Raptor-dependent mTOR (mammalian target of rapamycin)-mediated phosphorylation of insulin receptor substrate 1 (IRS-1) through Knockdown of Raptor as well as activators of the LKB1/AMPK pathway, such as the widely used antidiabetic compound metformin.
Abstract: Nutritional excess and/or obesity represent well-known predisposition factors for the development of non-insulin-dependent diabetes mellitus (NIDDM). However, molecular links between obesity and NIDDM are only beginning to emerge. Here, we demonstrate that nutrients suppress phosphatidylinositol 3 (PI3)-kinase/Akt signaling via Raptor-dependent mTOR (mammalian target of rapamycin)-mediated phosphorylation of insulin receptor substrate 1 (IRS-1). Raptor directly binds to and serves as a scaffold for mTOR-mediated phosphorylation of IRS-1 on Ser636/639. These serines lie close to the Y632MPM motif that is implicated in the binding of p85α/p110α PI3-kinase to IRS-1 upon insulin stimulation. Phosphomimicking mutations of these serines block insulin-stimulated activation of IRS-1-associated PI3-kinase. Knockdown of Raptor as well as activators of the LKB1/AMPK pathway, such as the widely used antidiabetic compound metformin, suppress IRS-1 Ser636/639 phosphorylation and reverse mTOR-mediated inhibition on PI3-kinase/Akt signaling. Thus, diabetes-related hyperglycemia hyperactivates the mTOR pathway and may lead to insulin resistance due to suppression of IRS-1-dependent PI3-kinase/Akt signaling.

Journal ArticleDOI
TL;DR: The data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that theheterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.
Abstract: The Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway participates in the control of many fundamental cellular processes including proliferation, survival, and differentiation. The pathway is deregulated in up to 30% of human cancers, often due to mutations in Ras and the B-Raf isoform. Raf-1 and B-Raf can form heterodimers, and this may be important for cellular transformation. Here, we have analyzed the biochemical and biological properties of Raf-1/B-Raf heterodimers. Isolated Raf-1/B-Raf heterodimers possessed a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimers between wild-type Raf-1 and B-Raf mutants with low or no kinase activity still displayed elevated kinase activity, as did heterodimers between wild-type B-Raf and kinase-negative Raf-1. In contrast, heterodimers containing both kinase-negative Raf-1 and kinase-negative B-Raf were completely inactive, suggesting that the kinase activity of the heterodimer specifically originates from Raf and that either kinase-competent Raf isoform is sufficient to confer high catalytic activity to the heterodimer. In cell lines, Raf-1/B-Raf heterodimers were found at low levels. Heterodimerization was enhanced by 14-3-3 proteins and by mitogens independently of ERK. However, ERK-induced phosphorylation of B-Raf on T753 promoted the disassembly of Raf heterodimers, and the mutation of T753 prolonged growth factor-induced heterodimerization. The B-Raf T753A mutant enhanced differentiation of PC12 cells, which was previously shown to be dependent on sustained ERK signaling. Fine mapping of the interaction sites by peptide arrays suggested a complex mode of interaction involving multiple contact sites with a main Raf-1 binding site in B-Raf encompassing T753. In summary, our data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that the heterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.

Journal ArticleDOI
TL;DR: The disrupted Phd genes indicate that among all three PHD proteins, PHD2 is uniquely essential during mouse embryogenesis.
Abstract: PHD1, PHD2, and PHD3 are prolyl hydroxylase domain proteins that regulate the stability of hypoxia-inducible factor α subunits (HIF-α). To determine the roles of individual PHDs during mouse development, we disrupted all three Phd genes and found that Phd2−/− embryos died between embryonic days 12.5 and 14.5 whereas Phd1−/− or Phd3−/− mice were apparently normal. In Phd2−/− mice, severe placental and heart defects preceded embryonic death. Placental defects included significantly reduced labyrinthine branching morphogenesis, widespread penetration of the labyrinth by spongiotrophoblasts, and abnormal distribution of trophoblast giant cells. The expression of several trophoblast markers was also altered, including an increase in the spongiotrophoblast marker Mash2 and decreases in the labyrinthine markers Tfeb and Gcm1. In the heart, trabeculae were poorly developed, the myocardium was remarkably thinner, and interventricular septum was incompletely formed. Surprisingly, while there were significant global increases in HIF-α protein levels in the placenta and the embryo proper, there was no specific HIF-α increase in the heart. Taken together, these data indicate that among all three PHD proteins, PHD2 is uniquely essential during mouse embryogenesis.

Journal ArticleDOI
TL;DR: It is suggested that MK2 inhibits the mRNA destabilizing activity of TTP and, in parallel, codegradation of TNP together, with the target mRNA resulting in increased cellular levels of T TP.
Abstract: The mitogen-activated protein kinase (MAPK) p38/MAPK-activated protein kinase 2 (MK2) signaling pathway plays an important role in the posttranscriptional regulation of tumor necrosis factor (TNF), which is dependent on the adenine/uridine-rich element (ARE) in the 3' untranslated region of TNF mRNA. After lipopolysaccharide (LPS) stimulation, MK2-deficient macrophages show a 90% reduction in TNF production compared to the wild type. Tristetraprolin (TTP), a protein induced by LPS, binds ARE and destabilizes TNF mRNA. Accordingly, macrophages lacking TTP produce large amounts of TNF. Here, we generated MK2/TTP double knockout mice and show that, after LPS stimulation, bone marrow-derived macrophages produce TNF mRNA and protein levels comparable to those of TTP knockout cells, indicating that in the regulation of TNF biosynthesis TTP is genetically downstream of MK2. In addition, we show that MK2 is essential for the stabilization of TTP mRNA, and phosphorylation by MK2 leads to increased TTP protein stability but reduced ARE affinity. These data suggest that MK2 inhibits the mRNA destabilizing activity of TTP and, in parallel, codegradation of TTP together, with the target mRNA resulting in increased cellular levels of TTP.

Journal ArticleDOI
TL;DR: Ch Chromatin immunoprecipitation experiments revealed that PRMT5 and MBD2 are recruited to CpG islands in a methylation-dependent manner in vivo and that H4R3, a substrate of PRMT, is methylated at these loci.
Abstract: The human genome contains a number of methyl CpG binding proteins that translate DNA methylation into a physiological response. To gain insight into the function of MBD2 and MBD3, we first applied protein tagging and mass spectrometry. We show that MBD2 and MBD3 assemble into mutually exclusive distinct Mi-2/NuRD-like complexes, called MBD2/NuRD and MBD3/NuRD. We identified DOC-1, a putative tumor suppressor, as a novel core subunit of MBD2/NuRD as well as MBD3/NuRD. PRMT5 and its cofactor MEP50 were identified as specific MBD2/NuRD interactors. PRMT5 stably and specifically associates with and methylates the RG-rich N terminus of MBD2. Chromatin immunoprecipitation experiments revealed that PRMT5 and MBD2 are recruited to CpG islands in a methylation-dependent manner in vivo and that H4R3, a substrate of PRMT, is methylated at these loci. Our data show that MBD2/NuRD and MBD3/NuRD are distinct protein complexes with different biochemical and functional properties.

Journal ArticleDOI
TL;DR: The results reveal the existence of a family of Kaiso-like proteins that bind methylated CpGs, like proteins of the MBD family, able to repress transcription in a methyl-dependent manner, yet their tissue-specific expression pattern suggests nonoverlapping functions.
Abstract: In vertebrates, densely methylated DNA is associated with inactive transcription. Actors in this process include proteins of the MBD family that can recognize methylated CpGs and repress transcription. Kaiso, a structurally unrelated protein, has also been shown to bind methylated CGCGs through its three Kruppel-like C2H2 zinc fingers. The human genome contains two uncharacterized proteins, ZBTB4 and ZBTB38, that contain Kaiso-like zinc fingers. We report that ZBTB4 and ZBTB38 bind methylated DNA in vitro and in vivo. Unlike Kaiso, they can bind single methylated CpGs. When transfected in mouse cells, the proteins colocalize with foci of heavily methylated satellite DNA and become delocalized upon loss of DNA methylation. Chromatin immunoprecipitation suggests that both of these proteins specifically bind to the methylated allele of the H19/Igf2 differentially methylated region. ZBTB4 and ZBTB38 repress the transcription of methylated templates in transfection assays. The two genes have distinct tissue-specific expression patterns, but both are highly expressed in the brain. Our results reveal the existence of a family of Kaiso-like proteins that bind methylated CpGs. Like proteins of the MBD family, they are able to repress transcription in a methyl-dependent manner, yet their tissue-specific expression pattern suggests nonoverlapping functions.

Journal ArticleDOI
TL;DR: The recruitment of two distinct classes of E3 ubiquitin ligases and a histone demethylase by BCOR suggests that BCOR uses a unique combination of epigenetic modifications to direct gene silencing.
Abstract: The corepressor BCOR potentiates transcriptional repression by the proto-oncoprotein BCL6 and suppresses the transcriptional activity of a common mixed-lineage leukemia fusion partner, AF9. Mutations in human BCOR cause male lethal, X-linked oculofaciocardiodental syndrome. We identified a BCOR complex containing Polycomb group (PcG) and Skp-Cullin-F-box subcomplexes. The PcG proteins include RING1, RYBP, NSPC1, a Posterior Sex Combs homolog, and RNF2, an E3 ligase for the mono-ubiquitylation of H2A. BCOR complex components and mono-ubiquitylated H2A localize to BCL6 targets, indicating that the BCOR complex employs PcG proteins to expand the repertoire of enzymatic activities that can be recruited by BCL6. This also suggests that BCL6 can target PcG proteins to DNA. In addition, the BCOR complex contains components of a second ubiquitin E3 ligase, namely, SKP1 and FBXL10 (JHDM1B). We show that BCOR coimmunoprecipitates isoforms of FBXL10 which contain a JmjC domain that recently has been determined to have histone H3K36 demethylase activity. The recruitment of two distinct classes of E3 ubiquitin ligases and a histone demethylase by BCOR suggests that BCOR uses a unique combination of epigenetic modifications to direct gene silencing.

Journal ArticleDOI
TL;DR: Examination of the function of the TCF family of transcription factors in ESC has found that Tcf3 is necessary to limit the steady-state levels of Nanog mRNA, protein, and promoter activity in self-renewing ESC.
Abstract: The dual function of stem cells requires them not only to form new stem cells through self-renewal but also to form lineage-committed cells through differentiation. Embryonic stem cells (ESC), which are derived from the blastocyst inner cell mass, retain properties of self-renewal and the potential for lineage commitment. To balance self-renewal and differentiation, ESC must carefully control the levels of several transcription factors, including Nanog, Sox2, and Oct4. While molecular mechanisms promoting transcription of these genes have been described, mechanisms preventing excessive levels in self-renewing ESC remain unknown. By examining the function of the TCF family of transcription factors in ESC, we have found that Tcf3 is necessary to limit the steady-state levels of Nanog mRNA, protein, and promoter activity in self-renewing ESC. Chromatin immunoprecipitation and promoter reporter assays showed that Tcf3 bound to a promoter regulatory region of the Nanog gene and repressed its transcriptional activity in ESC through a Groucho interaction domain-dependent process. The absence of Tcf3 caused delayed differentiation of ESC in vitro as elevated Nanog levels persisted through 5 days of embryoid body formation. These new data support a model wherein Tcf3-mediated control of Nanog levels allows stem cells to balance the creation of lineage-committed and undifferentiated cells.

Journal ArticleDOI
TL;DR: The phenotype of depletion of polo-like kinase 1 (Plk1) using RNA interference and showed that p53 is stabilized in Plk1-depleted cancer cells further support suggestions that Plk 1 may be a feasible cancer therapy target.
Abstract: We previously reported the phenotype of depletion of polo-like kinase 1 (Plk1) using RNA interference (RNAi) and showed that p53 is stabilized in Plk1-depleted cancer cells. In this study, we further analyzed the Plk1 depletion-induced phenotype in both cancer cells and primary cells. The vector-based RNAi approach was used to evaluate the role of the p53 pathway in Plk1 depletion-induced apoptosis in cancer cells with different p53 backgrounds. Although DNA damage and cell death can occur independently of p53, p53-deficient cancer cells were much more sensitive to Plk1 depletion than cancer cells with functional p53. Next, the lentivirus-based RNAi approach was used to generate a series of Plk1 hypomorphs. In HeLa cells, two weak hypomorphs showed only slight G2/M arrest, a medium hypomorph arrested with 4N DNA content, followed later by apoptosis, and a strong Plk1 hypomorph underwent serious mitotic catastrophe. In well-synchronized HeLa cells, a medium level of Plk1 depletion caused a 2-h delay of mitotic progression, and a high degree of Plk1 depletion significantly delayed mitotic entry and completely blocked cells at mitosis. In striking contrast, normal hTERT-RPE1 and MCF10A cells were much less sensitive to Plk1 depletion than HeLa cells; no apparent cell proliferation defect or cell cycle arrest was observed after Plk1 depletion in these cells. Therefore, these data further support suggestions that Plk1 may be a feasible cancer therapy target.

Journal ArticleDOI
TL;DR: KAP1 functions to coordinate activities that dynamically regulate changes in histone modifications and deposition of HP1 to establish a de novo microenvironment of heterochromatin, which is required for repression of gene transcription by KRAB-zfps.
Abstract: KAP1/TIF1β is proposed to be a universal corepressor protein for the KRAB zinc finger protein (KRAB-zfp) superfamily of transcriptional repressors. To characterize the role of KAP1 and KAP1-interacting proteins in transcriptional repression, we investigated the regulation of stably integrated reporter transgenes by hormone-responsive KRAB and KAP1 repressor proteins. Here, we demonstrate that depletion of endogenous KAP1 levels by small interfering RNA (siRNA) significantly inhibited KRAB-mediated transcriptional repression of a chromatin template. Similarly, reduction in cellular levels of HP1α/β/γ and SETDB1 by siRNA attenuated KRAB-KAP1 repression. We also found that direct tethering of KAP1 to DNA was sufficient to repress transcription of an integrated transgene. This activity is absolutely dependent upon the interaction of KAP1 with HP1 and on an intact PHD finger and bromodomain of KAP1, suggesting that these domains function cooperatively in transcriptional corepression. The achievement of the repressed state by wild-type KAP1 involves decreased recruitment of RNA polymerase II, reduced levels of histone H3 K9 acetylation and H3K4 methylation, an increase in histone occupancy, enrichment of trimethyl histone H3K9, H3K36, and histone H4K20, and HP1 deposition at proximal regulatory sequences of the transgene. A KAP1 protein containing a mutation of the HP1 binding domain failed to induce any change in the histone modifications associated with DNA sequences of the transgene, implying that HP1-directed nuclear compartmentalization is required for transcriptional repression by the KRAB/KAP1 repression complex. The combination of these data suggests that KAP1 functions to coordinate activities that dynamically regulate changes in histone modifications and deposition of HP1 to establish a de novo microenvironment of heterochromatin, which is required for repression of gene transcription by KRAB-zfps.

Journal ArticleDOI
TL;DR: DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and it is demonstrated here that this occurs through distinct mechanisms.
Abstract: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.

Journal ArticleDOI
TL;DR: The reduction of TG in the cytosol with concomitant accumulation ofTG in the microsome of Adfp−/− cells suggests that ADFP may facilitate the formation of new LDs, which are thought to form from the outer leaflet of the microsomal membrane.
Abstract: Adipose differentiation-related protein (ADFP; also known as ADRP or adipophilin), is a lipid droplet (LD) protein found in most cells and tissues. ADFP expression is strongly induced in cells with increased lipid load. We have inactivated the Adfp gene in mice to better understand its role in lipid accumulation. The Adfp-deficient mice have unaltered adipose differentiation or lipolysis in vitro or in vivo. Importantly, they display a 60% reduction in hepatic triglyceride (TG) and are resistant to diet-induced fatty liver. To determine the mechanism for the reduced hepatic TG content, we measured hepatic lipogenesis, very-low-density lipoprotein (VLDL) secretion, and lipid uptake and utilization, all of which parameters were shown to be similar between mutant and wild-type mice. The finding of similar VLDL output in the presence of a reduction in total TG in the Adfp-deficient liver is explained by the retention of TG in the microsomes where VLDL is assembled. Given that lipid droplets are thought to form from the outer leaflet of the microsomal membrane, the reduction of TG in the cytosol with concomitant accumulation of TG in the microsome of Adfp-/- cells suggests that ADFP may facilitate the formation of new LDs. In the absence of ADFP, impairment of LD formation is associated with the accumulation of microsomal TG but a reduction in TG in other subcellular compartments.

Journal ArticleDOI
TL;DR: Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.
Abstract: The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and gamma-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require gamma-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.

Journal ArticleDOI
TL;DR: It is shown that the stress-induced cytoplasmic accumulation of hnRNP A1 occurs in discrete phase-dense particles, the cytopolasmic stress granules (SGs), and this data are consistent with a model whereby hnNR NP A1 recruitment to SGs involves Mnk1/2-dependent phosphorylation of mRNA-bound hnGMP A1.
Abstract: hnRNP A1 is a nucleocytoplasmic shuttling protein that is involved in many aspects of mRNA metabolism. We have previously shown that activation of the p38 stress-signaling pathway in mammalian cells results in both hyperphosphorylation and cytoplasmic accumulation of hnRNP A1, affecting alternative splicing regulation in vivo. Here we show that the stress-induced cytoplasmic accumulation of hnRNP A1 occurs in discrete phase-dense particles, the cytoplasmic stress granules (SGs). Interestingly, mRNA-binding activity is required for both phosphorylation of hnRNP A1 and localization to SGs. We also show that these effects are mediated by the Mnk1/2 protein kinases that act downstream of p38. Finally, depletion of hnRNP A1 affects the recovery of cells from stress, suggesting a physiologically significant role for hnRNP A1 in the stress response. Our data are consistent with a model whereby hnRNP A1 recruitment to SGs involves Mnk1/2-dependent phosphorylation of mRNA-bound hnRNP A1.

Journal ArticleDOI
TL;DR: Novel insights are provided into the composition and structure of the spliceosome just prior to its catalytic activation and a potential role in activation for proteins recruited at this stage is suggested.
Abstract: The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis. Pre-mRNA splicing is catalyzed by a large RNP molecular machine, termed the spliceosome, which consists of the U1, U2, U4/U6, and U5 snRNPs and a multitude of non-snRNP proteins (reviewed in reference 48). The active site(s) responsible for the catalysis of pre-mRNA splicing is not preformed but, rather, is created anew during the highly dynamic process of spliceosome assembly. The latter is an ordered process during which several intermediates, termed E, A, B, and B*, can be detected in vitro (reviewed in reference 48). Assembly is initiated by the ATP-independent interaction of the U1 snRNP with the conserved 5 splice site of the pre-mRNA, forming the E complex. At this stage, the U2 snRNP is loosely associated with the pre-mRNA (11). In a subsequent step requiring ATP, the U2 snRNP stably interacts with the pre-mRNA’s branch site, leading to formation of the A complex (also called the prespliceosome). Spliceosome assembly culminates with the formation of the spliceosomal B complex, during which the preformed U4/U6.U5 tri-snRNP particle interacts with the A complex. The B complex thus contains a full set of U snRNAs in a precatalytic state. It subsequently undergoes major rearrangements, including destabilization or loss of the U1 and U4 snRNPs, leading to catalytic activation and the formation of the so-called activated spliceosome (B* complex).