Showing papers in "Nature Cell Biology in 2005"
TL;DR: It is demonstrated that Argonaute proteins — the signature components of the RNA interference (RNAi) effector complex, RISC — localize to mammalian P-bodies and suggested that translation repression by RISC delivers mRNAs to P- bodies, either as a cause or as a consequence of inhibiting protein synthesis.
Abstract: Small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs) can silence target genes through several different effector mechanisms. Whereas siRNA-directed mRNA cleavage is increasingly understood, the mechanisms by which miRNAs repress protein synthesis are obscure. Recent studies have revealed the existence of specific cytoplasmic foci, referred to herein as processing bodies (P-bodies), which contain untranslated mRNAs and can serve as sites of mRNA degradation. Here we demonstrate that Argonaute proteins--the signature components of the RNA interference (RNAi) effector complex, RISC--localize to mammalian P-bodies. Moreover, reporter mRNAs that are targeted for translational repression by endogenous or exogenous miRNAs become concentrated in P-bodies in a miRNA-dependent manner. These results provide a link between miRNA function and mammalian P-bodies and suggest that translation repression by RISC delivers mRNAs to P-bodies, either as a cause or as a consequence of inhibiting protein synthesis.
1,284 citations
TL;DR: Advances in various aspects of ERAD are summarized and new findings on how substrate dislocation is achieved are discussed, to help clarify the spatial separation between substrate selection and degradation in ERAD.
Abstract: Endoplasmic reticulum (ER)-associated protein degradation (ERAD) eliminates misfolded or unassembled proteins from the ER. ERAD targets are selected by a quality control system within the ER lumen and are ultimately destroyed by the cytoplasmic ubiquitin-proteasome system (UPS). The spatial separation between substrate selection and degradation in ERAD requires substrate transport from the ER to the cytoplasm by a process termed dislocation. In this review, we will summarize advances in various aspects of ERAD and discuss new findings on how substrate dislocation is achieved.
1,210 citations
TL;DR: Several enzymes involved in the ubiquitination and deubiquitination of signalling proteins that mediate IKK activation through a degradation-independent mechanism are revealed.
Abstract: The transcription factor NF-kappaB (nuclear factor kappa enhancer binding protein) controls many processes, including immunity, inflammation and apoptosis. Ubiquitination regulates at least three steps in the NF-kappaB pathway: degradation of IkappaB (inhibitor of NF-kappaB), processing of NF-kappaB precursors, and activation of the IkappaB kinase (IKK). Recent studies have revealed several enzymes involved in the ubiquitination and deubiquitination of signalling proteins that mediate IKK activation through a degradation-independent mechanism.
1,096 citations
TL;DR: A signalling pathway in which nitric oxide generation that follows apoptotic stimulation elicits S-nitrosylation of GAPDH, which triggers binding to Siah1 (an E3 ubiquitin ligase), nuclear translocation and apoptosis, which is prevented by NO deletion is reported.
Abstract: S -nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding
1,015 citations
TL;DR: Findings indicate an alternative mechanism for p53 to maintain genetic stability in ESCs, by inducing the differentiation of ESCs into other cell types that undergo efficient p53-dependent cell-cycle arrest and apoptosis.
Abstract: The tumour suppressor p53 becomes activated in response to upstream stress signals, such as DNA damage, and causes cell-cycle arrest or apoptosis. Here we report a novel role for p53 in the differentiation of mouse embryonic stem cells (ESCs). p53 binds to the promoter of Nanog, a gene required for ESC self-renewal, and suppresses Nanog expression after DNA damage. The rapid down-regulation of Nanog mRNA during ESC differentiation correlates with the induction of p53 transcriptional activity and Ser 315 phosphorylation. The importance of Ser 315 phosphorylation was revealed by the finding that induction of p53 activity is impaired in p53(S315A) knock-in ESCs during differentiation, leading to inefficient suppression of Nanog expression. The decreased inhibition of Nanog expression in p53(S315A) ESCs during differentiation is due to an impaired recruitment of the co-repressor mSin3a to the Nanog promoter. These findings indicate an alternative mechanism for p53 to maintain genetic stability in ESCs, by inducing the differentiation of ESCs into other cell types that undergo efficient p53-dependent cell-cycle arrest and apoptosis.
855 citations
TL;DR: It is proposed that the ECM controls the location of actin dynamics at the membrane, and thus the segregation of cortical components in interphase and mitosis and is maintained on the cortex of mitotic cells and used for spindle orientation.
Abstract: The cell division axis determines the future positions of daughter cells and is therefore critical for cell fate. The positioning of the division axis has been mostly studied in systems such as embryos or yeasts, in which cell shape is well defined. In these cases, cell shape anisotropy and cell polarity affect spindle orientation. It remains unclear whether cell geometry or cortical cues are determinants for spindle orientation in mammalian cultured cells. The cell environment is composed of an extracellular matrix (ECM), which is connected to the intracellular actin cytoskeleton via transmembrane proteins. We used micro-contact printing to control the spatial distribution of the ECM on the substrate and demonstrated that it has a role in determining the orientation of the division axis of HeLa cells. On the basis of our analysis of the average distributions of actin-binding proteins in interphase and mitosis, we propose that the ECM controls the location of actin dynamics at the membrane, and thus the segregation of cortical components in interphase. This segregation is further maintained on the cortex of mitotic cells and used for spindle orientation.
803 citations
TL;DR: It is identified that Plk4 is required — in cooperation with Cdk2, CP110 and Hs-SAS6 — for the precise reproduction of centrosomes during the cell cycle and this findings provide an attractive explanation for the crucial function of PlK4 in cell proliferation.
Abstract: The human Polo-like kinase 1 (PLK1) and its functional homologues that are present in other eukaryotes have multiple, crucial roles in meiotic and mitotic cell division. By contrast, the functions of other mammalian Polo family members remain largely unknown. Plk4 is the most structurally divergent Polo family member; it is maximally expressed in actively dividing tissues and is essential for mouse embryonic development. Here, we identify Plk4 as a key regulator of centriole duplication. Both gain- and loss-of-function experiments demonstrate that Plk4 is required--in cooperation with Cdk2, CP110 and Hs-SAS6--for the precise reproduction of centrosomes during the cell cycle. These findings provide an attractive explanation for the crucial function of Plk4 in cell proliferation and have implications for the role of Polo kinases in tumorigenesis.
797 citations
TL;DR: It is shown that transcriptional activation of cyclin B by FoxM1 is essential for timely mitotic entry, whereas CENP-F, another direct target of FoxM 1 identified here, isessential for precise functioning of the mitotic spindle checkpoint.
Abstract: Transcriptional induction of cell-cycle regulatory proteins ensures proper timing of subsequent cell-cycle events. Here we show that the Forkhead transcription factor FoxM1 regulates expression of many G2-specific genes and is essential for chromosome stability. Loss of FoxM1 leads to pleiotropic cell-cycle defects, including a delay in G2, chromosome mis-segregation and frequent failure of cytokinesis. We show that transcriptional activation of cyclin B by FoxM1 is essential for timely mitotic entry, whereas CENP-F, another direct target of FoxM1 identified here, is essential for precise functioning of the mitotic spindle checkpoint. Thus, our data uncover a transcriptional cluster regulated by FoxM1 that is essential for proper mitotic progression.
749 citations
TL;DR: The results support a functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of a target mRNA.
Abstract: In animals, the majority of microRNAs regulate gene expression through the RNA interference (RNAi) machinery without inducing small-interfering RNA (siRNA)-directed mRNA cleavage1. Thus, the mechanisms by which microRNAs repress their targets have remained elusive. Recently, Argonaute proteins, which are key RNAi effector components, and their target mRNAs were shown to localize to cytoplasmic foci known as P-bodies or GW-bodies2,3. Here, we show that the Argonaute proteins physically interact with a key P-/GW-body subunit, GW182. Silencing of GW182 delocalizes resident P-/GW-body proteins and impairs the silencing of microRNA reporters. Moreover, mutations that prevent Argonaute proteins from localizing in P-/GW-bodies prevent translational repression of mRNAs even when Argonaute is tethered to its target in a siRNA-independent fashion. Thus, our results support a functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of a target mRNA.
722 citations
TL;DR: It is shown that Argonaute 2, a key component of RISC, is not randomly distributed but concentrates in mRNA decay centres that are known as cytoplasmic bodies, providing new insight into the mechanism of RNAi function.
Abstract: RNA interference (RNAi) is an important means of eliminating mRNAs, but the intracellular location of RNA-induced silencing complex (RISC) remains unknown. We show here that Argonaute 2, a key component of RISC, is not randomly distributed but concentrates in mRNA decay centres that are known as cytoplasmic bodies. The localization of Argonaute 2 in decay centres is not altered by the presence or absence of small interfering RNAs or their targeted mRNAs. However, RNA is required for the integrity of cytoplasmic bodies because RNase eliminates Argonaute 2 localization. In addition, Argonaute 1, another Argonaute family member, is concentrated in cytoplasmic bodies. These results provide new insight into the mechanism of RNAi function.
679 citations
TL;DR: It is demonstrated that human c-Myc also directly enhances Pol I transcription of ribosomal RNA (rRNA) genes, and proposed that stimulation of rRNA synthesis by c- myc is a key pathway driving cell growth and tumorigenesis.
Abstract: c-Myc coordinates cell growth and division through a transcriptional programme that involves both RNA polymerase (Pol) II- and Pol III-transcribed genes. Here, we demonstrate that human c-Myc also directly enhances Pol I transcription of ribosomal RNA (rRNA) genes. rRNA synthesis and accumulation occurs rapidly following activation of a conditional MYC-ER allele (coding for a Myc-oestrogen-receptor fusion protein), is resistant to inhibition of Pol II transcription and is markedly reduced by c-MYC RNA interference. Furthermore, by using combined immunofluorescence and rRNA-FISH, we have detected endogenous c-Myc in nucleoli at sites of active ribosomal DNA (rDNA) transcription. Our data also show that c-Myc binds to specific consensus elements located in human rDNA and associates with the Pol I-specific factor SL1. The presence of c-Myc at specific sites on rDNA coincides with the recruitment of SL1 to the rDNA promoter and with increased histone acetylation. We propose that stimulation of rRNA synthesis by c-Myc is a key pathway driving cell growth and tumorigenesis.
TL;DR: It is established that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system, and that RAD51 is phosphorylated on Thr 309 in a ChK1-dependent manner, highlighting a crucial role for the Chk2 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.
Abstract: The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.
TL;DR: This work develops a model system of focal adhesion disassembly based on the finding that microtubule regrowth after nocodazole washout induces disassembly of focalAdhesion, and that this disassembly occurs independently of Rho and Rac, but depends on focaladhesion kinase (FAK) and dynamin.
Abstract: Imaging studies implicate microtubule targeting of focal adhesions in focal adhesion disassembly, although the molecular mechanism is unknown. Here, we develop a model system of focal adhesion disassembly based on the finding that microtubule regrowth after nocodazole washout induces disassembly of focal adhesions, and that this disassembly occurs independently of Rho and Rac, but depends on focal adhesion kinase (FAK) and dynamin. During disassembly, dynamin interacts with FAK and colocalizes with focal adhesions. Inhibition of dynamin prevents migration of cells with a focal adhesion phenotype. Our results show that focal adhesion disassembly involves microtubules, dynamin and FAK, and is not simply the reversal of focal adhesion formation.
TL;DR: It is shown that expression of a Paneth gene programme is critically dependent on TCF4 in embryonic intestine and that conditional deletion of the Wnt receptor Frizzled-5 abrogates expression of these genes in Paneth cells in the adult intestine, implying that Wnt signals in the crypt can separately drive a stem-cell/progenitor gene programme and aPaneth-cell maturation programme in intestinal cancer.
Abstract: Wnt signalling, which is transduced through beta-catenin/TCF4, maintains the undifferentiated state of intestinal crypt progenitor cells. Mutational activation of the pathway initiates the adenomacarcinoma sequence. Whereas all other differentiated epithelial cells migrate from the crypt onto the villus, Paneth cells home towards the source of Wnt signals--that is, the crypt bottom. Here, we show that expression of a Paneth gene programme is critically dependent on TCF4 in embryonic intestine. Moreover, conditional deletion of the Wnt receptor Frizzled-5 abrogates expression of these genes in Paneth cells in the adult intestine. Conversely, adenomas in Apc-mutant mice and colorectal cancers in humans inappropriately express these Paneth-cell genes. These observations imply that Wnt signals in the crypt can separately drive a stem-cell/progenitor gene programme and a Paneth-cell maturation programme. In intestinal cancer, both gene programmes are activated simultaneously.
TL;DR: A membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (Mr 80K) indicate that TRPC1 is a component of the vertebrate MSCCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels.
Abstract: The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na+, K+, Ca2+ and Mg2+) flux across the cell membrane, and is implicated in cell-volume regulation1, cell locomotion2, muscle dystrophy3 and cardiac arrhythmias4. However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp5. The oocyte was chosen because it expresses the prototypical MscCa (≥107MscCa/oocyte)6 that is preserved in cytoskeleton-deficient membrane vesicles7. We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (Mr 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1)8,9,10. Heterologous expression of the human TRPC1 resulted in a >1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels11.
TL;DR: Activation of the ATM kinase pathway at chromosome ends does not require overhang degradation or other overt DNA processing, and telomere structure after conditional deletion of mouse TRF2, the protective factor at telomeres is studied.
Abstract: Telomere attrition and other forms of telomere damage can activate the ATM kinase pathway What generates the DNA damage signal at mammalian chromosome ends or at other double-strand breaks is not known Telomere dysfunction is often accompanied by disappearance of the 3' telomeric overhang, raising the possibility that DNA degradation could generate the structure that signals Here we address these issues by studying telomere structure after conditional deletion of mouse TRF2, the protective factor at telomeres Upon removal of TRF2 from TRF2(F/-) p53-/- mouse embryo fibroblasts, a telomere damage response is observed at most chromosome ends As expected, the telomeres lose the 3' overhang and are processed by the non-homologous end-joining pathway Non-homologous end joining of telomeres was abrogated in DNA ligase IV-deficient (Lig4-/-) cells Unexpectedly, the telomeres of TRF2-/- Lig4-/- p53-/- cells persisted in a free state without undergoing detectable DNA degradation Notably, the telomeres retained their 3' overhangs, but they were recognized as sites of DNA damage, accumulating the DNA damage response factors 53BP1 and gamma-H2AX, and activating the ATM kinase Thus, activation of the ATM kinase pathway at chromosome ends does not require overhang degradation or other overt DNA processing
TL;DR: The results unmask KLf4 as a regulator of p53 that oncogenically transforms cells as a function of p21CIP1 status and provide a mechanistic explanation for the context-dependent oncogenic or tumour-suppressor functions of KLF4.
Abstract: KLF4 (GKLF/EZF) encodes a transcription factor that is associated with both tumour suppression and oncogenesis. We describe the identification of KLF4 in a functional genomic screen for genes that bypass RASV12-induced senescence. However, in untransformed cells, KLF4 acts as a potent inhibitor of proliferation. KLF4-induced arrest is bypassed by oncogenic RASV12 or by the RAS target cyclin-D1. Remarkably, inactivation of the cyclin-D1 target and the cell-cycle inhibitor p21CIP1 not only neutralizes the cytostatic action of KLF4, but also collaborates with KLF4 in oncogenic transformation. Conversely, KLF4 suppresses the expression of p53 by directly acting on its promoter, thereby allowing for RASV12-mediated transformation and causing resistance to DNA-damage-induced apoptosis. Consistently, KLF4 depletion from breast cancer cells restores p53 levels and causes p53-dependent apoptosis. These results unmask KLF4 as a regulator of p53 that oncogenically transforms cells as a function of p21CIP1 status. Furthermore, they provide a mechanistic explanation for the context-dependent oncogenic or tumour-suppressor functions of KLF4.
TL;DR: In this article, the authors used RNA interference to systematically screen the kinase and phosphatase component of the human genome and identified several new survival kinases. And they also identified a subset of phosphatases with tumour-suppressor-like activity.
Abstract: Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.
TL;DR: Tumour-derived PDGFRβ + (platelet-derived growth factor receptor β) progenitor perivascular cells (PPCs) that have the ability to differentiate into pericytes and regulate vessel stability and vascular survival in tumours are identified.
Abstract: The microvasculature consists of endothelial cells and their surrounding pericytes. Few studies on the regulatory mechanisms of tumour angiogenesis have focused on pericytes. Here we report the identification of tumour-derived PDGFRβ+ (platelet-derived growth factor receptor β) progenitor perivascular cells (PPCs) that have the ability to differentiate into pericytes and regulate vessel stability and vascular survival in tumours. A subset of PDGFRβ+ PPCs is recruited from bone marrow to perivascular sites in tumours. Specific inhibition of PDGFRβ signalling eliminates PDGFRβ+ PPCs and mature pericytes around tumour vessels, leading to vascular hyperdilation and endothelial cell apoptosis in pancreatic islet tumours of transgenic Rip1Tag2 mice.
TL;DR: It is concluded that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.
Abstract: Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.
TL;DR: It is demonstrated that DISC1 is a component of the microtubule-associated dynein motor complex and is essential for maintaining the complex at the centrosome, hence contributing to normal microtubular dynamics and suggesting that loss of DISC 1 function may underlie neurodevelopmental dysfunction in schizophrenia.
Abstract: Disrupted-In-Schizophrenia-1 (DISC1), originally identified at the breakpoint of a chromosomal translocation that is linked to a rare familial schizophrenia, has been genetically implicated in schizophrenia in other populations. Schizophrenia involves subtle cytoarchitectural abnormalities that arise during neurodevelopment, but the underlying molecular mechanisms are unclear. Here, we demonstrate that DISC1 is a component of the microtubule-associated dynein motor complex and is essential for maintaining the complex at the centrosome, hence contributing to normal microtubular dynamics. Carboxy-terminal-truncated mutant DISC1 (mutDISC1), which results from a chromosomal translocation, functions in a dominant-negative manner by redistributing wild-type DISC1 through self-association and by dissociating the DISC1-dynein complex from the centrosome. Consequently, either depletion of endogenous DISC1 or expression of mutDISC1 impairs neurite outgrowth in vitro and proper development of the cerebral cortex in vivo. These results indicate that DISC1 is involved in cerebral cortex development, and suggest that loss of DISC1 function may underlie neurodevelopmental dysfunction in schizophrenia.
TL;DR: It is demonstrated that endogenous Ago2 and transfected small interfering RNAs (siRNAs) are also present within these same cytoplasmic bodies and that the GW182 protein interacts with Ago2.
Abstract: The GW182 RNA-binding protein was initially shown to associate with a specific subset of mRNAs and to reside within discrete cytoplasmic foci named GW bodies (GWBs)1. GWBs are enriched in proteins that are involved in mRNA degradation2. Recent reports have shown that exogenously introduced human Argonaute-2 (Ago2) is also enriched in GWBs, indicating that RNA interference function may be somehow linked to these structures3,4. In this report, we demonstrate that endogenous Ago2 and transfected small interfering RNAs (siRNAs) are also present within these same cytoplasmic bodies and that the GW182 protein interacts with Ago2. Disruption of these cytoplasmic foci in HeLa cells interferes with the silencing capability of a siRNA that is specific to lamin-A/C. Our data support a model in which GW182 and/or the microenvironment of the cytoplasmic GWBs contribute to the RNA-induced silencing complex and to RNA silencing.
TL;DR: It is shown that RhoA and Cdc42, members of the Rho family of small GTPases, regulate the intracellular localization of PTEN in leukocytes and human transfected embryonic kidney cells, and that small G TPase-mediated regulation of PT EN has a significant role in the regulation of chemotaxis.
Abstract: PTEN (phosphatase and tensin homologue) is a phosphatase that dephosphorylates both protein and phosphoinositide substrates. It is mutated in a variety of human tumours and has important roles in a diverse range of biological processes1,2,3,4, including cell migration5,6,7 and chemotaxis8,9. PTEN's intracellular localization and presumably activity are regulated by chemoattractants in Dictyostelium10,11 and mouse neutrophils12. However, the mechanisms for its regulation remain elusive. Here we show that RhoA and Cdc42, members of the Rho family of small GTPases13,14,15,16, regulate the intracellular localization of PTEN in leukocytes and human transfected embryonic kidney cells. In addition, active RhoA is able to stimulate the phospholipid phosphatase activity of PTEN in human embryonic kidney cells and leukocytes, and this regulation seems to require RhoA's downstream effector, RhoA-associated kinase (Rock). Furthermore, we have identified key residues on PTEN that are required for its regulation by the small GTPase, and show that small GTPase-mediated regulation of PTEN has a significant role in the regulation of chemotaxis.
TL;DR: The results suggest that c-Myc coordinates the activity of all three nuclear RNA polymerases, and thereby plays a key role in regulating ribosome biogenesis and cell growth.
Abstract: The c-Myc oncoprotein regulates transcription of genes that are associated with cell growth, proliferation and apoptosis(1). c-Myc levels are modulated by ubiquitin/proteasome-mediated degradation( ...
TL;DR: It is shown that depletion of Par-3 in mammalian epithelial cells profoundly disrupts tight junction assembly, and a novel mechanism through which Par- 3 engages in the spatial regulation of Rac activity and establishment of epithelial polarity is revealed.
Abstract: The par (partitioning-defective) genes express a set of conserved proteins that function in polarization and asymmetric cell division. Par-3 has multiple protein-interaction domains, and associates with Par-6 and atypical protein kinase C (aPKC). In Drosophila, Par-3 is essential for epithelial cell polarization. However, its function in mammals is unclear. Here we show that depletion of Par-3 in mammalian epithelial cells profoundly disrupts tight junction assembly. Expression of a carboxy-terminal fragment plus the third PDZ domain of Par-3 partially rescues junction assembly, but neither Par-6 nor aPKC binding is required. Unexpectedly, Rac is constitutively activated in cells lacking Par-3, and the assembly of tight junctions is efficiently restored by a dominant-negative Rac mutant. The Rac exchange factor Tiam1 (ref. 7) binds directly to the carboxy-terminal region of Par-3, and knockdown of Tiam1 enhances tight junction formation in cells lacking Par-3. These results define a critical function for Par-3 in tight junction assembly, and reveal a novel mechanism through which Par-3 engages in the spatial regulation of Rac activity and establishment of epithelial polarity.
TL;DR: A simulation model of ERK signalling networks is developed by constraining in silico dynamics based on in vivo dynamics in PC12 cells that predicted and validated that transient ERK activation depends on rapid increases of epidermal growth factor and nerve growth factor but not on their final concentrations, whereas sustained ERKactivation depends on the final concentration of NGFbut not on the temporal rate of increase.
Abstract: To elucidate the hidden dynamics of extracellular-signal-regulated kinase (ERK) signalling networks, we developed a simulation model of ERK signalling networks by constraining in silico dynamics based on in vivo dynamics in PC12 cells. We predicted and validated that transient ERK activation depends on rapid increases of epidermal growth factor and nerve growth factor (NGF) but not on their final concentrations, whereas sustained ERK activation depends on the final concentration of NGF but not on the temporal rate of increase. These ERK dynamics depend on Ras and Rap1 dynamics, the inactivation processes of which are growth-factor-dependent and -independent, respectively. Therefore, the Ras and Rap1 systems capture the temporal rate and concentration of growth factors, and encode these distinct physical properties into transient and sustained ERK activation, respectively.
TL;DR: It is reported that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria, and clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo.
Abstract: Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities.
TL;DR: A shift in localization of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalization upon cell detachment, which mediates inhibition of Erk, PI(3)K and Rac, which defines a novel molecular mechanism for growth and tumour suppression by caveolin- 1.
Abstract: Growth of normal cells is anchorage dependent because signalling through multiple pathways including Erk, phosphatidylinositol-3-OH kinase (PI(3)K) and Rac requires integrin-mediated cell adhesion. Components of these pathways localize to low-density, cholesterol-rich domains in the plasma membrane named 'lipid rafts' or 'cholesterol-enriched membrane microdomains' (CEMM). We previously reported that integrin-mediated adhesion regulates CEMM transport such that cell detachment from the extracellular matrix triggers CEMM internalization and clearance from the plasma membrane. We now report that this internalization is mediated by dynamin-2 and caveolin-1. Internalization requires phosphorylation of caveolin-1 on Tyr 14. A shift in localization of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalization upon cell detachment, which mediates inhibition of Erk, PI(3)K and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1.
TL;DR: Evidence is provided that anti-apoptotic Bcl-2 proteins regulate the inositol 1,4,5-trisphosphate receptor (InsP3R) ER Ca2+ release channel resulting in increased cellular apoptotic resistance and enhanced mitochondrial bioenergetics and preserves survival.
Abstract: Members of the Bcl-2 protein family modulate outer mitochondrial membrane permeability to control apoptosis. However, these proteins also localize to the endoplasmic reticulum (ER), the functional significance of which is controversial. Here we provide evidence that anti-apoptotic Bcl-2 proteins regulate the inositol 1,4,5-trisphosphate receptor (InsP(3)R) ER Ca(2+) release channel resulting in increased cellular apoptotic resistance and enhanced mitochondrial bioenergetics. Anti-apoptotic Bcl-X(L) interacts with the carboxyl terminus of the InsP(3)R and sensitizes single InsP(3)R channels in ER membranes to low [InsP(3)], enhancing Ca(2+) and InsP(3)-dependent regulation of channel activity in vitro and in vivo, reducing ER Ca(2+) content and stimulating mitochondrial energetics. The pro-apoptotic proteins Bax and tBid antagonize this effect by blocking the biochemical interaction of Bcl-X(L) with the InsP(3)R. These data support a novel model in which Bcl-X(L) is a direct effector of the InsP(3)R, increasing its sensitivity to InsP(3) and enabling ER Ca(2+) release to be more sensitively coupled to extracellular signals. As a consequence, cells are protected against apoptosis by a more sensitive and dynamic coupling of ER to mitochondria through Ca(2+)-dependent signal transduction that enhances cellular bioenergetics and preserves survival.
TL;DR: It is shown that control of cholesterol and SM metabolism involves APP processing and also a functional basis for the link that has been observed between lipids and Alzheimer's disease (AD).
Abstract: Amyloid beta peptide (Aβ) has a key role in the pathological process of Alzheimer's disease (AD), but the physiological function of Aβ and of the amyloid precursor protein (APP) is unknown1,2. Recently, it was shown that APP processing is sensitive to cholesterol and other lipids3,4,5,6,7,8,9,10. Hydroxymethylglutaryl-CoA reductase (HMGR) and sphingomyelinases (SMases) are the main enzymes that regulate cholesterol biosynthesis and sphingomyelin (SM) levels, respectively. We show that control of cholesterol and SM metabolism involves APP processing. Aβ42 directly activates neutral SMase and downregulates SM levels, whereas Aβ40 reduces cholesterol de novo synthesis by inhibition of HMGR activity. This process strictly depends on γ-secretase activity. In line with altered Aβ40/42 generation, pathological presenilin mutations result in increased cholesterol and decreased SM levels. Our results demonstrate a biological function for APP processing and also a functional basis for the link that has been observed between lipids and Alzheimer's disease (AD).