Showing papers in "Nature Cell Biology in 2004"
TL;DR: Two distinct TOR complexes constitute a primordial signalling network conserved in eukaryotic evolution to control the fundamental process of cell growth.
Abstract: The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of cell growth. In budding yeast, TOR is found in structurally and functionally distinct protein complexes: TORC1 and TORC2. A mammalian counterpart of TORC1 (mTORC1) has been described, but it is not known whether TORC2 is conserved in mammals. Here, we report that a mammalian counterpart of TORC2 (mTORC2) also exists. mTORC2 contains mTOR, mLST8 and mAVO3, but not raptor. Like yeast TORC2, mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton. mTORC2 is not upstream of the mTORC1 effector S6K. Thus, two distinct TOR complexes constitute a primordial signalling network conserved in eukaryotic evolution to control the fundamental process of cell growth.
2,059 citations
TL;DR: It is shown that GSK-3β binds to and phosphorylates Snail at two consensus motifs to dually regulate the function of this protein and together function as a molecular switch for many signalling pathways that lead to EMT.
Abstract: The phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development. Snail, a zinc-finger transcription factor, triggers this process by repressing E-cadherin expression; however, the mechanisms that regulate Snail remain elusive. Here we find that Snail is highly unstable, with a short half-life about 25 min. We show that GSK-3beta binds to and phosphorylates Snail at two consensus motifs to dually regulate the function of this protein. Phosphorylation of the first motif regulates its beta-Trcp-mediated ubiquitination, whereas phosphorylation of the second motif controls its subcellular localization. A variant of Snail (Snail-6SA), which abolishes these phosphorylations, is much more stable and resides exclusively in the nucleus to induce EMT. Furthermore, inhibition of GSK-3beta results in the upregulation of Snail and downregulation of E-cadherin in vivo. Thus, Snail and GSK-3beta together function as a molecular switch for many signalling pathways that lead to EMT.
1,523 citations
TL;DR: Results indicate that the Bcl-2 family of proteins not only regulates apoptosis, but also controls non-apoptotic programmed cell death that depends on the autophagy genes.
Abstract: Programmed cell death can be divided into several categories including type I (apoptosis) and type II (autophagic death). The Bcl-2 family of proteins are well-characterized regulators of apoptosis, and the multidomain pro-apoptotic members of this family, such as Bax and Bak, act as a mitochondrial gateway where a variety of apoptotic signals converge. Although embryonic fibroblasts from Bax/Bak double knockout mice are resistant to apoptosis, we found that these cells still underwent a non-apoptotic death after death stimulation. Electron microscopic and biochemical studies revealed that double knockout cell death was associated with autophagosomes/autolysosomes. This non-apoptotic death of double knockout cells was suppressed by inhibitors of autophagy, including 3-methyl adenine, was dependent on autophagic proteins APG5 and Beclin 1 (capable of binding to Bcl-2/Bcl-x(L)), and was also modulated by Bcl-x(L). These results indicate that the Bcl-2 family of proteins not only regulates apoptosis, but also controls non-apoptotic programmed cell death that depends on the autophagy genes.
1,394 citations
TL;DR: Using quantitative assays, it is shown that kinases and adaptor molecules, including focal adhesion kinase, Src, p130CAS, paxillin, extracellular signal-regulated kinase and myosin light-chain kinase are critical for adhesion turnover at the cell front, a process central to migration.
Abstract: Cell migration is a complex, highly regulated process that involves the continuous formation and disassembly of adhesions (adhesion turnover). Adhesion formation takes place at the leading edge of protrusions, whereas disassembly occurs both at the cell rear and at the base of protrusions. Despite the importance of these processes in migration, the mechanisms that regulate adhesion formation and disassembly remain largely unknown. Here we develop quantitative assays to measure the rate of incorporation of molecules into adhesions and the departure of these proteins from adhesions. Using these assays, we show that kinases and adaptor molecules, including focal adhesion kinase (FAK), Src, p130CAS, paxillin, extracellular signal-regulated kinase (ERK) and myosin light-chain kinase (MLCK) are critical for adhesion turnover at the cell front, a process central to migration.
1,330 citations
TL;DR: The mapping of a protein interaction network around 32 known and candidate TNF-α/NF-κB pathway components is reported by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference.
Abstract: Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-α triggers a signalling cascade, converging on the activation of the transcription factor NF-κB, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-α/NF-κB pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-α/NF-κB pathway and is generally applicable to other pathways relevant to human disease.
956 citations
TL;DR: The salutary intersection of fundamental cell biology with the study of disease is well illustrated by the emerging elucidation of neurodegenerative disorders, where normally-soluble proteins accumulate, misfold and oligomerize, inducing cytotoxic effects that are particularly devastating in the post-mitotic milieu of the neuron.
Abstract: The salutary intersection of fundamental cell biology with the study of disease is well illustrated by the emerging elucidation of neurodegenerative disorders. Novel mechanisms in cell biology have been uncovered through disease-orientated research; for example, the discovery of presenilin as an intramembrane aspartyl protease that processes many diverse proteins within the lipid bilayer. A common theme has arisen in this field: normally-soluble proteins accumulate, misfold and oligomerize, inducing cytotoxic effects that are particularly devastating in the post-mitotic milieu of the neuron.
860 citations
TL;DR: The results indicate that in metazoa, di- and tri-methylation of K4/H3 is linked to active transcription and that significant differences exist in the genome-wide methylation pattern as compared with S. cerevisiae.
Abstract: Lysine residues within histones can be mono-, di - or tri-methylated. In Saccharomyces cerevisiae tri-methylation of Lys 4 of histone H3 (K4/H3) correlates with transcriptional activity, but little is known about this methylation state in higher eukaryotes. Here, we examine the K4/H3 methylation pattern at the promoter and transcribed region of metazoan genes. We analysed chicken genes that are developmentally regulated, constitutively active or inactive. We found that the pattern of K4/H3 methylation shows similarities to S. cerevisiae. Tri-methyl K4/H3 peaks in the 5' transcribed region and active genes can be discriminated by high levels of tri-methyl K4/H3 compared with inactive genes. However, our results also identify clear differences compared to yeast, as significant levels of K4/H3 methylation are present on inactive genes within the beta-globin locus, implicating this modification in maintaining a 'poised' chromatin state. In addition, K4/H3 di-methylation is not genome-wide and di-methylation is not uniformly distributed throughout the transcribed region. These results indicate that in metazoa, di- and tri-methylation of K4/H3 is linked to active transcription and that significant differences exist in the genome-wide methylation pattern as compared with S. cerevisiae.
787 citations
TL;DR: It is proposed that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.
Abstract: A fundamental question in stem cell research is whether cultured multipotent adult stem cells represent endogenous multipotent precursor cells. Here we address this question, focusing on SKPs, a cultured adult stem cell from the dermis that generates both neural and mesodermal progeny. We show that SKPs derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells. We demonstrate that these endogenous SKPs can first be isolated from skin during embryogenesis and that they persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. We propose that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.
761 citations
TL;DR: It is shown that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase, resulting in c- myc stabilization.
Abstract: The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.
761 citations
TL;DR: It is shown that, after cell stress, p53 interacts with the pro-apoptotic mitochondrial membrane protein Bak, which is consistent with a model in which p53 and Mcl1 have opposing effects on mitochondrial apoptosis by interacting with, and modulating the activity of, the death effector Bak.
Abstract: The tumour suppressor activity of the p53 protein has been explained by its ability to induce apoptosis in response to a variety of cellular stresses1,2. Thus, understanding the mechanism by which p53 functions in the execution of cell death pathways is of considerable importance in cancer biology. Recent studies have indicated that p53 has a direct signalling role at mitochondria in the induction of apoptosis3,4,5,6, although the mechanisms involved are not completely understood. Here we show that, after cell stress, p53 interacts with the pro-apoptotic mitochondrial membrane protein Bak. Interaction of p53 with Bak causes oligomerization of Bak and release of cytochrome c from mitochondria. Notably, we show that formation of the p53–Bak complex coincides with loss of an interaction between Bak and the anti-apoptotic Bcl2-family member Mcl1. These results are consistent with a model in which p53 and Mcl1 have opposing effects on mitochondrial apoptosis by interacting with, and modulating the activity of, the death effector Bak.
753 citations
TL;DR: It is shown that γ-H2AX foci (γ-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice, and accumulation of unrepairable DSBs may have a causal role in mammalian ageing.
Abstract: Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that gamma-H2AX foci (gamma-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.
TL;DR: It is suggested that SMYD3 has histone methyltransferase activity and plays an important role in transcriptional regulation as a member of an RNA polymerase complex and activation ofSMYD3 may be a key factor in human carcinogenesis.
Abstract: Colorectal and hepatocellular carcinomas are some of the leading causes of cancer deaths worldwide, but the mechanisms that underly these malignancies are not fully understood. Here we report the identification of SMYD3, a gene that is over-expressed in the majority of colorectal carcinomas and hepatocellular carcinomas. Introduction of SMYD3 into NIH3T3 cells enhanced cell growth, whereas genetic knockdown with small-interfering RNAs (siRNAs) in cancer cells resulted in significant growth suppression. SMYD3 formed a complex with RNA polymerase II through an interaction with the RNA helicase HELZ and transactivated a set of genes that included oncogenes, homeobox genes and genes associated with cell-cycle regulation. SMYD3 bound to a motif, 5′-CCCTCC-3′, present in the promoter region of downstream genes such as Nkx2.8. The SET domain of SMYD3 showed histone H3-lysine 4 (H3-K4)-specific methyltransferase activity, which was enhanced in the presence of the heat-shock protein HSP90A. Our findings suggest that SMYD3 has histone methyltransferase activity and plays an important role in transcriptional regulation as a member of
TL;DR: It is shown that the AT1 receptor can be activated by mechanical stress through an angiotensin-II-independent mechanism and this activation can be inhibited by an inverse agonist of the At1 receptor.
Abstract: The angiotensin II type 1 (AT1) receptor has a crucial role in load-induced cardiac hypertrophy. Here we show that the AT1 receptor can be activated by mechanical stress through an angiotensin-II-independent mechanism. Without the involvement of angiotensin II, mechanical stress not only activates extracellular-signal-regulated kinases and increases phosphoinositide production in vitro, but also induces cardiac hypertrophy in vivo. Mechanical stretch induces association of the AT1 receptor with Janus kinase 2, and translocation of G proteins into the cytosol. All of these events are inhibited by the AT1 receptor blocker candesartan. Thus, mechanical stress activates AT1 receptor independently of angiotensin II, and this activation can be inhibited by an inverse agonist of the AT1 receptor.
TL;DR: Results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.
Abstract: Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. We investigated whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functioned as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevented microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, was sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibited fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.
TL;DR: It is shown that hematopoietic stem cells become liver cells when cocultured with injured liver separated by a barrier and that HSCs contribute to the regeneration of injured liver by converting into functional hepatocytes without fusion.
Abstract: Both plasticity and cell fusion have been suggested to have a role in germ-layer switching1,2,3,4,5,6,7. To understand the mechanisms underlying cell fate changes, we have examined a highly enriched population of hematopoietic stem cells (HSCs)8,9,10 in vitro or in vivo in response to injury for liver-specific phenotypic and functional changes. Here we show that HSCs become liver cells when cocultured with injured liver separated by a barrier. Chromosomal analyses and tissue-specific gene and/or protein expression show that microenvironmental cues rather than fusion are responsible for conversion in vitro. We transplanted HSCs into liver-injured mice and observed that HSCs convert into viable hepatocytes with increasing injury. Notably, liver function was restored 2–7 d after transplantation. We conclude that HSCs contribute to the regeneration of injured liver by converting into functional hepatocytes without fusion.
TL;DR: Two functional screens for proteins required for the invasion of fibrosarcoma cells that identified the molecular chaperone heat shock protein 90 are described, indicating that cell-impermeant anti-hsp90 drugs might decrease invasiveness without the concerns inherent in inhibiting intracellular hsp90.
Abstract: Tumour cell invasiveness is crucial for cancer metastasis and is not yet understood. Here we describe two functional screens for proteins required for the invasion of fibrosarcoma cells that identified the molecular chaperone heat shock protein 90 (hsp90). The hsp90 alpha isoform, but not hsp90 beta, is expressed extracellularly where it interacts with the matrix metalloproteinase 2 (MMP2). Inhibition of extracellular hsp90 alpha decreases both MMP2 activity and invasiveness. This role for extracellular hsp90 alpha in MMP2 activation indicates that cell-impermeant anti-hsp90 drugs might decrease invasiveness without the concerns inherent in inhibiting intracellular hsp90.
TL;DR: It is demonstrated that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover.
Abstract: Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.
TL;DR: It is reported that growth factor stimulation initiates the rapid translocation of the transient receptor potential ion channel, TRPC5, from vesicles held in reserve just under the plasma membrane, resulting in tight spatial–temporal control of these Ca2+-permeant nonselective channels.
Abstract: The broadly expressed transient receptor potential (TRP) family of ion channels are permeant to cations, most resulting in increased intracellular calcium. However, their regulation and gating is not well understood. Here, we report that growth factor stimulation initiates the rapid translocation of the transient receptor potential ion channel, TRPC5, from vesicles held in reserve just under the plasma membrane. This process, which we term 'rapid vesicular insertion of TRP' (RiVIT), dramatically increases membrane-associated TRPC5 channels and functional TRPC5 current, resulting in tight spatial–temporal control of these Ca2+-permeant nonselective channels. Epidermal growth factor (EGF)-induced incorporation of functional TRP channels requires phosphatidylinositide 3-kinase (PI(3)K), the Rho GTPase Rac1 and phosphatidylinositol 4-phosphate 5-kinase (PIP(5)Kα). The increase in TRPC5 availability affects neurite extension rates in cultured hippocampal neurons, and may be a general mechanism for initiating Ca2+ influx and cell morphological changes in response to stimuli.
TL;DR: During the past fifteen years, a variety of peptides have been characterized for their ability to translocate into live cells, and most are efficient vectors that can internalize hydrophilic cargoes, and so provide a valuable biological (and potentially therapeutic) tool for targeting proteins into cells.
Abstract: During the past fifteen years, a variety of peptides have been characterized for their ability to translocate into live cells. Most are efficient vectors that can internalize hydrophilic cargoes, and so provide a valuable biological (and potentially therapeutic) tool for targeting proteins into cells. Furthermore, translocation of cell-permeable peptides across the plasma membrane and their subsequent access to the cytosol, even when fused to large hydrophilic proteins, is challenging the perception of the plasma membrane as an impermeable barrier.
TL;DR: FAPPs are essential components of a PtdIns(4)P- and ARF-regulated machinery that controls generation of constitutive post-Golgi carriers and overexpression of FAPP-PH impairs carrier fission.
Abstract: The molecular mechanisms underlying the formation of carriers trafficking from the Golgi complex to the cell surface are still ill-defined; nevertheless, the involvement of a lipid-based machinery is well established. This includes phosphatidylinositol 4-phosphate (PtdIns(4)P), the precursor for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). In yeast, PtdIns(4)P exerts a direct role, however, its mechanism of action and its targets in mammalian cells remain uncharacterized. We have identified two effectors of PtdIns(4)P, the four-phosphate-adaptor protein 1 and 2 (FAPP1 and FAPP2). Both proteins localize to the trans-Golgi network (TGN) on nascent carriers, and interact with PtdIns(4)P and the small GTPase ADP-ribosylation factor (ARF) through their plekstrin homology (PH) domain. Displacement or knockdown of FAPPs inhibits cargo transfer to the plasma membrane. Moreover, overexpression of FAPP-PH impairs carrier fission. Therefore, FAPPs are essential components of a PtdIns(4)P- and ARF-regulated machinery that controls generation of constitutive post-Golgi carriers.
TL;DR: It is shown that loss of Dicer results in cell death with the accumulation of abnormal mitotic cells that show premature sister chromatid separation and that Dicer-related RNA interference machinery is involved in the formation of the heterochromatin structure in higher vertebrate cells.
Abstract: RNA interference is an evolutionarily conserved gene-silencing pathway in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs1. The biological function of the RNAi-related pathway in vertebrate cells is not fully understood. Here, we report the generation of a conditional loss-of-function Dicer mutant in a chicken–human hybrid DT40 cell line that contains human chromosome 21. We show that loss of Dicer results in cell death with the accumulation of abnormal mitotic cells that show premature sister chromatid separation. Aberrant accumulation of transcripts from α-satellite sequences, which consist of human centromeric repeat DNAs, was detected in Dicer-deficient cells. Immunocytochemical analysis revealed abnormalities in the localization of two heterochromatin proteins, Rad21 cohesin protein and BubR1 checkpoint protein, but the localization of core kinetochore proteins such as centromere protein (CENP)-A and -C was normal. We conclude that Dicer-related RNA interference machinery is involved in the formation of the heterochromatin structure in higher vertebrate cells.
TL;DR: Filamins were discovered as the first family of non-muscle actin-binding proteins and are lage cytoplasmic proteins that cross-link cortical actin into a dynamic three-dimensional structure as discussed by the authors.
Abstract: Filamins were discovered as the first family of non-muscle actin-binding protein. They are lage cytoplasmic proteins that cross-link cortical actin into a dynamic three-dimensional structure. Filamins have also been reported to interact with a large number of cellular proteins of great functional diversity, suggesting that they are unusually versatile signalling scaffolds. More recently, genetic mutations in filamin A and B have been reported to cause a wide range of human diseases, suggesting that different diseases highlight distinct filamin interactions.
TL;DR: By removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore–microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either Kinetochores or spindle poles.
Abstract: For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.
TL;DR: PTOP binds to the carboxyl terminus of POT1 and recruits it to telomeres, and regulates POT 1 telomeric recruitment and telomere length in human cells.
Abstract: Telomere maintenance has been implicated in cancer and ageing, and requires cooperation between a multitude of telomeric factors, including telomerase, TRF1, TRF2, RAP1, TIN2, Tankyrase, PINX1 and POT1 (refs 1–12). POT1 belongs to a family of oligonucleotide-binding (OB)-fold-containing proteins that include Oxytricha nova TEBP, Cdc13, and spPot1, which specifically recognize telomeric single-stranded DNA (ssDNA)10,13,14,15,16,17,18,19. In human cells, the loading of POT1 to telomeric ssDNA controls telomerase-mediated telomere elongation12. Surprisingly, a human POT1 mutant lacking an OB fold is still recruited to telomeres. However, the exact mechanism by which this recruitment occurs remains unclear. Here we identify a novel telomere protein, PTOP, which interacts with both POT1 and TIN2. PTOP binds to the carboxyl terminus of POT1 and recruits it to telomeres. Inhibition of PTOP by RNA interference (RNAi) or disruption of the PTOP–POT1 interaction hindered the localization of POT1 to telomeres. Furthermore, expression of the respective interaction domains on PTOP and POT1 alone extended telomere length in human cells. Therefore, PTOP heterodimerizes with POT1 and regulates POT1 telomeric recruitment and telomere length.
TL;DR: A method to determine the dissociation rates of protein subunits from complexes that have a traceable localization inside single living cells is presented, which can be applied to many multi-protein complexes and will help to characterize the dynamic behaviour of complex networks of proteins in live cells.
Abstract: Most cellular activities are executed by multi-protein complexes that form the basic functional modules of their molecular machinery. Proteomic approaches can provide an evermore detailed picture of their composition, but do not reveal how these machines are organized dynamically to accomplish their biological function. Here, we present a method to determine the dissociation rates of protein subunits from complexes that have a traceable localization inside single living cells. As a case study, we systematically analysed the dynamic organization of vertebrate nuclear pore complexes (NPCs), large supramolecular complexes of about 30 different polypeptides. NPC components exhibited a wide range of residence times covering five orders of magnitude from seconds to days. We found the central parts of the NPC to be very stable, consistent with a function as a structural scaffold, whereas more peripheral components exhibited more dynamic behaviour, suggesting adaptor as well as regulatory functions. The presented strategy can be applied to many multi-protein complexes and will help to characterize the dynamic behaviour of complex networks of proteins in live cells.
TL;DR: A reciprocal regulatory interaction between the S LC26T DRA, SLC26A6 and CFTR markedly activates CFTR by increasing its overall open probablity (NPo) sixfold and provides a new understanding of epithelial chloride and bicarbonate transport.
Abstract: Chloride absorption and bicarbonate secretion are vital functions of epithelia, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR, which activates electrogenic chloride-bicarbonate exchange by SLC26T. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NP(o)) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.
TL;DR: Using the small interfering RNA (siRNA) strategy, it is demonstrated that the rapid activation of glycolysis by nitric oxide is dependent on phosphorylation of the energy charge-sensitive AMP-activated protein kinase, resulting in activation of PFK2 and protection of cells from apoptosis.
Abstract: After inhibition of cytochrome c oxidase by nitric oxide, astrocytes maintain energy production by upregulating glycolysis--a response which does not seem to be available to neurons Here, we show that in astrocytes, after inhibition of respiration by nitric oxide, there is a rapid, cyclic GMP-independent increase in the activity of 6-phosphofructo-1-kinase (PFK1), a master regulator of glycolysis, and an increase in the concentration of its most powerful positive allosteric activator, fructose-2,6-bisphosphate (F2,6P(2)) In neurons, nitric oxide failed to alter F2,6P(2) concentration or PFK1 activity This failure could be accounted for by the much lower amount of 6-phosphofructo-2-kinase (PFK2, the enzyme responsible for F2,6P(2) biosynthesis) in neurons Indeed, full activation of neuronal PFK1 was achieved by adding cytosol from nitric oxide-treated astrocytes Furthermore, using the small interfering RNA (siRNA) strategy, we demonstrated that the rapid activation of glycolysis by nitric oxide is dependent on phosphorylation of the energy charge-sensitive AMP-activated protein kinase, resulting in activation of PFK2 and protection of cells from apoptosis Thus the virtual absence of PFK2 in neurons may explain their extreme sensitivity to energy depletion and degeneration
TL;DR: A previously unidentified E3 complex is described: KPC (Kip1 ubiquitination-promoting complex), consisting of KPC1 and KPC2, which interacts with and ubiquitinates p27Kip 1 and is localized to the cytoplasm.
Abstract: The cyclin-dependent kinase inhibitor p27(Kip1) is degraded at the G0-G1 transition of the cell cycle by the ubiquitin-proteasome pathway. Although the nuclear ubiquitin ligase (E3) SCF(Skp2) is implicated in p27(Kip1) degradation, proteolysis of p27(Kip1) at the G0-G1 transition proceeds normally in Skp2(-/-) cells. Moreover, p27(Kip1) is exported from the nucleus to the cytoplasm at G0-G1 (refs 9-11). These data suggest the existence of a Skp2-independent pathway for the degradation of p27(Kip1) at G1 phase. We now describe a previously unidentified E3 complex: KPC (Kip1 ubiquitination-promoting complex), consisting of KPC1 and KPC2. KPC1 contains a RING-finger domain, and KPC2 contains a ubiquitin-like domain and two ubiquitin-associated domains. KPC interacts with and ubiquitinates p27(Kip1) and is localized to the cytoplasm. Overexpression of KPC promoted the degradation of p27(Kip1), whereas a dominant-negative mutant of KPC1 delayed p27(Kip1) degradation. The nuclear export of p27(Kip1) by CRM1 seems to be necessary for KPC-mediated proteolysis. Depletion of KPC1 by RNA interference also inhibited p27(Kip1) degradation. KPC thus probably controls degradation of p27(Kip1) in G1 phase after export of the latter from the nucleus.
TL;DR: T tandem mass spectrometry is used to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121, which is as active as theWAVE2–Abi1–Nap1–PIR121 complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo.
Abstract: WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.
TL;DR: It is shown that STAT3 is activated in the presence of active Notch, as well as the Notch effectors Hes1 and Hes5, thus promoting STAT3 phosphorylation and activation, and suppression of endogenous Hes1 expression reduces growth factor induction of STAT3osphorylation.
Abstract: Although the Notch and JAK–STAT signalling pathways fulfill overlapping roles in growth and differentiation regulation, no coordination mechanism has been proposed to explain their relationship. Here we show that STAT3 is activated in the presence of active Notch, as well as the Notch effectors Hes1 and Hes5. Hes proteins associate with JAK2 and STAT3, and facilitate complex formation between JAK2 and STAT3, thus promoting STAT3 phosphorylation and activation. Furthermore, suppression of endogenous Hes1 expression reduces growth factor induction of STAT3 phosphorylation. STAT3 seems to be essential for maintenance of radial glial cells and differentiation of astrocytes by Notch in the developing central nervous system. These results suggest that direct protein–protein interactions coordinate cross-talk between the Notch–Hes and JAK–STAT pathways.