Cell

SnapShot: PTEN signaling pathways.

Publication Date: 2008 May 2 PMID: 18455993
Authors: Carracedo, A. - Salmena, L. - Pandolfi, P. P.
Journal: Cell

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Application of active and kinase-deficient kinome collection for identification of kinases regulating hedgehog signaling.

Publication Date: 2008 May 2 PMID: 18455992
Authors: Varjosalo, M. - Bjorklund, M. - Cheng, F. - Syvanen, H. - Kivioja, T. - Kilpinen, S. - Sun, Z. - Kallioniemi, O. - Stunnenberg, H. G. - He, W. W. - Ojala, P. - Taipale, J.
Journal: Cell

To allow genome-scale identification of genes that regulate cellular signaling, we cloned >90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase activity-deficient mutants. To establish the utility of this resource, we tested the effect of expression of the kinases on three different cellular signaling models. In all screens, many kinases had a modest but significant effect, apparently due to crosstalk between signaling pathways. However, the strongest effects were found with known regulators and novel components, such as MAP3K10 and DYRK2, which we identified in a mammalian Hedgehog (Hh) signaling screen. DYRK2 directly phosphorylated and induced the proteasome-dependent degradation of the key Hh pathway-regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2 and the known Hh pathway component, GSK3beta. Our results establish kinome expression screening as a highly effective way to identify physiological signaling pathway components and genes involved in pathological signaling crosstalk.

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Identification of positionally distinct astrocyte subtypes whose identities are specified by a homeodomain code.

Publication Date: 2008 May 2 PMID: 18455991
Authors: Hochstim, C. - Deneen, B. - Lukaszewicz, A. - Zhou, Q. - Anderson, D. J.
Journal: Cell

Astrocytes constitute the most abundant cell type in the central nervous system (CNS) and play diverse functional roles, but the ontogenetic origins of this phenotypic diversity are poorly understood. We have investigated whether positional identity, a fundamental organizing principle governing the generation of neuronal subtype diversity, is also relevant to astrocyte diversification. We identified three positionally distinct subtypes of white-matter astrocytes (WMA) in the spinal cord, which can be distinguished by the combinatorial expression of Reelin and Slit1. These astrocyte subtypes derive from progenitor domains expressing the homeodomain transcription factors Pax6 and Nkx6.1, respectively. Loss- and gain-of-function experiments indicate that the positional identity of these astrocyte subtypes is controlled by Pax6 and Nkx6.1 in a combinatorial manner. Thus, positional identity is an organizing principle underlying astrocyte, as well as neuronal, subtype diversification and is controlled by a homeodomain transcriptional code whose elements are reutilized following the specification of neuronal identity earlier in development.

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Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons.

Publication Date: 2008 May 2 PMID: 18455990
Authors: Kurant, E. - Axelrod, S. - Leaman, D. - Gaul, U.
Journal: Cell

The removal of apoptotic cells by phagocytic neighbors is essential for metazoan development but remains poorly characterized. Here we report the discovery of a Drosophila phagocytosis receptor, Six-microns-under (SIMU), which is expressed in highly phagocytic cell types during development and required for efficient apoptotic cell clearance by glia in the nervous system and by macrophages elsewhere. SIMU is part of a conserved family of proteins that includes CED-1 and Draper (DRPR). Phenotypic analysis reveals that simu acts upstream of drpr in the same pathway and affects the recognition and engulfment of apoptotic cells, while drpr affects their subsequent degradation. SIMU strongly binds to apoptotic cells, presumably through its EMILIN-like domain, but requires no membrane anchoring, suggesting that it can function as a bridging molecule. Our study introduces an important factor in tissue-resident apoptotic clearance and underscores the prominent role of glia as "semiprofessional" phagocytes in the nervous system.

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The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development.

Publication Date: 2008 May 2 PMID: 18455989
Authors: Schenck, A. - Goto-Silva, L. - Collinet, C. - Rhinn, M. - Giner, A. - Habermann, B. - Brand, M. - Zerial, M.
Journal: Cell

During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. Here, we tested this hypothesis by investigating the role of the Rab5 effector Appl1, an endosomal protein that interacts with transmembrane receptors and Akt. We show that in zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3beta but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3beta, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. We propose that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development.

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Pirt, a phosphoinositide-binding protein, functions as a regulatory subunit of TRPV1.

Publication Date: 2008 May 2 PMID: 18455988
Authors: Kim, A. Y. - Tang, Z. - Liu, Q. - Patel, K. N. - Maag, D. - Geng, Y. - Dong, X.
Journal: Cell

Transient receptor potential vanilloid 1 (TRPV1) is a molecular sensor of noxious heat and capsaicin. Its channel activity can be modulated by several mechanisms. Here we identify a membrane protein, Pirt, as a regulator of TRPV1. Pirt is expressed in most nociceptive neurons in the dorsal root ganglia (DRG) including TRPV1-positive cells. Pirt null mice show impaired responsiveness to noxious heat and capsaicin. Noxious heat- and capsaicin-sensitive currents in Pirt-deficient DRG neurons are significantly attenuated. Heterologous expression of Pirt strongly enhances TRPV1-mediated currents. Furthermore, the C terminus of Pirt binds to TRPV1 and several phosphoinositides, including phosphatidylinositol-4,5-bisphosphate (PIP2), and can potentiate TRPV1. The PIP2 binding is dependent on the cluster of basic residues in the Pirt C terminus and is crucial for Pirt regulation of TRPV1. Importantly, the enhancement of TRPV1 by PIP2 requires Pirt. Therefore, Pirt is a key component of the TRPV1 complex and positively regulates TRPV1 activity.

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A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation.

Publication Date: 2008 May 2 PMID: 18455987
Authors: Erickson, J. R. - Joiner, M. L. - Guan, X. - Kutschke, W. - Yang, J. - Oddis, C. V. - Bartlett, R. K. - Lowe, J. S. - O'Donnell, S. E. - Aykin-Burns, N. - Zimmerman, M. C. - Zimmerman, K. - Ham, A. J. - Weiss, R. M. - Spitz, D. R. - Shea, M. A. - Colbran, R. J. - Mohler, P. J. - Anderson, M. E.
Journal: Cell

Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.

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Allosteric regulation of histidine kinases by their cognate response regulator determines cell fate.

Publication Date: 2008 May 2 PMID: 18455986
Authors: Paul, R. - Jaeger, T. - Abel, S. - Wiederkehr, I. - Folcher, M. - Biondi, E. G. - Laub, M. T. - Jenal, U.
Journal: Cell

The two-component phosphorylation network is of critical importance for bacterial growth and physiology. Here, we address plasticity and interconnection of distinct signal transduction pathways within this network. In Caulobacter crescentus antagonistic activities of the PleC phosphatase and DivJ kinase localized at opposite cell poles control the phosphorylation state and subcellular localization of the cell fate determinator protein DivK. We show that DivK functions as an allosteric regulator that switches PleC from a phosphatase into an autokinase state and thereby mediates a cyclic di-GMP-dependent morphogenetic program. Through allosteric activation of the DivJ autokinase, DivK also stimulates its own phosphorylation and polar localization. These data suggest that DivK is the central effector of an integrated circuit that operates via spatially organized feedback loops to control asymmetry and cell fate determination in C. crescentus. Thus, single domain response regulators can facilitate crosstalk, feedback control, and long-range communication among members of the two-component network.

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SRP keeps polypeptides translocation-competent by slowing translation to match limiting ER-targeting sites.

Publication Date: 2008 May 2 PMID: 18455985
Authors: Lakkaraju, A. K. - Mary, C. - Scherrer, A. - Johnson, A. E. - Strub, K.
Journal: Cell

SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted.

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Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex.

Publication Date: 2008 May 2 PMID: 18455984
Authors: Ciferri, C. - Pasqualato, S. - Screpanti, E. - Varetti, G. - Santaguida, S. - Dos Reis, G. - Maiolica, A. - Polka, J. - De Luca, J. G. - De Wulf, P. - Salek, M. - Rappsilber, J. - Moores, C. A. - Salmon, E. D. - Musacchio, A.
Journal: Cell

Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.

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TIPE2, a negative regulator of innate and adaptive immunity that maintains immune homeostasis.

Publication Date: 2008 May 2 PMID: 18455983
Authors: Sun, H. - Gong, S. - Carmody, R. J. - Hilliard, A. - Li, L. - Sun, J. - Kong, L. - Xu, L. - Hilliard, B. - Hu, S. - Shen, H. - Yang, X. - Chen, Y. H.
Journal: Cell

Immune homeostasis is essential for the normal functioning of the immune system, and its breakdown leads to fatal inflammatory diseases. We report here the identification of a member of the tumor necrosis factor-alpha-induced protein-8 (TNFAIP8) family, designated TIPE2, that is required for maintaining immune homeostasis. TIPE2 is preferentially expressed in lymphoid tissues, and its deletion in mice leads to multiorgan inflammation, splenomegaly, and premature death. TIPE2-deficient animals are hypersensitive to septic shock, and TIPE2-deficient cells are hyper-responsive to Toll-like receptor (TLR) and T cell receptor (TCR) activation. Importantly, TIPE2 binds to caspase-8 and inhibits activating protein-1 and nuclear factor-kappaB activation while promoting Fas-induced apoptosis. Inhibiting caspase-8 significantly blocks the hyper-responsiveness of TIPE2-deficient cells. These results establish that TIPE2 is an essential negative regulator of TLR and TCR function, and its selective expression in the immune system prevents hyperresponsiveness and maintains immune homeostasis.

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Tenets of PTEN tumor suppression.

Publication Date: 2008 May 2 PMID: 18455982
Authors: Salmena, L. - Carracedo, A. - Pandolfi, P. P.
Journal: Cell

Since its discovery as the elusive tumor suppressor gene at the frequently mutated 10q23 locus, PTEN has been identified as lost or mutated in several sporadic and heritable tumor types. A decade of work has established that PTEN is a nonredundant phosphatase that is essential for regulating the highly oncogenic prosurvival PI3K/AKT signaling pathway. This review discusses emerging modes of PTEN function and regulation, and speculates about how manipulation of PTEN function could be used for cancer therapy.

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A different TIPE of immune homeostasis.

Publication Date: 2008 May 2 PMID: 18455981
Authors: Freundt, E. C. - Bidere, N. - Lenardo, M. J.
Journal: Cell

Proteins with death effector domains (DED) are key signal transducers involved in cell death and inflammation. In this issue of Cell, Sun et al. (2008) describe TIPE2, a DED protein that negatively regulates both T cell receptor and Toll-like receptor signaling. These findings reveal a new element critical to the maintenance of homeostasis in both the adaptive and innate immune systems.

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Substrate selectivity APPLies to Akt.

Publication Date: 2008 May 2 PMID: 18455980
Authors: Zhao, S. - Guan, K. L.
Journal: Cell

The protein kinase Akt occupies a central position in multiple signaling pathways. Although numerous Akt substrates have been identified, less is known about the factors that regulate specific cellular responses to Akt signaling. In this issue, Schenck et al. (2008) demonstrate that the endosomal protein Appl1 modulates Akt's substrate selectivity to promote cell survival during zebrafish development.

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CaMKII: new tricks for an old dog.

Publication Date: 2008 May 2 PMID: 18455979
Authors: Griffith, L. C.
Journal: Cell

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a pivotal signaling molecule in both the brain and the heart. In this issue of Cell, Erickson et al. (2008) demonstrate a mechanism for CaMKII activation by reactive oxygen species that provides a direct link between kinase activation and cardiac dysfunction.

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Epigenome sequencing comes of age.

Publication Date: 2008 May 2 PMID: 18455978
Authors: Zhu, J. K.
Journal: Cell

Epigenetic states are responsive to developmental and environmental signals, and as a consequence a eukaryotic cell can have many different epigenomes. In this issue of Cell, Lister et al. (2008) present the floral epigenome of Arabidopsis using next-generation sequencing technology to analyze both DNA methylation at single-base resolution and the expression of small RNAs.

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Death in the CNS: six-microns-under.

Publication Date: 2008 May 2 PMID: 18455977
Authors: Elliott, M. R. - Ravichandran, K. S.
Journal: Cell

During embryonic development, large numbers of apoptotic cells are rapidly cleared by phagocytes. In this issue, Kurant et al. (2008) describe a new phagocytic receptor, called six-microns-under (SIMU), that promotes engulfment of apoptotic neurons by glial cells in the developing nervous system of Drosophila.

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Xenohormesis: sensing the chemical cues of other species.

Publication Date: 2008 May 2 PMID: 18455976
Authors: Howitz, K. T. - Sinclair, D. A.
Journal: Cell

Many plant molecules interact with and modulate key regulators of mammalian physiology in ways that are beneficial to health, but why? We propose that heterotrophs (animals and fungi) are able to sense chemical cues synthesized by plants and other autotrophs in response to stress. These cues provide advance warning about deteriorating environmental conditions, allowing the heterotrophs to prepare for adversity while conditions are still favorable.

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Highly integrated single-base resolution maps of the epigenome in Arabidopsis.

Publication Date: 2008 May 2 PMID: 18423832
Authors: Lister, R. - O'Malley, R. C. - Tonti-Filippini, J. - Gregory, B. D. - Berry, C. C. - Millar, A. H. - Ecker, J. R.
Journal: Cell

Deciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.

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SnapShot: The TGFbeta pathway interactome.

Publication Date: 2008 Apr 18 PMID: 18423207
Authors: Taylor, I. W. - Wrana, J. L.
Journal: Cell

MeSH Categories: Animals, Gene Regulatory Networks, Mammals, Protein Binding, *Signal Transduction, Transforming Growth Factor beta/*metabolism

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A quantitative spatiotemporal atlas of gene expression in the Drosophila blastoderm.

Publication Date: 2008 Apr 18 PMID: 18423206
Authors: Fowlkes, C. C. - Hendriks, C. L. - Keranen, S. V. - Weber, G. H. - Rubel, O. - Huang, M. Y. - Chatoor, S. - DePace, A. H. - Simirenko, L. - Henriquez, C. - Beaton, A. - Weiszmann, R. - Celniker, S. - Hamann, B. - Knowles, D. W. - Biggin, M. D. - Eisen, M. B. - Malik, J.
Journal: Cell

To fully understand animal transcription networks, it is essential to accurately measure the spatial and temporal expression patterns of transcription factors and their targets. We describe a registration technique that takes image-based data from hundreds of Drosophila blastoderm embryos, each costained for a reference gene and one of a set of genes of interest, and builds a model VirtualEmbryo. This model captures in a common framework the average expression patterns for many genes in spite of significant variation in morphology and expression between individual embryos. We establish the method's accuracy by showing that relationships between a pair of genes' expression inferred from the model are nearly identical to those measured in embryos costained for the pair. We present a VirtualEmbryo containing data for 95 genes at six time cohorts. We show that known gene-regulatory interactions can be automatically recovered from this data set and predict hundreds of new interactions.

MeSH Categories: Animals, Blastoderm, Drosophila melanogaster/*genetics/metabolism, Embryo, Nonmammalian/metabolism, Gene Expression Regulation, Developmental, *Gene Regulatory Networks, Genes, Insect, *Models, Genetic

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Sex-specific control and tuning of the pattern generator for courtship song in Drosophila.

Publication Date: 2008 Apr 18 PMID: 18423205
Authors: Clyne, J. D. - Miesenbock, G.
Journal: Cell

The differentially spliced transcription factors encoded by the fruitless (fru) gene are key determinants of sexual behavior in Drosophila. They are expressed in a minority of neurons with limited dimorphisms and regulate neural processes that remain largely unknown. Here, we use light-activated ion channels to stimulate fru-expressing neurons in the thoracic-abdominal ganglia, enabling direct functional comparisons of homologous circuitry between sexes. Optical stimulation of males or females initiates the unilateral wing vibrations that normally generate the male courtship song. The pattern-generating circuit operates differently in the two sexes, producing wing movement and sound in both but authentic songs only in males and in females expressing male fru product. A song-like motor program is thus present in females but lies dormant because the neural commands required for song initiation are absent. Supplying such commands artificially reveals fru-specific differences in the internal dynamics of the song generator and sets the stage for exploring their physiological basis.

MeSH Categories: Animals, Drosophila Proteins/genetics/physiology, Drosophila melanogaster/genetics/*physiology, Female, Ion Channels/physiology, Light, Male, Nerve Tissue Proteins/genetics/physiology, Neurons/physiology, *Sex Characteristics, *Sexual Behavior, Animal, Sound, Transcription Factors/genetics/physiology, Wing/metabolism

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Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling.

Publication Date: 2008 Apr 18 PMID: 18423204
Authors: Wu, X. - Tu, X. - Joeng, K. S. - Hilton, M. J. - Williams, D. A. - Long, F.
Journal: Cell

Canonical Wnt signaling critically regulates cell fate and proliferation in development and disease. Nuclear localization of beta-catenin is indispensable for canonical Wnt signaling; however, the mechanisms governing beta-catenin nuclear localization are not well understood. Here we demonstrate that nuclear accumulation of beta-catenin in response to Wnt requires Rac1 activation. The role of Rac1 depends on phosphorylation of beta-catenin at Ser191 and Ser605, which is mediated by JNK2 kinase. Mutations of these residues significantly affect Wnt-induced beta-catenin nuclear accumulation. Genetic ablation of Rac1 in the mouse embryonic limb bud ectoderm disrupts canonical Wnt signaling and phenocopies deletion of beta-catenin in causing severe truncations of the limb. Finally, Rac1 interacts genetically with beta-catenin and Dkk1 in controlling limb outgrowth. Together these results uncover Rac1 activation and subsequent beta-catenin phosphorylation as a hitherto uncharacterized mechanism controlling canonical Wnt signaling and may provide additional targets for therapeutic intervention of this important pathway.

MeSH Categories: 1-Phosphatidylinositol 3-Kinase/metabolism, Animals, Cell Line, Cell Nucleus/*chemistry/metabolism, Embryo, Mammalian/metabolism, Extremities/embryology, Intercellular Signaling Peptides and Proteins/metabolism, Mice, Mitogen-Activated Protein Kinase 9/metabolism, Neuropeptides/*metabolism, Phosphorylation, *Signal Transduction, Wnt Proteins/metabolism, beta Catenin/*analysis/genetics/metabolism, rac GTP-Binding Proteins/*metabolism, rho GTP-Binding Proteins/metabolism

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CASK Functions as a Mg2+-independent neurexin kinase.

Publication Date: 2008 Apr 18 PMID: 18423203
Authors: Mukherjee, K. - Sharma, M. - Urlaub, H. - Bourenkov, G. P. - Jahn, R. - Sudhof, T. C. - Wahl, M. C.
Journal: Cell

CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. Here we show, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, our data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, our study suggests that other pseudokinases (10% of the kinome) could also be catalytically active.

MeSH Categories: Animals, Cell Line, Cells, Cultured, Crystallography, X-Ray, Glycoproteins/*metabolism, Guanylate Kinase/*chemistry/*metabolism, Humans, Magnesium/metabolism, Mice, Models, Molecular, Neurons/metabolism, Neuropeptides/*metabolism, Nucleotides/metabolism, Protein Binding, Protein Structure, Tertiary, Rats, Sequence Homology, Amino Acid

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Upf1 phosphorylation triggers translational repression during nonsense-mediated mRNA decay.

Publication Date: 2008 Apr 18 PMID: 18423202
Authors: Isken, O. - Kim, Y. K. - Hosoda, N. - Mayeur, G. L. - Hershey, J. W. - Maquat, L. E.
Journal: Cell

In mammalian cells, nonsense-mediated mRNA decay (NMD) generally requires that translation terminates sufficiently upstream of a post-splicing exon junction complex (EJC) during a pioneer round of translation. The subsequent binding of Upf1 to the EJC triggers Upf1 phosphorylation. We provide evidence that phospho-Upf1 functions after nonsense codon recognition during steps that involve the translation initiation factor eIF3 and mRNA decay factors. Phospho-Upf1 interacts directly with eIF3 and inhibits the eIF3-dependent conversion of 40S/Met-tRNA(i)(Met)/mRNA to translationally competent 80S/Met-tRNA(i)(Met)/mRNA initiation complexes to repress continued translation initiation. Consistent with phospho-Upf1 impairing eIF3 function, NMD fails to detectably target nonsense-containing transcripts that initiate translation independently of eIF3 from the CrPV IRES. There is growing evidence that translational repression is a key transition that precedes mRNA delivery to the degradation machinery. Our results uncover a critical step during NMD that converts a pioneer translation initiation complex to a translationally compromised mRNP.

MeSH Categories: Animals, COS Cells, Cercopithecus aethiops, Codon, Nonsense, Hela Cells, Hepacivirus/metabolism, Humans, Phosphorylation, *Protein Biosynthesis, *RNA Stability, RNA, Messenger/*metabolism, Ribonucleoproteins/metabolism, Trans-Activators/*metabolism

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SKAR links pre-mRNA splicing to mTOR/S6K1-mediated enhanced translation efficiency of spliced mRNAs.

Publication Date: 2008 Apr 18 PMID: 18423201
Authors: Ma, X. M. - Yoon, S. O. - Richardson, C. J. - Julich, K. - Blenis, J.
Journal: Cell

Different protein complexes form on newly spliced mRNA to ensure the accuracy and efficiency of eukaryotic gene expression. For example, the exon junction complex (EJC) plays an important role in mRNA surveillance. The EJC also influences the first, or pioneer round of protein synthesis through a mechanism that is poorly understood. We show that the nutrient-, stress-, and energy-sensing checkpoint kinase, mTOR, contributes to the observed enhanced translation efficiency of spliced over nonspliced mRNAs. We demonstrate that, when activated, S6K1 is recruited to the newly synthesized mRNA by SKAR, which is deposited at the EJC during splicing, and that SKAR and S6K1 increase the translation efficiency of spliced mRNA. Thus, SKAR-mediated recruitment of activated S6K1 to newly processed mRNPs serves as a conduit between mTOR checkpoint signaling and the pioneer round of translation when cells exist in conditions supportive of protein synthesis.

MeSH Categories: Cell Line, Cell Nucleus/metabolism, Cytoplasm/metabolism, Eukaryotic Initiation Factor-4A/metabolism, Exons, Humans, Nuclear Cap-Binding Protein Complex/metabolism, Nuclear Proteins/*metabolism, *Protein Biosynthesis, Protein Kinases/*metabolism, RNA Splicing, RNA, Messenger/*metabolism, RNA-Binding Proteins/*metabolism, Ribonucleoproteins/metabolism, Ribosomal Protein S6 Kinases/*metabolism

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Yeast life span extension by depletion of 60s ribosomal subunits is mediated by Gcn4.

Publication Date: 2008 Apr 18 PMID: 18423200
Authors: Steffen, K. K. - MacKay, V. L. - Kerr, E. O. - Tsuchiya, M. - Hu, D. - Fox, L. A. - Dang, N. - Johnston, E. D. - Oakes, J. A. - Tchao, B. N. - Pak, D. N. - Fields, S. - Kennedy, B. K. - Kaeberlein, M.
Journal: Cell

In nearly every organism studied, reduced caloric intake extends life span. In yeast, span extension from dietary restriction is thought to be mediated by the highly conserved, nutrient-responsive target of rapamycin (TOR), protein kinase A (PKA), and Sch9 kinases. These kinases coordinately regulate various cellular processes including stress responses, protein turnover, cell growth, and ribosome biogenesis. Here we show that a specific reduction of 60S ribosomal subunit levels slows aging in yeast. Deletion of genes encoding 60S subunit proteins or processing factors or treatment with a small molecule, which all inhibit 60S subunit biogenesis, are each sufficient to significantly increase replicative life span. One mechanism by which reduced 60S subunit levels leads to life span extension is through induction of Gcn4, a nutrient-responsive transcription factor. Genetic epistasis analyses suggest that dietary restriction, reduced 60S subunit abundance, and Gcn4 activation extend yeast life span by similar mechanisms.

MeSH Categories: DNA-Binding Proteins/*physiology, Gene Deletion, Histone Deacetylases/physiology, Ribosomal Proteins/physiology, Ribosome Subunits, Large, Eukaryotic/*physiology, Saccharomyces cerevisiae/*physiology, Saccharomyces cerevisiae Proteins/*physiology, Silent Information Regulator Proteins, Saccharomyces cerevisiae/physiology, Sirtuins/physiology, Transcription Factors/*physiology

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Meiosis I is established through division-specific translational control of a cyclin.

Publication Date: 2008 Apr 18 PMID: 18423199
Authors: Carlile, T. M. - Amon, A.
Journal: Cell

In budding yeast, key meiotic events such as DNA replication, recombination, and the meiotic divisions are controlled by Clb cyclin-dependent kinases (Clb-CDKs). Using a novel synchronization procedure, we have characterized the activity of these Clb-CDKs and observed a surprising diversity in their regulation during the meiotic divisions. Clb1-CDK activity is restricted to meiosis I, and Clb3-CDK activity to meiosis II, through 5'UTR-mediated translational control of its transcript. The analysis of cells inappropriately producing Clb3-CDKs during meiosis I furthermore defines Clb3 as an inhibitor of the meiosis I chromosome segregation program. Our results demonstrate an essential role for Clb-CDK regulation in establishing the meiotic chromosome segregation pattern.

MeSH Categories: 5' Untranslated Regions/metabolism, Chromatids/metabolism, Cyclins/*metabolism, *Meiosis, *Protein Biosynthesis, Protein Kinases/metabolism, Saccharomyces cerevisiae/cytology, Saccharomyces cerevisiae Proteins/*metabolism

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The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions.

Publication Date: 2008 Apr 18 PMID: 18423198
Authors: Jhunjhunwala, S. - van Zelm, M. C. - Peak, M. M. - Cutchin, S. - Riblet, R. - van Dongen, J. J. - Grosveld, F. G. - Knoch, T. A. - Murre, C.
Journal: Cell

The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (V(H)), diversity (D(H)), joining (J(H)) and constant (C(H)) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triple-point angle measurements indicated the mean relative 3D positions of the V(H), D(H), J(H), and C(H) elements, showed compartmentalization and striking conformational changes involving V(H) and D(H)-J(H) elements during early B cell development. In pro-B cells, the entire repertoire of V(H) regions (2 Mbp) appeared to have merged and juxtaposed to the D(H) elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.

MeSH Categories: Animals, B-Lymphocytes/chemistry/metabolism, Cell Lineage, Cells, Cultured, *Genes, Immunoglobulin Heavy Chain, Mice, Mice, Inbred C57BL, Models, Molecular, Nucleic Acid Conformation, VDJ Exons

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Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency.

Publication Date: 2008 Apr 18 PMID: 18423197
Authors: Hanna, J. - Markoulaki, S. - Schorderet, P. - Carey, B. W. - Beard, C. - Wernig, M. - Creyghton, M. P. - Steine, E. J. - Cassady, J. P. - Foreman, R. - Lengner, C. J. - Dausman, J. A. - Jaenisch, R.
Journal: Cell

Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.

MeSH Categories: Animals, B-Lymphocytes/*cytology, *Cell Differentiation, Cell Nucleus/genetics, Embryonic Stem Cells/cytology, Humans, Mice, Pluripotent Stem Cells/*cytology, Transcription Factors/metabolism

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Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury.

Publication Date: 2008 Apr 18 PMID: 18423196
Authors: Imai, Y. - Kuba, K. - Neely, G. G. - Yaghubian-Malhami, R. - Perkmann, T. - van Loo, G. - Ermolaeva, M. - Veldhuizen, R. - Leung, Y. H. - Wang, H. - Liu, H. - Sun, Y. - Pasparakis, M. - Kopf, M. - Mech, C. - Bavari, S. - Peiris, J. S. - Slutsky, A. S. - Akira, S. - Hultqvist, M. - Holmdahl, R. - Nicholls, J. - Jiang, C. - Binder, C. J. - Penninger, J. M.
Journal: Cell

Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI.

MeSH Categories: Adaptor Proteins, Vesicular Transport/metabolism, Animals, Humans, Influenza, Human/metabolism, Interleukin-6/metabolism, Lung, Mice, Mice, Inbred C57BL, NADPH Oxidase/metabolism, NF-kappa B/metabolism, Orthomyxoviridae Infections/metabolism, *Oxidative Stress, Phospholipids/metabolism, Respiratory Distress Syndrome, Adult/*metabolism, Severe Acute Respiratory Syndrome/metabolism, Signal Transduction, Toll-Like Receptor 4/*metabolism

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Epidermal sensing of oxygen is essential for systemic hypoxic response.

Publication Date: 2008 Apr 18 PMID: 18423195
Authors: Boutin, A. T. - Weidemann, A. - Fu, Z. - Mesropian, L. - Gradin, K. - Jamora, C. - Wiesener, M. - Eckardt, K. U. - Koch, C. J. - Ellies, L. G. - Haddad, G. - Haase, V. H. - Simon, M. C. - Poellinger, L. - Powell, F. L. - Johnson, R. S.
Journal: Cell

Skin plays an essential role, mediated in part by its remarkable vascular plasticity, in adaptation to environmental stimuli. Certain vertebrates, such as amphibians, respond to hypoxia in part through the skin; but it is unknown whether this tissue can influence mammalian systemic adaptation to low oxygen levels. We have found that epidermal deletion of the hypoxia-responsive transcription factor HIF-1alpha inhibits renal erythropoietin (EPO) synthesis in response to hypoxia. Conversely, mice with an epidermal deletion of the von Hippel-Lindau (VHL) factor, a negative regulator of HIF, have increased EPO synthesis and polycythemia. We show that nitric oxide release induced by the HIF pathway acts on cutaneous vascular flow to increase systemic erythropoietin expression. These results demonstrate that in mice the skin is a critical mediator of systemic responses to environmental oxygen.

MeSH Categories: Animals, Blood Chemical Analysis, Epidermis/*physiology, Erythropoietin/metabolism, Humans, Hypoxia-Inducible Factor 1/genetics/metabolism, Keratinocytes/metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Nitric Oxide/blood, Oxygen/blood/*metabolism, Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism

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miRiad roles for the miR-17-92 cluster in development and disease.

Publication Date: 2008 Apr 18 PMID: 18423194
Authors: Mendell, J. T.
Journal: Cell

MicroRNAs (miRNAs) encoded by the miR-17-92 cluster and its paralogs are known to act as oncogenes. Expression of these miRNAs promotes cell proliferation, suppresses apoptosis of cancer cells, and induces tumor angiogenesis. New work reveals essential functions for these miRNAs not only in tumor formation but also during normal development of the heart, lungs, and immune system.

MeSH Categories: Animals, Embryo, Mammalian/*metabolism, Heart/embryology, Humans, Immune System/embryology, Lung/embryology, Mice, MicroRNAs/genetics/*metabolism, Multigene Family, Oncogenes, RNA, Neoplasm/genetics/*metabolism

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EJCs at the heart of translational control.

Publication Date: 2008 Apr 18 PMID: 18423193
Authors: Le Hir, H. - Seraphin, B.
Journal: Cell

In mammalian cells, the splicing machinery deposits the exon junction complex (EJC) on mRNA splice junctions. Two studies in this issue now link the EJC to different aspects of translational control. Ma et al. (2008) show that the EJC activates translation downstream of the mTOR signaling pathway, whereas Isken et al. (2008) establish that translation is repressed by partners of the EJC that are implicated in nonsense mediated decay (NMD).

MeSH Categories: Animals, Codon, Nonsense, *Exons, Humans, *Protein Biosynthesis, RNA Splicing, RNA Stability, Ribonucleoproteins/*metabolism

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Fly courtship song: triggering the light fantastic.

Publication Date: 2008 Apr 18 PMID: 18423192
Authors: Dornan, A. J. - Goodwin, S. F.
Journal: Cell

In a study in this issue, Clyne and Miesenbock (2008) apply an ingenious optogenetic technology to activate neurons that generate male-specific courtship song in flies. This work sheds new light on the neural circuitry underlying sexually dimorphic behaviors in Drosophila.

MeSH Categories: Animals, Drosophila melanogaster/*physiology, Female, Light, Male, Neurons/physiology, Sex Characteristics, *Sexual Behavior, Animal, Wing

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A TRIFfic perspective on acute lung injury.

Publication Date: 2008 Apr 18 PMID: 18423191
Authors: Martin, T. R. - Wurfel, M. M.
Journal: Cell

Acute lung injury (ALI) is a leading cause of death in people infected with H5N1 avian influenza virus or the SARS-coronavirus. Imai et al. (2008) now report that ALI is triggered by the signaling of oxidized phospholipids through Toll-like receptor 4 (TLR4) and the adaptor protein TRIF. These findings provide insight into the molecular pathogenesis of ALI, a condition for which treatment options are currently very limited.

MeSH Categories: Adaptor Proteins, Vesicular Transport/*metabolism, Animals, Humans, Mice, Oxidative Stress, Phospholipids/metabolism, Respiratory Distress Syndrome, Adult/*metabolism, Toll-Like Receptor 4/*metabolism

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O2 sensing: only skin deep?

Publication Date: 2008 Apr 18 PMID: 18423190
Authors: Semenza, G. L.
Journal: Cell

The transcription factor HIF-1 mediates adaptive responses to hypoxia, and its activity is negatively regulated by O2-dependent binding of the von Hippel-Lindau (VHL) protein. In this issue, Boutin et al. (2008) use conditional knockout mice to demonstrate that sensing of O2 by keratinocytes in the epidermis leads to alterations in cutaneous blood flow that affect the production of the hormone erythropoietin, thereby modulating red blood cell production and the O2-carrying capacity of blood.

MeSH Categories: Animals, Humans, Hypoxia-Inducible Factor 1/metabolism, Keratinocytes/metabolism, Mice, Oxygen/*metabolism, *Skin Physiology

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Rethinking pseudokinases.

Publication Date: 2008 Apr 18 PMID: 18423189
Authors: Kannan, N. - Taylor, S. S.
Journal: Cell

Pseudokinases lack conservation of one or more of the catalytic residues in the kinase core and as a consequence are typically thought to be catalytically inactive. New work by Mukherjee et al. (2008) challenges this assumption. They show that the pseudokinase domain of CASK (Ca2+/calmodulin activated serine-threonine kinase) adopts an active conformation and displays catalytic activity in vivo.

MeSH Categories: Guanylate Kinase/chemistry/genetics/*metabolism, Humans, Phosphorylation, Protein Conformation, Protein Structure, Tertiary

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India takes an open source approach to drug discovery.

Publication Date: 2008 Apr 18 PMID: 18423188
Authors: Singh, S.
Journal: Cell

Open source software may have been around for 17 years, but using an open source model to speed up drug discovery is a relatively new idea. This month, India is launching a new open source initiative for developing drugs to treat diseases such as tuberculosis, malaria, and HIV.

MeSH Categories: *Drug Design, Drug Industry/economics/*trends, Humans, India, Intellectual Property, International Cooperation, *Software

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