Thursday, September 1, 2011

Hot off the presses! Sep 02 Mol Cell

The Sep 02 issue of the Mol Cell is now up on Pubget (About Mol Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

  • Jonathan Widom 1955–2011
    - Mol Cell 43(5):691-692 (2011)
  • The Photomorphogenic Protein, DE-ETIOLATED 1, Is a Critical Transcriptional Corepressor in the Central Loop of the Arabidopsis Circadian Clock
    - Mol Cell 43(5):693-694 (2011)
    In this issue of Molecular Cell, Lau et al. (2011) demonstrate that DET1, a component of the COP10-DET1-DDB1 (CDD) complex, is a transcriptional corepressor recruited to the promoters of core clock genes via interaction with two MYB transcription factors, CCA1 and LHY.
  • A New Twist on Clock Protein Phosphorylation: A Conformational Change Leads to Protein Degradation
    - Mol Cell 43(5):695-697 (2011)
    Progressive phosphorylation of circadian clock proteins is a hallmark of time-keeping. In this issue of Molecular Cell, Querfurth et al. (2011) demonstrate that phosphorylation of Neurospora FRQ induces a conformational change, which can account for its temporally gated degradation.
  • HDAC3 at the Fulcrum of an Epithelial-Mesenchymal Balance
    - Mol Cell 43(5):697-698 (2011)
    In this issue of Molecular Cell, Wu et al. (2011) reveal an essential role for a chromatin modifier, histone deacetylase 3 (HDAC3), in hypoxia-induced epithelial-mesenchymal transition (EMT); HIF-activated HDAC3 integrates with WDR5 to impose chromatin modifications that culminate in EMT.
  • Translating a Low-Sugar Diet into a Longer Life by Maintaining Thioredoxin Peroxidase Activity of a Peroxiredoxin
    - Mol Cell 43(5):699-701 (2011)
    In this issue of Molecular Cell, Molin et al. (2011) reveal that caloric restriction alleviates PKA-dependent inhibition of sulfiredoxin translation, maintaining the thioredoxin peroxidase activity of a peroxiredoxin and increasing the hydrogen peroxide resistance and replicative life span of Saccharomyces cerevisiae.
  • Interaction of Arabidopsis DET1 with CCA1 and LHY in Mediating Transcriptional Repression in the Plant Circadian Clock
    - Mol Cell 43(5):703-712 (2011)
    The COP10-DET1-DDB1 (CDD) complex is an evolutionarily conserved protein complex discovered for its role in the repression of photomorphogenesis in Arabidopsis. It is important in many cellular and developmental processes in both plants and animals, but its molecular mode of action remains poorly understood. Here, we show that the CDD component DET1 possesses transcriptional repression activity and physically interacts with two closely related MYB transcription factors, CCA1 and LHY, which are core components of the plant circadian clock. DET1 associates with the promoter of CCA1/LHY target genes, such as TOC1, in a CCA1/LHY-dependent manner and is required for their repression, suggesting a recruitment of DET1 by the central oscillator components to regulate the clock. Our results reveal DET1 as a core transcriptional repression factor important for clock progression. Overall, the CDD complex may function as a transcriptional corepressor in diverse processes through d! irect interaction with distinct transcription factors.
  • Circadian Conformational Change of the Neurospora Clock Protein FREQUENCY Triggered by Clustered Hyperphosphorylation of a Basic Domain
    - Mol Cell 43(5):713-722 (2011)
    In the course of a day, the Neurospora clock protein FREQUENCY (FRQ) is progressively phosphorylated at up to 113 sites and eventually degraded. Phosphorylation and degradation are crucial for circadian time keeping, but it is not known how phosphorylation of a large number of sites correlates with circadian degradation of FRQ. We show that two amphipathic motifs in FRQ interact over a long distance, bringing the positively charged N-terminal portion in spatial proximity to the negatively charged middle and C-terminal portion of FRQ. The interaction is essential for the recruitment of casein kinase 1a (CK1a) into a stable complex with FRQ. FRQ-bound CK1a progressively phosphorylates the positively charged N-terminal domain of FRQ at up to 46 nonconsensus sites, triggering a conformational change, presumably by electrostatic repulsion, that commits the protein for degradation via the PEST1 signal in the negatively charged central portion of FRQ.
  • Rapid Phospho-Turnover by Receptor Tyrosine Kinases Impacts Downstream Signaling and Drug Binding
    - Mol Cell 43(5):723-737 (2011)
    Epidermal growth factor receptors (ErbB1–4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100–1000 times in the course of a typical immediate-early response to ligand. Rapid phospho-turnover is also observed for EGF-activated ErbB2 and ErbB3, unrelated RTKs, and multiple intracellular adaptor proteins and signaling kinases. Thus, the complexes formed on the cytoplasmic tail of active receptors and the downstream signaling kinases they control are highly dynamic and antagonized by potent phosphatases. We develop a kinetic scheme for binding of anti-ErbB1 drugs to receptors and show that rapid phospho-turnover significantly impacts th! eir mechanisms of action.
  • The Mechanism of Tail-Anchored Protein Insertion into the ER Membrane
    - Mol Cell 43(5):738-750 (2011)
    Tail-anchored (TA) proteins access the secretory pathway via posttranslational insertion of their C-terminal transmembrane domain into the endoplasmic reticulum (ER). Get3 is an ATPase that delivers TA proteins to the ER by interacting with the Get1-Get2 transmembrane complex, but how Get3's nucleotide cycle drives TA protein insertion remains unclear. Here, we establish that nucleotide binding to Get3 promotes Get3-TA protein complex formation by recruiting Get3 to a chaperone that hands over TA proteins to Get3. Biochemical reconstitution and mutagenesis reveal that the Get1-Get2 complex comprises the minimal TA protein insertion machinery with functionally critical cytosolic regions. By engineering a soluble heterodimer of Get1-Get2 cytosolic domains, we uncover the mechanism of TA protein release from Get3: Get2 tethers Get3-TA protein complexes into proximity with the ATPase-dependent, substrate-releasing activity of Get1. Lastly, we show that ATP enhances Get3 di! ssociation from the membrane, thus freeing Get1-Get2 for new rounds of substrate insertion.
  • Mechanisms Underlying the Dual-Mode Regulation of Microtubule Dynamics by Kip3/Kinesin-8
    - Mol Cell 43(5):751-763 (2011)
    The kinesin-8 family of microtubule motors plays a critical role in microtubule length control in cells. These motors have complex effects on microtubule dynamics: they destabilize growing microtubules yet stabilize shrinking microtubules. The budding yeast kinesin-8, Kip3, accumulates on plus ends of growing but not shrinking microtubules. Here we identify an essential role of the tail domain of Kip3 in mediating both its destabilizing and its stabilizing activities. The Kip3 tail promotes Kip3's accumulation at the plus ends and facilitates the destabilizing effect of Kip3. However, the Kip3 tail also inhibits microtubule shrinkage and is required for promoting microtubule rescue by Kip3. These effects of the tail domain are likely to be mediated by the tubulin- and microtubule-binding activities that we describe. We propose a concentration-dependent model for the coordination of the destabilizing and stabilizing activities of Kip3 and discuss its relevance to cellul! ar microtubule organization.
  • A Tethering Mechanism Controls the Processivity and Kinetochore-Microtubule Plus-End Enrichment of the Kinesin-8 Kif18A
    - Mol Cell 43(5):764-775 (2011)
    Metaphase chromosome positioning depends on Kif18A, a kinesin-8 that accumulates at and suppresses the dynamics of K-MT plus ends. By engineering Kif18A mutants that suppress MT dynamics but fail to concentrate at K-MT plus ends, we identify a mechanism that allows Kif18A to accumulate at K-MT plus ends to a level required to suppress chromosome movements. Enrichment of Kif18A at K-MT plus ends depends on its C-terminal tail domain, while the ability of Kif18A to suppress MT growth is conferred by the N-terminal motor domain. The Kif18A tail contains a second MT-binding domain that diffuses along the MT lattice, suggesting that it tethers the motor to the MT track. Consistently, the tail enhances Kif18A processivity and is crucial for it to accumulate at K-MT plus ends. The heightened processivity of Kif18A, conferred by its tail domain, thus promotes concentration of Kif18A at K-MT plus ends, where it suppresses their dynamics to control chromosome movements.
  • Mechanistic Analysis of Local Ori Melting and Helicase Assembly by the Papillomavirus E1 Protein
    - Mol Cell 43(5):776-787 (2011)
    Preparation of DNA templates for replication requires opening of the duplex to expose single-stranded (ss) DNA. The locally melted DNA is required for replicative DNA helicases to initiate unwinding. How local melting is generated in eukaryotic replicons is unknown, but initiator proteins from a handful of eukaryotic viruses can perform this function. Here we dissect the local melting process carried out by the papillomavirus E1 protein. We characterize the melting process kinetically and identify mutations in the E1 helicase and in the ori that arrest the local melting process. We show that a subset of these mutants have specific defects for melting of the center of the ori containing the binding sites for E1 and demonstrate that these mutants fail to untwist the ori DNA. This understanding of how E1 generates local melting suggests possible mechanisms for local melting in other replicons.
  • NBS1 Recruits RAD18 via a RAD6-like Domain and Regulates Pol η-Dependent Translesion DNA Synthesis
    - Mol Cell 43(5):788-797 (2011)
    Translesion DNA synthesis, a process orchestrated by monoubiquitinated PCNA, is critical for DNA damage tolerance. While the ubiquitin-conjugating enzyme RAD6 and ubiquitin ligase RAD18 are known to monoubiquitinate PCNA, how they are regulated by DNA damage is not fully understood. We show that NBS1 (mutated in Nijmegen breakage syndrome) binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Polη focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting domain of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting domain of RAD6. These domains of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously and are crucial for Polη-dependent UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in tr! anslesion DNA synthesis.
  • Context-Specific Regulation of NF-κB Target Gene Expression by EZH2 in Breast Cancers
    - Mol Cell 43(5):798-810 (2011)
    Both EZH2 and NF-κB contribute to aggressive breast cancer, yet whether the two oncogenic factors have functional crosstalk in breast cancer is unknown. Here, we uncover an unexpected role of EZH2 in conferring the constitutive activation of NF-κB target gene expression in ER-negative basal-like breast cancer cells. This function of EZH2 is independent of its histone methyltransferase activity but requires the physical interaction with RelA/RelB to promote the expression of NF-κB targets. Intriguingly, EZH2 acts oppositely in ER-positive luminal-like breast cancer cells and represses NF-κB target gene expression by interacting with ER and directing repressive histone methylation on their promoters. Thus, EZH2 functions as a double-facet molecule in breast cancers, either as a transcriptional activator or repressor of NF-κB targets, depending on the cellular context. These findings reveal an additional mechanism by which EZH2 promotes breast cancer progression and ! underscore the need for developing context-specific strategy for therapeutic targeting of EZH2 in breast cancers.
  • Interplay between HDAC3 and WDR5 Is Essential for Hypoxia-Induced Epithelial-Mesenchymal Transition
    - Mol Cell 43(5):811-822 (2011)
    Epithelial-mesenchymal transition (EMT) is important for organ development, metastasis, cancer stemness, and organ fibrosis. Molecular mechanisms to coordinately regulate hypoxia-induced EMT remain elusive. Here, we show that HIF-1α-induced histone deacetylase 3 (hdac3) is essential for hypoxia-induced EMT and metastatic phenotypes. Change of specific chromatin states is associated with hypoxia-induced EMT. Under hypoxia, HDAC3 interacts with hypoxia-induced WDR5, recruits the histone methyltransferase (HMT) complex to increase histone H3 lysine 4 (H3K4)-specific HMT activity, and activates mesenchymal gene expression. HDAC3 also serves as an essential corepressor to repress epithelial gene expression. Knockdown of WDR5 abolishes mesenchymal gene activation but not epithelial gene repression during hypoxia. These results indicate that hypoxia induces different chromatin modifiers to coordinately regulate EMT through distinct mechanisms.
  • Life Span Extension and H2O2 Resistance Elicited by Caloric Restriction Require the Peroxiredoxin Tsa1 in Saccharomyces cerevisiae
    - Mol Cell 43(5):823-833 (2011)
    Caloric restriction (CR) extends the life span of organisms ranging from yeast to primates. Here, we show that the thiol-dependent peroxiredoxin Tsa1 and its partner sulfiredoxin, Srx1, are required for CR to extend the replicative life span of yeast cells. Tsa1 becomes hyperoxidized/inactive during aging, and CR mitigates such oxidation by elevating the levels of Srx1, which is required to reduce/reactivate hyperoxidized Tsa1. CR, by lowering cAMP-PKA activity, enhances Gcn2-dependent SRX1 translation, resulting in increased resistance to H2O2 and life span extension. Moreover, an extra copy of the SRX1 gene is sufficient to extend the life span of cells grown in high glucose concentrations by 20% in a Tsa1-dependent and Sir2-independent manner. The data demonstrate that Tsa1 is required to ensure yeast longevity and that CR extends yeast life span, in part, by counteracting age-induced hyperoxidation of this peroxiredoxin.
  • A Biotin Switch-Based Proteomics Approach Identifies 14-3-3ζ as a Target of Sirt1 in the Metabolic Regulation of Caspase-2
    - Mol Cell 43(5):834-842 (2011)
    While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes14-3-3ζ dissociation from caspase-2 in both egg extract and human culture! d cells. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation.
  • Cross-Regulation between an Alternative Splicing Activator and a Transcription Repressor Controls Neurogenesis
    - Mol Cell 43(5):843-850 (2011)
    Neurogenesis requires the concerted action of numerous genes that are regulated at multiple levels. However, how different layers of gene regulation are coordinated to promote neurogenesis is not well understood. We show that the neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4) negatively regulates REST (NRSF), a transcriptional repressor of genes required for neurogenesis. nSR100 directly promotes alternative splicing of REST transcripts to produce a REST isoform (REST4) with greatly reduced repressive activity, thereby activating expression of REST targets in neural cells. Conversely, REST directly represses nSR100 in nonneural cells to prevent the activation of neural-specific splicing events. Consistent with a critical role for nSR100 in the inhibition of REST activity, blocking nSR100 expression in the developing mouse brain impairs neurogenesis. Our results thus reveal a fundamental role for direct regulatory interactions between a splicin! g activator and transcription repressor in the control of the multilayered regulatory programs required for neurogenesis.

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