Molecular Cell

The yeast 5'-3' exonuclease Rat1p functions during transcription elongation by RNA polymerase II.

Publication Date: 2010 Feb 26 PMID: 20188675
Authors: Jimeno-Gonzalez, S. - Haaning, L. L. - Malagon, F. - Jensen, T. H.
Journal: Mol Cell

Termination of RNA polymerase II (RNAPII) transcription of protein-coding genes occurs downstream of cleavage/polyadenylation sites. According to the "torpedo" model, the 5'-3' exonuclease Rat1p/Xrn2p attacks the newly formed 5' end of the cleaved pre-mRNA, causing the still transcribing RNAPII to terminate. Here we demonstrate a similar role of S. cerevisiae Rat1p within the gene body. We find that the transcription processivity defect imposed on RNAPII by the rpb1-N488D mutation is corrected upon Rat1p inactivation. Importantly, Rat1p-dependent transcription termination occurs upstream the polyadenylation site. Genetic and biochemical evidence demonstrate that mRNA capping is defective in rpb1-N488D cells, which leads to increased levels of Rat1p all along the gene locus. Consistently, Rat1p-dependent RNAPII termination is also observed in the capping-deficient ceg1-63 strain. Our data suggest that Rat1p serves to terminate RNAPII molecules engaged in the production of uncapped RNA, regardless of their position on the gene locus.

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Negative feedback loops involving small regulatory RNAs precisely control the Vibrio harveyi quorum-sensing response.

Publication Date: 2010 Feb 26 PMID: 20188674
Authors: Tu, K. C. - Long, T. - Svenningsen, S. L. - Wingreen, N. S. - Bassler, B. L.
Journal: Mol Cell

Quorum-sensing (QS) bacteria assess population density through secretion and detection of molecules called autoinducers (AIs). We identify and characterize two Vibrio harveyi negative feedback loops that facilitate precise transitions between low-cell-density (LCD) and high-cell-density (HCD) states. The QS central regulator LuxO autorepresses its own transcription, and the Qrr small regulatory RNAs (sRNAs) posttranscriptionally repress luxO. Disrupting feedback increases the concentration of AIs required for cells to transit from LCD to HCD QS modes. Thus, the two cooperative negative feedback loops determine the point at which V. harveyi has reached a quorum and control the range of AIs over which the transition occurs. Negative feedback regulation also constrains the range of QS output by preventing sRNA levels from becoming too high and preventing luxO mRNA levels from reaching zero. We suggest that sRNA-mediated feedback regulation is a network design feature that permits fine-tuning of gene regulation and maintenance of homeostasis.

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Direct activation of TACE-mediated ectodomain shedding by p38 MAP kinase regulates EGF receptor-dependent cell proliferation.

Publication Date: 2010 Feb 26 PMID: 20188673
Authors: Xu, P. - Derynck, R.
Journal: Mol Cell

Inflammatory stimuli activate ectodomain shedding of TNF-alpha, L-selectin, and other transmembrane proteins. We show that p38 MAP kinase, which is activated in response to inflammatory or stress signals, directly activates TACE, a membrane-associated metalloprotease that is also known as ADAM17 and effects shedding in response to growth factors and Erk MAP kinase activation. p38alpha MAP kinase interacts with the cytoplasmic domain of TACE and phosphorylates it on Thr(735), which is required for TACE-mediated ectodomain shedding. Activation of TACE by p38 MAP kinase results in the release of TGF-alpha family ligands, which activate EGF receptor signaling, leading to enhanced cell proliferation. Conversely, depletion of p38alpha MAP kinase activity suppresses EGF receptor signaling and downstream Erk MAP kinase signaling, as well as autocrine EGF receptor-dependent proliferation. Autocrine EGF receptor activation through TACE-mediated ectodomain shedding intimately links inflammation and cancer progression and may play a role in stress and conditions that relate to p38 MAP kinase activation.

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Interaction with AKAP79 modifies the cellular pharmacology of PKC.

Publication Date: 2010 Feb 26 PMID: 20188672
Authors: Hoshi, N. - Langeberg, L. K. - Gould, C. M. - Newton, A. C. - Scott, J. D.
Journal: Mol Cell

A-kinase anchoring proteins (AKAPs) coordinate cell signaling events. AKAP79 brings together different combinations of enzyme binding partners to customize the regulation of effector proteins. In neurons, muscarinic agonists mobilize an AKAP79-anchored pool of PKC that phosphorylates the KCNQ2 subunit of the M channel. This inhibits potassium permeability to enhance neuronal excitability. Using a dual fluorescent imaging/patch-clamp technique, we visualized AKAP79-anchored PKC phosphorylation of the kinase activity reporter CKAR concurrently with electrophysiological changes in KCNQ2 channels to show that AKAP79 synchronizes both signaling events to optimize the attenuation of M currents. AKAP79 also protects PKC from certain ATP-competitive inhibitors. Related studies suggest that context-dependent protein-protein interactions alter the susceptibility of another protein kinase, PDK1, to ATP analog inhibitors. This implies that intracellular binding partners not only couple individual molecular events in a cell signaling process but can also change the pharmacological profile of certain protein kinases.

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The matrix peptide exporter HAF-1 signals a mitochondrial UPR by activating the transcription factor ZC376.7 in C. elegans.

Publication Date: 2010 Feb 26 PMID: 20188671
Authors: Haynes, C. M. - Yang, Y. - Blais, S. P. - Neubert, T. A. - Ron, D.
Journal: Mol Cell

Genetic analyses previously implicated the matrix-localized protease ClpP in signaling the stress of protein misfolding in the mitochondrial matrix to activate nuclear-encoded mitochondrial chaperone genes in C. elegans (UPR(mt)). Here, we report that haf-1, a gene encoding a mitochondria-localized ATP-binding cassette protein, is required for signaling within the UPR(mt) and for coping with misfolded protein stress. Peptide efflux from isolated mitochondria was ATP dependent and required HAF-1 and the protease ClpP. Defective UPR(mt) signaling in the haf-1-deleted worms was associated with failure of the bZIP protein, ZC376.7, to localize to nuclei in worms with perturbed mitochondrial protein folding, whereas zc376.7(RNAi) strongly inhibited the UPR(mt). These observations suggest a simple model whereby perturbation of the protein-folding environment in the mitochondrial matrix promotes ClpP-mediated generation of peptides whose haf-1-dependent export from the matrix contributes to UPR(mt) signaling across the mitochondrial inner membrane.

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Mitochondrial disulfide bond formation is driven by intersubunit electron transfer in Erv1 and proofread by glutathione.

Publication Date: 2010 Feb 26 PMID: 20188670
Authors: Bien, M. - Longen, S. - Wagener, N. - Chwalla, I. - Herrmann, J. M. - Riemer, J.
Journal: Mol Cell

The disulfide relay system in the intermembrane space of mitochondria is of crucial importance for mitochondrial biogenesis. Major players in this pathway are the oxidoreductase Mia40 that oxidizes substrates and the sulfhydryl oxidase Erv1 that reoxidizes Mia40. To analyze in detail the mechanism of this oxidative pathway and the interplay of its components, we reconstituted the complete process in vitro using purified cytochrome c, Erv1, Mia40, and Cox19. Here, we demonstrate that Erv1 dimerizes noncovalently and that the subunits of this homodimer cooperate in intersubunit electron exchange. Moreover, we show that Mia40 promotes complete oxidation of the substrate Cox19. The efficient formation of disulfide bonds is hampered by the formation of long-lived, partially oxidized intermediates. The generation of these side products is efficiently counteracted by reduced glutathione. Thus, our findings suggest a role for a glutathione-dependent proofreading during oxidative protein folding by the mitochondrial disulfide relay.

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SUMOylation-dependent localization of IKKepsilon in PML nuclear bodies is essential for protection against DNA-damage-triggered cell death.

Publication Date: 2010 Feb 26 PMID: 20188669
Authors: Renner, F. - Moreno, R. - Schmitz, M. L.
Journal: Mol Cell

The IKK-related kinase IKKepsilon contributes to the antiviral response and can function as an oncogene that is frequently amplified in breast cancer. Here we report on an additional role of IKKepsilon as a mediator protecting from DNA-damage-induced cell death. Genotoxic stress allows for kinase-dependent entry of IKKepsilon into the nucleus, where IKKepsilon-dependent PML phosphorylation is a prerequisite for retention of this kinase in PML nuclear bodies. Within these subnuclear structures IKKepsilon inducibly colocalizes with TOPORS, which functions as a SUMO E3 ligase mediating SUMOylation of IKKepsilon at lysine 231. SUMO modification of IKKepsilon is required to trigger phosphorylation of nuclear substrates including NF-kappaB p65, thereby contributing to the antiapoptotic function of NF-kappaB in response to DNA damage.

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Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.

Publication Date: 2010 Feb 26 PMID: 20188668
Authors: Petermann, E. - Orta, M. L. - Issaeva, N. - Schultz, N. - Helleday, T.
Journal: Mol Cell

Faithful DNA replication is essential to all life. Hydroxyurea (HU) depletes the cells of dNTPs, which initially results in stalled replication forks that, after prolonged treatment, collapse into DSBs. Here, we report that stalled replication forks are efficiently restarted in a RAD51-dependent process that does not trigger homologous recombination (HR). The XRCC3 protein, which is required for RAD51 foci formation, is also required for replication restart of HU-stalled forks, suggesting that RAD51-mediated strand invasion supports fork restart. In contrast, replication forks collapsed by prolonged replication blocks do not restart, and global replication is rescued by new origin firing. We find that RAD51-dependent HR is triggered for repair of collapsed replication forks, without apparent restart. In conclusion, our data suggest that restart of stalled replication forks and HR repair of collapsed replication forks require two distinct RAD51-mediated pathways.

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Chaperoning of a replicative polymerase onto a newly assembled DNA-bound sliding clamp by the clamp loader.

Publication Date: 2010 Feb 26 PMID: 20188667
Authors: Downey, C. D. - McHenry, C. S.
Journal: Mol Cell

Cellular replicases contain multiprotein ATPases that load sliding clamp processivity factors onto DNA. We reveal an additional role for the DnaX clamp loader: chaperoning of the replicative polymerase onto a clamp newly bound to DNA. We show that chaperoning confers distinct advantages, including marked acceleration of initiation complex formation. We reveal a requirement for the tau form of DnaX complex to relieve inhibition by single-stranded DNA binding protein during initiation complex formation. We propose that, after loading beta(2), DnaX complex preserves an SSB-free segment of DNA immediately downstream of the primer terminus and chaperones Pol III into that position, preventing competition by SSB. The C-terminal tail of SSB stimulates reactions catalyzed by tau-containing DnaX complexes through a contact distinct from the contact involving the chi subunit. Chaperoning of Pol III by the DnaX complex provides a molecular explanation for how initiation complexes form when supported by the nonhydrolyzed analog ATPgammaS.

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A role for Gcn5 in replication-coupled nucleosome assembly.

Publication Date: 2010 Feb 26 PMID: 20188666
Authors: Burgess, R. J. - Zhou, H. - Han, J. - Zhang, Z.
Journal: Mol Cell

Acetylation of lysine residues at the H3 N terminus is proposed to function in replication-coupled (RC) nucleosome assembly, a process critical for the inheritance of epigenetic information and maintenance of genome stability. However, the role of H3 N-terminal lysine acetylation and the corresponding lysine acetyltransferase (KAT) in RC nucleosome assembly are not known. Here we show that Gcn5, a KAT that functions in transcription, works in parallel with Rtt109, the H3 lysine 56 KAT, to promote RC nucleosome assembly. Cells lacking both Gcn5 and Rtt109 are highly sensitive to DNA damaging agents. Moreover, cells lacking GCN5 or those that express N-terminal H3 mutants are compromised for deposition of new H3 onto replicating DNA and also show reduced binding of H3 to CAF-1, a histone chaperone involved in RC nucleosome assembly. These results demonstrate that Gcn5 regulates RC nucleosome assembly, in part, by promoting H3 association with CAF-1 via H3 acetylation.

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Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state.

Publication Date: 2010 Feb 26 PMID: 20188665
Authors: Skene, P. J. - Illingworth, R. S. - Webb, S. - Kerr, A. R. - James, K. D. - Turner, D. J. - Andrews, R. - Bird, A. P.
Journal: Mol Cell

MeCP2 is a nuclear protein with an affinity for methylated DNA that can recruit histone deacetylases. Deficiency or excess of MeCP2 causes severe neurological problems, suggesting that the number of molecules per cell must be precisely regulated. We quantified MeCP2 in neuronal nuclei and found that it is nearly as abundant as the histone octamer. Despite this high abundance, MeCP2 associates preferentially with methylated regions, and high-throughput sequencing showed that its genome-wide binding tracks methyl-CpG density. MeCP2 deficiency results in global changes in neuronal chromatin structure, including elevated histone acetylation and a doubling of histone H1. Neither change is detectable in glia, where MeCP2 occurs at lower levels. The mutant brain also shows elevated transcription of repetitive elements. Our data argue that MeCP2 may not act as a gene-specific transcriptional repressor in neurons, but might instead dampen transcriptional noise genome-wide in a DNA methylation-dependent manner.

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The case of the disappearing drug target.

Publication Date: 2010 Feb 26 PMID: 20188664
Authors: Prince, J. T. - Ahn, N. G.
Journal: Mol Cell

In this issue of Molecular Cell, Hoshi et al. (2010) report two examples in which small molecule inhibitors are rendered ineffective when their kinase targets are involved in protein-protein interactions, highlighting differences between in vivo and in vitro inhibition kinetics.

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StIKKing together: do multiple IKK pathways cooperate in the DNA-damage response?

Publication Date: 2010 Feb 26 PMID: 20188663
Authors: Allison, D. F. - Mayo, M. W.
Journal: Mol Cell

Although IKK-related kinases are known to augment immune pathways, their importance to DNA-damage response has not been previously elucidated; in this issue of Molecular Cell, Renner et al. (2010) show that genotoxic stress requires SUMOylated IKKvarepsilon to regulate NF-kappaB transcription and cell survival.

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A bird's-eye view of MeCP2 binding.

Publication Date: 2010 Feb 26 PMID: 20188662
Authors: Cohen, S. - Greenberg, M. E.
Journal: Mol Cell

In this issue of Molecular Cell, work from Skene, Bird, and colleagues reexamines MeCP2 function in the brain by characterizing the abundance and distribution of MeCP2 protein in mouse neurons and its role in regulating chromatin structure (Skene et al., 2010).

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Taking MLL through the MudPIT: identification of novel complexes that bring together MLL-fusion proteins and transcription elongation factors.

Publication Date: 2010 Feb 26 PMID: 20188661
Authors: Fromm, G. - Adelman, K.
Journal: Mol Cell

Recently in Molecular Cell, Lin et al. (2010) showed that multiple MLL-fusion proteins implicated in mixed-lineage leukemia (MLL) associate with AFF4, ELLs, and the positive transcription elongation factor P-TEFb, providing evidence that the dysregulated gene expression in MLL patients is due to aberrant transcription elongation.

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BACH1/FANCJ acts with TopBP1 and participates early in DNA replication checkpoint control.

Publication Date: 2010 Feb 12 PMID: 20159562
Authors: Gong, Z. - Kim, J. E. - Leung, C. C. - Glover, J. N. - Chen, J.
Journal: Mol Cell

Human TopBP1 plays a critical role in the control of DNA replication checkpoint. In this study, we report a specific interaction between TopBP1 and BACH1/FANCJ, a DNA helicase involved in the repair of DNA crosslinks. The TopBP1/BACH1 interaction is mediated by the very C-terminal tandem BRCT domains of TopBP1 and S phase-specific phosphorylation of BACH1 at Thr 1133 site. Interestingly, we demonstrate that depletion of TopBP1 or BACH1 attenuates the loading of RPA on chromatin. Moreover, both TopBP1 and BACH1 are required for ATR-dependent phosphorylation events in response to replication stress. Taken together, our data suggest that BACH1 has an unexpected early role in replication checkpoint control. A specific interaction between TopBP1 and BACH1 is likely to be required for the extension of single-stranded DNA regions and RPA loading following replication stress, which is a prerequisite for the subsequent activation of replication checkpoint.

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AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia.

Publication Date: 2010 Feb 12 PMID: 20159561
Authors: Lin, C. - Smith, E. R. - Takahashi, H. - Lai, K. C. - Martin-Brown, S. - Florens, L. - Washburn, M. P. - Conaway, J. W. - Conaway, R. C. - Shilatifard, A.
Journal: Mol Cell

Chromosomal translocations involving the MLL gene are associated with infant acute lymphoblastic and mixed lineage leukemia. There are a large number of translocation partners of MLL that share very little sequence or seemingly functional similarities; however, their translocations into MLL result in the pathogenesis of leukemia. To define the molecular reason why these translocations result in the pathogenesis of leukemia, we purified several of the commonly occurring MLL chimeras. We have identified super elongation complex (SEC) associated with all chimeras purified. SEC includes ELL, P-TEFb, AFF4, and several other factors. AFF4 is required for SEC stability and proper transcription by poised RNA polymerase II in metazoans. Knockdown of AFF4 in leukemic cells shows reduction in MLL chimera target gene expression, suggesting that AFF4/SEC could be a key regulator in the pathogenesis of leukemia through many of the MLL partners.

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Noncooperative interactions between transcription factors and clustered DNA binding sites enable graded transcriptional responses to environmental inputs.

Publication Date: 2010 Feb 12 PMID: 20159560
Authors: Giorgetti, L. - Siggers, T. - Tiana, G. - Caprara, G. - Notarbartolo, S. - Corona, T. - Pasparakis, M. - Milani, P. - Bulyk, M. L. - Natoli, G.
Journal: Mol Cell

A paradigm in transcriptional regulation is that graded increases in transcription factor (TF) concentration are translated into on/off transcriptional responses by cooperative TF binding to adjacent sites. Digital transcriptional responses underlie the definition of anatomical boundaries during development. Here we show that NF-kappaB, a TF controlling inflammation and immunity, is conversely an analog transcriptional regulator that uses clustered binding sites noncooperatively. We observed that increasing concentrations of NF-kappaB are translated into gradual increments in gene transcription. We provide a thermodynamic interpretation of the experimental observations by combining quantitative measurements and a minimal physical model of an NF-kappaB-dependent promoter. We demonstrate that NF-kappaB binds independently to adjacent sites to promote additive RNA Pol II recruitment and graded transcriptional outputs. These findings reveal an alternative mode of operation of clustered TF binding sites, which might function in biological conditions where the transcriptional output is proportional to the strength of an environmental input.

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Unconventional ubiquitin recognition by the ubiquitin-binding motif within the Y family DNA polymerases iota and Rev1.

Publication Date: 2010 Feb 12 PMID: 20159559
Authors: Bomar, M. G. - D'Souza, S. - Bienko, M. - Dikic, I. - Walker, G. C. - Zhou, P.
Journal: Mol Cell

Translesion synthesis is an essential cell survival strategy to promote replication after DNA damage. The accumulation of Y family polymerases (pol) iota and Rev1 at the stalled replication machinery is mediated by the ubiquitin-binding motifs (UBMs) of the polymerases and enhanced by PCNA monoubiquitination. We report the solution structures of the C-terminal UBM of human pol iota and its complex with ubiquitin. Distinct from other ubiquitin-binding domains, the UBM binds to the hydrophobic surface of ubiquitin centered at L8. Accordingly, mutation of L8A, but not I44A, of ubiquitin abolishes UBM binding. Human pol iota contains two functional UBMs, both contributing to replication foci formation. In contrast, only the second UBM of Saccharomyces cerevisiae Rev1 binds to ubiquitin and is essential for Rev1-dependent cell survival and mutagenesis. Point mutations disrupting the UBM-ubiquitin interaction also impair the accumulation of pol iota in replication foci and Rev1-mediated DNA damage tolerance in vivo.

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Regulation of translesion synthesis DNA polymerase eta by monoubiquitination.

Publication Date: 2010 Feb 12 PMID: 20159558
Authors: Bienko, M. - Green, C. M. - Sabbioneda, S. - Crosetto, N. - Matic, I. - Hibbert, R. G. - Begovic, T. - Niimi, A. - Mann, M. - Lehmann, A. R. - Dikic, I.
Journal: Mol Cell

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.

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Lipid-Induced conformational switch controls fusion activity of longin domain SNARE Ykt6.

Publication Date: 2010 Feb 12 PMID: 20159557
Authors: Wen, W. - Yu, J. - Pan, L. - Wei, Z. - Weng, J. - Wang, W. - Ong, Y. S. - Tran, T. H. - Hong, W. - Zhang, M.
Journal: Mol Cell

While most SNAREs are permanently anchored to membranes by their transmembrane domains, the dually lipidated SNARE Ykt6 is found both on intracellular membranes and in the cytosol. The cytosolic Ykt6 is inactive due to the autoinhibition of the SNARE core by its longin domain, although the molecular basis of this inhibition is unknown. Here, we demonstrate that unlipidated Ykt6 adopts multiple conformations, with a small population in the closed state. The structure of Ykt6 in complex with a fatty acid suggests that, upon farnesylation, the Ykt6 SNARE core forms four alpha helices that wrap around the longin domain, forming a dominantly closed conformation. The fatty acid, buried in a hydrophobic groove formed between the longin domain and its SNARE core, is essential for maintaining the autoinhibited conformation of Ykt6. Our study reveals that the posttranslationally attached farnesyl group can actively regulate Ykt6 fusion activity in addition to its anticipated membrane-anchoring role.

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The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes.

Publication Date: 2010 Feb 12 PMID: 20159556
Authors: Allaire, P. D. - Marat, A. L. - Dall'Armi, C. - Di Paolo, G. - McPherson, P. S. - Ritter, B.
Journal: Mol Cell

The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn/DENND1A DENN domain functions as a guanine nucleotide exchange factor (GEF) for Rab35 to regulate endosomal membrane trafficking. Loss of Rab35 activity causes an enlargement of early endosomes and inhibits MHC class I recycling. Moreover, it prevents early endosomal recruitment of EHD1, a common component of tubules involved in endosomal cargo recycling. Our data reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.

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A DNAJB chaperone subfamily with HDAC-dependent activities suppresses toxic protein aggregation.

Publication Date: 2010 Feb 12 PMID: 20159555
Authors: Hageman, J. - Rujano, M. A. - van Waarde, M. A. - Kakkar, V. - Dirks, R. P. - Govorukhina, N. - Oosterveld-Hut, H. M. - Lubsen, N. H. - Kampinga, H. H.
Journal: Mol Cell

Misfolding and aggregation are associated with cytotoxicity in several protein folding diseases. A large network of molecular chaperones ensures protein quality control. Here, we show that within the Hsp70, Hsp110, and Hsp40 (DNAJ) chaperone families, members of a subclass of the DNAJB family (particularly DNAJB6b and DNAJB8) are superior suppressors of aggregation and toxicity of disease-associated polyglutamine proteins. The antiaggregation activity is largely independent of the N-terminal Hsp70-interacting J-domain. Rather, a C-terminal serine-rich (SSF-SST) region and the C-terminal tail are essential. The SSF-SST region is involved in substrate binding, formation of polydisperse oligomeric complexes, and interaction with histone deacetylases (HDAC4, HDAC6, SIRT2). Inhibiting HDAC4 reduced DNAJB8 function. DNAJB8 is (de)acetylated at two conserved C-terminal lysines that are not involved in substrate binding, but do play a role in suppressing protein aggregation. Combined, our data provide a functional link between HDACs and DNAJs in suppressing cytotoxic protein aggregation.

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Asymmetric activation of the hsp90 dimer by its cochaperone aha1.

Publication Date: 2010 Feb 12 PMID: 20159554
Authors: Retzlaff, M. - Hagn, F. - Mitschke, L. - Hessling, M. - Gugel, F. - Kessler, H. - Richter, K. - Buchner, J.
Journal: Mol Cell

The chaperone Hsp90 is an ATP-dependent, dimeric molecular machine regulated by several cochaperones, including inhibitors and the unique ATPase activator Aha1. Here, we analyzed the mechanism of the Aha1-mediated acceleration of Hsp90 ATPase activity and identified the interaction surfaces of both proteins using multidimensional NMR techniques. For maximum activation of Hsp90, the two domains of Aha1 bind to sites in the middle and N-terminal domains of Hsp90 in a sequential manner. This binding induces the kinetically unfavored N terminally dimerized state of Hsp90, which primes for the hydrolysis-competent conformation. Surprisingly, this activation mechanism is asymmetric. The presence of one Aha1 molecule per Hsp90 dimer is sufficient to bridge the two subunits and to fully stimulate Hsp90 ATPase activity. This seems to functionalize the two subunits of the Hsp90 dimer in different ways, in that one subunit can be used for conformational ATPase regulation and the other for substrate protein processing.

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Swe1Wee1-dependent tyrosine phosphorylation of Hsp90 regulates distinct facets of chaperone function.

Publication Date: 2010 Feb 12 PMID: 20159553
Authors: Mollapour, M. - Tsutsumi, S. - Donnelly, A. C. - Beebe, K. - Tokita, M. J. - Lee, M. J. - Lee, S. - Morra, G. - Bourboulia, D. - Scroggins, B. T. - Colombo, G. - Blagg, B. S. - Panaretou, B. - Stetler-Stevenson, W. G. - Trepel, J. B. - Piper, P. W. - Prodromou, C. - Pearl, L. H. - Neckers, L.
Journal: Mol Cell

Saccharomyces WEE1 (Swe1), the only "true" tyrosine kinase in budding yeast, is an Hsp90 client protein. Here we show that Swe1(Wee1) phosphorylates a conserved tyrosine residue (Y24 in yeast Hsp90 and Y38 in human Hsp90alpha) in the N domain of Hsp90. Phosphorylation is cell-cycle associated and modulates the ability of Hsp90 to chaperone a selected clientele, including v-Src and several other kinases. Nonphosphorylatable mutants have normal ATPase activity, support yeast viability, and productively chaperone the Hsp90 client glucocorticoid receptor. Deletion of SWE1 in yeast increases Hsp90 binding to its inhibitor geldanamycin, and pharmacologic inhibition/silencing of Wee1 sensitizes cancer cells to Hsp90 inhibitor-induced apoptosis. These findings demonstrate that Hsp90 chaperoning of distinct client proteins is differentially regulated by specific posttranslational modification of a unique subcellular pool of the chaperone, and they provide a strategy to increase the cellular potency of Hsp90 inhibitors.

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SRC-3Delta4 mediates the interaction of EGFR with FAK to promote cell migration.

Publication Date: 2010 Feb 12 PMID: 20159552
Authors: Long, W. - Yi, P. - Amazit, L. - LaMarca, H. L. - Ashcroft, F. - Kumar, R. - Mancini, M. A. - Tsai, S. Y. - Tsai, M. J. - O'Malley, B. W.
Journal: Mol Cell

EGF induces signal transduction between EGFR and FAK, and FAK is required for EGF-induced cell migration. It is unknown, however, what factor mediates the interaction between EGFR and FAK and leads to EGF-induced FAK phosphorylation. Here, we identify SRC-3Delta4, a splicing isoform of the SRC-3 oncogene, as a signaling adaptor that links EGFR and FAK and promotes EGF-induced phosphorylations of FAK and c-Src. We identify three PAK1-mediated phosphorylations in SRC-3Delta4 that promote the localization of SRC-3Delta4 to the plasma membrane and mediate the interactions with EGFR and FAK. Importantly, overexpression of SRC-3Delta4 promotes MDA-MB231-induced breast tumor metastasis. Our findings identify phosphorylated SRC-3Delta4 as a missing adaptor between EGFR and its downstream signaling molecule FAK to coordinately regulate EGF-induced cell migration. Our study also reveals that a nuclear receptor coactivator can act in the periphery of a cell to directly mediate activation of an enzyme.

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Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis.

Publication Date: 2010 Feb 12 PMID: 20159551
Authors: Kohanski, M. A. - DePristo, M. A. - Collins, J. J.
Journal: Mol Cell

Antibiotic resistance arises through mechanisms such as selection of naturally occurring resistant mutants and horizontal gene transfer. Recently, oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here, we show that sublethal levels of bactericidal antibiotics induce mutagenesis, resulting in heterogeneous increases in the minimum inhibitory concentration for a range of antibiotics, irrespective of the drug target. This increase in mutagenesis correlates with an increase in ROS and is prevented by the ROS scavenger thiourea and by anaerobic conditions, indicating that sublethal concentrations of antibiotics induce mutagenesis by stimulating the production of ROS. We demonstrate that these effects can lead to mutant strains that are sensitive to the applied antibiotic but resistant to other antibiotics. This work establishes a radical-based molecular mechanism whereby sublethal levels of antibiotics can lead to multidrug resistance, which has important implications for the widespread use and misuse of antibiotics.

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The BCL-2 family reunion.

Publication Date: 2010 Feb 12 PMID: 20159550
Authors: Chipuk, J. E. - Moldoveanu, T. - Llambi, F. - Parsons, M. J. - Green, D. R.
Journal: Mol Cell

B cell CLL/lymphoma-2 (BCL-2) and its relatives comprise the BCL-2 family of proteins, which were originally characterized with respect to their roles in controlling outer mitochondrial membrane integrity and apoptosis. Current observations expand BCL-2 family function to include numerous cellular pathways. Here we will discuss the mechanisms and functions of the BCL-2 family in the context of these pathways, highlighting the complex integration and regulation of the BCL-2 family in cell fate decisions.

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The fast track to multidrug resistance.

Publication Date: 2010 Feb 12 PMID: 20159549
Authors: Kaufmann, B. B. - Hung, D. T.
Journal: Mol Cell

In this issue of Molecular Cell, Kohanski et al. (2010) demonstrate that even subinhibitory concentrations of bactericidal antibiotics result in the generation of reactive oxygen species, leading to an increase in mutation rate and the emergence of multidrug-resistant bacterial strains.

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Phosphotyrosine confers client specificity to Hsp90.

Publication Date: 2010 Feb 12 PMID: 20159548
Authors: Mayer, M. P.
Journal: Mol Cell

In this issue of Molecular Cell, Mollapour et al. (2010) report a new tyrosine phosphorylation site in Hsp90, which is essential for Hsp90's interaction with a subset of its client proteins, notably protein kinases.

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