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1.  Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis 
The EMBO Journal  2015;34(20):2506-2521.
The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin-binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin-binding site used by the E2∼Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2∼Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin.
doi:10.15252/embj.201592337
PMCID: PMC4609183  PMID: 26254304
enzyme mechanism; Parkinson's disease; phosphorylation; ubiquitination; ubiquitin ligase
2.  mTOR activates the VPS34–UVRAG complex to regulate autolysosomal tubulation and cell survival 
The EMBO Journal  2015;34(17):2272-2290.
Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient-sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome-lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3-phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3-kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process.
doi:10.15252/embj.201590992
PMCID: PMC4585463  PMID: 26139536
lysosome; mTOR; tubule; UVRAG; VPS34
3.  Ubiquitinated Fancd2 recruits Fan1 to stalled replication forks to prevent genome instability 
Science (New York, N.Y.)  2016;351(6275):846-849.
Mono-ubiquitination of Fancd2 is essential for repairing DNA inter-strand crosslinks (ICLs) but the underlying mechanisms are unclear. The Fan1 nuclease, also required for ICL repair, is recruited to ICLs by ubiquitinated (Ub)-Fancd2. This could in principle explain how Ub-Fancd2 promotes ICL repair, but we show recruitment of Fan1 by Ub-Fancd2 is dispensable for ICL repair. Instead Fan1 recruitment – and activity –restrains DNA replication fork progression, and prevent chromosome abnormalities from occurring, when DNA replication forks stall. Accordingly, Fan1 nuclease-defective knockin mice are cancer-prone. Moreover, we show that a Fan1 variant in high-risk pancreatic cancers abolishes recruitment by Ub-Fancd2, and causes genetic instability without affecting ICL repair. Therefore, Fan1 recruitment enables processing of stalled forks that is essential for genome stability and health.
doi:10.1126/science.aad5634
PMCID: PMC4770513  PMID: 26797144
4.  Erk5 Is a Key Regulator of Naive-Primed Transition and Embryonic Stem Cell Identity 
Cell Reports  2016;16(7):1820-1828.
Summary
Embryonic stem cells (ESCs) can self-renew or differentiate into any cell type, a phenomenon known as pluripotency. Distinct pluripotent states, termed naive and primed pluripotency, have been described. However, the mechanisms that control naive-primed pluripotent transition are poorly understood. Here, we perform a targeted screen for kinase inhibitors, which modulate the naive-primed pluripotent transition. We find that XMD compounds, which selectively inhibit Erk5 kinase and BET bromodomain family proteins, drive ESCs toward primed pluripotency. Using compound selectivity engineering and CRISPR/Cas9 genome editing, we reveal distinct functions for Erk5 and Brd4 in pluripotency regulation. We show that Erk5 signaling maintains ESCs in the naive state and suppresses progression toward primed pluripotency and neuroectoderm differentiation. Additionally, we identify a specialized role for Erk5 in defining ESC lineage selection, whereby Erk5 inhibits a cardiomyocyte-specific differentiation program. Our data therefore reveal multiple critical functions for Erk5 in controlling ESC identity.
Graphical Abstract
Highlights
•A kinase inhibitor screen identifies Erk5 as a key regulatory of ESC pluripotency•Erk5 suppresses transition to primed pluripotency and neural differentiation•Erk5 controls ESC identity by suppressing cardiomyocyte differentiation
Williams et al. combine chemical screening and genetic approaches to identify Erk5 kinase as a critical regulator of the naive-primed pluripotent transition and cardiomyocyte differentiation.
doi:10.1016/j.celrep.2016.07.033
PMCID: PMC4987282  PMID: 27498864
5.  The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids 
eLife  null;5:e12278.
The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1.
DOI: http://dx.doi.org/10.7554/eLife.12278.001
eLife digest
During digestion, proteins are broken down into their constituent parts called amino acids. Amino acids are transported in the bloodstream and are used to build up new cells and repair old ones. Optimal regulation of the cellular rates of amino acid uptake and protein synthesis is critical to the overall health of our bodies.
Inside each of our cells is a molecule called mammalian target of rapamycin (mTOR for short), which acts as a controller that receives information about amino acid availability. mTOR also senses how much of each amino acid the cell needs and calibrates the cell’s amino acid uptake and protein synthesis machineries accordingly.
When investigating an enzyme named ZNRF2, Hoxhaj et al. discovered that it interacts with mTOR on membranes inside cells. This raised questions about how ZNRF2 might work with mTOR to sense amino acid supplies and regulate cell growth.
Hoxhaj et al. found that when cells are provided with amino acids and growth-stimulating hormones, mTOR is activated and attaches a phosphate group to ZNRF2. This chemical modification promotes the release of ZNRF2 from membranes so that ZNRF2 separates from mTOR. In contrast, when cells are starved of amino acids, this phosphate group is removed from ZNRF2, which then returns to the membranes. On membranes, ZNRF2 also influences the activity of a pump called V-ATPase, which controls the internal acidity of the membrane-enclosed vesicles named lysosomes that help to recycle amino acids inside cells. The action of ZNRF2 on the pump may help to prime mTOR so that it is ready to sense amino acids.
These findings by Hoxhaj et al. suggest that ZNRF2 and mTOR may ‘tune’ each other, making constant to-and-fro adjustments to help ensure that levels of amino acid uptake and cell growth are set just right. However, many questions about ZNRF2 still remain to be addressed. For example, are genetic mutations in ZNRF2 involved in cancers, developmental disorders or growth syndromes? Is ZNRF2 most important in the brain, where it is particularly abundant? And how does ZNRF2 affect acidity within the lysosomes?
DOI: http://dx.doi.org/10.7554/eLife.12278.002
doi:10.7554/eLife.12278
PMCID: PMC4889327  PMID: 27244671
ZNRF2; mTORC1; amino acid sensing; V-ATPase; PP6; Ragulator; Human; Mouse
6.  Loss of iron triggers PINK1/Parkin‐independent mitophagy 
EMBO Reports  2013;14(12):1127-1135.
In this study, we develop a simple assay to identify mitophagy inducers on the basis of the use of fluorescently tagged mitochondria that undergo a colour change on lysosomal delivery. Using this assay, we identify iron chelators as a family of compounds that generate a strong mitophagy response. Iron chelation‐induced mitophagy requires that cells undergo glycolysis, but does not require PINK1 stabilization or Parkin activation, and occurs in primary human fibroblasts as well as those isolated from a Parkinson's patient with Parkin mutations. Thus, we have identified and characterized a mitophagy pathway, the induction of which could prove beneficial as a potential therapy for several neurodegenerative diseases in which mitochondrial clearance is advantageous.
A novel mitophagy assay uncovers a new PINK1/Parkin‐independent mitophagy pathway induced by a decrease in iron levels. This pathway is active in fibroblasts of Parkinson patients with Parkin mutations and could be exploited as a potential therapy.
doi:10.1038/embor.2013.168
PMCID: PMC3981094  PMID: 24176932
autophagy; iron/mitophagy; PINK1; Parkin; Molecular Biology of Disease; Metabolism
7.  mTOR activates the VPS34–UVRAG complex to regulate autolysosomal tubulation and cell survival 
The EMBO Journal  2015;34(17):2272-2290.
Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient-sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome-lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3-phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3-kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process.
doi:10.15252/embj.201590992
PMCID: PMC4585463  PMID: 26139536
lysosome; mTOR; tubule; UVRAG; VPS34
8.  Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis 
The EMBO Journal  2015;34(20):2506-2521.
The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin-binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin-binding site used by the E2∼Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2∼Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin.
doi:10.15252/embj.201592337
PMCID: PMC4609183  PMID: 26254304
enzyme mechanism; Parkinson’s disease; phosphorylation; ubiquitination; ubiquitin ligase
9.  Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders* 
The Journal of Biological Chemistry  2015;290(50):30030-30041.
Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser858 of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease.
doi:10.1074/jbc.M115.668103
PMCID: PMC4705965  PMID: 26499801
brain; cytoskeleton; insulin resistance; neurodegeneration; protein phosphorylation; MARKs; fasting; srGAP3
10.  K29-Selective Ubiquitin Binding Domain Reveals Structural Basis of Specificity and Heterotypic Nature of K29 Polyubiquitin 
Molecular Cell  2015;58(1):83-94.
Summary
Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages.
Graphical Abstract
Highlights
•Large-scale enzymatic assembly and purification of K29-linked polyubiquitin chains•K29 diubiquitin adopts extended conformation in crystal structure•Crystal structure of K29 diubiquitin in complex with selective binding domain•Presence of K29 chains within mixed/branched chains containing other linkages
Kristariyanto et al. find that K29-linked ubiquitin chains are present within ubiquitin chains containing other linkage types. They describe a method to assemble K29 chains, and they characterize a protein domain that selectively binds to these chains.
doi:10.1016/j.molcel.2015.01.041
PMCID: PMC4386640  PMID: 25752573
11.  Assembly and structure of Lys33-linked polyubiquitin reveals distinct conformations 
Biochemical Journal  2015;467(Pt 2):345-352.
Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys33-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys33 chains by combining the HECT (homologous to the E6–AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys33- and Lys11-linked Ub chains. Intriguingly, the crystal structure of Lys33-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys11-linked diUb. In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type.
Of the eight different polyubiquitin linkage types, very little is known about Lys33-linked polyubiquitin. Here the authors reveal that the HECT E3 ligase AREL1 assembles Lys33-linked polyubiquitin, and establish a method for large-scale assembly that enabled structural and biochemical studies.
doi:10.1042/BJ20141502
PMCID: PMC4390085  PMID: 25723849
deubiquitinase; homologous to the E6–AP C-terminus (HECT) E3 ligase; polyubiquitin; ubiquitin linkage; AREL1, apoptosis-resistant E3 ubiquitin protein ligase 1; ASU, asymmetric unit; Crn7, coronin-7; DUB, deubiquitinase; HECT, homologous to the E6–AP C-terminus; pRM, parallel reaction monitoring; PTM, post-translational modification; RBR, RING-between-RING; RING, really interesting new gene; TCR, T-cell antigen receptor; Ub, ubiquitin; UBD, ubiquitin-binding domain
12.  Mec1/Tel1–dependent phosphorylation of Slx4 stimulates Rad1–Rad10 dependent cleavage of non–homologous DNA tails 
DNA repair  2010;9(6):718-726.
Budding yeast Slx4 interacts with the Rad1–Rad10 endonuclease that is involved in nucleotide excision repair (NER), homologous recombination (HR) and single–strand annealing (SSA). We previously showed that Slx4 is dispensable for NER but is essential for SSA. Slx4 is phosphorylated by the Mec1 and Tel1 kinases after DNA damage on at least six Ser/Thr residues, and mutation of all six residues to Ala reduces the efficiency of SSA. In this study, we further investigated the role of Slx4 phosphorylation in SSA, specifically in regulating cleavage of 3′ non–homologous (NH) DNA tails by Rad1-Rad10 during SSA and HR. Slx4 became phosphorylated after induction of a single double–strand break (DSB) during SSA and dephosphorylation coincided approximately with completion of repair. Slx4 is recruited to 3′ NH tails during DSB repair, but this does not require phosphorylation of Slx4. However, we identified specific damage-dependent Mec1/Tel1 site of Slx4 phosphorylation, Thr 113, that is required for efficient cleavage of NH tails by Rad1–Rad10. Consistent with these data, deletion of both Mec1 and Tel1 severely reduces the efficiency of NH DNA tail cleavage during HR. These data show that phosphorylation of Slx4 by Mec1 and Tel1 plays an important role in facilitating NH DNA tail cleavage during HR.
doi:10.1016/j.dnarep.2010.02.013
PMCID: PMC4352089  PMID: 20382573
Slx4; Rad1; SSA; Mec1; Tel1; homologous recombination
13.  14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides 
Bioinformatics  2015;31(14):2276-2283.
Motivation: The 14-3-3 family of phosphoprotein-binding proteins regulates many cellular processes by docking onto pairs of phosphorylated Ser and Thr residues in a constellation of intracellular targets. Therefore, there is a pressing need to develop new prediction methods that use an updated set of 14-3-3-binding motifs for the identification of new 14-3-3 targets and to prioritize the downstream analysis of >2000 potential interactors identified in high-throughput experiments.
Results: Here, a comprehensive set of 14-3-3-binding targets from the literature was used to develop 14-3-3-binding phosphosite predictors. Position-specific scoring matrix, support vector machines (SVM) and artificial neural network (ANN) classification methods were trained to discriminate experimentally determined 14-3-3-binding motifs from non-binding phosphopeptides. ANN, position-specific scoring matrix and SVM methods showed best performance for a motif window spanning from −6 to +4 around the binding phosphosite, achieving Matthews correlation coefficient of up to 0.60. Blind prediction showed that all three methods outperform two popular 14-3-3-binding site predictors, Scansite and ELM. The new methods were used for prediction of 14-3-3-binding phosphosites in the human proteome. Experimental analysis of high-scoring predictions in the FAM122A and FAM122B proteins confirms the predictions and suggests the new 14-3-3-predictors will be generally useful.
Availability and implementation: A standalone prediction web server is available at http://www.compbio.dundee.ac.uk/1433pred. Human candidate 14-3-3-binding phosphosites were integrated in ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome database.
Contact: cmackintosh@dundee.ac.uk or gjbarton@dundee.ac.uk
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btv133
PMCID: PMC4495292  PMID: 25735772
14.  Improved Genome Editing in Human Cell Lines Using the CRISPR Method 
PLoS ONE  2014;9(10):e109752.
The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.
doi:10.1371/journal.pone.0109752
PMCID: PMC4193831  PMID: 25303670
15.  Two phases of inflammatory mediator production defined by the study of IRAK2 and IRAK1 knock-in mice 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(5):10.4049/jimmunol.1203268.
The roles of IL-1R-associated kinase (IRAK)2 and IRAK1 in cytokine production were investigated using immune cells from knock-in mice expressing the TNFR-associated factor 6 (TRAF6) binding-defective mutant IRAK2[E525A] or the catalytically inactive IRAK1[D359A] mutant. In bone marrow-derived macrophages (BMDMs), the IRAK2-TRAF6 interaction was required for the late (2-8 h) but not the early phase (0-2 h) of il6, and tnfa mRNA production and hence for IL-6 and TNF-α secretion by TLR agonists that signal via MyD88. Loss of the IRAK2-TRAF6 interaction had little effect on the MyD88-dependent production of anti-inflammatory molecules produced during the early phase, such as Dual Specificity Phosphatase 1, and a modest effect on IL-10 secretion. The LPS/TLR4-stimulated production of il6 and tnfa mRNA and IL-6 and TNF-α secretion was hardly affected, because the Toll/IL-1R domain-containing adapter-inducing IFN-β (TRIF) signaling pathway was used instead of the IRAK2-TRAF6 interaction to sustain late-phase mRNA production. IRAK1 catalytic activity was not rate-limiting for il6, tnfa or il10 mRNA production or the secretion of these cytokines by BMDMs, but IFN-β mRNA induction by TLR7 and TLR9 agonists was greatly delayed in plasmacytoid dendritic cells (pDCs) from IRAK1[D359A] mice. In contrast, IFN-β mRNA production was little affected in pDCs from IRAK2[E525A] mice, but subsequent IFN-α mRNA production and IFN-α secretion were reduced. IFN-β and IFN-α production were abolished in pDCs from IRAK1[D359A]×IRAK2[E525A] double knock-in mice. Our results establish that the IRAK2-TRAF6 interaction is rate limiting for the late, but not the early phase of cytokine production in BMDM and pDCs, and that the IRAK2-TRAF6 interaction is needed to sustain IκB-inducing kinase β activity during prolonged activation of the MyD88 signalling network.
doi:10.4049/jimmunol.1203268
PMCID: PMC3849919  PMID: 23918981
16.  ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome 
The dimeric 14-3-3 proteins dock onto pairs of phosphorylated Ser and Thr residues on hundreds of proteins, and thereby regulate many events in mammalian cells. To facilitate global analyses of these interactions, we developed a web resource named ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome, which integrates multiple data sets on 14-3-3-binding phosphoproteins. ANIA also pinpoints candidate 14-3-3-binding phosphosites using predictor algorithms, assisted by our recent discovery that the human 14-3-3-interactome is highly enriched in 2R-ohnologues. 2R-ohnologues are proteins in families of two to four, generated by two rounds of whole genome duplication at the origin of the vertebrate animals. ANIA identifies candidate ‘lynchpins’, which are 14-3-3-binding phosphosites that are conserved across members of a given 2R-ohnologue protein family. Other features of ANIA include a link to the catalogue of somatic mutations in cancer database to find cancer polymorphisms that map to 14-3-3-binding phosphosites, which would be expected to interfere with 14-3-3 interactions. We used ANIA to map known and candidate 14-3-3-binding enzymes within the 2R-ohnologue complement of the human kinome. Our projections indicate that 14-3-3s dock onto many more human kinases than has been realized. Guided by ANIA, PAK4, 6 and 7 (p21-activated kinases 4, 6 and 7) were experimentally validated as a 2R-ohnologue family of 14-3-3-binding phosphoproteins. PAK4 binding to 14-3-3 is stimulated by phorbol ester, and involves the ‘lynchpin’ site phosphoSer99 and a major contribution from Ser181. In contrast, PAK6 and PAK7 display strong phorbol ester-independent binding to 14-3-3, with Ser113 critical for the interaction with PAK6. These data point to differential 14-3-3 regulation of PAKs in control of cell morphology.
Database URL: https://ania-1433.lifesci.dundee.ac.uk/prediction/webserver/index.py
doi:10.1093/database/bat085
PMCID: PMC3914767  PMID: 24501395
17.  Loss of iron triggers PINK1/Parkin-independent mitophagy 
EMBO Reports  2013;14(12):1127-1135.
Loss of iron triggers PINK1/Parkin-independent mitophagy
A novel mitophagy assay uncovers a new PINK1/Parkin-independent mitophagy pathway induced by a decrease in iron levels. This pathway is active in fibroblasts of Parkinson patients with Parkin mutations and could be exploited as a potential therapy.
In this study, we develop a simple assay to identify mitophagy inducers on the basis of the use of fluorescently tagged mitochondria that undergo a colour change on lysosomal delivery. Using this assay, we identify iron chelators as a family of compounds that generate a strong mitophagy response. Iron chelation-induced mitophagy requires that cells undergo glycolysis, but does not require PINK1 stabilization or Parkin activation, and occurs in primary human fibroblasts as well as those isolated from a Parkinson's patient with Parkin mutations. Thus, we have identified and characterized a mitophagy pathway, the induction of which could prove beneficial as a potential therapy for several neurodegenerative diseases in which mitochondrial clearance is advantageous.
doi:10.1038/embor.2013.168
PMCID: PMC3981094  PMID: 24176932
autophagy; iron/mitophagy; PINK1; Parkin
18.  Cooperative Control of Holliday Junction Resolution and DNA Repair by the SLX1 and MUS81-EME1 Nucleases 
Molecular Cell  2013;52(2):221-233.
Summary
Holliday junctions (HJs) are X-shaped DNA structures that arise during homologous recombination, which must be removed to enable chromosome segregation. The SLX1 and MUS81-EME1 nucleases can both process HJs in vitro, and they bind in close proximity on the SLX4 scaffold, hinting at possible cooperation. However, the cellular roles of mammalian SLX1 are not yet known. Here, we use mouse genetics and structure function analysis to investigate SLX1 function. Disrupting the murine Slx1 and Slx4 genes revealed that they are essential for HJ resolution in mitotic cells. Moreover, SLX1 and MUS81-EME1 act together to resolve HJs in a manner that requires tethering to SLX4. We also show that SLX1, like MUS81-EME1, is required for repair of DNA interstrand crosslinks, but this role appears to be independent of HJ cleavage, at least in mouse cells. These findings shed light on HJ resolution in mammals and on maintenance of genome stability.
Graphical Abstract
Highlights
•Resolution of Holliday junctions in mouse cells requires the SLX1 nuclease•SLX1 acts cooperatively with MUS81-EME1 in HJ resolution and ICL repair•Mutations in SLX4 that prevent it binding to SLX1 and MUS81-EME1 abolish HJ resolution•DNA substrates of SLX1 and MUS81-EME1 in ICL repair appear to be different from HJs
doi:10.1016/j.molcel.2013.08.036
PMCID: PMC3808987  PMID: 24076219
19.  Localization-Dependent and -Independent Roles of SLX4 in Regulating Telomeres 
Cell Reports  2013;4(5):853-860.
Summary
SLX4, a scaffold for structure-specific DNA repair nucleases, is important for several types of DNA repair. Many repair proteins bind to sites of DNA damage, resulting in subnuclear “foci,” but SLX4 forms foci in human cells even without DNA damage. Using several approaches, we show that most, but not all, SLX4 foci localize to telomeres in a range of human cell lines irrespective of the mechanisms used to maintain telomere length. The SLX1 Holliday-junction-processing enzyme is recruited to telomeres by SLX4, and SLX4, in turn, is recruited by a motif that binds to the shelterin subunit TRF2 directly. We also show that TRF2-dependent recruitment of SLX4 prevents telomere damage. Furthermore, SLX4 prevents telomere lengthening and fragility in a manner that appears to be independent of telomere association. These findings reveal that SLX4 plays multiple roles in regulating telomere homeostasis.
Graphical Abstract
Highlights
•The SLX4 DNA repair complex binds to telomeres in different human cell lines•The SLX4 scaffold protein has a TRF2-binding motif responsible for telomere localization•SLX4 also has localization-independent roles at telomeres
SLX4 is a scaffold protein that regulates several DNA repair endonucleases and forms subnuclear foci in human cells that Rouse and colleagues identify as telomeres. SLX4 is recruited to telomeres by a motif that interacts with the shelterin subunit TRF2. Mutations in this motif prevent SLX4 from localizing at telomeres and cause telomeric DNA damage in human cells. SLX4 also has localization-independent roles at telomeres, such as preventing telomere overlengthening.
doi:10.1016/j.celrep.2013.07.033
PMCID: PMC3969258  PMID: 23994477
20.  PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development 
The EMBO Journal  2013;32(7):1008-1022.
Phosphoinositide-dependent kinase-1 (PDK1) controls the activation of a subset of AGC kinases. Using a conditional knockout of PDK1 in haematopoietic cells, we demonstrate that PDK1 is essential for B cell development. B-cell progenitors lacking PDK1 arrested at the transition of pro-B to pre-B cells, due to a cell autonomous defect. Loss of PDK1 decreased the expression of the IgH chain in pro-B cells due to impaired recombination of the IgH distal variable segments, a process coordinated by the transcription factor Pax5. The expression of Pax5 in pre-B cells was decreased in PDK1 knockouts, which correlated with reduced expression of the Pax5 target genes IRF4, IRF8 and Aiolos. As a result, Ccnd3 is upregulated in PDK1 knockout pre-B cells and they have an impaired ability to undergo cell-cycle arrest, a necessary event for Ig light chain rearrangement. Instead, these cells underwent apoptosis that correlated with diminished expression of the pro-survival gene Bcl2A1. Reintroduction of both Pax5 and Bcl2A1 together into PDK1 knockout pro-B cells restored their ability to differentiate in vitro into mature B cells.
PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development
The conditional knockout of PDK1 in haematopoietic cells reveals an essential role in cell-cycle arrest, differentiation, IgH recombination and the survival of B cells, which is mediated by the pro-survival factor Bcl2A1 and the transcription factor Pax5.
doi:10.1038/emboj.2013.40
PMCID: PMC3616287  PMID: 23463102
B cell; Pax5; PDK1; preB; VDJ recombination
21.  PGE2 Induces Macrophage IL-10 Production and a Regulatory-like Phenotype via a Protein Kinase A–SIK–CRTC3 Pathway 
The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE2, in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A–dependent pathway. Both TLR agonists and PGE2 promote the phosphorylation of the transcription factor CREB on Ser133. However, although CREB regulates IL-10 transcription, the mutation of Ser133 to Ala in the endogenous CREB gene did not prevent the ability of PGE2 to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser343, inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE2 on IL-10 production.
doi:10.4049/jimmunol.1202462
PMCID: PMC3620524  PMID: 23241891
22.  Identification of the Amino Acids 300–600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573 
PLoS ONE  2012;7(8):e43296.
Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300–600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300–600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573.
doi:10.1371/journal.pone.0043296
PMCID: PMC3422239  PMID: 22912850
23.  Evolution of signal multiplexing by 14-3-3-binding 2R-ohnologue protein families in the vertebrates 
Open Biology  2012;2(7):120103.
14-3-3 proteins regulate cellular responses to stimuli by docking onto pairs of phosphorylated residues on target proteins. The present study shows that the human 14-3-3-binding phosphoproteome is highly enriched in 2R-ohnologues, which are proteins in families of two to four members that were generated by two rounds of whole genome duplication at the origin of the vertebrates. We identify 2R-ohnologue families whose members share a ‘lynchpin’, defined as a 14-3-3-binding phosphosite that is conserved across members of a given family, and aligns with a Ser/Thr residue in pro-orthologues from the invertebrate chordates. For example, the human receptor expression enhancing protein (REEP) 1–4 family has the commonest type of lynchpin motif in current datasets, with a phosphorylatable serine in the –2 position relative to the 14-3-3-binding phosphosite. In contrast, the second 14-3-3-binding sites of REEPs 1–4 differ and are phosphorylated by different kinases, and hence the REEPs display different affinities for 14-3-3 dimers. We suggest a conceptual model for intracellular regulation involving protein families whose evolution into signal multiplexing systems was facilitated by 14-3-3 dimer binding to lynchpins, which gave freedom for other regulatory sites to evolve. While increased signalling complexity was needed for vertebrate life, these systems also generate vulnerability to genetic disorders.
doi:10.1098/rsob.120103
PMCID: PMC3411107  PMID: 22870394
Branchiostoma; Ciona; hereditary spastic paraplegia; RAB3GAP1; RAB3GAP2
24.  Polyubiquitin binding to ABIN1 is required to prevent autoimmunity 
The Journal of Experimental Medicine  2011;208(6):1215-1228.
The polyubiquitin-binding domain of ABIN1 limits TLR-induced MyD88 signaling to prevent spontaneous autoimmunity in mice.
The protein ABIN1 possesses a polyubiquitin-binding domain homologous to that present in nuclear factor κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of NF-κB (IκB) kinase (IKK) complex. To address the physiological significance of polyubiquitin binding, we generated knockin mice expressing the ABIN1[D485N] mutant instead of the wild-type (WT) protein. These mice developed all the hallmarks of autoimmunity, including spontaneous formation of germinal centers, isotype switching, and production of autoreactive antibodies. Autoimmunity was suppressed by crossing to MyD88−/− mice, demonstrating that toll-like receptor (TLR)–MyD88 signaling pathways are needed for the phenotype to develop. The B cells and myeloid cells of the ABIN1[D485N] mice showed enhanced activation of the protein kinases TAK, IKK-α/β, c-Jun N-terminal kinases, and p38α mitogen-activated protein kinase and produced more IL-6 and IL-12 than WT. The mutant B cells also proliferated more rapidly in response to TLR ligands. Our results indicate that the interaction of ABIN1 with polyubiquitin is required to limit the activation of TLR–MyD88 pathways and prevent autoimmunity.
doi:10.1084/jem.20102177
PMCID: PMC3173241  PMID: 21606507
25.  DNA Demethylase Activity Maintains Intestinal Cells in an Undifferentiated State Following Loss of APC 
Cell  2010;142(6):930-942.
Summary
Although genome-wide hypomethylation is a hallmark of many cancers, roles for active DNA demethylation during tumorigenesis are unknown. Here, loss of the APC tumor suppressor gene causes upregulation of a DNA demethylase system and the concomitant hypomethylation of key intestinal cell fating genes. Notably, this hypomethylation maintained zebrafish intestinal cells in an undifferentiated state which was released upon knock down of demethylase components. Mechanistically, the demethylase genes are directly activated by Pou5f1 and Cebpβ, and indirectly repressed by retinoic acid, which antagonizes Pou5f1 and Cebpβ. Apc mutants lack retinoic acid, due to the transcriptional repression of retinol dehydrogenase l1 via a complex that includes Lef1, Groucho2, Ctbp1, Lsd1 and Corest. Our findings imply a model wherein APC controls intestinal cell fating through a switch in DNA methylation dynamics. Wildtype APC and retinoic acid downregulate demethylase components, thereby promoting DNA methylation of key genes and helping progenitors commit to differentiation.
doi:10.1016/j.cell.2010.08.030
PMCID: PMC2943938  PMID: 20850014

Results 1-25 (33)