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1.  Fumarate induces redox-dependent senescence by modifying glutathione metabolism 
Nature communications  2015;6:6001.
Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalised and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.
doi:10.1038/ncomms7001
PMCID: PMC4340546  PMID: 25613188
2.  Dominant Suppression of Inflammation via Targeted Mutation of the mRNA Destabilizing Protein Tristetraprolin 
In myeloid cells, the mRNA-destabilizing protein tristetraprolin (TTP) is induced and extensively phosphorylated in response to LPS. To investigate the role of two specific phosphorylations, at serines 52 and 178, we created a mouse strain in which those residues were replaced by nonphosphorylatable alanine residues. The mutant form of TTP was constitutively degraded by the proteasome and therefore expressed at low levels, yet it functioned as a potent mRNA destabilizing factor and inhibitor of the expression of many inflammatory mediators. Mice expressing only the mutant form of TTP were healthy and fertile, and their systemic inflammatory responses to LPS were strongly attenuated. Adaptive immune responses and protection against infection by Salmonella typhimurium were spared. A single allele encoding the mutant form of TTP was sufficient for enhanced mRNA degradation and underexpression of inflammatory mediators. Therefore, the equilibrium between unphosphorylated and phosphorylated TTP is a critical determinant of the inflammatory response, and manipulation of this equilibrium may be a means of treating inflammatory pathologies.
doi:10.4049/jimmunol.1402826
PMCID: PMC4472942  PMID: 26002976
3.  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
4.  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
5.  Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells 
Highlights
•GYS1:GN1 complex expressed using bicistronic pFastBac-Dual vector in insect cells.•A large quantity of highly-pure stoichiometric GYS1:GN1 complex obtained.•Purified GYS1 is functional and heavily phosphorylated at several Ser/Thr residues.•GYS1:GN1 complex will be useful to reveal its structural and biochemical properties.
We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis.
doi:10.1016/j.pep.2014.12.007
PMCID: PMC4370744  PMID: 25527037
Energy metabolism; Glycogenesis; Glucosyltransferase; Phosphorylation
6.  Screening of DUB activity and specificity by MALDI-TOF mass spectrometry 
Nature Communications  2014;5:4763.
Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analysing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAB/MPN/Mov34 metalloenzyme DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs.
Deubiquitylases (DUBs) remove ubiquitin chains from proteins. Here the authors develop a mass spectrometry-based DUB activity screen using unmodified diubiquitin isomers to characterize substrate specificity for 42 human DUBs, and assess the potency and selectivity of 11 DUB inhibitors.
doi:10.1038/ncomms5763
PMCID: PMC4147353  PMID: 25159004
7.  Fumarate induces redox-dependent senescence by modifying glutathione metabolism 
Nature Communications  2015;6:6001.
Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.
Fumarate hydratase (FH) mutations are associated with renal cancer. Here, Zheng et al. use metabolomic and analytical chemistry approaches to reveal that fumarate accumulated due to FH loss covalently modifies intracellular glutathione, leading to oxidative stress and senescence.
doi:10.1038/ncomms7001
PMCID: PMC4340546  PMID: 25613188
8.  Screening of DUB activity and specificity by MALDI-TOF mass spectrometry 
Nature communications  2014;5:4763.
Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs.
doi:10.1038/ncomms5763
PMCID: PMC4147353  PMID: 25159004
Deubiquitylase; ubiquitin; quantification; MALDI-TOF; mass spectrometry; inhibitor screening
9.  Phosphorylation of Sli15 by Ipl1 Is Important for Proper CPC Localization and Chromosome Stability in Saccharomyces cerevisiae 
PLoS ONE  2014;9(2):e89399.
The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the ‘tension checkpoint’ – the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.
doi:10.1371/journal.pone.0089399
PMCID: PMC3928436  PMID: 24558497
10.  Cell fate decisions are specified by the dynamic ERK interactome 
Nature cell biology  2009;11(12):10.1038/ncb1994.
Extracellular signal-regulated kinase (ERK) controls fundamental cellular functions, including cell fate decisions1,2. In PC12, cells shifting ERK activation from transient to sustained induces neuronal differentiation3. As ERK associates with both regulators and effectors4, we hypothesized that the mechanisms underlying the switch could be revealed by assessing the dynamic changes in ERK-interacting proteins that specifically occur under differentiation conditions. Using quantitative proteomics, we identified 284 ERK-interacting proteins. Upon induction of differentiation, 60 proteins changed their binding to ERK, including many proteins that were not known to participate in differentiation. We functionally characterized a subset, showing that they regulate the pathway at several levels and by different mechanisms, including signal duration, ERK localization, feedback, crosstalk with the Akt pathway and differential interaction and phosphorylation of transcription factors. Integrating these data with a mathematical model confirmed that ERK dynamics and differentiation are regulated by distributed control mechanisms rather than by a single master switch.
doi:10.1038/ncb1994
PMCID: PMC3839079  PMID: 19935650
11.  Canonical and Kinase Activity-Independent Mechanisms for Extracellular Signal-Regulated Kinase 5 (ERK5) Nuclear Translocation Require Dissociation of Hsp90 from the ERK5-Cdc37 Complex 
Molecular and Cellular Biology  2013;33(8):1671-1686.
The mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase 5 (ERK5) plays a crucial role in cell proliferation, regulating gene transcription. ERK5 has a unique C-terminal tail which contains a transcriptional activation domain, and activates transcription by phosphorylating transcription factors and acting itself as a transcriptional coactivator. However, the molecular mechanisms that regulate its nucleocytoplasmatic traffic are unknown. We have used tandem affinity purification to identify proteins that interact with ERK5. We show that ERK5 interacts with the Hsp90-Cdc37 chaperone in resting cells, and that inhibition of Hsp90 or Cdc37 results in ERK5 ubiquitylation and proteasomal degradation. Interestingly, activation of cellular ERK5 induces Hsp90 dissociation from the ERK5-Cdc37 complex, leading to ERK5 nuclear translocation and activation of transcription, by a mechanism which requires the autophosphorylation at its C-terminal tail. Consequently, active ERK5 is no longer sensitive to Hsp90 or Cdc37 inhibitors. Cdc37 overexpression also induces Hsp90 dissociation and the nuclear translocation of a kinase-inactive form of ERK5 which retains transcriptional activity. This is the first example showing that ERK5 transcriptional activity does not require kinase activity. Since Cdc37 cooperates with ERK5 to promote cell proliferation, Cdc37 overexpression (as happens in some cancers) might represent a new, noncanonical mechanism by which ERK5 regulates tumor proliferation.
doi:10.1128/MCB.01246-12
PMCID: PMC3624243  PMID: 23428871
12.  Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target 
Background
Cucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology.
Findings
Cucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation.
Conclusions
The implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution.
doi:10.1186/1478-811X-11-58
PMCID: PMC3751690  PMID: 23945128
Actin; Cofilin; Cytoskeleton; Cucurbitacin
13.  Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells 
Biochemical Journal  2013;452(Pt 2):359-365.
Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells.
doi:10.1042/BJ20121873
PMCID: PMC3727213  PMID: 23557442
A20-binding inhibitor of nuclear factor κB 2 (ABIN-2); cancer Osaka thyroid (COT); nuclear factor κB 1 (NF-κB1); double-stranded-RNA-dependent protein kinase (PKR); tumour progression locus 2 (TPL-2); virus protein 35 (VP35); ABIN-2, A20-binding inhibitor of nuclear factor κB 2; BMDM, bone-marrow-derived macrophage; DM, decyl β-D-maltopyranoside; DTT, dithiothreitol; ERK, extracellular-signal-regulated kinase; GST, glutathione transferase; HA, haemagglutinin; HEK, human embryonic kidney; Hsp, heat-shock protein; IκB, inhibitor of NF-κB; IKK, IκB kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MKK, MAPK kinase; NF-κB, nuclear factor κB; PKR, double-stranded RNA-dependent protein kinase; TBK1, TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated NF-κB activator]-binding kinase 1; TCEP, tris-(2-carboxyethyl)phosphine; TNF, tumour necrosis factor; TPL-2, tumour progression locus 2; VP35, virus protein 35
14.  The AMPK-related kinase SIK2 is regulated by cAMP via phosphorylation at Ser358 in adipocytes 
Biochemical Journal  2012;444(Pt 3):503-514.
SIK2 (salt-inducible kinase 2) is a member of the AMPK (AMP-activated protein kinase) family of kinases and is highly expressed in adipocytes. We investigated the regulation of SIK2 in adipocytes in response to cellular stimuli with relevance for adipocyte function and/or AMPK signalling. None of the treatments, including insulin, cAMP inducers or AICAR (5-amino-4-imidazolecarboxamide riboside), affected SIK2 activity towards peptide or protein substrates in vitro. However, stimulation with the cAMP-elevating agent forskolin and the β-adrenergic receptor agonist CL 316,243 resulted in a PKA (protein kinase A)-dependent phosphorylation and 14-3-3 binding of SIK2. Phosphopeptide mapping of SIK2 revealed several sites phosphorylated in response to cAMP induction, including Ser358. Site-directed mutagenesis demonstrated that phosphorylation of Ser358, but not the previously reported PKA site Ser587, was required for 14-3-3 binding. Immunocytochemistry illustrated that the localization of exogenously expressed SIK2 in HEK (human embryonic kidney)-293 cells was exclusively cytosolic and remained unchanged after cAMP elevation. Fractionation of adipocytes, however, revealed a significant increase of wild-type, but not Ser358Ala, HA (haemagglutinin)–SIK2 in the cytosol and a concomitant decrease in a particulate fraction after CL 316,243 treatment. This supports a phosphorylation-dependent relocalization in adipocytes. We hypothesize that regulation of SIK2 by cAMP could play a role for the critical effects of this second messenger on lipid metabolism in adipocytes.
doi:10.1042/BJ20111932
PMCID: PMC3631101  PMID: 22462548
14-3-3; cAMP; insulin; phosphorylation; salt-induced kinase (SIK); 3T3-L1 adipocyte; AICAR, 5-amino-4-imidazolecarboxamide riboside; AMPK, AMP-activated protein kinase; CaMK, Ca2+/calmodulin-dependent kinase; COX, cytochrome c oxidase; CREB, cAMP-response-element-binding protein; CRTC2, CREB-regulated transcription co-activator-2; DMEM, Dulbecco's modified Eagle's medium; DSTT, Division of Signal Transduction Therapy; DTT, dithiothreitol; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione transferase; HA, haemagglutinin; HDAC, histone deacetylase; HEK, human embryonic kidney; HRP, horseradish peroxidase; HSL, hormone-sensitive lipase; IBMX, isobutylmethylxanthine; IP, immunoprecipitate; IRS1, insulin receptor substrate 1; LC, liquid chromatography; MAPK, mitogen-activated protein kinase; MS/MS, tandem MS; NLS, nuclear localization signal; PK, protein kinase; SIK, salt-inducible kinase; TBS-T, Tris-buffered saline containing 0.2% Tween 20
15.  The multifunctional poly(A)-binding protein (PABP) 1 is subject to extensive dynamic post-translational modification, which molecular modelling suggests plays an important role in co-ordinating its activities 
Biochemical Journal  2012;441(Pt 3):803-812.
PABP1 [poly(A)-binding protein 1] is a central regulator of mRNA translation and stability and is required for miRNA (microRNA)-mediated regulation and nonsense-mediated decay. Numerous protein, as well as RNA, interactions underlie its multi-functional nature; however, it is unclear how its different activities are co-ordinated, since many partners interact via overlapping binding sites. In the present study, we show that human PABP1 is subject to elaborate post-translational modification, identifying 14 modifications located throughout the functional domains, all but one of which are conserved in mouse. Intriguingly, PABP1 contains glutamate and aspartate methylations, modifications of unknown function in eukaryotes, as well as lysine and arginine methylations, and lysine acetylations. The latter dramatically alter the pI of PABP1, an effect also observed during the cell cycle, suggesting that different biological processes/stimuli can regulate its modification status, although PABP1 also probably exists in differentially modified subpopulations within cells. Two lysine residues were differentially acetylated or methylated, revealing that PABP1 may be the first example of a cytoplasmic protein utilizing a ‘methylation/acetylation switch’. Modelling using available structures implicates these modifications in regulating interactions with individual PAM2 (PABP-interacting motif 2)-containing proteins, suggesting a direct link between PABP1 modification status and the formation of distinct mRNP (messenger ribonucleoprotein) complexes that regulate mRNA fate in the cytoplasm.
doi:10.1042/BJ20111474
PMCID: PMC3298439  PMID: 22004688
mRNA translation; poly(A)-binding protein (PABP); poly(A)-binding-protein-interacting motif 2 (PAM2)–poly(A)binding protein C-terminal domain (PABC) interaction; posttranscriptional control; post-translational modification; RNA-binding protein; AdOX, adenosine dialdehyde; DTT, dithiothrietol; eEF, eukaryotic elongation factor; eIF, eukaryotic initiation factor; eRF, eukaryotic release factor; eRF3-N, N-terminal eRF3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G3BP, Ras GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein; HRP, horseradish peroxidase; MEF, mouse embryonic fibroblast; miRNA, microRNA; MLLE, methionine-leucine-leucine-glutamate motif-containing domain; MS/MS, tandem MS; PABC, poly(A)-binding protein 1 C-terminal domain; PABP, poly(A)-binding protein; PAIP, poly(A)-interacting protein; PAM, PABP-interacting motif; PAN, poly(A) nuclease; PCNA, proliferating cell nuclear antigen; PRMT, protein arginine N-methyltransferase; PTM, post-translational modification; RRM, RNA recognition motif; SG, stress granule; TD-NEM, transcription-dependent nuclear export motif; TOB, transducer of ERBB2; TSA, trichostatin A
16.  Pin1 and WWP2 regulate GluR2 Q/R site RNA editing by ADAR2 with opposing effects 
The EMBO Journal  2011;30(20):4211-4222.
Pin1 and WWP2 regulate GluR2 Q/R site RNA editing by ADAR2 with opposing effects
While the essential role of the adenosine deaminase ADAR2 in RNA editing is well established, how it is regulated remains largely unknown. Here, the prolyl isomerase Pin1 and the E3 ubiquitin ligase WWP2 are shown to play a role in regulating ADAR2 localisation and stability.
ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. We demonstrate that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1−/− mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins.
doi:10.1038/emboj.2011.303
PMCID: PMC3199391  PMID: 21847096
ADAR2; GluR2; Pin1; RNA editing; WWP2
17.  14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization 
Biochemical Journal  2010;430(Pt 3):393-404.
LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients, but still little is understood about how it is regulated or functions. In the present study we have demonstrated that 14-3-3 protein isoforms interact with LRRK2. Consistent with this, endogenous LRRK2 isolated from Swiss 3T3 cells or various mouse tissues is associated with endogenous 14-3-3 isoforms. We have established that 14-3-3 binding is mediated by phosphorylation of LRRK2 at two conserved residues (Ser910 and Ser935) located before the leucine-rich repeat domain. Our results suggests that mutation of Ser910 and/or Ser935 to disrupt 14-3-3 binding does not affect intrinsic protein kinase activity, but induces LRRK2 to accumulate within discrete cytoplasmic pools, perhaps resembling inclusion bodies. To investigate links between 14-3-3 binding and Parkinson's disease, we studied how 41 reported mutations of LRRK2 affected 14-3-3 binding and cellular localization. Strikingly, we found that five of the six most common pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) display markedly reduced phosphorylation of Ser910/Ser935 thereby disrupting interaction with 14-3-3. We have also demonstrated that Ser910/Ser935 phosphorylation and 14-3-3 binding to endogenous LRRK2 is significantly reduced in tissues of homozygous LRRK2(R1441C) knock-in mice. Consistent with 14-3-3 regulating localization, all of the common pathogenic mutations displaying reduced 14-3-3-binding accumulated within inclusion bodies. We also found that three of the 41 LRRK2 mutations analysed displayed elevated protein kinase activity (R1728H, ~2-fold; G2019S, ~3-fold; and T2031S, ~4-fold). These results provide the first evidence suggesting that 14-3-3 regulates LRRK2 and that disruption of the interaction of LRRK2 with 14-3-3 may be linked to Parkinson's disease.
doi:10.1042/BJ20100483
PMCID: PMC2932554  PMID: 20642453
cytoplasmic localization; 14-3-3 protein; leucine-rich repeat protein kinase 2 (LRRK2); Parkinson's disease; pathogenic mutation; phosphorylation; CDC, cell division cycle; DIG, digoxigenin; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; FBS, fetal bovine serum; GFP, green fluorescent protein; HEK-293, human embryonic kidney; Hsp90, heat-shock protein 90; IPI, International Protein Index; KLH, keyhole-limpet haemocyanin; LRRK2, leucine-rich repeat protein kinase 2; MARK3, microtubule affinity-regulating kinase 3; PD, Parkinson's disease; ROC, Ras of complex GTPase domain; COR, C-terminal of ROC; SILAC, stable isotope labelling of amino acids; TBST, Tris-buffered saline with Tween 20
18.  Prophossi: automating expert validation of phosphopeptide–spectrum matches from tandem mass spectrometry 
Bioinformatics  2010;26(17):2153-2159.
Motivation: Complex patterns of protein phosphorylation mediate many cellular processes. Tandem mass spectrometry (MS/MS) is a powerful tool for identifying these post-translational modifications. In high-throughput experiments, mass spectrometry database search engines, such as MASCOT provide a ranked list of peptide identifications based on hundreds of thousands of MS/MS spectra obtained in a mass spectrometry experiment. These search results are not in themselves sufficient for confident assignment of phosphorylation sites as identification of characteristic mass differences requires time-consuming manual assessment of the spectra by an experienced analyst. The time required for manual assessment has previously rendered high-throughput confident assignment of phosphorylation sites challenging.
Results: We have developed a knowledge base of criteria, which replicate expert assessment, allowing more than half of cases to be automatically validated and site assignments verified with a high degree of confidence. This was assessed by comparing automated spectral interpretation with careful manual examination of the assignments for 501 peptides above the 1% false discovery rate (FDR) threshold corresponding to 259 putative phosphorylation sites in 74 proteins of the Trypanosoma brucei proteome. Despite this stringent approach, we are able to validate 80 of the 91 phosphorylation sites (88%) positively identified by manual examination of the spectra used for the MASCOT searches with a FDR < 15%.
Conclusions:High-throughput computational analysis can provide a viable second stage validation of primary mass spectrometry database search results. Such validation gives rapid access to a systems level overview of protein phosphorylation in the experiment under investigation.
Availability: A GPL licensed software implementation in Perl for analysis and spectrum annotation is available in the supplementary material and a web server can be assessed online at http://www.compbio.dundee.ac.uk/prophossi
Contact: d.m.a.martin@dundee.ac.uk
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq341
PMCID: PMC2922888  PMID: 20651112
19.  Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production 
PLoS ONE  2010;5(7):e11465.
Background
Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 (Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages.
Methodology/Principal Findings
Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly I∶C). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-κB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase (P25911), the Rho guanine nucleotide exchange factor, Vav (P27870), and the adaptor protein Grb2 (Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo.
Conclusions/Significance
We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.
doi:10.1371/journal.pone.0011465
PMCID: PMC2903609  PMID: 20644716
20.  Constitutive phosphorylation of MDC1 physically links the MRE11–RAD50–NBS1 complex to damaged chromatin 
The Journal of Cell Biology  2008;181(2):227-240.
The MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin.
doi:10.1083/jcb.200709008
PMCID: PMC2315671  PMID: 18411308
21.  Purification and Identification of Secernin, a Novel Cytosolic Protein that Regulates Exocytosis in Mast Cells 
Molecular Biology of the Cell  2002;13(9):3344-3354.
After permeabilization with the pore-forming toxin streptolysin-O mast cells can be triggered to secrete by addition of both calcium and a GTP analogue. If stimulation is delayed after permeabilization, there is a progressive decrease in the extent of secretion upon stimulation, eventually leading to a complete loss of the secretory response. This loss of secretory response can be retarded by the addition of cytosol from other secretory tissues, demonstrating that the response is dependent on a number of cytosolic proteins. We have used this as the basis of a bioassay to purify Secernin 1, a novel 50-kDa cytosolic protein that appears to be involved in the regulation of exocytosis from peritoneal mast cells. Secernin 1 increases both the extent of secretion and increases the sensitivity of mast cells to stimulation with calcium.
doi:10.1091/mbc.E01-10-0094
PMCID: PMC124164  PMID: 12221138

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