===========================================================
.___ __ __
_________________ __ __ __| _/|__|/ |_
/ ___\_` __ \__ \ | | \/ __ | | \\_ __\
/ /_/ > | \// __ \| | / /_/ | | || |
\___ /|__| (____ /____/\____ | |__||__|
/_____/ \/ \/
grep rough audit - static analysis tool
v2.8 written by @Wireghoul
=================================[justanotherhacker.com]===
cl-xmls-3.0.2/tests/nxml/genetics-article.xml-135- </front>
cl-xmls-3.0.2/tests/nxml/genetics-article.xml:136: <body><sec><title>INTRODUCTION</title><p>Following transcription, RNA-binding proteins (RBPs) regulate post-transcriptional steps of gene expression, including pre-mRNA splicing, and mRNA transport, storage, stability and translation (<xref rid="gkt903-B1" ref-type="bibr">1</xref>,<xref rid="gkt903-B2" ref-type="bibr">2</xref>). Although some RBPs have general housekeeping functions on mRNAs [e.g. bind the mRNA 5′ cap or poly(A) tail], other specialized RBPs form ribonucleoprotein (RNP) interactions with discrete subsets of mRNAs which share specific sequence elements, and affect their post-transcriptional fate (<xref rid="gkt903-B3" ref-type="bibr">3</xref>). The latter group includes RBPs such as human antigen R (HuR), AU-binding factor 1 (AUF1), nucleolin and T-cell intracellular antigen (TIA)-1 and TIA-1-related (TIAR) proteins, which associate with subsets of target mRNAs and modulate their stability and/or translation rates (<xref rid="gkt903-B1" ref-type="bibr">1</xref>,<xref rid="gkt903-B2" ref-type="bibr">2</xref>). Specialized RBPs are directly involved in changing the patterns of expressed proteins in response to stress conditions, and such stress-response functions often require RBP post-translational modification (as reviewed in <xref rid="gkt903-B4" ref-type="bibr">4–6</xref>).</p><p>HuR has three RNA-recognition motifs (RRMs) through which it binds to a large collection of protein-coding and noncoding RNAs. Although it can interact with pre-mRNA intron sequences and has been linked to regulated splicing (<xref rid="gkt903-B7" ref-type="bibr">7–9</xref>), HuR is best known for stabilizing and modulating the translation of mature mRNAs with which it associates via the 3′-untranslated region (UTR), typically at U-rich sites (<xref rid="gkt903-B9" ref-type="bibr">9</xref>,<xref rid="gkt903-B10" ref-type="bibr">10</xref>). Through binding to subsets of mRNAs encoding proliferative, stress-response and cell survival proteins, HuR has been implicated in cellular processes, such as cell division, survival, senescence and the stress-response, and with pathologies such as cancer (<xref rid="gkt903-B11" ref-type="bibr">11</xref>,<xref rid="gkt903-B12" ref-type="bibr">12</xref>).</p><p>HuR function is regulated at the levels of protein abundance, localization and post-translational modification. HuR levels are reduced by specific microRNAs (e.g. miR-519 and miR-125), by ubiquitination in response to mild heat shock and by caspase-mediated cleavage in response to severe stress (reviewed in <xref rid="gkt903-B13" ref-type="bibr">13</xref>). HuR is predominantly localized in the nucleus, but its effects on mRNA stability and translation are linked to its transport to the cytoplasm, which requires the HuR nucleocytoplasmic shuttling domain (HNS) and transport proteins such as transportins 1 and 2, the chromosome region maintenance 1 and importin-1α (<xref rid="gkt903-B14" ref-type="bibr">14–17</xref>). The transport of HuR across the nuclear envelope is influenced by kinases including the cell cycle-dependent kinase (Cdk)1, AMP-activated protein kinase (AMPK), protein kinase (PK)C and the mitogen-activated protein kinase p38 (<xref rid="gkt903-B18" ref-type="bibr">18–21</xref>). The interaction of HuR with target transcripts is modulated through phosphorylation of serine and threonine residues by several kinases; phosphorylation by checkpoint kinase (Chk)2 generally reduced HuR interaction with mRNAs (<xref rid="gkt903-B22" ref-type="bibr">22</xref>,<xref rid="gkt903-B23" ref-type="bibr">23</xref>), whereas phosphorylation by activated p38 and PKC generally promoted HuR binding to mRNAs (<xref rid="gkt903-B4" ref-type="bibr">4</xref>,<xref rid="gkt903-B24" ref-type="bibr">24</xref>,<xref rid="gkt903-B25" ref-type="bibr">25</xref>).</p><p>Besides altering the ratio of cytoplasmic-to-nuclear HuR and the interaction of HuR with target mRNAs, a number of stress agents (e.g. heat shock, irradiation with ultraviolet light and treatment with hydrogen peroxide) can also enhance the aggregation of HuR in cytoplasmic RNP foci named stress granules (SGs) (<xref rid="gkt903-B14" ref-type="bibr">14</xref>,<xref rid="gkt903-B26" ref-type="bibr">26–29</xref>). SGs assemble in response to cell-damaging conditions to halt the translation of housekeeping mRNAs and to selectively allow stress-response and repair proteins to be translated (<xref rid="gkt903-B30" ref-type="bibr">30</xref>). Besides HuR, SGs also contain numerous other RBPs, such as poly(A)-binding protein (PABP), staufen, tristetraprolin, TIA-1, TIAR, RasGAP-associated endoribonuclease (G3BP), fragile X mental retardation syndrome, survival of motor neuron and cytoplasmic polyadenylation element binding proteins (<xref rid="gkt903-B30" ref-type="bibr">30</xref>). SGs are dynamic RNP structures that assemble rapidly when the cell encounters stress and disassemble in a timely manner after the stress discontinues. SGs are believed to be the sites of mRNA ‘triage’ where decisions are made on the stability of individual mRNAs while the global cellular translation is halted.</p><p>Despite the key role of HuR in the cellular stress-response, the mechanisms that control HuR localization in SGs and their possible impact on expression of HuR target stress-response mRNAs are unknown. Here, we report that in human cervical carcinoma cells, the arsenite-triggered accumulation of HuR in SGs is accompanied by increased HuR binding to target transcripts <italic>SIRT1</italic> and <italic>VHL</italic> mRNAs and by stabilization of these mRNAs. Unexpectedly, the accumulation of HuR in SGs was blocked by treatment with menadione, a drug that activated the tyrosine kinase Janus kinase 3 (JAK3). JAK3 phosphorylated three HuR tyrosine residues <italic>in vitro</italic>; mutagenesis to prevent HuR phosphorylation specifically at Y200 restored HuR accumulation in SGs, preserved HuR binding to <italic>SIRT1</italic> and <italic>VHL</italic> mRNAs and rescued their stability. These studies link HuR presence in SGs with the fate of target mRNAs, and highlight a novel function of tyrosine kinase JAK3 as regulator of HuR function.</p></sec><sec sec-type="materials|methods"><title>MATERIALS AND METHODS</title><sec><title>Cell culture, chemicals, transfection, small interfering RNAs and plasmids</title><p>Human HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and antibiotics. All plasmids were transfected using Lipofectamine-2000 (Invitrogen) and analyzed 48 h later. JAK3 and Chk2 siRNAs were from Santa Cruz Biotechnology. For mRNA stability assays, HeLa cells were treated with actinomycin D (2.5 μg/ml) to inhibit <italic>de novo</italic> transcription. Actinomycin D, arsenite (sodium arsenite) and menadione were from Sigma; pateamine A (used at 50 nM) was a gift from I.E. Gallouzi. A site-directed mutagenesis kit (Stratagene) was used to introduce point mutations in HuR expression vectors.</p></sec><sec><title>Western blot analysis</title><p>Whole-cell lysates, prepared in Radioimmunoprecipitation assay (RIPA) buffer, were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and transferred onto Polyvinylidene fluoride (PVDF) membranes (Invitrogen iBlot Stack). Primary antibodies recognizing HuR, PABP, TIA-1, JAK3, p(Y980)JAK3, p(T68)Chk2, Chk2, Tubulin, eIF2α and phosphorylated (p-)eIF2α were from Santa Cruz Biotechnology. Antibodies recognizing phosphotyrosine (pY) residues and Flag were from Cell Signaling and Sigma, respectively. HRP-conjugated secondary antibodies were from GE Healthcare.</p></sec><sec><title>Immunoprecipitation assays</title><p>For immunoprecipitation (IP) of endogenous RNP complexes from whole-cell extracts (<xref rid="gkt903-B22" ref-type="bibr">22</xref>), cells were lysed in 20 mM Tris-HCl at pH 7.5, 100 mM KCl, 5 mM MgCl<sub>2</sub> and 0.5% NP-40 for 10 min on ice and centrifuged at 10 000 <italic>g</italic> for 15 min at 4°C. The supernatants were incubated with protein A-Sepharose beads coated with antibodies that recognized HuR, Jak3 or Flag or with control IgG (Santa Cruz Biotechnology) for 1 h at 4°C. After the beads were washed with NT2 buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM MgCl<sub>2</sub> and 0.05% NP-40), the complexes were incubated with 20 U of RNase-free DNase I (15 min at 37°C) and further incubated with 0.1% sodium dodecyl sulphate/0.5 mg/ml proteinase K (15 min at 55°C) to remove DNA and proteins, respectively. The RNPs isolated from the IP materials were further assessed by reverse transcription (RT) using random hexamers and Maxima Reverse Transcriptase (Thermo Scientific) and real-time, quantitative (q) polymerase chain reaction (PCR) using gene-specific primers (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Table S1</ext-link>) as well as by western blot (WB) analysis.</p></sec><sec><title>RNA analysis</title><p>Trizol (Invitrogen) was used to extract total RNA, and acidic phenol (Ambion) was used to extract RNA for RIP analysis (<xref rid="gkt903-B22" ref-type="bibr">22</xref>). RT-qPCR analysis was performed using gene-specific primers (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Table S1</ext-link>) and SYBR green master mix (Kapa Biosystems), in an Applied Biosystems 7300 instrument. For polyribosome distribution analysis, cells were treated with cycloheximide (100 μg/ml, 15 min), and the resulting lysates (500 μl) were separated by ultracentrifugation through 10–50% linear sucrose gradients. The relative absorbance at UV 254 nm was recorded to trace the amount of RNAs throughout the gradients.</p></sec><sec><title>Biotin pulldown analysis</title><p>Recombinant maltose-binding protein (MBP)-HuR was incubated with a buffer containing 20 mM Tris-HCl at pH 7.5, 100 mM KCl, 5 mM MgCl<sub>2</sub> and 0.5% NP-40. Biotinylated <italic>SIRT1</italic> and <italic>GAPDH</italic> 3′-untranslated regions were synthesized by PCR amplification of cDNA using forward primers that contained the T7 RNA polymerase promoter sequence (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Table S1</ext-link>) in the presence of biotinylated CTP and T7 RNA polymerase, as described (<xref rid="gkt903-B22" ref-type="bibr">22</xref>,<xref rid="gkt903-B31" ref-type="bibr">31</xref>). Proteins present in the pulldown material were studied by WB analysis.</p></sec><sec><title><italic>In vitro</italic> kinase assay</title><p>To analyze the phosphorylation of HuR <italic>in vitro</italic>, MBP-HuR purified from <italic>Escherichia </italic><italic>coli</italic> was incubated with JAK3 protein immunoprecipitated from HeLa cells or purchased from Millipore. The assay was performed in kinase reaction buffer as described previously (<xref rid="gkt903-B31" ref-type="bibr">31</xref>).</p></sec><sec><title>Liquid chromatography-tandem mass spectrometry analysis</title><p>Protein samples were processed using the ‘Filter-Assisted Sample Preparation’ (FASP) method (<xref rid="gkt903-B32" ref-type="bibr">32</xref>). Briefly, protein samples were dissolved in urea (9 M) and subjected to reduction [5 mM Tris-(2-Carboxyethyl)phosphine, hydrochloride (TCEP), Sigma] at 60°C for 45 min and to alkylation (20 mM C<sub>2</sub>H<sub>4</sub>INO, Sigma) at 25°C for 15 min. Protein samples were cleaned using a 30-kDa Amicon Filter (UFC503096, Millipore) with urea (9 M) and NH<sub>4</sub>HCO<sub>3</sub> (30 mM). Samples were then proteolyzed with trypsin (Promega) and chymotrypsin (Roche) for 12 h at 37°C (1: 20 ratio). The digested peptides were desalted and eluted with 0.1% trifluoroacetic acid in 60% acetonitrile. Dry extracted peptides were resuspended in 7 µl 0.1% formic acid for Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis. Tandem mass spectrometry analysis of the peptides was conducted on LTQ-Orbitrap Velos interfaced with a 2D nanoLC system nanoACQUITY UltraPerformance LC System. Precursor and fragment ions were analyzed at 30 000 and 7500 resolutions, respectively. Peptide sequences were identified from isotopically resolved masses in MS and MS/MS spectra extracted with and without deconvolution using Thermo Scientific Xtract software. The data were analyzed using Proteome Discoverer 1.3 (Thermo Scientific) software configured with Mascot and Sequest search nodes and searched against Refseq version 46, human entries with oxidation on methionine, deamidation on residues N and Q, phosphorylation of Ser/Thr/Tyr residues as different variable modifications and carbamidomethyl group on cysteine residue as fixed modification. Mass tolerances on precursor and fragment masses were set to 15 ppm and 0.03 Da, respectively. Peptide validator node was used for peptide confirmation, and a 1% false discovery rate cutoff was used to filter the data.</p></sec><sec><title>Immunofluorescence assay</title><p>Cells were fixed with 2% (v/v) formaldehyde, permeabilized with 0.2% (v/v) Triton X-100, blocked with 5% (w/v) bovine serum albumin and incubated with primary antibodies recognizing HuR (Santa Cruz Biotechnology), TIA-1 (Santa Cruz Biotechnology), eIF3b (Santa Cruz Biotechnology), G3BP (BD biosciences) or Flag (Sigma). Alexa 488- or Alexa 568-conjugated secondary antibodies (Invitrogen) were used to detect primary antibody-antigen complexes with different color combinations as needed. Images were acquired using Axio Observer microscope (ZEISS) with AxioVision 4.7 Zeiss image-processing software or with LSM 510 Meta (ZEISS).</p></sec></sec><sec sec-type="results"><title>RESULTS</title><sec><title>JAK3 phosphorylates HuR and prevents its accumulation in SGs</title><p>HuR is normally a nuclear protein, as seen in HeLa cells (<xref ref-type="fig" rid="gkt903-F1">Figure 1</xref>A, control), but it can translocate to the cytoplasm on stress. In response to specific stress conditions, such as arsenite treatment, HuR was further mobilized to cytoplasmic SGs (<xref ref-type="fig" rid="gkt903-F1">Figure 1</xref>A). While performing experiments to test the presence of HuR in SGs after stress, we made the serendipitous discovery that 15 μM menadione (a chemotherapeutic agent that causes oxidative damage) enhanced HuR presence in the cytoplasm, but did not trigger HuR-positive SGs. Unexpectedly, menadione also prevented SG formation following exposure to 250 μM arsenite (<xref ref-type="fig" rid="gkt903-F1">Figure 1</xref>A, <ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S1A</ext-link>). The combined treatment with arsenite and menadione caused oxidative damage, as assessed by monitoring fluorescence after incubation with dihydrocalcein, an indicator of reactive oxygen species (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S1B</ext-link>). Although treatment with arsenite and menadione did not elicit immediate signs of apoptotic cell death by 4 h after treatment (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S1C</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">D</ext-link>), some cell loss and evidence of apoptosis were detectable by 24 h following treatment (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S1C</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">D</ext-link>). The formation of SGs appeared to be generally suppressed under these conditions, as other markers used to visualize SGs [e.g. G3BP and TIA-1 (<xref ref-type="fig" rid="gkt903-F1">Figure 1</xref>B)] similarly failed to aggregate in SGs. However, we could not exclude the possibility that SGs might have been visualized by testing for other SG markers, that SG formation was delayed or that SGs were too small for detection. Arsenite treatment blocked translation globally (<xref rid="gkt903-B33" ref-type="bibr">33</xref>); however, despite impairing SG formation, menadione did not rescue the translationally inhibited state, as evidenced by the fact that polysomes remained globally suppressed, eIF2α was still phosphorylated and HuR remained bound to PABP (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S2</ext-link>). To test whether menadione prevented the recruitment of HuR to SGs that formed in an eIF2α-dependent or -independent manner, we studied the effect of 50 nM pateamine A, a drug that induces SG formation independently of eIF2α phosphorylation (<xref rid="gkt903-B34" ref-type="bibr">34</xref>). As shown (<xref ref-type="fig" rid="gkt903-F1">Figure 1</xref>C), pateamine A-triggered SGs were not blocked by menadione treatment, suggesting that menadione blocked the recruitment of HuR to SGs triggered by eIF2α phosphorylation.
cl-xmls-3.0.2/tests/nxml/genetics-article.xml-137-<fig id="gkt903-F1" position="float"><label>Figure 1.</label><caption><p>Menadione prevents the accumulation of HuR in arsenite-triggered SGs. (<bold>A</bold>) HeLa cells were treated with sodium arsenite (250 μM) with or without menadione (15 µM) for 45 min, and SGs (arrowheads) were assessed by microscopy. HuR was visualized by immunofluorescence (green), and nuclei were visualized by staining with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (blue). (<bold>B</bold>) SG markers TIA-1 and G3BP were visualized by immunofluorescence staining (Materials and Methods) in cells treated as explained in (A); nuclei were visualized with DAPI. (<bold>C</bold>) HeLa cells were treated with menadione and/or pateamine A (50 nM), whereupon SG formation was assessed by immunofluorescence.</p></caption><graphic xlink:href="gkt903f1p"/></fig></p><p>To investigate the mechanisms underlying the dynamics of HuR assembly in SGs, we screened a library of kinase inhibitors (described in <xref rid="gkt903-B35" ref-type="bibr">35</xref>) for restoration of HuR-positive SGs. Among the compounds in the library, only the JAK3 inhibitor ZM 449829 was capable of reversing the effect of menadione and restoring SGs in cells treated concomitantly with arsenite and menadione (<xref ref-type="fig" rid="gkt903-F2">Figure 2</xref>A andB). Because inhibitors are not totally specific, we also tested whether reducing JAK3 levels [achieved by using small interfering (si)RNAs] influenced SG formation after arsenite and menadione treatments. As shown in <xref ref-type="fig" rid="gkt903-F2">Figure 2</xref>C, 48 h after transfecting JAK3 siRNA in HeLa cells, JAK3 abundance was substantially lower. Importantly, in these cells, menadione treatment no longer blocked arsenite-triggered HuR-containing SGs, whereas in control (Ctrl) siRNA-transfected cells, menadione continued to block the formation of arsenite-triggered SGs (<xref ref-type="fig" rid="gkt903-F2">Figure 2</xref>D). In contrast, another stress-activated kinase that can phosphorylate HuR, Chk2, was not found to be implicated in the effects of arsenite and/or menadione (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S3</ext-link>). The finding that JAK3 silencing mirrored the effect of inhibiting JAK3 lends further support to the notion that activation of JAK3 by menadione prevents the assembly of HuR-containing SGs.
##############################################
cl-xmls-3.0.2/tests/nxml/genetics-article.xml-142-<fig id="gkt903-F6" position="float"><label>Figure 6.</label><caption><p>Arsenite and menadione affect the levels and stability of HuR target mRNAs. (<bold>A</bold>) After treatment of arsenite and/or menadione, the half-lives (t<sub>1/2</sub>) of <italic>SIRT1</italic> and <italic>VHL</italic> mRNAs (top), as well as the half-lives of a control stable mRNA (<italic>GAPDH</italic> mRNAs) and a control labile mRNA (<italic>MYC</italic> mRNA) (bottom) were quantified by measuring the time required to achieve a 50% reduction in transcript levels after adding actinomycin D at time 0 h. (<bold>B</bold>) Forty-eight hours after transfecting HeLa cells with plasmids to express Flag-HuR(WT) or Flag-HuR(Y200F), the steady-state levels of <italic>SIRT1</italic> and <italic>VHL</italic> mRNAs were measured by RT-qPCR and normalized to the levels of <italic>GAPDH</italic> mRNA. The graphs represent the means and SD from three independent experiments.</p></caption><graphic xlink:href="gkt903f6p"/></fig></p><p>Given the documented levels of HuR association with <italic>SIRT1</italic> and <italic>VHL</italic> mRNAs (<xref ref-type="fig" rid="gkt903-F5">Figure 5</xref>) and the <italic>SIRT1</italic> and <italic>VHL</italic> mRNA half-lives (<xref ref-type="fig" rid="gkt903-F6">Figure 6</xref>A), we investigated the influence of non-phosphorylatable HuR Y200F mutant on the abundance of these mRNAs. When Flag-HuR(WT) was expressed in HeLa cells, the levels of <italic>SIRT1</italic> and <italic>VHL</italic> mRNAs rose after treatment with arsenite, but this increase was lost if cells were co-treated with menadione (<xref ref-type="fig" rid="gkt903-F6">Figure 6</xref>B). In contrast, when Flag-HuR(Y200F) was expressed, the arsenite-elicited increase in <italic>SIRT1</italic> mRNA and <italic>VHL</italic> mRNA levels was refractory to menadione treatment, and the mRNAs remained significantly elevated (<xref ref-type="fig" rid="gkt903-F6">Figure 6</xref>B). In sum, these results indicate that HuR tyrosine phosphorylation at Y200, which excludes HuR from SGs, also promotes the dissociation of HuR from target transcripts (<italic>SIRT1</italic> mRNA and <italic>VHL</italic> mRNA), or perhaps mobilizes HuR-<italic>SIRT1</italic> mRNA and HuR-<italic>VHL</italic> mRNA complexes away from SGs, accelerating their degradation (<xref ref-type="fig" rid="gkt903-F7">Figure 7</xref>).
cl-xmls-3.0.2/tests/nxml/genetics-article.xml:143:<fig id="gkt903-F7" position="float"><label>Figure 7.</label><caption><p>Schematic representation of the proposed influence of JAK3 on HuR localization and RNA-binding activity. See text for details.</p></caption><graphic xlink:href="gkt903f7p"/></fig></p></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><sec><title>Tyrosine-phosphorylation of HuR by JAK3</title><p>We have reported that tyrosine phosphorylation of HuR reduces its interaction with target mRNAs, leading to lower mRNA stability. The phosphorylation at a tyrosine was unexpected, as earlier work had only identified HuR as the substrate of serine and threonine phosphorylation by PKC, Chk2, p38 and Cdk1 [reviewed in (<xref rid="gkt903-B18" ref-type="bibr">18</xref>)]. In contrast to the earlier phosphorylation events, HuR tyrosine phosphorylation is found to influence mRNA fate linked to the absence of HuR in SGs. JAK3, identified here as a kinase responsible for the tyrosine phosphorylation of HuR, is best known in immune cells, where it is activated following exposure to cytokines (<xref rid="gkt903-B37" ref-type="bibr">37</xref>). However, JAK3 is also expressed in HeLa cells and its inhibition by ZM 449829 lowers pY-HuR levels. As identified by mass spec analysis, JAK3 phosphorylates three HuR residues (Y63, Y68, Y200), but it remains possible that other tyrosine kinases besides JAK3 can also phosphorylate HuR at tyrosines, although no such kinases have been identified to date. Because the ubiquitous HuR is abundant in immune cells, it will be interesting to test whether tyrosine phosphorylation of HuR at Y200 influences the response of immune cells to cytokines.</p><p>Treatment with arsenite or menadione for 45 min caused oxidative stress, and this effect was enhanced by joint treatment with both chemicals (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S1B</ext-link>). Nonetheless, by 4 h after the drugs were removed from the culture medium, assessments of cell numbers and annexin V-positive cells revealed little or no toxicity (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S1A</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">C</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">D</ext-link>). By 24 h after removing the drugs, cells treated with arsenite did not exhibit much toxicity, as measured by modest cell loss and the absence of annexin V-positive cells (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S1C</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">D</ext-link>); however, simultaneous addition of menadione to arsenite-treated cells did prove toxic, as evidenced by the enhanced cell loss and the high percentage of annexin V-positive cells (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S1C</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">D</ext-link>). These results indicate that SGs are a component of the stress-response program triggered by arsenite, which ultimately the cells survived. The concomitant treatment with menadione modified this stress-response program (in part by antagonizing the formation of HuR-positive SGs) and potentiated the toxicity of arsenite. It is plausible that the chemotherapeutic actions of menadione (<xref rid="gkt903-B38" ref-type="bibr">38</xref>) are linked to the cytotoxicity caused by menadione, as it interferes with the cellular response to stress conditions.</p></sec><sec><title>HNS phosphorylation affects HuR localization and binding to mRNAs</title><p>It was somewhat surprising to discover that HuR binding to <italic>SIRT1</italic> and <italic>VHL</italic> mRNAs was influenced by phosphorylation at Y200 (<xref ref-type="fig" rid="gkt903-F5">Figure 5</xref>), as this residue lies within the shuttling domain of HuR (the HNS) and not within one of the three RRMs. For example, previous reports had shown that phosphorylation at RRMs (S88 in RRM1, T118 in RRM2, S100 between RRM1 and RRM2 and S318 in RRM3) affected HuR binding to numerous mRNAs (<xref rid="gkt903-B18" ref-type="bibr">18</xref>), while phosphorylation in the HNS region (S202, S221, S242), generally altered HuR the relative abundance of HuR in the nucleus compared with the cytoplasm (<xref rid="gkt903-B19" ref-type="bibr">19–21</xref>). The finding that phosphorylation near the shuttling domain affects HuR binding suggests that pY200 could change the conformation of the RRMs in ways that lower their binding affinity for RNA. Alternatively, Y200 phosphorylation could mobilize HuR to areas of the cell that have reduced concentration of HuR target transcripts, and thus binding is reduced because mRNAs are unavailable. Distinguishing between these possibilities awaits further study.</p><p>The finding that the non-phosphorylatable HuR(Y200F) is found in SGs after arsenite + menadione, whereas the phosphorylatable counterpart, HuR(WT), is not, suggests that phosphorylation at Y200 actively excludes HuR from SGs. Although the molecular mediators of HuR exclusion from SGs are not identified in our experiments, we have evidence that menadione may block the assembly of other SG components, including TIA-1, G3BP and eIF3b (<xref ref-type="fig" rid="gkt903-F1">Figure 1</xref>B; <ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S6A</ext-link>; data not shown). In fact, it is possible that JAK3 may block the assembly of multiple SG components, perhaps by phosphorylating them in a coordinated manner. In this regard, JAK3 was capable of phosphorylating TIA-1 <italic>in vitro</italic> (<ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S6B</ext-link>). Therefore, it remains formally possible that in cells that form SGs, HuR is mobilized to SGs because the mRNAs that HuR associates with are actively recruited to, or ‘pulled to’, SGs. It is unknown at present whether JAK3 impairs the binding of HuR to mRNAs and for this reason, HuR is not mobilized to SGs, or instead JAK3 inhibits the mobilization of HuR to SGs and this in turn affects HuR binding to mRNAs locally enriched in SGs. Both possibilities agree with the notion that SGs are sites of mRNA reassortment and ‘triage’ (<xref rid="gkt903-B30" ref-type="bibr">30</xref>), where mRNA-binding factors form different RNPs to accomplish molecular decisions on mRNA turnover and translational status.</p></sec><sec><title>HuR binding to mRNAs increased by stress, linked to stabilization</title><p>The discovery that treatment with arsenite, a strong oxidant, increased HuR binding to <italic>SIRT1</italic> and <italic>VHL</italic> mRNAs was also against our expectation (<xref ref-type="fig" rid="gkt903-F5">Figure 5</xref>A and B; <ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Figure S5</ext-link>), as other stress agents (e.g. ionizing radiation and the oxidant hydrogen peroxide) instead triggered the dissociation of HuR from bound mRNAs (<xref rid="gkt903-B22" ref-type="bibr">22</xref>). As dissociation of mRNAs was linked to the phosphorylation of HuR by Chk2, it is possible that arsenite inhibits Chk2 activity, while menadione reverses this inhibition in HeLa cells. Of course, arsenite and/or menadione could also affect the phosphorylation of HuR by other kinases (p38, PKC), which influence HuR–mRNA interactions. Studies are underway to investigate these possibilities, particularly given earlier reports documenting an increase in HuR binding to some mRNAs in response to certain stresses [e.g. <italic>HIF1A</italic> mRNA after hypoxia, <italic>MKP1</italic> mRNA after hydrogen peroxide treatment (<xref rid="gkt903-B39" ref-type="bibr">39</xref>,<xref rid="gkt903-B40" ref-type="bibr">40</xref>)]. In sum, our findings add to a growing body of evidence that underscores the complex regulation of HuR by phosphorylation, and the impact of this modification on HuR localization, HuR binding to mRNAs and the fate of HuR target transcripts.</p></sec></sec><sec><title>SUPPLEMENTARY DATA</title><p><ext-link ext-link-type="uri" xlink:href="http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt903/-/DC1">Supplementary Data</ext-link> are available at NAR Online.</p></sec><sec><title>FUNDING</title><p>Funding for open access charge: <funding-source>National Institute on Aging-Intramural Research Program</funding-source>, <funding-source>National Institutes of Health</funding-source>.</p><p><italic>Conflict of interest statement</italic>. None declared.</p></sec>
cl-xmls-3.0.2/tests/nxml/genetics-article.xml-144-<sec sec-type="supplementary-material">