35 research outputs found

    Phosphorylation of CDK9 at Ser175 Enhances HIV Transcription and Is a Marker of Activated P-TEFb in CD4<sup>+</sup> T Lymphocytes

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    <div><p>The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification–tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb.</p></div

    Post-translational modifications (PTMs) of CDK9 Isoform 2 (117 amino acid extension at N-Terminus) identified by tandem mass spectrometry analysis.

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    <p>Fold-changes in PTM levels after PMA or TCR activation are relative to the non-stimulated condition. Analyses of the CDK9 isoform 2 was performed using mass spectrometry data from FLAG-CDK9 affinity isolates.</p

    Post-translational modifications (PTMs) of CDK9 Isoform 1 identified by tandem mass spectrometry analysis.

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    <p>Fold-changes in PTM levels after PMA or TCR activation are relative to the non-stimulated condition. Analyses of the CDK9 isoform 1 was performed using mass spectrometry data from FLAG-CDK9 affinity isolates.</p

    A Synchrotron-Based Hydroxyl Radical Footprinting Analysis of Amyloid Fibrils and Prefibrillar Intermediates with Residue-Specific Resolution

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    Structural models of the fibrils formed by the 40-residue amyloid-β (Aβ40) peptide in Alzheimer’s disease typically consist of linear polypeptide segments, oriented approximately perpendicular to the long axis of the fibril, and joined together as parallel in-register β-sheets to form filaments. However, various models differ in the number of filaments that run the length of a fibril, and in the topological arrangement of these filaments. In addition to questions about the structure of Aβ40 monomers in fibrils, there are important unanswered questions about their structure in prefibrillar intermediates, which are of interest because they may represent the most neurotoxic form of Aβ40. To assess different models of fibril structure and to gain insight into the structure of prefibrillar intermediates, the relative solvent accessibility of amino acid residue side chains in fibrillar and prefibrillar Aβ40 preparations was characterized in solution by hydroxyl radical footprinting and structural mass spectrometry. A key to the application of this technology was the development of hydroxyl radical reactivity measures for individual side chains of Aβ40. Combined with mass-per-length measurements performed by dark-field electron microscopy, the results of this study are consistent with the core filament structure represented by two- and three-filament solid state nuclear magnetic resonance-based models of the Aβ40 fibril (such as 2LMN, 2LMO, 2LMP, and 2LMQ), with minor refinements, but they are inconsistent with the more recently proposed 2M4J model. The results also demonstrate that individual Aβ40 fibrils exhibit structural heterogeneity or polymorphism, where regions of two-filament structure alternate with regions of three-filament structure. The footprinting approach utilized in this study will be valuable for characterizing various fibrillar and nonfibrillar forms of the Aβ peptide

    Post-translational modifications (PTMs) of CycT1 identified by tandem mass spectrometry analysis.

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    <p>Fold-changes in PTM levels after PMA or TCR activation are relative to the non-stimulated condition. Analyses of CycT1 was performed using mass spectrometry data from FLAG-CDK9 affinity isolates.</p

    Ser175 mediates the binding of Tat to CDK9 and BRD4.

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    <p>(A) Induction of Tat expression in latently infected Jurkat 2D10 cells. WCEs were prepared from 2D10 cells and treated for the indicated times with PMA (50 ng/ml), TNF-α (10 ng/ml), or a combination of anti-CD3 (0.125 µg/ml) and anti-CD28 (1 µg/ml) mAbs. The extracts were then subjected to Western blotting using a Tat monoclonal antibody.(B) Relative binding of Tat and BRD4 to P-TEFb. Top: Western blots of WCEs (left panels) or FLAG-CDK9 immunoprecipitates (right panels). Top two panels show an experiment detecting BRD4 association with FLAG-CDK9 while the bottom three panels show a separate experiment detecting CycT1 and Tat association with FLAG-CDK9. Graph shows the relative levels of co-precipitated BRD4, Tat, and CycT1 normalized to corresponding total CDK9 levels. Data are from three different experiments. Error bars: ± standard error of the mean. (C) Tat-dependent and signal-dependent dissociation of P-TEFb from 7SK snRNP. 293T cells stably expressing FLAG-tagged CDK9 were transiently transfected with HA-tagged Tat. Immunoprecipitation was performed using anti-FLAG antibody followed by immunoblotting using antibodies against CycT1, CDK9, HEXIM1, LARP7, and Tat.</p

    CDK9 phosphorylated at Ser175 is excluded from the 7SK snRNP complex.

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    <p>2D10 cells were engineered to stably express FLAG-HEXIM1. Left panels: WCEs were prepared from cells that were unstimulated or treated with PMA for 1 h with or without the inhibitor U0126. Right panels: FLAG-HEXIM1 complexes were isolated by anti-FLAG IP followed by elution with FLAG peptide. Immunoblotting was performed on whole cell extracts (input samples) and anti-FLAG immunoprecipitates using antibodies towards CycT1, CDK9, HEXIM1, LARP7, pT186 CDK9, and pSer175 CDK9. The result shown is representative of two different experiments. Note that HEXIM1- P-TEFb found in the HEXIM-associated complexes is devoid of phosphorylation at Ser175 whereas the modification is readily detected in WCEs and in FLAG-CDK9 immunoprecipates (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003338#ppat-1003338-g003" target="_blank"><b>Fig. 3</b></a>).</p

    Activated (CD25<sup>+</sup> CD69<sup>+</sup>) memory CD4<sup>+</sup> T-cells have elevated expression of pSer175 CDK9 and CycT1.

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    <p>(A) Resting memory CD4<sup>+</sup> T-cells isolated from a healthy donor stained for flourophore-conjugated antibodies against the T-cell activation markers (CD25 and CD69) and P-TEFb components (Cyclin T1 and pSer175 CDK9) components and then analyzed by multicolor flow cytometry. (B) Cells from the same donor activated for 16 hr with α-CD3 and α-CD28 mAbs.</p

    Rapid induction of Ser175 phosphorylation of CDK9 after stimulation of resting memory CD4+ T cells through the TCR.

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    <p>(A) Flow cytometry. Memory CD4<sup>+</sup> T-cells isolated from healthy donors were stimulated for 2 hr or 24 hr with α-CD3 and α-CD28 mAbs to activate the TCR, immunostained with the flourophore-conjugated antibodies against total CDK9, pThr186 CDK9, CycT1 and pSer175 CDK9 and analyzed by multicolor flow cytometry. Top panels: Representative data from Experiment 1 (<b>Figs. S5 and S6</b>). Bottom panels: Representative data from Experiment 2 (<b>Figs. S7 and S8</b>). (B) Kinetic analysis. Time course of P-TEFb activation in primary resting memory CD4<sup>+</sup> T-cells stimulated between 0 and 24 hrs with α-CD3 and α-CD28 mAbs or with PMA. Kinetic data from two TCR and one PMA activation (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003338#ppat.1003338.s009" target="_blank">Figs. S9</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003338#ppat.1003338.s010" target="_blank">S10</a></b>) experiments are shown. The fraction of positive cells staining for pThr186 (blue lines), CycT1 (red lines), pSer175 CDK9 (black lines), and total CDK9 (black lines) were measured by flow cytometry as shown in Panel A. Note that during the course of the experiment there is also a 2- to 10-fold increase in the mean fluorescent intensity for the CDK9 and CycT1 proteins that is not represented by the data for % positive cells.</p
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