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

<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.

<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

Ser175 is not required for P-TEFb formation and the assembly of the 7SK snRNP complex.

<p>(A) FLAG-CDK9 wildtype, S175A, S175D, T186A, or T186D were stably expressed in Jurkat 2D10 cells using the MSCV retroviral expression system. Western blotting analysis was performed on the whole cell extracts (left panels) and the corresponding anti-FLAG-CDK9 immunoprecipitates (right panels) using antibodies against CycT1, CDK9 and the 7SK RNP protein components HEXIM1 and LARP7. (B) Quantitative analysis of protein levels. Protein concentrations were estimated by densitometry of the Western blots and normalized against total immunoprecipitated CDK9. The data are from five independent experiments. Error bars: ± standard error of the mean.</p

Ser175 mediates the binding of Tat to CDK9 and BRD4.

<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

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

<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

CDK9 kinase activity does not require S175.

<p>Radioactive <i>in vitro</i> kinase assays perfomed using FLAG-CDK9 complexes isolated from Jurkat 2D10 cells stimulated for 20 h with 10 ng/mL TNF-α to induce Tat expression. Western blots of these IPs and the corresponding WCEs are shown in the lower panel of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003338#ppat-1003338-g005" target="_blank"><b>Fig. 5B</b></a>. The kinase assays were performed in the absence (A) or presence (B) of 250 ng of His-tagged full length human pol II CTD repeat substrate. Inhibition of activity by pretreatment with 100 nM flavopiridol confirms the activity in both assays to be CDK9 kinase. Note that the S175A and S175D mutations display wildtype kinase activity in both assays.</p

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

<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⁺ CD69⁺) memory CD4⁺ T-cells have elevated expression of pSer175 CDK9 and CycT1.

<p>(A) Resting memory CD4⁺ 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

Ser175 phosphorylation of CDK9 is rapidly induced by T-cell activation signals.

<p>(A) Affinity purification of FLAG-CDK9 complexes from Jurkat 2D10 cells and their identification by mass spectrometry. The percent values indicate the sequence coverage of the identified proteins. (B) Manually annotated MS/MS fragmentation spectra of the unmodified (upper) and phosphorylated (lower) CDK9 AFSLAK tryptic precursor peptides. (C) Ratio of phosphorylation of CDK9 at Ser175 and Thr186 in PMA stimulated (50 ng/ml) versus untreated cells with or without pretreatment with 20 µM U0126.</p

Signal-dependent phosphorylation of CDK9 at Ser175.

<p>(A) Detection of Ser175 phosphorylation by Western blotting after 1 h PMA (50 ng/mL) stimulation for wild type CDK9 and the T186A and T186D mutants. FLAG-CDK9 carrying the wild type sequence, or the S175A, S175D, T186A, or T186D mutations was stably expressed in latently infected Jurkat 2D120 cells using the MSCV retroviral expression system. Top panel: Whole cell extracts used for immunoprecipitation were immunoblotted for total CDK9. Note the slower migration of the ectopically expressed FLAG-CDK9 compared to the endogenous CDK9. Bottom three panels: Anti-FLAG-CDK9 immunoprecipitates were screened by immunoblotting for CDK9, pThr186, and pSer175 using a polyclonal antibody derived using a 19-residue peptide carrying a pSer175 epitope. (B) Validation of the epitope specificity of the pSer175 CDK9 antibody by peptide blocking. Purified antibody was pre-incubated overnight with pSer175 peptide epitope prior to immunoblotting anti-FLAG-CDK9 immunoprecipitates derived from control Jurkat T-cells, or 2D10 cells expressing FLAG-CDK9 before and after stimulation for 1 hr by PMA.</p