Protein Phosphatase-1 Activates CDK9 by Dephosphorylating Ser175

The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity is regulated by protein phosphatase-1 (PP1) which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by (32P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D – inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription

Respiratory Syncytial Virus Infection Sensitizes Cells to Apoptosis Mediated by Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease worldwide, especially in the pediatric population. For viruses in general, apoptotic death of infected cells is a mechanism for reducing virus replication. Apoptosis can also be an important factor in augmenting antigen presentation and the host immune response. We examined apoptosis in response to RSV infection of primary small airway cells, primary tracheal-bronchial cells, and A549 and HEp-2 cell lines. The primary cells and the A549 cell line gave generally similar responses, indicating their appropriateness as models in contrast to HEp-2 cells. With the use of RNase protection assays with probes representing 33 common apoptosis factors, we found strong transcriptional activation of both pro- and antiapoptotic factors in response to RSV infection, which were further studied at the protein level and by functional assays. In particular, RSV infection strongly up-regulated the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors death receptor 4 (DR4) and DR5. Furthermore, RSV-infected cells became highly sensitive to apoptosis induced by exogenous TRAIL. These findings suggest that RSV-infected cells in vivo are susceptible to killing through the TRAIL pathway by immune cells such as natural killer and CD4(+) cells that bear membrane-bound TRAIL. RSV infection also induced several proapoptotic factors of the Bcl-2 family and caspases 3, 6, 7, 8, 9, and 10, representing both the death receptor- and mitochondrion-dependent apoptotic pathways. RSV also mediated the strong induction of antiapoptotic factors of the Bcl-2 family, especially Mcl-1, which might account for the delayed induction of apoptosis in RSV-infected cells in the absence of exogenous induction of the TRAIL pathway

PP1 dephosphorylates T-loop derived CDK9 peptide phosphorylated on Ser 175.

<p>(<b>A</b>) <b>Phosphorylation of CDK9's T-loop derived peptides.</b> CDK7 or CDK9 T-loop derived peptides were phosphorylated by recombinant CDK2/cyclin E or CDK9/cyclin T1, resolved on 15% SDS Tric-Tricine gel and analyzed by Phosphor Imager. Lanes 1 and 2, phosphorylation of CDK7 derived T-loop peptides by CDK2/cyclin E. Lanes 4–7, phosphoryaltion of WT and mutant CDK9's T-loop-derived peptides by recombinant CDK9/cyclin T1. (<b>B</b>) <b>Dephosphorylation of CDK9's T-loop derived peptides by PP1.</b> CDK9-derived T loop WT peptides (lanes 1–3) or T186A mutant peptides (lanes 4–6) were phosphorylated by recombinant CDK9/cyclin T1, then CDK9 activity was blocked by 10 µM ARC and the peptides were incubated with the indicated amount of PP1. The peptides were resolved on 15% Tris-Tricine gel and analyzed by Phosphor Imager (upper panel) and also showed stained with Coomassie Blue (lower panel). Results are from a typical experiment of 3 performed.</p

CDK9 S175A activates HIV-1 transcription.

<p>(<b>A</b>) <b>Analysis of CDK9 mutants for the activation of HIV-1 transcription.</b> 293T cells were transfected with HIV-1 LTR-LacZ and Tat expression vectors along with the indicated WT or mutated Flag- CDK9. At 48 hours posttransfection β-galactosidase activity was analyzed using ONPG substrate. Results are averages of quadruplicates from a typical experiment of 3 performed. (<b>B</b>) <b>PP1 induction of HIV-1 transcription is reduced in the presence of CDK9 S175A mutant.</b> 293T cells were EGFP (control) or PP1-EGFP expression vector in combination with indicated WT or mutated Flag-tagged CDK9 and also with HIV-1 LTR-LacZ and Tat expression vectors. At 48 hours posttransfection β-galactosidase activity was analyzed using ONPG substrate. Results are from a typical experiment of 3 performed. (<b>C</b>) <b>Expression of CDK9, PP1 and Tat</b>. Protein expression from panels A and B was verified by immunoblotting.</p

CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

Abstract Background HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity. Results The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94) that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation. Conclusion Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1 provirus.</p

PP1 does not dephosphorylate CDK9 S175A mutant.

<p>293T cells were transfected with vectors expressing Flag-tagged CDK9 WT (lanes 1, 2 and 5) or CDK9 S175A mutant (lanes 3 and 4) and treated at 48 hours posttransfection with 100 nM okadaic acid and (³²P) orthophosphate. CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies and subjected to dephosphorylation by PP1 as indicated. Lane 5, immunoprecipitation with non-specific mouse IgG. The reactions were resolved on 10% SDS-PAGE and analyzed on Phosphor Imager and by immunoblotting with anti-CDK9 antibodies. On a lower panel quantitation of (³²P) phosphorylation of CDK9 is shown. Results are from a typical experiment of 2 performed.</p

Inhibition of PP1 prevents RNAPII CTD phosphorylation and inhibits CDK9 activity.

<p>(<b>A</b>) <b>High concentration of okadaic acid inhibits RNAPII phosphorylation </b><b><i>in vitro</i></b><b>.</b> HeLa cell nuclear extract was subjected to <i>in vitro</i> transcription without (lane 2) or with the addition of 10 nM okadaic acid (lane 3) or 1 µM okadaic acid (lane 4). Lane 1, untreated HeLa cell nuclear extract. RNAPII was resolved on 5% SDS-PAGE and analyzed with RNAPII CTD serine 2 phospho-epitope specific antibodies (Ser2). (<b>B</b>) <b>NIPP1 prevents RNAPII phosphorylation </b><b><i>in vitro</i></b>. HeLa cell nuclear extract was subjected to <i>in vitro</i> transcription without (lane 2) or with the addition of 5 µM NIPP1 (lane 3). Lane 1, untreated HeLa cell nuclear extract. RNAPII was resolved on 5% SDS-PAGE and analyzed with Ser2 phospho-epitope specific antibodies. (<b>C</b>) <b>Expression of cdNIPP1 prevents RNAPII phosphorylation in cultured cells.</b> 293T cells were transfected with vectors expressing wt cdNIPP1 (lane 2) or mutant cdNIPP1 (lane 3) or mock transfected (lane 1). At 48 hours post transfection, the cells were treated with 0.1 µM okadaic acid and pulsed with (³²P) orthophosphate for 3 hours. The cellular lysates were subjected to immunoprecipitation with 8WG16 antibodies against RNAPII CTD (lanes 1 to 3) or with non-specific mouse IgG2a (lane 4). Immunoprecipitated RNAPII was resolved on 5% SDS-PAGE and the gel was analyzed on Phosphor Imager. Separately, RNAPII was immunoprecipitated and analyzed by Western blotting (lower panel). (<b>D & E</b>) <b>Expression of cdNIPP1 inhibits enzymatic activity of CDK9.</b> Lysates of 293T cells (lane 1) or 293T cells continuously expressing cdNIPP1 (293T-cdNIPP1 cells) (lane 2) were immunoprecipitated with anti-CDK9 antibodies. Precipitated CDK9 was supplemented with γ-(³²P) ATP and purified yeast RNAPII (panel D) or GST-CTD (panel E) as substrates. GST-CTD and RNAPII were resolved on 10% and 7.5% SDS-PAGE gels and the gels were analyzed on Phosphor Imager. Immunoprecipitation of CDK9 was verified by immunoblotting (lower panel D). Also there was phosphorylation in the absence of substrate ((lower panel E) or when non-specific antibodies were used (panel E, lanes 3 and 4). Results are from a typical experiment of 2–4 performed.</p

Proposed model of CDK9/cyclin T1 activation in viral transcription.

<p>CDK9's Thr186 dephosphorylation by PP1/PIP1 complex leads to the dissociation of 7SK RNA and HEXIM1 protein and the release of inactive CDK9. CDK9 is phosphorylated by a cellular kinase, which may include active CDK9/cyclin T1 on Ser175 and Thr 186 that creates inactive CDK9/cyclin T1. This inactive phosphorylated CDK9/cyclin T1 is activated by PP1/PIP2 complex. The active CDK9/cyclin T1 can be recruited by Tat or re-associated with 7SK snRNP.</p

CDK9 is phosphorylated on Ser175 residue in cultured cells.

<p>(<b>A</b>) <b>MS/MS analysis of recombinant CDK9.</b> Recombinant CDK9/cyclin T1 was resolved on 10% SDS-PAGE. CDK9 was identified by Coomassie staining, in-gel digested with trypsin, and the eluted peptides were subjected to MS analysis on Thermo LTQ Orbitrap XL mass spectrometer as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018985#s4" target="_blank">Materials and Methods</a>. The SEQUEST search results are shown. Green, peptides identified with high probability by MS/MS analysis. Red, peptides identified with less probability. Black, peptides that were not detected. (<b>B</b>) <b>purification of (³²P)-labeled CDK9 for the peptide fingerprint analysis.</b> FLAG-tagged CDK9 was expressing in 293T cells and metabolically labeled in the presence of okadaic acid. CDK9 was immunoprecipitated, resolved on 10% SDS-PAGE and stained with colloidal Coomassie (upper panel), or exposed to Phosphor imager screen lower panel. Lane 1, mock-transfected cells. Lane 2, WT CDK9. Lane 3, CDK9 S175A mutant. (<b>C</b>) <b>Tryptic phosphopeptide mapping.</b> (³²P)-labeled CDK9 was trypsinized and resolved on Hunter thin layer peptide mapping electrophoresis system as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018985#s4" target="_blank">Materials and Methods</a>. WT CDK9 (upper panel) and CDK9 S175A (lower panel) are shown. Spots labeled as 1–3 were scraped and further analyzed by MS analysis. The results are representative from 2 experiments. (<b>D</b>) <b>Base peak chromatography of Spot 1.</b> Raw base peak chromatography data showing ion with mass 318.69 that matches to AFSLAK (M+2H)²⁺ peptide. Results are representative from 4 experiments. <b>E</b>. <b>MS/MS spectrum of derived from Spot 3.</b> The spectrum gives positive identification of GSQITQQSTNQSR peptide. Results are from a typical experiment of 3 performed.</p