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 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

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

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

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

Dephosphorylation of <i>in vivo</i> labeled CDK9 by PP1.

<p>(<b>A</b>) <b>PP1 dephosphorylates CDK9 (³²P)-phosphorylated in cultured cells.</b> 293T cells transfected with Flag-CDK9 vector were pulsed with (³²P) orthophosphate in the presence of 100 nM okadaic acid. Flag-CDK9 was immunoprecipitated with anti-Flag antibodies (lane 2) and subjected to dephosphorylation with PP1 (lanes 3 and 4), PP2A (lanes 5 and 6) or cdc25A (lanes 7 and 8). Lane 1, immunoprecipitation with non-specific mouse IgG. The reactions were resolved on 10% SDS-PAGE and analyzed on Phosphor Imager. (<b>B</b>) <b>Comparison of phosphatase activities of PP1 and PP2A.</b> Recombinant PP1 (0.1 U) and purified from human red blood cells PP2A (0.1 U) were assayed with the generic KR-pT-IRR substrate or phospho-Rb peptide and the reactions were stopped at indicated time points by the addition of malachite green solution. The amount of malachite green was quantified by the absorbance and recalculated into the phosphate concentration using phosphate standard curve. (<b>C</b>) <b>Dephosphorylation of CDK9's Thr186 by PP1.</b> 293T cells were transfected with vectors expressing Flag-CDK9 WT (lanes 1–4) or Flag-CDK9 T186A (lane 5) and treated at 48 hours posttransfection with 100 nM okadaic acid. CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies, subjected to dephosphorylation with PP1 (lane 2) or PP2A (lane 4) and analyzed by immunoblotting with phospho-specific CDK9 Thr186 or anti-CDK9 antibodies. (<b>D</b>) <b>Comparison of dephosphorylation of (³²P) CDK9 and Thr186-phosphorylated.</b> Combined results from three independent experiments shown as percent of CDK9 dephosphorylation. *P<0.05. Results are from a typical experiment of 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

PP1 loss impairs electrotaxis in HeLa cells.

<p><b>A.</b> Treatment of parental HeLa Tet-Off (HTO) cells with siRNA strongly depletes PP1 levels 48 h post transfection. Endogenous PP1 levels were visualized with PP1 antibodies that recognize all isoforms. <b>B.</b> Plot diagrams show that loss of PP1 impairs the ability of cells to migrate towards the cathode. Each line represents the migration trajectory of a single cell. The starting point for each cell migration track is at the origin. Cell tracks with end positions to the right appear in red (“C”, cathode) and those to the left appear in black (“A”, anode). EF-untreated cells were assayed as controls. Control siRNA cells migrate strongly towards the cathode; PP1 siRNA treated cells are unable to migrate in response to a DC EF. Scales show distance migrated in µm. <b>C.</b> PP1 depletion strongly reduces distance migrated, speed, and directedness in response to physiological DC EF. Error bars are S.E.M. <i>p</i> values for significant differences in distance, speed and directedness are shown. <b>D.</b> Localization of endogenous PP1 and distribution of filamentous-actin in control and PP1 depleted cells treated with DC EF. Endogenous PP1 levels were visualized with PP1 antibodies that recognize all isoforms (green) and polymerised actin was detected using rhodamine phalloidin (red). The nuclei have been stained with DAPI (blue). Arrows mark cells with a strong decrease in PP1 levels which correlate with defects in the formation of actin rich protrusions. Representative images are shown. Scale bar is 50 µm. <b>E.</b> Numbers of cells with filopodia were quantified by counting 100 cells. Error bars are S.E.M. <i>p</i> values for significant differences are shown. Images show a detail of cell protrusions in control siRNA and PP1 siRNA cells. Arrows mark numerous filopodia in control cells and outline areas with a major lack of filopodia at the cell edges in PP1 siRNA cells.</p

Cartoon showing the basic organization of the cervical epithelium and a mechanistic model to explain how PP1/NIPP1 may contribute to invasiveness of tumour cells.

<p>Cervical and vaginal epithelia have lumen potentials of about −25 to −50 mV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040769#pone.0040769-Boskey1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040769#pone.0040769-Szatkowski1" target="_blank">[66]</a>. Such a lumen potential would correspond to a transepithelial voltage gradients of 1.7 V/cm (170 mV/mm). In these electrophysiological conditions cervical epithelial cells would migrate towards the lumen as they turn over the epithelial lining layer (green arrow). Upregulation of NIPP1 and its recruitment to PP1 would reverse migration into the lumen, encouraging invasion of the surrounding tissue (red arrow).</p

Effect of pharmacological inhibition of Cdc42-GTPase on the HTO cells.

<p><b>A.</b> Effect of ML141 on Cdc42 GTPase activity in unstimulated cells cultured in complete medium and in EF-stimulated HTO cells overexpressing the FLAG-NIPP1 protein variants. Levels of Cdc42-GTP determined by G-LISA in parental, W.T-NIPP1, ΔC-NIPP1 and mRATA cells in the absence or presence of DC EF and in cells pre-treated with 10 µM of ML141 before electrical stimulation. <i>p</i> values parental to W.T-NIPP1 and parental to ΔC-NIPP1 in complete medium were 0.1 and 0.01, respectively; <i>p</i> values comparing samples in the absence and presence of ML141 were in all cases <0.01. <b>B.</b> Cdc42 inhibition rescues cathodal polarisation and this correlates with centrosome positioning. Directedness values for the migration of EF-treated cells incubated with ML141. Cdc42 inhibition rescues the positive cell directedness decreased by W.T-NIPP1 overexpression. The strongly negative directedness value displayed by ΔC-NIPP1 cells becomes closer to 0 when cells are pretreated with Cdc42 inhibitor. For simplification directedness values in the absence of EF of the parental, W.T-NIPP1, ΔC-NIPP1, and mNIPP1 with and without ML141 have not been included in the diagram. These were, without ML141, −0.07±0.04; 0.05±0.09; −0.08±0.05 and −0.01±0.04, respectively; with ML141 were −0.07±0.04; 0.09±0.05; −0.07±0.05 and −0.01±0.04, respectively. In the absence of EF values were in all cases very close to 0 and differences between the four lines were not statistically significant in any of the cases. Data was quantified from at least three experiments. Error bars are S.E.M. <i>p</i> values for significant differences in directedness are shown. Polarisation index of centrosomes calculated as explained in materials and methods. Polarisation index of W.T-NIPP1 and ΔC-NIPP1 cells becomes similar to the polarisation index of parental cells when cells are treated with the Cdc42 inhibitor ML141.</p