We explored whether PP2A or PP1 dephosphorylates CDK9 in vitro. CDK9 within the recombinant CDK9/cyclin T1 was autophosphorylated in the presence of γ-(P³²)-ATP. The kinase activity of CDK9 was blocked by the addition of 7 mM EDTA and (³²P) phosphorylated CDK9 was used as a substrate for PP1 or PP2A (Fig. 1A, lane 1). While PP2A efficiently dephosphorylated CDK9 (Fig. 1A, lanes 4 and 5), PP1 was approximately 10-time less efficient than PP2A in the dephosphorylation (Fig. 1A, lanes 2 and 3). Based on their activities towards the reference substrate, glycogen phosphorylase-a 27, PP1 was added at 1.5-fold higher activity than PP2A (Fig. 1B) and thus PP1 was even less efficient than PP2A, at least 20-time less efficient in dephosphorylation of CDK9.

Recently, CDK9 within the recombinant P-TEFb purified from insect cells was found to be phosphorylated on T186 28. We explore here whether CDK9 might be already in the phosphorylated state in our preparation of the recombinant P-TEFb. We asked whether dephosphorylation by either PP2A or PP1 of CDK9/cyclin T1 would enhance phosphorylation of CDK9 in the following kinase reaction. Recombinant CDK9/cyclin T1 was incubated with increasing concentrations of PP1 or PP2A followed by inhibition of the phosphatases with 1 μM okadaic acid. Then the autophosphorylation reaction was carried out in the presence of γ-(³²P)-ATP (Fig. 1C). Treatment with PP2A (Fig. 1C, lanes 4 and 5) but not with PP1 (Fig. 2, lanes 2 and 3) increased the efficiency of CDK9 autophosphorylation. This result indicates that recombinant CDK9 was already in partially phosphorylated state and that PP2A-mediated dephosphorylation of CDK9 enhanced subsequent phosphorylation of CDK9.

We next analyzed whether dephosphorylation of CDK9 by PP2A or by PP1 has an effect on formation of a complex between recombinant P-TEFb, HIV-1 Tat and TAR RNA. We utilized a biotinylated TAR RNA that was preincubated with recombinant Tat and recombinant CDK9/cyclin T1 and then precipitated with streptavidin agarose beads (Figs. 2A and 2B, lane 3). When TAR RNA was denatured or when Tat was omitted, CDK9/cyclin T1 was not precipitated with TAR RNA (Figs. 2A and 2B, lanes 1 and 2) indicating a specific P-TEFb:Tat:TAR RNA complex formation. Pre-treatment of CDK9/cyclin T1 with PP2A resulted in a significant decrease in the complex formation (Fig. 2A, lane 4). In contrast, pretreatment of CDK9/cyclin T1 with PP1 did not have an effect on P-TEFb: Tat: TAR RNA complex formation (Fig. 2B, lane 4). These results indicate that PP2A but not PP1 affects formation of the P-TEFb: Tat: TAR RNA complex in vitro.

Next we analyzed whether inhibition of PP2A has an effect on HIV-1 transcription in vitro. An HIV-1 LTR template that contains 308 nucleotides downstream of the transcription start was prepared by PCR using HIV-1 LTR-LacZ expression vector (see Methods). Purified Tat stimulated transcription on this template in the HeLa nuclear extract to approximately 5-fold (Fig. 3A). We used okadaic acid, which is a 100-fold more efficient in vitro inhibitor of PP2A than PP1 (Fig. 3B) to determine the effect of PP2A inhibition on HIV-1 transcription. Two different concentrations of okadaic acid were used: 10 nM – to inhibit PP2A and 1 μM – to inhibit PP1. Addition of either 10 nM or 1 μM concentrations of okadaic acid inhibited basal HIV-1 transcription (Fig. 3C, compare lanes 4 and 5 to lane 2) and also Tat-activated HIV-1 transcription (Fig. 3C, compare lanes 6 and 7 to lane 3). Thus this result indicates that inhibition of PP2A blocks both basal and Tat-activated transcription.

we cannot rule out the possibility that PP1 might also be involved in the HIV-1 transcription in vitro. We used recombinant NIPP1 protein which we previously used to inhibit PP1 in vitro 29. Similar to the experiment in the previous section, purified Tat stimulated transcription about 4-fold (Fig. 3D, compare lanes 2 and 3). Addition of NIPP1 inhibited Tat-activated transcription (Fig. 3D, lane 5), but did not affect basal HIV-1 transcription (Fig. 3D, lanes 4). This result indicates that PP1 might be involved in the Tat-activated transcription.

We next determined relative contribution of PP1 and PP2A to basal and Tat-activated HIV-1 transcription in cultured COS-7 cells using selective inhibition of PP2A and PP1. We used okadaic acid which selectively inhibits PP2A in vitro at concentrations below 1 nM (Fig. 3B) but which would inhibit both PP1 and PP2A at higher concentrations. COS-7 cells were co-transfected with Tat-expressing vector and HIV-1 LTR-LacZ (JK2) and expression of β-galactosidase was analyzed using quantitative ONPG-based assay 26. In these cells Tat potently stimulate transcription from HIV-1 LTR (Fig. 4A, compare lanes 1 and 2). Treatment of the transfected COS-7 cells with okadaic acid resulted in partial (about 30%) inhibition of Tat-induced transcription (Fig. 4A). The IC₅₀ of the okadaic acid-mediated inhibition was 4 nM (Fig. 4B). Surprisingly, okadaic acid had no inhibitory effect on HIV-1 basal transcription from a mutant HIV-1 LTR with a deletion of the fragment encoding TAR RNA (HIV-1 LTRΔTAR) (Fig. 4C). At the concentrations below 10 nM, treatment with okadaic acid did not affect viability of COS-7 cells (see supplemental Fig). These results indicate that PP2A has a moderate effect only on Tat-induced transcription in cultured cells. To analyze the contribution of PP1 to the control of HIV-1 transcription in COS-7 cells, vectors expressing NIPP1-EGFP WT or NIPP1-EGFP mutant (NIPP1 K193-197A/V201A/F203A/Y335D, NIPP1 mut) were transfected along with JK2 and Tat expression vector, as we previously described 26. In the mutant NIPP1 the PP1 binding sites in both the central and C-terminal domain of NIPP1 are mutated and it no longer interacts with PP1 30. Co-transfection of wild type, but not the mutant NIPP1-EGFP, inhibited Tat-activated transcription (Fig. 5A, lanes 3 and 4). In the absence of Tat, transcription from HIV-1 LTR or from a mutant HIV-1 LTR with a deletion of the fragment encoding TAR RNA (JK2ΔTAR) was inhibited about 50% by both NIPP1 and mutant NIPP1 (Fig. 5B), indicating that this effect of NIPP1 was not due to its ability to bind PP1. Taken together, inhibition of PP1 by over expression of NIPP1 reduces Tat-dependent HIV-1 transcription by 70%, in accord to our previous study 26, Thus PP1 and less likely PP2A might contribute to the regulation of Tat-dependent HIV-1 transcription.

We next analyzed whether CDK9 phosphorylation state is controlled by PP1 or by PP2A in cultured cells. HeLa cells were labelled with (³²P) orthophosphate in the absence and in the presence of okadaic acid and cellular extracts were immunoprecipitated with anti-cyclin T1 antibodies, resolved by 10% SDS-PAGE and transferred to PVDF membrane. Position of CDK9 was determined by probing the membrane with anti-CDK9 antibodies using 3,3'-Diaminobenzidine enhancer system (Fig. 6A). Precipitation of CDK9 from untreated cells and from the cells treated with okadaic acid showed that phosphorylation of CDK9 was increased in the presence of okadaic acid (Fig. 6B, lanes 1 and 2). To analyze whether this increase was due to inhibition of PP1 or PP2A, we utilized 293T cells that were stably transfected with the central domain of NIPP1 (residues 143–224) (293T-cdNIPP1 cells), an equally potent inhibitor of PP1 as full length NIPP1 30. Precipitation of endogenous CDK9 from untreated 293T-cdNIPP1 cells labeled with (³²P) in the absence or in the presence of 100 nM okadaic acid showed equal low level of CDK9 phosphorylation (Fig. 6C, lanes 1 and 2). To further investigate whether CDK9 phosphorylation was caused by PP1, we transiently express Flag- tagged CDK9 in 293T-cdNIPP1 cells, labeled cells with (³²P) and precipitated CDK9 with anti-Flag antibodies. Again treatment with 100 nM okadaic acid did not increase phosphorylation of CDK9 (Fig. 6D, lanes 1 and 2). Taken together these results indicate that CDK9 is dephosphorylated in vivo and that it is likely PP1 that dephosphorylates CDK9.

To further explore the CDK9 dephosphorylation, we analyzed phosphorylation of CDK9 mutants with mutation in Thr 186 (T186A mutant) or with mutations in Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354 residues (C8A mutant). 293T cells were transiently transfected with Flag-tagged CDK9, WT, T186A mutant or C8A mutant. Transfected cells were labeled with (³²P) without or with the addition of 100 nM okadaic acid. Precipitation of CDK9 with anti-Flag antibodies showed that while okadaic acid induced phosphorylation of WT CDK9 (Fig. 7A, lanes 2 and 3; and Fig. 7B), there was no further increase in phosphorylation of T186A or C8A mutant (Fig. 7A, lanes 4 to 7: and Fig. 7B). Interestingly, mutation of Thr 186 increases CDK9 phosphorylation level (Fig. 7A, lane 5 and Fig. 7B).

Taken together our results indicate that PP1 may potentially dephosphorylate Thr 186 as well as the C-terminal serines involved in the autophosphorylation of CDK9 and that dephosphorylation of CDK9 may have a regulatory effect in Tat-activated HIV-1 transcription.

COS-7 cells, 293T cells and HeLa cells were purchased from ATCC (Manassas, VA). 293T cells stably expressing NIPP1 (143–224) were generated by transfection of NIPP1-143-224-EGFP, and limited dilution cloning in the presence of geneticin (0.5 mg/ml) (Life Technologies, Rockville, MD). Human protein phosphatase PP2A was purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit protein phosphatase PP1 and recombinant NIPP1 were gifts from M. Bollen (Catholic University, Leuven, Belgium). Phosphorylase b was from Calzyme Laboratories (San Luis Obispo, CA). Protein (A) agarose was purchased from Sigma (Atlanta, GA). Human recombinant P-TEFb from baculovirus transfected Sf9 cells (NCCC) was purified as described in 34.

Rabbit polyclonal antibodies to CDK9 and goat polyclonal antibodies to cyclin T1 were purchased from Santa Cruz Biochemical (Santa Cruz, CA).

The reporter plasmid, pJK2, contained HIV-1 LTR (-138 to +82) followed by nuclear localization signal (NLS) and lacZ reporter gene (courtesy of Dr. Michael Emerman, Fred Hutchinson Cancer Institute, Seattle, WA). This plasmid expresses NLS-tagged β-galactosidase under the control of HIV-1 LTR 35. Tat expression plasmid was a gift from Dr. Ben Berkhout (University of Amsterdam) 36. The pGEM2Tat bacterial expression vector was obtained from NIH AIDS Research and Reference Reagents Program.

Recombinant CDK9/cyclin T1 (30 ng/reaction) was autophosphorylated in 20 μl reaction with 50 μM ATP (1 μCi of γ-(³²P)ATP) in kinase buffer (50 mM HEPES (pH 7.9), 10 mM MgCl₂, 6 mM EGTA and 2.5 mM DTT) for 1 hour at 30°C. The reaction was supplemented with 7 mM EDTA to inactivate the kinase, followed by addition of PP2A or PP1 and incubation for 30 min at 30°C. Dephosphorylation of recombinant CDK9/cyclin T1 was carried out in Tris-HCl buffer pH 8.0, 5 mM MgCl₂, 5 mM MnCl₂, 20 μM ZnSO₄ using indicated amount of PP1 or PP2A. The phosphatases were inhibited with 1 μM okadaic acid. The 250 nM ATP and 5 μCi γ-(³²P)ATP were added in the same buffer and incubated for 30 min at 30°C. Reactions were resolved on 10% SDS-PAGE and subjected to autoradiography and quantification with PhosphorImager Storm 860 (Molecular Dynamics).

10 mg of phosphorylase-b was dissolved in 300 μl BFA (10 mM glycerophosphate pH 7.4, 50 mM 2-mercaptoethanol) and dialyzed against the same buffer for 2 to 3 hours. Then 7.5 μl 500 mM Tris-HCl (pH 8.0) and 6 μl phosphorylase kinase were added and incubated 10 min 30°C followed by addition of 45 μl ATP-Mg mix (8.3 mM ATP, 83 mM MgCl₂, 75 μCi γ-(³²P)ATP) and incubation for 2 h at 30°C. Phosphorylase-a was precipitated with ammonium sulfate, resuspended in BFA and dialyzed against BFA for 1 to 2 day at 4°C. AG 501 × 8 (mixed anion and cation exchange) resin was placed in a separate dialysis bag to improve removal of unincorporated ATP and inorganic phosphate. Dialyzed phosphorylase-a was kept at 4°C. Approximately 0.2 nmol of phosphorylase-a was used as a substrate for PP1 or PP2A. The phosphorylase phosphatase assay was carried out for 10 min in a buffer containing 50 mM glycylglycine at pH 7.4, 0.5 mM dithiothreitol, and 5 mM β-mercaptoethanol as described 27.

Biotin-TAR RNA (51 nucleotides) was purchased from Molecula company http://www.molecula.com. To bind TAR RNA, streptavidin-agarose beads were washed with binding buffer (20 mM Tris-HCl, pH 7.5, 2.5 mM MgCl₂, 100 mM NaCl) and incubated with TAR RNA (5 μg/reaction) or formamide denatured TAR RNA for 30 min at 4°C. The beads were washed with the binding buffer and incubated with recombinant Tat protein (1.5 μg/reaction) for 30 min at 4°C. Beads were washed with binding buffer and then with TAK buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl₂, 5 mM MnCl₂, 10 μM ZnSO₄, 1 mM DTT) containing 100 mM NaCl. Then 64 μg of yeast tRNA and 100 μg of BSA were added per reaction to block the beads. Then recombinant CDK9/cyclin T1 (30 ng/reaction) was added in TAK buffer containing 100 mM NaCl. Protein phosphatases PP1 and PP2A were diluted in TAK buffer and added into reaction where indicated at 0.1U of PP1 or 0.04U of PP2A. Samples were incubated for 1 h on ice with occasional mixing. Then beads were washed 3 times with the binding buffer, and bound proteins and RNA were eluted in 1× SDS-loading buffer. Proteins were separated on 12% SDS-PAGE, transfer to PVDF membrane, immunoblotted with α-CDK9 (Santa Cruz Biotechnology) and α-Tat 4A4.8 (NIH, AIDS Research Program) antibodies. Biotin TAR RNA was detected by Ponceau S staining.

Biotinylated HIV-1 LTR DNA template which included-111 to +308 nucleotides of JK2 (HIV-1 LTR nucleotides-111 to +82) was amplified by PCR with the forward primer 5' biotinylated-TTCTACAAGGGACTTTCCGC-3' and the reverse primer 5'-CAGTACAGGCAAAAAGCAGC-3' (Life Technologies, Rockville, MD). Transcription reactions (20 μl) contained 75 μg of HeLa nuclear extract, 0.5 mM of each ATP, CTP and GTP, 20 μM UTP, 2 μCi [α³²P]UTP, 0.2–0.4 μg of the template in transcription buffer (20 mM HEPES at pH 7.9, 50 mM KCl, 6.25 mM MgCl₂, 0.5 mM EDTA, 2 mM DTT and 10% glycerol), RNAsin and a recombinant pGEM2 Tat72 for transactivation reaction. 10 nM okadaic acid has been used for inhibition of serine/threonine phosphatases. After 30 min incubation at 30°C, reactions were treated with proteinase K for 15 min at 55°C, and extracted with phenol-chloroform-isoamylalcohol(LifeTechnologies, Rockville, MD). RNA was precipitated from the water phase and resolved on 5% sequencing polyacrylamide gels containing 7 M urea. Mcp 1 digested PBR 322 plasmid (LifeTechnologies, Rockville, MD) labeled with Klenow fragment and ³²P-labeled dCTD served as molecular weight markers. The labeling was quantified by phosphor imaging (Packard Instruments).

COS-7 cells were cultured at 7 × 10⁵ cells/well in DMEM containing 10% fetal bovine serum. Co-transfections with Tat-expressing vector and HIV-1 LTR-LacZ or HIV-1 LTRΔTAR were performed at 75% confluency using a Ca²⁺ – phosphate protocol and the indicated reporter plasmids. After transfection the cells were cultured for an additional 48 hours and β-galactosidase activity was analyzed using quantitative ONPG-based assay 26. Where indicated, okadaic acid was added to transfected cells. Transfections were normalized using MTT assay (Sigma).

Cells were washed with phosphate-buffered saline (PBS) and lysed for 20 min at room temperature in 50 μl of lysis buffer, containing 20 mM HEPES at pH 7.9, 0.1% NP-40 and 5 mM EDTA. Subsequently, 100 μl of o-nitrophenyl-β-D-galactopyranoside (ONPG) solution (72 mM Na₂ PO₄ at pH 7.5, 1 mg/ml ONPG, 12 mM MgCl₂, 180 mM 2-mercaptoethanol) was added and incubated at room temperature until a yellow color was developed. The reaction was stopped by addition of 100 μl of 1 M Na₂CO₃. The 96-well plate was analyzed in a micro plate reader at 414 nm (Lab Systems Multiscan MS).

HeLa or 293T cells were incubated with phosphate-free DMEM media (Life Technologies, Rockville, MD) containing no serum for 1 hour. The media was changed to phosphate-free DMEM media supplemented with 0.5 mCi/ml of (³²P)-orthophosphate and cells were further incubated for 2 hours at 37°C. Where indicated, 0.1 μM okadaic acid (Sigma) was added to block cellular PPP-phosphatases. Cells were washed with PBS and lyzed in a buffer containing 50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1% NP-40, 0.1% SDS and protease cocktail (Sigma). After 10 min on ice, cellular material was scraped and then centrifuged at 14,000 rpm, 4°C for 30 min. The supernatant was recovered and used for immunoprecipitation. CDK9 was co-precipitated with anti-cyclin T1 antibodies coupled to protein A agarose for 2 h at 4°C in a TNN Buffer containing 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, and 1% NP-40. The immunoprecipitated P-TEFb was recovered by heating for 2 min at 100°C in Tris-SDS loading buffer, resolved on 10% SDS-PAGE (25) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Allen, TX). The membrane was analyzed with anti-CDK9 polyclonal antibodies using 3,3'-Diaminobenzidine enhancer system (Sigma) and was also exposed to Phosphor Imager screen (Packard Instruments, Wellesley, MA).