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. 2009 Sep 10;86(6):1345–1350. doi: 10.1189/jlb.0509309

Characterization of Cdk9 T-loop phosphorylation in resting and activated CD4+ T lymphocytes

Rajesh Ramakrishnan 1, Eugene C Dow 1, Andrew P Rice 1,1
PMCID: PMC2780919  PMID: 19741158

Abstract

The cellular kinase complex P-TEFb is composed of Cdk9 and cyclin T, and it is required for expression of most protein-coding genes by RNAP II. Cdk9 has been shown recently to be activated in cis by autophosphorylation of Thr186 in its T-loop. Using a phosphospecific Cdk9 antibody, we examined the level of Cdk9 T-loop phosphorylation in resting and activated CD4+ T lymphocytes. Cdk9 T-loop phosphorylation was found to be low-to-undetectable in resting CD4+ T lymphocytes, and upon activation by distinct stimuli, there is a rapid (<1 h) increase in pCdk9 that does not require protein synthesis. The low level of Cdk9 T-loop phosphorylation was not to be a result of the absence of an associated regulatory cyclin partner. These observations suggest that autophosphorylation of the Cdk9 T-loop is repressed in resting CD4+ T lymphocytes. The low level of T-loop phosphorylation in resting cells is also reflected in a low level of phosphorylation of Ser2 in the carboxyl terminal domain of RNAP II, suggesting that lack of Cdk9 T-loop autophosphorylation may limit RNAP II elongation in quiescent CD4+ T lymphocytes.

Keywords: P-TEFb, RNA polymerase II elongation, Cdk9, T lymphocyte activation

Introduction

RNAP II is a critical enzyme to the function of CD4+ T lymphocytes that functions to execute a program of transcriptional induction of hundreds of genes when resting cells are activated. By analogy to viral gene expression patterns, these genes have been roughly classified as immediate-early, early, and late genes based on whether they are independent of de novo protein synthesis, require protein synthesis but precede cell division, or are induced after cell division, respectively [1].

Transcription by RNAP II involves initiation and elongation to generate full-length transcripts, and both steps are carefully regulated. After initiation, RNAP II encounters blocks to elongation as a result of pausing or inefficient processivity. These blocks may function as a quality control to ensure capping of nascent mRNAs, thereby stabilizing the RNA for downstream processing events [2]. Additionally, these barriers might be important for genes requiring a rapid response to induction, such as heat shock proteins [2]. In fact, a recent study revealed that although most protein-coding genes undergo transcriptional initiation by RNAP II, only a subset produces full-length transcripts, indicating that many genes are severely defective in transcriptional elongation [3]. The block to elongation can be overcome by P-TEFb [4], which is a protein kinase that stimulates productive elongation of transcripts by hyperphosphorylating the CTD of RNAP II, phosphorylating the Spt5 subunit of 5,6-dichloro-1-β-D-ribofurano sylbenzimidazole sensitivity-inducing factor and the RD subunit of negative elongation factor [4]. The CTD of human RNAP II consists of 52 repeats of the heptapeptide sequence YSPTSPS, and a hallmark of P-TEFb kinase activity is the phosphorylation of Ser2 in these sequences [4].

The catalytic subunit of P-TEFb is Cdk9, a serine-threonine kinase with two isoforms: a major 42 kD and a minor 55 kD, which are differentially localized and regulated [5]. The regulatory subunit of P-TEFb is a cyclin protein that is cyclin T1, T2, or K. In cell types examined to date, cyclin T1 appears to be the major cyclin partner for P-TEFb. Cyclin T1 has been studied extensively, as the HIV-1 Tat protein targets cyclin T1 through a direct protein–protein interaction to activate proviral transcription, and this is essential for viral replication [6]. Additionally, P-TEFb function appears to be limiting for viral gene expression in long-lived memory CD4+ T lymphocytes that harbor latent HIV-1 provirus and are resistant to antiviral drugs [7].

About 50% of P-TEFb is found in a catalytically repressed snRNA complex, termed the 7SK snRNP complex, which contains 7SK snRNA, HEXIM1/2, LARP7, [8], and PIP7S [9] proteins. P-TEFb function is regulated in resting and activated CD4+ T lymphocytes. Cyclin T1 and to some extent, Cdk9 protein levels are induced when resting CD4+ T lymphocytes are activated with any of a number of stimuli: α-CD3 + α-CD28, PHA, prostratin, PMA + ionomycin, and a combination of the cytokines IL-2, IL-6, and TNF-α [10,11,12]. However, this induction occurs with relatively slow kinetics and is first observed at ∼14 h after T cell activation.

Given that the transcription of immediate-early genes in activated CD4+ T lymphocytes is likely dependent on P-TEFb, we were interested in examining if very early events after T cell activation were capable of up-regulating P-TEFb function. A potential site of regulation is that of the activation segment or T-loop of Cdk9, which is subject to multiple post-translational modifications [13, 14], the most critical being phosphorylation of Thr186, located at the tip of a flexible loop (T-loop) [15]. The phosphorylation of this threonine residue is the hallmark of activated Cdks, and it induces a major conformational change of the T-loop, allowing entry of the substrate and ATP into the kinase catalytic pocket [16]. The phosphorylation of Thr186 in Cdk9 is important, not only for its activation but also for its reversible association with the 7SK snRNP [15]. This association is another level of regulation that may keep active P-TEFb sequestered yet ready to function rapidly when the physiological demand of cells requires increased RNAP II transcription. It has been reported recently that Cdk9 is autophosphorylated in cis at Thr186 [17]. This mode of autoactivation of kinases is a common mechanism, as exemplified by glycogen synthase kinase-3 and p38α [18]. This autoactivation of Cdk9 raises interesting questions about the regulatory circuit for Cdk9 T-loop phosphorylation in resting and activated cells.

In this study, we found that the level of Cdk9 T-loop phosphorylation is very low in resting CD4+ T lymphocytes and is induced within 15–30 min after cells are activated by a process that does not require protein synthesis. The data indicate further that the low level of phosphorylation of the Cdk9 T-loop is not a result of the absence of an associated cyclin regulatory partner. We also found that increased phosphorylation of Ser2 in the CTD of RNAP II in activated lymphocytes correlates with the induction of T-loop phosphorylation, suggesting that repression of Cdk9 T-loop autophosphorylation in quiescent lymphocytes contributes to low levels of transcriptional elongation. This is the first report to demonstrate that Cdk9 T-loop phosphorylation is regulated in CD4+ T lymphocytes, and this regulation has implications for lymphocyte function, inflammatory diseases, and HIV-1 latent infection.

MATERIALS AND METHODS

Resting CD4+ T lymphocytes were isolated from healthy blood donors using the MACS CD4+ cell isolation kit II (Miltenyi Biotec, Germany) and anti-CD30 microbeads to deplete activated cells. Purity of CD4+ T lymphocytes and resting CD4+ T lymphocytes was determined by flow cytometry using a Beckman-Coulter XL-MCL cytometer with FITC-CD4, PE-CD3, PE-CD69, and FITC-CD25 antibodies. The resting status of CD4+ T lymphocytes was also confirmed by PI staining for DNA content cell cycle. For activation, resting CD4+ T lymphocytes were adjusted to 10 × 106 cells/ml in RPMI (Invitrogen, Carlsbad, CA, USA) with 10% FBS and 1% penicillin-streptomycin and activated with PHA (5 ug/ml), PMA (1 ng/ml), ionomycin (1 uM), PMA + ionomycin, or Dynabeads CD3/CD28 T cell expander (Invitrogen) at a 1:1 bead:cell ratio.

For immunoblots, cells were collected at time-points after activation, washed with PBS, and lysed with 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% (vol/vol) Nonidet P-40, 5 mM DTT, 4 mM MgCl2 buffer containing protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO, USA) and phosphatase inhibitor cocktail (Sigma Chemical Co.). A total of 50 ug protein was separated on 12% SDS-PAGE. Immunoblotting was performed using ECL Western blotting substrate (Pierce, Rockford, IL, USA) for detection. pCdk9, Cdk9, cyclin T1, cyclin T2a, Cdk7, and β-actin were probed as described earlier [10]. In some experiments, blots were blocked with 5% PhosphoBLOCKER blocking reagent (Cell Biolabs, San Diego, CA, USA), and in these experiments, primary (pCdk9) and secondary antibodies were diluted in 5% PhosphoBLOCKER blocking reagent. The RNAP II CTD, RNAP II CTD pSer2, and RNAP II CTD pSer5 were probed as described previously [19]. The blots were blocked in TBST with 3% w/v BSA. Antibody against pCdk9 (Cell Signaling, Beverly, MA, USA), RNAP II CTD (8WG16), pSer2 (H5), and pSer5 (H14; Covance Labs, Vienna, VA, USA) was used at 1:500, and antibodies against Cdk9, cyclin T1, cyclin T2a, Cdk7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (Sigma Chemical Co.) were used at 1:1000 dilutions. Immunoblots were quantified using ImageQuant software in a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA, USA).

For immunoprecipitations, lysates were precleared with True Blot anti-rabbit IgG beads (eBioScience, San Diego, CA, USA) and incubated with 5 μg anti-Cdk9 antibody (Santa Cruz Biotechnology) or control IgG antiserum for 2 h at 4°C. Samples were combined with 50 μl anti-rabbit IgG beads and incubated overnight on a rocking platform at 4°C. Beads were washed three times with 500 μl lysis buffer and then boiled in 30 μl Laemmli buffer (with 50 mM DTT). Proteins were resolved on 12% SDS-PAGE gels and immunoblotted as described above.

For immunofluorescence, CD4+ T lymphocytes were cytospun onto glass coverslips and fixed as described previously [20]. The cells were processed for immunofluorescence as described previously [21] using pCdk9 (1:50) or Cdk9 (1:500) antibodies for primary incubation. Cells were counterstained with DAPI, and deconvolution microscopy was conducted as described [21]. Images were then deconvolved, and representative single z-sections from each series were prepared for presentation using Adobe Photoshop (San Jose, CA, USA).

RESULTS

Cdk9 T-loop phosphorylation is induced rapidly in activated CD4+ T lymphocytes

To investigate regulation of P-TEFb function by phosphorylation of the Cdk9 T-loop in CD4+ T lymphocytes, we isolated resting CD4+ T lymphocytes from healthy blood donors by a negative selection method. Purity and resting status of CD4+ T lymphocyte preparations was found to be routinely >98%, as determined by flow cytometry using FITC-CD4 and PE-CD3 antibodies and PE-CD69 and FITC-CD25, respectively (data not shown). The resting status of CD4+ T lymphocytes was also confirmed by PI staining for DNA content, and >95% cells were found to be in the G0/G1 phase (data not shown).

In our first set of experiments, cells were activated with PHA, and cell extracts were prepared for immunoblot analysis. Phosphorylation of Thr186 in the T-loop of the major 42-kDa Cdk9 isoform was examined with a phosphorylated T-loop-specific antiserum. The specificity of this newly developed antiserum for recognizing phosphorylated Thr186 in Cdk9 has been described previously [22]. In resting CD4+ T lymphocytes, phosphorylation of the Cdk9 T-loop was low-to-undetectable (Fig. 1). In Donor 3, PHA activation induced a high level of Cdk9 T-loop phosphorylation within 1 h (Fig. 1A). In Donor 12, a significant increase in T-loop phosphorylation could be observed at 30 min postactivation. The total level of 42 kDa Cdk9 protein did not change in the relatively short time course of activation examined in Figure 1. In Donor 5 and additional donors, a significant induction of Cdk9 T-loop phosphorylation could be observed as early as 15–30 min after PHA activation (Fig. 1B, and data not shown). Phosphorylation of the T-loop of the 55-kDa minor isoform of Cdk9 was usually below the level of detection. However, in immunoblots in which it was detectable, its induction by T-lymphocyte activation paralleled that of the major Cdk9 42-kDa isoform (data not shown).

Figure 1.

Figure 1.

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Cdk9 T-loop phosphorylation is induced rapidly in activated CD4+ T lymphocytes.(A) Resting CD4+T lymphocytes (0) were activated with 5 ug/mL PHA, and cell extracts were prepared at the indicated time-points. The level of 42 kDa Cdk9 T-loop phosphorylation (p-Cdk9), total 42 kDa Cdk9, and β-actin (loading control) was measured in immunoblots. (B) Resting CD4+ T lymphocytes (0) were treated with and without cycloheximide (CHX; 200 μg/mL) for 30 min and activated with PHA (5 ug/mL) as indicated. Cell lysates were prepared and analyzed in immunoblots as described in A above.

To ascertain if protein synthesis is required for the rapid induction of Cdk9 T-loop phosphorylation, resting CD4+ T lymphocytes were treated with cycloheximide for 30 min and activated with PHA for 1 h. Immunoblot analysis revealed that inhibition of translation by cycloheximide did not affect Cdk9 T-loop phosphorylation (Fig. 1B). Metabolic labeling with 35S-methionine in the experiment shown in Figure 1B confirmed the inhibition of translation by cycloheximide (data not shown). We conclude from these data that de novo protein synthesis is not required for Cdk9 T-loop phosphorylation in CD4+ T lymphocytes. It has been reported recently that Cdk9 T-loop phosphorylation is autocatalytic [17], and this is consistent with our data. However, this autocatalytic phosphorylation appears to be repressed in resting CD4+ T lymphocytes.

Additive induction of Cdk9 T-loop phosphorylation in resting CD4+ T lymphocytes activated with PMA and ionomycin

We next investigated other T lymphocyte activation stimuli for effects on Cdk9 T-loop phosphorylation. Resting CD4+ T lymphocytes were activated with the phorbol ester PMA, the calcium ionophore ionomycin, or the combination of PMA+ ionomycin. Cell lysates were prepared at time-points after activation, and immunoblots were performed to measure levels of Cdk9 T-loop phosphorylation and total Cdk9 (Fig. 2). After normalization to the control protein Cdk7, there was an approximate tenfold induction of the T-loop-phosphorylated Cdk9 by PMA or ionomycin alone at 1.5 h. However, the combination of PMA + ionomycin induced Cdk9 T-loop phosphorylation 33-fold at 1.5 h, indicating an additive effect of the two activation stimuli.

Figure 2.

Figure 2.

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PMA and ionomycin show additive effects on Cdk9 T-loop phosphorylation in activated CD4+ T lymphocytes. Resting CD4+ T lymphocytes (0) were activated with PMA, ionomycin, or PMA + ionomycin. Cell lysates were prepared at the indicated time-points, and Cdk9 T-loop phosphorylation, total Cdk9, and Cdk7 were examined in immunoblots. After normalization to Cdk7, quantification of the immunoblot showed an induction of approximately tenfold in Cdk9 T-loop phosphorylation when cells were activated with PMA or ionomycin alone at 1.5 h. PMA + ionomycin induced T-loop phosphorylation 33-fold at 1.5 h.

Immunofluorescence analysis of Cdk9 T-loop phosphorylation in resting and activated CD4+ T lymphocytes

We also investigated the induction of Cdk9 T-loop phosphorylation by the independent technique of immunofluorescence. Resting CD4+ T lymphocytes were activated with PMA + ionomycin for 1 h and examined by immunofluorescence. Resting CD4+ T lymphocytes had a low level of pCdk9 (Fig. 3A, upper panel), although there was a relatively high level of total Cdk9 protein expression (Fig. 3B, upper panel). The induction of Cdk9 T-loop phosphorylation in activated CD4+ T lymphocytes as measured by immunofluorescence is in good agreement with the immunoblot analyses shown in Figures 1 and 2.

Figure 3.

Figure 3.

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Immunofluorescence analysis of Cdk9 Thr186 phosphorylation induction in resting and activated CD4+ T lymphocytes. Resting and PMA + ionomycin-activated (1 h) CD4+ T lymphocytes were cytospun onto glass coverslips, fixed, and stained with (A) pCdk9 (Thr186) or (B) Cdk9 antisera. Rectangular boxes indicate the areas that are enlarged (5×) in the panels displayed in the right column. Original scale bars are 5 μM.

Cdk9 T-loop and RNAP II CTD phosphorylation in resting CD4+ T lymphocytes activated with anti-CD3/CD28

In a physiological setting, T lymphocyte activation is brought about by simultaneous engagement of the TCR/CD3 complex and the CD28R on the T cell membrane by an APC. We therefore examined the kinetics of Cdk9 T-loop phosphorylation by activating resting CD4+ T lymphocytes with CD3/CD28-coated beads that mimic the biologically relevant dual signal process in vitro. In Donor 35, activation of resting CD4+ T lymphocytes with CD3/CD28 strongly induced Cdk9 T-loop phosphorylation in 2 h (Fig. 4A). For Donor 35, we also observed an induction in the level of cyclin T1, cyclin T2a, and HEXIM1 at 2 h. The level of total Cdk9 that was expressed at a high level was induced only at 48 h postactivation in agreement with our previous findings [10]. In Donor 37, activation of resting CD4+ T lymphocytes with CD3/CD28 for 2 h showed an induction in the levels of Cdk9 T-loop phosphorylation and cyclin T1 (Fig. 4A). Unlike Donor 35, however, cyclin T2a was not induced in Donor 37. We conclude from these data (Figs. 1234) that the level of Cdk9 T-loop phosphorylation is low in resting CD4+ T lymphocytes and is induced rapidly with similar kinetics by a variety of activation stimuli.

Figure 4.

Figure 4.

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Cdk9 T-loop and RNAP II CTD phosphorylation is induced rapidly in activated CD4+ T lymphocytes in response to activation by anti-CD3/CD28 signals. (A) Resting CD4+ T lymphocytes (0 h) were activated with Dynabeads CD3/CD28 T cell expander at a 1:1 ratio of bead:cell, and cell extracts were prepared at the indicated time-points. The level of 42 kDa Cdk9 T-loop phosphorylation, total 42 kDa Cdk9, cyclin T1, cyclin T2a, HEXIM1, and β-actin (loading control) was measured in immunoblots. (B) The level of total RNAP II CTD, pSer2, and pSer5 was measured in immunoblots for Donor 37 described in A. Following normalization to RNAP II CTD, quantification of the immunoblot showed an induction of approximately twofold in the pSer2 and pSer5 residues after activation of resting CD4+ T lymphocytes.

Phosphorylation of the CTD of the largest RNAP II subunit is intimately linked to transcriptional initiation and elongation. Cdk7, a component of the general transcription factor TFIIH, is largely responsible for pSer5 in the CTD, and this modification is believed to be involved in promoter clearance [23]. Cdk9, in complex with its cyclin partner, is responsible for pSer2 in the CTD, and this is involved in transcriptional elongation [6]. We investigated if the low level of Cdk9 T-loop phosphorylation in resting CD4+ T lymphocytes would be reflected in a low level of pSer2. The levels of total CTD and pSer5 and pSer2 CTD were examined in whole cell lysates from Donor 37 (Fig. 4B). After normalization to total CTD levels, pSer5 and pSer2 were found to be induced approximately twofold in activated cells relative to levels in resting cells. These data suggest that Cdk7 activity for Ser5 in the CTD is increased following T cell activation. Cdk9 activity for Ser2 in the CTD is also increased following activation, and this increase is correlated with an increase in Cdk9 T-loop phosphorylation. The enhanced pSer2 of the RNAP II CTD seen after 2 h of T cell activation is likely to reflect productive elongation of the immediate-early genes.

Repression of Cdk9 T-loop phosphorylation in resting CD4+ T lymphocytes is not a result of absence of an associated regulatory cyclin partner

The catalytic activity of Cdk9 is dependent on its association with a regulatory cyclin partner [4]. Based on our observations of the low level of Cdk9 T-loop phosphorylation in resting CD4+ T lymphocytes, we investigated if the potential repression of T-loop phosphorylation might be a result of the absence of a regulatory cyclin subunit. Also, as much of P-TEFb is in the 7SK snRNP, we examined the association between Cdk9 and cyclins T1 and T2a and HEXIM1 during this short time course of T cell activation. Cdk9 was immunoprecipitated from extracts of resting and activated CD4+ T lymphocytes, and association of cyclin T1, T2a, and HEXIM1 was evaluated in immunoblots. Similar levels of cyclin T1 and cyclin T2a were observed in Cdk9 immunoprecipitates from Donor 26 (Fig. 5). No Cdk9 was precipitated from extracts for Donor 36 with a control IgG antiserum, demonstrating the specificity of immunoprecipitates with the anti-Cdk9 antiserum. In Donor 36, an approximately equal amount of cyclin T1 was found to associate with Cdk9 in resting and anti-CD3/CD28-activated CD4+ T lymphocytes. This result indicates that the repression of Cdk9 T-loop phosphorylation in resting CD4+ T lymphocytes cannot be explained by the absence of a cyclin regulatory subunit. Additionally, a similar amount of HEXIM1 was observed in Cdk9 immunoprecipitates from resting and activated cells in Donor 26. This contrasts with previous results, where an increased level of HEXIM1 and Cdk9 association was seen when resting CD4 + T lymphocytes were activated for 48 h with prostratin [12].

Figure 5.

Figure 5.

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Low level of Cdk9 T-loop phosphorylation in resting CD4+ T lymphocytes is not a result of the absence of an associated cyclin partner. Resting CD4+ T lymphocytes were activated with 5 ug/mL PHA (Donor 26) or CD3/CD28 beads (Donor 36) for 2 h. Cell lysates were prepared from resting and activated CD4+ T lymphocytes. Immunoblots (IB) performed on whole cell lysates (Input) show the levels of pCdk9, total Cdk9, cyclin T1, cyclin T2a, HEXIM1, and β-actin (loading control). Immunoprecipitations (IP) were performed with 200 ug cell lysates using control IgG or Cdk9 antibody. Immunoblots were performed with a portion of the immunoprecipitates to confirm the presence of equivalent amounts of Cdk9 and for the levels of associated cyclin T1, cyclin T2a, and HEXIM1. The arrow points to the location of the specific cyclin T1 protein band.

We found here that Cdk9 T-loop phosphorylation is low in resting cells but can be induced within 15 min after activation without affecting overall Cdk9 protein levels, and this induction does not require protein synthesis. We also observed that Cdk9 is associated with a cyclin partner in resting cells, and this level of cyclin association does not change in 2 h after T lymphocyte activation. These observations suggest that repression of Cdk9 autophosphorylation regulates T-loop phosphorylation in resting CD4+ T lymphocytes.

DISCUSSION

What mechanisms might be involved in the repression of Cdk9 T-loop autophosphorylation in resting CD4+ T lymphocytes? Many EF-hand proteins such as calmodulin undergo a large, conformational change upon binding to Ca2+ following a calcium flux, and this affects the ability to interact with target proteins, resulting in activation or inactivation of these target proteins [24, 25]. For instance, Thr200 in calmodulin kinase IV must be phosphorylated for maximal activity, and this residue is masked in the full-length enzyme and is exposed for phosphorylation only upon binding to Ca2+/calmodulin [26]. Similarly, it is possible that different ionic concentrations in resting versus activated lymphocytes could affect the conformation of Cdk9, influencing T-loop autophosphorylation and thereby, its function.

Phosphatases play an important role in regulating kinase function. We reported recently that PPM1A and PPM1B negatively regulate Cdk9 T-loop phosphorylation under nonstress conditions in HeLa cells [22]. Another study reported that PP1α and PP2B are negative regulators of Cdk9 T-loop phosphorylation in HeLa cells in response to Ca2+ signaling and cellular stress by UV irradiation or high concentrations of hexamethylene bisacetamide [27]. The role of phosphatases in regulating Cdk9 T-loop phosphorylation in resting and activated CD4+ T lymphocytes warrants further investigation. Alternatively, it is possible that a repressor associates with Cdk9 in resting cells and inhibits T-loop autophosphorylation. A scenario in which an as-yet unidentified, activating kinase plays a role in relieving the repression of Cdk9 T-loop phosphorylation in resting cells cannot be ruled out.

P-TEFb is an essential host cofactor for HIV-1 replication, and CD4+ memory T lymphocytes are the major source of latently infected HIV-1 that preclude a cure of infection by existing antiviral drugs. The mechanisms involved in the establishment of latent infection are not well understood. In infected resting CD4+ T lymphocytes, latency appears to be regulated at the level of viral gene expression, suggesting that P-TEFb function is limiting [7]. It is likely that the level of Cdk9 T-loop phosphorylation is low in CD4+ T cells harboring latent HIV-1 provirus. Induction of Cdk9 T-loop phosphorylation could potentially activate the provirus in these cells. It is also conceivable that many chronic inflammatory diseases involve the dysregulation of P-TEFb, resulting in elevated transcription and subsequent overexpression of cytokines and other proteins. For example, a recent report found that patients showing the earliest clinical manifestation of multiple sclerosis had impaired regulation of CD4+ T lymphocyte quiescence [28]. Thus, it is possible that dysregulation of Cdk9 T-loop phosphorylation and its effects on T cell quiescence might contribute to inflammatory diseases. Future experiments can be directed toward investigating mechanisms that repress Cdk9 T-loop autophosphorylation in resting T lymphocytes, and this has implications for the pathology of inflammatory diseases and HIV-1 latent infection.

AUTHORSHIP

R. R. and E. C. D. performed experiments and analyzed data; R. R. and A. P. R. designed research and analyzed data; A. P. R. conceived the study; and R. R. and A. P. R. wrote the paper.

ACKNOWLEDGMENT

This work was supported by National Institutes of Health grant AI35381 to A. P. R.

DISCLOSURE

The authors declare no competing financial interests.

Footnotes

Abbreviations: Cdk9=cyclin-dependent kinase 9, CTD=carboxyl terminal domain, DAPI=4′,6-diamidino-2-phenylindole, HEXIM1=hexamethylene bis-acetamide-inducible 1, P-TEFb=positive transcription elongation factor-b, pCdk9=phosphorylated Cdk9, PI=propidium iodide, PP1=protein phosphatase 1, pSer2=phosphoserine 2, RNAP II=RNA polymerase II, Ser2=serine 2, snRNA/RNP=small nuclear RNA/ribonucleoprotein, Thr186=threonine 186

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