In addition to the transcription initiation factors, P-TEFb is tightly regulated in latently infected T cells (Fig.
). First, the expression of hCycT1 in resting CD4+
T cells is normally highly restricted. hCycT1 rapidly rise (within 1 h) upon activation of these cells with cytokines, protein kinase C agonists or through the T cell receptor [152
]. Nuclear factor 90 (NF90), a cellular RNA binding protein, has been recently reported to be an essential factor required for cyclin T1 translation initiation which is upregulated in activated T-cells [157
]. Similarly, hCycT1 expression can barely be detected in undifferentiated monocytes due to a microRNA miR-198 that represses hCycT1 protein synthesis [158
]. Monocyte differentiation into macrophages downregulates miR-198, thereby, permitting hCycT1 expression [158
Fig. (4) Model for the activation of P-TEFb in resting CD4+ T cells. In resting CD4+ T cells the majority of the P-TEFb in cells there are
only low levels of CycT1. Induction of the cells leads to new CycT1 synthesis and the assembly of the transcriptionally inactive (more ...)
There are two isoforms of CDK9 (CDK942
differs structurally from CDK942
by harboring an N-terminal 117-residue extension and is synthesized by a promoter that is upstream of the promoter for CDK942
]. Both CDK942
appear to be expressed at similar levels in human peripheral blood lymphocytes [161
] and both isoforms possess comparable kinase activity toward the C-terminal domain of the large subunit of RNAP II and can interact with Tat [160
]. While the basal expression of CDK942
is significantly elevated after ~1-2 days exposure to activation signals there is a reciprocal decrease in the levels of CDK955
Recent X-ray structures of the P-TEFb heterodimer (CDK9/hCycT1) and the Tat tri-molecular complex (CDK9/hCyT1/Tat) [64
] have now defined the interaction interfaces between each of the P-TEFb subunits and Tat (Fig.
). The association between CDK9 and hCycT1 triggers the phosphorylation of CDK9 at Thr-186 located within the flexible activation loop (T-loop) of the enzyme, which in turn induces its kinase activity [162
]. Tat binds to the phosphorylated P-TEFb and adopts a partially helical secondary structure after forming Zn-coordinated interactions with hCycT1. The Tat/P-TEFb X-ray structure also revealed that the N-terminal region of Tat forms two intermolecular hydrogen bonds with the T-loop region of CDK9 [64
Fig. (5) The CDK9 “T-loop” forms a critical part of the
interface between CDK9, CycT1 and Tat. In this structural model
based on Tahirov et al.  the CDK9 backbone is shown in red,
Tat is shown in purple and CycT1 is shown in green. The CDK9 (more ...)
T-loop phosphorylation of CDK9 is strictly required for P-TEFb molecules to be held within the 7SK snRNP complex; point mutations of Thr-186 that prevent T-loop phosphorylation (both T186A and the phospho-mimetic T186D) inhibit P-TEFb from associating with 7SK snRNA, HEXIM1, and LARP7 [163
]. It is not entirely clear how CDK9/hCycT1 heterodimerization triggers T-loop phosphorylation of CDK9. Baumli et al
] have identified Thr-186 to be a potential autophosphorylation site based on mass spectrometry analysis of in vitro
phosphorylated CDK9. However, a catalytically inactive D167N CDK9 mutant will not only heterodimerize with hCycT1 as efficiently as wild-type, but can also become incorporated into the 7SK snRNP complex [163
]. Additionally, in vitro
kinase and 7SK snRNP reconstitution assays performed by Zhou and colleagues using phosphatase-treated recombinant CDK9 as substrate and HeLa nuclear extracts as the source of kinase activity suggested that there is a novel, still unidentified, nuclear kinase that mediates Thr-186 phosphorylation [165
]. It seems likely that both autophosphorylation and external kinases are used to control Thr-186 phosphorylation.
Basal T-loop phosphorylation of CDK9 is extremely low in resting CD4+
], and this further restricts P-TEFb activity. Concomitant to hCycT1 induction, T-loop phosphorylation is significantly elevated upon brief (within 1 h) stimulation of these cells through the T-cell receptor. By contrast total cellular CDK9 levels are unchanged under these conditions [154
Transcriptional activity of P-TEFb in peripheral CD4 T cells is further controlled by its sequestration into the 7SK snRNP complex [166
]. Within this complex 7SK snRNA interacts directly with P-TEFb and its inhibitory protein HEXIM1 and acts as a scaffold to hold the complex together [168
]. This nuclear regulatory complex also comprises the 7SK snRNA 5’ capping enzyme MEPCE, and LARP7 which binds to the 3’ uridine-rich end of 7SK snRNA and protects it from degradation by nucleases [178
]. Although P-TEFb molecules that are held within 7SK snRNP are T-loop phosphorylated and therefore catalytically active [163
], they are transcriptionally inactive because they are physically unable to be accessed by genes. Nevertheless, P-TEFb-containing 7SK snRNP has been found to be conveniently localized in nuclear speckles in very close proximity to C-terminal hyperphosphorylated RNAP II which would be considered to be a marker of active transcription [152
]. A molecular signal or cue that would trigger disassembly of 7SK snRNP and release of P-TEFb is also likely to enable its recruitment to active genes. Therefore, the activation of P-TEFb in resting memory CD4 T cells may require multiple steps involving reversing the restriction on hCycT1 expression, P-TEFb complex formation and T-loop phosphorylation of CDK9, the initial assembly of P-TEFb molecules into 7SK snRNP, relocalization of the inactive complex into nuclear speckles, and the mobilization of active P-TEFb toward transcriptionally active genes (Fig.
The molecular mechanisms leading to the disassembly of 7SK snRNP and mobilization of P-TEFb toward active genes are not well understood. The bromodomain-containing protein Brd4 is able to remove P-TEFb from the 7SK complex and by virtue of its high affinity interaction with acetylated chromatin, can recruit P-TEFb to active cellular genes [164
]. Similarly, Tat has also been shown to directly mobilize P-TEFb from 7SK snRNP by outcompeting and physically displacing HEXIM1 from hCycT1 binding [174
]. These findings have led to a model that proposes that Tat and Brd4 extract P-TEFb from the inactive 7SK snRNP complex in a mutually exclusive manner.
It seems likely that post-translational modifications of components of the P-TEFb machinery mediate these mobilization events. Recently, we found that T-cell receptor (TCR) signaling in primary CD4 T lymphocytes and Jurkat T cells results in the immediate activation of transcription elongation from latent HIV proviruses, even at times when Tat levels are too low to sustain transcription elongation [35
]. This early increase in elongation is due to the activation of P-TEFb by the disruption of 7SK snRNP through the ERK pathway. Natarajan et al.
also reported that TCR signaling can enhance HIV transcription by stimulating P-TEFb dissociation from the RNP complex [183
In addition to this physiological pathway a variety of chemical inducers of HIV transcription appear to act by disrupting pTEFb. Zhou and colleagues [165
] have reported that treatment of HeLa cells with UV irradiation or the drug HMBA induced 7SK snRNP complex disruption through the activation of two phosphatase enzymes via
calcium signaling. According to their model, PP2B dephosphorylates HEXIM1 triggering a conformational change within the RNP that exposes the T-loop phosphate of Cdk9 to PP1α. Consequently, this sequential phosphatase activity disassembles the RNP complex. Finally Ott and colleagues [184
] have found that the acetylation of hCycT1 by p300 in HeLa cells induces P-TEFb release from 7SK snRNP.
In summary, the data support the model for P-TEFb activation in resting T cells involving both assembly of the 7SK snRNP complex and the subsequent mobilization of P-TEFb by cellular signaling and Tat (Fig. ). In the resting T cell there is relatively little P-TEFb assembled into active complexes or the 7SK snRNP complex, primarily because hCycT1 levels are limiting. After activation of the cells, hCycT1 rapidly rise due to new hCycT1 synthesis. The newly produced hCycT1 associates with pre-existing CDK9 and stimulates autophosphorylation of the T-loop and assembly into the 7SK RNP complex. If signaling is sustained, post-translation modifications direct a fraction of the P-TEFb away from the 7SK snRNP complex where it can then bind to Brd4, or if HIV in present, to Tat, and stimulate transcription.