Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation

Abstract

The HIV trans-activator Tat recruits the host transcription elongation factor P-TEFb to stimulate proviral transcription. Phosphorylation of Thr186 on the activation loop (T-loop) of cyclin-dependent kinase 9 (CDK9) is essential for its kinase activity and assembly of CDK9 and cyclin T1 (CycT1) to form functional P-TEFb. Phosphorylation of a second highly conserved Tloop site, Ser175, alters the competitive binding of Tat and the host recruitment factor bromodomain containing 4 (BRD4) to P-TEFb. Here, we investigated the intracellular mechanisms that regulate these key phosphorylation events required for HIV transcription. Molecular dynamics simulations revealed that the CDK9/CycT1 interface is stabilized by intramolecular hydrogen bonding of pThr186 by an arginine triad and Glu96 of CycT1. Arginine triad substitutions that disrupted CDK9/CycT1 assembly accumulated Thr186-dephosphorylated CDK9 associated with the cytoplasmic Hsp90/Cdc37 chaperone. The Hsp90/Cdc37/CDK9 complex was also present in resting T cells, which lack CycT1. Hsp90 inhibition in primary T cells blocked P-TEFb assembly, disrupted Thr186 phosphorylation, and suppressed proviral reactivation. The selective CDK7 inhibitor THZ1 blocked CDK9 phosphorylation at Ser175, and in vitro kinase assays confirmed that CDK7 activity is principally responsible for Ser175 phosphorylation. Mutation of Ser175 to Lys had no effect on CDK9 kinase activity or P-TEFb assembly but strongly suppressed both HIV expression and BRD4 binding. We conclude that the transfer of CDK9 from the Hsp90/Cdc37 complex induced by Thr186 phosphorylation is a key step in P-TEFb biogenesis. Furthermore, we demonstrate that CDK7-mediated Ser175 phosphorylation is a downstream nuclear event essential for facilitating CDK9 T-loop interactions with Tat.http://www.jbc.org/cgi

CDK9: a signaling hub for transcriptional control

“…As any other kinase, CDK9 contains an ATP binding site (Figure 1(b), green) and an activating T-loop containing the key T186 residue ( Figure 1(b), red). CDK9 activity and CycT interaction are governed by in cis auto-phosphorylation of T186 and three C-terminal phosphorylation sites [18,[27][28][29][30][31].…”

“…While T-loop phosphorylation at S175 was reported to increase under signal-regulated conditions (T-cell activation) to potentially control CDK9 interaction with other transcription elongation complexes (see CDK9 Delivery to Genome Regulatory Regions section below), this modification does not impact kinase activity [30]. In addition to evidence for in cis auto-phosphorylation, both T186 and S175 can be phosphorylated by other kinases, such as CDK7 in basal conditions [18].…”

“…In addition to evidence for in cis auto-phosphorylation, both T186 and S175 can be phosphorylated by other kinases, such as CDK7 in basal conditions [18]. However, it remains unclear whether these molecular events are important for global or pathway-specific transcription in basal-and/or signal-regulated conditions in key biological contexts [18,27,29,30].…”

“…While 7SK RNA is predominantly, if not exclusively, nuclear, this data is derived from RNA fluorescence in situ hybridization in which the probes used could, potentially, only detect the nuclear fraction of 7SK RNA and 7SK snRNP complex due to probe accessibility [42]. Similarly, various studies using immunofluorescence to detect the protein components of the snRNP have provided conflictive results (nuclear vs nuclear-cytoplasmic) potentially due to antibodies recognizing different 7SK component forms [30,[41][42][43]. Thus, rigorous studies examining the localization of the complex (and not its components in isolation) would be required to precisely address this conundrum and to further evaluate functional roles for the nuclear and cytoplasmic 7SK snRNP pools.…”

Blockade of CDK7 Reverses Endocrine Therapy Resistance in Breast Cancer

“…Thus, it is speculated that CDK7 inhibitor such as THZ1 inhibit cell proliferation and survival through multiple mechanisms. In this regard, the CAK-inhibitory activity of THZ1 [33] was associated with diminished phosphorylation/activation of CDK9, as well as CDK1 and 2 and arrest cell cycle progression in S-phase as well as G2/M arrest [32]. Additionally, tamoxifen-induced G1 arrest in both MCF-7 and LCC2 cells is similar to the findings of a previous study in which the percentage of cells at G0/G1 phase was markedly increased and the percentage of cells at S and G2/M phases were decreased, respectively [34].…”