Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation
Year: 2018
Type: article
Abstract: The HIV trans-activator Tat recruits the host transcription elongation factor P-TEFb to stimulate proviral transcription. Phosphorylation of Thr-186 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 T-loop site, Ser-175, 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 pThr-186 by an arginine triad and Glu-96 of CycT1. Arginine triad substitutions that disrupted CDK9/CycT1 assembly accumulated Thr-186–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 Thr-186 phosphorylation, and suppressed proviral reactivation. The selective CDK7 inhibitor THZ1 blocked CDK9 phosphorylation at Ser-175, and in vitro kinase assays confirmed that CDK7 activity is principally responsible for Ser-175 phosphorylation. Mutation of Ser-175 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 Thr-186 phosphorylation is a key step in P-TEFb biogenesis. Furthermore, we demonstrate that CDK7-mediated Ser-175 phosphorylation is a downstream nuclear event essential for facilitating CDK9 T-loop interactions with Tat.
Source: Journal of Biological Chemistry
Institution: Case Western Reserve University
Language: English
Topic: HIV Research and Treatment
Subfield: Virology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
Funders: amfAR, The Foundation for AIDS Research, National Institutes of Health, National Institute on Drug Abuse, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases, National Institute of Biomedical Imaging and Bioengineering, National Institute of Dental and Craniofacial Research
Awards: Multifaceted integration for estrogen receptor, Role of non-coding RNA in establishing and maintaining HIV latency, G350852447, HIV eradication by ADCC-activated NK cell killing, G5795173237, Drugs of abuse and the epigenetic and signaling pathways controlling HIV latency, Case Center for Synchrotron Bioscience, Identification and eliminationof HIV reservoirs in oral lymphoid tissues by engineered NK cells., Reversal of HIV latency by METH and Inflammation, Rustbelt Center for AIDS Research (Case/UHC-Pitt CFAR)
OpenAlex ID: W2802451173