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

J Biol Chem. 2018 Jun 29;293(26):10009-10025. doi: 10.1074/jbc.RA117.001347. Epub 2018 May 9.

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.

Keywords: 7SK snRNP; CDK activation loop; Cdc37; HIV latency; P-TEFb; Tat; bromodomain-containing protein 4 (BRD4); heat-shock protein 90 (Hsp90); human immunodeficiency virus (HIV); post-translational modification (PTM); proteomics; super-elongation complex; transcription elongation factor.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cyclin-Dependent Kinase 9 / chemistry
  • Cyclin-Dependent Kinase 9 / metabolism*
  • Enzyme Activation
  • HIV-1 / metabolism
  • HIV-1 / physiology*
  • HSP90 Heat-Shock Proteins / metabolism
  • Humans
  • Jurkat Cells
  • Phosphorylation
  • Positive Transcriptional Elongation Factor B / metabolism*
  • Protein Binding
  • Serine / metabolism
  • Threonine / metabolism
  • Virus Activation*
  • tat Gene Products, Human Immunodeficiency Virus / metabolism*

Substances

  • HSP90 Heat-Shock Proteins
  • tat Gene Products, Human Immunodeficiency Virus
  • Threonine
  • Serine
  • Positive Transcriptional Elongation Factor B
  • CDK9 protein, human
  • Cyclin-Dependent Kinase 9

Associated data

  • PDB/3MIA
  • PDB/3BLQ