Phosphorylated positive transcription elongation factor b (P-TEFb) is tagged for inhibition through association with 7SK snRNA

J Biol Chem. 2004 Feb 6;279(6):4153-60. doi: 10.1074/jbc.M310044200. Epub 2003 Nov 19.

Abstract

The positive transcription elongation factor b (P-TEFb), comprising CDK9 and cyclin T, stimulates transcription of cellular and viral genes by phosphorylating RNA polymerase II. A major portion of nuclear P-TEFb is sequestered and inactivated by the coordinated actions of the 7SK snRNA and the HEXIM1 protein, whose induced dissociation from P-TEFb is crucial for stress-induced transcription and pathogenesis of cardiac hypertrophy. The 7SK.P-TEFb interaction, which can occur independently of HEXIM1 and does not by itself inhibit P-TEFb, recruits HEXIM1 for P-TEFb inactivation. To study the control of this interaction, we established an in vitro system that reconstituted the specific interaction of P-TEFb with 7SK but not other snRNAs. Using this system, together with an in vivo binding assay, we show that the phosphorylation of CDK9, on possibly the conserved Thr-186 in the T-loop, was crucial for the 7SK.P-TEFb interaction. This phosphorylation was not caused by CDK9 autophosphorylation or the general CDK-activating kinase CAK, but rather by a novel HeLa nuclear kinase. Furthermore, the stress-induced disruption of the 7SK.P-TEFb interaction was not caused by any prohibitive changes in 7SK but by the dephosphorylation of P-TEFb, leading to the loss of the key phosphorylation important for 7SK binding. Thus, the phosphorylated P-TEFb is tagged for inhibition through association with 7SK. We discuss the implications of this mechanism in controlling P-TEFb activity during normal and stress-induced transcription.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cyclin T
  • Cyclin-Dependent Kinase 9 / genetics
  • Cyclin-Dependent Kinase 9 / metabolism
  • Cyclins / genetics
  • Cyclins / metabolism
  • HeLa Cells
  • Humans
  • In Vitro Techniques
  • Mutation
  • Phosphorylation
  • Positive Transcriptional Elongation Factor B / antagonists & inhibitors
  • Positive Transcriptional Elongation Factor B / genetics
  • Positive Transcriptional Elongation Factor B / metabolism*
  • Protein Binding
  • RNA Interference
  • RNA, Small Nuclear / genetics*
  • RNA, Small Nuclear / metabolism*
  • RNA-Binding Proteins / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Transcription Factors
  • Transcription, Genetic

Substances

  • CCNT1 protein, human
  • Cyclin T
  • Cyclins
  • HEXIM1 protein, human
  • RNA, Small Nuclear
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Transcription Factors
  • Positive Transcriptional Elongation Factor B
  • CDK9 protein, human
  • Cyclin-Dependent Kinase 9