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CDK9 autophosphorylation regulates high-affinity binding of the human immunodeficiency virus type 1 tat-P-TEFb complex to TAR RNA.

Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathione S-transferase-C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat-P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1-728)], but not truncated CycT1(1-303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.

Pubmed ID: 10958691

Authors

  • Garber ME
  • Mayall TP
  • Suess EM
  • Meisenhelder J
  • Thompson NE
  • Jones KA

Journal

Molecular and cellular biology

Publication Data

September 22, 2000

Associated Grants

  • Agency: NIAID NIH HHS, Id: AI644615

Mesh Terms

  • Adenosine Triphosphate
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Cyclic AMP-Dependent Protein Kinases
  • Cyclin H
  • Cyclin T
  • Cyclin-Dependent Kinase 9
  • Cyclin-Dependent Kinases
  • Cyclins
  • Gene Products, tat
  • Glutathione Transferase
  • HIV Long Terminal Repeat
  • HIV-1
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • Mutagenesis
  • Peptides
  • Phosphorylation
  • Positive Transcriptional Elongation Factor B
  • Protein Binding
  • Protein-Serine-Threonine Kinases
  • RNA Polymerase II
  • RNA, Viral
  • Recombinant Fusion Proteins
  • tat Gene Products, Human Immunodeficiency Virus