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.
Garber
M E
ME
Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Mayall
T P
TP
Suess
E M
EM
Meisenhelder
J
J
Thompson
N E
NE
Jones
K A
KA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D056748:Cyclin H; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus; D000095043:Cyclin-Dependent Kinase-Activating Kinase
C534751:CCNH protein, human; C503684:CCNT1 protein, human; D056748:Cyclin H; D056906:Cyclin T; D016213:Cyclins; D015696:Gene Products, tat; D010455:Peptides; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase