HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription.
HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246-256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.
Nekhai
Sergei
S
Department of Biochemistry and Molecular Biology, The George Washington University, School of Medicine, 2300 Eye Street N.W., Washington, DC 20037, USA.
Zhou
Meisheng
M
Fernandez
Anne
A
Lane
William S
WS
Lamb
Ned J C
NJ
Brady
John
J
Kumar
Ajit
A
D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D002460:Cell Line; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D005347:Fibroblasts / Q000821:virology; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D010766:Phosphorylation; D010957:Plasmids; D042925:Positive Transcriptional Elongation Factor B; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D018797:Cell Cycle Proteins; D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D011494:Protein Kinases; C060137:carboxy-terminal domain kinase; D011505:Protein-Tyrosine Kinases; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; C075840:TTK protein, human; D012319:RNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase