HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription

Abstract

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.

HIV-1 Tat Interaction with RNA Polymerase II C-terminal Domain (CTD) and a Dynamic Association with CDK2 Induce CTD Phosphorylation and Transcription from HIV-1 Promoter

“…Analysis of TTK by biochemical fractionation showed that TTK uniquely copurified with CDK2 and not with CDK9 or CDK7 (11). Tat induced the TTK and CDK2 to phosphorylate CTD, specifically at serine 2 residues (11), which was in contrast to the reported phosphorylation of Ser-5 by CDK9 in the presence of Tat (8).…”

“…The results suggest that CDK2/cyclin E phosphorylates Ser-2 and Ser-5 of CTD heptapeptide repeats in the absence of Tat. Tat Enhances CTD Phosphorylation by CDK2/Cyclin E-Tat has been reported to induce CTD phosphorylation by CDK7 (11,18,22,23) and CDK9 (8,24). We analyzed the effect of Tat on CTD phosphorylation by CDK2/cyclin E. In the absence of Tat, CDK2/cyclin E poorly phosphorylated recombinant GST-CTD (Fig.…”

“…/Cyclin E Phosphorylates Ser-2 and Ser-5 of the CTD Heptapeptide Repeat-We have recently demonstrated that CDK2/cyclin E phosphorylates CTD (11). CDK2 binds to a (S/T)PX(K/R) consensus phosphorylation site (19).…”

“…We have purified a Tat-associated T-cell-derived kinase (TTK) that phosphorylates CTD (9 -11). TTK stimulates Tat transactivation in vitro (11) and in vivo (10,11). TTK containes CDK2, which phosphorylates CDK7 (11).…”

SWI/SNF: The crossroads where extracellular signaling pathways meet chromatin

“…Recently, two independent studies identified cyclin E/cdk2 as a kinase complex able to interact and phosphorylate RNA polymerase II in vivo (Deng et al, 2002;Nekhai et al, 2002), suggesting another functional link between cyclin E and SWI/SNF, and defining a new role for cyclin E/cdk2 at the chromatin level in the transcription process.…”