CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

Abstract

BackgroundHIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity.ResultsThe siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94) that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation.ConclusionOur data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1 provirus.

Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

“…In a recent study, we demonstrated that HIV-1 transcription is regulated by CDK2, which phosphorylates the Ser90 amino acid residue of CDK9 (13). Dephosphorylation of this residue reduces the large P-TEFb complex and decreases HIV-1 transcription (13). Macrophages differentiated from induced pluripotent stem cells with stable CDK2 knockdown also exhibited the reduced susceptibility of these cells to HIV-1 infection (14), confirming our previous observation of CDK2 as a key regulator of HIV-1 transcription.…”

“…For preparation of whole-cell lysates, 293T cells were harvested in whole-cell lysis buffer (50 mM Tris-HCl buffer, pH 7.5, containing 0.5 M NaCl, 1% NP-40, 0.1% SDS, and a cocktail of protease inhibitors) as described above. Immunoprecipitation of CDK9 by use of an anti-CDK9 polyclonal antibody (Santa Cruz) was performed as previously described (13). Briefly, 400 g of lysate and 800 ng of antibody were incubated for 2 h at 4°C with 50 l of 50% protein A/G agarose suspended in 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl and 1% NP-40 (TNN buffer).…”

“…Separation of large and small P-TEFb complexes was performed using the methodology described in our recent publication (13), with slight modifications. Briefly, 293T cells in DMEM containing 10% fetal bovine serum were cultured for 48 h in the absence or presence of 1 M iron chelators.…”

“…While the kinase activity of CDK9 in the large P-TEFb complex is suppressed (10,11), this complex serves as the source of CDK9/cyclin T1 for recruitment by HIV-1 Tat (12). In a recent study, we demonstrated that HIV-1 transcription is regulated by CDK2, which phosphorylates the Ser90 amino acid residue of CDK9 (13). Dephosphorylation of this residue reduces the large P-TEFb complex and decreases HIV-1 transcription (13).…”

Kinase Control of Latent HIV-1 Infection: PIM-1 Kinase as a Major Contributor to HIV-1 Reactivation

“…Among other factors, PIM-1 is directly linked to NF-B, as well as to cyclin dependent kinase 2 (CDK2). CDK2 has recently been demonstrated to be important for HIV-1 transcription by regulating the phosphorylation of HIV-1 Tat and CDK9 (28)(29)(30). The direct functional proximity of PIM-1 on the PIN to these factors can be viewed as a descriptor of the importance of PIM-1 in the context of HIV-1 latency.…”