In this study, we provide evidence that the ubiquitylation of Cdk9 by Skp2 facilitates Tat transactivation. First, by using several independent systems, we found that Cdk9 is ubiquitylated in vivo. Second, not only was Skp2 required for Cdk9 ubiquitylation but it also contributed to optimal levels of Tat transactivation. Finally, the ubiquitylation of Cdk9 by Skp2 facilitated the formation of the ternary complex between P-TEFb, Tat, and TAR. Thus, Tat requires ubiquitylated Cdk9 to activate optimally the transcriptional elongation of HIV-1 genes.
These findings extend recent observations for the ubiquitin/proteosome system in the regulation of HIV-1 gene expression. Whereas the Hdm2 E3 ubiquitin ligase activity also facilitates Tat transactivation (1
), the role of Skp2 had not been addressed in this system. Rather, a negative role of Skp2 had been implicated in the function of the major histocompatibility complex class II transactivator (CIITA), which also depends on P-TEFb (13
). Therein, the expression of CycT1 which lacks the PEST sequence at its C terminus (CycT1ΔPEST) that binds Skp2 and mediates ubiquitylation of Cdk9, increased transcription activated by CIITA. In contrast, our biochemical and genetic evidence suggests that Skp2 acts as a coactivator for Tat transactivation. Indeed, the expression of the mutant Skp2ΔF protein or the absence of Skp2 in the MEF Skp2−/−
cells resulted in a loss of Cdk9 ubiquitylation. As a consequence, levels of Tat transactivation decreased. Consistently, the expression of the mutant CycT1ΔPEST protein decreased this activity in COS cells (data not presented). Overall, these observations indicate that multiple E3 ubiquitin ligases regulate one transcription unit. Moreover, ubiquitylation of Cdk9 could affect transcriptional units differently, which could stem from their distinct mechanisms of transcriptional activation. Indeed, CIITA recruits P-TEFb to activate transcriptional elongation of its target genes via the MHCII enhanceosome assembled on the DNA, whereas Tat achieves the same task by recruiting P-TEFb to TAR RNA (12
It remains to be established what fraction of Cdk9 is ubiquitylated in cells. Although we found slightly increased steady-state levels of Cdk9 in total cell lysates prepared from MEF Skp2−/−
cells compared to MEF Skp2+/+
cells, we did not observe any changes in levels of Cdk9 upon treatment of cells with the proteosomal inhibitor MG-132 or upon the expression of the Skp2ΔF protein. Likewise, a recent report by Garriga et al. suggested that levels of Cdk9 protein do not depend on Skp2 (8
). However, our functional data underscore the importance of Cdk9 ubiquitylation for the efficient formation of the ternary RNA-protein complex, which leads to optimal levels of Tat transactivation. In light of these observations, it is tempting to speculate that Cdk9 becomes ubiquitylated only when associated with the HIV-1 promoter or other cellular promoter elements. This scenario would resemble a mechanism of transcriptional activation by SREBP-1, c-Myc, and VP16, where the ubiquitylation of these activators occurs optimally on DNA (14
). Indeed, E3 ubiquitin ligase Skp2 associates with the cyclin D2 promoter in a c-Myc-dependent manner in cells (25
). It remains to be determined whether this situation holds true for the HIV-1 LTR as well.
How could the ubiquitylated Cdk9 affect Tat transactivation? In principle, ubiquitin moieties on Cdk9 could influence protein-protein or RNA-protein interactions. The evidence obtained from tethering experiments in which Skp2 did not affect transcription activated by heterologously tethered P-TEFb via DNA or RNA targets suggests that ubiquitylation of Cdk9 regulates positively neither its association with CycT1 to increase P-TEFb kinase activity nor its binding to Tat to increase the levels of P-TEFb on the HIV-1 promoter. Rather, by using an RNA capture assay, we found that the ubiquitylated form of Cdk9 facilitates the overall assembly of the ternary RNA-protein complex, possibly by increasing direct binding to TAR or by facilitating the relief of autoinhibitory intramolecular interactions within CycT1 (see below).
The recruitment of P-TEFb to the paused RNAPII by Tat is critical for stimulating transcriptional elongation of HIV-1 genes and viral replication. The significance of this decisive event in the course of the HIV-1 replicative cycle is reflected in the existence of several mechanisms, which regulate the formation of the ternary complex between P-TEFb, Tat, and TAR. The following model emerges from numerous studies. First, an inhibitory intramolecular interaction between the N- and C-terminal regions of CycT1, which prevents the ternary complex assembly, is relieved by the binding of the transcription elongation factor Tat-SF1 to the C-terminal region in CycT1 (4
). Second, Cdk9 phosphorylation is required for high-affinity binding of Tat and P-TEFb to TAR RNA (4
). In addition, the ubiquitylation of Cdk9 by Skp2 increases this RNA-protein complex assembly even further. Due to this sequence of events, high amounts of P-TEFb are concentrated in close proximity to its substrates, the CTD of RNAPII and the Spt5 subunit of DSIF. Moreover, P-TEFb targets the RD subunit of NELF, which dissociates from the lower stem of TAR RNA, thus contributing to the release of paused RNAPII (6
). Finally, the acetylation at a single lysine residue in the TAR RNA-binding domain of Tat by the transcriptional coactivator p300 enhances Tat transactivation, possibly by dissociating CycT1 from TAR RNA and transferring Tat onto the elongating RNAPII (11
). This model illustrates nicely how the assembly and disassembly of the multisubunit RNA-protein complex is a highly dynamic and temporally regulated process, which requires the participation of a variety of cellular cofactors, as well as multiple posttranslational modifications of its primary players.