This work describes a novel mechanism of P-TEFb regulation that involves the inhibition of CDK9 kinase activity by posttranslational modification of its catalytic core. Among the different HATs that were proven to acetylate CDK9, members of the GNAT family were found to be the most effective. In particular, GCN5 was found to acetylate one lysine (K48) that is conserved in the so-called subdomain II of almost all the eukaryotic protein kinases (conserved in over 95% of 370 sequences) and is hence essential for enzyme function (18
). Structural data on CDK2 indicate that the conserved subdomain II lysine is essentially involved in orienting the ATP phosphate residues as well as in magnesium binding within the catalytic pocket of the enzyme (7
). Not surprisingly, we found that the CDK9 K44,48R mutant had reduced affinity for the ATP analogue FSBA, which was previously used to map the ATP-binding sites of various proteins, including protein kinase A (56
). The FSBA reactive residues are indeed known to play crucial roles in the phosphotransfer reaction (24
). The residual kinase and FSBA-binding activities of the K44,48R mutant suggest that other lysines might inefficiently compensate for the K48 mutation, as occurs with analogous mutations in other kinases (15
Our experiments indicate that lysines 44 and 48 of CDK9 are the substrates for acetylation by the GNAT acetyltransferase family members GCN5 and P/CAF and, to a much lower extent, by p300. Very recent work has indeed shown that p300 might preferentially target K44 and that histone deacetylase proteins negatively regulate CDK9 function, thus implying a positive effect of acetylation (11
). The contradiction between those findings and the data presented here might be only apparent. Our work shows that GCN5-mediated acetylation, essentially occurring on K48, is repressive of transcription, whereas Fu et al. analyzed the effects of histone deacetylase 3, which presumably acts on K44. Thus, it might well be envisaged that CDK9, while kept in an inactive state on the transcriptionally silent provirus by GCN5- and P/CAF-mediated K48 acetylation, might become selectively acetylated on K44 by p300 only when the promoter becomes activated. Our previous ChIP experiments on the HIV-1 promoter have indeed revealed that p300, together with other HATs, is recruited onto the LTR upon transcriptional activation (23
). Other proteins, such as the viral transactivator Tat (21
), are known to be acetylated on different lysines by different HATs, with different functional consequences.
Protein modification by acetylation, as well as the function of cellular HATs, is commonly associated with increased transcriptional activity, mainly due to the positive effect that this modification has on chromatin. However, in several instances factor acetylation is inhibitory of transcription. The acetylation of NF-κB p65 (19
), HMGI(Y) (33
), IRF7 (3
), AFX (Foxo4) (13
), and Brm (2
) has been reported to repress gene expression; for some of these factors, acetylation might operate as a feedback mechanism to control the duration of transcription. In addition, GCN5-mediated acetylation has been recently shown to inhibit and relocalize the transcriptional coactivator PGC-1α (22
). Thus, CDK9 is not the only transcriptional regulator that is negatively regulated by this posttranslational modification.
Our ChIP experiments indicate that the latent state of HIV-1 is characterized by the binding, within the promoter region, of low levels of RNAPII, which is not phosphorylated on Ser2 since CDK9 is kept in an enzymatically inactive state by acetylation. Transcriptional activation of the latent provirus is paralleled by an increase in the total levels of recruited CDK9 and, most notably, in a marked reduction of its acetylated form. These results are consistent with the conclusion that acetylation of CDK9 participates in maintaining the transcriptional latency of the HIV-1 genome. Consistent with this notion, the latent state is also characterized by the presence, on the provirus, of detectable levels of the P/CAF and GCN5 HATs, which are likely responsible for CDK9 acetylation and thus cooperate to maintain promoter latency.
The observation that, in contrast to total CDK9, its acetylated form is detectable, both biochemically and by immunofluorescence, in a specific subnuclear compartment that corresponds to the insoluble nuclear fraction deserves further comments. This is the same compartment in which, together with the PML protein, both GCN5 (A. Sabò and M. Giacca, unpublished observations) and unphosphorylated RNAPII (46
) reside, along with a large fraction of cyclin T1, the main CDK9 partner (26
). Forcing protein accumulation into this compartment exerts a repressive role on HIV-1 transcription (26
). Based on these findings, we propose a model according to which the latent provirus is held in a transcriptionally inactive state when complexed with several of these interactors, which would modify its subnuclear localization and bring it into a transcriptionally repressive environment. According to this model, transcriptional activation would be concomitant with a modification of the subnuclear localization of proviral DNA with respect to the repressive matrix domain. This modification would be paralleled by the replacement of acetylated CDK9 with its unacetylated form, the consequent phosphorylation of RNPII, and the onset of processive transcription. In this scenario, the same HATs that exert a repressive role by acetylating CDK9 once in the repressive compartment would become transcriptional coactivators when outside this compartment by acetylating the proviral chromatin. Besides being compatible with all available experimental observations, this model would also eventually provide a molecular connection between the mechanisms that govern the rate of transcriptional initiation and those involved in regulating transcriptional elongation at the HIV-1 promoter. Further experiments will verify this testable hypothesis.
Finally, to our knowledge, this work reports the first example in which a eukaryotic HAT is found to specifically regulate the activity of a serine/threonine kinase by acetylating the conserved lysine positioned in its catalytic core. Indeed, only a couple of examples of HATs that regulate enzymatic function have very recently been discovered. In both Salmonella enterica
) and mammals (17
), acetyl coenzyme A synthetase is inhibited by the acetylation of the catalytic site of the enzyme. The Yersinia
effector protein YopJ acetylates conserved serine and threonine residues in the activation loop of eukaryotic mitogen-activated protein kinase kinase 6 (32
); in this case, acetylation inhibits kinase activation since these residues must be phosphorylated for the kinase to become active. The strict conservation of CDK9 K48 in all the CDKs suggests that this mechanism of regulation might play a broader role in controlling the function of other members of this kinase family. Preliminary observations in our laboratory indeed indicate that some CDKs involved in cell cycle control are also acetylated by GCN5.