Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

Abstract

P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.Transcription by RNA polymerase II (pol II) is a multistep process including preinitiation, initiation, promoter clearance, elongation, and termination (48). Phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of pol II plays a critical role in transition from transcriptional initiation to elongation as well as in coordinating transcription elongation and RNA maturation (7, 30). P-TEFb, a positive transcription elongation factor originally identified based on its ability to stimulate 5,6-dichloro-1-␤-D-ribofuranosyl-benzimidazole-sensitive transcription of long transcripts in vitro (36), stimulates transcription elongation by preferentially phosphorylating Ser2 of the heptapeptide repeat of the CTD of the largest subunit of pol II (35). P-TEFb is a heterodimeric complex comprised of cyclin-dependent kinase 9 (CDK9) and a regulatory cyclin subunit of the T family, namely T1, T2, or K1 (13, 43). CDK9 is a cdc2-related, ubiquitously expressed kinase protein (8). P-TEFb also enhances transcriptional elongation by phosphorylating and counteracting the inhibitory factors DSIF and NELF (5,24,51,54).In addition to its function as a global transcriptional elongation factor important for most pol II transcription (3, 47), activation of CDK9 kinase activity has also been linked to specific events such as human immunodeficiency virus (HIV) replication (20, 65), cardiac hypertrophy (45), and activation of lymphocytes (19). In this regard, P-TEFb is also known as TAK, an HIV Tat-associated kinase (20). Regulation of HIV transcription is primarily mediated by the HIV Tat protein, which recruits TAK/P-TEFb to enhance productive elongation of viral transcripts (11,12,64). Activation of P-TEFb induces heart hypertrophy in transgenic mice and induces myocyte enlargement in tissu...

HDAC3: taking the SMRT-N-CoRrect road to repression

“…Deacetylation of the acetyltransferases was shown to inhibit their function in the above examples. Finally, HDAC3 negatively regulates the activity of transcriptional elongation factor P-TEFb through deacetylation of CDK9 subunit (Fu et al, 2007). We expect that more and more proteins will be identified to be regulated through deacetylation by HDAC3 or its associated protein complexes.…”

Deleted in Breast Cancer 1, a Novel Androgen Receptor (AR) Coactivator That Promotes AR DNA-binding Activity

“…Generation of the AR-inducible Stable Cell Line-A stable FLAG-AR cell line was generated using the 293T Flp-In T-Rex cell line and Flp-In TM -T-REx TM system from Invitrogen according to the manufacturer's instructions essentially as described (31).…”

The TWIST/Mi2/NuRD protein complex and its essential role in cancer metastasis

“…The cells with empty vector were treated in the same way to serve as a control. npg and analyzed by mass spectrometry as described [47].…”

“…Methods for immunoprecipitation (IP) and immunoblotting were described previously [47,49]. TrueBlot IP/WB kit from eBioscience was used to eliminate the IgG bands in the immunoblotting analysis.…”

“…35 S-labeled TWIST, MTA2, HDAC2, Mi2β and RbAP46 proteins were synthesized using the TNT in vitro transcription-translation kit (Promega). The GST pull-down assays for protein-protein interactions between GST-fusion proteins and 35 S-labeled proteins were carried out as described [47].…”