BRD4 is an atypical kinase that phosphorylates Serine2 of the RNA Polymerase II carboxy-terminal domain

Abstract

The bromodomain protein, BRD4, has been identified recently as a therapeutic target in acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma, NUT midline carcinoma, colon cancer, and inflammatory disease; its loss is a prognostic signature for metastatic breast cancer. BRD4 also contributes to regulation of both cell cycle and transcription of oncogenes, HIV, and human papilloma virus (HPV). Despite its role in a broad range of biological processes, the precise molecular mechanism of BRD4 function remains unknown. We report that BRD4 is an atypical kinase that binds to the carboxyl-terminal domain (CTD) of RNA polymerase II and directly phosphorylates its serine 2 (Ser2) sites both in vitro and in vivo under conditions where other CTD kinases are inactive. Phosphorylation of the CTD Ser2 is inhibited in vivo by a BRD4 inhibitor that blocks its binding to chromatin. Our finding that BRD4 is an RNA polymerase II CTD Ser2 kinase implicates it as a regulator of eukaryotic transcription.B RD4 is a BET family protein that was identified originally as a ubiquitously expressed chromatin adapter that maintains epigenetic memory and regulates cell cycle progression (1). More recently, it has been characterized as a key determinant in acute myeloid leukemia, multiple myeloma, Burkitt's lymphoma, NUT midline carcinoma, colon cancer, and inflammatory disease (2-7). It suppresses tumor metastasis in mice, and its expression is a prognostic signature of breast cancer survival (8). BRD4 has been proposed to be a structural scaffold that regulates transcription indirectly by recruiting the elongation factor, PTEFb, to the transcription preinitiation complex (9).Productive transcription depends on the phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II). The Pol II CTD consists of consecutive repeats of the heptad Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , which vary in number between 26 in yeast and 52 in mammals (10). Phosphorylation of the CTD residues serine 5 (Ser5) and serine 2 (Ser2) is necessary for the recruitment of RNA capping and splicing factors, respectively (11). The order, pattern, and temporal separation of these phosphorylation events ensure an orderly transition from initiation to productive transcription elongation (11-13). CTD Ser5 residues are phosphorylated primarily by the CDK7 kinase component of TFIIH (14), whereas the subsequent Ser2 phosphorylation, currently attributed primarily to the CDK9 subunit of PTEFb and/or CDK12/13, releases Pol II from an early elongation block (15, 16). PTEFb nuclear localization and activation depends on BRD4, which is also known to interact with a variety of other factors, including the mediator complex (1,17). Despite the preponderance of evidence indicating a vital role for BRD4 in transcription, its precise molecular function is unknown.Here, we show that BRD4 is an atypical protein kinase that exhibits both auto-and transphosphorylation. Furthermore, BRD4 directly and specifically phosphorylates the Pol II CTD at the Ser2 position, and it is di...

Cross-talk Among RNA Polymerase II Kinases Modulates C-terminal Domain Phosphorylation

“…1B). The direct phosphorylation of CDK9 by BRD4 was further demonstrated by using flavopiridol, a potent and specific inhibitor of CDK9 kinase activity that does not affect BRD4 (9). Whereas the presence of flavopiridol completely abrogated CDK9 autophosphorylation, it did not inhibit the phosphorylation of CDK9 by BRD4 (Fig.…”

“…Plasmid Constructs-Murine FLAG-BRD4 WT and ⌬B1, ⌬B2, ⌬B1B2, ⌬N, and ⌬C mutants are as described before (9) and were a kind gift from K. Ozato (NICHD, NIH, Bethesda, MD). The human FLAG-tagged BRD4 WT and FAA-AAA mutant in the pCMV2 mammalian vector were a gift from E. Verdin (Gladstone Institute of Virology, University of California, San Francisco, CA).…”

“…Protein Purification-Recombinant FLAG-tagged BRD4 and associated mutant proteins were purified from Sf9 cells as described earlier (9). The FLAG peptide was eliminated on a microcon column (Millipore) and proteins were recovered in HKEG buffer (20 mM Hepes, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% (v/v) glycerol).…”

“…We recently identified a third CTD kinase, the bromodomain protein BRD4. BRD4, a candidate therapeutic target for multiple cancers, uniquely phosphorylates CTD Ser-2 sites (9). How the activities of these kinases are coordinated to ensure a proper temporal and spatial sequence of CTD phosphorylation remains unknown.…”

Controlling gene expression by DNA mechanics: emerging insights and challenges

“…2e). This activation is mediated by the carboxyl-terminal domain (CTD) of RNAPII, but requires that the CTD is phosphorylated by the kinase activity of the bromodomain chromatin factor BRD4 (Devaiah et al 2012) (Fig. 2d).…”