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.

Fig. 1.

BRD4 is an atypical kinase that binds to Pol II CTD. (A) BRD4 autophosphorylation in an in vitro kinase reaction (1 μg rBRD4, γ[³²P]ATP). CE, control extract. (B) BRD4 has intrinsic kinase activity. After denaturing gel electrophoresis and renaturation, BRD4 was subjected to an in-gel kinase reaction with γ[³²P]ATP. (C) BRD4 is an atypical kinase. (Upper) Map of putative kinase subdomains (III–IX), ATP binding site (★), and mutants. (Lower) Kinase assays of 1 μg BRD4 and equimolar amounts of BRD4 mutants. (D) BRD4 interacts with Pol II in vivo. BRD4 was immunoprecipitated from HeLa nuclear extract using α-BRD4 and immunoblotted with α-Pol II. (E) BRD4 interacts directly with Pol II CTD. BRD4 (0.25, 0.5, and 1 μg) was pulled down with 0.5 μg GST-CTD immobilized on GST-Sepharose beads. (F) CTD binds BRD4 kinase subdomains. (Upper) α-GST immunoblot of 0.2 μg GST-CTD (input) recovered by pull-down with 1 μg WT Flag-BRD4 or equimolar amounts of Flag-BRD4 mutants. (Lower) Quantitation of recovered GST-CTD.

Fig. 2.

BRD4 phosphorylates the Pol II CTD Ser2. (A) BRD4 phosphorylates CTD. In vitro kinase assay with 0.6 μg BRD4 and 0.25 μg GST-human CTD or GST peptide substrates. Equimolar amounts of PTEFb or Sf9 control extract (CE) were positive and negative kinase controls respectively. BRD4 autophosphorylation is not shown. (B) BRD4 requires Ser2 sites to phosphorylate CTD. GST-CTD (0.2 μg) with 25- or 16-heptad repeats was phosphorylated with 0.5 μg BRD4. The CTD proteins had unmodified sequences (WT) or alanine substitutions at every Thr4, Ser2, Ser5, Ser7, or both Ser2 and Ser5 sites. (C) BRD4 phosphorylates CTD Ser2. Immunoblots with α-Ser2P antibodies (3E10 and H5) after in vitro kinase assays of 0.1 μg GST-CTD with equimolar amounts of BRD4 (0.2 μg), PTEFb, or CE (−). Parallel kinase reactions with γ[³²P]ATP show total incorporation.

Fig. 3.

BRD4 kinase activity is distinct from other Pol II CTD kinases. (A) Effect of kinase inhibitors on BRD4. In vitro kinase assays with 0.25 μg GST-CTD, 0.6 μg BRD4, and inhibitors (1× and 5× IC₅₀) flavopiridol (50 and 250 nM), roscovitine (20 and 100 μM), NU 7441 (DNA-PK inhibitor; 14 and 70 nM), FR 180204 (MEK1/2 inhibitor; 0.4 and 2 μM), and apigenin (50 and 250 μM). (B) BRD4 kinase activity is distinct from PTEFb. GST-CTD (0.2 μg) was phosphorylated by recombinant murine BRD4 (0.2 μg), human BRD4 immunoprecipitated from HeLa nuclear extract, or PTEFb (0.1 μg) with or without 1 μM flavopiridol and 100 μM roscovitine. (C) BRD4 phosphorylates CTD Ser2 during in vitro transcription. In vivo transcription reactions incubated with flavopiridol (1 μM), roscovitine (100 μM), or apigenin (50 μM) were immunoblotted for Ser2P. (D) BRD4 phosphorylation of CTD is affected by prior phosphorylation. (Left) Experimental design. (Right) Autoradiograph of BRD4 phosphorylation of GST-CTD (0.2 μg) unphosphorylated, or prephosphorylated by PTEFb (0.1 μg), TFIIH (0.2 μg), or both.

Fig. 4.

BRD4 phosphorylates CTD Ser2 in vivo. (A) BRD4 overexpression increases Ser2P levels independent of PTEFb recruitment; 5 μg human Brd4, a BRD4 mutant incapable of binding PTEFb (FEE-AAA) or vector alone, were transfected into HeLa cells. Whole-cell extracts of these cells were immunoblotted for CTD Ser2P levels (Upper Right). Quantitation of averages ± SEM from two independent experiments (Upper Left). PTEFb components CDK9 and Cyclin T1 do not coimmunoprecipitate with overexpressed Flag-BRD4 mutant FEE-AAA (Lower). (B) Treatment with JQ1 derivative CPI203 inhibits BRD4 phosphorylation of CTD Ser2 in vivo. HeLa cells were transfected as above and treated with increasing concentrations of CPI203. Whole-cell extracts of these cells were immunoblotted for CTD Ser2P levels. Results were normalized to total Pol II levels (Lower). (C) In vivo CTD Ser2P levels are independent of CDK9/PTEFb in stem cells. CTD Ser2P, CDK9, and BRD4 levels in cell extracts from normal trabecular bone cells (NTB), human bone marrow stromal stem cells (BMSc), mature HeLa cells, MEFs or from MEFs transfected with control siRNA, BRD4, or CDK9-specific siRNA (Upper). Quantitation of averages ± SEM from two independent experiments (Lower).