The intrinsic kinase activity of BRD4 spans its BD2-B-BID domains

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• Bromodomain protein 4 (BRD4) is a key transcriptional and epigenetic regulator implicated in cancer and inflammatory diseases.
• BRD4 functions as both an active kinase and a scaffold, phosphorylating RNA polymerase II (Pol II) CTD, c-MYC, TAF7, and CDK9.
• Limited information exists regarding BRD4's biophysical properties and precise kinase activity.

Purpose of the Study:

• To characterize the biophysical properties and kinase activity of mouse Bromodomain protein 4 (BRD4).
• To identify the structural domains responsible for BRD4's dimerization, kinase activity, and substrate binding.
• To elucidate the mechanisms underlying BRD4's role in transcriptional regulation.

Main Methods:

• Analytical ultracentrifugation was used to determine BRD4's conformation and sedimentation coefficient.
• Site-directed mutagenesis was employed to delete specific motifs (B motif, A motif) and assess their impact on dimerization and activity.
• In vitro kinase assays were performed using purified Pol II CTD, TAF7, MLVIN, and NSD3 peptides as substrates.

Main Results:

• Mouse BRD4 (156 kD) exists as an extended dimer (∼6.7 S) with a high frictional ratio.
• The B motif (aa 503-548) is crucial for BRD4 dimerization, while the kinase activity resides in the BD2-B-BID domain (aa 351-598).
• BRD4 directly binds Pol II CTD, with the A motif essential for Pol II CTD phosphorylation but not TAF7 phosphorylation. BRD4 also phosphorylates MLVIN and NSD3 peptides.

Conclusions:

• BRD4 possesses a common kinase domain but multiple distinct substrate-binding sites, including the A motif for Pol II CTD.
• The dimeric conformation and specific motifs of BRD4 are critical for its kinase function and substrate interactions.
• These findings enhance our understanding of BRD4's structure-function relationship and its role in transcriptional regulation.