Kinase Family BRD
Kinase Classification: Group SAPPK: Family BRD
The BRD (Bromodomain) proteins have twin bromodomains, are chromatin associated, and have been reported to act as protein kinases on the C-terminal domain (CTD) of RNA polymerase.
Domain Structure
BRD proteins have twin bromodomains on the N-terminus and a C-terminus of about equal length that has no known domains.
Evolution
Mammals have four BRDs (BRD2/RING3, BRD3, BRD4 and BRDT), Drosophila has one (fs(1)h) and C. elegans has 3. A more distantly related homolog is found in fungi (S. cerevisiae Bdf1 and homologs) and in plants. Other basal lineages have proteins with single bromodomains that are not clearly orthologous to BRD, but may have related functions.
Evidence for Protein Kinase Activity
Human BRD2 was first identified as an autophosphorylating nuclear-specific protein in Hela extracts [1]. Recombinant BRD2 expressed in E. coli showed in vitro kinase activity. However, the kinase activity was only seen when the purified recombinant protein was first incubated with Hela cell extract and repurified. It was suggested that this incubation could allow BRD2 to be activated by phosphorylation, but it is also possible that this step allowed BRD2 to bind to a human kinase which was then responsible for the observed activity. Recombinant BRD2 purified from COS cells also showed in vitro kinase activity, without pre-incubation. Mutation of a C-terminal lysine (K578A) abolished the activity seen in E. coli, implicating it either in cryptic kinase activity or binding to an active human kinase.
If the BRD kinase activity is due to a tightly bound kinase, the most likely candidate is CDK9. It is not known if BRD2 binds CDK9, but BRD4, BRDT and the Drosophila homolog, fs(1)h are all reported to bind CDK9, and BRD4 may induce CDK9 phosphorylation [2, 3]. Bdf1, the yeast BRD is also known to bind to CK2 kinase [4].
Human BRD4 has also been shown to be a kinase with autophosphorylation and transphosphorylation activity [5]. Deletion mutations mapped the kinase activity to the N-terminal half, from AA 1-699. Deletion of either bromodomain with this region caused a partial loss of activity, and deletion of both bromodomains caused greater but incomplete loss of activity. Kinase activity was seen in protein purified from E. coli or from insect Sf9 cells, and survived an in-gel kinase assay after denaturation and renaturation. BRD4 phosphorylated Ser2 of the RNA polymerase C-terminal domain, with a pH profile that was distinct from other Ser2 kinases, and which was insensitive to CDK inhibitors.
References
- Denis GV and Green MR. A novel, mitogen-activated nuclear kinase is related to a Drosophila developmental regulator. Genes Dev. 1996 Feb 1;10(3):261-71. DOI:10.1101/gad.10.3.261 |
- Zhou M, Huang K, Jung KJ, Cho WK, Klase Z, Kashanchi F, Pise-Masison CA, and Brady JN. Bromodomain protein Brd4 regulates human immunodeficiency virus transcription through phosphorylation of CDK9 at threonine 29. J Virol. 2009 Jan;83(2):1036-44. DOI:10.1128/JVI.01316-08 |
- Bisgrove DA, Mahmoudi T, Henklein P, and Verdin E. Conserved P-TEFb-interacting domain of BRD4 inhibits HIV transcription. Proc Natl Acad Sci U S A. 2007 Aug 21;104(34):13690-5. DOI:10.1073/pnas.0705053104 |
- Sawa C, Nedea E, Krogan N, Wada T, Handa H, Greenblatt J, and Buratowski S. Bromodomain factor 1 (Bdf1) is phosphorylated by protein kinase CK2. Mol Cell Biol. 2004 Jun;24(11):4734-42. DOI:10.1128/MCB.24.11.4734-4742.2004 |
- Devaiah BN, Lewis BA, Cherman N, Hewitt MC, Albrecht BK, Robey PG, Ozato K, Sims RJ 3rd, and Singer DS. BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain. Proc Natl Acad Sci U S A. 2012 May 1;109(18):6927-32. DOI:10.1073/pnas.1120422109 |
