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

Abstract

Bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator that is a therapeutic target in many cancers and inflammatory diseases. BRD4 plays important roles in transcription as an active kinase, which phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II), the proto-oncogene c-MYC, and transcription factors TAF7 and CDK9. BRD4 is also a passive scaffold that recruits transcription factors. Despite these well-established functions, there has been little characterization of BRD4’s biophysical properties or its kinase activity. We report here that the 156 kD mouse BRD4 exists in an extended dimeric conformation with a sedimentation coefficient of ∼6.7 S and a high frictional ratio. Deletion of the conserved B motif (aa 503–548) disrupts BRD4’s dimerization. BRD4 kinase activity maps to amino acids 351 to 598, which span bromodomain-2, the B motif, and the BID domain (BD2-B-BID) and contributes to the in vivo phosphorylation of its substrates. As further assessed by analytical ultracentrifugation, BRD4 directly binds purified Pol II CTD. Importantly, the conserved A motif of BRD4 is essential for phosphorylation of Pol II CTD, but not for phosphorylation of TAF7, mapping its binding site to the A motif. Peptides of the viral MLV integrase (MLVIN) protein and cellular histone lysine methyltransferase, NSD3, which have been shown by NMR to bind to the extra-terminal (ET) domain, also are phosphorylated by BRD4. Thus, BRD4 has multiple distinct substrate-binding sites and a common kinase domain. These results provide new insights into the structure and kinase function of BRD4. Bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator that is a therapeutic target in many cancers and inflammatory diseases. BRD4 plays important roles in transcription as an active kinase, which phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II), the proto-oncogene c-MYC, and transcription factors TAF7 and CDK9. BRD4 is also a passive scaffold that recruits transcription factors. Despite these well-established functions, there has been little characterization of BRD4’s biophysical properties or its kinase activity. We report here that the 156 kD mouse BRD4 exists in an extended dimeric conformation with a sedimentation coefficient of ∼6.7 S and a high frictional ratio. Deletion of the conserved B motif (aa 503–548) disrupts BRD4’s dimerization. BRD4 kinase activity maps to amino acids 351 to 598, which span bromodomain-2, the B motif, and the BID domain (BD2-B-BID) and contributes to the in vivo phosphorylation of its substrates. As further assessed by analytical ultracentrifugation, BRD4 directly binds purified Pol II CTD. Importantly, the conserved A motif of BRD4 is essential for phosphorylation of Pol II CTD, but not for phosphorylation of TAF7, mapping its binding site to the A motif. Peptides of the viral MLV integrase (MLVIN) protein and cellular histone lysine methyltransferase, NSD3, which have been shown by NMR to bind to the extra-terminal (ET) domain, also are phosphorylated by BRD4. Thus, BRD4 has multiple distinct substrate-binding sites and a common kinase domain. These results provide new insights into the structure and kinase function of BRD4. BRD4 is a bromodomain and extra terminal (BET) family member that has pleiotropic functions throughout cell cycle. It interacts with chromatin modifying factors and transcription factors, among them the positive transcription elongation factor b (PTEFb), and recruits them to enhancers and promoters (1Devaiah B.N. Gegonne A. Singer D.S. Bromodomain 4: A cellular Swiss army knife.J. Leukoc. Biol. 2016; 100: 679-686Crossref PubMed Scopus (56) Google Scholar). BRD4 is a mitotic bookmark that remains associated with chromosomes throughout mitosis (2Dey A. Ellenberg J. Farina A. Coleman A.E. Maruyama T. Sciortino S. Lippincott-Schwartz J. Ozato K. A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G2-to-M transition.Mol. Cell. Biol. 2000; 20: 6537-6549Crossref PubMed Scopus (225) Google Scholar). It also functions in DNA repair and contributes to the regulation of alternative splicing (3Uppal S. Gegonne A. Chen Q. Thompson P.S. Cheng D. Mu J. Meerzaman D. Misra H.S. Singer D.S. The bromodomain protein 4 contributes to the regulation of alternative splicing.Cell Rep. 2019; 29: 2450-2460.e5Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Importantly, BRD4 is a kinase that regulates transcription, both directly and indirectly. It directly regulates transcription by phosphorylating the Ser2 of the RNA Pol II C-terminal domain (CTD) and facilitates the transition of RNA Pol II from initiation to elongation (4Devaiah B.N. Lewis B.A. Cherman N. Hewitt M.C. Albrecht B.K. Robey P.G. Ozato K. Sims R.J. Singer D.S. BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 6927-6932Crossref PubMed Scopus (247) Google Scholar). It also phosphorylates both the TFIID component, TAF7, and the transcription elongation factor, PTEFb/CDK9 (5Devaiah B.N. Singer D.S. Cross-talk among RNA polymerase II kinases modulates C-terminal domain phosphorylation.J. Biol. Chem. 2012; 287: 38755-38766Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). It indirectly regulates transcription through its phosphorylation of the transcription factor, c-MYC, leading to c-MYC degradation (6Devaiah B.N. Mu J. Akman B. Uppal S. Weissman J.D. Cheng D. Baranello L. Nie Z. Levens D. Singer D.S. MYC protein stability is negatively regulated by BRD4.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 13457-13467Crossref PubMed Scopus (36) Google Scholar). Thus, BRD4 is an active participant in transcription. Although the functions of BRD4, particularly as they relate to cancer, have been extensively studied, less is known about its biophysical properties in general and its kinase activity, specifically. Within the molecule, the N-terminal half of BRD4 is the better characterized (Fig. 1A). The kinase activity of BRD4, an atypical kinase, maps to the N-terminal half of the molecule. However, the position of the active site within the N-terminus has not been determined. In addition to the kinase domain, the N-terminal segment also encompasses two bromodomains, whose structures have both been resolved, which bind to acetylated lysines on histones and other proteins (7Filippakopoulos P. Picaud S. Mangos M. Keates T. Lambert J.-P. Barsyte-Lovejoy D. Felletar I. Volkmer R. Müller S. Pawson T. Histone recognition and large-scale structural analysis of the human bromodomain family.Cell. 2012; 149: 214-231Abstract Full Text Full Text PDF PubMed Scopus (1030) Google Scholar). The ET domain, whose structure has been determined, is one of the defining domains of the BET family within this region (8Lin Y.J. Umehara T. Inoue M. Saito K. Kigawa T. Jang M.K. Ozato K. Yokoyama S. Padmanabhan B. Güntert P. Solution structure of the extraterminal domain of the bromodomain-containing protein BRD4.Protein Sci. 2008; 17: 2174-2179Crossref PubMed Scopus (39) Google Scholar). Additional N-terminal elements A and B motifs, BID and SEED domains are largely uncharacterized, both structurally and functionally. The B motif has been reported to be important for chromatin binding; the BID domain is the target of phosphorylation by the kinase, casein kinase 2 (CK2) (9Garcia-Gutierrez P. Mundi M. Garcia-Dominguez M. Association of bromodomain BET proteins with chromatin requires dimerization through the conserved motif B.J. Cell Sci. 2012; 125: PubMed Scopus Google chromatin binding and for Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). is known about the structure of the C-terminal half of BRD4. It is to be is for both in and in vivo A. A. K. B.J. D.S. J. and PubMed Google Scholar). Despite the of the two of BRD4, the biophysical properties of the are The to to and the kinase domain within BRD4 and to its biophysical We report that BRD4 exists in as an extended whose structure is by the conserved B motif. We the BRD4 kinase activity within the BRD4 region the The BRD4 kinase activity with both to and to its and TAF7 substrates. In vivo phosphorylation of the Ser2 by BRD4 on the kinase activity. BRD4 has distinct binding sites for its substrates. Thus, BRD4 directly binds its Pol II and phosphorylation of the on the A motif, with that the binding In TAF7 phosphorylation not on the A motif, has distinct binding report that BRD4 phosphorylates both the viral integrase (MLVIN) and the cellular histone lysine methyltransferase, NSD3, which bind to the ET domain. these provide important new insights into BRD4 structure and domains of BRD4 have been The amino terminal half of the mouse BRD4, a molecule, two that bind acetylated histone BRD4 to as as other acetylated proteins (Fig. 1A). The carboxy-terminal half of BRD4 the domain, the and the binding site for as as other factors (Fig. B.N. Gegonne A. Chen Q. Meerzaman D. A. Ozato K. Singer D.S. BRD4 is a histone that from Biol. 2016; PubMed Scopus Google Scholar). analysis has been for the two and ET domain (7Filippakopoulos P. Picaud S. Mangos M. Keates T. Lambert J.-P. Barsyte-Lovejoy D. Felletar I. Volkmer R. Müller S. Pawson T. Histone recognition and large-scale structural analysis of the human bromodomain family.Cell. 2012; 149: 214-231Abstract Full Text Full Text PDF PubMed Scopus (1030) Google Y.J. Umehara T. Inoue M. Saito K. Kigawa T. Jang M.K. Ozato K. Yokoyama S. Padmanabhan B. Güntert P. Solution structure of the extraterminal domain of the bromodomain-containing protein BRD4.Protein Sci. 2008; 17: 2174-2179Crossref PubMed Scopus (39) Google Scholar). of BRD4’s structure that the half of the is the terminal half is extensively We have reported that BRD4 is a kinase that phosphorylates RNA polymerase II (Pol and the kinase domain to the amino terminal segment of BRD4 (4Devaiah B.N. Lewis B.A. Cherman N. Hewitt M.C. Albrecht B.K. Robey P.G. Ozato K. Sims R.J. Singer D.S. BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 6927-6932Crossref PubMed Scopus (247) Google Scholar). In to BRD4 is an atypical kinase that not have a active site (4Devaiah B.N. Lewis B.A. Cherman N. Hewitt M.C. Albrecht B.K. Robey P.G. Ozato K. Sims R.J. Singer D.S. BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 6927-6932Crossref PubMed Scopus (247) Google Scholar). We have shown that BRD4 phosphorylates the carboxy-terminal domain of RNA polymerase II which of of the The plays a in transcription initiation and elongation through the phosphorylation of its and by multiple BRD4 (4Devaiah B.N. Lewis B.A. Cherman N. Hewitt M.C. Albrecht B.K. Robey P.G. Ozato K. Sims R.J. Singer D.S. BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 6927-6932Crossref PubMed Scopus (247) Google D. M. The RNA polymerase II carboxy-terminal domain (CTD) PubMed Scopus Google A. K. kinases RNA polymerase II in a Chem. Biol. 2019; PubMed Scopus Google Scholar). BRD4 phosphorylates the of Pol II Ser2 and phosphorylates to of the L. D. K. B.N. R. K. RNA polymerase II regulates activity to 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). Although the kinase activity of BRD4 has been its have not been determined. the characterization of BRD4 kinase activity, the for two of its Pol II and BRD4 has both and (Fig. the of both Pol II and TAF7 as (5Devaiah B.N. Singer D.S. Cross-talk among RNA polymerase II kinases modulates C-terminal domain phosphorylation.J. Biol. Chem. 2012; 287: 38755-38766Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). of BRD4 in or of the TAF7 in with (Fig. both These that the binding of its substrates. to the BRD4 kinase activity in to the activity. We on to the of of In the of BRD4 with a of as assessed by the of (Fig. We a and of the that BRD4 for its the of two of BRD4’s the and BRD4 phosphorylation of the Pol II assessed an to which the phosphorylation with a of (Fig. an of but also the that a of the BRD4 phosphorylation of the C-terminal of TAF7, which its phosphorylation with a of of a of (Fig. The results the that BRD4 is an atypical kinase, BRD4 kinase activity a segment for activity, a of and and As the N-terminal half of BRD4 encompasses the two as as the A and B motifs, BID domain, ET and SEED kinase activity, the C-terminal half has kinase activity (4Devaiah B.N. Lewis B.A. Cherman N. Hewitt M.C. Albrecht B.K. Robey P.G. Ozato K. Sims R.J. Singer D.S. BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 6927-6932Crossref PubMed Scopus (247) Google Scholar). have extended these to further the kinase domain. with and phosphorylated (Fig. the active that BRD4 its kinase activity, through A further to a to segment activity, mapping the kinase activity within this region (Fig. of the SEED domain from BRD4 to activity and The for this is not further the kinase activity within the to a BRD4 of the segment 351 to which bromodomain 2 and the domains (BD2-B-BID) (Fig. Deletion of this region BRD4’s and phosphorylation by (Fig. that the domain is for BRD4’s kinase activity. However, within the the B motif is not for kinase activity (Fig. The indirectly the kinase domain. directly that the 351 to segment is for kinase activity, of BRD4 the to to and to the and for to and to the or TAF7 (Fig. to to and These results both the kinase domain and sites of phosphorylation within this The by is with of phosphorylation of the BID domain chromatin binding and for Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the or TAF7 as the the to the and both to and to TAF7 and these results that 351 to which the and BID is both and for kinase activity. The of the to to the CTD, but not TAF7, that the site maps to the to In the site maps to the region to The of binding sites of the and TAF7 on BRD4 is with in The that BRD4 bind to of the is with of BRD4 as a scaffold and report that binds to the C-terminal domain of BRD4 (6Devaiah B.N. Mu J. Akman B. Uppal S. Weissman J.D. Cheng D. Baranello L. Nie Z. Levens D. Singer D.S. MYC protein stability is negatively regulated by BRD4.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 13457-13467Crossref PubMed Scopus (36) Google Scholar). The ET domain of BRD4 is conserved among BET proteins and known to with the MLV integrase and NSD3, a histone lysine NMR of the BRD4 ET domain that BRD4 binds from both MLV integrase and S. M. of the ET domain to a C-terminal motif from a conserved of Natl. Acad. Sci. U. S. A. 2016; PubMed Scopus Google Q. L. T. of transcriptional regulator recognition by the ET domain of 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). These to these also be for BRD4 BRD4 phosphorylated both and Thus, BRD4 is of phosphorylating MLV and that bind to the ET domain, and TAF7 that bind to the N-terminal as as which binds to C-terminal domain, that BRD4 kinase activity is of the substrate-binding these results that the BRD4 kinase activity maps to a region 351 and and is of both and of a of binding to sites on the molecule. Although BRD4 phosphorylates the Pol II CTD, the of Pol II phosphorylation by BRD4 that a to a BRD4 and the be little is known about the biophysical properties of BRD4, is that the C-terminal half is (Fig. We of in a of 156 BRD4 be to from a with a factor of purified BRD4 as a with a factor of (Fig. to a of as by of BRD4 on there a BRD4 a also kD (Fig. These particularly in of that BRD4 as a with a of 156 kD in as be of a of (Fig. these BRD4 exists in an BRD4 in or BRD4 the BRD4 with the of and which and from an with and not the of BRD4 in that BRD4 not the of BRD4 with or which have been to the The of 2 with to the of BRD4 in (Fig. These results that BRD4 exists in an BRD4 is to be an protein, is to and a for a protein, which also be with its in and further the conformation of BRD4 in sedimentation analytical which to the properties of BRD4 the of factors of the of sedimentation of BRD4 is shown in of sedimentation from to in a sedimentation coefficient with a of S and a frictional of (Fig. and extended conformation the of BRD4 in and its in a or dimeric protein with of S or which with the frictional of or the frictional for BRD4 of and the in are with the that BRD4 is a in on the of the for dimerization to be in the further the structure of BRD4, both the and sedimentation in of two of BRD4, the region reported to dimerization of As shown in of the domain or the B motif in with factors of and with the BRD4 factor of is a be for BRD4 of and a in the sedimentation in of the two to that of on (Fig. these to the that BRD4 in exists as an extended Importantly, these further that dimerization is through by the B motif of the molecule. the biophysical properties of BRD4 the and of BRD4 and purified in by We the of the in In the with an of It in the with a factor of to a of (Fig. A and in a sedimentation coefficient of S and a frictional coefficient of a conformation (Fig. and these that the is a extended The sedimentation coefficient for and BRD4 that are and for (Fig. of BRD4 and an of are shown in be the two from a BRD4 and and S a and a from in the The is to in BRD4 but in the a The sedimentation coefficient (Fig. a of in the be to and the in the by ratio. a be in the of the sedimentation coefficient of the from S to S. results in of S for BRD4 to S in the is to the of the of S to to S and S as the to and (Fig. of sedimentation is with a binding by and The extended sedimentation coefficient of the of a of Thus, these results a BRD4 and CTD, of and by also the that the to with for both BRD4 and A. A. K. B.J. D.S. J. and PubMed Google M. M. T. P. RNA polymerase II through carboxy-terminal domain Biol. PubMed Scopus Google Scholar). The on in of BRD4 biophysical properties and kinase activity, leading to the of the in vivo of the BRD4 We that cellular of BRD4 but not of the kinase in phosphorylation of Ser2 of the Pol II CTD, and the this with BRD4, the to 598, or and the on in vivo of phosphorylation of Pol II and determined. As of BRD4 to of phosphorylation of (Fig. In of the to BRD4 into not of phosphorylation of the BRD4 the for the 351 to region for in vivo BRD4 kinase activity. the kinase activity of BRD4 is to its phosphorylation of in BRD4 contributes to the regulation of transcription, both as a scaffold that recruits transcriptional the transcription elongation factor and as an active kinase that transcriptional and the BRD4 phosphorylates the Pol II on a that is associated with and transcriptional Although other Ser2 kinases in transcriptional elongation in BRD4 is the Ser2 kinase in and (4Devaiah B.N. Lewis B.A. Cherman N. Hewitt M.C. Albrecht B.K. Robey P.G. Ozato K. Sims R.J. Singer D.S. BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 6927-6932Crossref PubMed Scopus (247) Google Scholar). BRD4 kinase also contributes to transcriptional elongation through its phosphorylation of the transcription factor TAF7 (5Devaiah B.N. Singer D.S. Cross-talk among RNA polymerase II kinases modulates C-terminal domain phosphorylation.J. Biol. Chem. 2012; 287: 38755-38766Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Importantly, BRD4 not transcription, also phosphorylates MYC protein leading to its degradation (6Devaiah B.N. Mu J. Akman B. Uppal S. Weissman J.D. Cheng D. Baranello L. Nie Z. Levens D. Singer D.S. MYC protein stability is negatively regulated by BRD4.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 13457-13467Crossref PubMed Scopus (36) Google Scholar). Thus, the by which BRD4 these functions is In this BRD4 kinase activity maps to a region the segment of the (Fig. have that substrate-binding sites the of the BRD4 molecule, the ET domain. We have also that BRD4 in exists in an extended that on the of the B motif and that directly interacts with its Pol II Although there have been on the in conformation of the of BRD4, are the to that BRD4 is an extended in with high frictional that as assessed by and These are with which reported dimerization of BRD4 in both by and with R. Q. J. J. Bromodomain protein associated with acetylated chromatin is important for of chromatin Biol. Chem. 2012; 287: Full Text Full Text PDF PubMed Scopus Google Scholar). that dimerization of BRD4 is on the B motif is also with an report that the B motif of a BET protein, contributes to dimerization (9Garcia-Gutierrez P. Mundi M. Garcia-Dominguez M. Association of bromodomain BET proteins with chromatin requires dimerization through the conserved motif B.J. Cell Sci. 2012; 125: PubMed Scopus Google Scholar). of the B motif not kinase activity in but in vivo (6Devaiah B.N. Mu J. Akman B. Uppal S. Weissman J.D. Cheng D. Baranello L. Nie Z. Levens D. Singer D.S. MYC protein stability is negatively regulated by BRD4.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 13457-13467Crossref PubMed Scopus (36) Google dimerization contributes to BRD4’s in vivo The that BRD4 is in an extended the N-terminus of BRD4 is two bromodomains, a with the and of BRD4’s and BRD4’s functions is its to a of substrates. In the have and the with one of its the Pol II CTD. The of of BRD4 with is a for proteins M. M. T. P. RNA polymerase II through carboxy-terminal domain Biol. PubMed Scopus Google Scholar). As by both and the the of the in is the that these not as in transcription in mapping of BRD4 kinase activity are with the binding to a segment of BRD4 amino acids and this region encompasses the A motif, which has been conserved is known about its We that the A motif contributes to BRD4’s regulation of transcription through an with the Pol II CTD, leading to its In to the CTD, TAF7 phosphorylation by BRD4 not on the A motif. Thus, a the segment of BRD4 and phosphorylates TAF7 but not the CTD. Although binding site on BRD4 has not been is to bind to the ET domain, MLV and of the ET domain not TAF7 The phosphorylation of the TAF7 to the CTD, its binding site and of this The of of BRD4 and the kinase activity to a region of BRD4 and which the active site and as The to segment is A BRD4 of 351 to has kinase activity, a from to is of The that the kinase activity maps to a segment of the is with the that BRD4 is an atypical the kinase domain bromodomain 2 which is a binding site for acetylated histone the kinase domain that the binding of BRD4 to chromatin its kinase activity. as have BRD4 is to MYC protein (6Devaiah B.N. Mu J. Akman B. Uppal S. Weissman J.D. Cheng D. Baranello L. Nie Z. Levens D. Singer D.S. MYC protein stability is negatively regulated by BRD4.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 13457-13467Crossref PubMed Scopus (36) Google Scholar). of the kinase domain to a of both Pol II and MYC phosphorylation in its In the have the kinase domain of BRD4 within its domain. The biophysical characterization of BRD4 that the of BRD4 structure to with and multiple which bind to within the molecule. These of the biophysical and of BRD4 to its function in vivo and to the of BRD4 and to on BRD4 in and into to viral The BRD4 protein into from on BRD4. The BRD4 as (6Devaiah B.N. Mu J. Akman B. Uppal S. Weissman J.D. Cheng D. Baranello L. Nie Z. Levens D. Singer D.S. MYC protein stability is negatively regulated by BRD4.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 13457-13467Crossref PubMed Scopus (36) Google the on BRD4. TAF7 with as A. Weissman J.D. M. Singer D.S. A transcription initiation Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). BRD4 purified cell from and with the (4Devaiah B.N. Lewis B.A. Cherman N. Hewitt M.C. Albrecht B.K. Robey P.G. Ozato K. Sims R.J. Singer D.S. BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 6927-6932Crossref PubMed Scopus (247) Google Scholar). on a and proteins in and by to the of The and of assessed on an and by analysis of and to purified as purified as S. M. phosphorylates the C-terminal domain of RNA polymerase II for transcription Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the protein and purified The by for the in 2 and The proteins on a and in and and purified that the not for kinase BRD4 in as but purified with the The of these protein as TAF7 and and purified and purified as with a of 2 N. A. J. N. of with integrase and PubMed Scopus Google A. N. A. N. BET proteins transcription Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and purified with a of The and of the BRD4, B and to and by on a on The and with 4 with a of with and The with factors to P. S. A for the analysis of protein and 2012; PubMed Scopus Google Scholar). In of purified BRD4 a to and the by to the in to the with an with an with to BRD4 a from to in and In into the of and of in the the to and and in in 2 with a of with the sedimentation coefficient in P. analysis of by sedimentation and J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). and to with the analysis of the to of the the to the and the frictional coefficient to the of from BRD4 with factors for of to the in with the by A. P. analytical J. PubMed Scopus Google Scholar). In kinase as (4Devaiah B.N. Lewis B.A. Cherman N. Hewitt M.C. Albrecht B.K. Robey P.G. Ozato K. Sims R.J. Singer D.S. BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 6927-6932Crossref PubMed Scopus (247) Google Scholar). in the kinase and with The kinase for The by in or in the of phosphorylation by a the to and in with of the and to or in the and cell with through on and for The II Pol and the and from are within the and the The that they have of with the of this The and Uppal for of the We also the for of We also Thompson and for and the of the Singer for throughout these A. K. J. D. B. N. and D. S. S. A. K. J. D. and D. S. S. A. K. J. D. B. N. and P. S. R. L. A. K. J. D. and D. S. S. R. L. by J. D. A. K. B. N. and D. S. S. by the of the for The is the of the and not the of the of

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