Cross-talk among RNA polymerase II kinases modulates C-terminal domain phosphorylation

Abstract

The RNA polymerase II (Pol II) C-terminal domain (CTD) serves as a docking site for numerous proteins, bridging various nuclear processes to transcription. The recruitment of these proteins is mediated by CTD phospho-epitopes generated during transcription. The mechanisms regulating the kinases that establish these phosphorylation patterns on the CTD are not known. We report that three CTD kinases, CDK7, CDK9, and BRD4, engage in cross-talk, modulating their subsequent CTD phosphorylation. BRD4 phosphorylates PTEFb/CDK9 at either Thr-29 or Thr-186, depending on its relative abundance, which represses or activates CDK9 CTD kinase activity, respectively. Conversely, CDK9 phosphorylates BRD4 enhancing its CTD kinase activity. The CTD Ser-5 kinase CDK7 also interacts with and phosphorylates BRD4, potently inhibiting BRD4 kinase activity. Additionally, the three kinases regulate each other indirectly through the general transcription factor TAF7. An inhibitor of CDK9 and CDK7 CTD kinase activities, TAF7 also binds to BRD4 and inhibits its kinase activity. Each of these kinases phosphorylates TAF7, affecting its subsequent ability to inhibit the other two. Thus, a complex regulatory network governs Pol II CTD kinases.

FIGURE 1.

BRD4 and CDK9 engage in cross-talk. A, BRD4 and CDK9 phosphorylate each other. BRD4 (0.3 μg), or equimolar amounts of BRD4 mutants were used in an in vitro kinase assay alone or with 0.15 μg of PTEFb. Maps of the BRD4 proteins used are shown (upper) with an autoradiograph of the phosphorylated proteins (lower). Numbers below each lane show the quantitation of total phosphorylation relative to autophosphorylation of each protein. B, BRD4 phosphorylates the CDK9 D167N kinase-dead mutant. Autoradiograph showing the results of a kinase reaction with 50 ng of wild type CDK9 or D167N mutant protein incubated with or without 0.2 μg of BRD4. C, BRD4 phosphorylates CDK9 in the presence of flavopiridol. Autoradiograph showing phosphorylation of BRD4 (0.2 μg) or PTEFb (0.1 μg) used in a kinase assay individually or together, with or without 1 μm flavopiridol. D, BRD4 phosphorylates CDK9 at both Thr-29 and Thr-186 sites in vivo. Kinase reactions were done in the presence or absence of flavopiridol (1 μm) with 0.15 μg of PTEFb alone or with 0.3 μg of BRD4 (first to third lanes). Reactions were also done with 0.15 μg of PTEFb and 0.15, 0.3, and 0.6 μg of BRD4 in the presence of flavopiridol (fourth to sixth lanes). The samples were immunoblotted with α-CDK9, phospho-specific α-CDK9 Thr(P)-186 or α-CDK9 Thr(P)-29. E, BRD4 is required for phosphorylation of CDK9 Thr-29 and Thr-186 sites in vivo. Whole cell extracts of HeLa cells transfected with control siRNA or BRD4-specific siRNA were immunoblotted with α-BRD4, α-CDK9, phospho-specific α-CDK9 Thr(P)-186, or α-CDK9 Thr(P)-29. F, BRD4 phosphorylates CDK9 at both Thr-29 and Thr-186 sites in vivo. Immunoprecipitates (IP) from HeLa cells untransfected or transfected with 2.5 μg of HA-D167N CDK9 alone or co-transfected with 1.0, 2.5, or 5.0 μg of BRD4 immunoblotted with α-CDK9, Thr(P)-29, or Thr(P)-186 α-phospho-CDK9 (upper). Immunoblots of whole cell extracts (WCE) show BRD4 levels relative to tubulin control (lower).

FIGURE 2.

CDK7 interacts with and phosphorylates BRD4. A, BRD4 associates with CDK7 in vivo. BRD4 was immunoprecipitated from HeLa nuclear extract using α-BRD4 and immunoblotted with α-CDK7. B, BRD4 interacts directly with CDK7. BRD4 (0.1, 0.4, and 0.6 μg) was pulled down with 0.2 μg of His-CDK7 (CAK) immobilized on Ni-NT beads. Beads alone or 0.6 μg of BRD4, loaded directly on the gel, are shown as negative and positive controls. C, CDK7 phosphorylates BRD4 in the presence of apigenin. Autoradiograph showing phosphorylation of BRD4 (0.3 μg) or CDK7/CAK (0.15 μg) in kinase assays individually or in combination, with or without 100 μm apigenin. D, CDK7 phosphorylates BRD4 at multiple sites. Autoradiograph of in vitro kinase assays of BRD4 (0.3 μg) or equimolar amounts of BRD4 mutants individually or with 0.12 μg of CAK. Numbers below each lane shows the quantitation of total phosphorylation relative to autophosphorylation of each protein. E, BRD4 is phosphorylated equally by the CAK complex and the holo-TFIIH complex. Autoradiograph showing wild type BRD4 (0.3 μg) and the BRD4 ΔN kinase-dead mutant (0.15 μg) phosphorylated by equimolar amounts of purified recombinant CAK (0.1 μg) or purified native TFIIH (0.26 μg). F, CDK7 phosphorylates BRD4 in vivo. HeLa cells were transfected with 3 μg of human BRD4 or a BRD4 mutant incapable of binding PTEFb (FEE-AAA) and were treated or not with (+)-JQ1. Equal amounts of BRD4 immunoprecipitated from these cells were immunoblotted with α-phosphoserine or α-BRD4 (upper). Quantitation of averages ± S.E. from two independent experiments (lower). G, auto- or transphosphorylation of BRD4 is not affected by the JQ1 inhibitor. Autoradiograph showing BRD4 (0.3 μg) either autophosphorylated, or transphosphorylated by PTEFb (0.1 μg) and CAK (0.1 μg), in the presence or absence of 250 nm JQ1.

FIGURE 3.

Cross-phosphorylation between CDK9, CDK9, and BRD4 affects their Pol II CTD kinase activities. A, the kinase activities of BRD4 and PTEFb exhibit dose-dependent synergy in phosphorylating the Pol II CTD. Autoradiograph of an in vitro kinase assay where GST-CTD (0.1 μg) was phosphorylated with either PTEFb alone (50, 100, or 200 ng; lanes 1–3), BRD4 alone (100, 200, or 400 ng; lanes 4–6), or 50 ng of PTEFb with BRD4 (100, 200, or 400 ng; lanes 7–9). Numbers below the lanes indicate quantitation of relative Pol II CTD phosphorylation. B, BRD4 activates or represses CDK9 phosphorylation of the Pol II CTD depending on the relative BRD4 concentration. Left, experimental design. Right, upper panel, autoradiograph of GST-CTD (0.1 μg) phosphorylated by 0.1 μg of PTEFb pre-phosphorylated by either 0.2 (left panel) or 0.6 μg (right panel) of BRD4. Right, lower panels, immunoblots with α-CDK9, α-Thr(P)-29, or α-Thr(P)-186 phospho-CDK9 antibodies, showing patterns of PTEFb phosphorylation following: 1 h of autophosphorylation in the absence of BRD4 or pre-phosphorylation by BRD4 for 10 min, 30 min, or 1 h. (Note that PTEFb alone generates both IIA and IIO forms of phosphorylated CTD, whereas BRD4 primarily generates the IIA form.) C, BRD4 CTD kinase activity is affected when it is phosphorylated by other kinases. BRD4 (0.3 μg) that was unphosphorylated, autophosphorylated, or pre-phosphorylated with CAK (0.1 μg), PTEFb (0.1 μg), or both was used to phosphorylate 0.1 μg of GST-CTD in the presence of 100 μm roscovitine and 1 μm flavopiridol. Numbers below the lanes indicate quantification of relative phosphorylation of the CTD. D, phosphorylation of BRD4 by CDK7 suppresses the CTD kinase activity of BRD4 in vitro. Top, experimental design; BRD4 (0.3 μg) was either autophosphorylated for 1 h or phosphorylated by CDK7/CAK (0.1 μg) for 10 min, 30 min, and 1 h in a kinase reaction with unlabeled ATP; control reactions were run in parallel with [γ-³²P]ATP (lower panel). The BRD4 pre-phosphorylated with unlabeled ATP was used in secondary kinase reactions with 0.1 μg of GST-CTD, [γ-³²P]ATP, and 100 μm roscovitine (upper panel). E, in vivo phosphorylation of BRD4 by CDK7 suppresses the Pol II CTD kinase activity of BRD4. Whole cell extracts (WCE) of HEK293 cells untransfected (control) or transfected with 5 μg of CDK7 (CDK7 OE) were immunoblotted with α-CTD Ser-2P, α-CTD Ser-5P, α-Pol II, α-BRD4, α-CDK9, α-CDK7, or α-tubulin (upper). Equal amounts of BRD4 immunoprecipitated (IP) from untransfected or CDK7-transfected cells were immunoblotted with α-phosphoserine or α-BRD4 (lower). Numbers below the lanes indicate quantitation of immunoblots. F, BRD4 phosphorylation of CDK9 is inhibited by TFIIH. In vitro kinase reactions were done with PTEFb (0.1 μg) in the presence of either TFIIH (0.4 μg), BRD4 (0.2 μg), or both and immunoblotted with α-CDK9, Thr(P)-29, and Thr(P)-186 α-phospho-CDK9.

FIGURE 4.

TAF7 binds and is phosphorylated by BRD4. A, TAF7 associates with BRD4 in vivo. BRD4 was immunoprecipitated (IP) from HeLa nuclear extract using α-BRD4 and immunoblotted with α-TAF7. B, TAF7 interacts directly with BRD4. BRD4 (0.5, 1.0, and 1.5 μg) was pulled down with 0.5 μg of GST-TAF7 immobilized on GST beads and immunoblotted with α-BRD4. BRD4 (1.5 μg) with GST beads alone, or insect cell control extract (CE) made in parallel with recombinant BRD4 were used as negative controls. C, TAF7 binds BRD4 kinase subdomains. Upper, map of BRD4 proteins used. Middle, α-TAF7 immunoblot of 0.3 μg of GST-TAF7 (input) recovered by pull-down with 1.0 μg of wild type FLAG-BRD4 or equimolar amounts of FLAG-BRD4 mutants. Lower, quantitation of recovered GST-TAF7. D, BRD4 interacts with the central serine-rich region of TAF7. Upper, α-BRD4 immunoblot of 0.6 μg of BRD4 recovered by pull-down with 1 μg of full-length GST-TAF7 or equimolar amounts of GST-TAF7 sections. Middle, quantitation of recovered BRD4. Lower, map of TAF7 proteins used showing interaction sites of kinases. E, TAF7 is phosphorylated by BRD4. Autoradiograph of a kinase assay with TAF7 (0.15 μg) alone (first lane) or with recombinant murine BRD4 (0.3 μg) or human BRD4 immunoprecipitated from HeLa nuclear extract, with or without 1 μm flavopiridol and 100 μm roscovitine. F, TAF7 phosphorylation is dependent on BRD4 kinase domains. Autoradiograph showing 0.1 μg of TAF7 phosphorylated by 0.3 μg of BRD4 WT or equimolar amounts of BRD4 mutants. G, BRD4 phosphorylates the C-terminal end of TAF7. Autoradiograph showing 0.75 μg of full-length GST-TAF7 or equimolar amounts of GST-TAF7 sections phosphorylated by 0.6 μg of BRD4 (additional band migrating at ∼98 kDa is most likely hyper-phosphorylated TAF7 accentuated in the 15% gel used here). H, phosphorylation of BRD4 modulates its ability to phosphorylate TAF7. BRD4 (0.3 μg) that was unphosphorylated, autophosphorylated, or pre-phosphorylated with CAK (0.1 μg), PTEFb (0.1 μg), or both was used to phosphorylate 0.1 μg of TAF7 in the presence of 100 μm roscovitine and 1 μm flavopiridol.

FIGURE 5.

TAF7 inhibits BRD4 phosphorylation of Pol II CTD. A, TAF7 inhibits BRD4 phosphorylation of Pol II CTD in vitro. Autoradiograph of a kinase assay with 0.1 μg of GST-CTD phosphorylated by 0.2 μg of BRD4 in the absence or presence of increasing amounts of TAF7 (0.1 and 0.2 μg). B, TAF7 and Pol II CTD do not compete in binding to BRD4. BRD4 (0.5 μg) was pulled down with 0.2 μg of GST-CTD immobilized on GST beads and 65 μm ATP in the absence or presence of increasing amounts of TAF7 (0.2 and 0.4 μg; ∼1:1 and 1:3 CTD:TAF7) and immunoblotted with α-BRD4. TAF7 bound to BRD4 was detected with α-TAF7. C, specific domains on TAF7 influence its inhibition of BRD4 CTD kinase activity. Autoradiograph showing 0.1 μg of GST-CTD phosphorylated by 0.2 μg of BRD4 in the absence or presence of 0.1 μg of full-length TAF7 or equimolar amounts of TAF7 AD, MID, and C-terminal sections. Numbers below the lanes indicate quantification of relative phosphorylation. D, TAF7 inhibits Pol II CTD Ser-2 phosphorylation by BRD4 in vivo. Five micrograms of human BRD4, BRD4 FEE-AAA mutant, or vector alone (control) were transfected into HeLa cells with or without co-transfection with increasing amounts of TAF7 (2.5 and 5.0 μg). Whole cell extracts of these cells were immunoblotted with α-CTD Ser-2P, α-TAF7, α-BRD4, and α-Tubulin control. E, phosphorylation of TAF7 by Pol II CTD kinases selectively affects its inhibitory potential. TAF7 (0.5 μg) was pre-phosphorylated or not by TAF1 (0.5 μg), BRD4 (0.5 μg), CAK (0.25 μg), or PTEFb (0.25 μg) for 1 h with unlabeled ATP. Nonphosphorylated or pre-phosphorylated TAF7, heat treated for 15 min at 70 °C, was added to kinase reactions with [γ-³²P]ATP, 0.1 μg of GST-CTD and CAK (0.1 μg; in the presence of 100 μm Apigenin), BRD4 (0.2 μg: in the presence of 100 μm roscovitine and 1 μm flavopiridol), or PTEFb (0.1 μg) (upper panels). Numbers below the lanes indicate quantitation of relative Pol II CTD phosphorylation. The α-TAF7 immunoblot (lower panel) documented equal amounts of unphosphorylated and pre-phosphorylated TAF7 in the reactions.

FIGURE 6.

Summary of Pol II CTD kinase cross-talk pathways. CDK7, a CTD Ser-5/7 kinase, phosphorylates BRD4 and inhibits its Pol II CTD kinase activity. BRD4, a CTD Ser-2 kinase, phosphorylates CDK9 either activating or inactivating its Pol II CTD kinase activity depending on their relative concentrations. CDK9, a CTD Ser-2/5 kinase, phosphorylates BRD4 modestly activating its kinase activity. All three kinases autophosphorylate as well as phosphorylate TAF7. Phosphorylation of TAF7 by each of the kinases affects its subsequent effect on the others. Phosphorylation, activation, and repression are indicated by stars (), arrowheads (), and cross-bars (⊣), respectively.