A kinase-independent activity of Cdk9 modulates glucocorticoid receptor-mediated gene induction

Abstract

A gene induction competition assay has recently uncovered new inhibitory activities of two transcriptional cofactors, NELF-A and NELF-B, in glucocorticoid-regulated transactivation. NELF-A and -B are also components of the NELF complex, which participates in RNA polymerase II pausing shortly after the initiation of gene transcription. We therefore asked if cofactors (Cdk9 and ELL) best known to affect paused polymerase could reverse the effects of NELF-A and -B. Unexpectedly, Cdk9 and ELL augmented, rather than prevented, the effects of NELF-A and -B. Furthermore, Cdk9 actions are not blocked either by Ckd9 inhibitors (DRB or flavopiridol) or by two Cdk9 mutants defective in kinase activity. The mode and site of action of NELF-A and -B mutants with an altered NELF domain are similarly affected by wild-type and kinase-dead Cdk9. We conclude that Cdk9 is a new modulator of GR action, that Ckd9 and ELL have novel activities in GR-regulated gene expression, that NELF-A and -B can act separately from the NELF complex, and that Cdk9 possesses activities that are independent of Cdk9 kinase activity. Finally, the competition assay has succeeded in ordering the site of action of several cofactors of GR transactivation. Extension of this methodology should be helpful in determining the site and mode of action of numerous additional cofactors and in reducing unwanted side effects.

Figure 1

Full length and chimeric NELF-B and full length NELF-A similarly influence GR transactivation by acting at two sites. Graphical analyses of competition assays with four amounts of chimeric NELF-B (A), full length wild-type NELF-B (B), and NELF-A (C) plasmids each with four concentrations of GREtkLUC were conducted in U2OS cells and a constant amount of transfected GR plasmid as described in Experimental Procedures. Plots of EC₅₀/A_max vs NELF were best fit with quadratic equations. Similar results were obtained in two additional independent experiments for chimeric NELF-B. Panels B (wild-type NELF-B) and C (NELF-A) are averaged data (±standard error of the mean) of four and six experiments, respectively, after normalization to the LUC activity with the smallest amount of GREtkLUC reporter and the largest amount of NELF plasmid.

Figure 2

Cdk9 is a decelerator that does not reverse the effects of NELF-A or NELF-B and is not inactivated by chemical inhibitors. (A–G) Cdk9 augments the inhibitory effects of NELF-A and NELF-B. Competition assays of Cdk9 with NELF-A (A–E) or NELF-B (F and G) were performed with the indicated amounts of Cdk9 and NELF plasmids. Graphs of A_max vs NELF-A (A) or NELF-B (F) illustrate the additive effects of Cdk9 with each NELF. The other graphs were plotted as in Figure 1. Similar results were obtained in five additional independent experiments. (H–K) Chemical inhibitors of Cdk9 do not block decelerator activity of Cdk9. Assays were performed as described in the legend of Figure 1 with the indicated amounts of Cdk9 and DRB (H and I) or flavopiridol (J and K) and plotted as A_max/EC₅₀ (H, I, and K) or 1/EC₅₀ (J). The amount of GREtkLUC reporter was 100 ng in all panels except for F and G, which used 30 ng. Similar results were obtained in two to five additional independent experiments.

Figure 3

Dominant negative kinase-dead Cdk9 has activity identical to that of wt Cdk9. (A) dnCdk9 augments the inhibitory effects of NELF-A. Competition assays with the indicated amounts of dnCdk9 and NELF-A plasmids were performed and plotted as in Figure 1. Similar results were obtained in four additional independent experiments. (B and C) Competition assays were performed as in Figure 1 with 0, 3, 6, and 10 ng each of wt Cdk9 and dnCdk9 plasmids and a constant amount of GR (0.5 ng) and GREtkLUC (100 ng) plasmids. After adjustment for the 3-fold higher level of protein expression of dnCdk9 compared to that of wt Cdk9, plots of A_max/EC₅₀ (A) and EC₅₀/A_max (B) vs the combined amounts of wt and dnCdk9 were constructed as described in the text. Similar results were obtained in a second independent experiment.

Figure 4

Kinase-defective Cdk9 mutants retain activity of wt Cdk9 with NELF-A and NELF-B in different cells. Competition assays were performed and plotted as in Figure 1 with the indicated amounts of dnCdk9 and NELF-A plasmids in U2OS cells (A) or 293 cells (B and C), with AcMtCdk9 and NELF-A plasmids in U2OS cells (D), with NELF-B in U2OS cells with dnCdk9 (E) or AcMtCdk9 (F), and with NELF-B in 293 cells with wt Cdk9 (G) or dnCdk9 (H). The amount of reporter plasmid was always 100 ng. The amount of GR plasmid used was 0.5 ng in U2OS cells and 5 ng in 293 cells. The range of linear expression of wt Cdk9 (≤40 ng), dnCdk9 (≤20 ng), and NELF-B (≤20 ng) is higher in 293 than in U2OS cells (data not shown). Similar results were obtained in one to four additional independent experiments except for that for NELF-B with AcMtCdk9 in U2OS cells (F), which was performed only once.

Figure 5

Wild-type and mutant NELF-A and -B activities are not altered by mutations that destroy Ckd9 kinase activity. Competition assays were performed with 0.3 or 0.5 ng of GR plasmid and 30 ng of GREtkLUC, and the results are plotted as in Figure 1 with the indicated amounts of wt Cdk9 and 4mtNELF-A (A and B) or wt NELF-A (C) plasmids or dnCdk9 and 4mtNELF-A (D and E) or wt NELF-A (F) plasmids, in U2OS cells. Similar results were obtained from five to eight independent experiments. (G and H) 4mtNELF-B has reduced potency, relative to that of wt NELF-B, in competition assays with dnCdk9. Competition assays were performed with 0.5 or 5 ng of GR plasmid and 100 ng of GREtkLUC, and A_max/EC₅₀ vs NELF-B was plotted as in Figure 1 with the indicated amounts of wt Cdk9 and wt NELF-A (G) or 4mtNELF-A (H) plasmids. The gray bar with the extending line represents the standard error of the mean and average amount of plasmid required for half-maximal reduction of A_max/EC₅₀ from six experiments. To compensate for the 2.09-fold more efficient expression of 4mtNELF-B protein, compared to that of wt NELF-B [as determined by Western blots (not shown)], the x-axis values of panel H should be multiplied by 2.09. Similar results were obtained from five additional independent experiments.

Figure 6

Cdk9 is a competitive decelerator acting after the site of action of GR and before that of the reporter gene. Competition assays with the indicated amounts of (A and B) Cdk9 and GREtkLUC reporter, (C) Cdk9 and GR, and (D) dnCdk9 and GR plasmids were performed in U2OS cells and the results plotted as in Figure 1. Similar results were obtained in five additional (for A and B) and two additional (for C and D) independent experiments.

Figure 7

Competitive decelerator activity of ELL that is augmented by Cdk9 in a manner independent of Cdk9 kinase activity and reversed by accelerators. Competition assays with 0.5 or 1 ng of GR plasmid, 100 ng of GREtkLUC, and the indicated amounts of (A and B) Cdk9 and ELL, (C and D) dnCdk9 and ELL, (E and F) CBP and ELL, and (G and H) TIF2 and ELL plasmids were performed in U2OS cells and the results plotted as in Figure 1. Wild-type Cdk9, dnCdk9, and ELL are linearly expressed with up to 20 ng of transfected plasmid; CBP is linearly expressed with up to 80 ng of plasmid, so no corrections are needed. TIF2 expression is corrected as described in the text. Similar results were obtained in five (A and B), two (C and D), one (E and F), and three (G and H) additional independent experiments.