Selective Kinase Inhibition Shows That Bur1 (Cdk9) Phosphorylates the Rpb1 Linker In Vivo

Abstract

Cyclin-dependent kinases play multiple roles in RNA polymerase II transcription. Cdk7/Kin28, Cdk9/Bur1, and Cdk12/Ctk1 phosphorylate the polymerase and other factors to drive the dynamic exchange of initiation and elongation complex components over the transcription cycle. We engineered strains of the yeast Saccharomyces cerevisiae for rapid, specific inactivation of individual kinases by addition of a covalent inhibitor. While effective, the sensitized kinases can display some idiosyncrasies, and inhibition can be surprisingly transient. As expected, inhibition of Cdk7/Kin28 blocked phosphorylation of the Rpb1 C-terminal domain heptad repeats at serines 5 and 7, the known target sites. However, serine 2 phosphorylation was also abrogated, supporting an obligatory sequential phosphorylation mechanism. Consistent with our previous results using gene deletions, Cdk12/Ctk1 is the predominant kinase responsible for serine 2 phosphorylation. Phosphorylation of the Rpb1 linker enhances binding of the Spt6 tandem SH2 domain, and here we show that Bur1/Cdk9 is the kinase responsible for these modifications in vivo.

Keywords: Cdk9; P-TEFb; RNA polymerase II; Rpb1; Spt6.

FIG 1

Construction of irreversibly sensitized (IS) kinase strains. (A) Sequence alignments of IS mutant positions (2–4) in human RSK2, Kin28, Bur1, and Ctk1 kinases. The residues mutated to create the “hole” or reactive cysteine are marked by asterisks. (B) Protein expression levels of wild-type and IS mutant kinases, before (0 min) or after 90 min of treatment with 50 μM CMK. Anti-HA blots show epitope-tagged kinases at the expected sizes of Kin28 (35 kDa), Bur1 (74 kDa), and Ctk1 (61 kDa). Rpb1 and Rpb3 are two RNA polymerase II subunits used as loading control bands. Strains used: YSB3216 (Kin28 WT), YSB3221 (Kin28 V21C, L83G), YSB3229 (Bur1 WT), YSB3232 (Bur1 V74C, L149G), YSB3235 (Ctk1 WT), and YSB3237 (Ctk1 V197C, F206G).

FIG 2

Growth inhibition of IS strains on CMK plates. Strains YF702 (wild type), YSB3356 (Kin28-IS), YSB3419 (Bur1-IS), and YSB3444 (Ctk1-IS) were spotted on YPD plates containing 10 μM CMK. Each row shows 3-fold dilutions. Plates were photographed after the indicated number of days at the indicated temperature.

FIG 3

Inhibition of CTD phosphorylations in the IS kinase strains. (A) Time course of CTD phosphorylation levels during CMK inhibition in vivo. The indicated yeast strains were grown to mid-log phase (OD of 1.0) at 30°C. Immediately after taking a sample to serve as the zero time point, CMK was added to a 20 μM final concentration. At each indicated time point, samples were taken and processed for immunoblotting as described in Materials and Methods section. Blots were probed with the indicated antibodies. (B) Transcription complexes assembled in vitro. Yeast nuclear extracts were prepared from the indicated strains and incubated with a DNA template carrying five Gal4-binding sites upstream of the CYC1 core promoter and a G-less cassette. Immobilized templates were isolated and analyzed by immunoblotting as described previously (28).The first lane in each set shows RNApII preinitiation complexes formed in the absence of NTPs. The second and third lanes show elongation complexes formed upon treatment with ATP, UTP, and CTP for 4 min, with the third lane showing the effect of CMK inhibition. The first and second lanes also received DMSO to control for the CMK vehicle.

FIG 4

Analysis of IS kinase inhibition with different CTD antibodies. The indicated yeast strains were analyzed before or after 15 min of inhibition with 20 μM CMK. Blots were probed with the indicated antibodies. TATA-binding protein (TBP) is shown as a loading control. Note that all blots were developed with Pierce SuperSignal West Pico chemiluminescent substrate, except for 4E12 (Ser7-P) and 3D12 (Tyr1-P), which required the Femto maximum-sensitivity substrate. No signal above background was detected with 6G7 (Thr4-P) (not shown). Results from combined multiple experiments are quantitated in Fig. S2 in the supplemental material.

FIG 5

Bur1 phosphorylates Rpb1 linker region residues that mediate interaction with the Spt6 tSH2 domain. (A) Phosphorylation by Bur1 or Ctk1 enhances in vitro binding of Spt6-tSH2 to an Rpb1 linker peptide. MBP fusions to the Rpb1 linker (residues 1456 to 1511) or mutated derivatives thereof were purified and then incubated with partially purified His₁₂-Bur1, His₁₂-Ctk1, or the corresponding mock purification fractions from an untagged strain. Proteins were split and separated by SDS-PAGE. One aliquot was stained with Coomassie blue (top panel) to show the amounts and purity of the MBP-Rpb1 linker proteins. A second aliquot was transferred to nitrocellulose, refolded, and then probed with GST-Spt6 tSH2, which was subsequently detected using antibodies against GST (middle panel, Far-WB). Strong binding to the WT linker peptide was observed after treatment with Bur1, but this was diminished in mutants lacking the sites previously shown to be important for this interaction (25) (S1493 and T1471). Ctk1 also supported binding at a lower level. The asterisk denotes a species found in the Bur1, mock, and, to a lesser extent, Ctk1 preparations that directly bound the anti-GST antibody, as can be seen in a parallel blot with GST-Spt6 tSH2 protein omitted (bottom panel, anti-GST). (B) The indicated yeast strains were analyzed before or after 15 min of inhibition with 20 μM CMK. Blots were probed with the antibodies specific for Rpb1 linker sites (see Fig. S3B in the supplemental material) or Spt5 and Spt6 as loading controls. Note that the Spt6 bands are all from the same exposure of a single blot, but intervening lanes were removed.