CK2 forms a stable complex with TFIIIB and activates RNA polymerase III transcription in human cells - PubMed (original) (raw)

CK2 forms a stable complex with TFIIIB and activates RNA polymerase III transcription in human cells

Imogen M Johnston et al. Mol Cell Biol. 2002 Jun.

Abstract

CK2 is a highly conserved protein kinase with growth-promoting and oncogenic properties. It is known to activate RNA polymerase III (PolIII) transcription in Saccharomyces cerevisiae and is shown here to also exert a potent effect on PolIII in mammalian cells. Peptide and chemical inhibitors of CK2 block PolIII transcription in human cell extracts. Furthermore, PolIII transcription in mammalian fibroblasts is decreased significantly when CK2 activity is compromised by chemical inhibitors, antisense oligonucleotides, or kinase-inactive mutants. Coimmunoprecipitation and cofractionation show that endogenous human CK2 associates stably and specifically with the TATA-binding protein-containing factor TFIIIB, which brings PolIII to the initiation site of all class III genes. Serum stimulates TFIIIB phosphorylation in vivo, an effect that is diminished by inhibitors of CK2. Binding to TFIIIC2 recruits TFIIIB to most PolIII promoters; this interaction is compromised specifically by CK2 inhibitors. The data suggest that CK2 stimulates PolIII transcription by binding and phosphorylating TFIIIB and facilitating its recruitment by TFIIIC2. CK2 also activates PolI transcription in mammals and may therefore provide a mechanism to coregulate the output of PolI and PolIII. CK2 provides a rare example of an endogenous activity that operates on the PolIII system in both mammals and yeasts. Such evolutionary conservation suggests that this control may be of fundamental importance.

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Figures

FIG. 1.

FIG. 1.

Human PolIII transcription is repressed specifically by peptide and chemical inhibitors of CK2. (A) Transcription using HeLa cell PC-B (2 μg) and PC-C (0.7 μg) fractions and a pLeu template (250 ng) after preincubation for 15 min at 30°C with buffer (lanes 1, 6, and 11) or 10, 20, 30, or 40 μg of CK2 phosphoacceptor peptide (RRREEETEEE; lanes 2 to 5, respectively), PKA phosphoacceptor peptide (LRRASLG; lanes 7 to 10, respectively), or Cdc2 phosphoacceptor peptide (RRRPMSPKKKA; lanes 12 to 15, respectively). (B) Transcription using a HeLa cell nuclear extract (15 μg) and a pVA1 template (250 ng) after preincubation for 15 min at 30°C with buffer (lanes 1, 3, and 6) or 30 μg of CK2 phosphoacceptor peptide (lanes 4 and 5) or PKA phosphoacceptor peptide (lanes 7 and 8). Reaction mixtures also contained 1 μl of recombinant CK2 (lanes 2, 5, and 8) or the corresponding buffer. (C) Transcription using a HeLa cell nuclear extract (15 μg) and a pLeu template (250 ng) after preincubation for 15 min at 30°C with buffer (lanes 1, 4, 5, and 8), with 6 and 12 mM 2,3-diphosphoglycerate (lanes 2 and 3, respectively), or with 50 and 100 μM quercetin (lanes 6 and 7, respectively).

FIG. 1.

FIG. 1.

Human PolIII transcription is repressed specifically by peptide and chemical inhibitors of CK2. (A) Transcription using HeLa cell PC-B (2 μg) and PC-C (0.7 μg) fractions and a pLeu template (250 ng) after preincubation for 15 min at 30°C with buffer (lanes 1, 6, and 11) or 10, 20, 30, or 40 μg of CK2 phosphoacceptor peptide (RRREEETEEE; lanes 2 to 5, respectively), PKA phosphoacceptor peptide (LRRASLG; lanes 7 to 10, respectively), or Cdc2 phosphoacceptor peptide (RRRPMSPKKKA; lanes 12 to 15, respectively). (B) Transcription using a HeLa cell nuclear extract (15 μg) and a pVA1 template (250 ng) after preincubation for 15 min at 30°C with buffer (lanes 1, 3, and 6) or 30 μg of CK2 phosphoacceptor peptide (lanes 4 and 5) or PKA phosphoacceptor peptide (lanes 7 and 8). Reaction mixtures also contained 1 μl of recombinant CK2 (lanes 2, 5, and 8) or the corresponding buffer. (C) Transcription using a HeLa cell nuclear extract (15 μg) and a pLeu template (250 ng) after preincubation for 15 min at 30°C with buffer (lanes 1, 4, 5, and 8), with 6 and 12 mM 2,3-diphosphoglycerate (lanes 2 and 3, respectively), or with 50 and 100 μM quercetin (lanes 6 and 7, respectively).

FIG. 1.

FIG. 1.

Human PolIII transcription is repressed specifically by peptide and chemical inhibitors of CK2. (A) Transcription using HeLa cell PC-B (2 μg) and PC-C (0.7 μg) fractions and a pLeu template (250 ng) after preincubation for 15 min at 30°C with buffer (lanes 1, 6, and 11) or 10, 20, 30, or 40 μg of CK2 phosphoacceptor peptide (RRREEETEEE; lanes 2 to 5, respectively), PKA phosphoacceptor peptide (LRRASLG; lanes 7 to 10, respectively), or Cdc2 phosphoacceptor peptide (RRRPMSPKKKA; lanes 12 to 15, respectively). (B) Transcription using a HeLa cell nuclear extract (15 μg) and a pVA1 template (250 ng) after preincubation for 15 min at 30°C with buffer (lanes 1, 3, and 6) or 30 μg of CK2 phosphoacceptor peptide (lanes 4 and 5) or PKA phosphoacceptor peptide (lanes 7 and 8). Reaction mixtures also contained 1 μl of recombinant CK2 (lanes 2, 5, and 8) or the corresponding buffer. (C) Transcription using a HeLa cell nuclear extract (15 μg) and a pLeu template (250 ng) after preincubation for 15 min at 30°C with buffer (lanes 1, 4, 5, and 8), with 6 and 12 mM 2,3-diphosphoglycerate (lanes 2 and 3, respectively), or with 50 and 100 μM quercetin (lanes 6 and 7, respectively).

FIG. 2.

FIG. 2.

Chemical inhibitors of CK2 reduce the expression of PolIII transcripts in proliferating murine fibroblasts. (A) Northern blot analysis of total RNA (10 μg) from A31 cells cultured in 0.5% serum (lanes 1, 3, and 5) or 20% serum (lanes 2, 4, and 6) and treated with 10 μM quercetin (lanes 3 and 4) or 10 μM DRB (lanes 5 and 6). The upper panel shows the blot probed with a B2 gene; the lower panel shows the same blot that has been stripped and reprobed with an ARPP P0 gene. (B) The B2 signals were quantified and normalized against the ARPP P0 signals. Means and standard deviations from three independent experiments are represented graphically.

FIG. 3.

FIG. 3.

Overexpression of kinase-inactive CK2 mutants reduces PolIII transcriptional activity. Transcription was analyzed with the pVA1 template (250 ng) and extracts (20 μg) of RS2.31 (lanes 1 and 2), RS3.22 (lanes 3 and 4), GV7.21 (lanes 5 and 6), and GV13.35 (lanes 7 and 8) cells grown in the presence (even-numbered lanes) or absence (odd-numbered lanes) of tetracycline (Tet). The CK2 catalytic subunit that is overexpressed in each sample is indicated; in each sample, CK2β is overexpressed in parallel. mut, mutant.

FIG. 4.

FIG. 4.

Depletion of CK2β with an antisense oligonucleotide results in decreased tRNA synthesis in human fibroblasts. (A) PCR amplification was carried out with tRNAArg (top), tRNATyr (middle), or glyceraldehyde phosphate dehydrogenase (GAPDH) (bottom) primers and cDNAs prepared from RNAs extracted from IMR-90 cells treated with sense (lane 1), antisense (lane 2), or no (lanes 3 and 4) oligonucleotides and grown in the presence (lanes 1 to 3) or absence (lane 4) of serum. (B) Extracts (30 μg) prepared from IMR-90 cells treated with sense (lane 1), antisense (lane 2), or no (lanes 3 and 4) oligonucleotides and grown in the presence (lanes 1 to 3) or absence (lane 4) of serum were resolved by SDS-polyacrylamide gel electrophoresis and then blotted with CK2β antibody C40420.

FIG. 5.

FIG. 5.

Preassembled PolIII transcription complexes are resistant to CK2 inhibitors. Transcription was analyzed with a HeLa cell nuclear extract (15 μg) and the pVA1 template (250 ng) in the presence of buffer (lanes 1, 4, 7, and 9), 20 μg of CK2 phosphoacceptor peptide (lanes 2 and 5), 20 μg of PKA phosphoacceptor peptide (lanes 3 and 6), or 100 μM quercetin (lanes 8 and 10). Lanes 1 to 3, 7, and 8 show reactions in which the extract was preincubated for 15 min at 30°C with peptide, buffer, or quercetin prior to the addition of pVA1 and nucleotides. Lanes 4 to 6, 9, and 10 show reactions in which the extract was preincubated for 15 min at 30°C with pVA1 before peptide, buffer, or quercetin was added with the nucleotides.

FIG. 6.

FIG. 6.

Endogenous TFIIIB is coimmunoprecipitated from HeLa cells with endogenous CK2. (A) HeLa cell extract (150 μg) was immunoprecipitated (IP) with anti-BRF antibody 128 (lane 1), anti-RB antibody C-15 (lane 2), anti-CK2α antibody H-286 (lane 3), or anti-TAFI48 antibody M-19 (lane 4). Precipitates were resolved by SDS-polyacrylamide gel electrophoresis and then analyzed by Western blotting with anti-BRF antibody 330. (B) HeLa cell extract (150 μg) was immunoprecipitated in the presence of buffer (lanes 1 and 4), 100 μM quercetin (lanes 2 and 5), or 400 μM DRB (lanes 3 and 6) with anti-CK2α antibody H-286 (lanes 1 to 3) or anti-TAFI48 antibody M-19 (lanes 4 to 6). Precipitates were resolved by SDS-polyacrylamide gel electrophoresis and then blotted with anti-BRF antibody 128. IgG, immunoglobulin G.

FIG. 7.

FIG. 7.

BRF is phosphorylated in vivo. (A) CHO cells in 10% FCS were transiently transfected with pcDNA3HA.BRF (10 μg) and labeled 48 h later with [32P]orthophosphate for 3 h in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 20 μM quercetin. Cells in reactions 1 and 3 were transferred to FCS-free medium 24 h before labeling. Transfected BRF was immunoprecipitated with anti-HA antibody F-7, resolved on an SDS-7.8% polyacrylamide gel, transferred to nitrocellulose, and then visualized by autoradiography (top) and Western blotting with F-7 (bottom). (B) Phosphorylated BRF was quantified and normalized against total immunoprecipitated BRF. Means and standard deviations from three independent experiments are represented graphically.

FIG. 8.

FIG. 8.

CK2 inhibitors specifically compromise the binding of TFIIIB to TFIIIC2. (A) A reticulocyte lysate (10 μl) containing in vitro-translated BRF was mixed with a HeLa cell extract (150 μg) in the presence of buffer (lanes 2 and 3), 100 μM quercetin (lane 4), 80 μM apigenin (lane 5), 40 μg of CK2 phosphoacceptor peptide (lane 6), or 40 μg of PKA phosphoacceptor peptide (control) (lane 7). The mixture was then immunoprecipitated (IP) with anti-TFIIICβ antiserum 4286 (lanes 3 to 7) or the corresponding preimmune serum (lane 2). Proteins retained after extensive washing were resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Lane 1 shows 10% of the input reticulocyte lysate. (B) Rat1A cells stably transfected with pcDNA3HA.BRF were lysed and immu-noprecipitated with anti-cyclin A (Cyc A) antibody (Ab) BF683 (lane 1) or anti-HA antibody F-7 (lanes 2 and 3). Immunoprecipitated material was resolved by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting with antiserum 4286 against TFIIIC2 (top), anti-BN51 antiserum against PolIII (middle), and antibody F-7 against the HA tag on transfected BRF (bottom). (C) Coprecipitated TFIIIC2 was quantitated by densitometry and normalized against the amount of immunoprecipitated HA-BRF. Nonspecific binding to control antibody was subtracted. The value obtained for vehicle-treated cells was arbitrarily assigned as 1.0. Values are the means of three experiments; error bars indicate standard deviations. (D) As for panel C, except that coprecipitated PolIII was quantitated instead of TFIIIC2.

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