5,6-dichloro-1-β-d-ribofuranosylbenzimidazole inhibits a hela protein kinase that phosphorylates an...

Vol. 159, No. 2, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS March 15, 1989 Pages 508-515

5,6-DICHLORO-l-/3-D-RIBO??DRANOSYLBENZIMIDAZOLE INHIBITS A HELA PROTEIN KINASE THAT PHOSPHORYLATES AN RNA POLYMERASE II-DERIVED PEPTIDE*

Audrey Stevens and Marilyn K. Maupin

Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-8077

Received January 23, 1989

SUMMARY: Aproteinkinasethatphosphorylates Lys(Tyr-Ser-Pro-Thr-Ser-Pro-Ser),, a synthetic peptidehomologous to the evolutionarily-conserved, tandemly-repeated heptapeptide sequence at the C-terminus of the large subunit of eukaryotic RNA polymerase II, has been detected in HeLa cell extracts and chromatographic fractions therefrom. The enzyme, which phosphorylates serine principally, can be distinguished from previously described major protein kinases which phosphorylate the peptide poorly, if at all. It is inhibited by the nucleoside analog, 5,6-dichloro-1-,9-D-ribofuranosylbenzimidazole. Results suggest that human placental RNA polymerase II is phosphorylated at the C-terminus of the large subunit by the partially-purified protein kinase and that the phosphorylation is also sensitive to the nucleoside analog.

The C-terminal domain of the large subunit of eukaryotic RNA polymerase

II consists of multiple repeats (52 in mammals) of a heptapeptide with the

consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser(l-3). Mutagenesis studies show

that the sequence is essential in vivo (4-6). Studies of Cadena and Dahmus (7)

show that RNA polymerase 110 is a form of the enzyme in which the C-terminal

sequence of the large subunit, 110 (M, - 240,000), is highly phosphorylated and

that the same enzyme is the transcriptionally active form in HeLa nuclei. RNA

polymerase IIA contains the IIa subunit (M, - about 210,000) which is poorly

phosphorylated at the C-terminus (7). Earlier studies of Bartholomew & &. (8)

with a HeLa cell extract show that the reactivity of RNA polymerase 110 in

specific transcription appears to be 10 times that of IIA. However, RNA

polymerase II of Drosophila, lacking the C-terminal sequence, does function in

a specific transcription system from Drosophila (9). The results suggest that

the C-terminal repeated sequence functions, possibly in a regulatory manner, to

modulate promoter utilization.

*Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc. Direct support was from a grant from the Exploratory Studies Program, Oak Ridge National Laboratory. Abbreviationq: DRB, 5,6-dichloro-1-/?-D-ribofuranosylbenzimidazole; TCA, tri- chloroacetic acid; H-7, 1-(5-isoquinolinylsulfonyl)-2 methylpiperazine; EGTA, ethylene glycol bis(p-aminoethyl ether)-N,N,N',N'-tetracetic acid.

DOO6-291x/89 $1.50

508

Vol. 159, No. 2, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Of particular interest with regard to RNA polymerase II0 being highly

phosphorylated is the question which is addressed in this paper -- what protein

kinases carry out the phosphorylation of the IIa and 110 subunits at the C-

terminal sequence? Previous studies of Dahmus (10) and Cadena and Dahmus (7)

showed that casein kinases I and II phosphorylate RNA polymerase II. Casein

kinase I phosphorylates predominantly the 110 form on the repeated C-terminal

sequence of the 110 subunit, and casein kinase II shows low level phosphorylation

of the C-terminal sequence of the IIa large subunit. Since casein kinase II is

inhibited by 5,6-dichloro-1-fi-D-ribofuranosylbenzimidazole (DRB) (11,12), an

inhibitor of mRNA synthesis (see reviews 13, 14), it has been suggested that the

inhibitory effect of DRB may be at the level of phosphorylation of a

transcription component, possibly RNA polymerase II.

In a search for protein kinase activity that could play an important

regulatory role involving RNA polymerase II phosphorylation, we have used as a

substrate a synthetic peptide, Lys(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4, homologous to

the C-terminal repeated sequence of the large subunit of RNA polymerase II. As

described here, we find that HeLa whole cell extracts and fractions derived

therefrom contain a peptide-phosphorylating activity which is inhibited by DRB

and which can be distinguished from previously described major protein kinases.

MATERIALS AND METHODS

Materials--The synthetic peptide, Lys(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4, was synthesized by the Analytical Services Facility of the University of Tennessee. The identity of the peptide was confirmed by amino acid composition and sequence analysis. DRB and H-7 were obtained from Sigma.

Preoaration of HeLa Whole Cell Extract--HeLa cells (American Type Culture Collection, strain S-3, CCL 2.2, mycoplasma-free) were grown, harvested, and processed into whole cell extracts by the procedures of Manley et al (15,16).

ProteinKinase Assavs--The reactionmixtures (40 ~1) for measuring proteinkinase activity contained 50 mM Tris-Cl buffer, pH 7.5, 10 mM MgCl,, 25 PM ATP (8.8 x lo6 cpm) (ICN Radiochemicals), 50 pg of substrate [synthetic peptide, phosvitin (Sigma), or histone (Sigma, Type IIIS)] and enzyme. Incubations were for 30 min at 27'C. For determination of phosphorylated peptide, the incubations were terminated by the addition of 0.8 ml of phosphate-buffered saline and 10 ~1 of 200 mM EDTA. The samples were dialyzed using Spectra/Par 6 (M, cutoff - 2000) (Spectrum) tubing for approximately 18 h against 400 volumes of phosphate- buffered saline. The dialysates were made 30% in acetic acid and applied to an AG l-X8 (BioRad) column (2 ml) as described by Kemp et al. (17). Radioactivity in the eluted peptide was determined by liquid scintillation counting. When phosvitin or histone was used as a substrate, reactions were stopped by the addition of lml of 5% TCA containing 100 mM sodium pyrophosphate. Radioactivity incorporated into proteinwas determined following filtration (WhatmanGF/A glass microfibre filters) and washing (5% TCA) of the samples. Protein Kinase C was assayed according to the procedure of Kraft and Anderson (18) as modified by Dawson and Cook (19).

Phosohoamino Acid Analvsis--[32P]Peptide from the AG l-X8 column was concentrated to dryness in vacua and hydrolyzed under nitrogen in 6 N HCl for 2 h at 110°C. Samples were analyzedby high-voltage paper electrophoresis using 0.5% pyridine, 5% acetic acid (pH 3.5) as the solvent system.

509


CNBr CleavaPe of Reaction Products--Incubations were terminated by the addition of TCA to lo%, and the acid-insoluble material was collected by centrifugation, washed with 5% TCA, and dissolved in 50 ~1 of formic acid. Five ~1 of CNBr (100 mg/ml) were added, and the reaction mixtures were incubated for 20 h at 25°C. At that time, 0.5 ml of water was added, and the samples were concentrated to dryness in vacua.

Purification of Human Placental RNA Polvmerase II. Casein Kinases I and II. and Protein Kinase C--RNA polymerase II of human term placenta was purified by slight modifications of the procedures of Hodo and Blatti (20) and Waechter &al. (21) through the phosphocellulose column. Instead of the stepwise-eluted DF,AE- cellulose columns describedby these authors, two DEAE-Sephadex A-25 columns were used in succession and eluted with linear gradients of 100 to 350 mM (NH,),SO, in the buffer of Waechter et al. (21). The two columns separated most of the protein kinase activity that contaminates RNA polymerase II. Casein kinases I and IIwere purifiedby DBAE-cellulose andphosphocellulose columnchromatography of extracts of calf thymus, as described by Dahmus (22) and Zandomeni et al. (12). Protein kinase C of pig brain was made as described by Woodgett and Hunter (23) through the DEAE-cellulose step.

RESULTS AND DISCUSSION

A synthetic peptide, Lys(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4, which is homologous

to the repeated heptapeptide consensus sequence at the C-terminus of the large

subunit of RNA polymerase II, has been used as a substrate to detect protein

kinase activity with HeLa whole cell extracts. An activity is present which is

comparable in amount to the histone and phosvitin phosphorylating activities

(Table IA). Fractionation of the peptide-phosphorylating activity of the HeLa

Table I

Features of Protein Kinase Activities Examined

Enzvme Substrate Other Addition I-r-32P1ATP Incoruorated

A. HeLa whole cell extract, 8 pg

B. Phosphocellulose- purified peptide kinase, 250 ng

C. Casein Kinase I, 50 ng

D: Casein Kinase II, 42 ng

E. Protein Kinase C, 5 pg

Peptide Phosvitin Histone

Peptide Phosvitin Histone Peptide Peptide Peptide Peptide Phosvitin Histone

Peptide None Phosvitin None

Peptide Phosvitin

None None

Peptide Histone Histone

None 14.6 None 57.0 None 8.1

None 3.6 None 0.9 None 0.6 EGTA, 0.25 mM 3.7 CAMP, 5 /bM 3.6 H-7, 100 j&J 1.9 DKB, 72 jd4 1.1 DRB, 72 fl 0.8 DEB, 72 pM 0.5

<0.2 7.4

<0.2 8.6

None <0.02 None 0.84 DEB, 72 j&l 0.82

(pmol)

The protein kinases were assayed as described under Materials and Methods.

510


-E \ p 0.2

2

0.1

FRACTION NO.

Fig. 1. DEAE-Sephadex (A) and phosphocellulose (B) column chromatography of the peptide kinase activity of HeLa whole cell extracts. (A) Two ml of a HeLa whole cell extract was diluted with 10% glycerol, 50 mM Tris-Cl buffer, pH 7.5, 1 mM EDTA, 1 mM DTT, to a KC1 concentration of 20 mM and applied to a DBAE-Sephadex A-25 column (10 ml). After washing with 15 ml of the same buffer containing 20 mM KCl, the column was eluted with a 120 ml linear gradient of 20 mM+300 mM KC1 in the same buffer. Fractions (3 ml) were collected and 10 ~1 of each were assayed for protein kinase activity with the synthetic peptide (M) or phosvitin M) as a substrate. Protein was determined by A,,, measurements e-a). (B) Following DEAE-Sephadex column chromatography of 4 ml of HeLa cell extract as described in (A), 4 peak fractions were combined, diluted with l/2 volume of the buffer described above without KCl, and applied to a phosphocellulose column (5 ml). After washing with 6 ml of the same buffer containing 100 mM KCl, the column was eluted with a 40 ml linear gradient of 100 mM+500 mM KC1 in the same buffer. Fractions (1 ml) were collected and 5 ~1 of each were assayed for kinase activity with the synthetic peptide (M, phosvitin M), or histone (M) as a substrate. A,,, , *--a.

whole cell extract on a DEAE-Sephadex column (Fig. lA), followed by a

phosphocellulose column (Fig. lB), shows that about 40% of the activity is found

in one dominant peak from the DEAE-Sephadex column and 70% of the latter

activity is recovered in one peak from phosphocellulose. The elution of the

dominantphosvitin-phosphorylatingactivities from the DEAE-Sephadex column (Fig.

1A) is similar to that reported by Zandomeni and Weinmann (11) and Dahmus (22)

for casein kinases I and II. Histone-phosphorylating activity is partially

separated from the peptide activity on both the DEAE-Sephadex column (data not

shown) and the phosphocellulose column (Fig. lB), and the ratios of both

phosvitin/peptide and histone/peptide are reduced considerably during the two-

step purification procedure (compare values in Table IB with those in Table 1A).

Casein kinases I and II, obtained by DEAE-Sephadex and phosphocellulose

chromatography of extracts of calf thymus as described by Dahmus (22) and

Zandomeni & &. (12), show very low peptide/phosvitin phosphorylating activity

(Table IC and ID). A partially-purified preparation of protein kinase C from

511


pig brain (23) has an almost undetectable activity with the peptide (Table IE).

The peptide-phosphorylating kinase is not inhibited by EGTA, is inhibited less

than 50% by the protein kinase inhibitor H-7 (100 PM) (24), and is not stimulated

by CAMP (Table IB), suggesting that it is not a Ca* or CAMP-requiring kinase.

The enzyme is inhibited by DRB (Table IB), a point which is addressed below.

Zandomeni and Weinmann (11) showed that casein kinase II is inhibited by DRB,

but casein kinase I and CAMP-dependent protein kinase are not. No inhibition

by DRB of protein kinase C (Table IE) or of the histone and phosvitin-

phosphorylating activities of the phosphocellulose-purified peptide kinase

fraction were found (Table IB).

The enzyme requires a divalent cation, Mg" being optimal at 10 mM. The

optimal pH is 7.5-8.0. The Km for ATP is 30 PM and for the synthetic peptide,

150 PM. The reaction is optimal at 27-2g"C, and linear with time to 30 min.

Phosphorylation of the peptide (M, - 3400) with the phosphocellulose

fraction shown in Fig. IB was demonstrated by Sephadex G-50 molecular sieve

chromatography (Fig. 2A) of the peptide itself and the 32P-labeled reaction

900

z600 s

P x

300

0, Blur Cykhreme c ACTH Insulin A dtrtron ((2 k0a ) (4.8 kDa) (2.4 kh)

\,I If------

.-e . - _ _ - . -

20 40 6 FRACTION NO.

0

P-ser -

P-thr -

P-tyr -

Incompletely Hydrolyzed

Peptide \

Origin -

2. Fig. Identification of ["Plpeptide and [32P]serine as reaction products of the partially-purified peptide kinase. (A) The 32P-labelled product of a reaction mixture containing peptide and phosphocellulose-purified kinase was collected from an AG l-X8 column. After evaporation to dryness $n vacua, the sample was dissolved in 50 ml4 Tris-Cl buffer, pH 7.5, 0.1 M NaCl, and chromatographed on a Sephadex G-50 column (50 ml) using the same buffer. Fractions (0.85 ml) were collected and counted for radioactivity M). Synthetic peptide (2 mg in the same buffer) was also applied and its elution measured by AzsO measurements M). Positions of marker proteins are shown. (B) Phosphoamino acid analysis was carried out as described under Materials and Methods. Standards were localizedby ninhydrfn staining (lane 1) and radioactive amino acids by autoradiography (lane 2).

512

Vol. 159, No. 2, 1989 BIOCHEMICAL AND BIOPHYBICAL RESEARCH COMMUNICATIONS

5b DR.9 hM 1

0

to- pM DRB

0.05 0:1 1/ATP (pM)

DREI inhibition of the HeLa peptide kinase and calf thymus casein kinase Fig. 3. II. (A) The effect of DRB concentration on the phosphorylating activity of the phosphocellulose-purified peptide kinase (0.25 rg) with 10 PM [7-32P]ATP and synthetic peptide (M). casein kinase II with 10 PM [r-"P]ATP and phosvitin (o--o) * and casein kinase II with 10 PM [T-~~P]GTP and phosvitin (A---A) was measured as described under Materials and Methods. (B) The nature of the inhibition of the peptide kinase by DRB was determined with the use of phosphocellulose-purified kinase (0.25 pg).

product isolated by AG l-X8 chromatography. That the peptide is phosphorylated

on serine was demonstrated by HCl hydrolysis of the same reaction product (Fig.

2B).

Fig. 3A shows that under the same reaction conditions the peptide kinase

is inhibited by DRB at approximately the same concentrations that inhibit casein

kinase II. About 40% inhibition of both kinases occurs with 9 fl DRB when the

ATP concentration is 10 FM. The slightly lower concentration of DRB (5 FM)

found by Zandomeni et al. (12) to give 50% inhibition of casein kinase II may

be due to the fact that the kinases in our hands are not as pure. We find that

the inhibition of casein kinase II with GTP replacing ATP is somewhat greater

(Fig. 3A). The inhibition of the peptide kinase by DRB is competitive with ATP

as shown by the Lineweaver-Burk plot in Fig. 3B.

The activity of the peptide as a protein kinase substrate suggested that

thehomologous sequence inRNApolymerase II could also be phosphorylated. Using

a partially purified preparation of RNA polymerase II of human placenta, its

phosphorylation by the phosphocellulose-purified peptide kinase was examined.

As Fig. 4A shows, both 210 KDa (subunit IIa) and 240 KDa (subunit 110) bands are

phosphorylated when the kinase and polymerase are incubated together (lane 3),

but not with either enzyme alone (lanes 1 and 2). Most of the other

phosphorylated protein bands are also present in the kinase preparation alone

(Fig. 4A, lane 1). Fig, 4A also shows (lane 4) that the phosphorylation of the

513


kDa

=a, IIa- 2001

68-

43-

kDa

200

68

kDa

200-

Fig. 4. Phosphorylation of the C-terminal sequence of the large subunit of human -al RNA polymerase by the peptide kinase and its inhibition by DRB. Phosphocellulose-purified peptide kinase (1-2 pg) was incubated with 2.1 ag of a partially purified fraction of placental RNApolymerase II under the conditions described under Materials and Methods except that the ATP concentration was 10 PM (specific activity - 4 x 104cpm/pmole). For (A), the reactions were stopped by the addition of TCA to lo%, and the acid-insoluble material was collected by centrifugation and dissolved in Laemmli loading buffer (25). For (B) and (C), the procedure described for CNBr cleavage of reaction products under Materials and Methods was followed. The products were dissolved in loading buffer and electrophoresed on an 8% polyacrylamide gel under the conditions of Laemmli (25). The gels were stained and destained, followed by autoradiography. (A) 1, kinase alone; 2, RNA polymerase II alone; 3, both; 4, both + 72 PM DRB. (B) The same as (A), except 4, both + 18 PM DRB, and 5, both + 72 pM DRB. (C) 1, kinase alone; 2, kinase + 72 PM DRFi; 3, RNA polymerase; 4, both; 5, both + 72 /d-l DR8. The positions of marker proteins are shown on the left.

210 KDa and 240 KDa bands is inhibited by DRB. Upon CNBr treatment of the

phosphorylated products prior to electrophoresis, bands in the range of 65-90

KDa are produced (Fig. 4B, lanes 3-5; Fig. 4C, lanes 4 and 5 -- two different

peptide kinase preparations from phosphocellulose were used for B and C). Cadena

and Dahmus (7) have shown that a 65 KDa fragment is produced by CNBr treatment

of the IIa subunit and 65+90 KDa fragments (due to multiple phosphorylation) from

the 110 subunit of RNA polymerase II. Counting of the 210 KDa and 240 KDa bands

as well as the 65+90 KDa bands shown in Fig. 4 showed that all the radioactivity

in the 210 KDa and 240 KDa bands was recovered in the smaller CNBr fragments.

No labeled CNBr fragments of this size are found with the kinase and RNA

polymerase II alone (lanes 1 and 2 in B and lanes l-3 in C). As shown in Fig.

4B, lanes 4 and 5, and Fig. 4C, lane 5, DRB inhibits strongly the phosphorylation

of the fragments derived by CNBr cleavage of the reaction products.

514


The protein kinase described here is the second protein kinase found to be

inhibited by DRB, and it seems likely that at least part of the DRB inhibition

of mRNA transcription (13,14) could be due to its inhibitory effect on

phosphorylation at the C-terminal repetitive sequence of the large subunit of

RNA polymerase II. The specificity of the kinase remains uncertain at this

stage of purification.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the assistance of: Dr. Charles L. Murphy, Director, Analytical Services Facility, University of Tennessee, for his work on the synthesis of the peptide; Margaret Yette for growing and harvesting the HeLa cells; Claude Stringer for amino acid sequence analysis of the synthetic peptide; and Michael Bast for the preparation of protein kinase C.

1. Allison, L. A., Moyle, M., Shales, M., and Ingles, C. J. (1985) Cell 42, 599-610.

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7. 8.

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10. 11. 12.

13. 14.

15.

16.

17.

18. 19. 20. 21.

22. 23. 24.

Corden, J. L., Cadena, D. L., Ahearn, J. M., Jr., and Dahmus, M. E. (1985) Proc. Natl. Acad. Sci. USA 82, 7934-7938. Ahearn, J. M., Jr., Bartolomei, M. S., West, M. L., Cisek, L. J.,and Corden, J. L.. (1987) J. Biol. Chem. 262, 10695-10705. Bartolomei, M. S., Halden, N. F., Cullen, C. R., and Corden, J. L. (1988) Mol. Cell Biol. 8, 330-339. Nonet, M., Sweetser, D., and Young, R. A. (1987) Cell 50, 909-915. Allison, L. A., Wong, J. K.-C., Fitzpatrick, V. D., Moyle, M., and Ingles, C. J. (1988) Mol. Cell. Biol. 8, 321-329. Cadena, D. L., and Dahmus, M. E. (1987) J. Biol. Chem. 262, 12468-12474. Bartholomew, B., Dahmus, M. E., and Meares, C. F. (1986) J. Biol. Chem. 261, 14226-14231. Zehring, W. A., Lee, J. M., Weeks, J. R., Jokerst, R. S., and Greenleaf, A. L. (1988) Proc. Natl. Acad. Sci. USA 85, 3698-3702. Dahmus, M. E. (1981) J. Biol. Chem. 256, 3332-3339. Zandomeni, R., and Weinmann, R. (1984) J. Biol. Chem. 259, 14804-14811. Zandomeni, R., Zandomeni, M. C., Shugar, D., and Weinmann, R. (1986) J.

Biol. Chem. 261, 3414-3419. Tamm, I., and Sehgal, P. (1978) Adv. Virus Res. 22, 187-258. Weinmann, R.. Ackerman, S., Bunick, D., Concino, M., and Zandomeni, R. (1983) Curr. Top. Microbial. Immunol. 109, 125-145. Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859. Manley, J. L., Fire, A., Samuels, M., and Sharp, P. A. (1983) Methods

Enzymol. 101, 568-582. Kemp, B. E., Benjamini, E., and Krebs, E. G. (1976) Proc. Natl. Acad. Sci. USA 73, 1038-1042. Kraft, A. J., and Anderson, W. B. (1982) J. Biol. Chem. 258, 9178-9183. Dawson, W. D., and Cook, J. S. (1987) J. Cell. Physiol. 132, 104-110. Hodo, H, G, III, and Blatti, S. P. (1977) Biochem. 16, 2334-2343. Waechter, D. E., Avignolo, C., Freund, E., Riggenbach, C. M., Mercer, W. E., McGuire, P. M., and Baserga, R. (1984) Mol. Cell. Biochem. 60, 77- 82. Dahmus, M. E. (1981) J. Biol. Chem. 256, 3319-3325. Woodgett, J. R., and Hunter, T. (1987) J. Biol. Chem. 262, 4836-4843. Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochem. 23, 5036-5041.

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REFERENCES

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5,6-dichloro-1-β-d-ribofuranosylbenzimidazole inhibits a hela protein kinase that phosphorylates an...

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