Inhibitory effect of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole on a protein kinase.

R Zandomeni,R Weinmann

doi:10.1016/s0021-9258(17)42674-3

Abstract

The adenosine analog 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits specific in vitro transcription initiation by RNA polymerase II. We report here that DRB inhibits a protein kinase present in an extract of HeLa cells and does not inhibit other protein kinases contained in the same extract. The protein kinase affected by DRB is cyclic AMP independent, prefers acidic protein substrates such as casein and phosvitin, and utilizes GTP as the phosphate donor almost as effectively as ATP in the phosphotransferase reaction. The DRB-sensitive protein kinase is also stimulated by polyamines and inhibited by quercitin and heparin. The biochemical and chromatographic properties of this enzyme correspond to those characteristic of casein kinase II. In HeLa cells, DRB is able to inhibit in vivo phosphorylation on some nuclear proteins. In HeLa cell extracts, in vitro phosphorylation of several proteins by [gamma-32P]GTP is inhibited by DRB. This protein kinase has a DRB sensitivity profile identical to the one previously reported for specific in vitro transcription by RNA polymerase II in a whole-cell extract (Zandomeni, R., Mittleman, B., Bunick, D., Ackerman, S., and Weinmann, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3167-3170). Thus we suggest that this protein kinase mediates DRB inhibition of specific RNA polymerase II transcription in vivo and in vitro.

Highlights

The adenosine analog 5,6-dichloro- l-fl-D-ribofuranosylbenzimidazole (DRB) inhibits specific invitro transcription initiation by RNA polymerase 11
We reported a requirement for hydrolysisof the By bond of we suggest that this protein kinase mediates DRB in- ATP for faithful initiationin vitro of whole-cell extracts (13, hibition of specific RNA polymerase I1 transcription in 14)
DEAE-Sepharose Chromatography-Whole-cell extracts active in thespecific in vitro transcription systemwere applied toDEAE-Sepharose,andproteins were elutedwith a salt gradient from 50 to 500 mM KC1 (Fig. lA).Using casein as a phosphate acceptor and [y3*P]ATP aasdonor, kinase activity was detected in theflowthrough and two major peaks (Fig. 1B)

Summary

Introduction

The adenosine analog 5,6-dichloro- l-fl-D-ribofuranosylbenzimidazole (DRB) inhibits specific invitro transcription initiation by RNA polymerase 11. Using [y3'P]GTP as thephosphate donor and casein as the acceptor, we found that half of the kinase activity of the extract could be inhibited by DRB at concentrations similar to those that affect in vitro specific transcription; at 60 pM DRB, phosphorylation on casein was reduced by 49.6% in a 30-min reaction at 30 "C.

Results

Conclusion