Cisplatin-induced ubiquitination of RNA polymerase II large subunit and suppression of induction by 7-hydroxystaurosporine (UCN-01).
Exposure of cells to DNA-damaging agents induces hyperphosphorylation of the C-terminal domain (CTD) of mammalian RNA polymerase II (RNAP II) large subunit (LS); the hyperphosphorylated RNAP II is then ubiquitinated. The purpose of this study was to verify that cisplatin-induced RNAP II ubiquitination is transcription dependent in living cells and to determine whether 7-hydroxystaurosporine (UCN-01) inhibits the ubiquitination induced by cisplatin. Cisplatin at clinically achievable concentrations (2.5-10 micro M) induced the ubiquitination of RNAP II in exponentially growing A2780 human ovarian tumor cells; the effect was drug-dose and exposure-time dependent. Such induction, however, was not observed in colcemid-selected mitotic cells. When detergent extraction was applied, the ubiquitinated RNAP II was recovered in the detergent-insoluble fraction, indicating that the protein was tightly bound to DNA. In an in vitro transcription reaction that consists of nuclear extracts and an immobilized DNA template containing a site-specific cisplatin lesion, the elongating RNAP II that was stalled at a cisplatin lesion site on the template was targeted by ubiquitins. Together, our results indicate that the ubiquitination is associated with transcription-coupled repair. We previously showed that the Ser/The kinase-inhibitor UCN-01 inhibits nucleotide excision repair. Here, we further determined the effect of UCN-01 on the phosphorylation and ubiquitination of RNAP II LS in a whole-cell system. Immunoblotting results showed that UCN-01 suppressed the cisplatin-induced ubiquitination and the cisplatin-induced shift from the hypophosphorylated IIa to the hyperphosphorylated IIo, without affecting the basal levels of the IIo and IIa forms of the RNAP II CTD, suggesting that UCN-01 acts by suppressing cisplatin-mediated induction of the one or more kinases that is responsible for the conversion of the IIo that is important for ubiquitination.
Yang
Li-Ying
LY
Department of Laboratory Medicine, Unit 54, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA. lyang@mdanderson.org
Jiang
Hong
H
Rangel
Kelly M
KM
Plunkett
William
W
D000970:Antineoplastic Agents / Q000494:pharmacology; D001710:Biotin / Q000737:chemistry; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008938:Mitosis; D010051:Ovarian Neoplasms / Q000188:drug therapy; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000378:metabolism*; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism*
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D025801:Ubiquitin; D001710:Biotin; C054852:7-hydroxystaurosporine; D012319:RNA Polymerase II; D019311:Staurosporine; D002945:Cisplatin