The Catalytic Subunit of <i>Schizosaccharomyces pombe</i> CK2 (Cka1) Negatively Regulates RNA Polymerase II Transcription through Phosphorylation of Positive Cofactor 4 (PC4)

Abstract

Positive cofactor 4 (PC4) is a transcriptional coactivator that plays important roles in transcription and DNA replication. In mammals, PC4 is phosphorylated by CK2, and this event downregulates its RNA polymerase II (RNAPII) coactivator function. This work describes the effect of fission yeast PC4 phosphorylation on RNAPII transcription in a cell extract, which closely resembles the cellular context. We found that fission yeast PC4 is strongly phosphorylated by the catalytic subunit of CK2 (Cka1), while the regulatory subunit (Ckb1) downregulates the PC4 phosphorylation. The addition of Cka1 to an in vitro transcription assay can diminish the basal transcription from the Ad-MLP promoter; however, the addition of recombinant fission yeast PC4 or Ckb1 can stimulate the basal transcription in a cell extract. Fission yeast PC4 is phosphorylated in a domain which has consensus phosphorylation sites for CK2, and two serine residues were identified as critical for CK2 phosphorylation. Mutation of one of the serine residues in PC4 does not completely abolish the phosphorylation; however, when the two serine residues are mutated, CK2 is no longer able to phosphorylate PC4. The mutant which is not phosphorylated is able to stimulate transcription even though it is previously phosphorylated by Cka1, while the wild type and the point mutant are inactivated by Cka1 phosphorylation, and they cannot stimulate transcription by RNAPII in cell extracts. Those results demonstrate that CK2 can regulate the coactivator function of fission yeast PC4 and suggests that this event could be important in vivo as well.

Keywords

• phosphorylation
• CK2
• transcription
• general transcription factors (GTFs)
• RNA polymerase II
• <i>Schizosaccharomyces pombe</i>