title: Identification of phosphorylation sites in the repetitive carboxyl-terminal domain of the mouse RNA polymerase II largest subunit.
abstract: The largest subunit of eukaryotic RNA polymerase II contains a carboxyl-terminal domain (CTD) which is comprised of repetitive heptapeptides with a consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We demonstrate here that the mouse CTD expressed in and purified from Escherichia coli can be phosphorylated in vitro by a p34cdc2/CDC28-containing CTD kinase from mouse ascites tumor cells. The product of this reaction, a phosphorylated form of the CTD, contains phosphoserine and phosphothreonine, but not phosphotyrosine. The same phosphoamino acid content is observed in the in vivo phosphorylated CTD from a mouse cell line. Synthetic peptides with naturally occurring non-consensus heptapeptide sequences can also be phosphorylated by CTD kinase in vitro. Phosphoamino acid analysis of these non-consensus heptapeptides together with direct sequencing of a phosphorylated heptapeptide reveals that serines (or threonines) at positions two and five are the sites phosphorylated by mouse CTD kinase. Thus, the -Ser(Thr)-Pro- motif common to p34cdc2/CDC28-containing protein kinases is the recognition site for mouse CTD kinase.
authors.affiliation: Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: J L
authors.initials: JL
authors.lastname: Corden
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000596:Amino Acids; D009842:Oligopeptides; D013912:Threonine; D011494:Protein Kinases; C060137:carboxy-terminal domain kinase; D012319:RNA Polymerase II