Zhang J, Corden J L
Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1991 Feb 5;266(4):2290-6.
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.
真核生物RNA聚合酶II的最大亚基包含一个羧基末端结构域（CTD），该结构域由具有一致序列Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7的重复七肽组成。我们在此证明，在大肠杆菌中表达并纯化的小鼠CTD在体外可被来自小鼠腹水肿瘤细胞的含p34cdc2/CDC28的CTD激酶磷酸化。该反应的产物，即CTD的磷酸化形式，含有磷酸丝氨酸和磷酸苏氨酸，但不含磷酸酪氨酸。在来自小鼠细胞系的体内磷酸化CTD中也观察到相同的磷酸氨基酸含量。具有天然存在的非一致七肽序列的合成肽在体外也可被CTD激酶磷酸化。对这些非一致七肽的磷酸氨基酸分析以及对磷酸化七肽的直接测序表明，第2位和第5位的丝氨酸（或苏氨酸）是小鼠CTD激酶磷酸化的位点。因此，含p34cdc2/CDC28的蛋白激酶共有的-Ser(Thr)-Pro-基序是小鼠CTD激酶的识别位点。
