Phosphorylation by Cak1 Regulates the C-Terminal Domain Kinase Ctk1 in Saccharomyces cerevisiae

Cyclin-dependent protein kinases (CDKs) are best known for their roles in cell cycle progression. CDK activity is regulated by phosphorylation, cyclin availability, and binding to inhibitory proteins. Full activation of CDKs requires phosphorylation on a conserved threonine residue within the so-called T loop (or activation segment) that is mediated by Cdk-activating kinases (CAKs) (23). The CAK in the budding yeast Saccharomyces cerevisiae is Cak1 (11, 22, 41). Cak1 phosphorylates the main cell cycle CDK in yeast, Cdc28, on Thr-169. This phosphorylation stabilizes cyclin binding and is essential for protein kinase activity (37). Cak1 functions as a monomer and does not require posttranslational modification (21). In addition to Cdc28, Cak1 phosphorylates a number of other CDKs (see below), including those that regulate transcription by RNA polymerase II (pol II). The largest subunit of RNA pol II contains a conserved C-terminal domain (CTD) composed of tandem repeats of the consensus heptapeptide sequence YSPTSPS (7, 9). Phosphorylation of Ser2 and Ser5 within the heptapeptide functions during transcriptional initiation, elongation, and RNA processing (5, 32, 34, 40). Phosphorylations of the two serine residues have essential nonredundant functions (45). Phosphorylation of Ser5 regulates the transition from transcriptional initiation to elongation and is limited to polymerases within the promoter region. In contrast, phosphorylation of Ser2 occurs after transcriptional initiation and remains high until transcriptional termination (4, 28). Phosphorylation of Ser2 increases the affinity of the polymerase for polyadenylation factors, enabling efficient processing of the pre-mRNA 3′ end (1, 38). Phosphorylated Ser5 and Ser2 also provide binding sites for the histone methyltransferases Set1 and Set2, respectively, facilitating passage of the polymerase through chromatin (25, 33, 43). Four different cyclin-dependent kinases (CDKs), Kin28, Srb10, Bur1, and Ctk1, phosphorylate the CTD in S. cerevisiae. Kin28 phosphorylates Ser5, and this phosphorylation is required for transcription of most RNA pol II genes (6, 13, 18). Srb10 phosphorylates Ser5 and Ser2, but the effect of this phosphorylation is inhibitory (3, 17). Ctk1 is the major kinase acting on Ser2; recent findings indicate that it also phosphorylates Ser5 (20, 30, 36). Dephosphorylation of Ser5 and Ser2 is accomplished by the Ssu72 and Fcp1 phosphatases at the end of the transcription cycle (27, 29). Phosphorylation of Ser2 by Ctk1 has been implicated in the control of transcript elongation (4, 19, 31). Recent studies revealed that it is also required for cotranscriptional recruitment of polyadenylation factors (1). In the absence of Ctk1, transcription termination and processing of RNA 3′ ends are compromised. Surprisingly, CTK1 is not an essential gene but its mutation confers a conditional cold-sensitive phenotype (30). Ctk1 assembles with Ctk2 and Ctk3 into an active protein kinase complex that phosphorylates RNA pol II on Ser2 (39). Ctk2 and Ctk3 are unstable proteins degraded through the ubiquitin-proteasome pathway (15, 16). Ctk1 activity and Ser2 phosphorylation of RNA pol II increase under a variety of stress conditions (35, 36). Like the cell cycle CDKs, most CDKs functioning during transcription undergo activating phosphorylation within their T loops. Thus, previous studies demonstrated that Cak1 phosphorylates Kin28 and Bur1 within their T loops on Thr162 and Thr240, respectively (12, 26, 44). These phosphorylations are not essential under normal conditions but provide growth advantages when CDK-cyclin interactions are weakened by additional mutations (24, 26). Substitutions within the T loops of Kin28 and Bur1 compromise CTD phosphorylation in vitro (24, 26, 44). We noticed that Ctk1 contains a typical T loop and examined whether it is phosphorylated. We found that Ctk1 was indeed phosphorylated on a conserved Thr-338 in vivo. Mutation of Thr-338 conferred a cold-sensitive phenotype and compromised the protein kinase activity of Ctk1. Several lines of evidence indicated that this phosphorylation was carried out by Cak1. We also found that Ctk1 phosphorylation and the increased phosphorylation of RNA pol II on Ser2 are essential to activate transcription of stationary-phase genes during the diauxic shift.