The C-terminal domain phosphatase and transcription elongation activities of FCP1 are regulated by phosphorylation

Abstract

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is heavily phosphorylated during the transition from transcription initiation to the establishment of an elongationcompetent transcription complex. FCP1 is the only phosphatase known to be specific for the CTD of the largest subunit of RNAPII, and its activity is believed to be required to reactivate RNAPII, so that RNAPII can enter another round of transcription. We demonstrate that FCP1 is a phosphoprotein, and that phosphorylation regulates FCP1 activities. FCP1 is phosphorylated at multiple sites in vivo. The CTD phosphatase activity of phosphorylated FCP1 is stimulated by TFIIF, whereas dephosphorylated FCP1 is not. In addition to its role in the recycling of RNAPII, FCP1 also affects transcription elongation. Phosphorylated FCP1 is more active in stimulating transcription elongation than the dephosphorylated form of FCP1. We found that only phosphorylated FCP1 can physically interact with TFIIF. We set out to purify an FCP1 kinase from HeLa cells and identified casein kinase 2, which, surprisingly, displayed a negative effect on FCP1-associated activities. N umerous studies have extensively documented the effects of phosphorylation on the transcriptional properties of RNA polymerase II (RNAPII) (1-5). The target of these regulatory phosphorylation events is the C-terminal domain (CTD) of the largest subunit of RNAPII. The hypophosphorylated form of RNAPII, designated RNAPIIA, was demonstrated to be essential for preinitiation complex formation and transcription initiation, whereas hyperphosphorylated RNAPII, designated RNAPIIO, is involved in transcription elongation (6, 7). During the transition from transcription initiation to elongation, the CTD of the largest subunit of the RNAPII is heavily phosphorylated at Ser-2 and Ser-5 within the multiply repeated consensus sequence YS 2 PTS 5 PS (2,4,8). The CTD of the mammalian protein contains 52 repeats, which are phosphorylated by a number of different protein kinases, including CDK7 (5, 9), CDK9 (10, 11), and CDK8 (3, 12). The CDK7 and CDK9 kinases are tightly linked to the transcription cycle. In contrast, the kinase activity of CDK8, a component of the Mediator complex, was shown to negatively regulate transcription initiation.Although multiple kinases target the CTD of RNAPII, the only protein phosphatase known to dephosphorylate the CTD is FCP1. FCP1 was identified in both mammalian cells (13-15) and Saccharomyces cerevisiae (16,17). Dephosphorylation of the CTD of the largest subunit of RNAPII by FCP1 can facilitate recycling of the hyperphosphorylated form of RNAPII, allowing RNAPII to enter another round of transcription (15). FCP1 dephosphorylates the CTD of the largest subunit of RNAPII in solution (14)(15)(16)(18)(19)(20) and when associated with transcription elongation complexes (15,21,22). The phosphatase activity of FCP1 is stimulated by the general transcription factor TFIIF; however, TFIIB inhibits this stimulation (14). Mapping of the interaction domains betwee...

Bromodomain Factor 1 (Bdf1) Is Phosphorylated by Protein Kinase CK2

“…Furthermore, it has recently been reported that CK2 is involved in transcription of Pol I-and Pol III-dependent genes (8,29). CK2 copurifies with several chromatin-related complexes and Fcp1, the RNA Pol II CTD phosphatase (6,25). In addition, CK2 appears central to many of the mechanisms that protect the cell against stress.…”

“…Furthermore, CK2 copurifies with several chromatin-related complexes, including FACT (Spt16/ Pob3) and Chd1 (17). CK2 also regulates the activity of Fcp1, the RNA Pol II CTD phosphatase (6,25). Thus, CK2 may play a widespread role in regulating eukaryotic gene expression and chromatin structure.…”

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

“…In contrast to the four kinases, there are two known CTD phosphatases in yeast, . Biochemical as well as genetic evidence indicates that both proteins may function as CTD phosphatases in the context of transcription (16,19,20,(25)(26)(27)(28)(29)(30)(31).It is the interplay between these CTD kinases and phosphatases that influences which factors associate with transcribing polymerase and when during the transcription cycle such associations occur. As mentioned, the recruitment of capping enzyme early in the transcription cycle is believed to be one consequence of CTD phosphorylation on Ser5 by Kin28 (6,7).…”

Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD

“…We think it does not, because the entire globular structure of Pol II (excluding the CTD) proved nonessential for the phosphatase function (see the discussion above). Rather, we speculate that Fcp1 interaction with the non-CTD site may play phosphatase-independent roles, such as stimulating elongation (39)(40)(41) by stabilizing the DNA͞RNA hybrid in the cleft.…”

“…We discuss the possibility that this interaction may cause a conformational change around the DNA͞RNA binding cleft of the polymerase. Such a CTD-independent interaction with Pol II could offer a structural basis for the stimulatory effect that Fcp1 has on transcription elongation (39)(40)(41). We stress that Fcp1 interaction with the non-CTD site does not play the role of molecular docking that was defined as a requirement for effective processing of the CTD (27), because the globular structure of Pol II proves nonessential for Fcp1 phosphatase activity.…”