Different Carboxyl-terminal Domain Kinase Activities Are Induced by Heat-shock and Arsenite.

121

128

CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, we tested the hypothesis that the kinases differ in their ability to phosphorylate CTD heptad arrays. Our data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO 4 . This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs.The three cyclin-dependent kinases, CDK7, CDK8, and CDK9, 1 have an established connection with transcription machinery and are regulated by constitutively expressed cyclins. These CDKs can hyperphosphorylate the CTD of the large subunit of RNA polymerase II (pol II) and therefore are CTD kinases (1). The CTD of mammalian pol II consists of 52 heptad repeats with the consensus sequence 1 YSPTSPS 7 , which is especially well-conserved in the first half (N-terminal) of the CTD. Of the phosphorylatable amino acid residues, serine 5 is the most conserved, with only one threonine replacement, indicating that it is likely to have a crucial role in the function of the CTD. Pol II CTD is highly phosphorylated in vivo, mostly at serine (positions 2 and 5) but also at threonine and tyrosine (2). Hyperphosphorylated CTD is correlated with transcription initiation and elongation, whereas the holoenzyme and preinitiation complexes contain pol II with hypophosphorylated CTD (3). In yeast cells, replacing either serine 2 or serine 5 with alanine in each of this organism's 26 CTD heptad repeats causes lethality (4).The functions of these kinases correlate with different stages of transcription. CDK7 is a subunit of the general transcription factor TFIIH and is the kinase that phosphorylates pol II CTD after the preinitiation complex is formed (5). CDK7 is the catalytic member of a three-subunit complex called CAK (CDKactivating kinase) composed of CDK7/cyclin H and MAT-1 (6, 7). CAK is also a subassembly of TFIIH (8). Comparison between the kinase activity of CAK and TFIIH revealed differences in substrate specificity: CAK shows a strong preference for CDK2, consistent with its function in the cell cycle, but cannot hyperphosphorylate pol II; TFIIH hyperphosphorylates pol II, but CDK2 i...

Section: Cdk7 -8 and -9 Differ In Phosphorylation Of Phospho-ctdsupporting

confidence: 58%

Three RNA Polymerase II Carboxyl-terminal Domain Kinases Display Distinct Substrate Preferences

Ramanathan

Rajpara

Reza

et al. 2001

121

128

“…Suppressor mutations that allow growth of cells with otherwise lethal serine to alanine or glutamate substitution mutations in position 2 do not suppress similar substitutions in position 5 (56). Finally, while Cdc2 kinase phosphorylates positions 2 and 5 (22), the TFIIH kinase activity and HS-CTD kinases are specific for position 5 (25,55). Together, these results indicate that serines in positions 2 and 5 are functionally different.…”

Section: Discussionmentioning

confidence: 54%

“…The major response to environmental stress is protein modification especially phosphorylation of a number of regulatory proteins by a cascade of stress-related protein kinases. In mammalian systems nutrient limitation or change in temperature induces the kinases belonging to mitogen-activated protein kinase pathway, c-JUN kinase pathways, and protein kinase C pathways (55,69,75). In S. cerevisiae the interplay between growth control and stress is quite intriguing but not well understood.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Growth-related Changes in Phosphorylation of Yeast RNA Polymerase II

Patturajan¹,

Schulte²,

Sefton³

et al. 1998

217

222

7 . Two forms of the largest subunit can be separated by SDS-polyacrylamide gel electrophoresis. The faster migrating form termed IIA contains little or no phosphate on the CTD, whereas the slower migrating II0 form is multiply phosphorylated. CTD kinases with different phosphoryl acceptor specificities are able to convert IIA to II0 in vitro, and different phosphoisomers have been identified in vivo. In this paper we report the binding specificities of a set of monoclonal antibodies that recognize different phosphoepitopes on the CTD. Monoclonal antibodies like H5 recognize phosphoserine in position 2, whereas monoclonal antibodies like H14 recognize phosphoserine in position 5. The relative abundance of these phosphoepitopes changes when growing yeast enter stationary phase or are heat-shocked. These results indicate that phosphorylation of different CTD phosphoacceptor sites are independently regulated in response to environmental signals.The largest subunit of RNA polymerase II (pol II) 1 contains a repetitive C-terminal domain (CTD) consisting of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-ProSer (1, 2). The CTD plays an essential (3-6) but as yet poorly understood role in mRNA synthesis with evidence indicating potential roles in initiation or promoter clearance (7-9), elongation (10 -15), and pre-mRNA processing (16 -20).Phosphorylation of the CTD is a key feature of CTD function. SDS gel electrophoresis separates the largest subunit into two species as follows: IIA contains a hypophosphorylated CTD and pol II0 is hyperphosphorylated on the CTD (21). Serine is the predominant in vivo phosphoacceptor with minor amounts of phosphothreonine and phosphotyrosine detected (22, 23). Although in vivo phosphorylation sites have not been mapped, in vitro studies have identified serines in both positions 2 and 5 (22, 24, 25) and tyrosine in position 1 (23) as potential phosphoryl acceptors. Mutation of these sites to unphosphorylatable alanine or phenylalanine residues in each yeast CTD repeat is lethal, suggesting a requirement for CTD phosphorylation in vivo (26).The preferential inclusion of pol IIA into preinitiation complexes (27-30) together with the observation that elongating pol II is phosphorylated on the CTD (31) led to the hypothesis that the CTD is reversibly phosphorylated with each transcription cycle (8). The unphosphorylated CTD has been shown to contact basal transcription factors TATA binding protein (32), TFIIE, and TFIIF (33), and these contacts, together with as yet undefined interactions with SRBs (34 -37), suggest that the CTD acts as a structural framework for the preinitiation complex (38). The pol II preinitiation complex also contains several protein kinases that are capable of phosphorylating the CTD (39 -45) suggesting that one role of this complex is to effect the conversion of pol IIA to pol II0 thereby releasing pol II from the initiation complex. Finally, CTD phosphatase is required to dephosphorylate pol II0 thus completing the CTD phosphorylation cycle (46, 47).Se...

“…Similar experiments were performed with another set of peptides to determine the influence of amino acids surrounding the phosphorylated site and to compare the specificity of the different CTD kinases. Using a similar method, we were recently able to distinguish the different CTD kinase activities that are induced by stress and heat-shock treatment (35).…”

Section: For Review)mentioning

confidence: 99%

Characterization of the Residues Phosphorylated in Vitro by Different C-terminal Domain Kinases

Trigon¹,

Serizawa²,

Conaway³

et al. 1998

Self Cite

108

102

The C-terminal part of the largest subunit of eukaryotic RNA polymerase II is composed solely of the highly repeated consensus sequence Tyr 1 -Ser 2 -Pro 3 -Thr 4 -Ser 5 -Pro 6 -Ser 7 . This domain, called the C-terminal domain (CTD), is phosphorylated mostly at serine residues during transcription initiation, but the precise role of this phosphorylation remains controversial. Several protein kinases are able to phosphorylate this sequence in vitro. The aim of this work was to define the positions of the amino acids phosphorylated by four of these CTD kinases (transcription factor (TF) IIH-kinase, DNAdependent protein kinase, and the mitogen-activated protein kinases ERK1 and ERK2) and to compare the specificity of these different protein kinases. We show that TFIIH kinase and the mitogen-activated protein kinases phosphorylate only serine 5 of the CTD sequence, whereas DNA-dependent protein kinase phosphorylates serines 2 and 7. Among the different CTD kinases, only TFIIH kinase is appreciably more active on two repeats of the consensus sequence than on one motif. These in vitro results can provide some clues to the nature of the protein kinases responsible for the in vivo phosphorylation of the RNA polymerase CTD. In particular, the ratio of phosphorylated serine to threonine observed in vivo cannot be explained if TFIIH kinase is the only protein kinase involved in the phosphorylation of the CTD.A characteristic feature of eukaryotic RNA polymerase II is the carboxyl-terminal domain (CTD) 1 of its largest subunit, composed of multiple repeats of the sequence Tyr 1 -Ser 2 -Pro 3 -Thr 4 -Ser 5 -Pro 6 -Ser 7 . The number of repeats differs according to the species. The RNA polymerase II CTD contains 26 repeats in yeast (1), 45 in Drosophila (2, 3), and 52 in mammals (4). The consensus sequence is highly conserved, and this domain is essential for cell viability (2, 3, 5). Partial deletions of the CTD alter the regulatory properties of distinct promoters in different ways. The CTD has been shown to interact with a multisubunit complex containing the TATA-binding protein, which is an integral part of the transcription initiation complex (6).The CTD motif is mainly composed of phosphorylatable amino acid residues, and the RNA polymerase II CTD is actually highly phosphorylated in vivo, mostly at serine and to a lesser extent at threonine and tyrosine (7)(8)(9)(10). This phosphorylation appears to play a role in transcription initiation (11-13), but its precise function remains to be established. The in vivo phosphorylation sites are not known, but this issue has been approached indirectly by comparing the ratio of phosphorylated serine and threonine. The predominance of serine phosphorylation in vivo (with a serine/threonine ratio of ϳ10:1) has been explained by phosphorylation at positions 2 and 5: the serine at position 2 is replaced by a threonine in one-fifth of the repeats, and position 5 has only one threonine out of 51 (9). Moreover, yeast strains in which CTDs have been modified by the substitution of these...