Three cyclin-dependent kinases preferentially phosphorylate different parts of the C-terminal domain of the large subunit of RNA polymerase II

Transcription

1 Eur. J. Biochem. 271, (2004) Ó FEBS 2004 doi: /j x Three cyclin-dependent kinases preferentially phosphorylate different parts of the C-terminal domain of the large subunit of RNA polymerase II Reena Pinhero, Peter Liaw, Kimberly Bertens and Krassimir Yankulov Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada The C-terminal domain (CTD) of the largest subunit of RNA polymerase II plays critical roles in the initiation, elongation and processing of primary transcripts. These activities are at least partially regulated by the phosphorylation of the CTD by three cyclin-dependent protein kinases (CDKs), namely CDK7, CDK8 and CDK9. In this study, we systematically compared the phosphorylation of different recombinant CTD substrates by recombinant CDK7/ CycH/MAT1, CDK8/CycC and CDK9/CycT1 kinases. We showed that CDK7, CDK8 and CDK9 produce different patterns of phosphorylation of the CTD. CDK7/CycH/ MAT1 generates mostly hyperphosphorylated full-length and truncated CTD peptides, while CDK8/CycC and CDK9/CycT1 generate predominantly hypophosphorylated peptides. Total activity towards different parts of the CTD also differs between the three kinases; however, these differences did not correlate with their ability to hyperphosphorylate the substrates. The last 10 repeats of the CTD can act as a suppressor of the activity of the kinases in the context of longer peptides. Our results indicate that the three kinases possess different biochemical properties that could reflect their actions in vivo. Keywords: carboxy-terminal domain; cyclin-dependent kinase; phosphorylation; RNA pol II. The C-terminus of the largest subunit of the eukaryotic RNA polymerase II consists of multiple repeats of a YSPTSPS consensus heptapeptide sequence [1,2]. This part of the polypeptide is referred to as CTD (C-terminal domain). In higher eukaryotes, the CTD consists of 52 heptapeptide repeats [1 3]. The N-terminal portion of the CTD contains mainly perfect YSPTSPS repeats; however, the repeats in the C-terminal portion significantly deviate from the consensus [2 5], probably reflecting a more specialized function of this part of the polypeptide. It has been demonstrated that the N-terminal half of the CTD supports RNA synthesis and capping of the primary transcript [6 8], whereas the C-terminal half supports splicing and 3 processing of the transcripts [6]. The importance of the C-terminal ISPDDSDEEN sequence of the CTD in the regulation of transcript processing has also been shown [9]. The CTD is phosphorylated at multiple Correspondence to K. Yankulov, Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. Fax: , Tel.: ext , yankulov@uoguelph.ca Abbreviations: CAK, CDK activating kinase; CDK, cyclin dependent kinase; CTD, C-terminal domain; Cyc, cyclin; MED, mediator; GST, glutathione S-transferase; MBP, myelin basic protein; MOI, multiplicity of infection; NAT, negative regulator of activated transcription; P-TEFb, positive transcriptional elongation factor b; SMCC, SRB/MED containing complex; SRB, suppressor of RNA polymerase B; TRAP, thyroid hormone receptor associated protein complex. (Received 18 November 2003, revised 9 January 2004, accepted 19 January 2004) sites, which leads to the production of two forms of RNA polymerase II in vivo: a hypophosphorylated form called IIa, and a hyperphosphorylated form called IIo [1,2,4,5]. It is well established that phosphorylation of the CTD regulates the transition of RNA polymerase II from initiation to elongation, the capping of primary transcripts and the efficiency of pol II elongation [1,2,10]. CTD phosphorylation has also been implicated in the cotranscriptional splicing and polyadenylation of nascent transcripts [1,2,10]. However, little is known about how the phosphorylation of different parts of the CTD contributes to these functions. At least three protein kinases are involved directly in the phosphorylation of the CTD and in the regulation of different stages of mrna synthesis [1,2]. Cyclin dependent kinase (CDK)7, in conjunction with cyclin (Cyc)H and MAT1, forms a tripartite complex known as CAK (CDKactivating kinase); however, a less abundant bipartite form (CDK7/CycH) has also been observed [11]. At the same time, CDK7/CycH/MAT1 has been identified as a component of the general pol II transcription factor, TFIIH [1,2], and of large protein complexes containing RNA polymerase II and general pol II transcription factors that are referred to as pol II holoenzyme complexes [12]. Another protein kinase, CDK8/CycC, has also been found in the pol II holoenzyme [13] and in other MED/SRB containing complexes such as TRAP/SMCC and NAT [14 17]. TRAP/SMCC and NAT both phosphorylate the CTD and repress activated, but not basal, transcription [17]. Another study indicates that NAT and TRAP/SMCC phosphorylate CycH of the TFIIH complex via its CDK8 kinase activity and inhibit TFIIH protein kinase activity [18]. Studies in Saccharomyces cerevisiae suggest that the

2 Ó FEBS 2004 Phosphorylation of pol II C-terminal domain (Eur. J. Biochem. 271) 1005 CDK8 homolog, Srb10p, phosphorylates the CTD prior to the formation of an initiation complex at promoters, which results in the repression of pol II transcription [19]. The third CTD kinase, CDK9, in complex with one of several homologous CycT molecules, has been initially identified as P-TEFb (positive transcription elongation factor-b) [20,21]. Independently, CDK9/CycT1 has been isolated as the HIVtat-associated kinase, TAK. It has been reported that the P-TEFb kinase activity operates in a CAK-independent manner [22]. Unlike CDK7/CycH/MAT1 and CDK8/ CycC, which are recruited to promoters prior to transcription initiation, P-TEFb is recruited to the elongating polymerase at a later stage of the transcription reaction [23 26]. The in vitro effects of P-TEFb on elongation cannot be replaced by TFIIH, thus suggesting that these complexes perform non-redundant functions [24]. Several studies have attempted to directly compare the phosphorylation of the CTD or synthetic CTD derivatives by CDK7/CycH/MAT1, CDK8/CycC and CDK9/ CycT1 [25,27 31]. CDK7/CycH/MAT1 and CDK8/CycC preferentially phosphorylate the S5 residue in the YSPTSPS repeat [25,27 29,31]. CDK7/CycH/MAT1 might also phosphorylate some S2 residues in the less conserved C-terminal portion of the CTD [32]. CDK9/CycT1 seems to preferentially phosphorylate the S2 residue of the YSPTSPS repeat on longer CTD substrates [21,25]; however, it can also phosphorylate S5 on short peptide substrates [28,31]. In addition, CDK9/CycT1 can shift its preference from S2 to S5 in the presence of the HIV-tat protein [25]. There is a greater uncertainty as to how these kinases phosphorylate full-length CTD and parts derived from it. One study demonstrates that the C-terminal portion of the CTD is phosphorylated more efficiently by CDK7/CycH/ MAT1 than by CDK8/CycC [27]. This effect is attributed to the frequent presence of K in position 7 of the heptad repeats in the C-terminal part of the CTD. Indeed, synthetic (YSPTSPK) 4 peptides are preferentially phosphorylated by CDK7/CycH/MAT1 as compared to CDK8/CycC [27]. Another study, using immunoprecipitated CDK7, CDK8 and CDK9, indicates that the three kinases phosphorylate equally well the N terminus of the CTD (repeats 1 29), but only CDK7 is able to produce the hyperphosphorylated IIo form of this substrate [28]. The C terminus (repeats 30 52) is efficiently phosphorylated by CDK7 only, but the hyperphosphorylated IIo form was not produced [28]. The authors conclude that the hyperphosphorylation, and the production of the IIo form of pol II and the CTD, is a result of phosphorylation of the first half of the CTD [28]. On fulllength CTD, the immunoprecipitated CDK7 has a much higher activity relative to CDK8 and CDK9. Surprisingly, both CDK7 and CDK9 produced the full-length hyperphosphorylated IIo form [28]. In this study, we systematically compared the phosphorylation pattern of recombinant CTD substrates by recombinant CDK7/CycH/MAT1, CDK8/CycC and CDK9/ CycT1 kinases. We showed that the three kinases do not dramatically differ in their activity towards the CTD in vitro; however, they displayed different abilities to hyperphosphorylate CTD substrates. Only CDK7/CycH/MAT1 was able to efficiently hyperphosphorylate the full-length CTD and produce the IIo form of this substrate. The N- and C-terminal portions of the CTD were differentially phosphorylated by CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. Finally, we showed data suggesting that certain CTD repeats in the context of larger polypeptides can suppress the activities of these kinases. Materials and methods Expression vectors The baculoviruses for the expression of CDK7, MAT1, CycC and His 6 -CDK9/CycT1 were as described previously [21,33,34]. The baculovirus for the expression of His-tagged CycH was produced by subcloning the human CycH into pbluebac (Invitrogen) and transfecting Sf9 cells according to the instructions of the manufacturer. The baculovirus containing His 6 -CDK8 was produced by subcloning the human CDK8 into pfastbachta and using the BACto-BAC recombination system (Life Technologies). The plasmids for the expression of glutathione S-transferase (GST)-CTD(1 52), GST-CTD(1 15, S5>A) and GST- CDK2 were as described previously [35]. Plasmids for the expression of GST-CTD(1 15), GST-CTD(1 25), GST- CTD(27 39), GST-CTD(27 42) and GST-CTD(27 52) were as described previously [6]. The plasmid for the expression of GST-CTD(42 52) was prepared by subcloning a PCR fragment, encompassing repeats 42 52, into pgex2t (Amersham). GST-CTD(1 52), GST-CTD(27 52) and GST-CTD(42 52) also contained the C-terminus ISPDDSDEEN peptide that is positioned next to the 52 heptad repeat in vertebrate RPB1. Expression and purification of recombinant kinases Recombinant kinases were expressed by infecting L of Sf9 cells ( cells per ml) with combinations of individual baculoviruses at a multiplicity of infection (MOI) of 5 for 48 h. The cells were harvested by centrifugation (275 g, 5min)at4 C and lysed in lysis buffer [10 mm Tris/HCl, ph 7.5, 10 mm NaCl, 2 mm 2-mercaptoethanol, 0.5 mm EDTA, 10 mm 2-glycerophosphate, 0.5 mm sodium vanadate, 2 mm NaF, 2 lgæml )1 leupeptin, 2 lgæml )1 aprotonin, 2 lgæml )1 pepstatin, 0.2% (v/v) Nonidet P-40, 50 lgæml )1 phenylmethanesulfonyl fluoride] by 10 strokes with the Dounce homogenizer. The proteins were extracted by adding NaCl to a final concentration of 0.4 M and then rocking for 30 min. The extract was clarified by spinning ( g, 30min)at4 C in an SW50.1 rotor (Beckman) and mixed with 1 ml of Ni 2+ nitrilotriacetic acid agarose beads (Qiagen) that had been equilibrated with 10 mm Tris/HCl, ph 7.6, containing 0.5 M NaCl, 5mM imidazole, 50 lgæml )1 phenylmethanesulfonyl fluoride, and 10% (v/v) glycerol. The beads were washed in the equilibration buffer and transferred to a column. Proteins were eluted in batch by buffers containing mm imidazole, 10 mm Tris/HCl, ph 7.6, 0.1 M NaCl, 50 lgæml )1 phenylmethanesulfonyl fluoride and 10% (v/v) glycerol. The fractions containing the recombinant protein kinases were pooled and the buffer was exchangedinpd10columns(bio-rad)to25mm sodium Hepes, ph 7.6, 0.1 mm EDTA, 1 mm dithiothreitol, 5% (v/v) glycerol. The proteins were then loaded onto a 5 ml

3 1006 R. Pinhero et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Econo-Pac Mono S cartridge (Bio-Rad) and eluted with a linear M NaCl gradient in 25 mm sodium Hepes, ph 7.6, 0.1 mm EDTA, 1 mm dithiothreitol, 5% (v/v) glycerol. The fractions containing recombinant protein kinases were identified by SDS/PAGE followed by silver staining, pooled and stored at )80 C. The identity of the recombinant proteins was confirmed by Western blot with antibodies against CDK7, CycH, MAT1, CDK8, CycC and CDK9. Expression and purification of recombinant substrates All GST-CTD fusion proteins and GST-CDK2 were expressed in BL21 cells using 0.5 mm isopropyl thio-b- D-galactoside (IPTG) for 3 h at 30 C. Cells were lysed by sonication in TEN buffer (20 mm Tris/HCl, ph 7.5, 5 mm EDTA, 200 mm NaCl, 1 lgæml )1 aprotonin, 1 lgæml )1 leupeptin, 1 lgæml )1 pepstatin, 2 mm benzamidine and 1mM phenylmethanesulfonyl fluoride). Triton-X-100 was added to 1% (v/v) and the extract was rocked for 20 min at 4 C and then spun at g in a JA20 rotor (Beckman) at 4 C. The supernatant was loaded onto glutathione sepharose 4B beads (Amersham). The bound proteins were eluted with 15 mm glutathione, 50 mm KCl, 20 mm Tris/ HCl, ph 8.0, 15% (v/v) glycerol, and stored at )80 C. Highly purified myelin basic protein (MBP) from bovine brain was a gift from G. Harauz (University of Guelph). Kinase assay Kinase reactions were performed in a 20 ll volume containing 50 mm KCl, 20 mm Tris/HCl, ph 8.0, 7 mm MgCl 2, 5 mm 2-glycerophosphate, 100 lgæml )1 BSA, 10 lm ATP, 2 lci ( Bq) [ 32 P]ATP[cP] (ICN), 40 lgæml )1 recombinant substrate and ngæml )1 purified kinase, or the same volume of control fractions from uninfected Sf9 cells. The kinase reactions were incubated for 30 min at 30 C, stopped by the addition of SDS/PAGE loading buffer, and analyzed on SDS/PAGE gels and by autoradiography. The separation of substrates from kinases after the kinase reaction was carried out as follows. The kinase reaction was stopped by adding 200 ll of ice-cold STOP buffer [10 mm sodium EDTA, ph 8, 50 mm KCl, 0.2% (v/v) Nonidet P-40] and incubated with 20 ll of glutathione sepharose 4B beads. The suspension was rocked for 20 min, the beads were washed three times in STOP buffer containing 200 mm NaCl, and the bound proteins were eluted by boiling in SDS/PAGE loading buffer. Quantification of levels of phosphorylation Levels of phosphorylation were measured by scanning exposed films on a Kodak DS 440CF image station using the KODAK 1D image analysis software. Relative signals along each lane in the gels were evaluated by using the grid option of the data analysis software. Quantification was conducted only with subsaturated films and only if the grids did not show saturation (flat) signals. Signals in each segment of the grid were corrected in Microsoft EXCEL by subtracting the corresponding signals from the identical segment in the grid from a sample without a substrate. Intensity curves were prepared in Microsoft EXCEL. Total phosphorylation of each substrate was calculated as the sum of signals in all segments corresponding to the substrate bands. Relative phosphorylation of individual substrates was calculated by measuring the signals from different substrates on the same X-ray film and normalizing them to a postulated value of 1 for the intensity of the phosphorylation of the GST-CTD(1 52) substrate. Average relative phosphorylation was calculated from these values. The incorporation of ATP in GST-CTD(1 52) and MBP (pmols of ATP min )1 Æmg )1 of protein) was determined according to the previously published procedure [36]. Results Expression, purification and characterization of recombinant CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1 Earlier studies have provided important information on the substrate preferences of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. However, a comprehensive description of their properties is far from complete. We therefore attempted a more systematic comparison of the activities of these kinases towards different substrates. To minimize variations resulting from different sources of material or purification procedures, we prepared the three recombinant kinases following the same expression/purification scheme. Briefly, CycH, CDK8 and CDK9 were cloned in baculovirus vectors as N-terminally 6-Histidine tagged proteins. CDK7, MAT1, CycC and CycT1 were expressed as untagged proteins. Sf9 cells were infected with combinations of CDK7/His 6 -CycH/MAT1, His 6 -CDK8/CycC and His 6 -CDK9/CycT1 baculoviruses. The kinases were subsequently purified by immobilized metal-affinity chromatography (IMAC) using Ni 2+ nitrilotriacetic acid agarose and then by ion-exchange chromatography on MonoS beads. This procedure purified the three kinases to nearhomogeneity, as determined by silver staining (Fig. 1A). The identities of the CDK7, CycH, MAT1, CDK8, CycC, and CDK9 bands in Fig. 1A were confirmed by Western blot (data not shown). All of these preparations displayed strong kinase activities towards the GST-CTD(1 52) and MBP (Figs 1B, 2 and 3). Typically, different preparations of CDK7/CycH/MAT1 and CDK9/CycT1 transferred between 0.3 and 1.6 nmols of ATP min )1 Æmg )1 of protein with both substrates. The CDK8/CycC preparations showed somewhat lower specific activities, of nmols of ATP min )1 Æmg )1 of protein. Importantly, when the Ni 2+ nitrilotriacetic acid agarose/monos fractions that correspond to the fractions with kinase complexes were isolated from uninfected cells, none showed detectable kinase activity towards these substrates (results not shown). We concluded that most, if not all, of the CTD- and MBPkinase activity in these preparations belonged to the expressed kinases. Next, we tested whether the three kinases would show substrate specificities that were reported by other groups. Kinase reactions were performed with MBP, GST- CDK2(K33>R) and GST-CTD15(S5>A). MBP is a common non-physiological kinase substrate that is rich in

4 Ó FEBS 2004 Phosphorylation of pol II C-terminal domain (Eur. J. Biochem. 271) 1007 Fig. 1. Characteristics of the recombinant CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1 kinases. (A) Samples from pooled Mono S chromatography fractions containing the three kinases were separated by SDS/PAGE (10% gel) and silver stained. The position of each individual recombinant polypeptide is shown on the left. The CycH/CDK7 band corresponds to a doublet of CDK7 and His 6 -CycH ( 40 kda). MAT1 is 36 kda. The CDK8 corresponds to a molecular mass of 53 kda and CycC 36 kda; CDK9 is 43 kda and CycT1, 81 kda. (B) Phosphorylation of the myelin basic protein (MBP), glutathione S-transferase (GST)-CDK2 and GST-C-terminal domain (CTD)(S5>A) 15 substrates by CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. Kinase reactions were performed with the combinations of kinase and substrate as indicated above each lane. The mobility of the substrate polypeptides are indicated on the left. The mobility of 60 and 20 kda molecular mass markers are indicated on the right. serine/threonine (17%) and lysine/arginine residues (19%). GST-CDK2(K33>R) is a catalytically inactive CDK2 molecule [37]. CDK2 is believed to be a physiological substrate of CDK7/CycH/MAT1 [11]. GST-CTD15(S5>A) contains 15 synthetic consensus YSPTAPS repeats [37]. As expected, all three kinases showed significant activity towards the generic MBP substrate (Fig. 1B, lanes 2, 6 and 10), transferring between and pmols of ATP per pmol of MBP per min (data not shown). Only CDK7/ CycH/MAT1 phosphorylated the GST-CDK2 (K33>R) substrate (Fig. 1B, lane 3). In agreement with previous studies [21,25,38], only CDK9/CycT1 phosphorylated the GST-CTD15(S5>A) substrate (Fig. 1B, lane 12), thus stressing the specificity of CDK7/CycH/MAT1 and CDK8/CycC for S5 of the YSPTSPS consensus and the preference of CDK9/CycT1 for S2. None of the substrates was phosphorylated in the absence of a kinase (Fig. 1B, lanes 13 16). We also noticed phosphorylated bands in the CDK8/CycC and CDK9/CycT1 samples that had the mobility of CDK8 (Fig. 1B, lanes 5 8) or CDK9, respectively (Fig. 1B, lanes 9 12). These bands probably represented the autophosphorylation of CDK8 and CDK9 that was reported previously [21,27,38]. In summary, we established that our recombinant kinases had properties that were similar or identical to the ones reported in previous studies for their native counterparts. We concluded that further comparison of the recombinant kinases was justified.

5 1008 R. Pinhero et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 2. CDK7/CycH/MAT1, but not CDK8/CycC and CDK9/CycT1 produce a hyperphosphorylated GST-CTD. (A) Kinase reactions were performed with the combinations of kinase and substrate, as indicated above each lane and as described in the Materials and methods. (B) Kinase reactions were performed with a fixed amount (800 ng) of GST-CTD(1 52) and serial 1 : 3 dilutions of the kinases, as indicated above each panel of lanes. The mobility of the hypophosphorylated GST-CTD(1 52)-IIa and the hyperphosphorylated GST-CTD(1 52)- IIo bands is indicated on the left. (C) Kinase reactions were performed with GST-CTD(1 52) and the kinases as indicated above each lane. The samples in the input lanes were loaded without further manipulations. The samples in the GST pull-down lanes were incubated with GSH-Separose 4B and the bound proteins were eluted from the washed beads. The mobility of the hypophosphorylated GST-CTD(1 52)-IIa and the hyperphosphorylated GST-CTD(1 52)-IIo bands are indicated on the left. The mobility of the 90 kda molecular mass markersisindicatedontheright. Fig. 3. Differential phosphorylation of parts of the C-terminal domain (CTD) by CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. (A) Kinase reactions were performed with the combinations of kinase and substrate, as indicated above each lane. The positions of the unphosphorylated substrate polypeptides (IIa) were derived from Coomassie stained gels without any kinase added (data not shown) and are marked by the asterisk in each lane. The bars above each lane represent relative levels of phosphorylation of the substrates. The signals of phosphorylation of the GST-CTD(1 52) were equalized between the three different kinases (lanes 2, 9, 16) and the signals of phosphorylation of the truncated CTD substrates were plotted relative to GST- CTD(1 52). The figure is representative of at least three independent kinase assays with each substrate/kinase combination. (B) The average ratios of phosphorylation of individual substrates relative to the GST- CTD(1 52) substrate were calculated and plotted. The bars represent at least three independent parallel experiments with each substrate and the three kinases. CDK7/CycH/MAT1 hyperphosphorylates CTD Next, we normalized the activity of the three kinases using MBP and compared their activity towards the full-length CTD substrate [GST-CTD(1 52)] (Fig. 2A). In these and all subsequent reactions, we used at least a 100-fold molar excess of CTD substrates vs. kinase. We did not notice major differences in the preference of the three kinases

6 Ó FEBS 2004 Phosphorylation of pol II C-terminal domain (Eur. J. Biochem. 271) 1009 towards MBP or GST-CTD(1 52). Therefore, in contrast to a previous report [28], we do not support the idea that there was a markedly higher CTD activity in CDK7/CycH/ MAT1 as compared to CDK8/CycC and CDK9/CycT1 (Fig. 2A, lanes 2, 5, 8). However, there was a substantial difference in the mobility of the phosphorylated GST- CTD(1 52) species that were generated by the three kinases. Whereas CDK8/CycC and CDK9/CycT1 produced mostly the higher mobility (hypophosphorylated) IIa form, CDK7/ CycH/MAT1 produced equal amounts of both the highermobility IIa and lower-mobility (hyperphosphorylated) IIo forms (Fig. 2A, compare lanes 2, 5 and 8). Most of the GST-CTD(1 52) retained the mobility of the unphosphorylated/hypophosphorylated band, as determined by Coomassie staining of the gels after the kinase reactions (data not shown). In addition, in the reactions with GST-CTD(1 52), the three kinases transferred between and pmols of ATP per pmol of GST-CTD(1 52) per min (data not shown). Thus, assuming only one phosphorylation per CTD molecule, a maximum of 6 20% of the GST-CTD molecules could be phosphorylated over the course of the reaction. It is therefore unlikely that the observed generation of the IIo band was a consequence of limiting substrate leading to high levels of phosphorylation. Nevertheless, to further test the possibility of limiting substrate, we titrated the kinases, thus reducing the kinase/substrate ratios. As indicated in Fig. 2B, titration of the kinases over a 24-fold range did not significantly alter the pattern of phosphorylation of the GST-CTD(1 52) substrate. Similarly, extending the incubation time of the kinase reactions did not produce a different pattern of phosphorylation of the GST- CTD (data not shown). Hence, the differential pattern of CTD phosphorylation does not appear to be solely a function of the level of kinase activity. A possible cause for the differential mobility of the GST- CTD substrate phosphorylated by the three kinases could be the contamination of the kinase reactions with the peptidy-prolyl isomerase, Pin1/Ess1 [39 42]. Pin1/Ess1 is a known modifier of the CTD structure that has been implicated in pol II transcription and RNA processing [39 42]. To test the possibility of Pin1/Ess1 involvement, we performed reactions with GST-CTD and the three kinases in the presence of the Pin1/Ess1 inhibitor, juglone [41,42]. Because we found no effect of juglone at concentrations up to 30 lm (data not shown), we believe it unlikely that the effects observed occurred as a result of Pin/Ess1 contamination. In all preparations of CDK8/CycC and CDK9/CycT1 we noticed the appearance of phosphorylated bands with similar mobility to the GST-CTD(1 52)-IIo band (Fig. 2B, lanes 5 12). These bands could potentially obscure the detection of the GST-CTD(1 52)-IIo form in the kinase reactions with CDK8/CycC and CDK9/CycT1. In order to circumvent this potential problem, we pulled out the GST- CTD(1 52) substrate molecules after completion of the kinase reactions and analyzed them separately. Briefly, kinase reactions were performed as usual and terminated by the addition of EDTA. Glutathione sepharose 4B beads (Amersham) were used to pull out GST-CTD(1 52) and elute it in SDS sample-loading buffer. In Fig. 2C (lanes 10 12), we clearly show that under conditions of nonlimiting substrate, only CDK7/CycH/MAT1 could produce substantial amounts of the hyperphosphorylated GST- CTD(1 52) IIo form. CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1 have preferences towards different parts of the CTD In a set of subsequent experiments, we analyzed the activity of the three kinases towards different parts of the CTD. CTD heptad repeats 1 15, 1 25, 27 39, 27 42, and (see Fig. 6) were expressed as GST fusion proteins. Kinase assays were performed exactly as with the full length GST-CTD(1 52) substrate. In these analyses, we first determined the relative levels of phosphorylation (IIa + IIo signals) of each of these substrates by the three kinases. The CTD repeats 27 39, and were definitely much better substrates for CDK7/CycH/MAT1 than repeats 1 15 and 1 25 (Fig. 3A, compare lanes 3 and 4 with lanes 5, 6 and 7). The best substrate for CDK7/CycH/ MAT1 appeared to be repeats (Fig. 3A, lane 6). Noticeably, repeats 1 25 and were less favored substrates than the shorter substrates represented by repeats 1 15 and 27 42, respectively (Fig. 3A, compare lanes 3 and 4 with lanes 6 and 7). CDK8/CycC phosphorylated repeats 1 25, and well (Fig. 3A, lanes 11 13), repeats 1 15 less well (Fig. 3A, lane 10) and repeats very poorly (Fig. 3A, lane 14). CDK9/CycT1 followed a pattern of total (IIo + IIa) phosphorylation that was similar to that of CDK8/CycC. However, the phosphorylation of repeats was comparable to that of repeats 1 15 (Fig. 3A, lanes 15 21). A comparison between the phosphorylation of individual CTD substrates by the three kinases is shown in Fig. 3B. CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1 phosphorylated repeats 1 15 and at comparable levels. CDK7/CycH/MAT1 consistently showed slightly higher activity; however, the difference in total phosphorylation (IIo + IIa) of these substrates in several independent experiments was not greater than twofold (Fig. 3B, graphs a, c). Repeats 1 25 were phosphorylated well by CDK8/ CycC and CDK9/CycT1 and only moderately by CDK7/ CycH/MAT1 (Fig. 3B, graph b). In contrast, repeats and were much better phosphorylated by CDK7/ CycH/MAT1 than CDK8/CycC and CDK9/CycT1 (Fig. 3B, graphs d, e). In the case of repeats 27 42, these differences were a result of the remarkably higher activity of CDK7/CycH/MAT1, while, in the case of repeats 27 52, the differences were caused by the modest-to-poor activity of CDK8/CycC and CDK9/CycT1 (Fig. 3A). In summary, we showed that the three kinases did not phosphorylate different parts of the CTD equally. Importantly, we outlined regions that enhance or suppress the activity of each kinase. Generation of the IIo form by different parts of the CTD We noticed substantial variations in the generation of the hyperphosphorylated IIo form of different CTD substrates by the three kinases. We decided to assess these variations by calculating the percentage of the signal in the IIo substrate bands. In order to do so, we measured the intensity of the radioactive signal along each lane of the gel and then subtracted the corresponding signals from the

7 1010 R. Pinhero et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 4. Generation of hyperphosphorylated GST-CTD substrates. (A) Kinase reactions were performed with the combinations of kinase and substrate, as indicated above each lane. The positions of the unphosphorylated substrate polypeptides (IIa) were derived from Coomassie stained gels without any kinase added (data not shown) and are marked by the asterisk in each lane. In order to obtain measurable signals, the kinase activity in lanes 4 9 was increased threefold as compared to the experiments in Fig. 3. (B) The intensity of the phosphorylation signal was determined along each lane in the gels of Fig. 3A. The signals from lanes 2 7, lanes 9 13 and lanes of the gels in Fig. 3A were plotted after subtracting the signals from lanes 1, 8 and 15, respectively. The graph representing the phosphorylation of GST-CTD(27 52) by CDK8/CycC was derived from lane 6 of (A), after subtracting the signal from lane 4. The asterisk indicates the position of the unphosphorylated/hypophosphorylated (IIa) substrate. The percentage of the signal in the IIo band is shown above each peak. lanes with samples that contained no substrate. After preparing a graph of the intensity of the signal from the substrates only, we calculated the percentage of signal in the IIo and IIa bands. Thus, assuming that the lower mobility bands corresponded to the hyperphosphorylated forms of the substrate, we evaluated the levels of production of hyperphosphorylated GST-CTD substrates by each kinase. In order to obtain measurable signals for all substrates and a comprehensive picture of the generation of the IIo form along the CTD, we performed kinase assays with the GST- CTD(1 15) and GST-CTD(27 52) substrates with higher amounts of the CDK8/CycC and CDK9/CycT1 (Fig. 4A). The results from these experiments and the calculations are presented in Fig. 4B. As in the case of the full length CTD (repeats 1 52), CDK7/CycH/MAT1 was very efficient in generating slowly migrating bands with all but the CTD(1 25) substrate (Fig. 3A, lanes 2 7, and Fig. 4B). We estimated that > 50% of the signal in these reactions was derived from the IIo-band of the substrates (Fig. 4B). In sharp contrast, CDK8/CycC did not generate considerable signals in the IIo band with repeats 1 25, and 27 42, despite the similar levels of phosphorylation with CDK7/ CycH/MAT1 (Fig. 3A, lanes and Fig. 4B). CDK9/ CycT1 produced a slightly higher percentage of signal from the IIo bands in these three substrates, but still the pattern of phosphorylation was similar to that observed with CDK8/ CycC (Fig. 3A, lanes and Fig. 4B). Surprisingly, CDK8/CycC and CDK9/CycT1 generated an ample percentage of signal in the IIo form of the CTD(1 15) and CTD(27 52) substrates, while total phosphorylation (IIo + IIa) was lower as compared to the other substrates (Fig. 3A, lanes 10, 14, 17, 21 and Fig. 4B). In the case of CDK9/CycT1, the GST-CTD(27 52) substrate generated 42% signal in the IIo band, which is comparable to that

8 Ó FEBS 2004 Phosphorylation of pol II C-terminal domain (Eur. J. Biochem. 271) 1011 produced by CDK7/CycH/MAT1 (Fig. 4B). In conclusion, we observed differential ability of the three kinases to hyperphosphorylate different parts of the CTD. This ability did not necessarily correlate to the levels of total phosphorylation of these parts. We also need to mention the mobility of the IIo forms of the different substrates. The IIo form of the N-terminal 1 15 repeats was only slightly retarded relative to the position of the unphosphorylated polypeptide (Fig. 3A, lanes 3, 10, 17, Fig. 4A). In comparison, the IIo forms of the C-terminal repeats 27 39, and were dramatically retarded, independently of the kinase that produced them (Figs 3A and 4A). The magnitude of mobility shift was not dependent on the number of repeats. For example, both CTD(1 15) and CTD(27 42) contain 15 heptad repeats, but their mobility shift was substantially different (Fig. 3A). Phosphorylation of GST-CTD(42 52) CDK8/CycC and CDK9/CycT1 phosphorylated GST- CTD(27 52) very weakly as compared to GST-CTD(27 42) (Fig. 3A). These observations suggested that repeats are a poor substrate for these kinases and that they contributed to the overall decrease of the phosphorylation of GST-CTD(27 52). We tested this possibility by performing kinase assays with a GST-CTD(42 52) substrate. In Fig. 5, we show that all three kinases poorly phosphorylated repeats as compared to the full length CTD (Fig. 5, compare lanes 2, 6 and 10 to 4, 8 and 12, respectively). CDK7/CycH/MAT1 phosphorylated repeats better than CDK8/CycC and CDK9/CycT1 (Fig. 5, lanes 4, 8, 12), but the overall signal was low. Thus, we obtained a Fig. 5. Phosphorylation of GST-CTD(42 52). Kinase reactions were performed with the combinations of kinase and substrate, as indicated above each lane. The position of the unphosphorylated GST-CTD(42 52) was derived from Coomassie stained gels without any kinase added (not shown) and is marked by asterisks. separate set of data, which indicated that repeats are a poor substrate for the three kinases and that they can influence the phosphorylation of the C-terminal portion of the CTD. Discussion In this study we performed a systematic comparison of the activity of CDK7/CycH/MAT1, CDK8/CycC and CDK9/ CycT1 towards recombinant CTD substrates. We expressed and purified all three recombinant kinases from insect cells following the same purification strategy. Our CTD substrates were portions of the natural mouse CTD. We worked under the conditions of non-limiting substrates and evaluated the relative activities of the kinases and the levels of production of hypophosphorylated (IIa) and hyperphosphorylated (IIo) forms for each of the substrates. This approach unveiled important differences between the three kinases that were not noticed in earlier studies [25,27 29,31]. First, we demonstrated that the three recombinant kinases transferred approximately equal amounts of phosphoryl groups to the full-length CTD substrate, yet they clearly produced different amounts of the hyperphosphorylated IIo form (Fig. 2A). CDK7/CycH/MAT1 generated approximately equal amounts of the hyperphosphorylated IIo and hypophosphorylated IIa forms, whereas CDK8/ CycC and CDK9/CycT1 produced predominantly the hypophosphorylated IIa form (Fig. 2). Titration of the kinases (Fig. 2b) and time-course experiments (data not shown) indicated that these specific patterns of phosphorylation were independent of the kinase/substrate ratio. Our observations strongly suggest that the three kinases act by different mechanisms on the pol II CTD substrate. Because the CTD contains multiple serine residues on the same polypeptide, it can be phosphorylated in two modes. In the disruptive mode, the kinase CTD complex will uncouple after the transfer of a phosphoryl group to the CTD, then the kinase will form a complex with another CTD molecule and phosphorylate it. Under the conditions of not-limiting substrate, a hypophosphorylated CTD (IIa) will be predominantly produced. If the kinase forms a complex with the CTD substrate and phosphorylates multiple S residues, then it acts by a processive mechanism. Under the conditions of not-limiting substrate, a hyperphosphorylated (IIo) form of the CTD will be produced. Another way of producing a hyperphosphorylated IIo form would be if (after the initial phosphorylation) the phosphorylated molecules become higher affinity substrates, leading to a multiple phosphorylation in a disruptive mode. Our results suggest that the CTD is phosphorylated in the disruptive mode by CDK8/CycC and CDK9/CycT1. However, CDK7/CycH/MAT1 operates by both processive and disruptive modes. Previous studies have shown that CDK7/CycH/MAT1 acts by the disruptive mechanism on short (YSPTSPS) 2 substrates [43] and that longer CTD substrates are much better phosphorylated [27]. Taken together, these and our observations suggest that the processive mechanism by CDK7/CycH/MAT1 needs more than two YSPTSPS repeats. Second, we demonstrated that different parts of the CTD are differentially phosphorylated by CDK7/CycH/MAT1. We showed that this kinase phosphorylates GST-CTD(27

9 1012 R. Pinhero et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 6. A model depicting the possible action of the three kinases on the C-terminal domain (CTD). The amino acid sequence of the mouse CTD is shown at the bottom. In the diagram, the heptad repeats, with N at position 7 of the YSPTSPS consensus, are shown as solid rectangles. Heptad repeats with K at position 7 are shown as halftone rectangles. Heptad repeats with T at position 7 are shown as striated rectangles. Bent arrows indicate processive phosphorylation. Straight arrows indicate disruptive phosphorylation. The three kinases seem to employ the processive mode of phosphorylation at the N-terminus of the CTD. The YSPTSPN repeats between the 20th and 30th repeats act as a suppressor of CDK7/CycH/MAT1 and could possibly prevent the spreading of the phosphorylation by this kinase into the N-terminal portion. The C-terminal subdomain is phosphorylated by the processive CDK7/CycH/MAT1 kinase via the cluster of YSPTSPKs around the 40th repeat. At the same time, CDK9/CycT1 is a weak processive kinase in this region. 42) significantly better than GST-CTD(27 39) (Fig. 3A). This minor extension of three heptad repeats creates a cluster of YSPTSPK repeats that leads to very high levels of phosphorylation of the rest of the molecule by CDK7/ CycH/MAT1 (Fig. 3A). Our observation is agreement with results of a previous study, which showed that a (YSPTSPK) 4 peptide is a better substrate for CDK7/CycH/ MAT1 than (YSPTSPS) 4 [27]. We therefore propose that the region encompassing repeats is the major site of CTD phosphorylation by CDK7/CycH/MAT1. At the same time, we showed that CDK7/CycH/MAT1 weakly phosphorylates GST-CTD(1 25) relative to the shorter GST-CTD(1 15) substrate (Fig. 3A). This difference between the two substrates applied only to CDK7/CycH/ MAT1. For CDK8/CycC and CDK9/CycT1, the better of the two substrates was GST-CTD(1 25) (Fig. 3A). Repeats contained two YSPTSPN repeats (Fig. 6). It has been shown that (YSPTSPN) 4 peptides are a less favored substrate of CDK7/CycH/MAT1 than (YSPTSPS) 4 [27]. We therefore propose that these repeats act as a suppressor of CDK7/CycH/MAT1 in the context of longer CTD substrates. Third, the last 10 C-terminal repeats (42 52) are a very poor substrate for all three kinases (Fig. 5). The presence of these repeats in the GST-CTD(27 52) substrate has a significantly negative effect on the activity of CDK8/CycC and CDK9/CycT1 and a moderately negative effect on the activity of CDK7/CycH/MAT1 (Fig. 3A). These results are consistent with the idea that repeats could act as a kinase suppressor in the context of the full-length CTD. The moderate effect on the activity of CDK7/CycH/MAT1 (Fig. 3A) could be attributed to the potent positive influence of the YSPTSPK repeats in It is noteworthy that the domain contains YSPTSPK repeats that alternate with an equal number of YSPTSPT repeats (see Fig. 6). In addition, both GST-CTD(27 52) and GST-CTD(42 52) contain the C-terminal ISPDDSDEEN sequence that is missing from GST-CTD(27 42). The importance of this peculiar alternating of the seventh amino acid in the C-terminal repeats and the ISPDDSDEEN sequence remains to be established. Fourth, we showed that the production of the hyperphosphorylated CTD substrates is not necessarily a result of their total phosphorylation. For example, total phosphorylation of GST-CTD(27 39) was approximately equal between the three kinases (Fig. 3B, column c). However, CDK7/CycH/MAT1 generated 62% of the signal in the IIo form, while CDK8/CycC and CDK9/CycT1 generated 2% and 5%, respectively (Fig. 4B, column d). At the same time, on the longer GST-CTD(27 52) substrate, CDK9/CycT1 produced 42% in the IIo form, yet total phosphorylation was very low (Figs 3A and 4B). The structure of the CTD provides little explanation for the basis of these differences. Nonetheless, it is clear that the ability of the kinases to produce hyperphosphorylated substrates is not related to their overall activity towards them. On a minor note, we noticed clear differences in the extent of retardation of the IIo band in SDS/PAGE between the C-terminal and the N-terminal parts of the CTD (Figs 3A, 4A and 5). The first 15 CTD repeats only slightly change their mobility upon hyperphosphorylation, independently of the phosphorylating kinase (Figs 3A and 4A), while the other CTD substrates display a dramatic retardation (Figs 3A, 4A and 5). We therefore suggest that the phosphorylation of the C-terminus of the CTD is responsible for the generation of the IIo form of pol II in vivo.an earlier study had reached the opposite conclusion [28]. This discrepancy might stem from the different substrates used. Furthermore, we used recombinant kinases, while the other group used immunoprecipitated CDK7 that might contain other kinase activities. Some of our conclusions and observations do not completely agree with separate pieces of evidence reported by other groups. Some of the differences can be explained by the fact that these studies used short synthetic CTD heptad peptides [25,27 29,31], while we used longer regions of the natural mouse CTD. For example, synthetic peptides were used to address the preference towards S2 or S5 and the effect of the seventh amino acid in YSPTSPS [27 29,31], but these substrates might have a limited use in assessing the preferences in the context of the natural CTD. Indeed, CDK9/CycT1 seems to phosphorylate well S5 of the CTD heptad consensus on short synthetic peptides [28,31], but it definitely prefers S2 on full length CTD [25]. In addition, in some of these studies, high (1 : 3) or unknown enzyme/ substrate ratios were used, thus posing the risk of masking

10 Ó FEBS 2004 Phosphorylation of pol II C-terminal domain (Eur. J. Biochem. 271) 1013 the differences in the kinase activity because of limiting substrates. The data in this report are summarized in the model presented in Fig. 6. All three kinases phosphorylate equally well the N-terminal repeats of the CTD. Even though to a different extent, all three kinases seem to employ the processive mode of phosphorylation in this region. The YSPTSPN repeats between the 20th and 30th repeats act as a suppressor of CDK7/CycH/MAT1 and may prevent the spreading of phosphorylation by this kinase into the C-terminal portion. Thus, the CTD seems to be separated into two subdomains. The C-terminal subdomain is mainly phosphorylated by the processive CDK7/CycH/MAT1 kinase via a focal point in the cluster of YSPTSPKs around the 40th repeat. At the same time, CDK9/CycT1 is a weak processive kinase in this region. As indicated previously [44,45], partially phosphorylated CTD is a better substrate for CDK9/CycT1 than unphosphorylated CTD. It is therefore possible that the CDK9/CycT1 activity could significantly increase upon initial phosphorylation of the CTD by another kinase. It is also possible that the potent phosphorylation of repeats by CDK7/CycH/MAT1 could spread partially into the last 10 repeats, thus triggering higher levels of processive activity by CDK9/CycT1. Such an idea is in agreement with the concept that TFIIH, which contains CDK7/CycH/MAT1, acts early in the transcription process [2,46]. P-TEFb, which contains CDK9/CycT1, acts after TFIIH [2,46]. In vivo, the function of the CTD is influenced by other modifications, including other phosphorylations, glycosylation and proline isomerization [1,2,5]. All these modifications and the corresponding enzymes could have additional effects on the substrate specificities of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. 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