From the Ecole Normale Superieure, Unité de
Génétique Moléculaire, 46, rue d'Ulm, 75230 Paris
Cedex 05, France, the ¶ Whitehead Institute for Biomedical
Research, Cambridge, Massachusetts 02142, the
Oklahoma Medical
Research Foundation, Oklahoma City, Oklahoma 73104, and the
** Wellcome/Cancer Research Campaign Institute, Tennis Court Road,
Cambridge CB2 1QR, United Kingdom
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ABSTRACT |
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The C-terminal part of the largest subunit of eukaryotic RNA polymerase II is composed solely of the highly repeated consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. 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, DNA-dependent 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.
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INTRODUCTION |
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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 Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. 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 (7-10). 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 ~10:1) 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 two serines are nonviable (14).
In the past 5 years, many protein kinases from various organisms have
been described as being able to phosphorylate the CTD in
vitro (15-22). Some of them are now well identified. -Kinase from rat (20) (also called factor b in yeast (16) and TFIIH or BTF2 in
humans (22)) is a multisubunit transcription factor that contains
DNA-dependent helicase activity, DNA repair activity, and
CTD kinase activity (see Ref. 23 for review). A subcomplex called TFIIK
is responsible for the CTD kinase activity and is composed of the
Cdk-related protein kinase MO15/Cdk7 in mammals (KIN28 in yeast),
associated with cyclin H (CCL1) and MAT-1 (24-27, 67, 68). The
kinase-cyclin pair MO15-cyclin H (KIN28-CCL1) is already known as a
cyclin-dependent kinase (CAK
(Cdc2-activating kinase)) in
vitro (28, 29). Other protein kinases have also been described as
able to phosphorylate the CTD. DNA-dependent protein kinase
(DNA-PK) is composed of a catalytic subunit (DNA-PKcs) and
a regulatory component corresponding to the Ku autoimmune antigen (30,
31). It acts as a CTD kinase when stimulated by linear double-stranded
DNA and by several transcriptional activators (30, 32). Recently,
DNA-PK has also been shown to phosphorylate the RNA polymerase I
transcriptional apparatus and to inhibit RNA polymerase I
transcription. Moreover, this protein kinase plays a major role in DNA
repair processes and recombination of immunoglobulin gene locus in the
cells of the immune system (33). MAP kinases are induced by mitogenic
stimuli and by heat shock. They are able to phosphorylate the CTD among
their many known in vitro substrates (34).
The aim of this study was to determine the sites phosphorylated in vitro in the CTD by TFIIH kinase, DNA-PK, and MAP kinases. Comparison of the results obtained in vitro with what is known about CTD phosphorylation in vivo gives some clues to the nature of the protein kinases involved in the in vivo phosphorylation of the RNA polymerase CTD. To determine which of the three serines is phosphorylated by the different CTD kinases, we synthesized a set of peptides containing one or two CTD motifs in which each serine was successively replaced by an alanine. We devised new electrophoretic conditions to be able to separate the phosphorylated peptides. 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).
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MATERIALS AND METHODS |
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Kinase Assays
Reaction mixtures (24 µl) contained (final concentrations) 40 mM -glycerophosphate, pH 7.4, 7.5 mM
MgCl2, 7.5 mM EGTA, 5% glycerol,
[
32-P]ATP (0.2 mM, 1 µCi), 50 mM NaF, 1 mM orthovanadate, 0.1% (v/v)
-mercaptoethanol, and the following peptides: hepta-3
(SPTSPSY)3; 0.4 mg/ml), hepta-2 ((SPTSPSY)2) or
modified hepta-2 (0.4 mg/ml), or hepta-1 (SPTSPSY) or modified hepta-1
(1.6 mg/ml) (unless otherwise mentioned in the figure legends).
Phosphorylation reactions were performed with 4 µl of human
ERK1 (agarose-conjugated ERK1; Upstate Biotechnology, Inc.) for 90 min
at 30 °C, with 0.4 units/ml murine recombinant ERK2 (from Prof. P. Cohen, University of Dundee, Dundee, United Kingdom) for 30 min at
30 °C, with ~40 ng of TFIIH kinase (TSK SP-5-PW fraction (36)) for
2.5 h at 30 °C in the presence of 1.6 mg/ml polyvinyl alcohol
and 5 mg/ml bovine serum albumin, or with 2 µl of DNA-PK (from Dr. G. Smith, Wellcome/Cancer Research Campaign Institute, Cambridge, United
Kingdom) in the presence of 200 µg/ml salmon sperm DNA for 30 min at
30 °C. The times used for in vitro reactions were chosen
to be in initial rate conditions. Peptides were synthesized by Dr. O. Siffert (Organic Chemistry Laboratory, Institut Pasteur). Reactions
were stopped by adding 1 volume of Laemmli sample buffer (37)
containing 5% -mercaptoethanol.
Gel Electrophoretic Conditions and Quantification
Hepta-3, Hepta-2, and Modified Hepta-2 Peptide Analysis-- Reaction samples were analyzed by 22% SDS-polyacrylamide gel electrophoresis performed according to Laemmli (37).
Hepta-1 Peptide Analysis and Modified Peptides-- Hepta-1 peptides were too small to be separated from the radiolabeled ATP front by the usual electrophoretic conditions. We acidified the buffer by replacing the Tris/glycine buffer with a phosphate buffer (38) as we previously described (35). Reaction samples were subjected to electrophoresis on 10% denaturing phosphate buffer gels at pH 6.0.
Gels were fixed in ethanol/acetic acid/trichloroacetic acid/water (3:1:1:5, v/v), dried, and submitted to autoradiography with an intensifying screen at 4 °C. Signals were quantified with a Fuji BAS reader, and measurements made with PC BAS.Phosphoamino Acid Analysis
The SPTTPSY peptide was phosphorylated by TFIIH kinase and analyzed as described above. The radiolabeled spot was cut out of the gel and washed rapidly in 500 µl of water. The phosphorylated peptide was eluted in 0.5 M ammonium acetate at 37 °C with gentle agitation overnight. Peptide hydrolysis and phosphoamino acid analysis were performed as described previously (39).
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RESULTS |
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TFIIH Kinase and the MAP Kinases ERK1 and ERK2 Phosphorylate Serine 5 of the CTD Motif-- Serizawa et al. (40) previously showed that TFIIH kinase phosphorylates the RNA polymerase II CTD at serine residues. To determine which of the three serines was phosphorylated by TFIIH kinase, we assayed a set of peptides in which serines were replaced by alanines. Fig. 1A shows that phosphorylation was completely lost when serine 5 was replaced by an alanine, whereas the two other serine substitutions had no effect on phosphorylation. Thus, only serine 5 of the CTD motif appears to be phosphorylated by TFIIH kinase. These results are in agreement with those reported by Roy et al. (25). Identical results were obtained with ERK1 (Fig. 1B) and ERK2 (data not shown).
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Phosphorylation by TFIIH Kinase and by ERK1 and ERK2 Reveals Differences in the Recognition of the Hepta-2 Peptide-- Within the entire CTD, all the repeats are contiguous, and all (but one) serines at positions 2 and 7 are close to the extremity of another repeat. Therefore, the absence of phosphorylation of these serines by TFIIH kinase (or MAP kinases) might be due to the absence of the amino acids surrounding these residues in the CTD. To rule out this possibility, we performed experiments with similar alanine replacements in hepta-2 peptides (Ser2-Pro3-Thr4-Ser5-Pro6-Ser7-Tyr8-Ser9-Pro10-Thr11-Ser12-Pro13-Ser14-Tyr1) (Fig. 2). These peptides were electrophoresed on a 22% denaturing polyacrylamide gel. Phosphorylation of the hepta-2 peptide by ERK1 or ERK2 revealed two separated bands with similar intensities. Phosphorylation efficiency was very low, and the peptide concentration that we used did not allow the characterization of multiple phosphorylations. The two bands correspond to phosphorylation in the left or right part of the peptide. Indeed, when serine 5 of the right part was replaced by an alanine, the upper band disappeared, and when the same substitution was made in the left side, there was no longer a lower band. Replacement of serine 9 had no effect on the phosphorylation. Therefore, as for hepta-1 peptides, only serine 5 of the CTD motif is phosphorylated in hepta-2 peptides by ERK1 and ERK2.
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Determination of the Consensus Sequence Recognized by TFIIH and MAP Kinases-- Another set of hepta-1 peptides was used to determine the involvement of each amino acid in the recognition by TFIIH and MAP kinases. At first glance, the comparison shows that the specificity of these different kinases is very similar. The hepta-1 peptide without any substitution was considered as the reference (Fig. 4A). C-terminal proline replacement by an alanine (SPTSASY) led to the abolition of peptide phosphorylation, a feature of the proline-dependent protein kinase family. Likewise, no significant peptide phosphorylation was observed when the N-terminal proline was displaced or removed (PSTSPSY, STSPSY, and STPSPSY). In conclusion, the two prolines of the motif are absolutely essential for TFIIH and MAP kinases. Phosphorylation was decreased by half when the tyrosine was replaced by an alanine. Thus, the tyrosine is important, but not essential. Replacement of the threonine at position 4 by a leucine or an arginine significantly increased the phosphorylation of the peptide, whereas glutamic acid had an opposite effect. Confirming the positive effect of a leucine at position 4, the N-terminal proline became nonessential when threonine 4 was replaced by a leucine (SALSPSY) (see "Discussion").
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Serines Phosphorylated by DNA-dependent Protein Kinase-- The same set of peptides used for TFIIH and MAP kinases was assayed to characterize the sites phosphorylated by DNA-PK in the CTD motif (Fig. 1C). None of the substitutions completely abolished the peptide phosphorylation. This suggested that more than one serine is phosphorylated by DNA-PK. However, serine 2 (APTSPSY) or serine 7 (SPTSPAY) replacement decreased peptide phosphorylation significantly. On the other hand, when serine 5 (SPTAPSY) was substituted, DNA-PK was even more efficient than with hepta-1. Another peptide was synthesized to determine the effect of the simultaneous substitution of serines 2 and 7 (peptide A2A7, APTSPAY). The introduction of these changes severely depressed phosphorylation by DNA-PK. We observed a residual phosphorylation after a long exposure that migrated like peptide A2A7 phosphorylated by ERK1, as if in the absence of its highest affinity sites, DNA-PK is able to weakly phosphorylate the serine located at position 5 (data not shown). In contrast, even after a long exposure, we never observed any phosphorylation of peptide A5 with the MAP kinases.
Phosphorylation of Modified Hepta-2 Peptides by DNA-PK-- Since the sites phosphorylated by DNA-PK were different from those phosphorylated by TFIIH and MAP kinases, two other modified hepta-2 peptides were synthesized. Four bands were observed by phosphorylating the hepta-2 peptide (Ser2-Pro3-Thr4-Ser5-Pro6-Ser7-Tyr8-Ser9-Pro10-Thr11-Ser12-Pro13-Ser14-Tyr1) with DNA-PK (Fig. 5). They may correspond to phosphorylation of serines 2, 7, 9, and 14, the four potential phosphorylation sites contained in the hepta-2 peptide. When serine at position 9 was replaced by an alanine (peptide A9), one of the bands disappeared, and when two serines were replaced (peptide A7A14), only two bands remained. These results support the hypothesis that each of the four bands corresponds to a dipeptide phosphorylated at only one site and that positions 2, 7, 9, and 14 are actually phosphorylated by DNA-PK.
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Influence of Substitution at Several Positions on Phosphorylation by DNA-PK-- Peptides identical to those used Fig. 4 were assayed with DNA-PK. A striking observation is that no substitution led to a complete loss of phosphorylation (Fig. 6). In half of the cases, the peptides were phosphorylated at least as well as the hepta-1 peptide. The replacement of the C-terminal proline (peptide A6) did not affect peptide phosphorylation. The same was true for the N-terminal proline with an increase in phosphorylation for peptide P4 (sequence STPSPSY); therefore, DNA-PK is not a proline-dependent protein kinase. Tyrosine replacement produced a decrease in phosphorylation; thus, this amino acid is important, but not essential. The peptide that contains arginine, a basic amino acid, at position 4 (SPRSPSY) was not a good substrate for DNA-PK. The two radiolabeled spots corresponding to this peptide that were separated on polyacrylamide gels may correspond to phosphorylation of either serine 2 or serine 7 (the amount of phosphorylated peptide reported on the histogram is the addition of the two values); the same is probably true for the other peptides, but they are not separated on the gel. In contrast, an acidic residue at position 4 (SPESPSY) led to very efficient peptide phosphorylation (Fig. 6, lower panel). No decrease in phosphorylation was observed when threonine 4 was substituted by a leucine, which is a neutral amino acid without a phosphate acceptor group, showing that this threonine is probably not phosphorylated by DNA-PK. Substitution of serine 5 by a threonine (SPTTPSY) decreased peptide phosphorylation. This result is surprising because this serine is not supposed to be phosphorylated by DNA-PK. Changes in charges or in the steric environment may explain the indirect effects on phosphorylation of serines 2 and 7.
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DISCUSSION |
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Models Concerning CTD Functions
Despite all the studies on the CTD and its phosphorylation, the
role of this domain of eukaryotic RNA polymerase II is still unclear. A
model frequently evoked for RNA polymerase II transcription initiation
is the recruitment of a hypophosphorylated RNA polymerase II to
the preinitiation complex and phosphorylation during the transition
from initiation to elongation (11-13). Possible pausing at an early
phase of transcription, dependent on proximal activating sequences
(42), might necessitate another phosphorylation for the release of RNA
polymerase. Therefore, a first phosphorylation might be necessary for
initiation and a second for relief of pausing. These two
phosphorylations might be carried out by different Ser/Thr or Tyr
protein kinases. By using immunofluorescence microscopy, Weeks et
al. (43) showed on polytene chromosomes that some specific genes
are transcribed by hyperphosphorylated polymerases, whereas hsp70
mRNAs are elongated by a mixture of hypo- and hyperphosphorylated forms. Similar results were obtained by in vivo protein-DNA
cross-linking assays (44). The CTD phosphorylation state during
transcription elongation may thus be different for the different types
of gene. Experiments in yeast suggest the involvement of several
protein kinases in CTD phosphorylation in vivo. Lee et
al. (45) have characterized the CTK1 gene encoding a
yeast nuclear CTD kinase, which presents some homologies to
cdc2/CDC28. CTK1 gene disruption decreases
in vivo phosphorylation of RNA polymerase II without abolishing it (45). The different sensitivity of CTD phosphorylation to
protein kinase inhibitors suggests that it is the same in higher eukaryotes: CTD phosphorylation in quiescent cells is decreased by the
protein kinase inhibitor
5,6-dichloro-1--D-ribofuranosylbenzimidazole, whereas it
is unaffected in serum-treated cells or heat-shocked cells (34, 46).
The number of protein kinases able to phosphorylate the CTD in vitro is still growing. A CTD kinase has been located in the yeast holoenzyme. This kinase is encoded by a new gene called SRB10, regulated by the cyclin SRB11 (47). Recently, another CTD kinase has been identified as a component of the positive transcription elongation factor of Drosophila. It has been suggested that this protein kinase could phosphorylate the CTD to release the RNA polymerase II from an abortive elongation state (48). In addition, McCracken et al. (69) showed recently that the CTD is required for efficient RNA processing. CTD phosphorylation might also be implicated in mRNA splicing. All these observations suggest that depending on the signal that induces specific gene transcription, the step in the transcription cycle or mRNA splicing, different enzymes might be involved in CTD phosphorylation. These protein kinases may have different site specificities in the CTD motif.
In Vivo Phosphorylation of the RNA Polymerase II CTD
Little is known about the exact phosphorylation state of RNA polymerase II in vivo. A shift on SDS-polyacrylamide gels revealed multiple CTD phosphorylation, and two-dimensional paper electrophoresis of in vivo labeled CTD showed a predominant phosphorylation of serine compared with threonine (ratio of ~10:1). This low level of threonine phosphorylation cannot be explained by threonine 4 phosphorylation because this position is highly conserved, and its phosphorylation would not lead to a correct ratio. However, it can be explained by phosphorylation at positions 2 and 5, which present some non-consensus threonines instead of serines (9).
Specificity of the Different Protein Kinases That Phosphorylate the CTD Motif
TFIIH and MAP Kinases Phosphorylate the Same Site in the CTD
Motif--
TFIIH kinase, ERK1, and ERK2 phosphorylate the same residue
in the repeated motif of the CTD. Surprisingly, the influence of each
amino acid of the motif seems very similar for TFIIH and MAP kinases.
They are proline-directed protein kinases. The consensus sequence
deduced from CTD peptides (PX(S/T)P) is identical to the
sequence determined for MAP kinases in the myelin basic protein and
epidermal growth factor receptor (41, 49, 50). For both kinases,
peptide phosphorylation is strongly enhanced by the replacement of
threonine 4 by a leucine. Moreover, the N-terminal proline, which is
essential for CTD sequence phosphorylation, is no longer necessary in
the presence of a leucine at position 4 (SALSPSY). When the site
phosphorylated by MAP kinases in whole proteins does not present a
proline at position 2, a hydrophobic amino acid at position
1 is
very often observed, confirming a strong influence of this residue on
peptide phosphorylation (51). One of the differences we found between
MAP and TFIIH kinase specificities in vitro is the more
stringent requirement of the latter for a serine at the phosphorylated
position. The second concerns the difference between hepta-1 and
hepta-2 peptide phosphorylation efficiencies. We observed a small and
progressive increase in phosphorylation of peptides containing one,
two, and three CTD motifs by ERK1 and ERK2. This is in agreement with
the data showing that the entire RNA polymerase II CTD is better
phosphorylated than a small peptide by most of the CTD protein kinases.
However, we showed that TFIIH kinase phosphorylates the hepta-2 peptide 13-fold better than the hepta-1 peptide, whereas the difference between
the hepta-3 and hepta-2 peptides is low and identical to that observed
for the other CTD kinases. Despite an identical phosphorylation site,
these experiments show that MAP and TFIIH kinase recognition sites are
not equivalent.
The Cdk2 Sequence Recognized by TFIIH Kinase Is Different from the
Consensus Sequence Determined with CTD Peptides--
TFIIH kinase is a
multisubunit complex containing several enzymatic activities. The CTD
kinase catalytic subunit of TFIIH kinase corresponds to the protein
kinase MO15/Cdk7 complexed with cyclin H (24-27, 67, 68). MO15 is
considered to be a Cdc2-like protein (52, 53). We showed in this study
that TFIIH kinase is also a proline-directed protein kinase like Cdc2.
However, definition of a general consensus sequence for TFIIH is
problematic. It was previously shown that this MO15-cyclin H complex,
also called CAK, was implicated in p34cdc2 and p33cdk2
activation by phosphorylation of threonine 161 for p34cdc2 and
threonine 160 for p33cdk2, whose surrounding sequences are
almost identical (RVYT*HEVVTLWYR) (54). There are no prolines, either
at position 2 or at position +1. One could imagine that a subunit
supplementary to MO15 and cyclin H would allow a change in specificity.
However, this hypothesis seems unlikely because Serizawa et
al. (67) showed that TFIIH kinase (identical to the factor we
used) phosphorylates both the RNA polymerase II CTD and Cdk2. One
similarity between CTD kinase and Cdc2 kinase is their relative
efficiency in phosphorylating serine and threonine. We showed that a
serine-containing peptide is a better substrate for TFIIH kinase than a
threonine-containing peptide. An identical conclusion was reached with
Cdk2 when Thr160 was replaced by a serine (54).
DNA-PK Is Able to Phosphorylate Two Serines in the CTD Motif-- Several proteins such as HSP90, Sp-1, p53, c-Jun, and SV40 large T antigen are in vitro substrates for DNA-PK. Studies of the sites phosphorylated by DNA-PK in these proteins defined a common minimal consensus sequence, which is Q(S/T) or (S/T)Q)(55-60), but these sequences are not present in the CTD, even in the non-consensus motifs. However, it has been previously noted that certain proteins such as c-Fos are also efficient substrates, but are apparently not phosphorylated on Q(S/T) or (S/T)Q motifs (61).
We showed in this study that in contrast to TFIIH and MAP kinases, DNA-PK does not phosphorylate serine 5 of the CTD motif, but rather the serines at positions 2 and 7. The substitution of each amino acid surrounding the phosphorylated serines did not lead to a complete loss of phosphorylation, suggesting that none of these amino acids is absolutely necessary. All the substitutions showed that prolines are not important for CTD phosphorylation by DNA-PK. For DNA-PK, the replacement leading to the most striking increase is the substitution of threonine 4 by a glutamic acid. The positive influence of glutamic acid was previously shown for p53 and c-Jun. One of the sites phosphorylated in vitro by DNA-PK is ES7Q for p53 and ES249QE for c-Jun (59, 60). The successive replacement of each of these glutamic acid residues in the c-Jun sequence decreased phosphorylation. The influence of glutamic acid 251 on serine 249 phosphorylation may be similar to the influence of glutamic acid 4 on serine 2 phosphorylation in the CTD sequence. This observation is consistent with the idea that acidic residues contribute to the recognition by DNA-PK, but such sequences are never found in the RNA polymerase II C-terminal domain, even in the non-consensus motifs.Tyrosine May Play a Role in DNA-Substrate Binding-- Tyrosine 1 replacement by an alanine significantly decreases peptide phosphorylation. Tyrosine may be important for enzyme-substrate binding or for DNA-substrate binding necessary for good recognition by DNA-PK. Most of the potential substrates phosphorylated by DNA-PK bind DNA, and the colocalization of the enzyme and the substrate on the same DNA fragment appears important. A unique CTD motif with a tyrosine at each end is able to bind DNA, but the case of a simple hepta-1 peptide has not been tested (62). More recently, West and Corden (14) showed that tyrosine replacement by a phenylalanine in yeast CTD is lethal for the cell. However, interpretation of the latter data remains difficult.
Physiological Significance of the in Vitro Phosphorylation Studies
The approach that we have chosen is open to criticism since it relies on the assumption that the peptides are recognized with the same efficiency when free in solution or inserted into a polypeptide chain. However, recent studies have determined the optimal consensus sequence phosphorylated by protein kinases by comparing their efficiency in degenerate peptide libraries (63). In the case of the CTD, this approach appears more valid as its highly repeated nature suggests that this domain has a repetitive three-dimensional structure. Indeed, this suggestion is supported by recent NMR studies (64). Therefore, it is likely that the conformation of 1- or 2-fold repeats of the motif is probably similar to the conformation of the same motif in the whole RNA polymerase II subunit.
The CTD kinases that we chose for this study are good candidates to participate in CTD phosphorylation in vivo. Cytoplasmic MAP kinases are activated by a broad range of agents and migrate into the nucleus only in the presence of inducers. Previous studies have shown that DNA-PK is present in preinitiation complexes and phosphorylates the CTD of endogenous RNA polymerase II (65). The sites phosphorylated by DNA-PK defined in this study are not in full agreement with the serine/threonine ratios already published for this protein kinase (66): the CTD phosphorylated by DNA-PK contains approximately equal amounts of phosphoserines and phosphothreonines. However, positions 2 and 7 indeed contain the highest number of substitutions of serines by threonines (64 Ser/14 Thr). Moreover, this ratio can decrease if DNA-PK preferentially phosphorylates the C-terminal end of the CTD, where the number of substitutions is higher, or if DNA-PK is more specific for threonines (a possibility that has not yet been tested). TFIIH kinase is a good candidate for in vivo CTD phosphorylation because of its presence in the holoenzyme. Moreover, we show in this study that its recognition site corresponds to two adjacent motifs, indicating a good specificity for CTD repeats.
All these protein kinases are located very close to the transcription apparatus, but we cannot conclude about their roles in CTD phosphorylation in vivo. If the CTD is phosphorylated by a single enzyme, neither TFIIH kinase nor DNA-PK and the MAP kinases can explain the in vivo ratio of serine to threonine (1:10) (9). In contrast to the former model, several protein kinases might be involved, inducing different CTD modifications. In both cases, a systematic determination of the specificity of each CTD kinase for the CTD motif remains useful.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Prof. P. Cohen for the gift of recombinant ERK2 protein. We thank C. Benes for advice about phosphoamino acid analysis and E. Thompson for critical reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from the Societe de Secours des Amis des Sciences.
To whom correspondence should be addressed. Tel.:
33-1-44-32-39-46; Fax: 33-1-44-32-39-41; E-mail:
morange{at}biologie.ens.fr.
1 The abbreviations used are: CTD, C-terminal domain; TF, transcription factor; DNA-PK, DNA-dependent protein kinase; MAP, mitogen-activated protein.
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REFERENCES |
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