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J Biol Chem, Vol. 274, Issue 32, 22387-22392, August 6, 1999


Kinase Activity and Phosphorylation of the Largest Subunit of TFIIF Transcription Factor*

Mireille RossignolDagger , Anne Keriel, Adrien Staub, and Jean-Marc Egly§

From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, B. P.163, 67404 Illkirch Cedex, Communaute Urbaine de Strasbourg, France

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The largest subunit of the human basal transcription factor TFIIFalpha (also called RAP74) was reported previously to be the target of some phospho/dephosphorylation process. We show that TFIIFalpha possesses a serine/threonine kinase activity, allowing an autophosphorylation of the two residues at position serine 385 and threonine 389. Mutation analysis strongly suggests that autophosphorylation of both sites regulates the transcription elongation process. Moreover we also evidence three additional phosphorylation sites located at positions 207-230, 271-283, and 335-344. These sites are phosphorylated by casein kinase II-like kinases and TAFII250, a component of TFIID.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The eukaryotic transcription factor IIF (TFIIF)1, purified as an RNA polymerase II-associated protein (RAP) (1), is also characterized as an integral component of RNA polymerase II (RNA pol II) holoenzyme (2-4). Human TFIIF is an alpha 2beta 2 heterotetramer consisting of two 28-kDa (RAP30, TFIIFbeta ) and two 58-kDa (RAP74, TFIIFalpha ) subunits, which are required at different steps of the transcription reaction: formation of the preinitiation complex, promoter opening, initiation, elongation, and recycling (reviewed in Refs. 5 and 6). TFIIF allows the integration of RNA pol II into the preinitiation complex containing TFIIA, TFIIB, and TFIID, through its beta  subunit (7-10). TFIIF stabilizes the preinitiation complex by interacting with the other transcription factors such as TFIIB (11-13), TFIID (12-15), and TFIIE (16). Being associated with RNA pol II during elongation (5, 6, 19), TFIIF prevents transient pausing and therefore may stimulate the in vitro RNA synthesis by RNA pol II to a rate corresponding to the rate observed in vivo (17, 18, 20-22).

Structure-function studies have evidenced the domains devoted to initiation and elongation functions. The carboxyl-terminal part of TFIIFbeta binds to DNA (19, 20) and is required essentially for initiation (21), whereas the central moity, which binds RNA pol II (22, 23), is devoted to an elongation function (21). TFIIFalpha contains three domains: the amino-terminal part (amino acids 1-217) that contacts the beta  subunit and is necessary for single round initiation and stimulation of elongation (17, 20, 24, 28); both the highly charged central part (amino acids 218-358) and the carboxyl-terminal part (amino acids 358-517) interact with TFIIB (11) and RNA pol II (25, 26). TFIIFalpha plays a role in multiple round transcription (24); it is involved in RNA pol II recycling by stimulating the CTD phosphatase, which uses the carboxyl-terminal domain of the RNA pol II largest subunit as a substrate at the end of each transcription cycle (27, 28).

TFIIF activity could be regulated by several enzymes such as acetylases (p300/CBP, p300/CBP-associated factor, and TAFII250) (29), poly(ADP-ribose) polymerase (30), and kinases (12, 31-33). Several studies have demonstrated that TAFII250 (12, 33) and TFIIH-associated kinase (31, 32) used TFIIFalpha as subtrate in vitro.

In the present study, we identify the various phosphorylation sites of TFIIFalpha . We demonstrate that TFIIFalpha possesses a serine threonine kinase activity, allowing an autophosphorylation of the two residues at position serine 385 and threonine 389. Mutation analysis strongly suggest that autophosphorylation of both sites regulate the transcription elongation process. Moreover we also evidence three phosphorylation sites located at positions 207-230, 271-283, and 335-344. These sites are phosphorylated by some CKII and TAFII250 kinases.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recombinant TFIIF-- The TFIIFalpha /pET15b and the TFIIFbeta /pVL1392 vectors were described previously (14, 34). Either serine 385 (TFIIFalpha S385A) or threonine 389 (TFIIFalpha T389A) or both residues (TFIIFalpha S385A/T389A) of TFIIFalpha were mutated to alanine by polymerase chain reaction amplification to generate SacI/PstI fragments, which were further inserted into the SacI/PstI-digested TFIIFalpha /pAcAB3 plasmid. The sequences of the 5' primers are: TFIIFalpha S385A, 5'-GGAGGGAGCTCAAGGGGCAACGCCCGCCCAGGCACGCCC-3'; TFIIFalpha T389A, 5'-GGAGGGAGCTCAAGGGGCAACAGCCGCCCAGGCGCCCCC-3'; TFIIFalpha S385A/T389A, 5'-GGAGGGAGCTCAAGGGGCAACGCCCGCCCAGGCGCCCCC3, and the sequence of the 3' primer is 5'-GCCGCAACCGCTTGGCTGCAGGC-3'.

Spodoptera frugiperda 9 cells (typically 2.5 × 108) were then coinfected with either wild type TFIIFalpha or TFIIFalpha -S385A, -T389A, or -S385A/T389A mutants and TFIIFbeta baculoviruses at a multiplicity of infection of 2 and 2 plaque-forming units/cell. Cells were collected 48 h postinfection, washed with 20 ml of ice-cold buffer A (20 mM Hepes, pH 7.9, 150 mM NaCl, and 1 mM dithiothreitol) and then dounced in 15 ml of buffer A containing 0.5 mM phenylmethylsulfonyl fluoride and 1 × protease inhibitor mixture (35). The extract was centrifuged (20,000 × g, 1 h) at 4 °C and loaded onto a 15-ml phosphocellulose P11 column (2.5 × 6 cm, flow rate: 0.25 ml/min) equilibrated in buffer A. Step gradient elutions were performed sequentially with 5 column volumes of buffer A containing 0.3, 0.6, and 1 M KCl. TFIIF eluted at 0.6 M KCl was dialyzed against buffer B (50 mM Tris-HCl, pH 7.9, 10% glycerol, 0.1 mM EDTA, 0.5 mM dithiothreitol) containing 0.1 M KCl before being applied onto a DEAE-5PW (Toso-Haas) (0.75 × 7.5 cm, flow rate: 0.6 ml/min). Proteins were eluted with a 12-ml linear gradient from 0.1 to 0.6 M KCl in buffer B. The TFIIF containing fractions of the DEAE-5PW column (0.3 ml, peak 0.25 M KCl) were pooled and used for subsequent experiments.

Kinase Assays-- Highly purified wild type (from HeLa cells or recombinant) or mutated TFIIF either free or immunoprecipitated with monoclonal antibodies designed against the alpha  subunit are incubated at 30 °C for 30 min in 20 µl of kinase buffer (20 mM Hepes, pH 7.9, 15 mM MgCl2, 30 mM KCl) containing 1 µCi of [gamma -32P]ATP. The reactions are stopped by the addition of protein loading buffer followed by SDS-PAGE and autoradiography. Phosphorylation of rTFIIF S385A/T389A and Escherichia coli TFIIF was in some case performed in the presence of 0.5 unit of casein kinase II (Roche Molecular Biochemicals) or HeLa whole cell extract.

To evaluate the intrinsic kinase activity of TFIIFalpha , an in-gel kinase assay was performed. In brief, rTFIIF (400 ng) is resolved by SDS-PAGE, and proteins are renatured by successive washes at room temperature for 30 min in 50 mM Tris-HCl, pH 7.9, and 2.5% Triton X-100; then in the kinase buffer containing 50 mM Tris-HCl, pH 7.9, and 2.5% Triton X-100; and finally in the kinase buffer. The kinase reaction was then initiated by addition of 35 µCi/ml [gamma -32P]ATP for 90 min at 30 °C. The gel was washed with 5% trichloroacetic acid and 1% sodium pyrophosphate, stained with Coomassie Blue, dried, and autoradiographied.

When indicated, phosphoamino acid analysis and phosphopeptide mapping by amino acid sequence analysis were performed on autophosphorylated rTFIIF (10 µg) or on rTFIIF S385A/T389A (10 µg) phosphorylated with 200 ng of HeLa WCE as described in Ref. 36.

Pulse Chase Assay of Transcription Elongation-- RNA polymerase II (10 µg) purified from HeLa cells (37) is incubated for 15 min at 28 °C in 50 mM Tris-HCl, pH 7.9, 10% glycerol, 0.1 mM EDTA, 50 mM KCl, 7 mM MgCl2 in the presence of 250 ng of oligo(dC)-tailed pGR220 (38), 3 units of recombinant RNasin (Promega), 50 µM ATP, 50 µM GTP, 2 µM CTP, and 10 µCi of [alpha -32P]CTP. The reaction was chased as indicated by addition of 100 µM cold CTP, 2 µM UTP, and 10 ng of either the wild type rTFIIF or rTFIIF mutants. Transcripts were analyzed by electrophoresis on 5% polyacrylamide, 7 M urea.

In vitro transcription assays using recombinant transcription factors and RNA pol II were used as described earlier (37).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A Serine/Threonine Kinase Is Associated with TFIIF-- Previous studies have shown that TFIIFalpha is phosphorylated in vitro as well as in vivo (12, 31, 32, 39, 40). On the other hand, we noticed that highly purified TFIIF was phosphorylated even in the absence of any additional kinase. To further analyze the potential enzymatic activity of TFIIF, in vitro kinase assays were performed with either purified endogenous TFIIF (hereafter referred to as eTFIIF) or purified recombinant TFIIF (hereafter referred to as rTFIIF). eTFIIF was purified from HeLa WCE on heparin-Ultrogel, DEAE-Spherodex, heparin-5PW and phenyl-5PW columns (37). The phenyl-5PW eluted fraction (0.65 M ammonium sulfate) was used for all subsequent experiments. rTFIIF was overexpressed in Sf9 insect cells coinfected with baculoviruses encoding the alpha  (TFIIFalpha ) and the beta  (TFIIFbeta ) subunits, respectively, and further purified from cytoplasmic cell extract on phosphocellulose and DEAE columns (Fig. 1A). The baculovirus expression system was preferred to expression in E. coli because post-translational modifications, such as phosphorylation, are similar to those observed in mammalian system.


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  		Fig. 1.   A serine/threonine kinase is associated with TFIIF. A, eTFIIF and rTFIIF purified from HeLa cells whole cell extract (phenyl fraction (37)) or baculovirus-infected cells (DEAE fraction), respectively, were immunoprecipitated with Ab-TFIIFalpha and subjected to an in vitro kinase assay. The reactions were resolved on SDS-PAGE followed by Coomassie Blue staining (Coomassie) and autoradiography of the same gel (Autoradiogram). The position of the alpha  and beta  subunits is indicated. H, immunoglobulin heavy chain; L, immunoglobulin light chain. B, phosphoamino acid analysis of rTFIIF. The in vitro autophosphorylated 32P-rTFIIF was resolved on SDS-PAGE. The radiolabeled TFIIFalpha was subjected to phosphoamino acid analysis. The positions of the standard phosphoamino acids (P-Ser, phosphoserine; P-Thr, phosphothreonine; and P-Tyr, phosphotyrosine) and free phosphate (Pi) are indicated.

To further eliminate contaminating enzymatic activities, eTFIIF and rTFIIF were immunoprecipitated with monoclonal antiboby raised against the alpha  subunit (Ab-TFIIFalpha ) cross-linked to protein G-Sepharose beads, and the immuno-adsorbed proteins were extensively washed at high salt concentration (1 M KCl). The immobilized proteins were assayed for kinase activity in the presence of [gamma -32P]ATP and in the absence of any additional substrate, before being resolved on SDS-PAGE (41). The antibody directed toward the alpha  subunit is able to precipitate not only rTFIIFalpha , but also rTFIIFbeta in a stoichiometric amount (Fig. 1A, compare lane 2 with lane 1). This immunoprecipitation is highly specific, since no other additional polypeptide, except the heavy and the light chain of immunoglobulin, are present. Moreover as shown on the autoradiogram, a radiolabeled polypeptide having the same electrophoretic mobility as TFIIFalpha is observed, suggesting that TFIIF is phosphorylated (Fig. 1, compare lanes 1 and 2 with lanes 3 and 4, respectively).

Phosphoamino acid analysis demonstrates that the phosphorylation occurs at serine and threonine residues on both rTFIIFalpha and eTFIIFalpha (Fig. 1B and data not shown). In order to identify phosphoserine and phosphothreonine residues, a phosphopeptide mapping analysis was performed. In vitro phosphorylated 32P-rTFIIFalpha was trypsin-digested, and peptides were resolved by high performance liquid chromatography. Microsequencing of the single radiolabeled oligopeptide, encompassing residues 383-402 of TFIIFalpha , evidenced the two autophosphorylation sites (Fig. 2, AuPS). A careful analysis of the dehydro-amino acid derivative resulting from Edman degradation reaction reveals that serine and threonine at position 385 (Ser-385) and 389 (Thr-389) are phosphorylated. Together, these experiments suggest that TFIIFalpha is efficiently phosphorylated and that this phosphorylation is catalyzed by either a serine/threonine protein kinase tightly associated with TFIIF or by one of the subunits of TFIIF.


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  		Fig. 2.   Diagram illustrating the organization and the phosphorylation sites of TFIIFalpha ; the NH2-terminal domain has most of the functions required for accurate initiation and elongation. The central region was shown to be a serum responsive factor (SRF) activation sequence. The COOH-terminal region is targeted by TFIIB and RNA pol II and is required for CTD phosphatase stimulation and multiple round transcription stimulation. Sequence alignments with the Xenopus laevis TFIIFalpha (XTFIIFalpha ), the Drosophila melanogaster factor 5a (df5a), and Saccharomyces cerevisiae Ssu1p/Tfg1 (ySsu71) of the four microsequenced phosphopeptides obtained are shown. Identical and similar residues are in bold. The kinases consensus sequences are boxed.

TFIIFalpha Is a Serine/Threonine Kinase-- To further demonstrate the presence of some kinase activity associated with TFIIF, we performed an in-gel kinase assay. This assay discriminates between intrinsic TFIIF-mediated kinase activity versus contaminating kinase copurifying with TFIIF. After SDS-PAGE, TFIIF subunits were renatured by repeated washings of the gel and then subjected to kinase reaction in the gel in the presence of [gamma -32P]ATP (see "Experimental Procedures" and Fig. 3A). This allows detecting a single radioactive polypeptide that possesses the same apparent molecular weight as TFIIFalpha , revealing that TFIIFalpha autophosphorylates independently of the presence of TFIIFbeta . This latter result was further confirmed by measuring the in vitro kinase activity of either rTFIIF (composed of alpha  and beta  subunits) or the alpha  subunit (rTFIIFalpha ) overproduced in insect cells and purified as described above. Under these conditions, TFIIFalpha is phosphorylated regardless of the presence of TFIIFbeta (Fig. 3B, compare lane 3 with lane 4). However, we consistently observed a higher autophosphorylation rate of TFIIFalpha when TFIIFbeta was not present in the kinase reaction. Indeed, quantification of the radioactive signals corresponding to TFIIFalpha reveals that in the absence of TFIIFbeta the kinase activity of TFIIFalpha is about 2.5 times greater.


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  		Fig. 3.   TFIIFalpha is a kinase. A, purified rTFIIF (400 ng) was immunoprecipitated with Ab-TFIIFalpha , resolved on SDS-PAGE, and renatured in the kinase buffer before being submitted to an in-gel kinase assay in the presence of [alpha -32P]ATP. The position of the TFIIFalpha and TFIIFbeta is indicated. B, autophosphorylation of rTFIIFalpha immunoprecipitated with Ab-TFIIFalpha in the presence or absence of TFIIFbeta as indicated at the top of each panel.

Characterization of TFIIF Kinase Activity-- To characterize the autophosphorylation activity of TFIIFalpha , time course and dose-response experiments were performed. The autophosphorylation activity is linear between 1 and 30 min and reaches a plateau after 30 min (Fig. 4A). This activity exhibits a linear dependence during 15-min reaction (Fig. 4B). We also found that ATP, dATP, and GTP all act as cofactors for the TFIIFalpha kinase in competition assays, while CTP or cAMP had no effect on the phosphorylation of TFIIFalpha (data not shown).


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  		Fig. 4.   Characterization of TFIIFalpha kinase activity. A, in vitro kinase reactions were performed for the indicated time point at 30 °C using 50 ng of highly purified rTFIIF. B, increasing amounts of highly purified rTFIIF were used in a autophosphorylation assay for 15 min at 30 °C. The reactions were terminated by addition of Laemmli buffer followed by SDS-PAGE. TFIIFalpha autophosphorylation was quantitated using a Fuji bas 2000 analyzer, and the activity is represented in diagrams in the analyzer units. C, in vitro kinase reactions were performed with 50 ng of highly purified rTFIIF in the presence of 0, 2.5, 5, 10, 25, and 50 µM DRB. The result of one representative experiment is presented. D, TFIIFalpha autophosphorylation was quantitated, and activity is represented as a percentage of the activity observed in absence of DRB.

Next, we tested the potential transphosphorylation activity of TFIIFalpha by using either standard serine/threonine substrates such as histone H1 and casein or a variety of substrates involved in basal transcription such as TFIIB, TBP, TFIID, TFIIEalpha , TFIIEbeta , TFIIH, and the carboxyl-terminal domain of RNA pol II. Under our experimental conditions, none of these substrates was phosphorylated by TFIIFalpha (data not shown).

Since the adenosine analogue 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole (DRB) is known to inhibit RNA pol II elongation and several kinases such as P-TEFb (42) and TFIIH-associated kinase (43), we tested whether it could be used as an inhibitor of TFIIF kinase activity. Kinase assays were carried out with highly purified rTFIIF in the presence of increasing amounts of DRB (Fig. 4C, lanes 2-6). The autophosphorylation activity of TFIIF is very sensitive to DRB, with a 50% inhibition point (IC50) of 7 mM (Fig. 4, C and D).

TFIIF Autophosphorylation Modulates the Elongation Activity-- We have demonstrated that TFIIFalpha is phosphorylated on Ser-385 and Thr-389; we next tested whether the phosphorylation of these two residues has any effect on the transcriptional activity of TFIIF. Therefore Ser-385 and Thr-389 residues were, individually or together, mutated to alanine, giving rise to three recombinant baculoviruses encoding rTFIIFalpha /S385A, rTFIIFalpha /T389A, and the double mutant rTFIIFalpha S385A/T389A. These three baculoviruses were coinfected separately with the baculovirus encoding TFIIFbeta . Wild type rTFIIF and rTFIIF mutants were purified and tested in an in vitro kinase assay as described above. None of the rTFIIFalpha mutants are phosphorylated, whereas the wild type rTFIIFalpha is phosphorylated (Fig. 5A, compare lane 1 with lanes 2-4), demonstrating that phosphorylation requires the presence of both Ser-385 and Thr-389 residues to occur.


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  		Fig. 5.   TFIIFalpha autophosphorylation modulates elongation process. A, in vitro autophosphorylation of rTFIIF (15 ng of either wild type rTFIIF or rTFIIF mutants) were performed before being subjected to SDS-PAGE, autoradiography (Autoradio), and immunoblotting with Ab-TFIIFalpha (WB). B, transcription reactions were performed on AdML promoter-containing template (run-off, 309 nt), in the presence of either wild type rTFIIF (20 ng) or rTFIIF mutants (20 ng) or in the absence of TFIIF (-) as indicated at the top of the panel. The presence of the specific transcripts (run-off, 309 nt) was detected by autoradiography. The average and standard deviation (sd) of the quantification of four independent RNA synthesis experiments are indicated at the bottom of the panel. C, transcription elongation assays were performed as designed (lower panel) on oligo(dC)-tailed pGR220 template with RNA pol II either alone or in the presence of wild type rTFIIF (10 ng) or rTFIIF S385A/T389A mutant (10 ng). The chase phase was performed for the indicated time.

The rTFIIF mutants were then tested in an in vitro run-off transcription assay in the presence of the adenovirus major late promoter as a DNA template, RNA pol II, the basal transcription factors TBP, TFIIB, TFIIE, TFIIH, and either wild type rTFIIF or mutated rTFIIF (as indicated at the top of Fig. 5B). Transcriptions were performed in the presence of equal amounts of each mutated protein in subsaturating concentration of wild type rTFIIF, as judged in titration experiments (data not shown). Neither the single nor the double mutations at position Ser-385 and Thr-389 of TFIIFalpha appear to modify the transcription activity of TFIIF (Fig. 5B, compare lane 1 with lanes 2-4, and see the quantification of three independent experiments at the lowest part of the panel).

Since TFIIF is a basal transcription factor that functions in both transcription initiation and elongation, we tested whether TFIIFalpha mutations affect its elongation activity, using a standard "tailed template" assay (38). This assay allows the synthesis of defined RNA transcripts in the absence of factors required for promoter-specific initiation. The transcription template is a linearized double-stranded plasmid that has been modified by addition of a 3' oligo(dC) tail to one of its termini. RNA pol II binds the oligo(dC) tail and initiates transcription rapidly and specifically within the first 6 (dC) residues adjacent to the duplex DNA terminus (44). The increase in size of the RNA transcripts is directly proportional to the rate of RNA chain elongation by RNA pol II. Transcription of the oligo(dC)-tailed plasmid template was initiated by RNA pol II, in the absence of UTP, but in the presence of ATP, GTP, and 2 µM [alpha -32P]CTP. As diagrammed in Fig. 5C, the first and second groups of nontemplate strand (dT) residues are ~135 and ~250 nt downstream of the oligo(dC) tail, respectively. During the pulse phase, transcripts of ~135 nt accumulate, and after 15 min, these transcripts were chased with 100 µM cold CTP and 2 µM UTP in the absence or the presence of subsaturating amounts of wild type rTFIIF or equal amounts of rTFIIF mutants. In the absence of TFIIF or in the presence of each of the TFIIF subunits (data not shown), transcripts of ~250 nt are synthesized at a very slow rate, starting at t = 16 min (Fig. 5, lane 7), whereas in the presence of wild type rTFIIF, transcripts are accumulated more rapidly at t = 4 min (lane 12) and chased into longer products (lanes 15-28), indicating that TFIIF increased the rate of RNA chain elongation by RNA pol II through these sites. In the presence of wild type rTFIIF, synthesis of 280 nt (lane 15) and 309 nt transcripts (lane 17) occurs after 12 and 20 min chase, respectively, whereas in the presence of rTFIIF S385A/T389A the 280-nt (lane 23) and the 309-nt transcripts (lane 25) appear earlier at 8- and 16-min chase, respectively. In this case (that repeatedly was reproduced), the rate of elongation by RNA pol II is slightly increased, thus demonstrating that the autophosphorylation of TFIIF may down-regulate the RNA pol II elongation activity.

TFIIFalpha Can Be Phosphorylated by CKII-- It was demonstrated that the transcriptional activity of TFIIF can be regulated by phosphorylation of the alpha  subunit (40). We demonstrate here that residues S385 and T389 of TFIIFalpha are the targets of a phosphorylation event. To further evaluate whether the regulation of TFIIF transcription activity involves other phosphorylation sites than the two presently identified, in vitro kinase assays were carried out in the presence or in the absence of HeLa WCE using the mutated rTFIIF S385A/T389A as a substrate (which was previously immunoprecipitated with Ab-TFIIFalpha ). After 30 min of incubation, the beads were extensively washed, and the immunoabsorbed proteins were resolved by SDS-PAGE and detected by autoradiography. As shown in Fig. 6A, a radiolabeled polypeptide having the same electrophoretic mobility as TFIIFalpha is observed, indicating that in such conditions, TFIIF is efficiently phosphorylated (compare lanes 2 and 3). The radiolabeled polypeptide does not correspond to endogenous TFIIFalpha present in HeLa WCE, since it is not observed in the presence of HeLa WCE alone (Fig. 6A, compare lanes 1 and 3). In order to identify these phosphorylation sites, a phosphopeptide mapping analysis was performed on the radiolabeled rTFIIFaS385A/T389A after digestion by either trypsin or Glu-C endoproteinase. Microsequencing analysis shows that the 32P labeling is found within three oligopeptides 207-230, 271-283, and 335-344 of TFIIFalpha (Fig. 2, PS); each of them contains a CKII consensus phosphorylation motif, which is well conserved among different species. Indeed, we observed that CKII is able to phosphorylate both rTFIIF S385A/T389A mutant (Fig. 6A, lane 4), as well as TFIIFalpha overexpressed in E. coli (Fig. 6B). Trypsin digests of both radiolabeled polypeptides further confirm that the CKII consensus motifs are effectively phosphorylated (data not shown). Moreover, using partially purified TFIID (37), we observed a weak but significant phosphorylation of TFIIFalpha (Fig. 6C, lane 2). This may be explained by the fact that TAFII250 selectively phosphorylates the PS1 site (33). It should be also noticed that TFIIFalpha overproduced in E. coli is weakly autophosphorylated (Fig. 6B, lane 1), thus suggesting a potential cooperative effect between the various phosphorylation processes.


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  		Fig. 6.   TFIIFalpha is a substrate for casein kinase II (CKII). A, purified rTFIIF S385/T389 (DEAE fraction) was immunoprecipitated with Ab-TFIIFalpha and subjected to an in vitro kinase assay in the presence of either HeLa whole cell extract (WCE) or casein kinase II (CKII). Reaction were then resolved on SDS-PAGE, followed by autoradiography of the gel. B, purified E. coli rTFIIFalpha (34) was immunoprecipitated with Ab-TFIIFalpha and incubated with radioactive ATP either alone or in the presence of HeLa WCE or CKII. After extensive washing with a buffer containing 1 M KCl, immunoprecipitated rTFIIFa was subjected to an in vitro kinase assay with [gamma -32P].


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the course of this study, we have determined that the largest subunit of the basal transcription factor TFIIFalpha is the target of several phosphorylation processes. First, according to the in-gel kinase assay and microsequencing analysis, TFIIFalpha , which possesses a serine/threonine kinase activity, autophosphorylates at position Ser-385 and Thr-389. Careful screening of the TFIIFalpha amino acid sequence, however, did not reveal any known consensus kinase motifs. Second, we identified three other additional phosphorylated domains of TFIIFalpha . These oligopeptides at positions 207-230, 271-283, and 335-344 (Fig. 2), which contain the CKII consensus phosphorylation motif (SXXE/D), are indeed the target of the CKII kinase as well as of kinases present in HeLa WCE, as shown by in vitro kinase assays followed by microsequencing analysis. Interestingly, using purified TFIID, we observed a weak but significant phosphorylation of TFIIFalpha , likely due to the presence of TAFII250 (12, 33, 35). TAFII250, the largest component of the TFIID protein complex, and another component of the basal transcription machinery, indeed possesses a kinase activity (45). TAFII250 autophosphorylates and transphosphorylates TFIIFalpha with which it interacts (12). Phosphorylation of rTFIIFalpha S385A/T389A and E. coli TFIIFalpha by TAFII250 is much weaker than by HeLa WCE (data not shown and Fig. 6C, lane 1), thus likely explaining the specific phosphorylation of the PS1 oligopeptide motif previously identified (33). In this case, we failed to map the phosphorylated site(s), since TFIIFalpha was weakly phosphorylated by both TFIID and TAFII250.

We also show here that abrogation of the two autophosphorylation sites of TFIIFalpha results in an up-regulation of the transcription reaction. Compared with wild type TFIIF, addition of rTFIIFalpha S385A/T389A, which cannot autophosphorylate, increased the rate of elongation of the transcription and as such may participate to the initial burst of RNA synthesis, further followed by a lower rate of synthesis likely associated with RNA pol II recycling as hypothesized by Lei et al. (24). Indeed, they have shown that the COOH-terminal domain of TFIIFalpha (from amino acid 358 to 517) stimulates the CTD phosphatase (which uses the CTD of RNA pol II as a substrate), upon TFIIF/RNA pol II interaction (46). It thus is possible that TFIIF autophosphorylation, which decreases the elongation rate, could be one of the first steps implied in the RNA pol II recycling, through some modification of RNA pol II/TFIIF interaction. In such hypothesis, the fact that the autophosphorylation of TFIIFalpha occurs at the end of the elongation remains to be further established. It cannot be excluded, however, that the variations in the elongation rate (24) might be related to some changes in the overall conformation of the transcription complexes. Whether or not the other identified phosphorylation sites play some role in the transcription initiation step through TAFII250 and/or CKII-like kinases function is still an open question. Indeed, TFIIFalpha is targeted by TFIIB another basal transcription factor and RNA pol II (27, 46).

Knowing that TFIIFalpha has distinct functions in bringing RNA pol II through the various steps of the transcription process (29, 41, 47, and this study), it will be of great interest to further characterize all the components involved in the phospho/dephosphorylation process orchestrating the formation of the preinitiation, the initiation, the elongation, and the termination transcription complexes throughout the gene expression regulatory cascade. Further investigations to understand the connection between autophosphorylation and phosphorylation of TFIIFalpha with consequence of some changes in the elongation rate and/or in the activation of transcription (46) are also needed.

    ACKNOWLEDGEMENTS

We are thankful to J. Acker and C. Kedinger for the gift of TFIIFalpha -pAcAB3 and TFIIFbeta -pVL1392 baculovirus transfer vectors; to C. Rochette-Egly and to L. Tora for fruitful discussion; and to J. W. Conaway, R. C. Conaway, and J. Bradsher and F. Tirode for critical reading of the manuscript. We thank J. M. Chipoulet for expertise in protein purification, C. Braun and A. Fery for excellent technical assistance, I. Kolb-Cheynel for baculovirus expression, and P. Eberling for peptides synthesis.

    FOOTNOTES

* This work was supported by grants from the INSERM, the CNRS, the Ministère de la Recherche et de l'Enseignement Supérieur, the Ligue Nationale contre le Cancer, the Association pour la Recherche sur le Cancer, and the Human Frontier Science Program.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.

Dagger Supported by a CNRS/Région Alsace fellowship. Present address: Surgical Research, Children's Hospital, Harvard Medical School, 300 Longwood Ave., Boston, MA 02115.

§ To whom correspondence should be addressed. Tel.: 33-3-88-65-34-47; Fax: 33-3-88-65-32-01; E-mail: egly@igbmc.u-strasbg.fr.

    ABBREVIATIONS

The abbreviations used are: TFIIF, transcription factor IIF; RAP, RNA polymerase II-associated protein; pol II, polymerase II; PAGE, polyacrylamide gel electrophoresis; WCE, whole cell extract; DRB, 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole; nt, nucleotide(s); CKII, casein kinase II; CTD, carboxyl-terminal domain of RNA polymerase II.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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