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Heat shock of HeLa cells inactivates a nuclear protein phosphatase specific for dephosphorylation of the C-terminal domain of RNA polymerase II
Introduction
Materials And Methods
Cells and cytoplasts
CTD phosphatase assay
Cellular extraction
CTD phosphatase binding to RAP74 coupled beads
Electrophoresis/western blots/antibodies
Immunofluorescence
Results
Altered phosphorylation of the RNAP II largest subunit during heat shock
Decreased CTD phosphatase activity in extracts from heat-shocked cells
The FCP1 subunit of CTD phosphatase and the RAP74 subunit of TFIIF are nuclear proteins extracted in low salt buffers
CTD phosphatase activity and FCP1 protein interact with RAP74 in vitro
Heat shock results in the aggregation of CTD phosphatase
Discussion
Acknowledgements
References
Heat shock of HeLa cells inactivates a nuclear protein phosphatase specific for dephosphorylation of the C-terminal domain of RNA polymerase II
ABSTRACT
INTRODUCTION
The C-terminal domain (CTD) of the largest RNA polymerase II (RNAP II) subunit is comprised of multiple repeats of the heptapeptide sequence YSPTSPS (1,2). The CTD plays a key role in gene expression and its activity at different steps in the transcription cycle is regulated by multisite phosphorylation (3-7). The underphosphorylated form of RNAP II, designated RNAP IIA, assembles into a preinitiation complex on the promoter. Phosphorylation of the CTD occurs concomitantly with initiation of transcription. Accordingly, elongation is catalyzed by the phosphorylated form of RNAP II, designated RNAP IIO. The largest subunits of RNAPs IIA and IIO are designated IIa and IIo, respectively. The underphosphorylated CTD mediates multiple protein-protein interactions involved in assembly of the preinitiation complex whereas the phosphorylated CTD facilitates the association of the various enzymatic complexes involved in processing of the primary transcript (3-8).
Several cyclin-dependent kinases (CDK) have been shown to phosphorylate the CTD (9). One of these, CDK7, and its partner, cyclin H, are subunits of the general transcription factor TFIIH, a component of the preinitiation complex. This kinase is essential to ensure proper elongation of the transcript (10). As RNAP IIO cannot assemble into a preinitiation complex, the CTD must be dephosphorylated by phosphatase(s) to regenerate RNAP IIA and complete the cycle. The major CTD phosphatase present in both yeast and human cell extracts has been purified (11,12). This phosphatase is highly specific as the CTD in an intact RNAP II is its only known substrate. The docking site on RNAP II is localized outside the CTD. CTD phosphatase from HeLa cells is comprised of a single subunit of 150 kDa (13). The activity of this phosphatase is modulated by the general transcription factors TFIIB and TFIIF: it is strongly stimulated by the RAP74 subunit of TFIIF. Utilizing RAP74 as bait in a two-hybrid screen, an essential subunit of this CTD phosphatase, designated FCP1, has recently been identified (14,15). FCP1 corresponds to the 150 kDa subunit of CTD phosphatase.
As a consequence of the antagonist action of CTD kinases and CTD phosphatase(s), the IIa and IIo subunits coexist in cells in a dynamic equilibrium (16). In yeast cells, inactivation of ts-mutants of either the CDK7 homolog, KIN28, or its associated cyclin, CCL1, leads to a rapid overall dephosphorylation of the IIo subunit (17,18). However, in human cells submitted to severe heat shocks, although CDK7 bound to TFIIH is inactivated (19), the ratio of IIa to IIo increases (20). Such an increase in IIo subunit could in principle result from stress activation of a specific CTD kinase (21) and/or stress inactivation of a CTD phosphatase. To specifically examine the latter possibility, the CTD phosphatase activity has been investigated in HeLa cells submitted to heat shocks.
MATERIALS AND METHODS
Cells and cytoplasts
Monolayers of HeLa or L929 cells were propagated on tissue culture dishes or tubes in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (DMEM; Gibco BRL, NY). Heat shocks were performed by immersing sealed dishes or tubes in a water bath adjusted to 0.1°C at the appropriate temperature.
To obtain enucleated cells (cytoplasts), L929 fibroblasts were seeded on small round plastic slides, ultracentrifuged in warm (33°C) DMEM containing cytochalasin B (10 µg/ml) and cycloheximide (10 µg/ml) during 40 min at 9000 g. The enucleated cytoplasts remained attached to the slides and were lysed in 1× Laemmli buffer [1× Laemmli buffer composition: 60 mM Tris-HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate (SDS), 1% 2-mercaptoethanol and 0.002% bromophenol blue]. The absence of nuclei in the cytoplasts was checked by Hoechst staining of duplicate slides allowed to recover for 3 h at 37°C in fresh medium.
CTD phosphatase assay
Cells were lysed in buffer P [50 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 10 mM KCl, 0.1 mM ethylenediaminetetraacetatic acid (EDTA), 10% glycerol, 0.5 mM dithiothreitol] supplemented with 0.5% Nonidet P-40, and the lysate was fractionated by centrifugation at 10 000 g into a supernatant and a pellet. CTD phosphatase was assayed in buffer P as described (11). 32P-labeled RNAP IIO served as substrate and was prepared as previously described (22). The most C-terminal serine of the largest RNAP II subunit was phosphorylated by casein kinase II in the presence of [[gamma]-32P]ATP. Subsequently, 32P-labeled RNAP IIA was converted to RNAP IIO by phosphorylation with CTDK1 in the presence of 2 mM cold ATP. 32P-labeled RNAP IIO (1000-5000 c.p.m., ~50 fmol) was incubated with extracts (1 µl) and recombinant RAP74 (0.17 µg) at 30°C. Dephosphorylation was arrested at various times by adding 20 µl of 2× Laemmli buffer. Samples were electrophoresed on a 5% polyacrylamide gel and the distribution of labeled bands visualized by autoradiography. To determine CTD phosphatase activities, the amounts in subunit IIa were quantified with an Imager (Fuji BAS1000).
Cellular extraction
HeLa cells were processed through sequential extractions and low speed centrifugations following a procedure set up to isolate the core filaments of the nuclear matrix (23). In brief, after a wash in phosphate-buffered saline (PBS), HeLa cells were lysed at 4°C in cytoskeleton buffer: 10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA and 0.5% Triton X-100. The cytoskeletal frameworks and nuclei were pelleted by centrifugation at 600 g for 3 min. This first pellet was resuspended in digestion buffer (cytoskeleton buffer containing 50 mM NaCl instead of 100 mM, and 50 µg/ml DNase I) and left for 30 min at room temperature. Ammonium sulfate was added to a final concentration of 0.25 M, for 10 min at room temperature. A second pellet was recovered after centrifugation at 600 g for 3 min This pellet was resuspended and further extracted with digestion buffer containing 2 M NaCl at 4°C.
CTD phosphatase binding to RAP74 coupled beads
Histidine tagged RAP74 recombinant protein was expressed and purified using a variation of published procedures (24). In brief, bacteria expressing RAP74 were lysed and sonicated in 4 M urea, 20 mM HEPES, pH 7.9, 100 mM KCl, 50 mM EDTA, 10 mM EGTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 4 µg/ml lysozyme and loaded on a P11 column and eluted with KCl gradient. RAP74 was further purified by chromatography on Mono Q and Mono S. Purified RAP74 was coupled to HiTrap NHS beads according to the manufacturer’s instructions (Pharmacia). Mock HiTrap beads were prepared by a similar procedure except that they were not incubated with protein.
Aliquots (5 µl) of low salt extract were added to 45 µl of buffer P and incubated with 50 µl of beads (mock or RAP74 coupled) for 30 min at 4°C. Supernatants from the beads were recovered after centrifugation. The beads were washed twice in buffer P and resuspended in 50 µl of buffer P. CTD phosphatase activity was assayed in either supernatants or bead resuspensions.
Electrophoresis/western blots/antibodies
Non-denaturing extracts generated by cell fractionation or in vitro reactions were supplemented with 2× Laemmli buffer; whole cells, cytoplasts and centrifugation pellets were dissolved in 1× Laemmli buffer. All samples were heated for 5 min at 95°C before loading on SDS-polyacrylamide gels. Western blots were visualized using either anti-mouse or anti-rabbit IgG horseradish peroxidase conjugates (Promega) and chemiluminescence (Pierce).
The POL 3/3 monoclonal antibody recognizes the RNAP II largest subunit at an evolutionary conserved epitope located outside the CTD and was provided by Ekkerhard Bautz (25). The CC-3 monoclonal antibody, provided by Michel Vincent, recognizes a particular phosphoepitope within the CTD (19). The anti-FCP1 antiserum was obtained from rabbits immunized with a portion of the FCP1 open reading frame (amino acids 443-669) and was provided by Jack Greenblatt. The anti-RAP74 rabbit antiserum was provided by Zachary Burton (26). The anti-histone H1 rabbit antiserum was provided by Stefan Dimitrov, the mouse anti-tubulin monoclonal antibody was from Amersham.
Immunofluorescence
Following the procedure of Grande and co-workers (27), cells grown on coverslips for 48 h were fixed in 2% formaldehyde in PBS for 15 min at room temperature, permeabilized for 10 min in 0.5% Triton X-100 in PBS, incubated for 10 min in 100 mM glycine in PBS, blocked with 5% bovine serum albumin (BSA) in PBS for 15 min, followed by 5 min in PBG buffer [0.5% BSA and 0.1% w/v fish skin gelatin (Sigma) in PBS]. Incubations with the primary antibody (anti FCP1) in PBG were performed overnight at 4°C. The coverslips were subsequently washed four times in PBG and incubated for 1 h at room temperature with Cy3-conjugated goat anti-rabbit IgG (Amersham). Next, coverslips were washed twice in PBG and twice in PBS. Coverslips were mounted in Vectashield (Vector Labs)
RESULTS
Altered phosphorylation of the RNAP II largest subunit during heat shock
The phosphorylation state of RNAP II was investigated as a function of the severity of heat shock. HeLa cells were heated at various temperatures and the whole cell lysates were analyzed by immunoblotting. Cells grown at 37°C contain nearly equimolar amounts of RNAPs IIO and IIA. This is supported by the observation that extracts probed with monoclonal antibody POL 3/3, which is directed against the core domain of the largest subunit, show equal intensities of the hyperphosphorylated (IIo) and underphosphorylated (IIa) largest subunit (Fig.
Figure 1. Phosphorylation of the RNAP II largest subunit after heat shock. HeLa cells were heat shocked for 1 h at various temperatures ranging from 41 to 45°C. Whole cell lysates were electrophoresed on a 5% polyacrylamide-SDS gel and analyzed by western blot with either the POL 3/3 (epitope outside the CTD) or the CC-3 (phosphoepitope within the CTD) antibodies. The hyperphosphorylated (IIo) and underphosphorylated (IIa) subunits of the largest RNAP II subunit are indicated. To test whether changes in CTD phosphorylation might result from changes in CTD phosphatase activity, HeLa cells were lysed in a low salt buffer and fractionated by centrifugation into a pellet and a supernatant. CTD phosphatase activity was determined in the extract using as a substrate, RNAP IIO, labeled with 32P on the unique casein kinase II site at the C-terminus of the largest subunit and outside the consensus CTD repeat (29). Dephosphorylation results in a mobility shift of the largest subunit from the position of subunit IIo to the position of subunit IIa, without a loss of label (Fig.
Figure 2. Decreased CTD phosphatase activity in extracts from heat-shocked HeLa cells. (A) 32P-labeled RNAP IIO, with the labeled phosphate at the C-terminal casein kinase II site, was incubated at 30°C for 30 min without (-) or with a low salt extract from control (C) or heat-shocked cells (HS). (B and C) Time-courses of RNAP IIO dephosphorylation: RNAP IIO was incubated at 30°C for the times indicated with low salt extracts from control cells or cells heat shocked at 45°C for 15, 30 or 60 min (B) or with extracts from cells heat shocked for 60 min at 37, 41, 43 and 45°C (C). CTD dephosphorylation was analyzed by electrophoresis followed by autoradiography. The radioactivity in subunit IIa was quantified (arbitrary units) and plotted as a function of reaction time. The kinetics of dephosphorylation, catalyzed by extracts of cells heat shocked for 15, 30 or 60 min at 45°C, is shown in Figure 
Decreased CTD phosphatase activity in extracts from heat-shocked cells
A
B, C
The FCP1 subunit of CTD phosphatase and the RAP74 subunit of TFIIF are nuclear proteins extracted in low salt buffers
A highly specific CTD phosphatase has been previously charac-terized in HeLa cells and reported to be resistant to okadaic acid (11). In agreement with these studies, the CTD phosphatase activity found in low salt extracts was processive and was not affected by okadaic acid (data not shown). Although the activity of the previously characterized CTD phosphatase is strongly enhanced by the RAP74 subunit of the TFIIF general transcription factor (11), the CTD phosphatase activity detected in the low salt extracts was not affected by the addition of recombinant RAP74 (not shown). This is consistent with the observation that these extracts contain substantial amounts of RAP74 (Fig. Figure 3. Solubilization and nuclear localization of the FCP1 subunit of CTD phosphatase and of the RAP74 subunit of TFIIF. (A) A low salt extract from control HeLa cells was fractionated by centrifugation into a supernatant (Sup) and a pellet (Pel) which were analyzed by western blot using anti-RAP74 or anti-FCP1 antibodies. (B) Extracts from whole cells (WC) or from enucleated cytoplasts (Cyt) of murine L929 cells were analyzed by western blot. Tubulin (TUB) was taken as a control for the partitioning of cytoplasmic proteins and RNAP II largest subunit (RPB1) and histone H1 (H1) were taken as controls for nuclear proteins; specific antibodies were used for detection. A 150 kDa protein, designated FCP1, had been identified as an essential subunit of the previously characterized CTD phosphatase (14,15). Therefore, it was attempted to link the FCP1 protein to the CTD phosphatase activity. In unstressed cells, FCP1 anti-serum detected a 150 kDa protein in the extracts used for CTD phosphatase assay (Fig. As transcription is a nuclear process and transcription factors are generally recovered in high salt nuclear extracts, such a finding was unexpected. Therefore, the intracellular localization of FCP1 was investigated first by immunofluorescence. A weak but clear nuclear staining excluded from nucleoli was observed with anti-FCP1 antiserum on unstressed HeLa cells (Fig. Figure 4. Immunofluorescence localization of the FCP1 phosphatase subunit. HeLa cells unstressed (C) or heat shocked at 45°C for 30 min (HS) were fixed in formaldehyde then reacted with anti-FCP1 antibodies and stained with fluorescent goat anti-rabbit antibodies. The nucleoplasm appears brighter than cytoplasm and nucleoli in control cells. However, there is a relative decrease in the intensity of nucleoplasmic staining in heat-shocked cells. To establish the relationship between FCP1 and the CTD phosphatase activity present in the extracts, their abilities to bind to RAP74 were examined. A low salt extract from unstressed cells was incubated either with Hi-Trap beads covalently coupled to recombinant RAP74 or with mock control beads and fractionated by centrifugation. Results presented in the lower panel of Figure Heat shock leads to the inactivation of numerous enzymes either through post-translational modifications decreasing their specific activity or through aggregation and loss of solubility (31). To test the latter possibility, the distribution of the FCP1 subunit of CTD phosphatase in the low salt extracts and in the pelleted insoluble material from control (C) or heat-shocked (HS) cells was examined. As shown above (see also lane 0 of Fig. Figure 5. The CTD phosphatase activity and FCP1 are both retained on beads coupled to recombinant RAP74 protein. A low salt extract from unstressed HeLa cells was incubated either with RAP74 coupled to Hi-Trap beads (RAP74, lanes 3 and 4) or with uncoupled control beads (control, lanes 5 and 6) and fractionated by centrifugation into material bound to the beads (B) or free (F). In the upper part of the figure, 32P-labeled RNAP IIO (lane 1) was incubated with either the extract (E, lane 2), the beads (B, lanes 4 and 6) or bead supernatants (F, lanes 3 and 5); in the lower part of the figure, FCP1 was detected in the same fractions by western blot.
Figure 6. CTD phosphatase activity and amounts of FCP1 present in extracts from heat-shocked cells. (A, C and D) Western blot using anti RAP74 or anti-FCP1 antibodies or anti-histone H1 antibodies. Within each panel, the material loaded in each lane originated from the same amount of cells. (A) Heating time course at 45°C. (B) CTD phosphatase reaction rates determined in Figure 2B ([closed circle]); amounts of FCP1 in the supernatants ([open square]) or pellets ([open triangle]) shown in (A) evaluated by densitometry, were plotted versus heat shock duration at 45°C and fitted with exponential curves. Phosphatase rates and FCP1 amounts in the supernatants were normalized relative to control cells (100%). (C) HeLa cells, unstressed (C) or heat shocked at 45°C for 1 h, were lysed immediately (0) or allowed to recover during 5 or 18 h at 37°C in the presence of cycloheximide (50 µg/ml). (A and C) HeLa cells were lysed in a low salt buffer, fractionated by centrifugation into a supernatant (Sup) and a pellet (Pel). (D) HeLa cells were heat-shocked for 1 h at 45°C then lysed in a low salt buffer and fractionated at low speed into a supernatant (S1) and a pellet (P1). The P1 pellet was resuspended and incubated with DNase I and ammonium sulfate and refractionated into a second supernatant (S2) and a second pellet (P2). The P2 pellet was resuspended in a high salt (2 M NaCl) buffer and refractionated into a third supernatant (S3) and a pellet (P3). To characterize the insolubilized FCP1 protein, heat-shocked HeLa cells were processed through sequential extractions and low speed centrifugations following a procedure set up to isolate the core filaments of the nuclear matrix (23). As previously described, FCP1 from heat-shocked cells remained insoluble in a low salt buffer containing non-ionic detergent (Fig. 
CTD phosphatase activity and FCP1 protein interact with RAP74 in vitro
Heat shock results in the aggregation of CTD phosphatase
A
B
C, D
DISCUSSION
CTD phosphatase activity is readily detectable in low salt extracts from unstressed HeLa cells. This activity is specific in that dephosphorylation of the CTD is processive and the phosphate labeled by casein kinase II on the most C-terminal serine, outside the consensus CTD repeat, is not removed. The major CTD phosphatase present in cell extracts has been previously purified, it is insensitive to okadaic acid and interacts with RAP74 (11). The FCP1 protein is an essential subunit of the CTD phosphatase (14). Here we show that the FCP1 protein is readily extracted from unstressed cells in a low salt buffer, but cell enucleation indicates that it is strictly nuclear in agreement with previous experiments using tagged-recombinant FCP1 (14). A leakage from the nucleus during cell lysis is not uncommon (see, for example, 32).
CTD phosphatase activity is diminished in extracts prepared from HeLa cells heat shocked at temperatures as mild as 41°C. Similar observations were obtained with murine cells, NIH 3T3 and L929 (data not shown). The decrease in CTD phosphatase activity reflects a decrease in FCP1 protein. However, the FCP1 subunit of CTD phosphatase is not degraded, it is recovered in the nuclear pellet fraction. The FCP1 protein remains insoluble in a high salt buffer after DNase I digestion, like the core filaments of the nuclear matrix. No relocalization is detected by immunofluorescence. The exponential decay in CTD phosphatase activity and FCP1 solubility argues in favor of a passive process such as heat-denaturation. During recovery at 37°C, FCP1 solubility is restored. Such behavior during heat shock has been described for many proteins mostly nuclear (32-36). However, it should be emphasized that not all nuclear proteins aggregate in these conditions. For example, the solubilities of RAP74 and histone H1 remain unaffected by heat shock.
In unstressed cells, phosphate within the CTD turns over rapidly: the hypophosphorylated subunit IIa accumulates rapidly upon knocking out the KIN28 yeast CDK7 homolog (17,18) or exposure of mammalian cells to CTD kinase inhibitors (16). Such turnover results from the antagonistic action of CTD kinases and CTD phosphatases. Upon severe stress (45°C), the bulk RNAP II CTD is hyperphosphorylated. Accordingly, there must be a change in the relative activity of CTD kinases and/or CTD phosphatase(s) in favor of CTD phosphorylation. This could result from the inhibition of CTD phosphatase activity and/or the activation of a heat-resistant CTD kinase insensitive to the usual kinase inhibitors (20). Results presented here indicate that the RAP74 stimulated CTD phosphatase is inactivated during heat shock in mammalian cells.
Despite the CTD phosphatase impairment, the CTD has a tendency to dephosphorylate in HeLa cells at mild temperatures. Such heat shock induced CTD dephosphorylation is even more pronounced in cells from other species such as flies or mice (21). To interpret the overall CTD phosphorylation pattern as the consequence of a CTD kinase/CTD phosphatase imbalance will require a careful analysis of the temperature dependence of CTD kinase and CTD phosphatase activities during heat shock. The multiplicity of CTD kinases is expected to render suchanalysis difficult. Indeed, in Saccharomyces cerevisiae, the UME3/SSN8/SRB11 C-type cyclin associated to CDK8/SRB10 is destroyed during heat shock (37) and in previous work, we have shown that in mammalian cells CDK7 associated to the TFIIH general transcription factor is heat-inactivated (19).
Overall changes in enzyme activities and CTD phosphorylation may not concern the pool of molecules directly involved in gene expression. CDK7 is detected on transcribed genes in polytene chromosomes from unstressed Chironomus tentans salivary glands (38). However, CDK7 is not present on the transcribed heat-shock genes on polytene chromosomes from stressed glands. Nevertheless, in heat-shocked D.melanogaster and C.tentans salivary glands, phosphorylated RNAP IIO is associated with the elongation complexes (38-40) despite an overall dephosphorylation of the CTD in fly cells (21). However, it should be noted that the phosphorylated CTD is less phosphorylated on transcribed genes from heat-shocked glands than from unstressed glands (39). A decreased CTD phosphatase activity might be required to allow the phosphorylation of RNAP II upon entry into elongation of transcription on the heat-shock genes and compensate a decreased CTD kinase activity.
The various CTD kinases phosphorylate distinct amino acid residues on the CTD (41). Furthermore, the various residues on the CTD may have distinct CTD phosphatase sensitivity. As a result, the immunoreactivity of the phosphorylated CTD from severely heat-shocked cells is distinct from that of control cells (19 and this work). As different phosphorylation sites on the CTD have different functions (42), overall changes in CTD kinase and CTD phosphatase activities are likely to influence the properties of the phosphorylated RNAP II. During severe heat shocks, the transcription of most genes is shut off, but that of heat-shock genes remains extremely efficient. Heat-inactivation of CTD kinases and CTD phosphatases might contribute to this selective transcription (43). Indeed, the yeast heat-shock genes show a lower requirement than other genes for active KIN28 protein, the yeast CDK7 homolog (44). Furthermore, in contrast to CDK7 which acts as a general activator of transcription, the yeast CDK8/SRB10 protein represses gene expression (45); its heat-inactivation may contribute to relieve heat-shock gene repression (37). Phosphorylation of the CTD plays a pivotal role in the generation of pre-mRNAs. The enzymes involved in pre-mRNA processing interact with the phosphorylated CTD (6,7). Heat-shock temperatures as mild as 41°C, which lead to the activation of heat-shock responsive genes, result in an alteration in the CTD phosphorylation pattern. This alteration might also contribute to the reported impaired splicing and polyadenylation. During heat shock, splicing and polyadenylation are arrested (36,46,47). However, the major heat-shock genes, which code for the inducible HSP70, contain no introns. Thus, the heat-inactivation of both CTD phosphatase and CTD kinases might contribute to the selective synthesis of heat-shock mRNAs.
ACKNOWLEDGEMENTS
We are indebted to Drs Ekkehard Bautz, Zachary Burton, Stefan Dimitrov, Jack Greenblatt and Michel Vincent for providing antibodies.This work was supported by grants from the Association pour la Recherche sur le Cancer, ARC 6250 (to O.B.) and the National Institutes of Health, GM33300 (to M.E.D.).
REFERENCES
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