From the
Department of Medicine, University of
California San Diego, La Jolla, California 92093-0650 and the
Section of Molecular and Cellular Biology,
Division of Biological Sciences, University of California Davis, Davis,
California 95616
Received for publication, February 19, 2003 , and in revised form, April 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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FCP1 is a class C phosphatase containing a BRCT domain that is required for
interaction with RNAP II and dephosphorylation of the CTD
(15,
16). FCP1 interacts with and
is stimulated by RAP74, the larger subunit of TFIIF
(16,
17). Class C phosphatases are
resistant to inhibitors that block other classes of Ser/Thr phosphatases and
bind Mg2+ or Mn2+ in the binuclear metal center of the
catalytic site (18,
19). The
DXDX(T/V)
motif (where
represents a hydrophobic residue) present in the FCP1 homology domain
characterizes a subfamily of class C phosphatases, with both Asp residues
being essential for activity
(20,
21).
Synthetic lethality is observed between mutant FCP1 and reduced levels of RNAP II in Saccharomyces cerevisiae and Schizosaccharomyces pombe, indicating that FCP1 is an essential gene (21, 22). It remains uncertain whether the activity of yeast FCP1 accounts for the dephosphorylation in vivo of both Ser5 and Ser2 and whether this is the sole activity that catalyzes CTD dephosphorylation. Mutations in FCP1 lead to increased phosphorylation of Ser2, suggesting that it functions in vivo in the dephosphorylation of Ser2 (13). Yeast FCP1 appears more specific for Ser2 phosphate when synthetic peptides are used as substrate (23). However, mammalian FCP1 dephosphorylates both Ser2 and Ser5 in vitro in the context of native RNAP II (24). Furthermore, modifications such as that catalyzed by the Ess1/Pin1 peptidyl prolyl isomerase may alter the activity and specificity of FCP1 (2527). Given the importance of CTD phosphorylation in gene expression (28), it is essential to know whether additional CTD phosphatases exist and, if so, their specificity and the mechanisms by which they are targeted to RNAP II at discrete stages of the transcription cycle.
Although FCP1 is the only reported CTD phosphatase, examination of the data bases reveals additional genes that consist principally of a domain with homology to the CTD phosphatase domain of FCP1. Three closely related human genes encoding small proteins with CTD phosphatase domain homology, but lacking a BRCT domain, have been identified. In the present study, we show that a gene located on chromosome 2 encodes a nuclear CTD phosphatase. This protein preferentially dephosphorylates Ser5 within the CTD of RNAP II and is stimulated by RAP74. Expression of this small CTD phosphatase (SCP1) inhibits activated transcription from a variety of promoter-reporter gene constructs, whereas expression of a mutant lacking phosphatase activity enhances transcription. This newly identified small CTD phosphatase appears to play an important role in the regulation of RNAP II transcription.
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EXPERIMENTAL PROCEDURES |
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Preparation and Purification of 32P-Labeled RNAP IIO
Isozymes and [32P]-GST-CTDoCalf thymus RNAP IIA was
purified by the method of Hodo and Blatti
(31) with modifications as
described by Kang and Dahmus
(32). Specific isozymes of
32P-labeled RNAP IIO were prepared by phosphorylation at the most
C-terminal serine (CKII site) in the largest subunit of purified RNAP IIA with
recombinant CKII and [-32P]ATP
(17,
33), followed by CTD
phosphorylation in the presence of 2 mM ATP with either purified
CTDK1/CTDK2, TFIIH, P-TEFb, recombinant MAPK2/ERK2, or recombinant Cdc2
kinase. The RNAP IIO isozymes were individually purified over a DE53 column
with a step elution of 500 mM KCl
(29). Because only the most
C-terminal serine is labeled with 32P and lies outside the
consensus repeat, dephosphorylation by CTD phosphatase results in an
electrophoretic mobility shift in SDS-PAGE of subunit IIo to the position of
subunit IIa without the loss of label. GST-CTD was prepared and purified as
previously described (34).
32P-Labeled GST-CTDo was subsequently prepared from GST-CTDa by
CKII followed by MAPK2/ERK2. GST-CTDo was purified over a glutathione-agarose
column with a step elution of 15 mM glutathione.
Phosphatase AssaysPNP reaction mixtures (200 µl)
containing 50 mM Tris acetate, pH 5.5, 10 mM
MgCl2, 0.5 mM DTT, 10% glycerol, 20 mM
PN
P, and recombinant proteins were incubated at 30 °C for 1 h. The
reactions were quenched by adding 800 µl of 0.25 N NaOH. Release
of para-nitrophenyl was determined by measuring
A410.
N-terminal biotinylated CTD phosphopeptides, composed of four tandem repeats YSPTSPS and containing phosphoserine at position 2 or 5, were synthesized (Alpha Diagnostics, San Antonio, TX). Phosphatase reaction mixtures (50 µl) containing 50 mM Tris acetate, pH 5.5, 10 mM MgCl2, 0.5 mM DTT, 10% glycerol, 25 µM phosphopeptide, and wild type or mutant SCP1 were incubated for 60 min at 37 °C. The reactions were quenched by adding 0.5 ml of malachite green (Biomol). Phosphate release was measured at A620 and quantified relative to a phosphate standard curve.
CTD phosphatase assays utilizing RNAP IIO and GST-CTDo as substrate were performed as described previously (35) with minor modifications. Reactions were performed in 20 µl of CTD phosphatase buffer (50 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 20% glycerol, 0.025% Tween 80, 0.1 mM EDTA, 5 mM DTT) in the presence of 20 mM KCl. Each reaction contained specified amounts of GST-CTDo and/or RNAP IIO and was carried out in the presence of 7 pmol of RAP74. Reactions were initiated by the addition of FCP1 or SCP1 and incubated at 30 °C for 30 min. Assays were terminated by the addition of 5x Laemmli buffer, and RNAP II subunits and GST-CTD were resolved on a 5% SDS-PAGE gel. The gel images were developed by autoradiography and scanned by an Amersham Biosciences Image Scanner Storm 860 in the phosphor screen mode. Data were quantitatively analyzed by ImageQuant software.
Tissue Culture and TransfectionsHuman 293, COS-7, and CV1
cells were grown at 37 °C in Dulbecco's modified Eagle's medium
supplemented with 10% normal calf serum (Invitrogen). Subconfluent cells were
transfected in six-well tissue culture dishes using Effectene (Qiagen)
according to the manufacturer's instructions. Reporter and activator plasmids
(100 ng each) and FLAG SCP1 (80 ng) or its mutant were used per well. For T3
and PPAR transfections, 20 ng of retinoid X receptor plasmid was also
added. The amounts of ligands used were as follows: 100 nM T3, 1
µM PPAR
609843 (Ligand Pharmaceuticals), and 100
nM dexamethasone. LMX1 and E47 expression plasmids (100 ng) were
cotransfected with 100 ng of the rat insulin promoter-luciferase reporter
construct with the indicated concentrations of SCP1 and phosphatase-deficient
SCP1 expression plasmids. The total amounts of transfected DNA were kept
constant by the addition of empty vector. Cells were harvested 48 h after
transfections, and cellular extracts were assayed for luciferase activity
using the luciferase assay system (Promega) according to the manufacturer's
instructions.
ImmunofluorescenceCells grown on coverslips were fixed in 2% paraformaldehyde, neutralized, and blocked using 2.5% fetal calf serum/phosphate-buffered saline. Rabbit polyclonal IgG 6703 was used at 1:100 dilution, followed by goat anti-rabbit IgG H+L chains conjugated to Alexa Fluor 488 (1:250) (Molecular Probes, Inc., Eugene, OR). Mouse anti EEA1 was used at 1:1000, followed by goat anti-mouse IgG conjugated to Alexa Fluor 594 (1:250). Omission of primary antibodies was used as a negative control. The coverslips were viewed using the Zeiss Axiophot, which is equipped with a Hamamatsu Orca ER firewire camera that runs on Improvision Openlab 3.0.9 software.
ImmunoprecipitationsFor immunoprecipitation experiments, 75% confluent COS-7 cells from a 10-cm dish were harvested in lysis buffer (phosphate-buffered saline containing 1% Nonidet P-40, 1 mM DTT, and protease inhibitors). Lysates were incubated with 20 µl of Sepharose-conjugated anti-SCP1 (6703) IgG at 4 °C for 6 h. Beads were washed with phosphate-buffered saline, and the complexes were evaluated by Western blotting using specific anti-RNAP II antibodies. Rabbit anti-SNX1 antibody was used as control IgG.
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RESULTS |
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To determine whether SCP1 has phosphatase activity, the protein was
expressed as a GST fusion and both SCP1 261 and SCP1 214 were assayed using
PNP as substrate. As reported for FCP1 from S. pombe
(23) utilizing PN
P as
substrate, the pH optimum for SCP1 phosphatase activity is near 5
(Fig. 2A). Phosphatase
activity was Mg2+-dependent and resistant to the phosphatase
inhibitors okadaic acid and microcystin
(Fig. 2B).
Ca2+ could not substitute for Mg2+. Mutations of
Asp96 to Glu (D96E) had little to no effect on phosphatase activity
(data not shown), whereas mutating Asp98 to Asn (D98N) in
conjunction with the D96E mutation completely abolished phosphatase activity
(Fig. 2B). SCP1 is
thus a class 2C phosphatase whose activity is dependent on acidic residues in
the conserved DXD motif. SCP2 exhibited similar phosphatase activity
(Fig. 2B).
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To determine whether GST-SCP1 214 has CTD phosphatase activity, GST-CTDo
and RNAP IIO were utilized as substrates, and the activity of SCP1 was
compared directly with that of FCP1. Recombinant CTDo (rCTDo) and RNAP IIO
utilized as substrate in these experiments were prepared by the
phosphorylation of purified GST-CTDa or RNAP IIA with casein kinase II (CKII)
in the presence of [-32P]ATP, followed by phosphorylation
with MAPK2/ERK2 in the presence of excess unlabeled ATP. MAPK2/ERK2 was used
in these initial experiments, because it phosphorylates both GST-CTDa and RNAP
IIA with comparable efficiency
(24). As expected, FCP1
efficiently converts RNAP IIO to RNAP IIA in a processive manner
(Fig. 2C, lanes
712) (24,
35). Even high concentrations
of FCP1 did not result in measurable dephosphorylation of rCTDo
(Fig. 2C, lanes
16) consistent with the idea that a docking site on RNAP II is
required for activity (17).
GST-SCP1 214 catalyzed the dephosphorylation of both RNAP IIO and GST-CTDo
with comparable efficiency (Fig.
2D). In contrast to FCP1, the SCP1-catalyzed
dephosphorylation of RNAP IIO appears nonprocessive in that a number of
phosphorylated intermediates are visible in SDS-PAGE. SCP1 is specific for
dephosphorylation of the consensus repeat in that the phosphate at the CKII
site is not removed. As shown below, the pattern of dephosphorylation varies
depending on the CTD kinase used in the preparation of RNAP IIO. Mutant SCP1
(D96E,D98N) lacked activity on either substrate (data not shown). SCP1 is thus
a CTD phosphatase that acts on both RNAP IIO and rCTDo. SCP2 exhibits
comparable CTD phosphatase activity when RNAP IIO is utilized as substrate
(see Fig. 4).
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SCP1 Preferentially Dephosphorylates Ser5 of the CTD Heptad RepeatTo determine the specificity of SCP1 with respect to its ability to dephosphorylate specific positions within the consensus repeat, RNAP IIO isozymes were prepared in vitro by the phosphorylation of RNAP IIA with CTD kinases of known specificity. TFIIH, P-TEFb, and MAPK2/ERK2 preferentially phosphorylate Ser5 when synthetic peptides serve as substrate (39, 40), whereas Cdc2 kinase phosphorylates Ser2 and Ser5 (41). Although the specificity appears relaxed when RNAP II serves as substrate, RNAP IIO prepared with Cdc2 kinase is clearly distinct from RNAP IIO generated by other CTD kinases (24). Results presented in Fig. 3A indicate that RNAP IIO, prepared by the phosphorylation of RNAP IIA with distinct CTD kinases, exhibit a differential sensitivity to dephosphorylation with SCP1. SCP1 most efficiently dephosphorylates RNAP IIO generated by TFIIH and was unable to dephosphorylate RNAP IIO prepared with Cdc2 kinase. SCP1 was also unable to dephosphorylate RNAP IIO generated by Abl tyrosine kinase (data not shown). The dephosphorylation of RNAP IIO isozymes prepared with P-TEFb, MAPK2/ERK2, and CTDK1/CTDK2 occurred at a reduced rate relative to that of RNAP IIO prepared with TFIIH. Furthermore, whereas the dephosphorylation reaction appears processive for RNAP IIO prepared by TFIIH, it is clearly nonprocessive for RNAP IIO generated by MAPK2/ERK2. In contrast, FCP1 shows no preference for RNAP IIO generated by TFIIH and efficiently dephosphorylates RNAP IIO generated by Cdc2 kinase (24). These results suggest SCP1 differs from FCP1 in substrate specificity, showing relative preference for the dephosphorylation of Ser5 in the heptad repeat.
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To investigate the relative reactivity of SCP1 for Ser2 and Ser5, a synthetic 28-aa peptide containing four heptad repeats phosphorylated exclusively on Ser2 or on Ser5 was dephosphorylated in the presence of increasing amounts of SCP1. As shown in Fig. 3B, SCP1 preferentially dephosphorylates the Ser5 phosphopeptide compared with the Ser2 phosphopeptide. This substrate specificity contrasts to that reported for FCP1 from S. pombe, which preferentially dephosphorylate the Ser2 phosphopeptide (23). Mammalian FCP1, within a comparable concentration range, did not act on either phosphopeptide (data not shown). These results using synthetic phosphopeptide substrates confirm that SCP1 preferentially dephosphorylates Ser5 phosphate of the CTD.
Effect of RAP74 on the Activity of SCP1The RAP74 subunit of
TFIIF stimulates CTD phosphatase activity of FCP1
(17). Furthermore, the domains
of FCP1 that bind RAP74 are required for FCP1-dependent viability in S.
cerevisiae (15).
Therefore, it was of interest to determine whether RAP74 can also influence
the activity of SCP. CTD phosphatase activity was measured at low enzyme
concentrations to more readily detect stimulatory effects of RAP74. As shown
in Fig. 4, RAP74 shifted the
dose-response curve for SCP1-catalyzed dephosphorylation of RNAP IIO to an
10-fold lower concentration. The CTD phosphatase activity of the GST
fusion forms of SCP1 261, SCP1 214, and SCP2 were also enhanced by RAP74. The
presence of comparable concentrations of bovine serum albumin does not
stimulate the CTD phosphatase activity of SCP1. In support of the conclusion
that RAP74 stimulates the activity of SCPs, RAP74 bound directly to GST-SCP1
but not to GST (data not shown). The binding and stimulatory effects of RAP74
suggest that TFIIF is important for optimal CTD phosphatase activity for both
FCP1 and SCP1.
SCP1 Is Located in the Nucleus Associated with RNAP II Although SCP1 lacks an obvious nuclear localization sequence, it is found in the nucleus. Immunofluorescence microscopy using a rabbit polyclonal anti-SCP1 antibody demonstrated nuclear localization of endogenous SCP1 in COS-7 cells (Fig. 5B). Co-staining with 4',6-diamidino-2-phenylindole for nuclear identification and with the early endosomal marker EEA1 for cellular detail confirmed the specific localization of SCP1 in nuclei (Fig. 5, A and B).
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Co-immunoprecipitation was used to assess the association of SCP1 with RNAP II. Sepharose-immobilized anti-SCP1 IgG 6703 was used to immunoisolate SCP1 from COS-7 cells. Immunoisolates were resolved by SDS-PAGE and blotted with anti-RNAP II antibodies. As shown in Fig. 5C, RNAP II was present in SCP1 immunoprecipitates, indicating that SCP1 and RNAP II either interact directly or are in the same macromolecular complex. To determine whether SCP1 preferentially associated with either Ser2 or Ser5 phosphorylated RNAP IIO, lysates were prepared in the presence of EDTA, to inhibit phosphatase activity. SCP1 immunoprecipitates were then blotted with monoclonal antibodies specific for Ser2 phosphate (H5) and Ser5 phosphate (H14). Both forms of RNAP IIO were present in COS-7 cell lysates. Ser5 phosphate-enriched RNAP IIO appeared to be preferentially associated with SCP1 in immunoprecipitates as indicated by the ratios of co-immunoprecipitated RNAP IIO relative to the amount of RNAP IIO contained in the extract (Fig. 5C).
SCP1 Affects RNAP II Transcription in VivoTo assess the effect of SCP1 on transcription in vivo, the activity of a variety of luciferase reporter gene constructs was examined in the presence or absence of cotransfected SCP1. Targeting a Gal 4-DNA binding domain SCP1 fusion or the phosphatase-inactive Gal 4-SCP1 mutant upstream of a thymidine kinase promoter-luciferase reporter (Gal 4-TK-Luc) had no significant effect on transcriptional activity (Fig. 6A). Interestingly, untethered SCP1 in the presence of several reporter constructs had no significant effect on reporter gene expression, whereas the inactive mutant resulted in a significant stimulation of expression from the E1A-Luc, pGL3-Luc, and Gal 4-TATA-Luc constructs (Fig. 6B). The phosphatase-deficient SCP1 mutant increased luciferase activity 1.56-fold.
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In contrast, wild type or phosphatase-inactive SCP1 affected reporter gene expression from a variety of regulated promoters. Luciferase activity from a Gal 4-TK-Luc reporter that was strongly stimulated by co-expressing a Gal 4-VP16 fusion protein (30-fold stimulation) was strongly inhibited by SCP1 (Fig. 6C). In contrast, phosphatase-inactive SCP1 enhanced Gal 4-VP16-stimulated activity about 2-fold.
Opposing effects of active SCP1 and the inactive mutant SCP1 were observed
using a number of inducible promoter-reporter constructs. Ligand-activated
T3 receptor activity on a DR+4 TRE-TK-Luc reporter gene was
inhibited by active SCP1 and enhanced by inactive SCP1
(Fig. 6D). Similar
results were obtained when the C terminus of T3R was fused to
the Gal 4-DNA binding domain (Gal 4-T3R
C) and targeted to Gal
4-TK-Luc. SCP1 also inhibited dexamethasone-stimulated glucocorticoid receptor
activity on a GRE-TK-Luc construct, whereas mutant SCP1 significantly enhanced
activity (Fig. 6D).
Finally, SCP1 inhibited ligand-activated PPAR
receptor activity assayed
on a PPAR
promoter response element, and mutant SCP1 enhanced activity
(Fig. 6D). A similar
response pattern was observed when the Gal 4-DNA binding domain was fused to
the C terminus of PPAR
and targeted to Gal 4-TK-Luc (data not shown).
The same pattern of responses was observed in HEK293, COS-7, and CV-1 cells
(data not shown).
Although there was variability in the extent of inhibition and stimulation of transcription observed with SCP1 and mutant SCP1, respectively, the overall patterns were similar using additional inducible systems such as estrogen and retinoic acid receptor-responsive promoters (data not shown). The extent of inhibition and stimulation probably results from variable expression levels of transfected proteins as well as potentially different rate-limiting steps in transcription from specific promoters.
The competing effects of SCP1 and mutant SCP1 were further examined using a
rat insulin promoter-luciferase construct. There is strong synergy on this
promoter between the basic helix-loop-helix protein E47 and the
LIM-homeodomain protein LMX1, which bind to adjacent DNA target sites
(42,
43). Co-expression of LMX1 and
E47 enhanced luciferase activity 25-fold from the rat insulin 1 promoter
(Fig. 6E). When
phosphatase-inactive SCP1 was held constant, increasing amounts of SCP1
inhibited luciferase expression (Fig.
6E). In the presence of mutant SCP1, the SCP1 inhibition
curve was right-shifted (20% inhibition with 40 ng of SCP1 plasmid plus
20 ng of mutant SCP1 plasmid versus >90% inhibition with 40 ng of
SCP1 plasmid alone (Fig.
6E)). With a constant input of SCP1 plasmid, increasing
amounts of mutant SCP1 not only blocked the inhibitory effects of SCP1 but
enhanced activity significantly. The stimulatory effects of transfected
phosphatase-inactive SCP1 are thus consistent with it acting as a dominant
negative.
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DISCUSSION |
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SCP1 preferentially catalyzes dephosphorylation of RNAP IIO phosphorylated by TFIIH. RNAP IIO phosphorylated by P-TEFb and MAPK2/ERK2 are also dephosphorylated by SCP1 but at a reduced rate. RNAP IIO phosphorylated by Cdc2 kinase, which preferentially phosphorylates Ser2 with some phosphorylation at Ser5, is not a substrate for SCP1. The preferential dephosphorylation of Ser5 by SCP1 was confirmed using a four-heptad repeat peptide substrate. Interestingly, the specificity of SCP1 contrasts with that of S. pombe FCP1, which prefers Ser2 with a similar peptide substrate (23). However, FCP1 dephosphorylates Ser2 phosphates and Ser5 phosphates with comparable efficiency when native RNAP IIO serves as substrate in vitro (24). The relative specificity of FCP1 also differs from SCP1 in that, unlike FCP1, SCP1 shows preference for the dephosphorylation of TFIIH-phosphorylated RNAP IIO.
It is clear from the results presented in Fig. 2, C and D, that, when RNAP IIO phosphorylated with MAPK2/ERK2 is utilized as substrate, the specific activity of SCP1 is substantially lower than that of FCP1. This difference in specific activity is in part due to the fact that MAPK2/ERK2 phosphorylated RNAP IIO is an especially poor substrate for SCP1 (Fig. 3), whereas FCP1 dephosphorylates different isozymes of RNAP IIO with comparable efficiency (24). The amount of SCP1 required to dephosphorylate MAPK2/ERK2-phosphorylated RNAP IIO is 50100-fold higher than that required to dephosphorylate TFIIH phosphorylated RNAP IIO. Furthermore, the BRCT domain and the C terminus of FCP1 that may facilitate its interaction with RNAP II (16) are absent from SCP1. Most importantly, without a better understanding of the factors that influence the recruitment of SCP1 to complexes containing RNAP IIO, it is difficult to interpret differences in specific activity in a binary reaction. The finding that SCP1 dephosphorylates rCTDo and RNAP IIO with comparable efficiency, whereas FCP1 does not dephosphorylate rCTDo even at concentrations 100 times higher than that required to dephosphorylate RNAP IIO, suggests that the interaction of SCP1 and FCP1 with the CTD require different molecular interactions.
During the transcription cycle, protein complexes assemble and disassemble on the CTD in a dynamic and regulated manner. Ser5 phosphorylation is detected primarily at the promoter region, whereas Ser2 phosphorylation is seen in coding regions (12). Phosphorylation of Ser5 facilitates recruitment of the capping enzymes and allosterically activates their activity (59, 48, 49). Given the preference of SCP1 for phosphoserine 5, SCPs are candidates for acting early in the transcription cycle, perhaps facilitating the transition from initiation and capping to promoter clearance and processive transcript elongation.
Like FCP1, SCP1 phosphatase activity is found in a complex with RNAP II, and its activity is stimulated by RAP74. Mapping studies indicate that the C-terminal domain of FCP1 distal to the phosphatase domain interacts with TFIIF (15, 16). A region near the C terminus of SCP1 shares homology with the putative RAP74 interaction domain in FCP1. Additional studies are necessary to establish whether this region of SCP1 mediates its interaction with RAP74. These observations suggest that RAP74 plays an important role in regulating phosphate turnover at both Ser2 and Ser5, consistent with the involvement of TFIIF in assembling RNAP II into preinitiation complexes and in stimulating the rate of transcript elongation.
The results of reporter gene assays indicate that overexpression of either wild type or mutant SCP1 can influence gene expression. The overexpression of mutant SCP1 activates transcription severalfold from nearly all promoters examined (Fig. 6, BD), whereas overexpression of wild type SCP1 appears to selectively inhibit activated transcription from a variety of inducible promoter-reporter gene constructs (Fig. 6D). Because mutant SCP1 is competitive with SCP1, the stimulatory effects are consistent with partial inhibition of endogenous SCP. The stimulatory effects of the phosphatase-inactive mutant suggest that the Ser5 dephosphorylation cycle is highly dynamic and probably carefully regulated. Based on the assumption that mutant SCP1 is recruited to the promoter, overexpression would shift the equilibrium in the direction of increased Ser5 phosphorylation. This could in principle lead to an increase in the efficiency of cap formation and promoter clearance. The inhibitory effects of overexpressed SCP1 are consistent with excessive dephosphorylation of the CTD, perhaps leading to a decrease in the efficiency of cap formation, promoter clearance, and/or transcript elongation. Because of the close sequence homologies between SCP1, SCP2, and SCP3, mutant SCP1 may interfere with all three family members. The basis for the apparent selectivity in inhibiting activated transcription is not clear. This could in principle result from an increased efficiency in the recruitment of SCP1 to a given promoter and/or a difference in the rate-limiting step during initiation or promoter clearance.
The present studies do not exclude the possibility that SCP1 influences the state of phosphorylation of substrates other than the CTD; nor do they address the function of related family members that may function in a variety of pathways including those located at the cell surface and nuclear membrane (50, 51). SCP1 co-immunoprecipitates with the nuclear LIM interactor of LIM homeodomain proteins (52) as well as with transcription factors such as the T3 receptor, Lhx1, Mesp1, and Olig2, indicating that it is not a specific nuclear LIM-interacting factor (data not shown). SCP2/0S4 was initially identified in a chromosomal region frequently amplified in sarcomas and brain tumors (37). The ectopic expression of the Xenopus ortholog induced mesoderm marker expression and formation of a secondary dorsal axis. Mutation of the DXDX(T/V) motif abrogated the latter effect (53), suggesting that phosphatase activity is essential for duplicate axis formation. In contrast, SCP3/HYA22 was initially identified in a genomic region homozygously deleted in a lung carcinoma cell line (38). The level of expression of each SCP family member may thus affect gene transcription and have overlapping but also diverse biological effects. SCP1, -2, and -3 are widely expressed in human tissues, with the highest expression observed in skeletal muscle and the lowest expression in brain (36, 37).
SCP1 and related Ser5 phosphatases may thus complement FCP1 to control the orderly cycle of phosphorylation and dephosphorylation of the CTD during the transcription cycle. An understanding of the precise role that specific CTD phosphatases play in the transcription cycle is dependent on defining the specific point in the cycle at which they act and the mechanism by which they are recruited to RNAP II-containing complexes. Given the importance of the CTD in mediating the synthesis and processing of the primary transcript, an understanding of the dynamics of CTD phosphorylation is central to elucidating the mechanisms and regulation of gene expression.
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FOOTNOTES |
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* These studies were supported by National Institutes of Health Grants
DK13149 (to G. N. G.) and GM33300 (to M. E. D.). The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0650. E-mail: ggill{at}ucsd.edu.
1 The abbreviations used are: RNAP, RNA polymerase; CKII, casein kinase II;
CTD, C-terminal domain of RNA polymerase II; FCP1, TFIIF-associated CTD
phosphatase; PNP, para-nitrophenylphosphate; P-TEFb, positive
transcription elongation factor b; RAP74, RNA polymerase II-associated protein
of 74 kDa (larger subunit of TFIIF); SCP, small CTD phosphatase; GST,
glutathione S-transferase; MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase; DTT, dithiothreitol; PPAR
,
peroxisome proliferator-activated receptor
; rCTDo, recombinant CTDo;
TK, thymidine kinase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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