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J. Biol. Chem., Vol. 280, Issue 45, 37681-37688, November 11, 2005
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1
From the
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, Department of Horticultural Sciences, Texas A&M University, College Station, Texas 77843, and the £Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854
Received for publication, May 13, 2005 , and in revised form, August 31, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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The information content of the CTD reflects the instantaneous balance between the activities of CTD kinases and CTD phosphatases at either some or all of the serine phosphorylation sites. A subject of intense interest (and debate) is whether and how CTD kinases and phosphatases are themselves responsive to specificity determinants within the CTD. Among the CTD phosphatases, some enzymes fail to discriminate between Ser2-PO4 and Ser5-PO4 substrates (9, 10), whereas others display a preference (2- to 10-fold) for either Ser2-PO4 or Ser5-PO4 (11, 12). Recently, we described two types of CTD phosphatases that act exclusively on Ser5-PO4: (i) the paralogous plant enzymes CPL1 and CPL2, which were identified genetically as regulators of osmotic stress and abscisic acid-responsive transcription in Arabidopsis thaliana (13, 14), and (ii) yeast Ssu72, which interacts with TFIIB and with proteins involved in RNA 3'-end formation (15-20).
CPL1/2 and Ssu72 belong to different enzyme families. The plant CPL1/2 proteins, like the prototypal CTD phosphatase Fcp1, are members of the DXDXT superfamily of metal-dependent phosphotransferases that act via an aspartyl-phosphoenzyme intermediate (8, 11, 13, 21-23). Ssu72 belongs to the CXXXXXR superfamily of metal-independent phosphohydrolases that act via a cysteinyl phosphoenzyme intermediate (15, 24, 25). This situation, whereby nature has selected two entirely different structural solutions to perform the same site-specific chemical transformation of the CTD, provides an opportunity to address key questions about how the CTD code is read. Here we demonstrate that CPL1/2 and Ssu72 rely on different positional cues in the CTD primary structure to recognize and hydrolyze Ser5-PO4.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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-D-galactopyranoside and 2% ethanol, and incubation was continued for 20 h at 17 °C. Cells were harvested by centrifugation and stored at -80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria were resuspended in 25 ml of buffer A (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10% glycerol). Phenylmethylsulfonyl fluoride and lysozyme were added to final concentrations of 500 µM and 100 µg/ml, respectively. After incubation on ice for 30 min, Triton X-100 was added to a final concentration of 0.1%, and the lysate was sonicated to reduce viscosity. Insoluble material was removed by centrifugation. The soluble extracts were mixed for 30 min with 1 ml of Ni2+- nitrilotriacetic acid-agarose (Qiagen) that had been equilibrated with buffer A containing 0.1% Triton X-100. The resin was recovered by centrifugation, resuspended in buffer A, and poured into columns. The columns were washed with 10 ml of 20 mM imidazole in buffer A and then eluted stepwise with 1.5 ml of buffer A containing 50, 100, 250, and 500 mM imidazole. The polypeptide compositions of the column fractions were monitored by SDS-PAGE. The recombinant His10Smt3-CPL polypeptides were recovered predominantly in the 250 mM imidazole fractions. The 250 mM imidazole eluates were dialyzed against buffer A containing 2 mM dithiothreitol and 0.01% Triton X-100 and then stored at -80 °C. The CPL1 and CPL2 concentrations were determined by SDS-PAGE analysis of serial dilutions of the CPL preparations in parallel with serial dilutions of a BSA standard. The gels were stained with Coomassie Blue, and the staining intensities of the His10Smt3-CPL and BSA polypeptides were quantified using a Fujifilm FLA-5000 digital imaging and analysis system. CPL1 and CPL2 concentrations were calculated by interpolation to the BSA standard curve. Wild-type Ssu72 and the C15S mutant were produced in E. coli as glutathione S-transferase fusions and purified from soluble bacterial extracts by glutathione-Sepharose affinity chromatography as described (15). Protein concentration was measured with the Bio-Rad dye reagent using BSA as the standard.
Velocity Sedimentation—Aliquots (40 µg) of the nickel-agarose preparations of CPL1 and CPL2 were mixed with catalase (45 µg), BSA (45 µg), and cytochrome c (45 µg), and the mixtures were applied to 4.8-ml 15-30% glycerol gradients containing 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 1 mM EDTA, 2 mM dithiothreitol, 0.01% Triton X-100. The gradients were centrifuged in a SW50 rotor at 50,000 rpm for 17 h at 4 °C. Fractions (
0.26 ml) were collected from the bottoms of the tubes.
CTD Phosphopeptides—CTD Ser-PO4 peptides were synthesized and purified by the Sloan-Kettering Microchemistry Core Laboratory as described previously (2, 8). The peptides were dissolved in 10 mM Tris-HCl (pH 7.4), 1 mM EDTA and stored at 4 °C. The molar concentrations of the phosphopeptides were initially estimated from the absorbance at 274 nM using an extinction coefficient of 1.4 x 103 M-1 for tyrosine. The content of Ser-PO4 was then determined for each peptide, measuring the release of inorganic phosphate after digestion with calf intestinal phosphatase as described (8).
CTD Phosphatase Assay—Reaction mixtures (25 µl) containing 50 mM Tris acetate (pH 5.5), 10 mM MgCl2, CTD phosphopeptide, and CPL1 or CPL2 were incubated for 60 min at 37 °C. Reaction mixtures (25 µl) containing 50 mM Tris acetate (pH 7.0), 5 mM dithiothreitol, CTD phosphopeptide, and Ssu72 as specified were incubated for 120 min at 30 °C. The reactions were quenched by adding 0.5 ml of malachite green reagent (BIOMOL Research Laboratories, Plymouth Meeting, PA). Release of phosphate was determined by measuring A620 and interpolating the value to a phosphate standard curve. The amounts of CTD substrate in the CTD phosphatase reactions are expressed as input phosphoserine, as determined by CIP digestion performed in parallel with each assay.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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10 kDa larger than its predicted size. The CPL1 and CPL2 preparation contained several polypeptides in the 60-90-kDa range and a cluster of smaller polypeptides migrating at 20-27 kDa (the latter correspond to His10Smt3- and His10Smt3-peptide fusions arising via proteolysis) (Fig. 1A and data not shown).
The quaternary structure of recombinant CPL1 and CPL2 was investigated by zonal velocity sedimentation through a 15-30% glycerol gradient (Fig. 1, B and C). Marker protein catalases (native size 248 kDa), BSA (66 kDa), and cytochrome c (12 kDa) were included as internal standards in the gradient. The tagged CPL1 and CPL2 proteins sedimented as discrete peaks coincident with BSA. CPL1 and CPL2 were clearly separated from the cluster of low molecular weight polypeptides, which cosedimented with cytochrome c. The CTD Ser5 phosphatase activity profile (measured by the release of Pi from a synthetic phosphopeptide) paralleled the abundance of the intact CPL1 and CPL2 polypeptides. These results are consistent with a monomeric quaternary structure for the catalytic domains of CPL1 and CPL2.
CPL1- and CPL2-catalyzed release of Pi from a 28-amino acid tetraheptad CTD Ser5-PO4 substrate was proportional to enzyme concentration (expressed as the amount of full-length CPL fusion protein); 95% of the input Ser5-PO4 residues were hydrolyzed at saturating CPL levels (Fig. 2A). From the slope of the titration curves we estimated that CPL1 and CPL2 hydrolyzed a 6400- and 9300-fold molar excess of Ser5-PO4/enzyme, respectively, during the 60-min reaction. Neither CPL1 nor CPL2 catalyzed Pi release from a tetraheptad CTD Ser2-PO4 substrate (Fig. 2A). A kinetic analysis is shown in Fig. 2B of the reaction of CPL2 with the tetraheptad CTD-Ser5-PO4 substrate at two different levels of input enzyme. Product accumulated steadily with a pseudo-first order profile. The initial rate was proportional to enzyme concentration; the turnover number was 13 s-1.
Reaction of CPL1 and CPL2 with a 14-amino acid diheptad CTD Ser5-PO4 substrate YSPTSPSYSPTSPS resulted in Pi release proportional to the amount of input enzyme (Fig. 2C). 85% of the input phosphoserine was hydrolyzed at saturating enzyme levels (Fig. 2C and data not shown). CPL1 and CPL2 released an 11,000-fold molar excess of Pi/enzyme during the 60-min reaction. In contrast, CPL1 and CPL2 were inert in hydrolyzing a diheptad CTD Ser2-PO4 substrate YSPTSPSYSPTSPS (Fig. 2C). This experiment showed that two heptad repeats suffice for Ser5-specific CTD phosphatase activity.
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CTD Ser5 Phosphatase Activity of Ssu72—The specificity of Ssu72 was characterized using a recombinant glutathione S-transferase-Ssu72 fusion protein purified from a soluble bacterial lysate by glutathione-affinity chromatography (Fig. 3D). Ssu72 hydrolyzed Pi from the tetraheptad Ser5-PO4 substrate; activity reached a plateau at 2.5-5 µg of input protein (Fig. 3A), at which point 55% of the input Ser5-PO4 residues had been hydrolyzed. Product accumulated steadily during a 3-h reaction (Fig. 3B), which signified that the low end point was not dictated by inactivation of the enzyme during the incubation. Additional experiments showed that, although adding more Ssu72 to the reaction after the end point had been achieved did not result in further Pi release, adding more peptide without more enzyme did result in hydrolysis of 50% of the additional substrate (not shown). Ssu72 failed to release Pi from the tetraheptad Ser2-PO4 substrate (Fig. 3, A and B).
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CPLs and Ssu72 Recognize Different Specificity Determinants in the CTD—To probe the role of CTD primary structure in Ser5 phosphatase activity, we tested mutated versions of the tetraheptad CTD Ser5-PO4 peptide (YSPTSPS)4, wherein every Tyr1, Ser2, Pro3, Thr4, or Pro6 was replaced by alanine. The titration profiles of the reaction of CPL1 with the S2A, T4A and P6A substrates were similar to that of the wild-type CTD (Fig. 4A). However, the CPL1 activity profiles with the P3A and Y1A substrates displayed a shift to the right (Fig. 4A). The specific activities of CPL1 with the P3A and Y1A substrates were 16 and 4%, respectively, of the activity with the wild-type (YSPTSPS)4 substrate. CPL2 hydrolyzed the S2A and T4A CTDs as well as the wild-type CTD. Activity was reduced modestly by the P6A change (to 27% of wild-type) and drastically by the P3A (3%) and Y1A (1%) CTD mutations (Fig. 4B). These experiments demonstrate that Tyr1 and Pro3 are the critical determinants of CPL phosphatase activity at position Ser5.
Ssu72 responded quite differently to changes in CTD primary structure (Fig. 4C). The P3A change that was deleterious to the CPLs had virtually no ill effect on Ssu72, whereas the T4A mutation, which was well tolerated by the CPLs, was inimical to Ssu72, reducing its specific activity to 3% of the wild-type CTD level (Fig. 4C). Moreover, the P6A lesion abolished Ser5 phosphatase activity of Ssu72, though it had no effect on CPL1 and only a modest effect on CPL2. Ssu72 activity was reduced severely (to 4% of wild-type) by the Y1A change and was unaffected by S2A. We conclude that: (i) Tyr1, Thr4, and Pro6 are the critical determinants of Ssu72 phosphatase, and (ii) different CTD phosphatases with the same exquisite specificity for Ser5-PO4 achieve their specificities by recognizing different structural cues in the CTD.
By titrating the wild-type (YSPTSPS)4 substrate against fixed amounts of CPL1, CPL2, and Ssu72, we determined apparent Km values of 60, 140, and 280 µM, respectively (not shown). These values are higher than the substrate concentrations (30 µM) used in the specific activity determinations in Fig. 4. Thus, we retested the activity of CPL1 and CPL2 with the wild-type, Y1A, P3A, and P6A tetraheptad Ser5-PO4 peptides at substrate concentrations (240-260 µM) in excess of the Km for wild-type peptide (Fig. 5). The hierarchy of mutational effects at higher substrate was similar to what was observed at limiting substrate. The extents of phosphate release by CPL1 with the P6A, P3A, and Y1A substrates were 96, 27, and 13%, respectively, of the activity with the wild-type (YSPTSPS)4 substrate (Fig. 5, left panel). Phosphate release by CPL2 with the P6A, P3A, and Y1A substrates was 64, 7, and 4% of activity with the wild-type peptide (middle panel). Thus, raising the CTD concentration above Km only slightly mitigated the effects of the Y1A or P3A changes on CPL1 activity, or the effects of Y1A, P3A, and P6A on CPL2 function, vis-à-vis their activity with the wild-type tetraheptad substrate. A parallel experiment with Ssu72 at the higher substrate concentrations showed that phosphate release with the Y1A, T4A, and P6A substrates was 8, 12, and 3% of its activity with the wild-type peptide (Fig. 5, right panel).
Effects of a Lysine Substitution at Position 7 of the CTD Heptad—The identity of the amino acid at position 7 of the CTD is either highly conserved (e.g. all serines in the Schizosaccharomyces pombe CTD (GenBankTM accession code CAB57941 [GenBank] ) and Encephalitozoon cuniculi CTD (GenBankTM CAD26175 [GenBank] )) or subject to variation from one heptad to another within the same CTD. In the case of human RNA polymerase II (GenBankTM P24928 [GenBank] ), one-half (26/52) of the heptads have a consensus Ser7, and the others are substituted, most commonly with Lys7 (7 heptads) or Asn7 (5 heptads). The CTD of the malaria parasite Plasmodium falciparum (GenBankTM Z98551 [GenBank] ) is dominated by Lys7-containing heptads (11/15 repeats).
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30% as active with the S7K CTD substrate as it was with the wild-type CTD (Fig. 6C). Thus, neither the CPLs nor Ssu72 depend on Ser7 for their Ser5-specific phosphatase activity.
Different Phasing of Specificity Determinants for CPLs and Ssu72— Further insight into the location of the specificity determinants emerged from an analysis of the reaction of CPLs and Ssu72 with diheptad monophosphorylated CTDs, which contained Ser5-PO4 in either the amino-terminal heptad (YSPTSPSYSPTSPS) or the carboxyl-terminal heptad (YSPTSPSYSPTSPS). Ssu72 displayed a 10-fold preference for hydrolysis of Ser5-PO4 within the amino-terminal heptad (Fig. 7C). This result implies that the recognition of Tyr1 by Ssu72 occurs on the carboxyl side of the Ser5-PO4 moiety that is being hydrolyzed, i.e. Ssu72 has higher activity on the proximal repeat because Ssu72 recognizes the Thr4 and Pro6 flanking the Ser5-PO4 and the Tyr1 in the distal repeat (see Fig. 9). Ssu72 is only weakly active on the distal repeat because there is no Tyr1 available on the carboxyl side of the phosphoserine. From a substrate titration experiment at a fixed level of Ssu72, we determined an apparent Km of 850 µM for the YSPTSPSYSPTSPS peptide (data not shown); thus, the affinity of the enzyme for a tetraheptad CTD with four phosphoserines is 3-fold higher than for a diheptad with a single phosphoserine.
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Properties of the CTD Code—Here we have begun to illuminate how the CTD code is read by two classes of protein phosphatases that specifically dephosphorylate Ser5. The instructive findings are that the plant CPLs and yeast Ssu72 recognize entirely different constellations of specificity determinants in the CTD. Whereas the CPLs rely on Tyr1 and Pro3 located on the upstream side of the Ser5-PO4 target site, Ssu72 recognizes Thr4 and Pro6 flanking the target Ser5-PO4 plus the downstream Tyr1 residues of the adjacent heptad (Fig. 9). The two classes of phosphatases not only see different amino acids, they see them in entirely different registers with respect to Ser5-PO4.
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-like extended conformation, whereby every other residue is oriented toward or away from the protein surface (Fig. 9).
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The Ser5-PO4, Pro6, and Tyr1 moieties that Ssu72 recognizes are located on the protein side of the interface in CDS1, whereas Ser2 and Pro3, to which Ssu72 is indifferent, project away from the protein surface (Fig. 9). We therefore speculate that the CTD adopts a CDS1-like conformation in its interaction with Ssu72. Although Thr4 does not contribute to the CTD-protein interface at CDS1 of the capping enzyme, the Thr O
is in position to donate a hydrogen bond to the vicinal phosphate group at Ser5 (4).
The finding of at least two CTD coding elements that signal specific hydrolysis of Ser5-PO4 has implications for understanding CTD information content. First, the functional "footprints" of CPL1/2 and Ssu72 on the CTD primary structure collectively span more than one heptad repeat, a state of affairs that is consistent with recent genetic evidence that the essential functional unit of the CTD is contained with a contiguous diheptad pair (26). Second, there is no simple rule for what is recognized. The CTD phosphorylation sites are located within Ser-Pro dipeptides. With respect to CTD Ser5-specific dephosphorylation, Ssu72 is identified here as a stringently Pro6-directed phosphatase, whereas CPL1 is Pro6-independent and CPL2 is only modestly affected by elimination of Pro6. Third, even though the CTD is structurally plastic, there are likely to exist a set of CTD conformational states that are utilized reiteratively by different CTD-binding proteins, even where the proteins are themselves not structurally related (e.g. the CTD phosphatases and the mRNA capping enzyme). Fourth, the information content of the CTD can be amplified by blending multiple distinct CTD coding elements (such as those seen here for the CPLs and Ssu72) to form a bipartite, or even higher order, recognition site for CTD-associated factors (such as the capping enzyme).
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FOOTNOTES |
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1 To whom correspondence should be addressed. E-mail: s-shuman{at}ski.mskcc.org.
2 The abbreviations used are: CTD, carboxyl-terminal domain; CDS, CTD-docking site; BSA, bovine serum albumin.
3 S. Hausmann, unpublished information.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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