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• W2048103661 abstract "The cyanobacterial circadian oscillator can be reconstituted in vitro; mixing three clock proteins (KaiA, KaiB, and KaiC) with ATP results in an oscillation of KaiC phosphorylation with a periodicity of ∼24 h. The hexameric ATPase KaiC hydrolyzes ATP bound at subunit interfaces. KaiC also exhibits autokinase and autophosphatase activities, the latter of which is particularly noteworthy because KaiC is phylogenetically distinct from typical protein phosphatases. To examine this activity, we performed autodephosphorylation assays using 32P-labeled KaiC. The residual radioactive ATP bound to subunit interfaces was removed using a newly established method, which included the dissociation of KaiC hexamers into monomers and the reconstitution of KaiC hexamers with nonradioactive ATP. This approach ensured that only the signals derived from 32P-labeled KaiC were examined. We detected the transient formation of [32P]ATP preceding the accumulation of 32Pi. Together with kinetic analyses, our data demonstrate that KaiC undergoes dephosphorylation via a mechanism that differs from those of conventional protein phosphatases. A phosphate group at a phosphorylation site is first transferred to KaiC-bound ADP to form ATP as an intermediate, which can be regarded as a reversal of the autophosphorylation reaction. Subsequently, the ATP molecule is hydrolyzed to form Pi. We propose that the ATPase active site mediates not only ATP hydrolysis but also the bidirectional transfer of the phosphate between phosphorylation sites and the KaiC-bound nucleotide. On the basis of these findings, we can now dissect the dynamics of the KaiC phosphorylation cycle relative to ATPase activity. The cyanobacterial circadian oscillator can be reconstituted in vitro; mixing three clock proteins (KaiA, KaiB, and KaiC) with ATP results in an oscillation of KaiC phosphorylation with a periodicity of ∼24 h. The hexameric ATPase KaiC hydrolyzes ATP bound at subunit interfaces. KaiC also exhibits autokinase and autophosphatase activities, the latter of which is particularly noteworthy because KaiC is phylogenetically distinct from typical protein phosphatases. To examine this activity, we performed autodephosphorylation assays using 32P-labeled KaiC. The residual radioactive ATP bound to subunit interfaces was removed using a newly established method, which included the dissociation of KaiC hexamers into monomers and the reconstitution of KaiC hexamers with nonradioactive ATP. This approach ensured that only the signals derived from 32P-labeled KaiC were examined. We detected the transient formation of [32P]ATP preceding the accumulation of 32Pi. Together with kinetic analyses, our data demonstrate that KaiC undergoes dephosphorylation via a mechanism that differs from those of conventional protein phosphatases. A phosphate group at a phosphorylation site is first transferred to KaiC-bound ADP to form ATP as an intermediate, which can be regarded as a reversal of the autophosphorylation reaction. Subsequently, the ATP molecule is hydrolyzed to form Pi. We propose that the ATPase active site mediates not only ATP hydrolysis but also the bidirectional transfer of the phosphate between phosphorylation sites and the KaiC-bound nucleotide. On the basis of these findings, we can now dissect the dynamics of the KaiC phosphorylation cycle relative to ATPase activity. The circadian clock is an endogenous timing mechanism in living organisms that coordinates various biological activities with daily environmental changes. The circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942 is the sole example that can be reconstituted in vitro; the phosphorylation state of KaiC shows robust circadian rhythms by mixing KaiA, KaiB, KaiC, and ATP in a test tube (1.Nakajima M. Imai K. Ito H. Nishiwaki T. Murayama Y. Iwasaki H. Oyama T. Kondo T. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro.Science. 2005; 308: 414-415Crossref PubMed Scopus (789) Google Scholar). Eukaryotic circadian clocks have long been understood as transcriptional-translational feedback loops in which clock gene products repress their own transcription (2.Lakin-Thomas P.L. Transcriptional feedback oscillators: maybe, maybe not ….J. Biol. Rhythms. 2006; 21: 83-92Crossref PubMed Scopus (103) Google Scholar). Recently, however, protein-based circadian oscillations have been found in human red blood cells (3.O'Neill J.S. Reddy A.B. Circadian clocks in human red blood cells.Nature. 2011; 469: 498-503Crossref PubMed Scopus (589) Google Scholar) and in microalgae (4.O'Neill J.S. van Ooijen G. Dixon L.E. Troein C. Corellou F. Bouget F.Y. Reddy A.B. Millar A.J. Circadian rhythms persist without transcription in a eukaryote.Nature. 2011; 469: 554-558Crossref PubMed Scopus (387) Google Scholar), suggesting that protein-based circadian clocks exist ubiquitously in living organisms. Among the many experimental systems for studying circadian oscillations, the cyanobacterial in vitro system is the simplest one and is expected to be the best model to understand the principle underlying the protein-based oscillations. KaiC belongs to the P-loop NTPase superfamily, which includes both NTPases and kinases characterized by a strongly conserved nucleotide-binding motif, the P-loop (5.Leipe D.D. Koonin E.V. Aravind L. Evolution and classification of P-loop kinases and related proteins.J. Mol. Biol. 2003; 333: 781-815Crossref PubMed Scopus (223) Google Scholar). KaiC is a double-domain P-loop ATPase consisting of the N-terminal CI and C-terminal CII domains (6.Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. Physical interactions among circadian clock proteins KaiA, KaiB, and KaiC in cyanobacteria.EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (140) Google Scholar). KaiC forms a double doughnut-like homohexamer with an ATP molecule at each subunit interface in the two rings (7.Pattanayek R. Wang J. Mori T. Xu Y. Johnson C.H. Egli M. Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.Mol. Cell. 2004; 15: 375-388Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 8.Xu Y. Mori T. Pattanayek R. Pattanayek S. Egli M. Johnson C.H. Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13933-13938Crossref PubMed Scopus (117) Google Scholar). KaiC exhibits ATPase and autokinase activities (9.Terauchi K. Kitayama Y. Nishiwaki T. Miwa K. Murayama Y. Oyama T. Kondo T. ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16377-16381Crossref PubMed Scopus (175) Google Scholar, 10.Nishiwaki T. Iwasaki H. Ishiura M. Kondo T. Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 495-499Crossref PubMed Scopus (152) Google Scholar). KaiA activates both of these activities, whereas KaiB inhibits the effect of KaiA (9.Terauchi K. Kitayama Y. Nishiwaki T. Miwa K. Murayama Y. Oyama T. Kondo T. ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16377-16381Crossref PubMed Scopus (175) Google Scholar, 11.Iwasaki H. Nishiwaki T. Kitayama Y. Nakajima M. Kondo T. KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 15788-15793Crossref PubMed Scopus (232) Google Scholar, 12.Kitayama Y. Iwasaki H. Nishiwaki T. Kondo T. KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system.EMBO J. 2003; 22: 2127-2134Crossref PubMed Scopus (228) Google Scholar). The active sites for ATP hydrolysis reside at the subunit interfaces of CI and CII (9.Terauchi K. Kitayama Y. Nishiwaki T. Miwa K. Murayama Y. Oyama T. Kondo T. ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16377-16381Crossref PubMed Scopus (175) Google Scholar), whereas the autokinase activity is located only in CII (13.Hayashi F. Iwase R. Uzumaki T. Ishiura M. Hexamerization by the N-terminal domain and intersubunit phosphorylation by the C-terminal domain of cyanobacterial circadian clock protein KaiC.Biochem. Biophys. Res. Commun. 2006; 348: 864-872Crossref PubMed Scopus (47) Google Scholar). The autophosphorylation sites, Ser-431 and Thr-432 (14.Nishiwaki T. Satomi Y. Nakajima M. Lee C. Kiyohara R. Kageyama H. Kitayama Y. Temamoto M. Yamaguchi A. Hijikata A. Go M. Iwasaki H. Takao T. Kondo T. Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13927-13932Crossref PubMed Scopus (161) Google Scholar), face the ATP molecule on the neighboring protomer (8.Xu Y. Mori T. Pattanayek R. Pattanayek S. Egli M. Johnson C.H. Identification of key phosphorylation sites in the circadian clock protein KaiC by crystallographic and mutagenetic analyses.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13933-13938Crossref PubMed Scopus (117) Google Scholar). In addition to these activities, KaiC exhibits autophosphatase activity (15.Xu Y. Mori T. Johnson C.H. Cyanobacterial circadian clockwork: roles of KaiA, KaiB, and the kaiBC promoter in regulating KaiC.EMBO J. 2003; 22: 2117-2126Crossref PubMed Scopus (190) Google Scholar), despite a lack of homology with the Ser/Thr and Tyr-specific protein phosphatases (16.Shi Y. Serine/threonine phosphatases: mechanism through structure.Cell. 2009; 139: 468-484Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar, 17.Tabernero L. Aricescu A.R. Jones E.Y. Szedlacsek S.E. Protein-tyrosine phosphatases: structure-function relationships.FEBS J. 2008; 275: 867-882Crossref PubMed Scopus (121) Google Scholar). The reaction mechanism for KaiC autodephosphorylation has not yet been elucidated. To address this issue, we performed an autodephosphorylation assay using KaiC labeled with 32P. Prior to the assay, we removed the residual radioactive ATP bound to subunit interfaces using a newly established method, which included the dissociation of KaiC hexamers into monomers and the reconstitution of KaiC hexamers with nonradioactive ATP. This approach ensured that only the signals derived from 32P-labeled KaiC were examined. Surprisingly, we detected a transient elevation of [32P]ATP preceding the formation of 32Pi, suggesting that KaiC dephosphorylates itself through a novel sequential mechanism, i.e. KaiC generates ATP as a reaction intermediate, followed by its hydrolysis. The first step of the reaction is regarded as a reversal of autophosphorylation. On the basis of our observations, we demonstrate that the dephosphorylation of KaiC is catalyzed by the active site of ATPase through a completely different mechanism from those of typical Ser/Thr and Tyr phosphatases. We used Escherichia coli BL21 cells as the host strain for expressing recombinant Kai proteins. Recombinant KaiA was expressed as described previously (6.Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. Physical interactions among circadian clock proteins KaiA, KaiB, and KaiC in cyanobacteria.EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (140) Google Scholar) and purified according to a previously reported method (14.Nishiwaki T. Satomi Y. Nakajima M. Lee C. Kiyohara R. Kageyama H. Kitayama Y. Temamoto M. Yamaguchi A. Hijikata A. Go M. Iwasaki H. Takao T. Kondo T. Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13927-13932Crossref PubMed Scopus (161) Google Scholar). Recombinant KaiB was expressed and purified as described previously (6.Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. Physical interactions among circadian clock proteins KaiA, KaiB, and KaiC in cyanobacteria.EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (140) Google Scholar, 11.Iwasaki H. Nishiwaki T. Kitayama Y. Nakajima M. Kondo T. KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 15788-15793Crossref PubMed Scopus (232) Google Scholar, 18.Nishiwaki T. Satomi Y. Kitayama Y. Terauchi K. Kiyohara R. Takao T. Kondo T. A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria.EMBO J. 2007; 26: 4029-4037Crossref PubMed Scopus (177) Google Scholar). Full-length or CII-truncated KaiC hexamers were expressed and purified as described previously (6.Iwasaki H. Taniguchi Y. Ishiura M. Kondo T. Physical interactions among circadian clock proteins KaiA, KaiB, and KaiC in cyanobacteria.EMBO J. 1999; 18: 1137-1145Crossref PubMed Scopus (140) Google Scholar, 9.Terauchi K. Kitayama Y. Nishiwaki T. Miwa K. Murayama Y. Oyama T. Kondo T. ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16377-16381Crossref PubMed Scopus (175) Google Scholar, 14.Nishiwaki T. Satomi Y. Nakajima M. Lee C. Kiyohara R. Kageyama H. Kitayama Y. Temamoto M. Yamaguchi A. Hijikata A. Go M. Iwasaki H. Takao T. Kondo T. Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13927-13932Crossref PubMed Scopus (161) Google Scholar, 18.Nishiwaki T. Satomi Y. Kitayama Y. Terauchi K. Kiyohara R. Takao T. Kondo T. A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria.EMBO J. 2007; 26: 4029-4037Crossref PubMed Scopus (177) Google Scholar). Protein concentrations were determined using the Bradford method and bovine serum albumin as a standard. [γ-32P]ATP (NEG002Z; 370 MBq/ml, 222 TBq/mmol) and [α-32P]ATP (NEG003H; 370 MBq/ml, 111 TBq/mmol) were purchased from PerkinElmer Life Sciences. Unless stated otherwise, the phosphorylation rhythm of KaiC was reconstituted and examined as reported previously (1.Nakajima M. Imai K. Ito H. Nishiwaki T. Murayama Y. Iwasaki H. Oyama T. Kondo T. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro.Science. 2005; 308: 414-415Crossref PubMed Scopus (789) Google Scholar). Briefly, 3.5 μm KaiC was incubated at 30 °C in the presence of 1.2 μm KaiA, 3.5 μm KaiB, and 1 mm ATP in 20 mm Tris (pH 8.0), 150 mm NaCl, 5 mm MgCl2, and 1 mm DTT. Aliquots of the reaction mixture were taken every 4 h and subjected to SDS-PAGE (gels with 11% T and 0.67% C) (18.Nishiwaki T. Satomi Y. Kitayama Y. Terauchi K. Kiyohara R. Takao T. Kondo T. A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria.EMBO J. 2007; 26: 4029-4037Crossref PubMed Scopus (177) Google Scholar). After electrophoresis, gels were subjected to Coomassie Brilliant Blue staining using Quick CBB Plus (Wako Pure Chemical Industries, Ltd.) and scanned using an ImageScanner III system (GE Healthcare). Densitometry of the gels was performed using ImageJ 1.41 software (National Institutes of Health). The levels of phosphorylated KaiC relative to those of total KaiC were plotted against time. 32P-Labeled KaiC hexamers were obtained using a previously described method with modifications (19.Ito H. Kageyama H. Mutsuda M. Nakajima M. Oyama T. Kondo T. Autonomous synchronization of the circadian KaiC phosphorylation rhythm.Nat. Struct. Mol. Biol. 2007; 14: 1084-1088Crossref PubMed Scopus (86) Google Scholar). KaiC hexamers (5–10 mg/ml) were incubated on ice for 24 h in buffer A (20 mm Tris (pH 8.0), 150 mm NaCl, 5 mm MgCl2, and 2 mm DTT) in the presence of 1 mm [γ-32P]ATP. The specific activity of ATP in the reaction mixture was 12 GBq/mmol. To remove [γ-32P]ATP bound to the subunit interfaces of KaiC hexamers, 32P-labeled KaiC hexamers were dissociated into monomers. The reaction mixture was passed twice through Micro Bio-Spin P-30 columns (Bio-Rad) equilibrated with buffer A containing 0.1 mm ADP, and samples were incubated on ice for 24 h. KaiC monomer fractions were then obtained via gel filtration chromatography using a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with buffer A containing 0.1 mm ADP. The autodephosphorylation reaction was initiated by replacing 0.1 mm ADP with 1 mm ATP using Micro Bio-Spin P-30 columns and incubating the resulting solution, which contained ∼1 mg/ml KaiC, at 30 °C. Aliquots of the reaction mixture were collected at predetermined time points, and reactions were stopped by adding an equal volume of Laemmli sample buffer (62.5 mm Tris (pH 6.8), 2% SDS, 25% glycerol, and 0.01% bromphenol blue; Bio-Rad) supplemented with 5% (v/v) 2-mercaptoethanol. The resulting samples were subjected to either SDS-PAGE or thin-layer chromatography on PEI-cellulose F plates (Merck) using 0.75 m KH2PO4 as the mobile phase (20.Yegutkin G.G. Samburski S.S. Jalkanen S. Novak I. ATP-consuming and ATP-generating enzymes secreted by pancreas.J. Biol. Chem. 2006; 281: 29441-29447Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). 32P-Derived signals were detected via autoradiography using BAS IP MS 2040E imaging plates (GE Healthcare) and a Typhoon 9400 image analyzer (GE Healthcare). Signals were quantified using the Toolbox module of ImageQuant TL 7.0 software (GE Healthcare). Background signals were subtracted using the local median method performed by the software. The standard curves used to calculate concentrations of 32P-labeled molecules from autoradiograms were obtained using serial dilutions of 1 mm [γ-32P]ATP solution (12 GBq/mmol). Radioactivity was detected at the positions corresponding to ATP and Pi markers as well as at the origin on the chromatogram (supplemental Fig. S1). We calculated the concentrations of the molecules that stayed at the thin-layer chromatography origin and of phosphorylated KaiC separated using SDS-PAGE and plotted the data against time. We concluded that the radioactivity at the origin was derived from 32P-labeled KaiC because the two traces almost completely overlapped (supplemental Fig. S2). Therefore, in this experiment, we analyzed all three 32P-labeled species on a single chromatogram. The kinetic analysis was performed using Global Kinetic Explorer 2.5 software (KinTek Corp.) (21.Johnson K.A. Simpson Z.B. Blom T. Global Kinetic Explorer: a new computer program for dynamic simulation and fitting of kinetic data.Anal. Biochem. 2009; 387: 20-29Crossref PubMed Scopus (421) Google Scholar). Errors for each parameter were estimated using the FitSpace Explorer module in the software (22.Johnson K.A. Simpson Z.B. Blom T. FitSpace Explorer: an algorithm to evaluate multidimensional parameter space in fitting kinetic data.Anal. Biochem. 2009; 387: 30-41Crossref PubMed Scopus (240) Google Scholar) by calculating the lower and upper boundaries. The calculated boundaries reflect fits within minimum χ2 multiplied by 1.2. Nucleotides bound to either full-length or CII-truncated KaiC were analyzed by reconstituting KaiC hexamers from monomers in the presence of 1 mm [α-32P]ATP. The specific activity of ATP in the reaction mixture was 6.0 GBq/mmol. The resulting solution, which contained ∼1 mg/ml protein, was incubated at 30 °C. Aliquots were collected at predetermined time points and applied twice to Micro Bio-Spin P-30 columns to remove unbound nucleotides. The eluate was mixed with an equal volume of Laemmli sample buffer containing 5% (v/v) 2-mercaptoethanol to release KaiC-bound nucleotides from the proteins. Samples were spotted onto PEI-cellulose plates and subjected to thin-layer chromatography to separate ATP and ADP using KH2PO4 as the mobile phase. To monitor the autodephosphorylation of KaiC, we prepared 32P-labeled KaiC phosphorylated at Ser-431 and Thr-432. At 30 °C, KaiC is markedly phosphorylated in the presence of KaiA (11.Iwasaki H. Nishiwaki T. Kitayama Y. Nakajima M. Kondo T. KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 15788-15793Crossref PubMed Scopus (232) Google Scholar) and dephosphorylated in its absence (15.Xu Y. Mori T. Johnson C.H. Cyanobacterial circadian clockwork: roles of KaiA, KaiB, and the kaiBC promoter in regulating KaiC.EMBO J. 2003; 22: 2117-2126Crossref PubMed Scopus (190) Google Scholar). For autodephosphorylation assays, however, it is desirable to phosphorylate KaiC without KaiA because residual KaiA might interfere with autodephosphorylation. Ito et al. (19.Ito H. Kageyama H. Mutsuda M. Nakajima M. Oyama T. Kondo T. Autonomous synchronization of the circadian KaiC phosphorylation rhythm.Nat. Struct. Mol. Biol. 2007; 14: 1084-1088Crossref PubMed Scopus (86) Google Scholar) showed that the ratio of phosphorylated KaiC to total KaiC reached ∼0.7 after 30 h of incubation at 4 °C, suggesting that incubation at low temperature promotes autophosphorylation without KaiA. We incubated KaiC in the presence of [γ-32P]ATP on ice and monitored the incorporation of radioactive phosphate for 24 h. After 24 h of incubation, ∼30% of KaiC was newly phosphorylated based on Coomassie Brilliant Blue staining (Fig. 1A, upper panel). KaiC-specific radioactive signals increased together with the increasing intensity of phosphorylated KaiC bands (Fig. 1A, lower panel). Based on the autoradiogram, ∼0.3 mol of phosphate was incorporated into 1 mol of KaiC monomers. The 32P-labeled KaiC hexamer obtained as described above contained residual [γ-32P]ATP at the subunit interface. Next, we eliminated KaiC-bound [γ-32P]ATP to ensure that only signals derived from 32P-labeled KaiC were examined during the autodephosphorylation assay. We expected that [γ-32P]ATP bound to the subunit interface would be released when 32P-labeled KaiC hexamers were dissociated into monomers. To date, the procedure for the monomerization of Synechococcus KaiC hexamers has not been established. Hayashi et al. (23.Hayashi F. Suzuki H. Iwase R. Uzumaki T. Miyake A. Shen J.R. Imada K. Furukawa Y. Yonekura K. Namba K. Ishiura M. ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC.Genes Cells. 2003; 8: 287-296Crossref PubMed Scopus (108) Google Scholar) showed that Thermosynechococcus KaiC hexamers dissociated into monomers in the absence of ATP. Therefore, we removed ATP from the reaction mixture and incubated the resulting solution to prepare Synechococcus KaiC monomers. Because KaiC formed aggregates in the absence of ATP at temperatures higher than ∼10 °C, we incubated the reaction mixture on ice. We added 0.1 mm ADP to stabilize the KaiC monomers during the incubation period. After 24 h of incubation, the KaiC monomers were separated using gel filtration chromatography (Fig. 1B). To verify that the KaiC monomers were functional, we used them to recreate the KaiC phosphorylation rhythm. The KaiC monomers were allowed to re-form hexamers by replacing 0.1 mm ADP with 1 mm ATP, and the resulting KaiC hexamers were incubated at 30 °C in the presence of KaiA and KaiB. It took <5 min for KaiC hexamers to form from monomers (supplemental Fig. S3). As shown in Fig. 1C, this approach did not affect the KaiC phosphorylation rhythm. As expected, the radioactive ATP that had been bound to KaiC was almost completely removed with this procedure (ATP-derived signals at time 0 in Fig. 2A). Autodephosphorylation was initiated by adding 1 mm nonradioactive ATP to 32P-labeled KaiC monomers and incubating the resulting 32P-labeled KaiC hexamers at 30 °C. At the indicated time points, an aliquot of the reaction mixture was withdrawn and subjected to analysis. Surprisingly, we observed a transient elevation in the ATP concentration prior to an increase in the Pi levels (Fig. 2A). The sum of the concentrations of these molecules was assumed to be constant throughout the reaction (Fig. 2A). These results suggest that KaiC autodephosphorylation occurs through a previously unknown two-step mechanism. First, KaiC forms ATP as an intermediate during autodephosphorylation using ADP as a phosphate acceptor. This reaction can be regarded as the reversal of the autophosphorylation reaction. Subsequently, [32P]ATP is hydrolyzed by KaiC ATPase to form 32Pi. We normalized the total concentration of these molecules to 1 and plotted the relative concentration of each molecule over time (Fig. 2B) The small standard deviations observed in four independent experiments indicate that the autodephosphorylation process was highly reproducible. Although the reversal of kinase reactions requires ADP as a phosphate acceptor, no reports have previously shown that KaiC binds ADP. To address this issue, we examined the time-dependent changes in the levels of adenine nucleotides bound to KaiC. We reconstituted KaiC hexamers from monomers in the presence of 1 mm [α-32P]ATP and incubated the samples at 30 °C. The number of adenine nucleotides bound to each KaiC protomer remained nearly constant at two (supplemental Fig. S4), indicating that the nucleotide-binding sites of both CI and CII were occupied. The ratio of ADP to the total bound nucleotides increased, reaching ∼0.7 after 4 h of incubation (Fig. 2C, closed circles). These results suggest that KaiC hexamers contain both ADP and ATP. We also examined the nucleotides bound to CII-truncated KaiC (Fig. 2C, open circles). In contrast to full-length KaiC, >95% of the nucleotides bound to CII-truncated KaiC were ATP. It is possible that the CII domain is responsible for ADP binding to KaiC. To test our hypothesis of the reaction mechanism, we modeled the autodephosphorylation process (see (Eq. 1), (Eq. 2), (Eq. 3) below) and fit our data to the rate equations derived from (Eq. 1), (Eq. 2), (Eq. 3). Autodephosphorylation is an intersubunit reaction that occurs at CII subunit interfaces. To simplify the model, we assumed that the hypothetical reaction unit consisted of two subunits facing each other; one contained a nucleotide-binding site, and the other contained a phosphorylation site. We defined KaiCa as the subunit containing the nucleotide-binding site, and KaiCb as the subunit containing the 32P-labeled phosphorylation site (Fig. 3). KaiC-bound ADP should be a product of the hydrolysis of bound ATP and should not be incorporated from the reaction mixture because the reaction mixture contained little ADP. We first modeled the hydrolysis of nonradioactive ATP bound to KaiCa, which provides the phosphate acceptor ADP. k1 is the rate constant of ATP hydrolysis, and k−1 is the overall rate constant of the opposing reaction, including the reversal of ATP hydrolysis, the release of products, and the incorporation of nonradioactive ATP (Equation 1).KaiCa⋅ATP⇄k1k−1KaiCa⋅ADP⋅Pi(Eq. 1) We modeled the transfer of phosphate from 32P-labeled KaiCb to ADP on KaiCa. We expected that the amount of both 32P-labeled KaiCb and KaiCa-bound ADP would affect the rate of this process (Equation 2).32P-KaiCb+KaiCa⋅ADP⇄k2k−2KaiCb+KaiCa⋅[32P]ATP(Eq. 2) [32P]ATP is hydrolyzed to form 32Pi (Equation 3). The forward and reverse rate constants of ATP hydrolysis are k3 and k−3, respectively. The reincorporation of [32P]ATP into the KaiC hexamer was not considered because residual [32P]ATP was removed prior to autodephosphorylation (Equation 3).KaiCa⋅[32P]ATP⇄k3k−3KaiCa⋅ADP⋅32Pi(Eq. 3) We performed analyses using Global Kinetic Explorer (21.Johnson K.A. Simpson Z.B. Blom T. Global Kinetic Explorer: a new computer program for dynamic simulation and fitting of kinetic data.Anal. Biochem. 2009; 387: 20-29Crossref PubMed Scopus (421) Google Scholar), a program that allows for the numeric integration of rate equations and the fitting of experimental data based on nonlinear regression analysis. We set the initial values of 32P-labeled KaiCb and the sum of nonradioactive KaiCa-bound nucleotides to 1 and assumed that the initial values of [32P]ATP and 32Pi were 0 (Fig. 2B). We estimated that the ratio of KaiCa-bound ADP at time 0 was 0.10 by subtracting the amount of ADP bound to CII-truncated KaiC (Fig. 2C, open circles) from the amount bound to full-length KaiC (Fig. 2C, closed circles). We allowed k1 and k3 to float together, whereas k−1 and k−3 varied independently. The solid lines in Fig. 2B represent the best fit to the data of (Eq. 1), (Eq. 2), (Eq. 3), the χ2 value of which is 76.4 with 91 degrees of freedom. The rate constants and associated errors for the parameters calculated using FitSpace Explorer (22.Johnson K.A. Simpson Z.B. Blom T. FitSpace Explorer: an algorithm to evaluate multidimensional parameter space in fitting kinetic data.Anal. Biochem. 2009; 387: 30-41Crossref PubMed Scopus (240) Google Scholar) are shown in Table 1. The forward rate constants are well constrained by the experimental data. Reverse rate constants of k−1 and k−2 cannot be accurately resolved on the time scale of the experiment, and only the upper limits are defined.TABLE 1Kinetic parameters for KaiC autodephosphorylationRate constantValueLower boundaRange of parameters is for fits within 20% increase in χ2 values.Upper boundaRange of parameters is for fits within 20% increase in χ2 values.h−1h−1h−1k+1 and k+3bParameters were constrained to the same value during the global fitting process.1.191.161.22k−1cOnly upper limits of these parameters were defined.0.258k+20.7930.7550.866k−2cOnly upper limits of these parameters were defined.0.217k−30.007380.005900.00935a Range of parameters is for fits within 20% increase in χ2 values.b Parameters were constrained to the same value during the global fitting process.c Only upper limits of these parameters were defined. Open table in a new tab We have shown that KaiC undergoes autodephosphorylation via a previously unknown mechanism that differs from the reactions catalyzed by standard Ser/Thr and Tyr phosphatases (16.Shi Y. Serine/threonine phosphatases: mechanism through structure.Cell. 2009; 139: 468-484Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar, 17.Tabernero L. Aricescu A.R. Jones E.Y. Szedlacsek S.E. Protein-tyrosine phosphatases: structure-function relationships.FEBS J. 2008; 275: 867-882Crossref PubMed Scopus (121) Google Scholar). This mechanism includes generation of an ATP intermediate, followed by hydrolysis of this molecule (Fig. 3). The former step is regarded as the reversal of autophosphorylation. Previously, we (18.Nishiwaki T. Satomi Y. Kitayama Y. Terauchi K. Kiyohara R. Takao T. Kondo T. A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria.EMBO J. 2007; 26: 4029-4037Crossref PubMed Scopus (177) Google Scholar) and another group (24.Rust M.J. Markson J.S. Lane W.S. Fisher D.S. O'Shea E.K. Ordered phosphorylation governs oscillation of a three-protein circadian clock.Science. 2007; 318: 809-812Crossref PubMed Scopus (276) Google Scholar) proposed that the phosphorylation cycle of KaiC was understood as the sequential transition among the four phosphorylation states derived from Ser-431 and Thr-432. This model was insufficient to explain the phosphorylation cycle because two independent autokinase and autophosphatase activities were assumed to exist in KaiC. In this study, we have demonstrated that both the phosphorylation and dephosphorylation of KaiC are mediated by a single phosphotransfer reaction catalyzed by the active site of ATP hydrolysis in CII. On the basis of our findings, we propose a revision of the previous model: the KaiC phosphorylation rhythm is sustained by a periodical shift in the equilibrium of the intersubunit phosphate transfer reaction between KaiC-bound nucleotides and phosphorylation sites (Equation 2 and Fig. 3). It is possible that the direction in which the intersubunit phosphate transfer reaction proceeds is determined by KaiA, which functions as an autokinase activator. The reaction favors ATP formation in the absence of KaiA at 30 °C. In the presence of KaiA, the reaction proceeds to phosphorylated KaiC formation (11.Iwasaki H. Nishiwaki T. Kitayama Y. Nakajima M. Kondo T. KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 15788-15793Crossref PubMed Scopus (232) Google Scholar). The local structure of the active site may be affected by the presence or absence of KaiA, which determines the direction of phosphate transfer. In agreement with this idea, Kim et al. (25.Kim Y.I. Dong G. Carruthers Jr., C.W. Golden S.S. LiWang A. The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12825-12830Crossref PubMed Scopus (93) Google Scholar) suggested that KaiA regulated the orientation of ATP bound at the active site through a structural change in a looped segment near the C terminus. KaiB inactivates KaiA by forming a ternary complex with KaiA and Ser-431-phosphorylated KaiC (18.Nishiwaki T. Satomi Y. Kitayama Y. Terauchi K. Kiyohara R. Takao T. Kondo T. A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria.EMBO J. 2007; 26: 4029-4037Crossref PubMed Scopus (177) Google Scholar), which should result in a circadian change in the direction of phosphate transfer. We previously proposed that the phosphorylation cycle was coupled with the ATPase activity of KaiC (9.Terauchi K. Kitayama Y. Nishiwaki T. Miwa K. Murayama Y. Oyama T. Kondo T. ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16377-16381Crossref PubMed Scopus (175) Google Scholar). The precise relationship was not elucidated because the autodephosphorylation mechanism was not understood correctly. How, then, is the KaiC phosphorylation cycle regulated by the ATPase? The ATPase activity of CII has two critical roles: one is the degradation of the ATP intermediate of autodephosphorylation and the other is providing ADP, a substrate for the reversal of the autokinase reaction. We believe that ADP bound to the active site was not incorporated from the reaction mixture but was a product of the hydrolysis of bound ATP because the reaction mixture contained little ADP, if any. In agreement with this idea, Rust et al. (26.Rust M.J. Golden S.S. O'Shea E.K. Light-driven changes in energy metabolism directly entrain the cyanobacterial circadian oscillator.Science. 2011; 331: 220-223Crossref PubMed Scopus (146) Google Scholar) reported that increasing the ADP/ATP ratio in the reaction mixture did not affect autodephosphorylation. It is possible that ADP is not released immediately after ATP hydrolysis, which suggests that ADP release is the rate-limiting step in the CII ATPase reaction. The ATPase activity of CII should regulate the phosphorylation cycle as a partial reaction of autodephosphorylation, whereas the ATPase activity of CI may function as a pacemaker of circadian oscillation (9.Terauchi K. Kitayama Y. Nishiwaki T. Miwa K. Murayama Y. Oyama T. Kondo T. ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16377-16381Crossref PubMed Scopus (175) Google Scholar). We anticipate that other as-yet unknown proteins that catalyze both forward and reverse kinase reactions exist to regulate reversible or cyclic physiological processes. One candidate is E. coli AceK, which exhibits kinase, phosphatase, and ATPase activities (27.Laporte D.C. Stueland C.S. Ikeda T.P. Isocitrate dehydrogenase kinase/phosphatase.Biochimie. 1989; 71: 1051-1057Crossref PubMed Scopus (30) Google Scholar). Although direct evidence has not yet been reported, the phosphatase activity of AceK has been postulated to result from a reversal of the kinase reaction (27.Laporte D.C. Stueland C.S. Ikeda T.P. Isocitrate dehydrogenase kinase/phosphatase.Biochimie. 1989; 71: 1051-1057Crossref PubMed Scopus (30) Google Scholar). AceK phosphorylates and dephosphorylates isocitrate dehydrogenase, reversibly controlling the flux of metabolites at branching points in metabolic pathways (27.Laporte D.C. Stueland C.S. Ikeda T.P. Isocitrate dehydrogenase kinase/phosphatase.Biochimie. 1989; 71: 1051-1057Crossref PubMed Scopus (30) Google Scholar). We thank Dr. Tetsuya Mori (Vanderbilt University) and Drs. Shuji Akiyama, Yohko Kitayama, Yasuhiro Onoue, Atsushi Mukaiyama, and Naoki Takai (Nagoya University) for helpful discussion and advice and Akira Morita (Nagoya University) for reconstitution of KaiC hexamers. Download .zip (2.9 MB) Help with zip files" @default.
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