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• W2008556260 abstract "CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of Pi/mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr1037 located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5′-p-fluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades. CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of Pi/mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr1037 located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5′-p-fluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades. carbamoyl phosphate synthetase aspartate transcarbamoylase dihydroorotase 5′-p-fluorosulfonylbenzoyladenosine mitogen-activated protein kinase cAMP-dependent protein kinase A phosphoserine phosphothreonine 5-phosphoribosyl-1-pyrophosphate 5′-adenylyl-β,γ-imidodiphosphate CAD (1Mori M. Ishida H. Tatibana M. Biochemistry. 1975; 14: 2622-2630Crossref PubMed Scopus (79) Google Scholar, 2Shoaf W.T. Jones M.E. Biochemistry. 1973; 12: 4039-4051Crossref PubMed Scopus (143) Google Scholar, 3Coleman P. Suttle D. Stark G. J. Biol. Chem. 1977; 252: 6379-6385Abstract Full Text PDF PubMed Google Scholar) is a multifunctional protein that catalyzes the first three steps of the de novo pyrimidine biosynthetic pathway in mammalian cells. The protein consists of multiple copies of a 243-kDa polypeptide organized into discrete domains (Fig. 1 A) that have glutamine-dependent carbamoyl phosphate synthetase (CPSase),1 aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase) activities. The CPSase activity, the first committed and rate-limiting step in the pathway, is the locus of control. Most CPSases share a common catalytic mechanism (4Meister A. Adv. Enzymol. Relat. Areas Mol. Biol. 1989; 62: 315-374PubMed Google Scholar) in which the synthesis of carbamoyl phosphate proceeds through a complex series of partial reactions catalyzed by different functional domains.Glutamine+H2O→glutamate+NH3HCO3−+ATP→carboxyphosphate+ADPCarboxyphosphate+NH3→carbamate+PiCarbamate+ATP→carbamoyl phosphate+ADPREACTIONS1–4CPSases also have homologous domain structures. Glutamine hydrolysis (Reaction 1) occurs on the 40-kDa glutaminase domain or subunit. The synthetase (CPS) domain or subunit consists of two homologous 60-kDa subdomains, CPS.A and CPS.B. The activation of bicarbonate and the subsequent reaction with NH3 (Reactions 2 and 3) occurs on CPS.A, whereas the phosphorylation of carbamate to form carbamoyl phosphate (Reaction 4) occurs on CPS.B (5Post L.E. Post D.J. Raushel F.M. J. Biol. Chem. 1990; 265: 7742-7747Abstract Full Text PDF PubMed Google Scholar). Lusty recognized that the CPS domain (6Nyunoya H. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4629-4633Crossref PubMed Scopus (123) Google Scholar) evolved by gene duplication, translocation, and fusion of an ancestral kinase gene. CAD CPSase also regulates the flux of metabolites through the de novo pyrimidine pathway (7Chen J.J. Jones M.E. J. Biol. Chem. 1979; 254: 2697-2704Abstract Full Text PDF PubMed Google Scholar) via a complex network of interacting control mechanisms. The CPS domain is subject to allosteric activation by 5-phosphoribosyl-1-pyrophosphate (PRPP) and feedback inhibition by UTP (8Tatibana M. Ito K. J. Biol. Chem. 1969; 244: 5903-5913Abstract Full Text PDF Google Scholar, 9Hager S.E. Jones M.E. J. Biol. Chem. 1967; 242: 5667-5673Abstract Full Text PDF PubMed Google Scholar, 10Hager S.E. Jones M.E. J. Biol. Chem. 1967; 242: 5674-5680Abstract Full Text PDF PubMed Google Scholar, 11Levine R.L. Hoogenraad N.J. Kretchmer N. Biochemistry. 1971; 10: 3694-3699Crossref PubMed Scopus (56) Google Scholar, 12Tatibana M. Shigesada K. Biochem. Biophys. Res. Commun. 1972; 46: 491-497Crossref PubMed Scopus (41) Google Scholar, 13Mally M.I. Grayson D.R. Evans D.R. J. Biol. Chem. 1980; 255: 11372-11380Abstract Full Text PDF PubMed Google Scholar, 14Shaw S.M. Carrey E.A. Eur. J. Biochem. 1992; 207: 957-965Crossref PubMed Scopus (26) Google Scholar). Domain swapping experiments (15Liu X. Guy H.I. Evans D.R. J. Biol. Chem. 1994; 269: 27747-27755Abstract Full Text PDF PubMed Google Scholar, 16Sahay N. Guy H.I. Xin L. Evans D.R. J. Biol. Chem. 1998; 273: 31195-31202Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) showed that both allosteric ligands bind to the extreme carboxyl end of CPS.B (Fig.1 A), the B3 regulatory subdomain. Carrey (14Shaw S.M. Carrey E.A. Eur. J. Biochem. 1992; 207: 957-965Crossref PubMed Scopus (26) Google Scholar, 17Carrey E.A. Campbell D.G. Hardie D.G. EMBO J. 1985; 4: 3735-3742Crossref PubMed Scopus (58) Google Scholar, 18Irvine H.S. Shaw S.M. Paton A. Carrey E.A. Eur. J. Biochem. 1997; 247: 1063-1073Crossref PubMed Scopus (30) Google Scholar) discovered that purified CAD is phosphorylated by cAMP-dependent protein kinase A (PKA). Phosphorylation does not alter the catalytic activity of CPSase or any of the other CAD activities but results in the loss of sensitivity to the allosteric inhibitor, UTP. There are two PKA phosphorylation sites, one located within the B3 regulatory subdomain (Ser1406) and a second (Ser1859) in the interdomain linker that connects the ATC and DHO domains (Fig. 1 A). Desensitization to UTP results from phosphorylation of Ser1406 in the regulatory subdomain (17Carrey E.A. Campbell D.G. Hardie D.G. EMBO J. 1985; 4: 3735-3742Crossref PubMed Scopus (58) Google Scholar, 19Guy H.I. Evans D.R. Adv. Exp. Med. Biol. 1994; 370: 729-733Crossref PubMed Scopus (3) Google Scholar, 20Banerjei L.C. Davidson J.N. Somat. Cell. Mol. Genet. 1997; 23: 37-49Crossref PubMed Scopus (10) Google Scholar). Sahay et al. (16Sahay N. Guy H.I. Xin L. Evans D.R. J. Biol. Chem. 1998; 273: 31195-31202Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) subsequently discovered that the response to PRPP is also diminished by PKA phosphorylation. CAD was also found (21Graves L.M. Guy H.I. Kozlowski P. Huang M. Lazarowski E. Pope R.M. Collins M.A. Dahlstrand E.N. Earp III, H.S. Evans D.R. Nature. 2000; 403: 328-332Crossref PubMed Scopus (173) Google Scholar) to be regulated both in vivo andin vitro by the MAPK cascade. MAPKs (22Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1662) Google Scholar) are ubiquitous components of the mitogen activated cascade that is involved in cellular proliferation in response to growth factors and have also been shown to be activated by oncogene products (23Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar). MAPK phosphorylates Thr456 in the A1 subdomain of the CAD CPS (Fig.1 A). MAPK-mediated phosphorylation, like that of PKA, abolishes UTP inhibition; however, PRPP activation is markedly stimulated. Both the loss of sensitivity to UTP and increased sensitivity to PRPP would be expected to activate CPSase and thus is likely to be important for regulation of pyrimidine biosynthesis. We have recently found (24Sigoillot F.D. Evans D.R. Guy H.I. J. Biol. Chem. 2002; 277: 15745-15751Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) that the growth-dependent activation and down-regulation of pyrimidine biosynthesis is a consequence of the sequential action of MAPK and PKA. The CPSase activity of CAD is up-regulated by the MAPK cascade when cells enter the proliferative phase and is subsequently down-regulated in resting cells by PKA-mediated phosphorylation. During her seminal studies of PKA phosphorylation of CAD, Carrey noticed (17Carrey E.A. Campbell D.G. Hardie D.G. EMBO J. 1985; 4: 3735-3742Crossref PubMed Scopus (58) Google Scholar) that radiolabeled phosphate was incorporated into CAD when it was incubated with [γ-32P]ATP in the absence of exogenous kinases, a phenomenon that was attributed to either autophosphorylation or the presence of contaminating kinases. Among the estimated 1000 protein kinases in mammalian cells (25Hunter T. Cell. 1987; 50: 823-829Abstract Full Text PDF PubMed Scopus (774) Google Scholar), intra- or intermolecular autophosphorylation has been found to be a common theme. Moreover, there are many examples, including cyclic nucleotide-dependent protein kinases, calcium/calmodulin-dependent protein kinases and protein kinase C (26Smith J.A. Francis S.H. Corbin J.D. Mol. Cell. Biochem. 1993; 127: 51-70Crossref PubMed Scopus (80) Google Scholar), where autophosphorylation is known to play an important role in the regulation of the kinase. While autophosphorylation is a common, perhaps universal characteristic of protein kinases (26Smith J.A. Francis S.H. Corbin J.D. Mol. Cell. Biochem. 1993; 127: 51-70Crossref PubMed Scopus (80) Google Scholar), it is less frequently observed in kinases that phosphorylate small molecules. Autophosphorylation of yeast hexokinase PII (27Fernandez R. Herrero P. Fernandez E. Fernandez T. Lopez-Boado Y. Moreno F. J. Gen. Microbiol. 1988; 134: 2493-2498PubMed Google Scholar, 28Heidrich K. Otto A. Behlke J. Rush J. Wenzel K. Kriegel T. Biochemistry. 1997; 36: 1960-1964Crossref PubMed Scopus (32) Google Scholar) results in the inactivation of the enzyme. Creatine kinase was initially reported to be autophosphorylated (29Hemmer W. Furter-Graves E. Frank G. Wallimann T. Furter R. Biochim. Biophys. Acta. 1995; 1251: 81-90Crossref PubMed Scopus (23) Google Scholar), but this observation was subsequently shown (30David S. Haley B. Biochemistry. 1999; 38: 8492-8500Crossref PubMed Scopus (8) Google Scholar) to be due to the incorporation of the entire nucleotide into the active site of the enzyme. Here we show that incubation of CAD with ATP resulted in incorporation of the γ-phosphate of the nucleotide into a specific threonine residue in CPS.B, that the reaction was catalyzed by CAD itself and was not the result of contaminating kinases, and that autophosphorylation resulted in increased CPSase activity and a modulation of the effects of the allosteric ligands, UTP and PRPP. CAD was isolated from BHK 165-23 (31Wahl G.M. Padgett R.A. Stark G.R. J. Biol. Chem. 1979; 254: 8679-8689Abstract Full Text PDF PubMed Google Scholar), a baby hamster kidney cell line derived from BHK-21 in which the CAD gene was amplified by exposure to the ATCase inhibitor,N-phosphonacetyl-l-aspartate. The protein was purified to homogeneity by 40% ammonium sulfate precipitation followed by gel filtration chromatography on a Bio-Gel A-5m column (Bio-Rad) as described previously (3Coleman P. Suttle D. Stark G. J. Biol. Chem. 1977; 252: 6379-6385Abstract Full Text PDF PubMed Google Scholar). In some instances, CAD was further purified by affinity chromatography on anN-phosphonacetyl-l-aspartate column (32Purcarea C. Herve G. Ladjimi M.M. Cunin R. J. Bacteriol. 1997; 179: 4143-4157Crossref PubMed Google Scholar). CAD was also isolated from Escherichia coli transformed with pCKCAD10, a plasmid expressing the full-length CAD polypeptide as described previously (33Guy H.I. Evans D.R. J. Biol. Chem. 1994; 269: 23808-23816Abstract Full Text PDF PubMed Google Scholar). Escherichia coli CPSase was purified from E. coli pLLK12 transformants following the protocol described by Guillou et al. (34Guillou F. Rubino S.D. Markovitz R.S. Kinney D.M. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8304-8308Crossref PubMed Scopus (37) Google Scholar). Protein quantitation was performed by the Lowry method (35Lowry O. Rosenbrough N. Farr A. Randall R. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) and by scanning stained SDS-polyacrylamide gels calibrated with known amounts of bovine serum albumin using the software UNSCAN-IT (Silk Scientific Corp.). The phosphoserine (Z-PS1) and phosphothreonine (Z-PT1) rabbit polyclonal antibodies from Zymed Laboratories Inc. were used for immunoblotting at a dilution of 1:1000 to 1:2000. The rabbit polyclonal CAD serum prepared as previously described (36Grayson D.R. Lee L. Evans D.R. J. Biol. Chem. 1985; 260: 15840-15849Abstract Full Text PDF PubMed Google Scholar) was used at a 1:5000 dilution. CAD at a concentration of 20–200 μg/ml in a kinase buffer consisting of 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 0.48 mg/ml bovine serum albumin was reacted at 30 °C with ATP at the concentrations indicated. The extent of phosphorylation was determined by immunoblotting or by measuring the incorporation of [γ-32P]inorganic phosphate. SDS-polyacrylamide gel electrophoresis on 5 or 10% gels was carried out as described by Laemmli (37Laemmli U. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207129) Google Scholar). The samples were heated at 100 °C for 4 min in loading buffer prior to electrophoresis. The proteins were separated and transferred onto a 0.45-μm nitrocellulose membrane (Bio-Rad) as described by the manufacturer. The analysis was performed using the ECL reagents (Amersham Biosciences) as described previously (24Sigoillot F.D. Evans D.R. Guy H.I. J. Biol. Chem. 2002; 277: 15745-15751Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Signals were visualized using Biomax ML film (Eastman Kodak Co.) and quantitated by scanning the immunoblots with a HP ScanJet 4c and the software UNSCAN-IT (Silk Scientific). Care was taken to ensure that all exposures fell within the linear response range of the film. The glutamine-dependent CPSase assay was carried out as previously described (33Guy H.I. Evans D.R. J. Biol. Chem. 1994; 269: 23808-23816Abstract Full Text PDF PubMed Google Scholar). The assay mixture (1 ml) contained 20–60 μg of protein, 100 mmTris-HCl, pH 8.0, 100 mm KCl, 7.5% Me2SO, 2.5% glycerol, 1 mm dithiothreitol, 3.5 mmglutamine, 20.2 mm aspartate, 1.5 mm ATP, 3.5 mm MgCl2, and 5 mm sodium [14C]bicarbonate (1.6 × 106 μCi/mol). When UTP or PRPP were included, the concentration of MgCl2was adjusted in order to maintain a 2 mm excess over the sum of the concentrations of ATP, UTP, and PRPP. The reaction was performed at 37 °C for 15 min and quenched by the addition of 1 ml of 40% trichloroacetic acid and heated at 100 °C for 15 min. Approximately 0.2 g of dry ice was added to the vials to eliminate the excess CO2 generated during the reaction. The sample was then heated at 100 °C for an additional 15 min prior to counting in a Beckman-Coulter counter. The ATCase and DHOase activities were determined using the previously described colorimetric method (38Prescott L.M. Jones M.E. Anal. Biochem. 1969; 32: 408-419Crossref PubMed Scopus (333) Google Scholar,39Pastra-Landis S.C. Foote J. Kantrowitz E.R. Anal. Biochem. 1981; 118: 358-363Crossref PubMed Scopus (111) Google Scholar). The ATCase assay mixture contained 5 mm carbamoyl phosphate and 12 mm aspartate in a buffer consisting of 100 mm Tris-HCl, pH 8.0, 100 mm KCl, 7.5% Me2SO, 2.5% glycerol, and 1 mm dithiothreitol. The DHOase activity was assayed by monitoring the conversion of 1 mm dihydroorotate to carbamoyl aspartate in 25 mm HEPES, pH 7.5, 5% glycerol. The on-membrane kinase assay was carried out according to Ferrell et al. (40Ferrell J.E., Jr. Martin G.S. J. Biol. Chem. 1989; 264: 20723-20729Abstract Full Text PDF PubMed Google Scholar). Briefly, purified CAD (30–65 μg) was separated on a 5% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The protein was then incubated for 1 h at room temperature with gentle rocking in a buffer containing 7 m guanidine hydrochloride, 50 mm Tris-HCl, pH 8.3, 50 mm dithiothreitol, and 2 mm EDTA. The protein was renatured on the membrane for 16–18 h at 4 °C by incubation in a buffer containing 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 2 mm dithiothreitol, 2 mm EDTA, 1% (w/v) bovine serum albumin (Sigma), and 0.1% Igepal CA-630 (Sigma). The nitrocellulose membrane was then washed with a buffer containing 5% (w/v) bovine serum albumin, as a blocking agent, in 30 mmTris-HCl, pH 7.5, at room temperature for 1 h. The membrane was then incubated with 150 μCi of [γ-32P]ATP (ICN) (4500 mCi/μmol) in 10 ml of kinase buffer consisting of 30 mmTris-HCl, pH 7.5, 10 mm MgCl2, 2 mmMnCl2, for 5 h. The reaction was quenched by washing the membrane twice in 30 mm Tris-HCl, pH 7.5. The membrane was further washed once with a buffer containing 30 mmTris-HCl, pH 7.5, 0.05% Igepal CA-630 and once in a buffer containing 30 mm Tris-HCl, pH 7.5, for 10 min each at room temperature. The membrane was then washed 10 min at room temperature in 1 m KOH and rinsed several times in 10% (v/v) acetic acid. The membrane was subjected to autoradiography using a Cyclone Storage phosphor screen, and the signals were captured with the Packard Cyclone Storage phosphor system. The membrane was also incubated after the blocking step above with 0.5 mm cold ATP in the kinase buffer and then subjected to Thr(P) immunoblotting and quantitation as described above. The phosphorylated residues were identified by liquid chromatography-mass spectrometry. CAD (330 μg) was subjected to SDS-polyacrylamide gel electrophoresis. After staining and destaining, the CAD bands were excised from the gel and pooled. The gel slices were washed twice with 50% acetonitrile in water and then frozen at −20 °C. The protein was digested in situ with trypsin, and sequence analysis was performed at the Harvard Microchemistry Facility by microcapillary reverse-phase high pressure liquid chromatography nanoelectrospray tandem mass spectrometry (μLC/MS/MS) on a Finnigan LCQ DECA quadrupole ion trap mass spectrometer. The stoichiometry of CAD autophosphorylation was determined by the method described by Carrey et al. (17Carrey E.A. Campbell D.G. Hardie D.G. EMBO J. 1985; 4: 3735-3742Crossref PubMed Scopus (58) Google Scholar). Purified CAD (21.3 μg) was incubated in kinase buffer with 200 μm [γ-32P]ATP (200 μCi/μmol) in a final volume of 100 μl for 120 min. Bovine serum albumin (20 μg) was added as a carrier, and the protein was precipitated by the addition of 50 μl of 25% trichloroacetic acid. After 30 min on ice, the sample was centrifuged at 24,000 ×g at 4 °C for 30 min. The trichloroacetic acid precipitate was washed twice with 25% trichloroacetic acid and resuspended in 1 n NaOH. Scintillation mixture (RPI) was added (10 ml), and the sample was counted in a Beckman Coulter LS 6500 scintillation counter. While investigating the phosphorylation of purified CAD by PKA and MAPK, we noticed a persistent background phosphorylation. To further characterize this phenomenon, CAD was incubated with 0.5 mm ATP, and samples were withdrawn at the indicated times (Fig.2). Analysis by immunoblotting using antibodies directed against Thr(P) gave a strong signal and showed that the phosphorylation occurred slowly over a period of 2 h (Fig.2 A). No phosphorylation could be detected with phosphoserine (Ser(P)) antibodies (Fig. 2 B, lanes 1–8), although control samples (lanes 9 and 10) using CAD phosphorylated with PKA indicated that the antibodies reacted strongly and specifically with the serine residues known to be phosphorylated by this kinase. The intensity of the signals was quantitated by scanning the immunoblots. The progress curve for Thr phosphorylation (Fig. 2 C) was distinctly sigmoidal and began to level off after about 1 h. A time course extended over a period of 7 h (not shown) indicated that no additional phosphorylation occurred after 2 h. Quantitation of the extent of phosphorylation resulting from six different phosphorylation experiments, determined by measuring the incorporation of either [γ-32P]ATP or [γ-33P]ATP into CAD during a 2-h reaction period (see “Experimental Procedures”), revealed that there was 0.97 ± 0.16 mol of phosphate incorporated/mol of CAD monomer. A series of reactions (Fig.3) were carried out to assess the requirements for phosphorylation. In contrast to the extensive threonine phosphorylation observed following incubation of CAD with 0.5 mm ATP (Fig. 3 A, lane 3), immunoblotting of unreacted CAD (lane 1) or of CAD incubated in the absence of ATP (lane 2) revealed only very low levels of threonine and serine phosphorylation. In the presence of the serine/threonine-specific protein phosphatase 1, the extent of phosphorylation was markedly reduced (lane 4). No phosphorylation occurred when CAD was heat-denatured (lane 5) prior to incubation with ATP or if the reaction was carried out in the presence of EGTA to complex Mg2+ (lane 6). Phosphorylation proceeded apace even in the presence of the other CPSase substrates, bicarbonate and glutamine (lane 7). Threonine phosphorylation also occurred when PKA was present in the reaction mixture (lanes 8 and 9). Phosphoryl transfer did not occur using UTP, GTP, or PRPP as the phosphate-donating substrate (Fig. 3 B, lanes 3–8). The lower levels of phosphorylation that occurred in the presence of both 0.5 mm ATP and either 0.5 or 4 mm ADP (Fig. 3 B, lanes 10and 11), suggested that the nucleotide diphosphate is a competitive inhibitor of the phosphorylation reaction. Interestingly, the presence of PKA (lane 9) in the reaction mixture appeared to reduce the extent of threonine phosphorylation during a 60-min reaction period (Fig. 3 B, lane 9; cf. lane 2) but had much smaller effect during the longer 2-h reaction times (Fig.3 A, lanes 8 and 9;cf. lane 3). This result suggests that the rate but not the extent of autophosphorylation may be influenced by the presence of protein kinase A. In summary, threonine but not serine residues are phosphorylated, the reaction is specific for MgATP, and the native CAD structure is required for phosphorylation to occur. It was possible that the phosphorylation of CAD was catalyzed by a contaminating kinase that copurified with the protein, although several preparations of CAD isolated from BHK165-23 cells exhibited the same extent of phosphorylation. To investigate this possibility, an “on-membrane” kinase assay, devised by Ferrell and Martin (40Ferrell J.E., Jr. Martin G.S. J. Biol. Chem. 1989; 264: 20723-20729Abstract Full Text PDF PubMed Google Scholar), was carried out. CAD was subjected to SDS gel electrophoresis and then transferred to a nitrocellulose membrane. The rationale for the method is that SDS gel electrophoresis should denature the proteins and separate CAD from any kinase that may be present and disrupt any putative CAD-kinase complexes. CAD was then renatured on the nitrocellulose membrane and incubated with [γ-32P]ATP. Autoradiography clearly showed that CAD is phosphorylated and that the extent of phosphorylation increased linearly (Fig.4) with the amount of CAD applied to the SDS gel. Alternatively, the membrane was incubated with unlabeled ATP and then probed with Thr(P) and Ser(P) antibodies (not shown). This approach confirmed that phosphate was incorporated into threonine residues. CAD isolated from E. coli cells transformed with pCKCAD10 (33Guy H.I. Evans D.R. J. Biol. Chem. 1994; 269: 23808-23816Abstract Full Text PDF PubMed Google Scholar), a plasmid expressing the full-length CAD polypeptide, was also found to be phosphorylated to the same extent as the CAD isolated from mammalian cells (data not shown). The relative paucity of Ser/Thr kinases in bacterial cells also argues against the involvement of contaminating kinases in the CAD phosphorylation. In contrast, purifiedE. coli CPSase exhibited no phosphorylation despite the conservation of the target threonine residue described below (Fig.1 B). Collectively, these experiments provide strong support for an intrinsic CAD kinase activity and suggest that phosphate incorporation is the result of an autophosphorylation reaction. To determine the site of autophosphorylation, CAD (0.3 mg) was reacted for 6 h with 0.5 mm ATP and subjected to SDS-polyacrylamide gel electrophoresis. The protein bands were excised, exhaustively digested with trypsin, and analyzed by liquid chromatography-mass spectrometry at the Harvard Microchemistry Facility. The analysis clearly revealed two major phosphopeptides (Table I). Phosphopeptide 1 was located in the CPS.B B1 subdomain close to the junction with the B2 subdomain (Fig. 1 A). The sequence of this peptide revealed tandem threonine and serine residues. The analysis favored phosphoserine, although phosphorylation of the adjacent threonine residue could not be ruled out. Phosphopeptide 2 corresponded to the serine residue located in the interdomain linker connecting the DHO and ATC domains shown previously (17Carrey E.A. Campbell D.G. Hardie D.G. EMBO J. 1985; 4: 3735-3742Crossref PubMed Scopus (58) Google Scholar) to be phosphorylated by PKA. There was an indication of a third phosphopeptide located within the glutaminase domain (phosphopeptide 3), although the evidence for this phosphopeptide was not compelling. For reasons discussed below, Thr1037 is considered to be the probable autophosphorylation site.Table IMass spectrometry analysis of autophosphorylated CADPeptideTarget residueDomainCAD residue nos.Sequence1Thr/SerCPS.B11034–1048VLGTSPEAIDSAENR2SerDA linker1855–1869IHRASDPGLPAEEPK31-aLow confidence assignment but possible phosphopeptide.SerGLN1-bGlutaminase domain.138–168LVQSGTEPSTLPFVDPNARPLAPEVSIKTPRAutophosphorylation sites are underlined.1-a Low confidence assignment but possible phosphopeptide.1-b Glutaminase domain. Open table in a new tab Autophosphorylation sites are underlined. The well characterized reaction of CAD with 5′-FSBA results in the selective modification of the ATP binding site of both the CPS.A and CPS.B domains (42Kim H.S. Lee L. Evans D.R. Biochemistry. 1991; 30: 10322-10329Crossref PubMed Scopus (21) Google Scholar). To confirm the suspected involvement of the active sites of CPSase in autophosphorylation, CAD (1.3 μm) was reacted with 2.2 mm FSBA for 2 h as described previously (42Kim H.S. Lee L. Evans D.R. Biochemistry. 1991; 30: 10322-10329Crossref PubMed Scopus (21) Google Scholar). The excess reagent was removed by dialysis, and the extent of autophosphorylation was determined by immunoblotting using the Thr(P) antibodies. No autophosphorylation could be detected (data not shown). Since FSBA modification abolished the overall synthesis of carbamoyl phosphate and both ATP-dependent partial reactions, this result is consistent with the interpretation that ATP bound to the active site of either CPS.A or CPS.B or both is responsible for autophosphorylation. CAD was incubated at 30 °C for 30 min with various ATP concentrations, and the extent of autophosphorylation was determined by scanning immunoblots probed with Thr(P) antibodies (Fig. 5). The resulting ATP saturation curve was hyperbolic, although significant substrate inhibition was apparent at ATP concentrations exceeding 2 mm. The ATP saturation curve of CPS.B exhibits similar inhibition at high ATP concentrations (41Guy H.I. Evans D.R. J. Biol. Chem. 1996; 272: 13762-13769" @default.
• W2008556260 created "2016-06-24" @default.
• W2008556260 creator A5024934066 @default.
• W2008556260 creator A5038332935 @default.
• W2008556260 creator A5049448904 @default.
• W2008556260 date "2002-07-01" @default.
• W2008556260 modified "2023-09-30" @default.
• W2008556260 title "Autophosphorylation of the Mammalian Multifunctional Protein That Initiates de Novo Pyrimidine Biosynthesis" @default.
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