Growth-dependent regulation of mammalian pyrimidine biosynthesis by the protein kinase A and MAPK signaling cascades.

The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo pyrimidine biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of pyrimidine biosynthesis, the activity of the de novo pyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in pyrimidine biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of pyrimidine biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.

The rate of de novo pyrimidine biosynthesis parallels the growth rate of the cell, and there is good evidence (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) that the activation of the pathway is necessary for proliferation of tumor and neoplastic cells. In mammalian cells, the pathway consists of six steps (Fig. 1A) that result in the formation of UMP. The flux of metabolites through the pathway (14) is controlled by carbamoyl phosphate synthetase (CPSase), 1 the enzyme that catalyzes the first committed and rate-limiting step of the pathway. Mammalian CPSase is part of a large multifunctional protein called CAD (15)(16)(17) that also carries aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase) activities, enzymes that catalyze the second and third steps of the pathway, respectively. The 243-kDa CAD polypeptide (Fig. 1B) is organized (18 -20) into multiple domains, subdomains, and linkers, each with a specific function.
Carrey and co-workers (26,28,29) 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 and a second in the interdomain linker that connects the ATC and DHO domains (Fig. 1B). Desensitization to UTP correlates with the phosphorylation of Ser 1406 in the regulatory subdomain. Banerjei and Davidson (30) showed that in transfected cells, replacement of Ser 1406 with glutamate mimics the effects of in vitro phosphorylation of CAD. The phosphorylation of Ser 1859 in the interdomain linker has no effect on allosteric regulation but has been implicated in carbamoyl phosphate channeling (31). PKA-mediated phosphorylation was considered an activation mechanism that could explain growth-dependent changes in pyrimidine biosynthesis in vivo because the constraints imposed on the pathway by UTP inhibition are effectively abolished.
More recently, it was discovered (32) that the response to PRPP is also diminished by PKA phosphorylation. The maximum allosteric activation is unchanged, but the affinity of PRPP for CPSase is substantially weakened. Thus, depending on the intracellular concentration of allosteric ligands, PKAmediated phosphorylation might be expected to either up-or down-regulate the activity of the pathway. Although cAMP is required for the growth of yeast, in most mammalian cells (about 75% of the cases examined), it is considered (33) antagonistic to cell growth and proliferation. Therefore, cAMP may serve in many instances as a counter-regulator to signals generated by insulin and growth factors. Thus, there are likely to be other mechanisms, in addition to PKA phosphorylation, that override the allosteric constraints on CAD activity when cells enter the proliferative phase.
Recently, CAD was found (34) to be regulated both in vivo and in vitro by the MAPK cascade. MAPKs (35), such as extracellular signal-regulated kinases (Erks) 1 and 2, are ubiquitous components of the mitogen-activated cascade that result in cellular proliferation in response to growth factors. Erk1 and Erk2 are also activated by oncogene products (36). MAPK phosphorylates a Thr 456 in the A1 subdomain of the CAD CPS (Fig.  1B). Although far from the regulatory domain in the linear sequence, molecular modeling 2 based on the known structure of Escherichia coli CPSase (37) suggests that it lies in close proximity to the B3 regulatory subdomain. 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 are thus likely to be important for regulation of pyrimidine biosynthesis.
These observations suggest that mammalian CPSase is likely to be controlled in vivo by the interplay of allosteric effectors and two different signaling cascades. Here, we show that activation of pyrimidine biosynthesis in rapidly growing cells is associated with phosphorylation by MAPK and that PKA phosphorylation is involved in its down-regulation as cell growth is arrested by contact inhibition.

EXPERIMENTAL PROCEDURES
Cell Culture-BHK 165-23 (38) is 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 (PALA). The cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% dialyzed fetal bovine serum and 2 g/ml gentamicin (Invitrogen). Two million cells were plated in 75-cm 2 flasks containing 25 ml of media. The media were changed every 2 days. The cells were judged to be confluent when nearly all of the attached cells were in contact with one another, and cell growth had ceased. Cells were counted using a hemocytometer, and viability was assessed by trypan blue staining. Although CAD is overexpressed in these cells, the growth-dependent variation in the rate of pyrimidine biosynthesis is identical to that observed in other cell types (39 -41), and the elevated levels of protein greatly facilitate quantitation of the phosphorylation state.
Pyrimidine Biosynthesis Assay-The rate of de novo pyrimidine biosynthesis was measured as described by Huisman et al. (1) and Fairbanks et al. (42). A suspension of cells (0.16 -1.6 ϫ 10 6 ) in 90 l of Dulbecco's modified Eagle's medium/F-12 without bicarbonate was placed in a 1-ml microfuge tube. A parallel assay mixture was prepared that was identical, except that it contained 1 mM PALA (a gift of Drs. V. Narayanan and L. Kedda of the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, NIH, Silver Spring, MD), a potent bisubstrate inhibitor of ATCase that completely blocks (43) the flux through the de novo pyrimidine biosynthetic pathway. The reaction was initiated by the addition of 10 l of 9.1 mM sodium [ 14 C]bicarbonate (220 Ci/mol; ICN Inc.). The cell suspension was incubated at 37°C for 5, 10, 20, 30, 45, and 60 min and then pelleted by centrifugation at 1000 ϫ g for 1 min in a microfuge. The cells were washed twice in ice-cold phosphate-buffered saline and then lysed by the addition of 200 l of 10% trichloroacetic acid. The trichloroacetic acid extract was pelleted by centrifugation at 12,000 ϫ g for 1 min. The supernatant was counted, and in some instances, the trichloroacetic acid was removed by back extraction with water-saturated diethyl ether, pH 5.0, for high pressure liquid chromatography analysis. The incorporation of [ 14 C]bicarbonate into RNA and DNA was measured by counting the trichloroacetic acid pellets after dissolving them in 200 l of 0.1 M NaOH. The progress curves were linear over a 60-min period. The rate of de novo pyrimidine biosynthesis was taken as the difference between the rate of bicarbonate incorporation into acid-soluble metabolites in the presence and absence of PALA.
Preparation of Cell Extracts-The cells were washed twice with icecold phosphate-buffered saline containing 0.1 mM phenylmethylsulfonyl fluoride and harvested in 4 ml of ice-cold phosphate-buffered salinephenylmethylsulfonyl fluoride by scraping. Cells were collected, and the flasks were rinsed with 4 ml of ice-cold phosphate-buffered salinephenylmethylsulfonyl fluoride. The cell suspension was centrifuged at 10,000 ϫ g at 4°C for 5 min, and a lysis buffer consisting of 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol, and 1% Triton supplemented with mammalian protease inhibitor mixture and mammalian phosphatase inhibitor mixture (Sigma) was added (200 l/3 ϫ 10 6 cells). The cell suspension was vortexed to dissociate cell clumps, sonicated for 10 s six times in an ice bath, and centrifuged at 24,000 ϫ g at 4°C for 30 min. The supernatants were collected, and aliquots were stored at Ϫ80°C. In some instances, CAD was isolated by affinity chromatography on a PALA column (44), and its identity was verified by immunoblotting using CAD-specific antibodies. Protein quantitation was performed by using the method of Lowry et al. (45).
Immunoblotting-SDS-PAGE on 5% or 10% gels was carried out as described by Laemmli (47). The samples were heated at 100°C for 4 min in loading buffer before electrophoresis. The proteins were sepa-rated and transferred onto a 0.45 m nitrocellulose membrane (Bio-Rad) as described by the manufacturer. The analysis was performed using the ECL reagents according to the manufacturer's protocol (Amersham Biosciences). Signals were visualized using Biomax ML film (Kodak) and quantitated by scanning the immunoblots with a Hewlett Packard ScanJet 4c and UNSCAN-IT software (Silk Scientific Corp.). Care was taken to ensure that all exposures fell within the linear response range of the film.
CAD Enzyme Assays-The glutamine-dependent CPSase assay was carried out as described previously (48). The assay mixture (1 ml) contained 60 g of protein, 100 mM Tris-Cl, 100 mM KCl, 7.5% Me 2 SO, 2.5% glycerol, adjusted at pH 8.0, 1 mM dithiothreitol, 3.5 mM glutamine, 20.2 mM aspartate, 1.5 mM ATP, 3.5 mM MgCl 2 , and 5 mM sodium [ 14 C]bicarbonate (1.6 ϫ 10 6 Ci/mol). When UTP and/or PRPP were included, the concentration of MgCl 2 was adjusted to maintain a 2 mM excess over the sum of the concentration of ATP, UTP, and PRPP. The reaction was performed at 37°C for 15 min, 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 CO 2 generated during the reaction, and then the vials were heated at 100°C for an additional 15 min before counting in a Beckman-Coulter counter.
ATCase and DHOase Assays-The ATCase and DHOase activities were determined using the previously described colorimetric method (49,50). The ATCase assay mixture contained 5 mM carbamoyl phosphate and 12 mM aspartate in a buffer consisting of 100 mM Tris-Cl, 100 mM KCl, 7.5% Me 2 SO, 2.5% glycerol, adjusted at pH 8.0, 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.
cAMP-dependent PKA Assays-The PKA activity in the cell extract (20 -150 g of protein) was determined using the PKA assay kit (Invitrogen) following the manufacturer's recommendations. The assay is based on the phosphorylation of the specific PKA substrate kemptide by FIG. 2. Growth curve and pyrimidine biosynthesis. BHK165-23 cells were seeded at 2 ϫ 10 6 cells/T75 flask and grown as described under "Experimental Procedures." Flasks were harvested each day, and the cell number (E) was determined by counting. The rate of de novo pyrimidine biosynthesis was assayed by measuring the incorporation of [ 14 C]bicarbonate into the acid-soluble fraction (q) containing the pathway intermediates and nucleotides.

FIG. 3. Growth-dependent phosphorylation of CAD.
A, cells were harvested on the indicated days, and the relative phosphorylation of CAD was determined by immunoblotting using antibodies directed specifically against phosphothreonine. Enzyme assays and immunoblotting with anti-CAD antibodies showed that the concentration of CAD remained constant throughout the growth curve. B, the phosphorylation of CAD on threonine (q) and serine (E) residues in cells harvested at the indicated growth stage was determined by scanning the immunoblot as described under "Experimental Procedures." [␥-32 P]ATP. A second method, the PepTag PKA assay kit (Promega), was also employed. This assay relies on a change in the electrophoretic mobility of a fluorescent kemptide derivative upon phosphorylation. The phosphorylation of the kemptide could also be measured by electrophoresis on a 20% SDS-PAGE followed by immunoblotting using anti-phosphoserine antibodies. The relative intensity of the signals was quantitated as described above. The PKA inhibitor H89 was purchased from Sigma.
MAPK Assays-The p44/p42 (Erk1/Erk2) activities were assayed using the assay kit from New England Biolabs following the manufacturer's protocol. The phosphorylation of the MAPK substrate GST-Elk1 (2 g) was followed by Western blotting using specific anti-phospho-Elk1 antibodies. Alternatively, Erk1/Erk2 were assayed using phospho-Erk1/Erk2 antibodies that recognize only the activated kinases (Promega). MAPK could be inactivated in vivo by inhibiting MEK with compound PD98059 purchased from Sigma.

RESULTS
Growth Phase Dependence of Pyrimidine Biosynthesis-Growth curves were obtained by seeding BHK 165-23 cells at a concentration of 2 ϫ 10 6 cells/25 ml in 75-cm 2 T flasks and monitoring cell number (Fig. 2) over a period of 5-6 days. After an initial lag period, the cells entered an exponential growth phase during which the cell number increased to 17 ϫ 10 6 over a period of 4 days. As the cells became confluent, there was strong contact inhibition that arrested growth. Thereafter, the cell number began to gradually decline.
The rate of pyrimidine biosynthesis was assayed by measuring the time-dependent incorporation of [ 14 C]bicarbonate into nucleotides and pathway intermediates in the presence and absence of 1 mM N-phosphonacetyl-L-aspartate, a highly specific bisubstrate ATCase inhibitor that blocks entry into the pyrimidine biosynthetic pathway. The specific flux through the pyrimidine pathway corresponds to the difference in the rate of total incorporation and the incorporation in the presence of PALA. Typically, pyrimidine biosynthesis corresponds to 60% of the total incorporation. Incorporation proceeded linearly for at least 1 h.
In resting cells, the rate of pyrimidine biosynthesis was found to be 0.5 pmol/min/10 6 cells. During exponential growth, the rate increased to 4 pmol/min/10 6 cells and then dropped precipitously to the basal level when the cells entered the confluent growth phase. Immunoblots using CAD-specific antibodies as well as CAD isolation by affinity chromatography on PALA-Sepharose columns (data not shown) demonstrated that the intracellular concentration of CAD remained constant throughout the growth curve. These results demonstrate that the activity of the de novo pyrimidine biosynthetic pathway closely mirrors the growth rate of the cells.
Phosphorylation of CAD-As described above, the allosteric response of CAD CPSase activity is regulated by phosphorylation of Ser 1406 by PKA and phosphorylation of Thr 456 by MAPK. Consequently, growth state-dependent CAD phosphorylation was monitored using antibodies directed specifically against phosphoserine and phosphothreonine that could discriminate between these two phosphorylation states. These antibodies have been used to quantitate the phosphorylation of purified CAD by the isolated MAPK and PKA (data not shown). The intensity of the signals on the immunoblots correlated well with the extent of incorporation of [␥-32 P]ATP into purified CAD catalyzed by the two kinases.
Immunoblots of CAD from cells harvested in the early stages of cell growth showed that the level of phosphothreonine was low (Fig. 3A) but increased appreciably during exponential growth and then declined abruptly to the basal level when the cells became confluent. Changes in the extent of serine and threonine phosphorylation were quantitated by scanning the immunoblots (Fig. 3B). Whereas CAD threonine phosphorylation increased 2.5-fold during the exponential phase, the level of serine phosphorylation remained low and began to increase only toward the end of the exponential growth phase. Serine phosphorylation reached a maximum increase of 2.5-fold in fully confluent cells and then began to decline. These results are consistent with the interpretation that phosphorylation of CAD by MAPK is highest during exponential growth and declines when growth is arrested, whereas the level of CAD phosphorylation by PKA is low during the exponential growth phase and subsequently peaks when the cells become confluent.
Growth-dependent Changes in Kinase Activity-The activity of MAPK, measured by the specific phosphorylation of the substrate Elk1, was very low in newly seeded cells and in the early exponential growth phase (Fig. 4) but increased rapidly as the cells began to grow. MAPK activity peaked in the midexponential cells and then began to decline as the cells approached confluence. The relative MAPK activity in the midexponential growth phase cells was ϳ40-fold higher than the values observed in resting or confluent cells. In contrast, the activity of PKA (Fig. 4), assayed by phosphate incorporation into the specific PKA kemptide substrate, increased only slightly during the exponential phase of growth. The alternative assay methods described under "Experimental Procedures" gave comparable results. Thus, whereas MAPK was activated in rapidly growing cells, the activity of PKA did not change appreciably as a function of growth state.
Growth-dependent Changes in Allosteric Regulation-The growth-dependent changes in the rate of pyrimidine biosynthe- FIG. 4. Growth-dependent changes in kinase activity. A, the activity of MAPK in cells harvested in different stages of growth was determined by reaction of MAPK with Elk1, and the extent of phosphorylation of the substrate was monitored by Western blotting using phospho-Elk1-specific antibodies. B, the MAPK activity (q), shown in A, and the PKA activity (E), determined using the Promega electrophoretic assay kit described under "Experimental Procedures," were quantitated by gel scanning. sis must be a consequence of changes in the activity of CPSase in the cell. CAD was isolated and assayed in vitro in the presence and absence of allosteric effectors to determine how the function of the molecule is altered in different phases of growth. As expected, in the absence of allosteric effectors, the CPSase, ATCase, activities of CAD did not change significantly in the different growth stages (Table I) because the intracellular concentration of the protein remained constant. However, there were progressive changes in the allosteric regulation of the CPSase component as growth proceeded. The extent of activation by 50 M PRPP increased during exponential growth and then returned to the basal level when cells became confluent. Similarly, 1 mM UTP inhibited CPSase activity in early exponential and, to some extent, confluent cells but was a modest activator in rapidly growing cells.
A more detailed analysis of CPSase sensitivity to allosteric effectors was obtained by measuring the response of isolated CAD to increasing concentrations of PRPP (Fig. 5). The maximum PRPP activation of CAD from newly seeded cells was 10-fold. However, the extent of activation increased as growth continued, reaching a maximum of 22-fold in mid-exponential cells. As the cells became confluent, there was a gradual decline in PRPP activation of CAD. By day 6, when the cells were fully confluent, the maximum activation of CPSase was only 6-fold.
In contrast, the response to UTP was rather small (Table I), so that the predominant growth state-dependent changes in CAD regulation are associated with changes in sensitivity to the allosteric activator, PRPP. The relative insensitivity of CPSase to pyrimidine nucleotides may have important consequences in the cell because increases in the intracellular pool of UTP, such as the 2-fold increase observed in EGF-stimulated cells (34), would not appreciably reduce the rate of pyrimidine biosynthesis. UTP inhibition and PRPP activation of purified CAD are antagonistic (32,51) in the sense that UTP reduces PRPP activation and vice versa. Similar results were obtained for CAD isolated from mid-exponential cells (Fig. 6). However, it is significant that PRPP strongly activated CPSase even in the presence of UTP at concentrations of up to 1 mM, suggesting that the nucleotide cannot effectively counter the increased PRPP activation that develops in rapidly growing cultures. DISCUSSION Previous studies (34) showed that CAD isolated from EGFstimulated cells exhibited changes in allosteric regulation that would be expected to promote de novo pyrimidine biosynthesis. The formation of carbamoyl phosphate by the CPSase domain of CAD is the first committed, rate-limiting step in the pathway. PRPP activation of CPSase was stimulated, and UTP was converted from an inhibitor to a weak allosteric activator in response to EGF. The modulation of these allosteric transitions resulted from the phosphorylation of Thr 456 by the MAPK cascade. However, the actual rate of flux through the de novo pyrimidine biosynthetic pathway was not measured. Moreover, the changes in CAD phosphorylation state, allosteric regulation, and pyrimidine biosynthesis associated with the arrest of cell growth by contact inhibition are unknown. In this report, we show that the flux through the de novo pyrimidine pathway increases 8-fold when resting cells enter the exponential growth phase and then decreases to basal levels when the cells become confluent and growth ceases.
PKA phosphorylates two serine residues of CAD (Ser 1406 and FIG. 5. Growth state-dependent changes in the allosteric regulation of CAD. The CPSase activity in the absence of allosteric effectors remained unchanged throughout the growth curve, but there were appreciable changes in the response to PRPP. The PRPP response curve of CAD was measured in cells harvested immediately after passage (day 1, ‚) and in early exponential (day 2, OE), exponential (day 3, f; day 4, E), and confluent cells (day 5, q; day 6, Ⅺ).  a CPSase activity was assayed at 3.5 mM glutamine, 5.0 mM bicarbonate, and substaturing ATP (1.5 mM) to allow measurement of the allosteric response. The ATCase activities were assayed with saturating concentrations of substrates (5 mM carbamoyl phosphate and 12 mM aspartate).
Ser 1859 ), whereas MAPK phosphorylates a specific threonine residue (Thr 456 ). Consequently, the phosphorylation state could be measured using specific antibodies directed against phosphothreonine and phosphoserine. We cannot rule out with certainty the participation of additional kinases because the size and complexity of the CAD molecule complicate the direct determination of all phosphorylation sites. 3 However, we are confident for several reasons that the observed changes in phosphorylation are due, at least in large part, to MAPK and PKA: (a) the maximum CAD threonine phosphorylation was observed in rapidly growing, mid-exponential cells, corresponding to the peak MAPK activity in the cell, (b) the maximum CAD threonine phosphorylation by MAPK occurs when the cells begin to grow exponentially after EGF stimulation (34), (c) MEK inhibitor PD98059 and PKA inhibitor H89 partially reversed threonine and serine phosphorylation, respectively (data not shown), (d) the allosteric response to PRPP and UTP of CAD with high threonine phosphorylation was identical to the results obtained after MAPK phosphorylation of CAD both in vivo and in vitro (34), and (e) when CAD phosphoserine was elevated, the response to PRPP was reduced, a response similar to that observed when CAD is phosphorylated by PKA in vitro. Thus, the growth-dependent changes in the CAD phosphorylation state are likely to be mediated by MAPK and PKA.
CAD phosphothreonine increased appreciably during exponential growth and then decreased when the cells became confluent. These changes correlate with the growth-dependent up-and down-regulation of de novo pyrimidine biosynthesis and with the MAPK activity in the cell. It is interesting that the increase in serine phosphorylation of CAD in confluent cultures was not associated with increased PKA activity but rather with a decrease in the MAPK activity. We have found with purified CAD 4 that prior phosphorylation with MAPK impairs phosphorylation of the protein by PKA. Thus, a plausible explanation of this result is that a decrease in MAPK activity in confluent cells leads to dephosphorylation of Thr 456 that now permits phosphorylation of Ser 1406 by PKA. We cannot distinguish between PKA-mediated phosphorylation of Ser 1406 and Ser 1859 ; however, because phosphorylation of the latter site has no effect on the allosteric properties of CAD, for the purposes of our working hypothesis we assume that the relevant modification occurs on Ser 1406 .
The observed changes in the allosteric response to UTP and PRPP are similar to the results obtained with EGF-stimulated cells (34) and with purified CAD upon phosphorylation with MAPK and PKA (28,32). In exponentially growing cells, CAD was activated 11-fold by 50 M PRPP compared with 3.2-fold for cells in the early exponential growth phase (Table I). UTP activated the CPSase from exponentially growing cells by 119% but was an inhibitor of CAD isolated from early exponential cells (38%). When the cells became confluent, PRPP activation decreased; there was only a 2-fold activation by 50 M PRPP, which is less than the activation observed in early exponential cells, a result consistent with the diminished sensitivity of CPSase to the activator after PKA phosphorylation. UTP, although no longer an activator, did not inhibit the CPSase activity of confluent cells to any great extent (14%), which was as expected because PKA-mediated phosphorylation, like MAPK-mediated phosphorylation, diminishes inhibition by the nucleotide. Therefore, the dominant factor responsible for regulating the CPSase activity in vivo appears to be the response of the enzyme to PRPP.
Depending on the intracellular concentration of the effectors, the growth-dependent modulation of the allosteric response could fully account for the observed changes in the rate of de novo pyrimidine biosynthesis. For example, in the presence of 50 M PRPP and 1 mM UTP, the CPSase activity from cells harvested in the early exponential growth stage (day 1) was 0.62 nmol/min/mg, compared with a value of 3.78 nmol/min/mg for CAD from exponentially growing cultures (day 4). This 6-fold increase in CAD CPSase activity would be expected to result in a proportional increase in the rate of de novo pyrimidine biosynthesis, close to the experimentally observed value. Experiments are under way to determine the growth-dependent changes in the intracellular concentration of UTP and PRPP.
A model depicting the growth-dependent regulation of CAD CPSase and the de novo pyrimidine biosynthetic pathway is shown schematically in Fig. 7. In newly seeded cells, the level of phosphorylation of CAD CPSase is low, as is the flux through the pyrimidine pathway. As the culture progresses to the exponential phase, MAPK is activated, Thr 456 of CAD is phosphorylated, and CPSase is activated. This modification leads to an appreciable increase in pyrimidine biosynthesis. Ser 1406 remains unphosphorylated because CAD-Thr 456 ϳP is a relatively poor substrate for PKA. Once the culture enters the confluent stage, MAPK is deactivated, Thr 456 is dephosphorylated by a phosphatase (probably protein phosphatase I), and Ser 1406 is phosphorylated by PKA. The combination of phosphorylation of the PKA site and dephosphorylation of the MAPK site leads to a decrease in PRPP sensitivity, lower CPSase activity, and a down-regulation of pyrimidine biosynthesis. Many aspects of this model remain to be tested. Nevertheless, it is clear that the CPSase activity of CAD and the flux through the de novo pyrimidine biosynthetic pathway are governed by the sequential action of two central signaling cascades. 3 CAD more than 217 predicted tryptic peptides, and only microgram amounts of the protein can be recovered from cell extracts. However, we are attempting to analyze the complete profile of CAD phosphorylation by LC-mass spectroscopy experiments using the Harvard Microchemistry Facility. We were successful in determining the phosphorylation state of purified CAD, but the analysis required 0.3 mg of the protein.
We are now attempting to scale up the isolation of CAD from cells in various growth states. 4 7. Model for growth state-dependent regulation of pyrimidine biosynthesis. The transition from resting to exponential growth phase is accompanied by the activation of MAPK and the phosphorylation of Thr 456 of CAD. PRPP activation increases, UTP becomes a weak activator, and there is an appreciable increase in CPSase activity and flux through the de novo pyrimidine biosynthetic pathway. When the cells enter the confluent stage and growth begins to slow, MAPK activity decreases, Thr 456 is dephosphorylated, and Ser 1406 is phosphorylated by PKA. Sensitivity to PRPP decreases, CPSase activity is downregulated, and the rate of pyrimidine biosynthesis returns to the basal level.