Growth-dependent Regulation of Mammalian Pyrimidine Biosynthesis by the Protein Kinase A and MAPK Signaling Cascades

Abstract

The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, ratelimiting 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 mitogenactivated 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 downregulation 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 (1-13) 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 (15-17) 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.The CPSase activity of CAD is allosterically regulated by the inhibitor UTP and the activator PRPP (13,(21)(22)(23)(24)(25)(26). A domain swapping experiment (27) clearly showed that the allosteric ligands bind to a regulatory subdomain (B3) at the extreme carboxyl end of the CPS.B domain of CAD (Fig. 1B).Carrey and co-workers (26, 28, 29) discovered that pur...

Cell Cycle-dependent Regulation of Pyrimidine Biosynthesis

“…The MAP kinase (p44/p42 or Erk1/Erk2) activities were assayed using the New England Biolabs kit following the manufacturer's protocol. The phosphorylation of the MAP kinase substrate, glutathione S-transferase-Elk1 (2 g), was followed by Western blotting using specific anti-phospho-Elk1 antibodies (18). Alternatively, Erk1/Erk2 were assayed using phosphoErk1/2 antibodies that recognize only the activated kinases (Promega).…”

“…The amount of the radiolabeled nucleotides were calculated from the specific radioactivity of the 14 C-labeled sodium bicarbonate. Immunoblotting-For immunoblotting the following antibodies were used at the previously described dilution (18) (Pharmingen). The rabbit polyclonal CAD serum was prepared as previously described (38).…”

“…Briefly, 10 T-75 flasks of exponentially growing cells (60 -80% confluent) were serum-starved in DMEM, 0.1% fetal bovine serum for 24 h. The cells were then stimulated by the addition of 25 ml of DMEM supplemented with 10% fetal bovine serum and incubated at 37°C. The cultures were harvested at 2-h intervals over a period of 20 -24 h for pyrimidine biosynthesis assays and the preparation of cell extracts as previously described (18).…”

“…The rabbit polyclonal CAD serum was prepared as previously described (38). Immunoblotting and quantitation of the immunoblots were carried out as previously described (18).…”

“…We have recently shown (18) that the activation of pyrimidine biosynthesis in exponentially growing BHK 165-23 cells is a consequence of the MAP kinase-mediated phosphorylation of CAD and that its subsequent down-regulation as the culture became confluent is associated with dephosphorylation of the MAP kinase site and phosphorylation by PKA. To more precisely define the mechanism of growth-related activation and down-regulation of pyrimidine biosynthesis, the activity of the pathway and the role of the diverse control mechanisms was examined as BHK 165-23 cells traversed the cell cycle.…”