Protein kinase C modulates the up-regulation of the pyrimidine biosynthetic complex, CAD, by MAP kinase.

The multifunctional protein CAD initiates de novo pyrimidine biosynthesis in mammalian cells. CAD is activated by MAP kinase (Erk1/2) just prior to the S phase of the cell cycle, when the demand for pyrimidine nucleotides is greatest, and down-regulated as the cells emerge from S phase by protein kinase A (PKA) phosphorylation. MAP kinase phosphorylates Thr456, while PKA phosphorylates Ser1406 and Ser1859, although only Ser1406 is involved in regulation. LC/mass spectrometry showed that Ser1873, a residue that lies within a putative protein kinase C (PKC) consensus sequence is also phosphorylated. Purified CAD was reacted with ATP and a panel of eight PKC isozymes. Most isozymes resulted in limited CAD phosphorylation, but the delta and epsilon isozymes were most effective. While the level of Thr456 phosphorylation is very low in confluent cells, exposure of stationary BHK 165-23 cells to the PKC activator, phorbol 12-myristate-13-acetate (PMA) resulted in a 3-fold increase in the modification of this residue. The stimulation of Thr456 phosphorylation was blocked by PKC inhibitors. The PKA inhibitor, H-89, also stimulated PMA-induced Thr456 modification probably because PKA mediated phosphorylation of CAD Ser1406 antagonizes the MAP kinase phosphorylation. Thus, the extent of Thr456 phosphorylation and the activation of pyrimidine biosynthesis depend on the synergistic and antagonistic interactions of three signaling pathways, MAP kinase, PKC and PKA. Deletions mutants lacking the putative PKC site, Ser1873 do not exhibit PMA induced Thr456 phosphorylation. We conclude that the activating MAP kinase phosphorylation of CAD proceeds through a PKC dependent pathway.

Protein kinase A phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade.

The flux through the de novo pyrimidine biosynthetic pathway is controlled by the multifunctional protein CAD, which catalyzes the first three steps. The cell cycle dependent regulation of pyrimidine biosynthesis is a consequence of sequential phosphorylation of CAD Thr456 and Ser1406 by the MAP kinase and PKA cascades, respectively. Coordinated regulation of the pathway requires precise timing of the two phosphorylation events. These studies show that phosphorylation of purified CAD by PKA antagonizes MAP kinase phosphorylation, and vice versa. Similar results were observed in vivo. Forskolin activation of PKA in BHK-21 cells resulted in a 8.5 fold increase in Ser1406 phosphorylation and severely curtailed the MAP kinase mediated phosphorylation of CAD Thr456. Moreover, the relative activity of MAP kinase and PKA was found to determine the extent of Thr456 phosphorylation. Transfectants expressing elevated levels of MAP kinase resulted in a 11-fold increase in Thr456 phosphorylation, whereas transfectants that overexpress PKA reduced Thr456 phosphorylation 5-fold. While phosphorylation of one site by one kinase may induce conformational changes that interfere with phosphorylation by the other, the observation that both MAP kinase and PKA form stable complexes with CAD suggest that the mutual antagonism is the result of steric interference by the bound kinases. The reciprocal antagonism of CAD phosphorylation by MAP kinase and PKA provides an elegant mechanism to coordinate the cell cycle-dependent regulation of pyrimidine biosynthesis ensuring that signals for up- and down-regulation of the pathway do not conflict.

Nuclear localization and mitogen-activated protein kinase phosphorylation of the multifunctional protein CAD.

CAD is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. The activation of the pathway required for cell proliferation is a consequence of the phosphorylation of CAD Thr-456 by mitogen-activated protein (MAP) kinase. Although most of the CAD in the cell was cytosolic, cell fractionation and fluorescence microscopy showed that Thr(P)-456 CAD was primarily localized within the nucleus in association with insoluble nuclear substructures, including the nuclear matrix. CAD in resting cells was cytosolic and unphosphorylated. Upon epidermal growth factor stimulation, CAD moved to the nucleus, and Thr-456 was found to be phosphorylated. Mutation of the CAD Thr-456 and inhibitor studies showed that nuclear import is not mediated by MAP kinase phosphorylation. Two fluorescent CAD constructs, NLS-CAD and NES-CAD, were prepared that incorporated strong nuclear import and export signals, respectively. NLS-CAD was exclusively nuclear and extensively phosphorylated. In contrast, NES-CAD was confined to the cytoplasm, and Thr-456 remained unphosphorylated. Although alternative explanations can be envisioned, it is likely that phosphorylation occurs within the nucleus where much of the activated MAP kinase is localized. Trapping CAD in the nucleus had a minimal effect on pyrimidine metabolism. In contrast, when CAD was excluded from the nucleus, the rate of pyrimidine biosynthesis, the nucleotide pools, and the growth rate were reduced by 21, 36, and 60%, respectively. Thus, the nuclear import of CAD appears to promote optimal cell growth. UMP synthase, the bifunctional protein that catalyzes the last two steps in the pathway, was also found in both the cytoplasm and nucleus.

Flow-based amperometric detection of dopamine in an immobilized cell reactor.

A protocol is described for immobilizing PC 12 cells on to the lumen of fused silica microbore tubing having an inside diameter of 250 microm and coated with a thin layer of poly-L-lysine. Optimization studies of the immobilization procedure indicated that a 50 microg ml(-1) solution of poly-L-lysine was the best material for cell adhesion to the fused silica tubing. In addition, it was found that the cells become attached to the poly-L-lysine in approximately 2 h, after which they could be maintained inside of the tubing for a period up to 5 days. Importantly, the immobilized cells ability to release neurotransmitters was evident by measuring the Ca(2+)-induced release of dopamine with an in column amperometric detection scheme involving a Nafion modified platinum ultramicroelectrode.

Detection of ATP-induced nitric oxide in a biomimetic circulatory vessel containing an immobilized endothelium.

Conditions for the adhesion of bovine pulmonary artery endothelial cells (bPAECs) in microbore tubing of 250-microm i.d. are described. When immobilized to the lumen of microbore tubing, these cells represent a mimic of a circulatory vessel's endothelium. The microbore tubing is coated with 100 microg mL(-1) fibronectin in order to promote bPAEC adhesion to the lumen of the tubing. A series of micrographs of the cells inside of the tubing indicates that approximately 3.5 h is necessary for cell adhesion. In this study, adenosine triphosphate (ATP) is used to induce the release of nitric oxide from the endothelium mimic. The endothelium-derived NO is detected amperometrically at a parallel flow cell containing a glassy carbon working electrode modified with Nafion. Results indicate that detectable amounts of NO are only produced by the endothelium mimic when ATP is present in the buffer. The typical concentration of NO produced by the endothelium mimic upon the introduction of 100 microM ATP is approximately 0.80 microM. Based on the injection volume of ATP and the estimated number of cells on the tubing lumen, this value corresponds to approximately 1 amol of NO/cell. Moreover, shear stress alone does not provide the agonistic effect required for NO production in the submicromolar range.

Cell cycle-dependent regulation of pyrimidine biosynthesis.

De novo pyrimidine biosynthesis is activated in proliferating cells in response to an increased demand for nucleotides needed for DNA synthesis. The pyrimidine biosynthetic pathway in baby hamster kidney cells, synchronized by serum deprivation, was found to be up-regulated 1.9-fold during S phase and subsequently down-regulated as the cells progressed through the cycle. The nucleotide pools were depleted by serum starvation and were not replenished during the first round of cell division, suggesting that the rate of utilization of the newly synthesized nucleotides closely matched their rate of formation. The activation and subsequent down-regulation of the pathway can be attributed to altered allosteric regulation of the carbamoyl-phosphate synthetase activity of CAD (carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase), a multifunctional protein that initiates mammalian pyrimidine biosynthesis. As the culture approached S-phase there was an increased sensitivity to the allosteric activator, 5-phosphoribosyl-1-pyrophosphate, and a loss of UTP inhibition, changes that were reversed when cells emerged from S phase. The allosteric regulation of CAD is known to be modulated by MAP kinase (MAPK) and protein kinase A (PKA)-mediated phosphorylations as well as by autophosphorylation. CAD was found to be fully autophosphorylated in the synchronized cells, but the level remained invariant throughout the cycle. Although the MAPK activity increased early in G(1), the phosphorylation of the CAD MAPK site was delayed until just before the onset of S phase, probably due to antagonistic phosphorylation by PKA that persisted until late G(1). Once activated, pyrimidine biosynthesis remained elevated until rephosphorylation of CAD by PKA and dephosphorylation of the CAD MAPK site late in S phase. Thus, the cell cycle-dependent regulation of pyrimidine biosynthesis results from the sequential phosphorylation and dephosphorylation of CAD under the control of two important signaling cascades.