Abstract
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.
Highlights
The first three steps of mammalian de novo pyrimidine biosynthesis are catalyzed by CAD1 [1,2,3], a 1.4-MDa multifunc
Dihydroorotate synthesized by the CAD complex is oxidized by mitochondrial dihydroorotate dehydrogenase (DHOdhase) to orotate, which is subsequently converted to UMP by the cytoplasmic bifunctional protein UMP synthase [4, 5] (Reactions 4 – 6)
carbamoyl phosphate synthetase (CPSase) is subject to feedback inhibition by the end product, UTP, and allosteric activation by PRPP, a substrate of both UMP synthase and glutamine PRPP amidotransferase, the enzyme catalyzing the first step in the purine biosynthetic pathway (6 – 8)
Summary
The first three steps of mammalian de novo pyrimidine biosynthesis are catalyzed by CAD1 [1,2,3], a 1.4-MDa multifunc-. The activity of CPSase is stimulated, and the flux through the pathway increases, providing precursors for the synthesis of DNA, RNA, and the glycoproteins needed for membrane bioclear export sequence; PMSF, phenylmethylsulfonyl fluoride; PRPP, 5-phospho-D-ribosyl-1-pyrophosphate; UMP synthase, the bifunctional protein that has orotate phosphoribosyltransferase and orotidine-5Јphosphate decarboxylase activities and catalyzes the last two steps in the de novo pyrimidine biosynthetic pathway; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; ECFP, enhanced cyan fluorescent protein; BHK, baby hamster kidney; GFP, green fluorescent protein. The growth-dependent up- and down-regulation of the pathway is governed by the sequential phosphorylation of CAD by the MAP kinase and cAMP-protein kinase A cascades In accord with this interpretation, the pyrimidine pathway is persistently activated in MCF7 breast cancer cells [15] as a consequence of elevated MAP kinase activity that results in the continuous phosphorylation of CAD Thr-456, whereas CAD Ser-1406 remains unphosphorylated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
