title: Regulation of carbamoyl phosphate synthetase by MAP kinase.
abstract: The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. The rate-limiting step in this pathway is catalysed by carbamoyl phosphate synthetase (CPS II), part of the multifunctional enzyme CAD. Here we describe the regulation of CAD by the mitogen-activated protein (MAP) kinase cascade. When phosphorylated by MAP kinase in vitro or activated by epidermal growth factor in vivo, CAD lost its feedback inhibition (which is dependent on uridine triphosphate) and became more sensitive to activation (which depends upon phosphoribosyl pyrophosphate). Both these allosteric regulatory changes favour biosynthesis of pyrimidines for growth. They were accompanied by increased epidermal growth factor-dependent phosphorylation of CAD in vivo and were prevented by inhibition of MAP kinase. Mutation of a consensus MAP kinase phosphorylation site abolished the changes in CAD allosteric regulation that were stimulated by growth factors. Finally, consistent with an effect of MAP kinase signalling on CPS II activity, epidermal growth factor increased cellular uridine triphosphate and this increase was reversed by inhibition of MAP kinase. Hence these studies may indicate a direct link between activation of the MAP kinase cascade and de novo biosynthesis of pyrimidine nucleotides.
authors.affiliation: Department of Pharmacology, Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599-7365, USA. lmg@med.unc.edu
authors.forename: L M
authors.initials: LM
authors.lastname: Graves
authors.forename: H I
authors.initials: HI
authors.lastname: Guy
authors.forename: P
authors.initials: P
authors.lastname: Kozlowski
authors.forename: M
authors.initials: M
authors.lastname: Huang
authors.forename: E
authors.initials: E
authors.lastname: Lazarowski
authors.forename: R M
authors.initials: RM
authors.lastname: Pope
authors.forename: M A
authors.initials: MA
authors.lastname: Collins
authors.forename: E N
authors.initials: EN
authors.lastname: Dahlstrand
authors.forename: H S
authors.initials: HS
authors.lastname: Earp
authors.forename: D R
authors.initials: DR
authors.lastname: Evans
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004080:Dihydroorotase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D008647:Mesocricetus; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D010766:Phosphorylation; D011742:Pyrimidine Nucleotides / Q000096:biosynthesis; D051381:Rats; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: C033626:CAD trifunctional enzyme; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009097:Multienzyme Complexes; D011742:Pyrimidine Nucleotides; D010754:Phosphoribosyl Pyrophosphate; D001221:Aspartate Carbamoyltransferase; D020928:Mitogen-Activated Protein Kinases; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D014544:Uridine Triphosphate