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.
Kotsis
Damian H
DH
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Masko
Elizabeth M
EM
Sigoillot
Frederic D
FD
Di Gregorio
Roberto
R
Guy-Evans
Hedeel I
HI
Evans
David R
DR
D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004080:Dihydroorotase / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D010766:Phosphorylation; D021122:Protein Subunits / Q000378:metabolism
C033626:CAD trifunctional enzyme; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D005576:Colforsin; D004815:Epidermal Growth Factor; D001221:Aspartate Carbamoyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D004080:Dihydroorotase; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)