Autophosphorylation of the Mammalian Multifunctional Protein That Initiates de Novo Pyrimidine Biosynthesis

Sigoillot, Frederic; Evans, David R.; Guy, Hedeel I.

doi:10.1074/jbc.m203512200

Cited by 10 publications

(6 citation statements)

References 44 publications

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“…This gene encodes a multifunctional 2230 amino acid protein (Fig. 2C) whose activities comprise the first three steps in de novo pyrimidine biosynthesis (Sigoillot et al, 2002) and which is responsible for the rate‐limiting step in this pathway. The observed deletion excises base pairs 510–528 (corresponding to aa 170–175 of the wild‐type protein), resulting in a frame‐shift into an alternative reading frame that contains a stop codon 105 bp downstream of the deletion (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This gene encodes a multifunctional 2230 amino acid protein (Fig. 2C) whose activities comprise the first three steps in de novo pyrimidine biosynthesis (Sigoillot et al, 2002) and which is responsible for the rate-limiting step in this pathway. The observed deletion excises base pairs Dashed arrow shows normal hindbrain plexus, where the sVII, sIX and sX nerves terminate.…”

Section: Sl23 Is a Truncated Cadmentioning

confidence: 99%

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Novel role for carbamoyl phosphate synthetase 2 in cranial sensory circuit formation

Cox

LaMora²,

Johnson

et al. 2013

Intl J of Devlp Neuroscience

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In zebrafish, cranial sensory circuits form by 4 days post-fertilization. We used a forward genetic screen to identify genes involved in the formation of these circuits. In one mutant allele, sl23, axons arising from the epibranchial sensory ganglia do not form their stereotypical terminal fields in the hindbrain. These embryos also had small eyes and deformed jaws, suggesting a pleiotropic effect. Using positional cloning, a 20-nucleotide deletion in the carbamoyl-phosphate-synthetase2-aspartate-transcarbamylase-dihydroorotase (cad) gene was found. Injection of a CAD morpholino phenocopied the mutant and mutants were rescued by injection of cad RNA. Cad activity is required for pyrimidine biosynthesis, and thus is a prerequisite for nucleic acid production and UDP-dependent protein glycosylation. Perturbation of nucleic acid biosynthesis can result in cell death. sl23 mutants did not exhibit elevated cell death, or gross morphological changes, in their hindbrains. To determine if defective protein glycosylation was involved in the aberrant targeting of sensory axons, we treated wild type embryos with tunicamycin, which blocks N-linked protein glycosylation. Interference with glycosylation via tunicamycin treatment mimicked the sl23 phenotype. Loss of cad reveals a critical role for protein glycosylation in cranial sensory circuit formation.

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Section: Resultsmentioning

confidence: 99%

“…This gene encodes a multifunctional 2230 amino acid protein (Fig. 2C) whose activities comprise the first three steps in de novo pyrimidine biosynthesis (Sigoillot et al, 2002) and which is responsible for the rate-limiting step in this pathway. The observed deletion excises base pairs Dashed arrow shows normal hindbrain plexus, where the sVII, sIX and sX nerves terminate.…”

Section: Sl23 Is a Truncated Cadmentioning

confidence: 99%

Novel role for carbamoyl phosphate synthetase 2 in cranial sensory circuit formation

Cox

LaMora²,

Johnson

et al. 2013

Intl J of Devlp Neuroscience

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“…CAD Phosphorylation-Previous studies show (18,41) that the extent of the phosphorylation of the MAP kinase site, Thr-456, could be quantitated by immunoblotting with phosphothreonine antibodies corrected for autophosphorylation. Similarly, the extent of phosphorylation of the CAD PKA sites (Ser-1406 and Ser-1859) could be quantitated using the phosphoserine antibodies.…”

Section: Methodsmentioning

confidence: 99%

Cell Cycle-dependent Regulation of Pyrimidine Biosynthesis

Sigoillot

Berkowski

Sigoillot

et al. 2003

Journal of Biological Chemistry

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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 upregulated 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 downregulation 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.The activity of the de novo pyrimidine biosynthetic pathway closely parallels the growth rate of the cell and is highest during periods of rapid proliferation (1-13). Cells growing in culture synthesize pyrimidines at a rapid rate during the exponential growth phase, but the pathway is much less active when the cells enter the stationary phase or have their growth arrested by serum deprivation (14 -18). In an earlier study, Mitchell and Hoogenraad (19) show that pyrimidine biosynthesis is maximally activated during the S phase of the cell cycle. Thus, the de novo biosynthetic pathway plays a dominant role in providing precursors for the synthesis of DNA that accompanies cell division. Furthermore, there is strong evidence (7,11,12) that the activation of the pathway is a prerequisite for the proliferation of tumor and neoplastic cells.The flux through the pathway is governed (20, 21) by the activity of carbamoyl-phosphate synthetase II (CPSase), 1 a component of CAD (Fig. 1), the multifunctional protein (22-24) that init...

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“…Moreover, a specific Thr residue (Fig. 2, Thr1037) in the B1 subdomain was found to be autophosphorylated both in vivo and in vitro (59), resulting in a selective increase in CPSase activity and modulation of the allosteric transitions.…”

Section: Regulation Of Pyrimidine Biosynthesismentioning

confidence: 99%