Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.1.6 (CAD)
 4,420 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The multienzyme polypeptide CAD is phosphorylated at two sites by cyclic AMP (cAMP)-dependent protein kinase. Site 2 has two interesting features: it is located in a 'linking region' between two discretely folded enzyme domains, and a histidine, instead of the more usual arginine, is found three positions N-terminal to the phosphorylated serine. A synthetic peptide corresponding to the sequence around site 2 has an extended or random structure in solution, and the proton n.m.r. chemical shift of the histidine residues can be titrated against pH in the range 6.0-8.0. The peptide is phosphorylated more rapidly by cAMP-dependent protein kinase at lower pH values, indicating that the protonated histidine side chain corresponds to the arginine in the consensus recognition sequence for the kinase. Kemptide, a specific synthetic substrate for the kinase, was phosphorylated with a higher affinity and at a similar rate at all pH values. CAD was a better substrate than the synthetic peptide, and labelling was not affected by the pH of the incubation conditions. The results indicate that the phosphorylation site in the interdomain linker is sufficiently exposed to the solvent to ensure accessibility to the kinase, but that secondary or tertiary structure in the intact protein allows the histidine residue to remain protonated at physiological pH and enhances recognition of the phosphorylatable serine residue.
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PMID:A protonated histidine residue in a phosphorylation site for cyclic AMP-dependent protein kinase. Comparison of a synthetic peptide with the exposed linking region in the multienzyme polypeptide CAD. 135 77

CAD codes for a trifunctional protein involved in the catalysis of the first three enzymatic activities in the de novo pyrimidine biosynthetic pathway, namely, carbamoyl-phosphate synthetase II (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). CAD regulation was studied in the human promyelocyte leukemic line HL-60 as it differentiated into monocytic or granulocytic lineages after induction by 12-O-tetradecanoylphorbol-13-acetate or trans-retinoic acid and dibutyryl cyclic AMP, respectively. Within 12 h of induction of HL-60 cells with either inducer, total cellular levels of CAD RNA essentially disappeared. On the other hand, no apparent decreases in beta-actin RNA levels were seen even 48 h after HL-60 cells were induced, as compared with untreated cells. With nuclear runoff assays, it was clearly shown that the inactivation of CAD gene expression during the induction of HL-60 cells with either inducer was at the transcriptional level. The nuclear runoff experiments also demonstrated that the CAD gene expression was shut down in less than 4 h after induction, well before morphological changes were observed in these cells. At the enzymatic level, the activity of aspartate transcarbamylase, one of the three enzymes encoded by the CAD gene, decreased by about half within 24 h of induction, suggesting a CAD protein half-life of 24 h in differentiating HL-60 cells. Nevertheless, this means that significant levels of aspartate transcarbamylase activity remained even after the cells have stopped proliferating. From the RNA data, it is clear that CAD gene expression is rapidly turned off as promyelocytes begin to terminally differentiate into macrophages and granulocytes. We suspect that the inactivation of the CAD gene in induced HL-60 cells is a consequence of the differentiating cells leaving the cell cycle and becoming nonproliferating.
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PMID:Transcriptional regulation of the human CAD gene during myeloid differentiation. 288 43

We have studied the regulation of expression of the carbamoyl-phosphate synthetase II-aspartate transcarbamylase-dihydroorotase gene in F9 teratocarcinoma cells during their differentiation into parietal endoderm cells by induction with a combination of retinoic acid and dibutyryl cyclic AMP. Steady-state levels of CAD mRNA decreased by 7-fold in F9 cells following 120 h of retinoic acid and dibutyryl cyclic AMP induction as compared to levels in uninduced cells. Conversely, no apparent changes were found in the steady-state levels of beta-actin mRNA between induced and uninduced cells. Despite a 7-fold decrease in the steady-state levels of CAD mRNA, its rate of transcription remained the same between induced and uninduced cells, indicating a role for posttranscriptional mechanisms for its down regulation during retinoic acid- and dibutyryl cyclic AMP-induced differentiation of F9 cells. The cellular growth rate of F9 cells as determined by [3H]thymidine uptake and parallel cell counting decreased markedly during their induction with retinoic acid and dibutyryl cyclic AMP. Taken together, it is apparent that the expression of the CAD gene is cell-growth-dependent and its regulation in this system is at the posttranscriptional level.
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PMID:Posttranscriptional regulation of the expression of CAD gene during differentiation of F9 teratocarcinoma cells by induction with retinoic acid and dibutyryl cyclic AMP. 289 7

We have examined the domain organization, and the locations of the sites phosphorylated by the cyclic-AMP-dependent protein kinase, in the multifunctional polypeptide of the pyrimidine-biosynthetic protein, CAD. Fragments produced after limited proteolysis by elastase or trypsin were separated by SDS/polyacrylamide gel electrophoresis and transferred onto nitrocellulose. The blots were probed with antibodies raised against the core aspartate carbamoyltransferase (ACTase) and dihydroorotase (DHOase) fragments to locate fragments containing these domains, and we also examined the locations of the phosphorylation sites by complete tryptic digestion of blotted, 32P-labelled fragments, followed by analytical isoelectric focussing. Our results are consistent with the domain order glutaminase(GLNase)-carbamoyl-phosphate synthetase-(CPSase)-DHOase-ACTase, as suggested by recently reported homologies between the predicted amino acid sequence for the Drosophila rudimentary gene product, and monofunctional CPSases/ACTases/DHOases. In particular, the finding of a 95-kDa elastase fragment which cross-reacted with both anti-DHOase and anti-ACTase antibodies rules out the previously suggested domain order: DHOase-GLNase-CPSase-ACTase. Phosphorylation by cyclic-AMP-dependent protein kinase accelerates cleavage of native CAD by both elastase and trypsin, and abolishes the protective effect of UTP. Site 1 is located close to the C-terminal end of the 160-kDa GLNase/CPSase region. Comparison with the predicted amino acid sequence of the Drosophila rudimentary gene revealed a strong homology between the tryptic peptide containing site 1 from hamster CAD, and a region at the extreme C-terminal end of the CPSase II domain of the Drosophila enzyme. Alignment of the Drosophila sequence and that of rat liver CPSase I, which is not phosphorylated by cyclic-AMP-dependent protein kinase, revealed that this putative site 1 region is missing in CPSase I. Site 2 could not be located with certainty, either from the limited proteolysis data, or from comparison of the sequence around this site and the sequence of the rudimentary gene. There were also one or more previously undetected minor phosphorylation site(s) located in the protease-sensitive hinge region between the DHOase and ACTase domains.
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PMID:Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine-biosynthetic protein CAD. 334 46

Recently, a tyrosine hydroxylase (TH)-expressing CNS-derived cell line, CAD, was obtained that is capable of undergoing reversible morphological differentiation. The isolation of the CAD line allowed us to ask whether different DNA regulatory elements direct TH transcription when cells are growing and undifferentiated versus postmitotic and differentiated. To this end, we compared expression of a transiently transfected bacterial chloramphenicol acetyltransferase reporter gene under the transcriptional control of TH 5' flanking DNA in CAD cells grown in the presence and absence of serum. Mutational analysis indicates that CAD cells differently regulate TH transcription depending on their state of differentiation. In both states, the cyclic AMP response element and AP1 site each activate transcription. However, in undifferentiated cells, the dyad/E-box element represses expression by approximately 2.7-fold, whereas it modestly activates transcription in differentiated cells. The role of the dyad/ E-box as a repressor correlates well with the two- to threefold lower amount of endogenous TH protein present in the undifferentiated CAD cells. This study demonstrates the differential use of TH DNA regulatory elements in proliferating, undifferentiated and nonproliferating, differentiated immortalized neuronal cells.
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PMID:Differentiation of a catecholaminergic CNS cell line modifies tyrosine hydroxylase transcriptional regulation. 964 50

Acute activation of Galpha(i/o)-coupled D2 dopamine receptors inhibits A2A adenosine receptor stimulation of adenylate cyclase. This antagonistic interaction between D2 dopamine and A2A adenosine receptors has been well documented; however, the effects of persistent activation of D2 dopamine receptors on subsequent A2A adenosine receptor signaling have not been explored. The present study investigated the effects of short-term (3-h) and long-term (18-h) activation of D2L dopamine receptors on subsequent A2A adenosine receptor stimulation of adenylate cyclase in CAD-D2L and NS20Y-D2L neuroblastoma cells. Short- and long-term activation of D2L dopamine receptors markedly increased 5'-N-methylcarboxamidoadenosine (MECA)-stimulated cyclic AMP accumulation 1.4-fold and 1.7-fold, respectively. D2L receptor-induced sensitization of A2A-stimulated cyclic AMP accumulation was blocked by the D2 antagonist spiperone and pertussis toxin pretreatment. In addition, persistent activation of A2A adenosine receptors resulted in 50% desensitization of subsequent MECA-stimulated cyclic AMP accumulation; however, MECA-induced desensitization of A2A adenosine receptors did not prevent completely quinpirole-induced sensitization of adenylate cyclase. These studies revealed a novel mode of regulation between D2L dopamine and A2A adenosine receptors and suggest a cooperative interaction in the regulation of cyclic AMP signaling.
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PMID:Sensitization of neuronal A2A adenosine receptors after persistent D2 dopamine receptor activation. 1456 8

Mechanisms coordinating neural progenitor cell cycle exit and differentiation are incompletely understood. The cyclin-dependent kinase inhibitor p27(Kip1) is transcriptionally induced, switching specific neural progenitors from proliferation to differentiation. However, neuronal differentiation-specific transcription factors mediating p27(Kip1) transcription have not been identified. We demonstrate the homeodomain transcription factor Phox2a, required for central nervous system (CNS)- and neural crest (NC)-derived noradrenergic neuron differentiation, coordinates cell cycle exit and differentiation by inducing p27(Kip1) transcription. Phox2a transcription and activation in the CNS-derived CAD cell line and primary NC cells is mediated by combined cyclic AMP (cAMP) and bone morphogenetic protein 2 (BMP2) signaling. In the CAD cellular model, cAMP and BMP2 signaling initially induces proliferation of the undifferentiated precursors, followed by p27(Kip1) transcription, G(1) arrest, and neuronal differentiation. Small interfering RNA silencing of either Phox2a or p27(Kip1) suppresses p27(Kip1) transcription and neuronal differentiation, suggesting a causal link between p27(Kip1) expression and differentiation. Conversely, ectopic Phox2a expression via the Tet-off expression system promotes accelerated CAD cell neuronal differentiation and p27(Kip1) transcription only in the presence of cAMP signaling. Importantly, endogenous or ectopically expressed Phox2a activated by cAMP signaling binds homeodomain cis-acting elements of the p27(Kip1) promoter in vivo and mediates p27(Kip1)-luciferase expression in CAD and NC cells. We conclude that developmental cues of cAMP signaling causally link Phox2a activation with p27(Kip1) transcription, thereby coordinating neural progenitor cell cycle exit and differentiation.
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PMID:Homeodomain transcription factor Phox2a, via cyclic AMP-mediated activation, induces p27Kip1 transcription, coordinating neural progenitor cell cycle exit and differentiation. 1698 76

In noradrenergic progenitors, Phox2a mediates cell cycle exit and neuronal differentiation by inducing p27(Kip1) transcription in response to activation of the cyclic AMP (cAMP) pathway. The mechanism of cAMP-mediated activation of Phox2a is unknown. We identified a cluster of phosphoserine-proline sites in Phox2a by mass spectrometry. Ser206 appeared to be the most prominent phosphorylation site. A phospho-Ser206 Phox2a antibody detected dephosphorylation of Phox2a that was dependent on activation of the cAMP pathway, which occurred prior to neuronal differentiation of noradrenergic CAD cells. Employing serine-to-alanine and serine-to-aspartic acid Phox2a substitution mutants expressed in inducible CAD cell lines, we demonstrated that the transcriptional activity of Phox2a is regulated by two sequential cAMP-dependent events: first, cAMP signaling promotes dephosphorylation of Phox2a in at least one site, Ser206, thereby allowing Phox2a to bind DNA and initiate p27(Kip1) transcription; second, following dephosphorylation of the phosphoserine cluster (Ser202 and Ser208), Phox2a becomes phosphorylated by protein kinase A (PKA) on Ser153, which prevents association of Phox2a with DNA and terminates p27(Kip1) transcription. This represents a novel mechanism by which the same stimulus, cAMP signaling, first activates Phox2a by dephosphorylation of Ser206 and then, after a built-in delay, inactivates Phox2a via PKA-dependent phosphorylation of Ser153, thereby modulating onset and duration of p27(Kip1) transcription.
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PMID:Time-dependent activation of Phox2a by the cyclic AMP pathway modulates onset and duration of p27Kip1 transcription. 1963 7

Cardiovascular disease (CVD), including myocardial infarction (MI) and peripheral or coronary artery disease (PAD, CAD), remains the number one killer of individuals in the United States and worldwide, accounting for nearly 18 million (>30%) global deaths annually. Despite considerable basic science and clinical investigation aimed at identifying key etiologic components of and potential therapeutic targets for CVD, the number of individuals afflicted with these dreaded diseases continues to rise. Of the many biochemical, molecular, and cellular elements and processes characterized to date that have potential to control foundational facets of CVD, the multifaceted cyclic nucleotide pathways continue to be of primary basic science and clinical interest. Cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP) and their plethora of downstream protein kinase effectors serve ubiquitous roles not only in cardiovascular homeostasis but also in the pathogenesis of CVD. Already a major target for clinical pharmacotherapy for CVD as well as other pathologies, novel and potentially clinically appealing actions of cyclic nucleotides and their downstream targets are still being discovered. With this in mind, this review article focuses on our current state of knowledge of the cyclic nucleotide-driven serine (Ser)/threonine (Thr) protein kinases in CVD with particular emphasis on cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKG). Attention is given to the regulatory interactions of these kinases with inflammatory components including interleukin 6 signals, with G protein-coupled receptor and growth factor signals, and with growth and synthetic transcriptional platforms underlying CVD pathogenesis. This article concludes with a brief discussion of potential future directions and highlights the importance for continued basic science and clinical study of cyclic nucleotide-directed protein kinases as emerging and crucial controllers of cardiac and vascular disease pathologies.
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PMID:Cyclic Nucleotide-Directed Protein Kinases in Cardiovascular Inflammation and Growth. 2936 84