Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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

The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometrically by cyclic AMP-dependent protein kinase. Phosphorylation activates the ammonia-dependent carbamyl phosphate synthetase activity of the complex by reducing the apparent Km for ATP. This effect is particularly marked in the presence of the allosteric feedback inhibitor, UTP, when the apparent Km is reduced by greater than 4-fold. Inhibition by physiological concentrations of UTP is substantially relieved by phosphorylation. Cyclic AMP-dependent protein kinase phosphorylates two serine residues on the protein termed sites 1 and 2, and the primary structures of tryptic peptides containing these sites have been determined: Site 1: Arg-Leu-Ser(P)-Ser-Phe-Val-Thr-Lys Site 2: Ile-His-Arg-Ala-Ser(P)-Asp-Pro-Gly-Leu-Pro-Ala-Glu-Glu-Pro-Lys During the phosphorylation reaction, activation of the carbamyl phosphate synthetase shows a better correlation with occupancy of site 1 rather than site 2. Both phosphorylation and activation can be reversed using purified preparations of the catalytic subunits of protein phosphatases 1- and -2A, and inactivation also correlates better with dephosphorylation of site 1 rather than site 2. We believe this to be the first report that a key enzyme in nucleotide biosynthesis is regulated in a significant manner by reversible covalent modification. The physiological role of this phosphorylation in the stimulation of cell proliferation by growth factors and other mitogens is discussed.
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PMID:Phosphorylation and activation of hamster carbamyl phosphate synthetase II by cAMP-dependent protein kinase. A novel mechanism for regulation of pyrimidine nucleotide biosynthesis. 409 95

Here we review the different apoptotic DNases. From a functional point of view, DNases implicated in apoptosis may be classified into three groups: the Ca2+/Mg2+ endonucleases, the Mg2+-endonucleases, and the cation-independent endonucleases. The first group includes DNase I which has no specificity for the linker region, DNase gamma which has some homology with DNase I, and other DNases which cleave DNA in the linker region. Both DNase I and DNase gamma have been cloned. The other nucleases of this category have dispersed molecular weights. Their sequences are unknown and it is difficult to determine their role(s) in apoptosis. It seems that different pathways are present and that these nucleases may be activated either by caspases or serine proteases. The caspase 3 activated DNase (CAD, CPAN, or DFF40) belongs to the Mg2+-dependent endonucleases. DNase II belongs to the third group of acid endonucleases or cation-independent DNases. We have shown the involvement of DNase II in lens cell differentiation. Recently, the molecular structure of two different enzymes has been elucidated, one of which has a signal peptide and appears to be secreted. The other, called L-DNase II, is an intracellular protein having two enzymatic activities; in its native form, it is an anti-protease, and after posttranslational modification, it becomes a nuclease.
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PMID:DNases and apoptosis. 1101 79

Beta-amyloid precursor protein (APP) is implicated in the pathobiology of Alzheimer's disease (AD). To gain insight into its function, we have investigated the proteolytic processing and post-translational modification of APP in relation to its intracellular traffic and localization. The proteolytic processing that generates the amyloid beta-peptide (Abeta) also releases into the cytoplasm the carboxy-terminal fragment of APP, Cgamma. Using the catecholaminergic cell line, CAD, and an antibody to a form of APP that is phosphorylated at Thr668 (pAPP; numbering for APP695), we show that a phosphorylated, carboxy-terminal fragment of APP, probably Cgamma, is present in the nucleus, where it localizes to subnuclear particles. The labeling with anti-pAPP antibody co-localizes with proteins that define the splicing factor compartment (SFC) [e.g. the small nuclear ribonucleoprotein (snRNP), U2B, and serine/arginine-rich (SR) proteins], but is excluded from the coiled bodies and the gems. This distribution of pAPP epitopes was found in CAD cells independent of their state of differentiation, as well as in primary cortical neurons, epithelial cells and fibroblasts. We further show that exogenously expressed Cgamma becomes phosphorylated, and distributes throughout the cell. A fraction of this Cgamma is translocated into the nucleus, where it co-localizes with endogenous pAPP epitopes. Finally, we show that the APP binding, scaffolding protein, Fe65 co-localizes with pAPP epitopes and with expressed Cgamma at intranuclear speckles. These results suggest that phosphorylated Cgamma accumulates at the SFC. Thus, APP may play a role in pre-mRNA splicing, and Fe65 and APP phosphorylation may regulate this function.
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PMID:A phosphorylated, carboxy-terminal fragment of beta-amyloid precursor protein localizes to the splicing factor compartment. 1472 57

Low-energy CAD product-ion spectra of various molecular species of phosphatidylserine (PS) in the forms of [M-H]- and [M-2H+Alk]- in the negative-ion mode, as well as in the forms of [M+H]+, [M+Alk]+, [M-H+2Alk]+, and [M-2H+3Alk]+ (where Alk=Li, Na) in the positive-ion mode contain rich fragment ions that are applicable for structural determination. Following CAD, the [M-H]- ion of PS undergoes dissociation to eliminate the serine moiety (loss of C3H5NO2) to give a [M-H-87]- ion, which equals to the [M-H]- ion of a phoshatidic acid (PA) and give rise to a MS3-spectrum that is identical to the MS2-spectrum of PA. The major fragmentation process for the [M-2H+Alk]- ion of PS arises from primary loss of 87 to give rise to a [M-2H+Alk-87]- ion, followed by loss of fatty acid substituents as acids (RxCO2H, x=1,2) or as alkali salts (e.g., RxCO2Li, x=1,2). These fragmentations result in a greater abundance of [M-2H+Alk-87-R2CO2H]- than [M-2H+Alk-87-R1CO2H]- and a greater abundance of [M-2H+Alk-87-R2CO2Li]- than [M-2H+Alk-87-R1CO2Li]-; while further dissociation of the [M-2H+Alk-87-R2(or 1)CO2Li]- ions gives a preferential formation of the carboxylate anion at sn-1 (R1CO2-) over that at sn-2 (R2CO2-). Other major fragmentation process arises from differential loss of the fatty acid substituents as ketenes (loss of Rx'CH=CO, x=1,2). This results in a more prominent [M-2H+Alk-R2'CH=CO]- ion than [M-2H+Alk-R1'CH=CO]- ion. Ions informative for structural characterization of PS are of low abundance in the MS2-spectra of both the [M+H]+ and the [M+Alk]+ ions, but are abundant in the MS3-spectra. The MS2-spectrum of the [M+Alk]+ ion contains a unique ion corresponding to internal loss of a phosphate group probably via the fragmentation processes involving rearrangement steps. The [M-H+2Alk]+ ion of PS yields a major [M-H+2Alk-87]+ ion, which is equivalent to an alkali adduct ion of a monoalkali salt of PA and gives rise to a greater abundance of [M-H+2Alk-87-R1CO2H]+ than [M-H+2Alk-87-R2CO2H]+. Similarly, the [M-2H+3Alk]+ ion of PS also yields a prominent [M-2H+3Alk-87]+ ion, which undergoes consecutive dissociation processes that involve differential losses of the two fatty acyl substituents. Because all of the above tandem mass spectra contain several sets of ion pairs involving differential losses of the fatty acid substituents as ketenes or as free fatty acids, the identities of the fatty acyl substituents and their positions on the glycerol backbone can be easily assigned by the drastic differences in the abundances of the ions in each pair.
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PMID:Studies on phosphatidylserine by tandem quadrupole and multiple stage quadrupole ion-trap mass spectrometry with electrospray ionization: structural characterization and the fragmentation processes. 1602 63

Pluripotent neural crest (NC) cells differentiate to diverse lineages, including the neuronal, sympathoadrenal lineage. In primary NC cultures, bone morphogenetic protein 2 (BMP2) requires moderate activation of cAMP signaling for induction of the sympathoadrenal lineage. However, the mechanism by which cAMP signaling synergizes with BMP2 to induce the sympathodrenal lineage is unknown. Herein, we demonstrate that moderate activation of cAMP signaling induces both transcription and activity of proneural transcription factor Phox2a. In NC cultures inhibition of cAMP-response element-binding protein (CREB)-mediated transcription by expression of dominant-negative CREB suppresses Phox2a transcription and sympathoadrenal lineage development. Interestingly, the constitutively active CREB(DIEDML), despite inducing Phox2a transcription, is insufficient for sympathoadrenal lineage development, requiring activation of the cAMP pathway. Because CREB(DIEDML)-mediates cAMP-dependent transcription without requiring activation by the cAMP-dependent protein kinase A (PKA), these results identify PKA activation as necessary in sympathoadrenal lineage development. Treatment of NC cultures with the PKA inhibitor H89 or 1-10 nm okadaic acid (OA), a serine/threonine PP2A-like phosphatase inhibitor, suppresses sympathoadrenal lineage development. Likewise, OA treatment of the CNS-derived catecholaminergic CAD cell line inhibits cAMP-mediated neuronal differentiation. Specifically, OA inhibits cAMP-mediated Phox2a dephosphorylation, cAMP-dependent Phox2a DNA binding in vitro, and cAMP- and Phox2a-dependent dopamine-beta-hydroxylase-luciferase reporter expression. Together, these results support cAMP-dependent Phox2a dephosphorylation is required for its activation. We conclude that moderate activation of cAMP signaling has dual inputs in catecholaminergic, sympathoadrenal lineage development; that is, regulation of both Phox2a transcription and activity. These results provide the first mechanistic understanding of how moderate activation of the cAMP pathway in synergy with BMP2 promotes sympathoadrenal lineage development.
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PMID:The cAMP pathway regulates both transcription and activity of the paired homeobox transcription factor Phox2a required for development of neural crest-derived and central nervous system-derived catecholaminergic neurons. 1620 40

The involvement of caspase-3 and its failure in the induction of DNA fragmentation during erythropoiesis were investigated with TF-1 cells. During erythroid differentiation, caspase-3 activation and cleavage of caspase-3 substrates such as ICAD (inhibitor of caspase-activated DNase) were detected without concomitant phosphatidyl-serine (PS) externalization and DNA fragmentation. These observations are in contrast to our understanding that DNA is degraded by CAD (caspase-activated DNase) when ICAD is cleaved by caspase-3. Our study demonstrates that CAD is downregulated at the mRNA and protein level during the erythroid differentiation in TF-1 cells. This provides a mechanism for the first time how cells avoid DNA fragmentation with activated caspase-3.
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PMID:Erythropoietin activates caspase-3 and downregulates CAD during erythroid differentiation in TF-1 cells - a protection mechanism against DNA fragmentation. 1652 48

We have previously reported that the pro-apoptotic pyrrolobenzoxazepine, PBOX-6, induces apoptosis in chronic myelogenous leukaemia (CML) cells which is accompanied by oligonucleosomal DNA fragmentation. In this study we show that PBOX-6-induced oligonucleosomal DNA fragmentation occurs in the absence of caspase and CAD activation in CML cells. Dissection of the signalling pathway has revealed that induction of apoptosis requires the upstream activation of a trypsin-like serine protease that promotes the phosphorylation and inactivation of anti-apoptotic Bcl-2. In addition, in this system chymotrypsin-like serine proteases are dispensable for high molecular weight DNA fragmentation, however are required for the activation of a relatively small manganese-dependent acidic endonuclease that is responsible for oligonucleosomal fragmentation of DNA. Furthermore, we demonstrate mitochondrial involvement during PBOX-6-induced apoptosis and suggest the existence of unidentified mitochondrial effectors of apoptosis.
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PMID:Caspase-activated DNase (CAD)-independent oligonucleosomal DNA fragmentation in chronic myeloid leukaemia cells; a requirement for serine protease and Mn2+-dependent acidic endonuclease activity. 1682 Sep 64

Calcium ions have been implicated in apoptosis for many years, however the precise role of this ion in the cell death process remains incomplete. We have extensively examined the role of Ca(2+) on nuclear degradation in vitro using highly purified nuclei isolated from non-apoptotic rat thymocytes. We show that these nuclei are devoid of CAD (caspase-activated DNase), and DNA degradation occurs independent of caspase activity. Serine proteases rather than caspase-3 appear necessary for this Ca(2+) -dependent DNA degradation in nuclei. We analyzed nuclei treated with various concentrations of Ca(2+) in the presence of both a physiological (140 mM) and apoptotic (40 mM) concentration of KCl. Our results show that a 5-fold increase in Ca(2+) is required to induce DNA degradation at the physiological KCl concentration compared to the lower, apoptotic concentration of the cation. Ca(2+) -induced internucleosomal DNA degradation was also accompanied by the release of histones, however the apoptotic-specific phosphorylation of histone H2B does not occur in these isolated nuclei. Interestingly, physiological concentrations of K(+) inhibit both Ca(2+) -dependent DNA degradation and histone release suggesting that a reduction of intracellular K(+) is necessary for this apoptosis-associated nuclear degradation in cells. Together, these data define an inherent caspase-independent catabolic pathway in thymocyte nuclei that is sensitive to physiological concentrations of intracellular cations.
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PMID:An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: antagonism by physiological concentrations of K(+) ions. 1829 29

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


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