Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribonucleotide reductase is a highly regulated, cell cycle-controlled activity that plays an important role in DNA synthesis and repair. Recent studies have shown that elevated expression of the rate-limiting R2 component of ribonucleotide reductase increases Raf-1 protein activation and mitogen-activated protein kinase activity and acts as a novel malignancy determinant in cooperation with activated oncogenes like H-ras. We show that hydroxyurea-resistant mouse L cells with elevated R2 gene expression and increased ribonucleotide reductase activity exhibit significantly decreased sensitivities to the chemotherapeutic compounds N-(phosphonacetyl)-L-aspartate (PALA) and methotrexate (MTX). Furthermore, BALB/c 3T3 cells containing a retroviral expression vector encoding the R2 sequence also showed decreased sensitivity to PALA and MTX when compared to cells containing the same vector but without the R2 coding region. Colonies that developed in the presence of PALA or MTX contained amplifications of the CAD or dihydrofolate reductase genes and exhibited wild-type p53 function as determined in sequence-specific p53 binding activity assays. NIH-3T3 cells containing the R2 retroviral expression vector also showed significantly decreased sensitivity to hydroxyurea and MTX but not to PALA. Furthermore, NIH-3T3 cells transfected with a vector containing the R2 sequence in antisense orientation exhibited increased sensitivity to hydroxyurea, PALA, and MTX. Similarly, mouse 10T1/2 cells that are highly transformed and drug resistant due to alterations in H-ras and a mutant oncogenic form of p53 exhibited significant increases in sensitivity to hydroxyurea, PALA, and MTX when transfected with a vector containing the R2 sequence in antisense orientation and compared to cells containing the same vector without the antisense sequence. These results indicate that altered expression of the R2 component is capable of significantly modifying drug sensitivity properties of tumor cells. We hypothesize that this occurs, at least in part, through a mechanism of increased genetic instability that is independent of direct p53 mutation or loss and involves R2 stimulation of the mitogen-activated protein kinase signal pathway.
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PMID:Ribonucleotide reductase R2 gene expression and changes in drug sensitivity and genome stability. 935 52

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.
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PMID:Regulation of carbamoyl phosphate synthetase by MAP kinase. 1065 30

The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo pyrimidine biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of pyrimidine biosynthesis, the activity of the de novo pyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in pyrimidine biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of pyrimidine biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.
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PMID:Growth-dependent regulation of mammalian pyrimidine biosynthesis by the protein kinase A and MAPK signaling cascades. 1187 54

CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of P(i)/mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr(1037) located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades.
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PMID:Autophosphorylation of the mammalian multifunctional protein that initiates de novo pyrimidine biosynthesis. 1198 31

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 up-regulated 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 down-regulation 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.
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PMID:Cell cycle-dependent regulation of pyrimidine biosynthesis. 1243 17

Hypoxia-inducible factors (HIF) are a family of heterodimeric transcriptional regulators that play pivotal roles in the regulation of cellular utilization of oxygen and glucose and are essential transcriptional regulators of angiogenesis in solid tumor and ischemic disorders. The transactivation activity of HIF complexes requires the recruitment of p300/CREB-binding protein (CBP) by HIF-1 alpha and HIF-2 alpha that undergo oxygen-dependent degradation. HIF activation in tumors is caused by several factors including mitogen-activated protein kinase (MAPK) signaling. Here we investigated the molecular basis for HIF activation by MAPK. We show that MAPK is required for the transactivation activity of HIF-1 alpha. Furthermore, inhibition of MAPK disrupts the HIF-p300 interaction and suppresses the transactivation activity of p300. Overexpression of MEK1, an upstream MAPK activator, stimulates the transactivation of both p300 and HIF-1 alpha. Interestingly, the C-terminal transactivation domain of HIF-1 alpha is not a direct substrate of MAPK, and HIF-1 alpha phosphorylation is not required for HIF-CAD/p300 interaction. Taken together, our data suggest that MAPK signaling facilitates HIF activation through p300/CBP.
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PMID:MAPK signaling up-regulates the activity of hypoxia-inducible factors by its effects on p300. 1258 75

The activity of the de novo pyrimidine biosynthetic pathway in the MCF7 breast cancer cells was 4.4-fold higher than that in normal MCF10A breast cells. Moreover, while pyrimidine biosynthesis in MCF10A was tightly regulated, increasing as the culture matured and subsequently down-regulated in confluency, the biosynthetic rate in MCF7 cells remained elevated and invariant in all growth phases. The flux through the pathway is regulated by carbamoyl phosphate synthetase, a component of the multifunctional protein, CAD. The intracellular CAD concentration was 3.5- to 4-fold higher in MCF7 cells, an observation that explains the high rate of pyrimidine biosynthesis but cannot account for the lack of growth-dependent regulation. In MCF10A cells, up-regulation of the pathway in the exponential growth phase resulted from MAP kinase phosphorylation of CAD Thr456. The pathway was subsequently down-regulated by dephosphorylation of P approximately Thr456 and the phosphorylation of CAD by PKA. In contrast, the CAD P approximately Thr456 was persistently phosphorylated in MCF7 cells, while the PKA site remained unphosphorylated and consequently the activity of the pathway was elevated in all growth phases. In support of this interpretation, inhibition of MAP kinase in MCF7 cells decreased CAD P approximately Thr456, increased PKA phosphorylation and decreased pyrimidine biosynthesis. Conversely, transfection of MCF10A with constructs that elevated MAP kinase activity increased CAD P approximately Thr456 and the pyrimidine biosynthetic rate. The differences in the CAD phosphorylation state responsible for unregulated pyrimidine biosynthesis in MCF7 cells are likely to be a consequence of the elevated MAP kinase activity and the antagonism between MAP kinase- and PKA-mediated phosphorylations.
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PMID:Breakdown of the regulatory control of pyrimidine biosynthesis in human breast cancer cells. 1499 69

Ischemic preconditioning affords the most powerful protection to a heart submitted to a prolonged ischemia-reperfusion. During the past decade, a huge amount of work allowed to better understand the features of this protective effect as well as the molecular mechanisms. Ischemic preconditioning reduces infarct size and improves functional recovery; its effects on arrhythmias remain debated. Triggering of the protection involves cell surface receptors that activate pro-survival pathways including protein kinase C, PI3-kinase, possibly Akt and ERK1/2, whose downstream targets remain to be determined. Much attention has been recently focused on the role of mitochondrial K(+)ATP channels and the permeability transition pore that seem to play a major role in the progression toward irreversible cellular injury. Based on these experimental studies attempts have been made to transfer preconditioning from bench to bedside. Human experimental models of ischemic preconditioning have been set up, including cardiac surgery, coronary angioplasty or treadmill exercise, to perform pathophysiological studies. Yet, protecting the heart of CAD (coronary artery disease) patients requires a pharmacological approach. The IONA trial has been an example of the clinical utility of preconditioning. It helped to demonstrate that chronic administration of nicorandil, a K(+)ATP opener that mimics ischemic preconditioning in experimental preparations, improves the cardiovascular prognosis in CAD patients. Recent experimental studies appear further encouraging. It appears that "postconditioning" the heart (i.e. performing brief episodes of ischemia-reperfusion at the time of reperfusion) is as protective as preconditioning. In other words, a therapeutic intervention performed as late as at the time of reflow can still significantly limit infarct size. Further work is needed to determine whether this may be transferred to the clinical practice.
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PMID:[How to use the paradigm of ischemic preconditioning to protect the heart?]. 1519 Apr 69

CAD is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. The activation of the pathway required for cell proliferation is a consequence of the phosphorylation of CAD Thr-456 by mitogen-activated protein (MAP) kinase. Although most of the CAD in the cell was cytosolic, cell fractionation and fluorescence microscopy showed that Thr(P)-456 CAD was primarily localized within the nucleus in association with insoluble nuclear substructures, including the nuclear matrix. CAD in resting cells was cytosolic and unphosphorylated. Upon epidermal growth factor stimulation, CAD moved to the nucleus, and Thr-456 was found to be phosphorylated. Mutation of the CAD Thr-456 and inhibitor studies showed that nuclear import is not mediated by MAP kinase phosphorylation. Two fluorescent CAD constructs, NLS-CAD and NES-CAD, were prepared that incorporated strong nuclear import and export signals, respectively. NLS-CAD was exclusively nuclear and extensively phosphorylated. In contrast, NES-CAD was confined to the cytoplasm, and Thr-456 remained unphosphorylated. Although alternative explanations can be envisioned, it is likely that phosphorylation occurs within the nucleus where much of the activated MAP kinase is localized. Trapping CAD in the nucleus had a minimal effect on pyrimidine metabolism. In contrast, when CAD was excluded from the nucleus, the rate of pyrimidine biosynthesis, the nucleotide pools, and the growth rate were reduced by 21, 36, and 60%, respectively. Thus, the nuclear import of CAD appears to promote optimal cell growth. UMP synthase, the bifunctional protein that catalyzes the last two steps in the pathway, was also found in both the cytoplasm and nucleus.
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PMID:Nuclear localization and mitogen-activated protein kinase phosphorylation of the multifunctional protein CAD. 1589 Jun 48

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.
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PMID:Protein kinase A phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade. 1720 80


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