Chemical Features of the Protein Kinase CK2 Polyamine Binding Site<sup>,</sup>

Leroy, Didier; Filhol, Odile; Delcros, Jean‐Guy; Pares, S.; Chambaz, E.M.; Cochet, Claude

doi:10.1021/bi961949u

Cited by 50 publications

(23 citation statements)

References 41 publications

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“…Although there is no information related to the effect of hypoxia on CK2, hypoxia is known to increase the levels of polyamines in the lung (34), which in turn are strong activators of CK2 (35,36). Our finding that the CK2 inhibitor partially blocks hypoxia-stimulated XDH/XO phosphorylation and hypoxia-stimulated XDH/XO activity strongly suggests a role for this kinase in mediating the effects of hypoxia on XDH/XO.…”

Section: Table I Percent Of Inhibition Of Xdh/xo Phospholabelingmentioning

confidence: 62%

Phosphorylation of Xanthine Dehydrogenase/Oxidase in Hypoxia

Kayyali

Donaldson

Huang

et al. 2001

Journal of Biological Chemistry

110

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The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre-and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Posttranslational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased (ϳ50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an ϳ4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.Xanthine dehydrogenase is the rate-limiting step in the catabolism of purines, where it catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid. In this reaction, XDH 1 utilizes NAD ϩ preferentially as the electron acceptor. However, when XDH is converted to XO, the preferred electron acceptor becomes molecular oxygen resulting in the formation of superoxide and hydrogen peroxide. This generation of reactive oxygen species is thought to be the basis of XDH/XO involvement in various pathological conditions such as ischemia-reperfusion injury. Reversible conversion of XDH to XO can occur after the oxidation of eight cysteine residues in the molecule into four cystines by agents such as pyrimidines or oxidized glutathione (1, 2). This conversion may be reversed upon the addition of reducing agents such as dithiothreitol. XDH can also be converted into XO irreversibly through proteolysis (3). Experimental proteolysis by trypsin has allowed the identification of three different parts of the molecule: a 20-kDa N-terminal fragment, a 40-kDa flavin-binding fragment, and an 80-kDa molybdopterin-binding fragment, all of which remain attached after proteolysis (3). It is believed that both reversible and irreversible conversion of XDH to XO are due to conformational changes in the molecule that...

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Section: Table I Percent Of Inhibition Of Xdh/xo Phospholabelingmentioning

confidence: 62%

Phosphorylation of Xanthine Dehydrogenase/Oxidase in Hypoxia

Kayyali

Donaldson

Huang

et al. 2001

Journal of Biological Chemistry

110

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“…Reduction of polyamine levels alters the association of ER with cellular proteins such that estradiol appears to be unable to activate gene expression and facilitate cell growth. Changes in the phosphorylation status of ER or other proteins may also contribute to the decrease in protein-protein interactions, since polyamines are known to increase the activity of casein kinase II (Cochet & Chambez 1983, Leroy et al 1997. However, exogenous addition of spermidine, prior to GST pull down assays, was able to restore ER association of proteins, suggesting that the differences in ER-associated proteins observed in our studies are mostly due to changes in the level of spermidine.…”

Section: Discussionmentioning

confidence: 59%

Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells

Shah¹,

Klinge²,

Faaland³

et al. 1999

Journal of Molecular Endocrinology

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We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 µM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells.

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“…After their uptake by enterocytes, polyamines may act directly on their own. Polyamine-protein interactions between the positive charges of spermine and the negative charges of acidic amino-acids have been described for the polyamine binding sites of some enzymes (45). But it seems unlikely that polyamines have a direct effect on the ␣-1,2-fucosyltransferase protein, because polyamines do not modify enzyme activity when added to an acellular medium in vitro (unpublished data).…”

Section: Discussionmentioning

confidence: 93%

Polyamine Participation in the Maturation of Glycoprotein Fucosylation, but not Sialylation, in Rat Small Intestine

Biol-N’garagba

2002

Pediatric Research

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The aim of this study was to determine the role of polyamines in the diet-related maturation of the intestinal glycoprotein glycosylation during postnatal development in the rat. The activity of ␣-2,6-sialyltransferase and the sialylated forms of glycoproteins in the intestinal brush-border membranes were found to decrease considerably after weaning, in parallel with the intestinal level of putrescine. By contrast, the activity of ␣-1,2-fucosyltransferases, the mRNA levels for two ␣-1,2-fucosyltransferase genes, FTA and FTB, and the fucosylated forms of glycoproteins all increased after weaning, in parallel with the levels of spermidine and spermine. These results suggest a possible role of polyamines in the evolution of glycosylation. The treatment of suckling rats with spermidine or spermine reproduced the high intestinal levels of these polyamines corresponding to those normally found after weaning. After these treatments, a rise in the activity of the ␣-1,2-fucosyltransferase was observed, associated with a fall in ␣-L-fucosidase activity. The ␣-1,2-fucosyltransferase FTB gene was found to be regulated at the transcriptional level, but not by its inhibitor, fuctinin. The result of these variations was the precocious appearance of several ␣-1,2-fucoproteins, which are normally found in brushborder membranes after weaning. The treatment of suckling rats with putrescine, which induced only a transitory rise in intestinal putrescine, had a similar but weaker effect on the fucosylation process than spermidine or spermine, and treatment with ornithine was ineffective. ␣-2,6-Sialylation was not affected by any of the treatments. Spermidine and spermine turned out to be more effective than putrescine for intestinal glycoprotein fucosylation, but did not affect their sialylation. Spermidine and spermine, whose intestinal levels where found to increase at weaning time, may have been partly responsible for the natural evolution of the intestinal glycoprotein fucosylation that occurred during this period. The rat small intestine is immature until the end of the third week of life, when the weaning period begins. Intestinal maturation is associated with morphologic changes, an increase in mucus production, immunologic adaptation to new microbial and nutritional antigenic contents (1), and digestive adaptation to new nutriments (2).Two types of glycoproteins play important functional roles in the small intestine. Mucins (secreted by the goblet cells) are different in composition between neonatal and mature rats, especially as regards their glycan chains (3). In the neonate, these changes may affect the permeability (4) and the barrier function of the intestine, and consequently may favor infections (5) and food allergies (6). At the apical enterocyte level, in the brush-border membranes, most of the digestive enzymes (lactase, sucrase, maltase, aminopeptidase, alkaline phosphatase) are glycoproteins whose activity changes a great deal at weaning time to enable the animal to cope with the adult solid diet (2). A shift from...

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Chemical Features of the Protein Kinase CK2 Polyamine Binding Site^,

Cited by 50 publications

References 41 publications

Phosphorylation of Xanthine Dehydrogenase/Oxidase in Hypoxia

Phosphorylation of Xanthine Dehydrogenase/Oxidase in Hypoxia

Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells

Polyamine Participation in the Maturation of Glycoprotein Fucosylation, but not Sialylation, in Rat Small Intestine

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Chemical Features of the Protein Kinase CK2 Polyamine Binding Site,

Cited by 50 publications

References 41 publications

Phosphorylation of Xanthine Dehydrogenase/Oxidase in Hypoxia

Phosphorylation of Xanthine Dehydrogenase/Oxidase in Hypoxia

Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells

Polyamine Participation in the Maturation of Glycoprotein Fucosylation, but not Sialylation, in Rat Small Intestine

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Chemical Features of the Protein Kinase CK2 Polyamine Binding Site^,