Processive Post-translational Modification

Morris, Daniel P.; Stevens, Robert; Wright, David J.; Stafford, Darrel W.

doi:10.1074/jbc.270.51.30491

Cited by 103 publications

(84 citation statements)

References 22 publications

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“…5B), only a limited amount of the fIX was shifted to the higher molecular mass form. This limited conversion is consistent with the results obtained by Morris et al (5); when bovine liver carboxylase was incubated with a fIX derivative substrate and the products were analyzed, most of the fIX product was undercarboxylated (i.e., 1-11 glas per mol fIX), and only a small amount of fully carboxylated fIX was observed (5). The contrast in fIX carboxylation that we observed in the two assays is consistent with differences in processivity, and these differences correlate with the carboxylation of the carboxylase.…”

Section: Discussionsupporting

confidence: 80%

“…This multiplicity raises the question of whether the carboxylase is a processive enzyme, i.e., effecting all modifications as a result of a single binding event. When purified bovine liver carboxylase was coincubated with an excess of a substrate derived from the factor IX (fIX) propeptide plus gla domain and the extent of fIX carboxylation was analyzed, some fully carboxylated fIX was observed among many intermediate forms, suggesting some degree of processivity (5).…”

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confidence: 99%

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Vitamin K-dependent carboxylation of the carboxylase

Berkner

Pudota

1998

Proc. Natl. Acad. Sci. U.S.A.

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Vitamin K-dependent (VKD) proteins require modification by the VKD-␥-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated in vitro with 14 CO 2 and then analyzed after SDS͞PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of in vitro-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla͞mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the in vitro reaction. Carboxylase carboxylation also was detected in baculovirus-(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated in vivo. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla͞mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation in vitro suggest a possible role for carboxylase carboxylation in factor IX turnover, and in vivo studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.Vitamin K-dependent (VKD) proteins undergo an unusual posttranslational modification required for their biological activity, namely the carboxylation of clusters of glu residues to ␥-glutamyl glus, or glas, in a region of the VKD proteins called the gla domain (1, 2). Carboxylation of the VKD proteins effects their Ca 2ϩ -mediated interaction with phospholipid bilayers and is carried out by an integral membrane endoplasmic reticulum enzyme, the VKD-␥-glutamyl carboxylase. The carboxylase modifies VKD substrates by using CO 2 , O 2 , and vitamin K hydroquinone as cofactors, and the carboxylase is also an epoxidase, converting the vitamin K hydroquinone to vitamin K 2,3-epoxide. Subsequent regeneration of the vitamin K hydroquinone cofactor is carried out by a reductase that has been characterized but not yet identified (3, 4). Carboxylation of the gla domain involves the modification of multiple glus, ranging from 3 to 12 for the different VKD proteins. This multiplicity raises the question of whether the carboxylase is a processive enzyme, i.e., effecting all modifications as a result of a single binding event. When purified bovine liver carboxylase was coincubated...

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Section: Discussionsupporting

confidence: 80%

mentioning

confidence: 99%

Vitamin K-dependent carboxylation of the carboxylase

Berkner

Pudota

1998

Proc. Natl. Acad. Sci. U.S.A.

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“…The activation of p38 MAPK by PKG as demonstrated here indicates a possible role for this enzyme in transcriptional regulation. This notion is supported by the finding that NO synthesis in several systems can cause cGMP-dependent alterations in transcription, including activation of immediate early gene expression (3,19,38). There are widespread reports that demonstrate transcriptional up-regulation of inducible nitric oxide synthase by cytokines, a process in which p38 MAPK activation is essential (39 -42).…”

Section: Discussionsupporting

confidence: 52%

Nitric Oxide Activation of p38 Mitogen-activated Protein Kinase in 293T Fibroblasts Requires cGMP-dependent Protein Kinase

Browning¹,

McShane

Marty

et al. 2000

Journal of Biological Chemistry

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An increase in cellular levels of cyclic nucleotides activates serine/threonine-dependent kinases that lead to diverse physiological effects. Recently we reported the activation of the p38 mitogen-activated protein kinase (MAPK) pathway in neutrophils by a cGMP-dependent mechanism. In this study we demonstrated that exogenously supplied nitric oxide leads to activation of p38 MAPK in 293T fibroblasts. Phosphorylation of p38 corresponded with an increase in ATF-2-dependent gene expression. The effect of nitric oxide was mimicked by addition of 8-bromo-cGMP, indicating that activation of soluble guanylyl cyclase was involved. The importance of cGMP-dependent protein kinase in the activation of p38 MAPK by nitric oxide in 293T cells was assessed in a transfection based assay. Overexpression of cGMP-dependent protein kinase-1␣ caused phosphorylation of p38 in these cells and potentiated the effectiveness of cGMP. Overexpression of a catalytically inactive mutant form of this enzyme (T516A) blocked the ability of both nitric oxide and 8-bromo-cGMP to activate p38 as measured by both p38 phosphorylation and ATF-2 driven gene expression. Together, these data demonstrate that nitric oxide stimulates a novel pathway leading to activation of p38 MAPK that requires activation of cGMPdependent protein kinase.Nitric oxide (NO) is an important signaling molecule that can affect many tissues to regulate diverse physiological processes, including smooth muscle relaxation, inflammation, platelet activation, apoptosis, and neuronal function (1-5). Several mechanisms have been described for NO function, including nitrosylation of key thiol groups of susceptible proteins, formation of reactive peroxynitrite, stimulation of ADP-ribosylation, and activation of soluble guanylyl cyclase (6 -12). Guanylyl cyclase converts available GTP into cGMP that behaves as a second messenger by activating cyclic nucleotide dependent protein kinases (12). Increases in either cAMP or cGMP are capable of activating both protein kinase A (PKA) 1 and protein kinase G (PKG), but the former is responsive to lower levels of cAMP and the latter is more sensitive to cGMP. In the past, the lower expression relative to PKA has made it difficult to identify processes regulated specifically by PKG, although specific inhibitors such as KT5823 have often been used to suggest the importance of this enzyme.It has been reported that some effects of NO, including activation of gene expression and apoptosis, may be mediated by mitogen activated kinases (MAPK) (12)(13)(14)(15)(16)(17)(18)(19)(20). The MAP-kinases including extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase (JNK) and p38 represent a family of proteins that are activated by phosphorylation by upstream MAPK kinases (MEKs), which are activated in response to growth factors, cytokines, or various forms of cellular stress (21-23). MAPKs regulate fundamental cellular processes such as growth and differentiation by activating transcription factors (24,25). Several studies have addressed th...

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“…Previous studies indicate that all or nearly all of the Glu residues to be carboxylated are modified during a single substrate binding event (9,10). The loss of as few as three carboxylations can markedly decrease the activities of vitamin K-dependent proteins (8).…”

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confidence: 99%

Binding of the Factor IX γ-Carboxyglutamic Acid Domain to the Vitamin K-dependent γ-Glutamyl Carboxylase Active Site Induces an Allosteric Effect That May Ensure Processive Carboxylation and Regulate the Release of Carboxylated Product

Lin

Straight

Stafford

2004

Journal of Biological Chemistry

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Propeptides of the vitamin K-dependent proteins bind to an exosite on ␥-glutamyl carboxylase; while they are bound, multiple glutamic acids in the ␥-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.The vitamin K-dependent ␥-glutamyl carboxylase is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the post-translational modification of specific glutamic acid residues to ␥-carboxylglutamic acid in a number of vitamin K-dependent proteins. In these vitamin K-dependent proteins, multiple glutamic acid residues in the amino-terminal Gla 1 domain are modified (1-3). The existence of these multiple ␥-carboxylglutamic acid residues allows the Gla domain to form the calcium-dependent conformation required for the activity of these vitamin K-dependent proteins (4, 5). Under-carboxylated vitamin K-dependent proteins cannot form this calcium-dependent structure and, as a result, possess poor affinities for phospholipid surfaces, endothelial cells, or activated platelets (6 -8).Previous studies indicate that all or nearly all of the Glu residues to be carboxylated are modified during a single substrate binding event (9, 10). The loss of as few as three carboxylations can markedly decrease the activities of vitamin K-dependent proteins (8). Therefore, because the most frequently used anticoagulant, warfarin, causes under-carboxylation by reducing vitamin K concentration, understanding the mechanism by which processivity yields functional enzymes is important.A tethered model has been proposed to account for how carboxylase accomplishes full carboxylation of pro-factor IX (9, 11). In this hypothesis, the primary interaction between carboxylase and its substrates is mediated by a propeptide sequence of 18 amino acids. Presumably, the propeptide anchors the Gla domain of the substrate near the carboxylase active site for a time sufficient for modification of all or most of the Glu residues of the Gla domain.Several lines of evidence are consistent with the idea that full carboxylation is the result of a stochastic, processive mechanism, i.e. the degree of carboxylation is controlled by the balance between the carboxylation rate of the substrate and the dissociation rate of the product. First, bone Gla protein, whose propeptide has an extremely low affinity (K i Ͼ 500 M) for the carboxylase and therefore a rapid dis...

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Processive Post-translational Modification

Cited by 103 publications

References 22 publications

Vitamin K-dependent carboxylation of the carboxylase

Vitamin K-dependent carboxylation of the carboxylase

Nitric Oxide Activation of p38 Mitogen-activated Protein Kinase in 293T Fibroblasts Requires cGMP-dependent Protein Kinase

Binding of the Factor IX γ-Carboxyglutamic Acid Domain to the Vitamin K-dependent γ-Glutamyl Carboxylase Active Site Induces an Allosteric Effect That May Ensure Processive Carboxylation and Regulate the Release of Carboxylated Product

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