scholarly journals The regulatory protein of glucokinase binds to the hepatocyte matrix, but, unlike glucokinase, does not translocate during substrate stimulation

1995 ◽  
Vol 309 (3) ◽  
pp. 711-713 ◽  
Author(s):  
L Agius ◽  
M Peak ◽  
E Van Schaftingen
Keyword(s):  
Binding Site ◽  
Regulatory Protein ◽  
Kinetic Properties ◽  
Cell Matrix ◽  
Elevated Glucose ◽  
Dependent Mechanism

The kinetic properties of hepatic glucokinase (hexokinase IV) are modulated by binding to a regulatory protein. This study shows that, in hepatocytes incubated with 5 mM glucose as sole carbohydrate substrate, both glucokinase and its regulatory protein bind to the cell matrix by a Mg(2+)-dependent mechanism. After incubation with an elevated [glucose] or with fructose, glucokinase, but not its regulatory protein, translocates from the Mg(2+)-dependent binding site. It is suggested that the regulatory protein acts as a receptor for anchoring glucokinase to the hepatocyte matrix and inhibiting its activity in metabolically quiescent conditions.

scholarly journals Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site.

1987 ◽  
Vol 7 (12) ◽  
pp. 4400-4406 ◽  
Author(s):  
K D Breunig ◽  
P Kuger
Keyword(s):  
Protein Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Protein Binding Site ◽  
Regulatory Sequence ◽  
Functional Homology ◽  
Beta Galactosidase

As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M. Ruzzi, K.D. Breunig, A.G. Ficca, and C.P. Hollenberg, Mol. Cell. Biol. 7:991-997, 1987). Here we demonstrate that the region of homology specifically binds a K. lactis regulatory protein. The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography. These extracts could be used directly for DNase I and exonuclease III protection experiments. A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor. The homology of LAC9 protein with GAL4 (J.M. Salmeron and S. A. Johnston, Nucleic Acids Res. 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription.

scholarly journals Assessment of the Role of Activator Protein-1 on Transcription of the Mouse Steroidogenic Acute Regulatory Protein Gene

2004 ◽  
Vol 18 (3) ◽  
pp. 558-573 ◽  
Author(s):  
Pulak R. Manna ◽  
Darrell W. Eubank ◽  
Douglas M. Stocco
Keyword(s):  
Binding Site ◽  
Gene Transcription ◽  
Dna Sequences ◽  
Regulatory Protein ◽  
Activator Protein 1 ◽  
Transcriptional Responses ◽  
Activator Protein ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene

Abstract cAMP-dependent mechanisms regulate the steroidogenic acute regulatory (StAR) protein even though its promoter lacks a consensus cAMP response-element (CRE, TGACGTCA). Transcriptional regulation of the StAR gene has been demonstrated to involve combinations of DNA sequences that provide recognition motifs for sequence-specific transcription factors. We recently identified and characterized three canonical 5′-CRE half-sites within the cAMP-responsive region (−151/−1 bp) of the mouse StAR gene. Among these CRE elements, the CRE2 half-site is analogous (TGACTGA) to an activator protein-1 (AP-1) sequence [TGA(C/G)TCA]; therefore, the role of the AP-1 transcription factor was explored in StAR gene transcription. Mutation in the AP-1 element demonstrated an approximately 50% decrease in StAR reporter activity. Using EMSA, oligonucleotide probes containing an AP-1 binding site were found to specifically bind to nuclear proteins obtained from mouse MA-10 Leydig and Y-1 adrenocortical tumor cells. The integrity of the sequence-specific AP-1 element in StAR gene transcription was assessed using the AP-1 family members, Fos (c-Fos, Fra-1, Fra-2, and Fos B) and Jun (c-Jun, Jun B, and Jun D), which demonstrated the involvement of Fos and Jun in StAR gene transcription to varying degrees. Disruption of the AP-1 binding site reversed the transcriptional responses seen with Fos and Jun. EMSA studies utilizing antibodies specific to Fos and Jun demonstrated the involvement of several AP-1 family proteins. Functional assessment of Fos and Jun was further demonstrated by transfecting antisense c-Fos, Fra-1, and dominant negative forms of Fos (A-Fos) and c-Jun (TAM-67) into MA-10 cells, which significantly (P < 0.01) repressed transcription of the StAR gene. Mutation of the AP-1 site in combination with mutations in other cis-elements resulted in a further decrease of StAR promoter activity, demonstrating a functional cooperation between these factors. Mammalian two-hybrid assays revealed high-affinity protein-protein interactions between c-Fos and c-Jun with steroidogenic factor 1, GATA-4, and CCAAT/enhancer binding protein-β. These findings demonstrate that Fos and Jun can bind to the TGACTGA element in the StAR promoter and provide novel insights into the mechanisms regulating StAR gene transcription.

scholarly journals Synergistic activation of ADH2 expression is sensitive to upstream activation sequence 2 (UAS2) orientation, copy number and UAS1-UAS2 helical phasing.

1995 ◽  
Vol 15 (6) ◽  
pp. 3442-3449 ◽  
Author(s):  
M S Donoviel ◽  
N Kacherovsky ◽  
E T Young
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Reporter Gene ◽  
Copy Number ◽  
Glucose Repression ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Upstream Regulatory Region ◽  
Specific Sequence

The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression. Two cis-acting upstream activation sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified. UAS1 is the binding site for the transcriptional regulator Adr1p. UAS2 has been shown to be important for ADH2 expression and confers glucose-regulated, ADR1-independent activity to a heterologous reporter gene. An analysis of point mutations within UAS2, in the context of the entire ADH2 upstream regulatory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein. In the context of the ADH2 upstream regulatory region, including UAS1, working in concert with the ADH2 basal promoter elements, UAS2-dependent gene activation was dependent on orientation, copy number, and helix phase. Multimerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression. In contrast, UAS2-dependent expression of a reporter gene containing the ADH2 basal promoter and coding sequence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation. The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1. Inhibition of UAS2-dependent gene expression by Adr1p was also observed with a UAS2-dependent ADH2 reporter gene. This inhibition increased with ADR1 copy number and required the DNA-binding activity of Adr1p. Specific but low-affinity binding of Adr1p to UAS2 in vitro was demonstrated, suggesting that the inhibition of UAS2-dependent gene expression observed in vivo could be a direct effect due to Adr1p binding to UAS2.

Different mechanisms in the attachment of cells to native and denatured collagen

1979 ◽  
Vol 38 (1) ◽  
pp. 267-281
Author(s):  
S.L. Schor ◽  
J. Court
Keyword(s):  
Cell Attachment ◽  
Experimental Conditions ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Independent Mechanism ◽  
Cell Matrix Interactions ◽  
Serum Dependent ◽  
Kinetics Of ◽  
Dependent Mechanism

The attachment of cells to collagen has been reported previously to require the presence of serum and the particular serum protein involved in this process, variously known as CIG, CAP or fibronectin, has been isolated. This conclusion that cell attachment to collagen requires serum (or more precisely, fibronectin) is based on experiments measuring the kinetics of cell attachment to films of collagen. We have measured the kinetics of attachment of HeLa and attachment to films of collagen-containing substrata under a variety of experimental conditions and present evidence that the serum-dependent mechanism of cell attachment described by others is actually only the case for films of denatured collagen, while cell attachment to native collagen fibres occurs by a different, serum-independent, mechanism. The possible relevance of these findings to cell-matrix interactions in vivo is discussed.

scholarly journals The mechanism by which the human apolipoprotein B gene reducer operates involves blocking of transcriptional activation by hepatocyte nuclear factor 3.

1993 ◽  
Vol 13 (3) ◽  
pp. 1534-1546 ◽  
Author(s):  
B Paulweber ◽  
F Sandhofer ◽  
B Levy-Wilson
Keyword(s):  
Binding Site ◽  
Transcriptional Activation ◽  
Apolipoprotein B ◽  
Regulatory Protein ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Apo B ◽  
Human Apolipoprotein ◽  
Colon Carcinoma Cells ◽  
Mediator Protein

Previously, we showed that when a DNA fragment extending from -3067 to -2734 of the human apolipoprotein B (apo-B) gene is inserted immediately upstream of an apo-B promoter segment (-139 to +121), transcription from this promoter is reduced by about 10-fold in cultured colon carcinoma cells (CaCo-2) but not in cultured hepatoma cells (HepG2). We postulated that this reducer operates by a mechanism involving active repression of a transcriptional activator that binds to the segment from -111 to -88 of the apo-B promoter (B. Paulweber and B. Levy-Wilson, J. Biol. Chem. 266:24161-24168 1991). In the current study, the reducer element has been localized to a 24-bp sequence from -2801 to -2778 of the apo-B gene that contains a binding site for the negative regulatory protein ARP-1. Furthermore, we have demonstrated that the transcription factor hepatocyte nuclear factor 3 alpha (HNF-3 alpha) binds to the sequence 5'-TGTTTGCTTTTC-3' from -95 to -106 of the apo-B promoter, to stimulate transcription. Transcriptional activation by HNF-3 is repressed when the reducer sequence is inserted immediately upstream of the HNF-3 binding site, suggesting a mechanism by which the reducer-bound protein blocks the activation promoted by HNF-3. Data from cotransfection experiments in which ARP-1 is overexpressed in the absence of its binding site suggest that ARP-1 interacts either directly or via a mediator protein with proteins recognizing the HNF-3 site and that this interaction is sufficient to repress transcriptional activation by HNF-3. Because transcriptional activation by Sp1 is not affected by the reducer, it is unlikely that the reducer interacts directly with basic components of the transcriptional machinery.

scholarly journals Specific Features for the Competent Binding of Substrates at the FMN Adenylyltransferase Site of FAD Synthase from Corynebacterium ammoniagenes

2019 ◽  
Vol 20 (20) ◽  
pp. 5083 ◽  
Author(s):  
Sonia Arilla-Luna ◽  
Ana Serrano ◽  
Milagros Medina
Keyword(s):  
Binding Site ◽  
Thermodynamic Parameters ◽  
Spectroscopic Properties ◽  
Open Cavity ◽  
Flavin Mononucleotide ◽  
Kinetic Properties ◽  
Biophysical Properties ◽  
Conserved Motif ◽  
Corynebacterium Ammoniagenes ◽  
Optimal Geometry

Bifunctional FAD synthases (FADSs) catalyze FMN (flavin mononucleotide) and FAD (flavinadenine dinucleotide) biosynthesis at their C-riboflavin kinase (RFK) and N-FMN:adenylyltransferase (FMNAT) modules, respectively. Biophysical properties and requirements for their FMNAT activity differ among species. Here, we evaluate the relevance of the integrity of the binding site of the isoalloxazine of flavinic substrates for FMNAT catalysis in Corynebacterium ammoniagenes FADS (CaFADS). We have substituted P56 and P58, belonging to a conserved motif, as well as L98. These residues shape the isoalloxazine FMNAT site, although they are not expected to directly contact it. All substitutions override enzyme ability to transform substrates at the FMNAT site, although most variants are able to bind them. Spectroscopic properties and thermodynamic parameters for the binding of ligands indicate that mutations alter their interaction modes. Substitutions also modulate binding and kinetic properties at the RFK site, evidencing the crosstalk of different protomers within CaFADS assemblies during catalysis. In conclusion, despite the FMNAT site for the binding of substrates in CaFADS appearing as a wide open cavity, it is finely tuned to provide the competent binding conformation of substrates. In particular, P56, P58 and L98 shape the isoalloxazine site to place the FMN- and FAD-reacting phosphates in optimal geometry for catalysis.

The uptake of atropine and related drugs by intestinal smooth muscle of the guinea-pig in relation to acetylcholine receptors

1965 ◽  
Vol 163 (990) ◽  
pp. 1-44 ◽  
Keyword(s):  
Smooth Muscle ◽  
Binding Site ◽  
Equilibrium Constant ◽  
Rate Constant ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Kinetic Properties ◽  
Kinetic Behaviour ◽  
Kinetics Of ◽  
Bound Drug

In an attempt to study the properties of acetylcholine receptors in intestinal smooth muscle, measurements have been made of the uptake of tritium-labelled atropine and methylatropinium, and of 14 C-labelled methylfurmethide by the longitudinal muscle of guinea-pig small intestine in vitro . Substantial amounts of atropine were taken up from very dilute solutions, a clearance of 160 ml. per g tissue (wet weight) being achieved at the lowest concentration tested (1.5 × 10 -10 M). Analysis of the curve relating atropine uptake at equilibrium to the bath concentration, which was explored over a concentration range 1.5 × 10 -10 M to 2.5 × 10 -3 M, enabled three components to be distinguished: (1) A binding site with a capacity of 180 pmoles/g, and equilibrium constant 1.1 × 10 -9 M. (2) A binding site of capacity about 1000 pmoles/g and equilibrium constant about 5 × 10 -7 M. (3) A compartment with a clearance of 4.7 ml./g (nonsaturable). The equilibrium constant of the first binding site agreed exactly with that measured for acetylcholine antagonism in the same tissue. Methylatropinium was taken up in rather smaller amounts than atropine, and analysis of the uptake curve showed a binding site of capacity about 90 pmoles/g with an equilibrium constant 6.5 × 10 -10 M, an ill-defined series of binding sites with much higher equilibrium constants, and a constant clearance of about 0.4 ml. /g. Analysis of this curve was much less clear cut than that of atropine. The equilibrium constant for blockade of acetylcholine receptors by methylatropinium was 4.7 × 10 -10 M. Atropine was not taken up appreciably by striated muscle, nerve or tendon of the guineapig; hydrolysed atropine was not taken up by smooth muscle (and lacks atropinic activity); cocaine and d -tubocurarine in high concentrations did not affect atropine uptake; lachesine and benzhexol blocked atropine uptake competitively at low concentrations, and with lachesine the equilibrium constant for this interaction agreed with that measured for acetylcholine antagonism (1.4 × 10 -9 M). These findings suggested that the atropine taken up could be related to receptor-bound drug. The kinetics of atropine uptake and washout were studied over the concentration range 0.5-5 × 10 -9 M. Uptake and washout took place approximately exponentially between 2½ and 50 min, and the rate constant was 4.5-5 × 10 -4 s -1 for both uptake and washout. The uptake rate constant did not increase with concentration. This contrasted with the kinetics of receptor blockade, which took place much faster, with a rate constant which increased linearly with concentration, in accordance with the theoretical kinetic behaviour of a single binding site. This finding precluded a simple identification of atropine taken up with receptor-bound drug. Studies with various metabolic inhibitors suggested that no metabolic energy was required for the accumulation of atropine, and by dialysis experiments, the atropine taken up was shown to be bound in homogenized tissue. A theoretical study, using an analogue computer, was made of the kinetic properties of three passive binding systems, in order to see whether the observed kinetic behaviour could be simulated. It was found that a system of four binding sites in series, with only one communicating directly with the surrounding medium, could show these kinetic properties, and the outermost binding site could still show the kinetic behaviour of receptors. Experimental testing of this model demands more accurate kinetic measurements than can be made by the method used in this study. The acetylcholine-like stimulant, methylfurmethide, was taken up very slowly (taking more than 24 h to reach equilibrium), reaching a clearance of about 5 ml. /g after 6 h. This uptake was unaffected by atropine in a concentration sufficient to block 80% of acetylcholine receptors, but was blocked by depolarization in high potassium solution, suggesting that it was behaving passively as a slowly permeant cation. No uptake referable to acetylcholine receptors was detected. These findings are discussed in relation to the abundance and chemical behaviour of acetylcholine receptors in smooth muscle, and in relation to current theories of drug action.

Inhibition of glucokinase translocation by AMP-activated protein kinase is associated with phosphorylation of both GKRP and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

2008 ◽  
Vol 294 (3) ◽  
pp. R766-R774 ◽  
Author(s):  
Mohammed H. Mukhtar ◽  
Victoria A. Payne ◽  
Catherine Arden ◽  
Andrew Harbottle ◽  
Salmaan Khan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Regulatory Protein ◽  
Subcellular Location ◽  
Inhibitory Effect ◽  
Maximal Effect ◽  
Glucokinase Activator ◽  
Elevated Glucose ◽  
Glucose Phosphorylation ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The rate of glucose phosphorylation in hepatocytes is determined by the subcellular location of glucokinase and by its association with its regulatory protein (GKRP) in the nucleus. Elevated glucose concentrations and precursors of fructose 1-phosphate (e.g., sorbitol) cause dissociation of glucokinase from GKRP and translocation to the cytoplasm. In this study, we investigated the counter-regulation of substrate-induced translocation by AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), which is metabolized by hepatocytes to an AMP analog, and causes activation of AMP-activated protein kinase (AMPK) and depletion of ATP. During incubation of hepatocytes with 25 mM glucose, AICAR concentrations below 200 μM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100–140 μM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR also counteracted translocation induced by a glucokinase activator and partially counteracted translocation by sorbitol. However, AICAR did not block the reversal of translocation (from cytoplasm to nucleus) after substrate withdrawal. Inhibition of glucose-induced translocation by AICAR was greater than inhibition by glucagon and was associated with phosphorylation of both GKRP and the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32. Expression of a kinase-active PFK2 variant lacking ser-32 partially reversed the inhibition of translocation by AICAR. Phosphorylation of GKRP by AMPK partially counteracted its inhibitory effect on glucokinase activity, suggesting altered interaction of glucokinase and GKRP. In summary, mechanisms downstream of AMPK activation, involving phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and GKRP are involved in the ATP-independent inhibition of glucose-induced glucokinase translocation by AICAR in hepatocytes.

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