Cloning and expression in <i>Escherichia coli</i> of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase

Takazawa, Kazunaga; Vandekerckhove, Joël; Dumont, Jacques Emile; Erneux, Christophé

doi:10.1042/bj2720107

Cited by 89 publications

(82 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the range of apparent half-maximal substrate concentration (about 1 M), 9 M of the product, Ins(1,3,4,5)P 4 , was present, acting as a competitive type of product inhibitor. Although the derived K m Ј values thus were higher than the true K m (ϳ0.1 M), the assay was adequate to rapidly analyze influence of inhibitors on the K m for Ins(1,4,5)P 3 .…”

Section: Methodsmentioning

confidence: 99%

Antiproliferative Plant and Synthetic Polyphenolics Are Specific Inhibitors of Vertebrate Inositol-1,4,5-trisphosphate 3-Kinases and Inositol Polyphosphate Multikinase

Mayr

Windhorst

Hillemeier

2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

1 an enzyme ubiquitously found in all mammalian tissues (1), plays a central role in the anabolic routes of inositol phosphates in vertebrate cells because it phosphorylates the second messenger Ins(1,4,5)P 3 as the starting substrate of these reactions. The IP3K family consists of three isoenzymes referred to as IP3K-A (2-4), IP3K-B (5), and IP3K-C (6) differing in their degree of activation by Ca 2ϩ -CaM, affinity for Ins(1,4,5)P 3 (reviewed in Ref. 7), tissue and intracellular distribution. IP3K-B is expressed in nearly all human tissues and is localized at actin filaments (8, 9) and the endoplasmic reticulum (9, 10) whereas IP3K-A was identified mainly in brain and testis (9, 11) and shows an exclusive F-actin localization (12). IP3K-C is also widely expressed in mammalian cells and in rat it was shown to shuttle actively between nucleus and cytoplasm (13). An additional Ins(1,4,5)P 3 kinase enzyme, inositol polyphosphate multikinase (IPMK), was identified in yeast, rat, man, and plants (14 -18). IPMK exhibits nuclear localization and evidently plays a role in nuclear InsP metabolism (16). In vertebrates, the Ins(1,3,4,5)P 4 product of these enzymes is the starting substrate to be metabolized by different InsP kinases and phosphatases leading to Ins(1,3,4,5,6)P 5 , InsP 6 and the pyrophosphorylated isoforms of InsP 5 and InsP 6 (19,20). InsP 6 is normally the most abundant InsP in eukaryotic cells and appears to be important in several processes; in yeast it is essential for intact vesicle structures (21), mRNA export (22, 23) and is involved in chromatin remodeling (24). For mammalian cells a stimulation of DNA repair (25), endocytosis (26), and exocytosis (27) in pancreatic ␤-cells, Ca 2ϩ channel activity (28, 29), an involvement in synaptic vesicle trafficking (30,31), and other effects were reported for InsP 6 (reviewed in Refs. 19 and 32).To ease studies of the importance of higher InsPs for cell proliferation or other cellular functions in vivo, one possible approach is to block their biosynthesis by pharmacological inhibition of IP3K isoforms and IPMK. Other approaches are knock-out or RNAi experiments shutting down enzymes generating Ins(1,3,4,5)P 4 or higher InsPs. An individual knock-out of IP3K-A in mice resulted in a very weak CNS phenotype in the former case (34). IP3K-B knockout revealed an essential role of the enzyme or its products for T-cell immunity (35). That deletion of the broadly expressed IP3K-B did not generate further phenotypes may be caused by rescue phenomena based on the fact that a total of four enzymes are able to convert Ins(1,4,5)P 3 to Ins(1,3,4,5)P 4 . These considerations led us to start a search for pharmacological IP3K and IPMK inhibitors. We first examined herbal and synthetic compounds belonging to the flavonoids, anthraquinones, coumarins, triphenylmeth-* This work was supported by Grants Ma989 and GRK 336 (to G. W. M.) from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This articl...

show abstract

Section: Methodsmentioning

confidence: 99%

Antiproliferative Plant and Synthetic Polyphenolics Are Specific Inhibitors of Vertebrate Inositol-1,4,5-trisphosphate 3-Kinases and Inositol Polyphosphate Multikinase

Mayr

Windhorst

Hillemeier

2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…isoform was stimulated up to 10-fold [8,9,16]. The specificity of Ins(1,4,5)P $ phosphorylation by recombinant Ins(1,4,5)P $ 3-kinases A and B at the 3-position of the inositol ring has been resolved by HPLC [17][18][19].…”

Section: Introductionmentioning

confidence: 99%

Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C

Dewaste

Pouillon

Moreau

et al. 2000

Biochemical Journal

View full text Add to dashboard Cite

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P3 to Ins(1,3,4,5)P4. cDNAs encoding two isoenzymes of Ins(1,4,5)P3 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P3 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75.207kDa and a Km for Ins(1,4,5)P3 of 6µM. Northern-blot analysis showed the presence of a transcript of approx. 3.9kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5αF′ bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P3 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin–Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P3 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin–Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin.

show abstract

“…After a 30-min centrifugation at 15,000 ϫ g, the supernatant was applied to a CaM-affinity column which was eluted as described in Ref. 20 P]ATP (3000 Ci/mmol), and 20 l of enzyme, was incubated for 1 h at 37°C, and the reaction was terminated by the addition of 0.8 ml of 10 mM EDTA followed by 2 min of boiling. The sample was applied to a HPLC Partisphere-SAX column, eluted with a nonlinear gradient made of water and 1.0 M NH 4 H 2 PO 4 (pH 3.8).…”

mentioning

confidence: 99%

A Novel, Rapid, and Highly Sensitive Mass Assay for Phosphatidylinositol 3,4,5-Trisphosphate (PtdIns(3,4,5)P3) and Its Application to Measure Insulin-stimulated PtdIns(3,4,5)P3 Production in Rat Skeletal Muscle in Vivo

Kaay¹,

Batty²,

Cross³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

The pivotal role of phosphatidylinositol 3-kinase (PI 3-kinase) in signal transduction has been well established in recent years. Receptor-regulated forms of PI 3-kinase are thought to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) at the 3-position of the inositol ring to give the putative lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ). Cellular levels of PtdIns(3,4,5)P 3 are currently measured by time-consuming procedures involving radiolabeling with high levels of 32 PO 4 , extraction, and multiple chromatography steps. To avoid these lengthy and hazardous procedures, many laboratories prefer to assay PI 3-kinase activity in cell extracts and/or appropriate immunoprecipitates. Such approaches are not readily applied to measurements of PtdIns(3,4,5)P 3 in extracts of animal tissues. Moreover, they can be misleading since the association of PI 3-kinases in molecular complexes is not necessarily correlated with the enzyme's activity state. Direct measurements of PtdIns(3,4,5)P 3 would also be desirable since its concentration may be subject to additional control mechanisms such as activation or inhibition of the phosphatases responsible for PtdIns(3,4,5)P 3 metabolism. We now report a simple, reproducible isotope dilution assay which detects PtdIns(3,4,5)P 3 at subpicomole sensitivity, suitable for measurements of both basal and stimulated levels of PtdIns(3,4,5)P 3 obtained from samples containing approximately 1 mg of cellular protein. Total lipid extracts, containing PtdIns(3,4,5)P 3 , are first subjected to alkaline hydrolysis which results in the release of the polar head group Ins(1,3,4,5)P 4 . The latter is measured by its ability to displace [ 32 P]Ins(1,3,4,5)P 4 from a highly specific binding protein present in cerebellar membrane preparations. We show that this assay solely detects PtdIns(3,4,5)P 3 and does not suffer from interference by other compounds generated after alkaline hydrolysis of total cellular lipids. Measurements on a wide range of cells, including rat-1 fibroblasts, 1321N1 astrocytoma cells, HEK 293 cells, and rat adipocytes, show wortmannin-sensitive increased levels of PtdIns(3,4,5)P 3 upon stimulation with appropriate agonists. The enhanced utility of this procedure is further demonstrated by measurements of PtdIns(3,4,5)P 3 levels in tissue derived from whole animals. Specifically, we show that stimulation with insulin increases PtdIns(3,4,5)P 3 levels in rat skeletal muscle in vivo with a time course which parallels the activation of protein kinase B in the same samples.PI 3-kinases 1 represent a family of enzymes which phosphorylate phosphoinositides on the 3-position of the inositol ring (1). The role of PI 3-kinases in mitogenic signaling, membrane ruffling, trafficking, cell motility, inflammatory and immune cell responses, activation of neutrophils, and the metabolic effects of insulin (2-8) has been well established using biochemical, pharmacological, and genetic approaches. At least two major classes of PI 3-kinas...

show abstract

Cloning and expression in Escherichia coli of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase

Cited by 89 publications

References 27 publications

Antiproliferative Plant and Synthetic Polyphenolics Are Specific Inhibitors of Vertebrate Inositol-1,4,5-trisphosphate 3-Kinases and Inositol Polyphosphate Multikinase

Antiproliferative Plant and Synthetic Polyphenolics Are Specific Inhibitors of Vertebrate Inositol-1,4,5-trisphosphate 3-Kinases and Inositol Polyphosphate Multikinase

Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C

A Novel, Rapid, and Highly Sensitive Mass Assay for Phosphatidylinositol 3,4,5-Trisphosphate (PtdIns(3,4,5)P3) and Its Application to Measure Insulin-stimulated PtdIns(3,4,5)P3 Production in Rat Skeletal Muscle in Vivo

Contact Info

Product

Resources

About