Clonage moléculaire des isoformes B et C de l'inositol 1, 4, 5-trisphosphate 3-kinase et influence de leur surexpression sur la réponse calcique

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Clonage moléculaire des isoformes B et C de l'inositol 1, 4, 5-trisphosphate 3-kinase et influence de leur surexpression sur la réponse calcique

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Inositol 1, 4, 5-trisphosphate 3-kinase and 5-phosphatase

info:eu-repo/semantics/publishe

Inositol 1, 4, 5-trisphosphate 3-kinase and 5-phosphatase

info:eu-repo/semantics/publishe

Calcium-Calmodulin-dependent protein kinase II and protein kinase C-mediated phosphorylation and activation of D-myo-inositol 1,4,5-trisphosphate 3-kinase B in astrocytes

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase catalyzes the production of D-myo-inositol 1,3,4,5-tetrakisphosphate from the second messenger Ins (1,4,5)P3. Transient and okadaic acid-sensitive activation of Ins(1,4,5)P3 3-kinase by 8-10-fold is observed in homogenates prepared from rat cortical astrocytes after incubation with either carbachol or UTP. 12-O-Tetradecanoylphorbol-13-acetate provokes the activation of Ins(1,4,5)P3 3-kinase by 2-fold in both cell systems. The kinase was purified by calmodulin-Sepharose from the two cell systems. Enzyme activity corresponding to the silver-stained 88-kDa protein could be regenerated after SDS-polyacrylamide gel electrophoresis. Antibodies to two distinct peptides chosen in the primary structure of human Ins(1,4,5)P3 3-kinase B recognized the astrocytic native isoform. In [32P]orthophosphate-preincubated cells, a major phosphorylated 88-kDa enzyme could be purified and identified in cells in response to receptor activation or 12-O-tetradecanoylphorbol-13-acetate treatment. Calmodulin kinase II inhibitors (i.e. KN-93 and KN-62) and a protein kinase C inhibitor (i.e. calphostin C) prevented the phosphorylation of the 88-kDa isoenzyme. In addition to enzyme activation, a redistribution of Ins(1,4,5)P3 3-kinase from soluble to particulate fraction of astrocytes was observed. In vitro phosphorylation of the purified enzyme by calmodulin kinase II and protein kinase C added together resulted in a maximal 60-70-fold activation.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Inositol polyphosphate kinase activity of Arg82/ArgRIII is not required for the regulation of the arginine metabolism in yeast

Arg82, a nuclear regulator of diverse cellular processes in yeast, is an inositol polyphosphate kinase. Some defects such as the regulation of arginine metabolism observed in an arg82Δ, result from a lack of Mcm1 and Arg80 stability. We show here that neither the kinase activity of Arg82 nor inositol phosphates are required for the control of arginine metabolism. Arg82 mutations keeping kinase active affect the expression of arginine genes, whereas mutations in the kinase domain do not impair this metabolic control. Copyright (C) 2000 Federation of European Biochemical Societies.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

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

Inositol 1,4,5-trisphosphate (InsP(3)) 3-kinase catalyzes the phosphorylation of InsP(3) to inositol 1,3,4,5-tetrakisphosphate (InsP(4)). cDNAs encoding three isoenzymes of InsP(3) 3-kinase (3-kinases A, B, and C) have been previously reported; however, a demonstrably full-length cDNA encoding human InsP(3) 3-kinase B was still lacking. Here we report the cloning of a full-length 2841-bp cDNA encoding human InsP(3) 3-kinase B. Northern blot analysis shows the presence of an ubiquitous transcript of approximately 7.2 kb in a large number of human tissues. InsP(3) 3-kinase activity measured in COS-7 cells transfected with InsP(3) 3-kinase B shows an activity that was 8-fold increased upon the addition of Ca(2+)/calmodulin in the assay mixture.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Inositol 1,3,4,5-tetrakisphosphate is essential for T lymphocyte development.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) is phosphorylated by Ins(1,4,5)P(3) 3-kinase, generating inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). The physiological function of Ins(1,3,4,5)P(4) is still unclear, but it has been reported to be a potential modulator of calcium mobilization. Disruption of the gene encoding the ubiquitously expressed Ins(1,4,5)P(3) 3-kinase isoform B (Itpkb) in mice caused a severe T cell deficiency due to major alterations in thymocyte responsiveness and selection. However, we were unable to detect substantial defects in Ins(1,4,5)P(3) amounts or calcium mobilization in Itpkb(-/-) thymocytes. These data indicate that Itpkb and Ins(1,3,4,5)P(4) define an essential signaling pathway for T cell precursor responsiveness and development.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

The three isoenzymes of human inositol-1,4,5-trisphosphate 3-kinase show specific intracellular localization but comparable Ca2+ responses on transfection in COS-7 cells.

Inositol 1,4,5-trisphosphate [Ins(1,4,5) P3] 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate. cDNAs encoding three human isoenzymes of InsP3 3-kinase (A, B and C) have been reported previously [Choi, Kim, Lee, Moon, Sim, Kim, Chung and Rhee (1990) Science 248, 64-66; Dewaste, Pouillon, Moreau, Shears, Takazawa and Erneux (2000) Biochem. J. 352, 343-351; Dewaste, Roymans, Moreau and Erneux (2002) Biochem. Biophys. Res. Commun. 291, 400-405; Takazawa, Perret, Dumont and Erneux (1991) Biochem. Biophys. Res. Commun. 174, 529-535]. The localization of InsP3 3-kinase isoenzymes fused at their N-terminus to the green fluorescent protein has been studied by confocal microscopy. The A isoform appeared to associate with the cytoskeleton, whereas the C isoform was totally cytoplasmic. The B isoform had a more complex localization: it appeared in the plasma membrane, cytoskeleton and in the endoplasmic reticulum. The three human isoenzymes of InsP3 3-kinase can thus be distinguished by their N-terminal sequence, sensitivity to Ca2+/calmodulin and localization on transfection in COS-7 cells. We have compared the cytosolic Ca2+ responses induced by ATP in COS-7 cells transfected with the three isoenzymes. Cells expressing high levels of any of the three isoforms no longer respond to ATP, whereas cells expressing low levels of each enzyme showed a reduced response consisting of one to three Ca2+ spikes in response to 100 microM ATP. These effects were seen only in wild-type InsP3 3-kinase-transfected cells. 3-Kinase-dead mutant cells behaved as vector-transfected cells. The results highlight the potential role of the three isoforms of InsP3 3-kinase as direct InsP3 metabolizing enzymes and direct regulators of Ca2+ responses to extracellular signals