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
Autor: | Françoise Bex, Colette Moreau, Ludwig Missiaen, Christophe Erneux, Humbert De Smedt, Florence De Smedt, Frank Wuytack, Valérie Dewaste |
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Rok vydání: | 2003 |
Předmět: |
Gene isoform
Calmodulin Recombinant Fusion Proteins Green Fluorescent Proteins CHO Cells Endoplasmic Reticulum Transfection Biochemistry Cell membrane chemistry.chemical_compound Cricetinae Chlorocebus aethiops medicine Animals Humans Inositol Molecular Biology Cytoskeleton DNA Primers COS cells Base Sequence biology Kinase Endoplasmic reticulum Cell Membrane Cell Biology Molecular biology Isoenzymes Luminescent Proteins Phosphotransferases (Alcohol Group Acceptor) medicine.anatomical_structure chemistry COS Cells embryonic structures Mutagenesis Site-Directed biology.protein Calcium Research Article Subcellular Fractions |
Zdroj: | Biochemical Journal. 374:41-49 |
ISSN: | 1470-8728 0264-6021 |
Popis: | 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 μM 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. |
Databáze: | OpenAIRE |
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