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.
Dewaste
Valérie
V
Institute of Interdisciplinary Research, Université Libre de Bruxelles, Campus Erasme, Bldg. C, 808 route de Lennik, B-1070 Brussels, Belgium.
Moreau
Colette
C
De Smedt
Florence
F
Bex
Françoise
F
De Smedt
Humbert
H
Wuytack
Frank
F
Missiaen
Ludwig
L
Erneux
Christophe
C
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003599:Cytoskeleton / Q000201:enzymology*; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection
D017931:DNA Primers; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium