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

Abstract

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.

Human Inositol 1,4,5-Trisphosphate 3-Kinase Isoform B (IP3KB) Is a Nucleocytoplasmic Shuttling Protein Specifically Enriched at Cortical Actin Filaments and at Invaginations of the Nuclear Envelope

“…Thus, Ins(1,4,5)P 3 is being converted before it can activate Ins(1,4,5)P 3 receptors and thereby stimulate Ca 2ϩ release from the lumen of the endoplasmic reticulum. A similar effect was observed by Erneux and co-workers (19) in ATP-stimulated COS-7 cells overexpressing IP3KB. In contrast to the results reported by Erneux and co-workers (19), who observed Ca 2ϩ signals in low expressing cells, in our experiments, Ca 2ϩ signals were inhibited in all observed cells independent of the IP3KB/ EGFP expression level.…”

“…A similar effect was observed by Erneux and co-workers (19) in ATP-stimulated COS-7 cells overexpressing IP3KB. In contrast to the results reported by Erneux and co-workers (19), who observed Ca 2ϩ signals in low expressing cells, in our experiments, Ca 2ϩ signals were inhibited in all observed cells independent of the IP3KB/ EGFP expression level. These differences might be due to the use of different transfection methods, cell lines, and agonists.…”

“…Recent studies described additional functions of IP3KB in selection and survival of B cells (15,16), signaling of neutrophils (17), and modulation of myelopoiesis (18). In mammalian cells, multiple intracellular localizations of IP3KB, namely to the plasma membrane, cytoskeleton, and endoplasmic reticulum, have been observed (19). Most likely, these intracellular targeting specificities of IP3KB are of regulatory importance for all processes mediated by IP3KB and its product Ins(1,3,4,5)P 4 .…”

Regulation of innate immunity by inositol 1,3,4,5-tetrakisphosphate

“…A and B isoforms were found to be localized in both cytoskeleton and cytosolic, indicating a connection between cytoskeleton structure and InsP3K function. 5,[55][56][57] Consistent with this, similar with F-actin, InsP3KB is highly localized in the leading edge of polarized neutrophils. 16 In addition, since all three isoforms contain a calmodulin binding motif, the InsP3KB activity in neutrophils might also be regulated by calcium.…”

Inositol 1,4,5-trisphosphate 3-kinases: functions and regulations

“…They are different in their molecular masses, Ca 2+ /calmodulin (Ca 2+ /CaM) sensitivity, intracellular distribution and tissue expression [23,27] (Tab. 1).…”