Abstract
Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) 3-kinases (IP(3)Ks) are a group of calmodulin-regulated inositol polyphosphate kinases (IPKs) that convert the second messenger Ins(1,4,5)P(3) into inositol 1,3,4,5-tetrakisphosphate. However, what they contribute to the complexities of Ca(2+) signaling, and how, is still not fully understood. In this study, we have used a simple Ca(2+) imaging assay to compare the abilities of various Ins (1,4,5)P(3)-metabolizing enzymes to regulate a maximal histamine-stimulated Ca(2+) signal in HeLa cells. Using transient transfection, we overexpressed green fluorescent protein-tagged versions of all three mammalian IP(3)K isoforms, including mutants with disrupted cellular localization or calmodulin regulation, and then imaged the Ca(2+) release stimulated by 100 microm histamine. Both localization to the F-actin cytoskeleton and calmodulin regulation enhance the efficiency of mammalian IP(3)Ks to dampen the Ins (1,4,5)P(3)-mediated Ca(2+) signals. We also compared the effects of the these IP(3)Ks with other enzymes that metabolize Ins(1,4,5)P(3), including the Type I Ins(1,4,5)P(3) 5-phosphatase, in both membrane-targeted and soluble forms, the human inositol polyphosphate multikinase, and the two isoforms of IP(3)K found in Drosophila. All reduce the Ca(2+) signal but to varying degrees. We demonstrate that the activity of only one of two IP(3)K isoforms from Drosophila is positively regulated by calmodulin and that neither isoform associates with the cytoskeleton. Together the data suggest that IP(3)Ks evolved to regulate kinetic and spatial aspects of Ins (1,4,5)P(3) signals in increasingly complex ways in vertebrates, consistent with their probable roles in the regulation of higher brain and immune function.
Highlights
IP3Ks may initiate a chain of reactions responsible for the production of higher inositol phosphates, having diverse intracellular roles (10), this view has been challenged (11, 12)
For example, IP3KA is concentrated in dendritic spines by way of an interaction with the filamentous actin (Factin) cytoskeleton (39), a localization that is subject to dynamic regulation by Ca2ϩ (40)
IP3KB can associate with F-actin (41), but it can be targeted to the endoplasmic reticulum (24), a targeting that may be dynamically regulated by proteases (25)
Summary
CDNA Constructs cDNA clones encoding each of the enzymes of interest were used as PCR templates, and the amplified inserts were subcloned into the pEGFP vectors (Clontech) for expression as GFP fusion proteins or the pQE30 vector for expression in bacteria. InsP3 3-Kinase B—The full-length cDNA of rat InsP3 3-kinase B (44), a gift from Dr G Banting (Department of Biochemistry, University of Bristol, Bristol, UK) was amplified by PCR, and the purified PCR product was subcloned into the HindIII and BamHI sites of the pEGFPC1 vector. Sequencing of this clone revealed a single point mutation at T220C in the open reading frame, which was corrected using QuikChange (Stratagene) mutagenesis, as described in Ref. 25. The kinase-dead mutant of IP3KA was the double mutant D260A/K262A
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