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J. Biol. Chem., Vol. 282, Issue 13, 9526-9535, March 30, 2007

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Regulation of Inositol 1,4,5-Trisphosphate 3-Kinases by Calcium and Localization in Cells^*

Samantha M. Lloyd-Burton¹, Jowie C. H. Yu², Robin F. Irvine³, and Michael J. Schell⁴

From the Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom and the Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) 3-kinases (IP₃Ks) are a group of calmodulin-regulated inositol polyphosphate kinases (IPKs) that convert the second messenger Ins(1,4,5)P₃ into inositol 1,3,4,5-tetrakisphosphate. However, what they contribute to the complexities of Ca²⁺ signaling, and how, is still not fully understood. In this study, we have used a simple Ca²⁺ imaging assay to compare the abilities of various Ins (1,4,5)P₃-metabolizing enzymes to regulate a maximal histamine-stimulated Ca²⁺ signal in HeLa cells. Using transient transfection, we overexpressed green fluorescent protein-tagged versions of all three mammalian IP₃K isoforms, including mutants with disrupted cellular localization or calmodulin regulation, and then imaged the Ca²⁺ release stimulated by 100 µM histamine. Both localization to the F-actin cytoskeleton and calmodulin regulation enhance the efficiency of mammalian IP₃Ks to dampen the Ins (1,4,5)P₃-mediated Ca²⁺ signals. We also compared the effects of the these IP₃Ks with other enzymes that metabolize Ins(1,4,5)P₃, including the Type I Ins(1,4,5)P₃ 5-phosphatase, in both membrane-targeted and soluble forms, the human inositol polyphosphate multikinase, and the two isoforms of IP₃K found in Drosophila. All reduce the Ca²⁺ signal but to varying degrees. We demonstrate that the activity of only one of two IP₃K isoforms from Drosophila is positively regulated by calmodulin and that neither isoform associates with the cytoskeleton. Together the data suggest that IP₃Ks evolved to regulate kinetic and spatial aspects of Ins (1,4,5)P₃ signals in increasingly complex ways in vertebrates, consistent with their probable roles in the regulation of higher brain and immune function.

Received for publication, November 2, 2006 , and in revised form, January 11, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a Studentship from Merck Sharp & Dohme.

² Supported by the Croucher Foundation.

⁴ Supported by the Case Fellowship from the Royal Society and by Uniformed Services University of the Health Sciences Grant RO75LN.

³ Supported by the Royal Society and the Wellcome Trust. To whom correspondence should be addressed: Dept. of Pharmacology, University of Cambridge, Tennis Ct. Rd., Cambridge CB2 1PD, United Kingdom. Tel.: 44-1223-339683; Fax: 44-1223-334040; E-mail: rfi20{at}cam.ac.uk.

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