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Essential role of the EF-hand domain in targeting sperm phospholipase Cζ to membrane phosphatidylinositol 4,5-bisphosphate (PIP2)

Nomikos, Michail, Sanders, Jessica Rose, Parthimos, Dimitris, Buntwal, Luke, Calver, Brian Lewis, Stamatiadis, Panagiotis, Smith, Adrian, Clue, Matthew, Sideratou, Zili, Swann, Karl and Lai, Francis Anthony 2015. Essential role of the EF-hand domain in targeting sperm phospholipase Cζ to membrane phosphatidylinositol 4,5-bisphosphate (PIP2). Journal of Biological Chemistry 290 (49) , pp. 29519-29530. 10.1074/jbc.M115.658443

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Abstract

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca2+ oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP2) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca2+ oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP2 hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP2, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca2+ oscillation inducing activity and in vitro interaction with PIP2 without affecting its Ca2+ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca2+ frequency and the binding ability of different PLCζ mutants to PIP2. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP2, leading to complete abolishment of its Ca2+ oscillation inducing activity.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Medicine
Subjects: R Medicine > R Medicine (General)
Publisher: American Society for Biochemistry and Molecular Biology
ISSN: 0021-9258
Date of First Compliant Deposit: 30 March 2016
Date of Acceptance: 1 October 2015
Last Modified: 21 Jun 2019 20:53
URI: http://orca-mwe.cf.ac.uk/id/eprint/83928

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