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Phospholipase C binding to PtdIns(4,5)P2 requires the XY-linker region

Nomikos, Michail, Elgmati, Khalil, Theodoridou, Maria, Calver, Brian Lewis, Nounesis, George, Swann, Karl and Lai, Francis Anthony 2011. Phospholipase C binding to PtdIns(4,5)P2 requires the XY-linker region. Journal of Cell Science 124 (15) , pp. 2582-2590. 10.1242/jcs.083485

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Abstract

Phospholipase C-zeta (PLCζ) is a strong candidate for the mammalian sperm-derived factor that triggers the Ca2+ oscillations required for egg activation at fertilization. PLCζ lacks a PH domain, which targets PLCδ1 to the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) substrate in the plasma membrane. Previous studies failed to detect PLCζ in the plasma membrane, hence the means of PLCζ binding to PtdIns(4,5)P2 is unclear. We find that the PLCζ XY linker, but not the C2 domain, exhibits robust binding to PtdIns(4,5)P2 or to liposomes containing near-physiological levels of PtdIns(4,5)P2. The role of positively charged residues within the XY linker was addressed by sequentially substituting alanines for three lysine residues, K374, K375 and K377. Microinjection of these mutants into mouse eggs enabled their Ca2+ oscillation-inducing activities to be compared with wild-type PLCζ. The XY-linker mutant proteins were purified and the in vitro PtdIns(4,5)P2 hydrolysis and binding properties were monitored. Successive reduction of net positive charge within the PLCζ XY linker significantly affects both in vivo Ca2+-oscillation-inducing activity and in vitro PtdIns(4,5)P2 interaction of mouse PLCζ. Our data suggest that positively charged residues within the XY linker play an important role in the PLCζ interaction with PtdIns(4,5)P2, a crucial step in generating the Ca2+ activation signal that is essential for fertilization in mammals.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
R Medicine > R Medicine (General)
Uncontrolled Keywords: Phospholipase C; Phosphoinositide signalling; Calcium oscillations; Egg activation; Fertilization
Publisher: The Company of Biologists Ltd
ISSN: 0021-9533
Last Modified: 02 May 2019 11:37
URI: http://orca-mwe.cf.ac.uk/id/eprint/29671

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