Phospholipid binding properties and functional characterization of a sea urchin phospholipase Cd in urchin and mouse eggs Kevin Coward a,1 , Helen Owen b,1 , Richard Tunwell d , Karl Swann c , John Parrington a, * a Department of Pharmacology, University of Oxford, Oxford OX1 3QT, UK b Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK c Department of Obstetrics and Gynaecology, Cardiff University, Cardiff CF14 4XN, UK d Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK Received 3 April 2007 Available online 18 April 2007 Abstract We recently identified a novel phospholipase Cd isoform, PLC-dsu, in sea urchin gametes, whose precise functional role during fer- tilization and early embryogenesis remains unknown. Here, we characterized the binding of the PLC-dsu PH domain to different phos- phatidylinositol (PI) phospholipids and studied changes in its localization during fertilization. The PLC-dsu PH domain bound most strongly to PI(3,4)P 2 and PI(3,5)P 2 phospholipids, in contrast to the PLCd1 PH domain which bound predominantly to PI(4,5)P 2 .A green fluorescent protein tagged PLC-dsu PH domain localized to the plasma membrane and its localization increased at fertilization and following addition of a Ca 2+ ionophore. However, recombinant PLC-dsu failed to cause Ca 2+ signals like those seen at fertilization, in mouse and sea urchin eggs. Our findings suggest that PLC-dsu is unlikely to be directly involved in the process of egg activation but may play a role in mediating extracellular signals transmitted via the PI 3 0 -kinase pathway. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Calcium; Phospholipase C; PLCd; PH domain; Egg; Embryo; Sea urchin Calcium (Ca 2+ ) is a key intracellular messenger involved in numerous cellular processes [1]. One key developmental event mediated by intracellular Ca 2+ is the activation of the egg at fertilization [2,3]. The Ca 2+ rise witnessed at fertil- ization varies significantly between species. In sea urchins, frogs and fish, a single explosive Ca 2+ wave is observed [2,3]. In contrast, mammals, nemertean worms and ascidi- ans exhibit a series of periodic Ca 2+ oscillations [2,3]. Ca 2+ signals also accompany the first mitotic division during sea urchin embryogenesis and may regulate events such as nuclear envelope breakdown and the metaphase–anaphase transition [3]. In sea urchins, Ca 2+ has also been suggested to play a role in regulating vegetal cell fate [4]. The phosphoinositide (PI) signalling pathway plays a central role during fertilization in sea urchins, as demon- strated by biochemical measurements indicating an increased turnover of phospholipids at this time [5,6] and the fact that the phospholipase C (PLC) inhibitor U73122 inhibits both Ca 2+ release and egg activation [6]. Six subfamilies of PLC have been identified: b, c, d, e [7], f [8] and g [9]. PLCs vary in the specific combination of regulatory domains present in each isoform. PH domains bind polyphosphoinositides like PIP 2 and PIP 3 , thus target- ing the protein to the plasma membrane [10,11], and medi- ate interaction with G protein bc subunits [12,13]. A number of studies have suggested that sea urchin PLCc plays a central role in triggering egg activation fol- lowing its activation by a tyrosine kinase of the Src family [14] and may also exert influence during early embryogen- esis [15]. PLCb has recently been identified in sea urchin eggs, exhibiting strong homology with mammalian PLCb4 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.04.050 * Corresponding author. Fax: +44 (0) 1865 271853. E-mail address: john.parrington@pharm.ox.ac.uk (J. Parrington). 1 These authors contributed equally to this work. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 357 (2007) 964–970