Phospholipase C and D regulation of Src, calcium release and membrane fusion during Xenopus laevis development

Abstract

This review emphasizes how lipids regulate membrane fusion and the proteins involved in three developmental stages: oocyte maturation to the fertilizable egg, fertilization and during first cleavage. Decades of work show that phosphatidic acid (PA) releases intracellular calcium, and recent work shows that the lipid can activate Src tyrosine kinase or phospholipase C during Xenopus fertilization. Numerous reports are summarized to show three levels of increase in lipid second messengers inositol 1,4,5-trisphosphate and sn 1,2-diacylglycerol (DAG) during the three different developmental stages. In addition, possible roles for PA, ceramide, lysophosphatidylcholine, plasmalogens, phosphatidylinositol 4-phosphate, phosphatidylinositol 5-phosphate, phosphatidylinositol 4,5-bisphosphate, membrane microdomains (rafts) and phosphatidylinositol 3,4,5-trisphosphate in regulation of membrane fusion (acrosome reaction, sperm-egg fusion, cortical granule exocytosis), inositol 1,4,5-trisphosphate receptors, and calcium release are discussed. The role of six lipases involved in generating putative lipid second messengers during fertilization is also discussed: phospholipase D, autotaxin, lipin1, sphingomyelinase, phospholipase C, and phospholipase A2. More specifically, proteins involved in developmental events and their regulation through lipid binding to SH3, SH4, PH, PX, or C2 protein domains is emphasized. New models are presented for PA activation of Src (through SH3, SH4 and a unique domain), that this may be why the SH2 domain of PLCγ is not required for Xenopus fertilization, PA activation of phospholipase C, a role for PA during the calcium wave after fertilization, and that calcium/calmodulin may be responsible for the loss of Src from rafts after fertilization. Also discussed is that the large DAG increase during fertilization derives from phospholipase D production of PA and lipin dephosphorylation to DAG.

Keywords: Cleavage; Fertilization; Oocyte maturation; Phosphatidic acid; Phospholipase D; Protein–lipid binding; SH3 domain.

Fig. 1

Early Xenopus development. Oocyte maturation: progesterone induces the oocyte nuclear envelope breakdown (producing a white spot in the animal pole), progression to metaphase II of meiosis, and an increase IP₃ receptor number and clustering in the cortex to produce the fertilizable egg. Fertilization is associated with an increase in [Ca⁺²]_i that stimulates cortical granule exocytosis, gravitational rotation, the completion of meiosis, followed by mitotic division and first cleavage.

Fig. 2

(A) IP₃ mass increases during oocyte maturation, fertilization and first cleavage. The average of numerous determinations is shown for simplification (see original publications for error bars and statistics)(Stith et al., 1992a; Stith et al., 1994; Stith et al., 1993; Stith et al., 1992b). Progesterone induces oocyte maturation to the egg, fertilization is noted as time zero, and first cleavage occurs at 100 min after fertilization. White spot represents appearance of the nucleus near the cell surface and this event occurs just before nuclear envelope breakdown. (B) DAG mass increases during oocyte maturation, fertilization and first cleavage. As measured by the DAG kinase assay (Stith et al., 1992b; Stith et al., 1997), note that the largest increase in DAG occurs during first cleavage, an intermediate increase occurs during fertilization (time zero) and smaller increases occur during progesterone-induced oocyte maturation to the egg.

Fig. 3

IP₃ degradation is inhibited by lowering [Ca⁺²]_i. After injection of ³H-IP₃ with or without calcium buffer BAPTA (Stith et al., 1994), activated eggs were homogenized and IP₃ purified by HPLC and radioactivity was determined by liquid scintillation.

Fig. 4

After blocking cleavage, an IP₃ mass increase still occurs at proper time. Injection (10 nL) of colchicine (1 mg/ml) or a sham injection (“CLEAVAGE”) of groups of 20 zygotes at 25 min after fertilization blocked cleavage. Eggs without treatment or zygotes were homogenized relative to the time for the completion of first cleavage (100 min after insemination) and IP₃ mass determined as noted (Stith et al., 1993).

Fig. 5

A model for Xenopus Fertilization. The model notes three pathways to [Ca⁺²]_i release: through Src and PLCγ (35% of [Ca⁺²]_i release), phosphatidic acid (PA) action on PLC (52%), and an unknown pathway (13%). The model is based on the action of inhibitors (“T” symbol, versus activation denoted by an arrow), lipid and enzyme analysis (Bates et al., 2014; Petcoff et al., 2008; Stith et al., 1997) (C. Fees, J. Stafford, and B. J. Stith, unpublished results). Sperm activate PLD1b at ~1 min after insemination and elevated PA may have multiple roles in fertilization: (1) PA binds and stimulates Src which in turn activates PLCγ leading to a rapid IP₃ increase beginning at ~1 min, (2) PA directly induces a slow activation of PLC to produce a peak IP₃ value at ~5 min, (3) PA dephosphorylation by lipin1 is believed to be responsible for the large, late increase in DAG at 10 min, (4) PA may play a role in the membrane fusion events of fertilization such as sperm-egg merger or cortical granule exocytosis, and (5) PA may activate PI4 5’kinase to double the mass of PI45P2 during fertilization. The cause of the small (13%) release of [Ca⁺²]_i, that is independent of the PLD1b pathway and PA production, is sufficient to induce fertilization with a 12 min delay (Bates et al., 2014). This other pathway could involve a messenger from sperm (sperm PA is elevated during the acrosome reaction), ceramide, Ca⁺², PI4P, or a G protein (Bates et al., 2014; Kline et al., 1991; Morrison et al., 2000; Sato et al., 2003; Tokmakov et al., 2014).

Fig. 6

A model for phosphatidic acid (PA) activation of Src. The tight configuration of Src is largely stabilized by the binding of multiple prolines found in the SH2-SH1 linker region to the SH3 domain and, secondly, by the SH2 domain binding the phosphorylated tyrosine 527 (red circle near c terminus). Furthermore, the active site is blocked by the activation loop containing unphosphorylated tyrosine 416/418 and its phosphorylation relieves inhibition by moving the activation loop out of the active site. Thus, inactive Src would be anchored to membrane rafts by a myristoylated tail and perhaps by anionic lipid binding to the SH4 and the unique lipid binding region (ULBR). As PA binds to Src, anionic lipids are known to bind to the SH4, ULBR and SH3 domains, PA elevation (dark red phospholipid) would lead to new membrane binding to the SH3 domain, and perhaps an enhancement of SH4 and ULBR binding. Thus, PA would lead to a “loosening” of Src configuration, autophosphorylation and Src activation and PA would mimic known Src activating proteins. We are currently studying the effect of PA on phosphorylation of tyrosine 527, and the binding of Src SH3 to polyproline should be quantified as a function of PA containing vesicles.

Fig. 7

Elevation of [Ca⁺²] stimulates Xenopus tyrosine kinase and PLC activity. Plasma membrane-cortex preparation was obtained from Xenopus oocytes and IP₃ mass and tyrosine kinase activity measured (Morrison et al., 2000) after [Ca⁺²] was buffered to various levels with BAPTA (Stith et al., 1994).