Evidence that phosphatidylinositol 3-kinase is involved in sperm-induced tyrosine kinase signaling in Xenopus egg fertilization

Protein-tyrosine phosphorylation plays an important role in egg activation signaling at fertilization. We show that in Xenopus, fertilization stimulates a rapid and transient tyrosine phosphorylation of egg proteins, as revealed by immunoblotting with anti-phosphotyrosine antibody. Immunofluorescent microscopic analysis demonstrated that the phosphorylation occurs in cortical area of the egg animal hemisphere. To further characterize subcellular compartment for fertilization-dependent tyrosine kinase signaling, we isolated low density detergent-insoluble membrane (LD-DIM) fraction from Xenopus eggs. The egg LD-DIM was enriched in cholesterol and GM1 ganglioside. It also contained signaling molecules such as Xyk (Xenopus egg Src), Gqα, Ras, integrin β1 and CD9. Fertilization stimulated tyrosine phosphorylation of Xyk and some other LD-DIM proteins. Remarkably, sperm stimulated tyrosine phosphorylation of the LD-DIM proteins in vitro. The sperm-dependent phosphorylation was sensitive to the tyrosine kinase inhibitors PP2 and genistein. We found that pretreatment of eggs with methyl-β-cyclodextrin, a cholesterol-binding substance, led to a decrease in cholesterol, Xyk and sperm-induced tyrosine phosphorylation in LD-DIM. In methyl-β-cyclodextrin-treated eggs, sperm-induced Ca2+ transient and first cell division were also inhibited. These findings suggest that the egg LD-DIM might serve as subcellular microdomain for tyrosine kinase signaling in Xenopus egg fertilization.

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The Class II Phosphatidylinositol 3-Phosphate Kinase PIK3C2A Promotes Shigella flexneri Dissemination through Formation of Vacuole-Like Protrusions

Infection and Immunity ◽

10.1128/iai.03138-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1695-1704 ◽

Cited By ~ 19

Author(s):

Ana-Maria Dragoi ◽

Hervé Agaisse

Keyword(s):

Plasma Membrane ◽

Tyrosine Kinase ◽

Shigella Flexneri ◽

Class Ii ◽

Kinase Signaling ◽

Tyrosine Kinase Signaling ◽

Content Dissemination ◽

Content Type ◽

Infected Cells ◽

Phosphatidylinositol 3

Intracellular pathogens such asShigella flexneriandListeria monocytogenesachieve dissemination in the intestinal epithelium by displaying actin-based motility in the cytosol of infected cells. As they reach the cell periphery, motile bacteria form plasma membrane protrusions that resolve into vacuoles in adjacent cells, through a poorly understood mechanism. Here, we report on the role of the class II phosphatidylinositol 3-phosphate kinase PIK3C2A inS. flexneridissemination. Time-lapse microscopy revealed that PIK3C2A was required for the resolution of protrusions into vacuoles through the formation of an intermediate membrane-bound compartment that we refer to as a vacuole-like protrusion (VLP). Genetic rescue of PIK3C2A depletion with RNA interference (RNAi)-resistant cDNA constructs demonstrated that VLP formation required the activity of PIK3C2A in primary infected cells. PIK3C2A expression was required for production of phosphatidylinositol 3-phosphate [PtdIns(3)P] at the plasma membrane surrounding protrusions. PtdIns(3)P production was not observed in the protrusions formed byL. monocytogenes, whose dissemination did not rely on PIK3C2A. PIK3C2A-mediated PtdIns(3)P production inS. flexneriprotrusions was regulated by host cell tyrosine kinase signaling and relied on the integrity of theS. flexneritype 3 secretion system (T3SS). We suggest a model ofS. flexneridissemination in which the formation of VLPs is mediated by the PIK3C2A-dependent production of the signaling lipid PtdIns(3)P in the protrusion membrane, which relies on the T3SS-dependent activation of tyrosine kinase signaling in protrusions.

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