Effect of GST-Fyn-SH2 on the sperm-induced calcium transient. Zebrafish eggs were injected with a mixture of calcium green–dextran together with the control GST protein (upper panel) or with the GST-FYN-SH2 fusion protein (lower panel) at intracellular concentrations of 8 μM. The eggs were mixed with sperm, and aquarium water was added (arrow) to activate the sperm and induce fertilization. Fluorescence was recorded at 10-s intervals over a 70-micron optical section through the equator of the egg.

Fertilization induces transient activation of Src-family PTKs. The effect of fertilization on Src-family PTK activity was measured using a single cell assay (left panel). Individual zebrafish eggs were collected before and at different times after fertilization, transferred to a micro pipet tip then crushed at the time (minutes) indicated at the top of the Figure and the PTK activity in each egg was assayed as described in materials and methods. The resultant ³²PO₄-labeled proteins were analyzed by SDS-PAGE and subjected to partial alkaline hydrolysis to reduce contamination by ³²PO₄-Ser and ³²PO₄-Thr. The remaining phosphoproteins were detected by autoradiography. The position of the 60-kDa band indicative of Src-family PTK activity is indicated by the arrow. The effect of the PTK inhibitor PP2 on this type of assay was examined in a second experiment (right panel). Eggs collected at 5 min postinsemination were assayed in the presence of different concentrations of the PTK inhibitor PP2 or the inactive analog PP3. The concentration of each inhibitor (nanomolar) is indicated at the top.

Effect of GST-Fyn-SH2 on Src-family PTK activity in vitro. Immunoprecipitates prepared from the particulate fraction of solubilized zebrafish zygotes (5 min postinsemination) using the Pan-Src antibody (Src-2) to pull down Src family kinases. Kinase activity in the immunoprecipitates was measured as described in materials and methods. Kinase reactions contained either no addition (A) or 1 μM (B), 4 μM (C), or 8 μM GST-FYN-SH2 fusion protein (D) to test the effect of this fusion protein on kinase activity in vitro. The position of the autophosphorylated Src-family kinase band is indicated by the arrow.