5 M A23187, 10 mM H2O2, or 5 U/ml cathepsin B. Protein samples were immunoprecipitated with the anti p85 subunit of PI 3 kinase antibody and the immunoprecipitates were analyzed by immunoblotting with the same antibody or with the anti phosphotyrosine antibody. Aster isks indicate the positions of the p85 bands selleck chem Ponatinib in each panel. LD DIM Inhibitors,Modulators,Libraries fractions were prepared from Xenopus eggs that had been inseminated for the periods indicated. Pro tein samples were analyzed for the presence of PLC?, p85 subunit of PI 3 kinase, or tyrosine phosphorylated UPIII by immunoprecipitation and immunoblotting as described in Methods. Triton X 100 solubilized extracts of Xenopus unfertilized eggs were also analyzed for the presence of phosphorylated MAPK by direct immunoblotting as in Figure 1C.

Asterisks indicate the positions of the protein bands of interest. strated that PIP3 was capable of activating the autophos phorylation of Src. Inositol phospholipids other than PIP3. namely PI, PIP, and Inhibitors,Modulators,Libraries PIP2, did not show such a Src activating property. These results indicate PIP3 to be a specific and direct activator for Src tyrosine kinase in Xenopus eggs. Discussion In Xenopus eggs, the sperm induced activation of Src tyro sine kinase plays an essential role in Ca2 transient and egg activation. The present study has indicated the activity of PI 3 kinase to connect egg sperm interaction/ fusion with the activation of Src, because 1 LY294002 inhibits the sperm induced activation of Src, suggesting that PI 3 kinase acts as an upstream activator for Src, and 2 tyrosine phosphorylation of the p85 subunit of PI 3 kinase, a well known phenomenon in several cellular sys tems, in which one or more tyrosine kinase is activated, is not stimulated Inhibitors,Modulators,Libraries in fertilized eggs.

If so, Inhibitors,Modulators,Libraries what is the mecha nism for Src activation by PI 3 kinase activity One possi bility is, as demonstrated in the present study, the kinase activating interaction of PIP3 with Src. The Src homology 2 domain of the Src protein is capable of binding PIP3, supporting Inhibitors,Modulators,Libraries the possibility that interaction with PIP3 would displace the negative regulatory intramolecu lar interaction involving the SH2 domain, leading to the Src kinases activation. In our experimental system, Wortmannin, another potent inhibitor for PI 3 kinase, is not inhibitory to the sperm induced egg activation in Xenopus in eggs injected with Wortmannin at up to 20 M, sperm could promote Src activation, p85 translocation, cortical contraction, and most importantly Akt phosphorylation, all of which are events of egg activation.

There is a pos sibility that, in Xenopus eggs, Wortmannin binds preferen tially to molecules other than sellckchem PI 3 kinase. This idea is supported by the finding of Carnero and Lacal that wortmannin is able to induce meiotic maturation in Xeno pus oocytes at doses slightly higher that those required for complete inhibition of PI 3 kinase.