We detected immediate phosphorylation of 3 phosphoinositide dependent kinase 1, coincid ing with phosphorylation of Akt selleckbio at Thr308. phosphoryl ation of Akt at Ser473 was detected after 15 minutes. Phosphorylation of several ty rosines in PI3K regulatory subunits by Inhibitors,Modulators,Libraries PI3K agonists has been previously demonstrated and phosphorylation of the inter SH2 domain was suggested to mediate recep tor specificity and p110 catalytic activity. We probed for phosphorylation of PI3K regulatory subunit iSH2 using a pTyr specific antibody, which detects a conserved Tyr within the iSH2 domain. This antibody has been previously used to probe for PI3K activation in response to Src. To discriminate between BMP2 effects on iSH2 Tyr phosphorylation of p55�� and p85, equal amounts of flag tagged p55�� and HA tagged p85 were expressed in HEK293T cells.

BMP2 stimulation resulted in a time dependent phosphoryl ation of p55�� Tyr199 after 15 minutes, whereas p85 phosphorylation appeared less affected. Subsequently, we investigated whether BMP2 induced PI3K signalling is p55�� dependent. For this, we per formed siRNA mediated knock Inhibitors,Modulators,Libraries down of endogenous p55��. As expected, siRNA mediated knock down of p55�� signifi cantly Inhibitors,Modulators,Libraries impaired BMP2 induced Akt phosphorylation at Thr308 compared to a scrambled siRNA control. In addition, we investigated the effect of p55�� overexpression on BMP2 induced Akt phosphoryl ation. We found that p55�� overexpression exerts a domin ant negative effect on BMP2 induced Akt phosphorylation, a phenomenon that has been previously reported to under lie an unbalanced ratio between the regulatory and cata lytic subunits.

Taken together, these results demonstrate that p55�� specifically links BMP2 with the activation of PI3K signalling. BMP2 induced PIP3 production is dependent on p55�� Inhibitors,Modulators,Libraries We then analysed whether BMP2 induced PIP3 produc tion requires p55�� by performing a PI3K activity assay. For this, C2C12 cells were stimulated with BMP2 follow ing pull down of p55�� or p85. Subsequently, we analysed in vitro lipid kinase activity of precipitated complexes using a competitive ELISA system. Precipi tates of p55�� revealed increased PIP3 production after BMP2 stimulation for 15 minutes, which further increased at the 60 minute time point. By contrast, pre treatment with the PI3K inhibitor LY 294002 or pull down of p85 gained PIP3 levels comparable to levels in non stimulated p55�� precipitates. Inhibitors,Modulators,Libraries The pull down of p85 only resulted in ele vated PIP3 levels when cells merely were stimulated with insulin. This further underlines the role of p85 in other pathways, but not BMP signalling. Pull down controls for both regulatory subunits and the co immunoprecipitated p110 are shown.