Figure 4

BMP2-induced PI3K signalling is specifically mediated via p55γ. (A) Phospho-kinetics of PI3K effector proteins in C2C12 upon stimulation with 10 nM BMP2 for the indicated time. Phosphorylation of PDK1 (Ser241), Akt (Thr308) and Akt (Ser473) was analysed. (B) BMP2-dependent tyrosine phosphorylation at the inter-SH2 domain of PI3K regulatory subunits p55γ and p85α. HEK293T cells were transfected with equal amounts of p55γ-flag and p85α-HA and stimulated with 10 nM BMP2 for the indicated time. Upper panel depicts BMP2-dependent phosphorylation of conserved Tyr458 of p85α (see arrowheads, double band at approximately 100 kDa) corresponding to Tyr199 of p55γ (see arrowhead at approximately 55 kDa). The detected signals migrated accordingly to the signals of p85α-HA and p55γ-flag in the expression control (arrowheads, lower panel). (C) Knock-down of p55γ reduces BMP2-induced Akt-Thr308 phosphorylation of C2C12 cells upon 60 minutes’ stimulation with BMP2. The relative phospho-Akt-Thr308 to GAPDH levels were determined. (D) Overexpression of p55γ-flag in C2C12 cells reduces BMP2-induced Akt-Thr308 phosphorylation upon 60 minutes’ stimulation with BMP2. The relative phospho-Akt-Thr308 to GAPDH levels were determined. For experiments C and D: error bars represent standard deviation from three independent experiments. P-values from one-way analysis of variance with post-hoc Bonferroni-test are indicated. (See also Additional file 4: Figure S4.)