Figure 2

PI3K regulatory subunit p55γ interacts with BMPRII and p110α. (A) p55γ-specific peptides obtained after GST-BMPRII-pull-down from C2C12 lysates [20] are shown in yellow. (B) Scheme depicting BMPRII and truncations, including all tyrosines (black lines) and the ones serving as putative p55γ-SH2 domain binding sites (*). Tyrosines identified by alignment with known SH2 binding peptides (*) and oriented peptide library technique (**). Lines indicate localisation of 24 intracellular tyrosines in BMPRII. On the right, lanes 1 to 8 show input controls (top) and co-immunoprecipitation of BMPRII-LF, -SF or TC3-8 with p55γ from transfected HEK293T cells. The expected molecular weights of BMPRII and truncations are marked by white arrowheads. (C) Co-immunoprecipitation of endogenous BMPRII-LF and -SF (black arrowheads) with endogenous p55γ, but not p85α upon stimulation with 10 nM BMP2 for indicated time. The depicted western blots are representative of three independent experiments. (D) Immunocytochemical staining of C2C12 cells displaying co-localisation of endogenous p55γ, but not p85α, with overexpressed HA-tagged BMPRII-LF. Enlarged region of interest depicts co-localisation of p55γ but not p85α (green) with HA-tagged BMPRII-LF (red) at the cell periphery. Scale bar: 10 μm. (E) Co-immunoprecipitation from C2C12 lysates of endogenous BMPRII and endogenous p110α upon BMP2 stimulation for indicated time. (F) BMP2-induced tyrosine phosphorylation of BMPRII-LF. HEK293T cells transfected with HA-tagged BMPRII-LF were treated with 10 nM BMP2 for indicated time and subjected to immunoprecipitation with anti-HA antibody. Input controls of HA-tagged BMPRII-LF and BMP2-induced Smad 1/5/8 phosphorylation kinetics are shown (upper two panels). Fourth panel indicates BMPRII tyrosine phosphorylation through incubation of α-HA precipitates with pan anti-pTyr antibody. Dotted lines in F and B indicate deletion of non-relevant lanes. (See also Additional file 1: Figure S1C). (ECD) extracellular domain; (KD) kinase domain; (LF) long form; (SF) short form; (TC) truncation; (IgG) immunoglobulin G.