Upon p55�� precipitation we con firmed an interaction with wild type BMPRII LF and all BMPRII selleck chem inhibitor truncations as well as BMPRII SF. To validate the interaction of p55�� with both splice forms, we performed studies in C2C12 cells by pull down of either endogenous p55�� or endogenous p85. We then probed for co precipitated en dogenous BMPRII by use of a BMPRII specific antibody recognising an extracellular epitope. As shown in lanes 1 to 3, endogenous p55��, but not p85, co immunoprecipitated with BMPRII LF and BMPRII SF, with the receptor association to p55�� increasing over time during BMP2 treatment. Furthermore, we detected the class Ia catalytic subunit p110 in p55�� precipitates, suggesting that BMP2 activates PI3K heterodimers of p55�� and p110.

Since co immunoprecipitation in C2C12 cells confirmed a p55�� but not p85 interaction with BMPRII, we compared their respective co localisation patterns in intact cells. For this, C2C12 cells were transiently transfected with Human influenza hemagglutinin tagged BMPRII LF Inhibitors,Modulators,Libraries and stained by use of antibodies binding to regulatory subunits and the HA tag. Epifluorescence microscopy revealed strong co localisation of p55��, but only partial co localisation of p85, with BMPRII LF within C2C12 cell protrusions. Co localisation was quantified defining a fixed Inhibitors,Modulators,Libraries region of interest. Mean Pearsons coefficient of three sets of independent experiments revealed 0. 933 0. 092 for co localisation of p55�� and 0. 741 0. 093 for p85 with BMPRII LF. We then con firmed that p110 indeed specifically binds to BMPRII by precipitation of endogenous p110 which co immunoprecipitated BMPRII in a BMP2 dependent manner.

Together, these data dem onstrate that p55�� specifically binds to BMPRII irre spective of the presence of the C terminal tail and is part of a p110 containing Inhibitors,Modulators,Libraries PI3K complex. BMPRII becomes tyrosine phosphorylated in a BMP2 dependent manner Class Ia PI3Ks interact with activated growth factor recep tors via pTyr motifs recognised by the SH2 domains of the regulatory subunit. BMPRII is a serinethreonine kinase and its tyrosine phosphorylation has not been investigated Inhibitors,Modulators,Libraries to Inhibitors,Modulators,Libraries our knowledge. The cytosolic part of BMPRII LF contains 24 tyrosines. the majority of ty rosines are located within the kinase domain, a few in the C terminal tail and none in the juxtamembrane region selleck catalog preceding the kinase domain. An in silico alignment of the BMPRII cytosolic domain with known SH2 domain binding peptides and analysis using ScanSite oriented peptide library technique identified five poten tial tyrosines that could act as SH2 domain docking sites. To first analyse BMP2 dependent tyrosine phosphorylation of BMPRII, we transfected HEK293T cells with HA tagged BMPRII LF, followed by immunoprecipitation using anti HA antibody.