BMP-6 induces phosphorylation of Smad1/5/8
Upon ligand binding, the type II receptor transphosphorylates and activates the type I receptor. Type I receptors can signal via several pathways. We examined the effect of BMP-6 on Smad phosphorylation, as the activation of Smad is considered to be a major signalling pathway for BMPs [17]. B cells were cultured in serum-free media over night and then treated with BMP-6 for various time points. Total protein lysates were prepared, and the amounts of the phosphorylated forms of Smad1/5/8 were determined by western blot analysis. Interestingly, treatment with 500 ng/ml BMP-6 induced phosphorylation of Smad. The BMP-6 induced phosphorylation was high at the earliest time point tested (15 minutes), and remained high for at least 48 hours (Figure 5). A similar phosphorylation was observed in Ramos cells, but not in HL60 cells (Figure 6). Furthermore, we also tested whether other known downstream signalling pathways of BMP-6 could be triggered by BMP-6 in human B cells. However, we did not observe any significant changes in the level of phospho-STAT3 or phospho-p38 upon BMP-6 treatment of B cells (data not shown).