Mutations that prevent phosphorylation of the BMP4 prodomain impair proteolytic maturation of homodimers leading to lethality in mice.

Bone morphogenetic protein4 (BMP4) plays numerous roles during embryogenesis and can signal either alone as a homodimer, or together with BMP7 as a more active heterodimer. BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments. In humans, heterozygous mutations within the prodomain of BMP4 are associated with birth defects. We studied the effect of two of these mutations (p.S91C and p.E93G), which disrupt a conserved FAM20C phosphorylation motif, on ligand activity. We compared the activity of ligands generated from BMP4, BMP4 or BMP4 in embryos and found that these mutations reduce the activity of BMP4 homodimers but not BMP4/7 heterodimers. We generated and knock-in mice and found that mice die by E11.5 and display reduced BMP activity in multiple tissues including the heart. Most mice die before weaning and mutants die prenatally with reduced or absent eyes, heart and ventral body wall closure defects. Mouse embryonic fibroblasts (MEFs) isolated from and embryos show accumulation of BMP4 precursor protein, reduced levels of cleaved BMP ligand and reduced BMP activity relative to MEFs from wild type littermates. Because is not expressed in MEFs, the accumulation of unprocessed BMP4 precursor protein in mice carrying these mutations most likely reflects an inability to cleave BMP4 homodimers, leading to reduced levels of ligand and BMP activity in vivo. Our results suggest that phosphorylation of the BMP4 prodomain is required for proteolytic activation of BMP4 homodimers, but not heterodimers.