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ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. In the current studies, we demonstrate that mice carrying a knock-in point mutation that causes simultaneous rather than sequential cleavage of both prodomain sites show loss of BMP4 function and die during mid-embryogenesis. Levels of mature BMP4 are severely reduced in mutants, although levels of precursor and cleaved prodomain are unchanged compared with wild type. Our biochemical analysis supports a model in which the transient prodomain/ligand complex that forms during sequential cleavage plays an essential role in prodomain-mediated stabilization of the mature ligand until it can acquire protection from degradation by other means. By contrast, simultaneous cleavage causes premature release of the ligand from the prodomain, leading to destabilization of the ligand and loss of signaling in vivo.
Fig. S1. Generation of Bmp4S2KHAmyc mice. (A) Genomic organization of the wild type Bmp4
allele, the targeting vector and the Bmp4S2KHAMycNeo allele. The positions of the external probe used
for Southern analysis, and primers (arrows) surrounding the myc (primer pair a + b) and HA
(primer pair c + d) tag that were used for PCR based genotyping are indicated. The Bmp4HAmycNeo
allele lacks the isoleucine to lysine substitution and BglII site but is otherwise identical. (B)
Southern blot analysis of genomic DNA from targeted or non-targeted (Bmp4+/+) ES cells.
Genomic DNA was digested with BamHI and probed with the genomic fragment shown in panel
A. (C) Western blot showing equivalent levels of pSmad1 and total Smad1 in explants isolated
from the dorsal side of early gastrula stage (st. 10) Xenopus embryos injected with RNA
encoding untagged, myc tagged or HA and myc tagged BMP4.
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