Site-specific Cleavage of BMP4 by Furin, PC6, and PC7

Sylvia M Nelsen,Jan L Christian

doi:10.1074/jbc.m109.028506

Abstract

Bone morphogenetic proteins (BMPs) require proteolytic activation by members of the proprotein convertase (PC) family. Pro-BMP4 is initially cleaved at a site adjacent to the mature ligand domain (S1) and then at an upstream site (S2) within the prodomain. Cleavage at the S2 site, which appears to occur in a tissue-specific fashion, regulates the activity and signaling range of mature BMP4. To test the hypothesis that tissue-specific cleavage of pro-BMP4 is regulated by differential expression of a site-specific protease, we identified the PCs that cleave each site in vivo. In Xenopus oocytes, furin and PC6 function redundantly to cleave both the S1 and S2 sites of pro-BMP4, as evidenced by the results of antisense-mediated gene knockdown and the use of the furin- and PC6-selective inhibitor alpha(1)-PDX. By contrast, alpha(1)-PDX blocked cleavage of the S2 but not the S1 site of pro-BMP4 in embryos, suggesting the existence of a developmentally regulated S1 site-specific convertase. This protease is likely to be PC7 based on knowledge of its required substrate cleavage motif and resistance to alpha(1)-PDX. Consistent with this prediction, an alpha(1)-PDX variant engineered to target PC7, in addition to furin and PC6, completely inhibited cleavage of BMP4 in oocytes and embryos. Further studies showed that pc7 transcripts are expressed and polyadenylated, and that the PC7 precursor protein undergoes efficient autocatalytic activation in both oocytes and embryos. These results suggest that PC7, or a convertase with similar substrate specificity, functions to selectively cleave the S1 site of pro-BMP4 in a developmentally regulated fashion.

Highlights

The bioactivity of BMP4 is regulated post-translationally, at the level of proteolytic activation
Injected oocytes were cultured for 3 days to allow for turnover of endogenous proprotein convertase (PC) proteins and injected with limiting for cleavage, levels of precursor monomer were not affected by the depletion of endogenous convertases over the short time course of this analysis
Levels of dimerized BMP4 precursor increased significantly in oocytes depleted of furin, and this effect was accompanied by a corresponding decrease in the levels of both the fully cleaved prodomain and mature BMP4 (Fig. 2C)

Summary

Introduction

The bioactivity of BMP4 is regulated post-translationally, at the level of proteolytic activation. To begin to test potential mechanisms for tissue-specific regulation of S2 cleavage, we used loss-of-function approaches along with protein-based inhibitors to ask which PCs cleave the S1 and S2 sites of BMP4 in vivo.

Results

Conclusion