Cleavages within the Prodomain Direct Intracellular Trafficking and Degradation of Mature Bone Morphogenetic Protein-4

Abstract

Pro bone morphogenetic protein-4 (BMP-4) is initially cleaved at a consensus furin motif adjacent to the mature ligand domain (the S1 site), and this allows for subsequent cleavage at an upstream motif (the S2 site). Previous studies have shown that S2 cleavage regulates the activity and signaling range of mature BMP-4, but the mechanism by which this occurs is unknown. Here, we show that the pro-and mature domains of BMP-4 remain noncovalently associated after S1 cleavage, generating a complex that is targeted for rapid degradation. Degradation requires lysosomal and proteosomal function and is enhanced by interaction with heparin sulfate proteoglycans. Subsequent cleavage at the S2 site liberates mature BMP-4 from the prodomain, thereby stabilizing the protein. We also show that cleavage at the S2, but not the S1 site, is enhanced at reduced pH, consistent with the possibility that the two cleavages occur in distinct subcellular compartments. Based on these results, we propose a model for how cleavage at the upstream site regulates the activity and signaling range of mature BMP-4 after it has been released from the prodomain. INTRODUCTIONBone morphogenetic protein-4 (BMP-4) is a signaling molecule that acts as a morphogen to influence cell fate in a concentration-dependent manner. BMP-4 was originally identified as a protein that is capable of inducing ectopic bone formation, but more recent studies have shown that it plays many different roles during embryonic development and in adults (Hogan, 1996).BMP-4 function is essential for normal embryogenesis as illustrated by the fact that mice homozygous for a null allele of BMP-4 form little or no mesoderm and die near the time of gastrulation (Winnier et al., 1995). BMP-4 heterozygous mutant mice are viable but display a variety of birth defects, including reduced numbers of primordial germ cells, polydactyly, and kidney, eye, and craniofacial abnormalities (Dunn et al., 1997;Lawson et al., 1999;Miyazaki et al., 2000;Chang et al., 2001). These data indicate that control of BMP-4 gene dosage is essential for normal embryonic patterning.Excess BMP-4 activity also leads to birth defects. Mice mutant for the BMP antagonists gremlin, noggin, and/or chordin show early lethality and/or defects in the spinal cord, forebrain, somites, skeleton, and kidney (Brunet et al., 1998;McMahon et al., 1998;Gong et al., 1999;Khokha et al., 2003). In humans, mutations in the noggin gene are responsible for multiple synostoses syndrome, a genetic disease characterized by fusion of the joints (Gong et al., 1999), and abnormally high levels of BMP-4 protein are a key feature of fibrodysplasia ossificans progressiva, a crippling hereditary disorder in which ectopic bone forms throughout the body (Kaplan and Shore, 1998).The requirement for strict regulation of BMP-4 dosage is met by controlling BMP-4 activity at multiple levels. At the extracellular level, BMP-4 is regulated by binding proteins, such as chordin and noggin, that block activation of cell surface receptors, and by the prote...

Xenopus Lefty requires proprotein cleavage but not N‐linked glycosylation to inhibit nodal signaling

“…Alternatively, the secondary structure of Xlefty, or the inherent protease sensitivity at each site, may differ between oocytes and embryonic tissues, such that CS2 cleavage does not occur in embryonic tissues. A general conclusion from our studies, therefore, despite the numerous reported experiments using Xenopus oocytes to infer the mechanisms regulating the biochemical processing of secreted proproteins (Dale et al, 1989;Thomsen and Melton, 1993;Jones et al, 1996;Cui et al, 1998Cui et al, , 2001Eimon and Harland, 2002;Degnin et al, 2004), is that only embryonic cells should be used for future studies of Xlefty.…”

“…Cleavage at site 1 (S1) separates the ligand from the prodomain and subsequent cleavage within the prodomain (at S2) seems to be required in order to disrupt non-covalent prodomain/ligand interactions, which releases the BMP-4 ligand for productive receptor engagement (Cui et al, 2001). When the BMP-4 ligand remained prodomain-associated, the complex seems to be targeted for efficient degradation via the lysosomal/proteosomal pathway (Degnin et al, 2004). Therefore, Degnin et al (2004) suggested that tissue-specific cleavage at S2 might contribute to the spatiotemporal regulation of BMP-4 activity.…”

“…When the BMP-4 ligand remained prodomain-associated, the complex seems to be targeted for efficient degradation via the lysosomal/proteosomal pathway (Degnin et al, 2004). Therefore, Degnin et al (2004) suggested that tissue-specific cleavage at S2 might contribute to the spatiotemporal regulation of BMP-4 activity. This hypothesis was validated by mutating the cleavage motif at S2 in the endogenous locus of mice, which led to defects being detected in those tissues that require the highest levels of BMP signaling (Goldman et al, 2006).…”

“…Vg1 and xBMP-4 have been shown to undergo N-linked glycosylation, but we are uncertain if such a modification is a general mechanism for the stabilization of the ligand domain, or if this effect is specific for a subset of the TGF␤ family (Dale et al, 1989;Degnin et al, 2004). We are currently testing the role of glycosylation in regulating the protein stability and signaling strength of the Xenopus Nodalrelated proteins.…”

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo

“…1A) (15). The BMP4 ligand forms a transient, noncovalent complex with the prodomain following cleavage at the S1 site, but subsequent cleavage at the S2 site disrupts the complex, freeing the ligand from the prodomain (20).…”